Poster Sessions

IMMUNOLOGY

ABSTRACTS

Poster Sessions Poster Session: Authophagy P0001 Apoptotic parasites silence macrophages by misusing the autophagy machinery P. Crauwels,* S. Gottwalt,* F. Ja¨ckel,* M. Thomas,* E. Bank,* P. Walther, M. Bastianà & G. van Zandbergen* *Immunology, Paul-Ehlich-Institut, Langen, Germany, Electronmicrosà copy Facility, University Ulm, Ulm, Germany, Veterinary Medicine, Paul-Ehlich-Institut, Langen, Germany Purpose/Objective: An appropriate T cell response to Leishmania (Lm) infection is critical for an effective immune response. Human macrophages (MF) can present antigen to T lymphocytes and at the same time serve as host cells. Upon macrophage infection the virulent inoculum of Lm promastigotes consists of apoptotic and viable promastigotes. The viable promastigotes enter a maturing phagolysosome were they can survive and grow as amastigotes; the fate of apoptotic parasites is unclear. Materials and methods: In this study, we hypothesize that the apoptotic promastigotes use the MFs«autophagy machinery to down regulate MF antigen presentation and T cell activation. Results: Upon promastigote uptake by human primary MFs, we found apoptotic promastigotes to enter a compartment positive for the autophagy marker LC3. This LC3 compartment matured over time and became LAMP positive. 24 h later the compartment resolved after highly efficient parasite degradation. When co-incubated with autologous T lymphocytes, MFs infected with viable promastigotes induced a strong CD4-positive T cell proliferation. Compared to viable parasites a significantly lower T cell reactivity was observed in response to MFs inoculated with apoptotic or a mixed population of apoptotic and viable parasites. Subsequently, preliminary results suggest that only in the presence of apoptotic promastigotes and human T cells Lm infection could be sustained in human MF over a period of 7 days. Conclusions: We found that apoptotic promastigotes enter a maturing LC3 compartment. Our data suggest that degradation of parasites in this compartment could be involved in a down regulation of T cell activation. We now further investigate and characterize the proliferating T cell subsets and how the autophagy machinery and apoptotic promastigotes may dampen immune responses in human primary macrophages.

P0002 Autophagy is activated in the B cells of patients with SLE and correlates with disease activity A. J. Clarke, U. Ellinghaus & T. J. Vyse Medical and Molecular Genetics, King’s College London, London, UK Purpose/Objective: Autophagy is increasingly appreciated as an important immune surveillance and effector mechanism, but understanding of its dynamic function in human autoimmune disease is limited. We sought to evaluate its role in the B and T lymphocytes of patients with SLE compared with healthy controls.

Materials and methods: Patient samples were collected with informed consent, and disease activity quantified by the SELENA-SLEDAI index. Multispectral imaging flow cytometry was performed using an Amnis ImageStreamX instrument. LC3-positive autophagosomes were enumerated in viable, non-apoptotic cells using the Bright Detail Intensity algorithm implemented in IDEAS 6. Autophagic flux was determined by incubation with chloroquine. As an alternative measure of autophagy, uptake of the novel autophagosomotropic dye CytoID (Enzo) was analysed using conventional flow cytometry. Autophagy was assayed in negatively selected B cells stimulated with combinations of anti-IgM and anti-CD40 antibodies, and interferon-a. Results: Autophagy was significantly increased in the CD19+ B cells of patients with SLE compared with healthy controls (P = 95% of larvae are killed by a host granulomatous response within the first 2 weeks of infection. We investigated the early immune events that govern resistance to primary filarial infection. Materials and methods: Experimental B. malayi infections were undertaken by infection with 50 L3 larvae into the peritoneal cavities of interleukin(IL)-4 receptor alpha-/- IL-5-/- mice, Severe Combined ImmunoDeficient (SCID) mice or WT mice all on a BALB/c

2012 The Author(s) 2012 Blackwell Publishing Ltd, Immunology, 137 (Suppl. 1), 185 772

200 Poster Sessions background. Administration of anti-C-C chemokine receptor 3 (CCR3) antibody and clodronate encapsulated liposomes 1 day before infection selectively depleted eosinophils and macrophage populations, respectively. Larvae and peritoneal exudate cells were recovered at between+2 and+14 days. More long-term infections (+12 weeks) were used to examine impact of manipulation of the host response on the establishment of patent adult infections. Results: We report that severely impaired type 2 signalling (IL-4, -5 and -13) in IL-4Ra-/-/IL-5-/- mice produces a susceptible phenotype able to host long-term B. malayi fecund infections both in the peritoneal cavity and lymphatics accompanied by a complete ablation of eosinophils at the infection site. Resistance is dictated by early immune events as significantly higher parasite burdens can be discerned in IL-4Ra-/-/IL-5-/- mice as little as+2d after infection. By tracking larval attrition during the first 2 weeks following temporal ablation of eosinophils or macrophages, we show both cell types are necessary for resistance to early larval establishment. Increased susceptibility is also apparent in SCID mice following anti-CCR3 targeted ablation of eosinophils indicating the mechanism of early larval attrition is independent of adaptive immunity. Conclusions: The presence of both CCR3+ granulocytes and macrophages are important for optimum resistance to larval B. malayi infection. We speculate that recruited CCR3+ eosinophilic granulocytes may mediate larval killing by regulating macrophage effector function.

P0043 Expression of HPV16-E7 oncoprotein in skin exacerbates the contact hypersensitivity response to DNCB A. S. Bergot,* S. Le Tran,* D. Mittal,* M. A. Grimbaldeston & I. H. Frazer* *Epithelial Cancer Division, University of Queensland Diamantina Institute, Brisbane, Qld, Australia, Division of Human Immunology, Centre for Cancer Biology, Adelaide, SA, Australia Purpose/Objective: Cervical cancer is the second most common cancer of women worldwide and is the first cancer described to be entirely induced by a virus, the human papillomavirus. This study is aimed to better understand the immunopathology of pre-cancerous skin lesions associated with HPV infection. Materials and methods: We use C57BL/6 mice expressing HPV16-E7 oncoprotein as a transgene from a keratin 14 promoter in mouse skin (E7 mice). The inflammatory response to DNCB was investigated after application to ear skin of E7 and control mice. Results: E7 mouse ears undergo increased swelling relative to control ears within the first 8 h (early) following DNCB exposure, and also exhibit increased swelling at day 5 (late) which resolves by10 days after DNCB exposure. E7 skin has two to three-times more mast cells than control, and there were more degranulated mast cells in the basal layer of the epidermis. Mast cells in E7 mouse skin were found to have a greater capacity to degranulate during a passive cutaneous anaphylaxis response. The early response induced by DNCB was accompanied by substantial mast cell degranulation in E7 mouse ear skin, which was not observed in control ear skin. The increased late response to DNCB in E7 mice was accompanied by elevated levels of IL-1b and IL-19, but not IL-33, in the skin. Conclusions: E7 transgenic skin shows exaggerated swelling in response to a contact sensitising agent, in association with mast cell degranulation and IL-19 release.We are now investigating a possible link between mast cells and IL-19 in the immunopathology of HPV infected skin, particularly as mast cells have been associated with HPV positive intraepithelial neoplasia in humans, and as IL-19 plays a role in psoriasis lesions where skin thickening similar to that in HPV16-E7 skin is observed.

P0044 Functional responses of basophils to endogenous danger signals C. Stoeckle, A. Thomet, U. Gurzeler, T. Rabachini de Almeida, T. Kaufmann & H. U. Simon Institute of Pharmacology, University of Bern, Bern, Switzerland Purpose/Objective: Cellular injury, necrosis or stress that might occur during inflammatory responses or mechanical injury leads to release of damage associated molecular patterns (DAMPs), intracellular molecules that are normally retained inside the cell. These molecules include ATP, uric acid, S100A proteins and HMGB1, which are interpreted as danger signals by the immune system when present in the extracellular milieu. Several DAMPs have been shown to have differential (usually pro-inflammatory) effects on immune cells, including neutrophils and eosinophils, but whether and how basophils respond to endogenous danger signals is unknown. Since basophils are important players in allergic immune responses and these often involve cellular damage, we aimed to understand how human and mouse basophils react to different DAMPs and how this might contribute to an inflammatory response. Materials and methods: We cultured human ex vivo isolated or mouse in vitro differentiated basophils in the presence or absence of different DAMPs and assessed viability, chemotaxis, surface expression of activation markers CD63 and CD203c, and chemotactic responses. Results: We found that basophils are capable of responding to a variety of different DAMPs and that different DAMPs affect a variety of basophil functions, including survival, migration and cytokine release. Conclusions: Our results suggest that endogenous danger signals modulate inflammation also through their action on basophils.

P0045 Hsp70 and Hsc70 dynamics in human neutrophils under heat shock conditions A. Boyko, A. Sapozhnikov & E. Kovalenko Lab of Cell Interactions, Immunology Department, ShemiakinOvchinnikov Inst of Bioorganic Chemistry RAS, Moscow, Russia Purpose/Objective: Neutrophils are short lived cells characterized by low biosynthetic activity, however they are inducible for synthesis of heat shock proteins 70 kDa (HSP70s) possessing cell protective functions. The aim of this study was to analyze the dynamics of HSP70 content in human neutrophils in response to heat shock (HS), and to investigate factors affecting the dynamics. Materials and methods: Neutrophils isolated by density gradient from heparinized venous blood of healthy volunteers were stained intracellularly with monoclonal antibodies (mAbs) directed to constitutive Hsc70 (SPA815, Stressgen) or inducible Hsp70 (SPA810, Stressgen) proteins, or with HSP70 antibody not distinguished between Hsc70 and Hsp70 (BRM-22, Sigma). Detection of intracellular HSP70 levels was performed by flow cytometry. Results: In most cases after short-term heating (43C, 10 min) we registered an increase and following decrease in 15 30 min after the end of HS of HSP70 levels with all three mAbs (BRM-22, SPA810, SPA815). We did not detect any increase phase by Western blotting. Pre-incubation with cycloheximide, inhibitor of protein synthesis, did not change the dynamics of intracellular HSP70 levels revealed by flow cytometry. This fact indicates that the HSP70 increase phase is not mediated by de novo protein synthesis, but is possibly related to the availability of epitopes of HSP70s for binding with mAbs. The decrease phase of HSP70 level was confirmed by Western blotting normalized with b-actin. This phase can be connected with release of HSP70s into extracellular space phenomenon described mostly for Hsp70. Treatment of neutrophils with glybenclamide, ATP sensitive potassium


Poster Sessions 201 channel blocker, resulted in the HSP70 decrease stage reduction detected with all mAbs suggesting that Hsc70 but not only Hsp70 was released from the cells. Treatment of neutrophils with NH4Cl, lysosomotropic compound, resulted in greater increase of intracellular HSP70 level, possibly, through impairment of vesicle maturation. Conclusions: Thus, our data suggest that both Hsp70 and Hsc70 are released from neutrophils under the HS conditions through the endolysosomal pathway involving ATP-sensitive mechanism.

P0046 Human neutrophils establish cellular interaction with natural killer cells to enhance their interferon-gamma production A. Micheletti,* C. Costantini,* F. Calzetti,* W. Vermi & M. A. Cassatella* *Pathology and Diagnostics, University of Verona, Verona, Italy, Pathology, University of Brescia, Brescia, Italy Purpose/Objective: The role of neutrophils in the orchestration of immune responses has been increasingly recognised by their functional interactions with various immune cells, such as dendritic cells, monocytes as well as lymphocytes. Herein, we aimed at defining whether human neutrophils establish a functional cross-talk with natural killer (NK) cells too. In fact, it has been clearly recognised that NK cell activation is finely tuned by accessory cells which provide them with activating signals such as membrane molecules or soluble factors. Based on these premises, the aims of this study were: (i) to evaluate the ability of human neutrophils to activate NK cells in terms of IFN-c production; (ii) to characterize the cell-cell interactions that could eventually occur between human neutrophils and NK cells. Materials and methods: Human neutrophils and autologous NK cells were isolated from buffy-coats of healthy donors. NK cells were then cultured alone or with neutrophils at 1:1 ratio and stimulated in the presence of LPS in combination with IL-15 and IL-18, for 18 h. IFN-c production was then assessed by ELISA. Cell-cell interactions and cell identity were characterized by confocal microscopy and immunohistochemistry. Results: NK cells showed an enhanced capacity to secrete IFN-c when cultured in the presence of neutrophils under our stimulatory conditions. Such an increased IFN-c production by NK cells was found to be dependent on the contact between the two cell types. By the use of specific neutralizing antibodies, we could then identify the specific membrane molecules involved in the NK cell-neutrophil interactions, specifically the CD11d/CD18 integrin on the NK cell, and the ICAM-3 immunoglobulin family member on neutrophil side. The potential in vivo occurrence of a neutrophil/NK cell cross-talk was uncovered in the lesions of patients with psoriasis, Crohn’s disease and Sweet’s syndrome disease, in which a neutrophil-NK cell colocalization is present. Conclusions: The present work reports the capacity of human neutrophils to potentiate the IFN-c release by NK cells via contactdependent mechanisms. These data may have potential implications in the pathogenesis of diseases where a neutrophil/NK cell colocalization is observed.

P0047 Mast cell-specific NFATc1-deficiency drives overexpression of the transcription factor NFATc2 in murine bone marrow-derived mast cells P. Friedrich, A. Michel, G. Manz & M. Stassen Institute for Immunology, University Medical Center Johannes Gutenberg University, Mainz, Germany Purpose/Objective: To investigate the function of the transcription factor NFATc1 in murine mast cells we generated mast cell-specific

NFATc1-deficient mice by crossing Mcpt5Cre mice with NFATc1fl/fl animals. Materials and methods: We produced bone marrow-derived mast cells from these animals and looked after their ability to express the cytokines IL-6 and IL-9 in vitro. For this we used qRT PCR and ELISA. Via Western Blotting we checked the expression of the transcription factors IRF-4 and NFATc2. Results: According to the expectations based on our previous work, the expression of IL-6 was unaffected in the absence of NFATc1. However, the production of IL-9 was enhanced. Detailed analyses revealed the overexpression of the closely related NFATc2 in NFATc1deficient mast cells. Concomitantly, the expression of the transcription factor IRF-4 and of IL-1b was also strongly enhanced. Thus, the increased expression of IL-9 in the absence of NFATc1 might be explained by the autocrine effect of IL-1b or by IRF-4-mediated transactivation of the IL-9 promotor. How NFATc1 modulates the expression of NFATc2 and the influence of the latter on the production of IRF-4 and IL-1b is currently unknown. Conclusions: The model used in this study to examine the specific function of NFATc1 in mast cells is not suitable, since the absence of this transcription factor can be compensated by an overexpression of NFATc2. These results, however, show a possible influence of NFATc1 on the production of NFATc2 as part of an intracellular network modulating the expression of IL-9.

P0048 Mast cells phagocyte Candida albicans via Toll-like 2 receptor without TNF-a and IL-10 production K. H. Pinke, H. G. Lima & V. S. Lara Department of Stomatology, Area Pathology, Bauru School of Dentistry, University of Saõ Paulo, Bauru, Brazil Purpose/Objective: The pattern recognition receptors of innate immunity are important in defense against pathogens by recognizing of surface molecules and triggering important signaling pathways. Candida albicans (C. albicans) is recognized by cells of innate immunity by Toll-like 2 receptors. Mast cells have this receptor on cellular membrane and its immune mechanisms include synthesis and secretion of mediators, antigen presentation, as well as phagocytic and microbicidal activities. So, this study evaluated in vitro the TNF-a and IL-10 production and phagocytosis by mast cells challenged with C. albicans and the involvement of TLR2 in these mechanisms. Materials and methods: Murine bone marrow cells (BMMC) wild type (TLR2+ /+) or TLR2 knockout (TLR2-/-) were cultured for 21 days in presence of stem cell factor (SCF) and interleukin-3 (IL-3). Mast cells were challenged with FITC-labeled C. albicans by 30 or 60 min and the phagocytosis analyzed by confocal laser scanning microscopy. TNF-a and IL-10 production was measured by ELISA after 24 h of challenge with C. albicans. Results: BMMC TLR2+ /+ did not produce TNF-a or IL-10 after stimulation or not with C. albicans. However, these cells challenged with zymosan produced detectable levels of TNF-a, but not IL-10. After 30 min, 30% of BMMC TLR2+ /+ showed internalized C. albicans, approximately four yeasts per cell. Besides, the BMMC TLR2-/- did not produced TNF-a or IL-10 irrespective of experimental conditions and their phagocytosis was also impaired. After 30 and 60 min, 98.4% and 99.6% of mast cells, respectively, had not internalized fungi. The percentage of zymosan phagocytosis by mast cells was also very low (2.7%). Conclusions: Data demonstrated the phagocytic capacity of BMMC against C. albicans yeasts and the strong involvement of the TLR2 receptor in this mechanism. Still, the absence of TNF-a and IL-10 production after fungal challenge may represent an immune escape of the C. albicans after their internalization by mast cells.


202 Poster Sessions P0049 Neutrophil chemotaxis is impaired in celiac disease K. M. Lammers Mucosal Biology Research Center, University of Maryland School of Medicine, Baltimore, MD, USA Purpose/Objective: Celiac disease (CD) is triggered by the ingestion of gliadin, the immunogenic component of gluten-containing grains. We have observed that exposure to gliadin induces rapid and massive influx of neutrophils to the murine gut mucosa, suggesting it is a chemoattractant factor for neutrophils. Aims of this study; has gliadin also chemoattractant properties for human neutrophils, and do neutrophils from healthy individuals (HC) and CD patients respond differently to gliadin. Materials and methods: Neutrophils were isolated from venous blood of HC and CD patients and applied in an under-agarose assay to monitor neutrophil chemotaxis to pepsin-trypsin-digested gliadin (PTG) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) as a positive control. Resting neutrophils and PTG- or fMLP-stimulated neutrophils were analyzed by flow cytometry for CD62L surface expression. Results: Human neutrophils migrated towards PTG and fMLP. However, compared to the chemotactic response of HC neutrophils to PTG (5.7 ± 1.3 net neutrophil migration), the chemotactic response of CD neutrophils was markedly reduced (0.4 ± 0.3 net neutrophil migration, P < 0.001). A similar, albeit non-significant difference was also observed in CD versus HC neutrophil migration to fMLP (4.8 ± 1.1 versus 11.9 ± 2.9 net neutrophil migration, respectively, P = 0.067). The percentage of CD62L-expressing neutrophils (resting 67 ± 15) diminished after fMLP- (52 ± 22) and PTG-stimulation (44 ± 19) in HC, but remained unchanged in CD (resting 86 ± 5, fMLP (61 ± 24), PTG (90 ± 6). Conclusions: These results suggest that PTG is also a chemoattractant factor for human neutrophils. Compared to HC, the CD neutrophil chemotactic response to PTG and fMLP is impaired in the underagarose assay, suggesting that CD neutrophils have reduced chemotactic potential that possibly involves L-selectin.

Granulocytes; Basophils, Mast Cells Oesinophils P0050 Redox regulation of degranulation in human neutrophils N. Vorobjeva Department Immunology, Lomonosov Moscow State University, Moscow, Russia Purpose/Objective: The NADPH oxidase of neutrophilic granulocytes is a complex enzyme consisting of membrane and cytosolic subunits. After activation of the cells the cytosolic subunits translocate to the membrane where the functioning oxidase complex is formed. The active enzyme carries out the transfer of one electron from cytosolic NADPH oxidase to molecular oxygen, generating thereby superoxide anion in the exterior of the cell or in the phagocytic vacuoles and H+, NADPH+ in the cytosol. It was demonstrated by Jankowski and Grinstein (1999) that NADPH oxidase is able to transfer electrons against a potential gradient. Thus, the activity of NADPH oxidase is not only restricted to destruction of invading microorganisms, but it can rapidly change the membrane potential causing a depolarization. The degranulation plays a principal role in most proinflammatory functions of neutrophils. However the relationships between a membrane potential and degranulation was not investigated completely. In order to investigate the regulatory role of redox potential on degranulation, we tested the inhibitors of NADPH oxidase, diphenylene iodonium (DPI) and apocynin, in the in vitro model of CB/fMLPactivated human neutrophils.

Materials and Methods: Exocytic insertion of CD63 and CD66b into the cell membrane was determined by flow cytometry. Radical oxygen species (ROS) production was measured using luminol chemiluminescence method. Results: Activation of neutrophils with CB/fMLP resulted in a high release of azurophil and specific granules. Adding the DPI in several concentrations before activation of the cells resulted in inhibition of azurophil and specific granules release, while a specific inhibitor of enzyme complex, apocynin, caused a dose-dependent suppression of degranulation of CB/fMLP-activated cells. Conclusions: The present study showed that inhibition of NADPH oxidase, responsible for the depolarization of membranes, resulted in downregulation of degranulation of activated cells that can be caused by the deterioration of fusion proteins interaction with the membranes.

P0051 Role of the CREB transcription factor in inflammatory cytokine production by human neutrophils P. McDonald, F. A. Simard, T. Z. Mayer & A. Cloutier Medicine Faculty Pulmonary Division/Research, Universite´ de Sherbrooke, Sherbrooke, QC, Canada Purpose/Objective: Neutrophils influence innate and adaptative immunity by generating numerous mediators whose regulation largely depends on transcription factors typically associated with inflammation, such as NF-kB and C/EBP factors. Here, we show that this functional response also involves the CREB transcription factor. Materials and methods: Protein analyses by immunoblot and ELISA; gene regulation analyses by EMSA, qPCR, ChIP assays, and luciferase assays. Most studies in primary neutrophils, except those featuring transient overexpressions or luciferase assays, which were carried out in DMSO-differentiated, granulocytic human PLB-985 cells. Results: Neutrophil stimulation with physiological agonists (LPS, TNF) led to a rapid and transient CREB DNA-binding activity, and concurrent phosphorylation of CREB1 on S133. Accordingly, the same stimuli elicited the transactivation of a CREB-driven luciferase reporter in neutrophil-like PLB-985 cells. Functionally, overexpression of a dominant negative CREB mutant (K-CREB) or of a point mutant (S133A) resulted in a decreased ability of neutrophilic PLB-985 cells to generate inflammatory cytokines (CXCL8, CCL3, CCL4, TNFa). In primary neutrophils, CREB DNA binding and S133 phosphorylation were found to occur downstream of the p38 MAPK/MSK1 axis. The parallel phosphorylation of another bZIP transcription factor, C/EBPb, was similarly affected. Inhibition of p38 MAPK or MSK1 prevented cytokine generation in neutrophils, as well as the recruitment of either P-CREB or P-C/EBPb to chemokine promoters in ChIP assays. Conclusions: Collectively, our data show the involvement of CREB in neutrophil cytokine production, the key role of its S133 residue, some of the molcular mechanisms involved, some of the upstream signaling events, and the parrallel activation of another bZIP factor. Our study also identifies potential molecular targets that could be exploited in the context of several chronic inflammatory diseases that prominently feature neutrophils and their products.


Poster Sessions 203 P0052 Signal Inhibitory Receptor on Leukocytes-1 (SIRL-1) negatively regulates the oxidative burst in human phagocytes

P0054 T4 phages head proteins do not induce production of reactive oxygen species by granulocytes

T. A. M. Steevels,* K. van Avondt,* G. H. A. Westerlaken,* J. Walk, L. Bont,à P. J. Cofferà & L. Meyaard* *Immunology, UMC Utrecht, Utrecht, The Netherlands, Pediatrics, UMC Utrecht, Utrecht, The Netherlands, àPediatrics and Immunology, UMC Utrecht, Utrecht, The Netherlands

P. Miernikiewicz, K. Dabrowska, A. Piotrowicz, K. Hodyra, B. Owczarek, G. Figura, A. Kopciuch, K. Macegoniuk & A. Gorski Bacteriophage Laboratory, Institute of Immunology and Experimental Therapy, Wroclaw, Poland

Purpose/Objective: The production of reactive oxygen species (ROS) is an important effector mechanism mediating intracellular killing of microbes by phagocytes. Inappropriate or untimely ROS production can lead to tissue damage, thus tight regulation is essential. We recently characterized Signal Inhibitory Receptor on Leukocytes-1 (SIRL-1) as an inhibitory receptor expressed by human phagocytes. We now set out to study its role in the regulation of these cells. Materials and methods: Primary human monocytes and neutrophils were used to study bacterial killing, oxidative burst and SIRL-1 expression upon activation with various stimuli. SIRL-1 signalling was analyzed in transfectants of wild type and mutated SIRL-1. In a cohort of RSV-infected infants, we studied SIRL-1 expression on neutrophils in BAL and peripheral blood. Results: We demonstrate that ligation of SIRL-1 dampens Fc receptorinduced ROS production in primary human phagocytes. In accordance, SIRL-1 engagement on these cells impairs microbicidal activity of neutrophils, without affecting phagocytosis. The inhibition of ROS production may result from reduced activation of extracellular signalregulated kinase (ERK), since co-ligation of Fc receptors and SIRL-1 on phagocytes inhibited phosphorylation of ERK. Importantly, we demonstrate that microbial and inflammatory stimuli cause rapid down-regulation of SIRL-1 expression on the surface of primary neutrophils and monocytes. In accordance, SIRL-1 expression levels on neutrophils in bronchoalveolar lavage fluid from patients with neutrophilic airway inflammation are greatly reduced. Conclusions: We propose that SIRL-1 on phagocytes sets an activation threshold to prevent inappropriate production of oxygen radicals. Upon infection, SIRL-1 expression is down-regulated, allowing microbial killing and clearance of the pathogen.

P0053 Subpopulations of blood cells forming the mast cells cytes rosettes

lympho-

T. Sydorenko, Y. U. Serezhko & O. Melnikov Immunology and Pathophysiology, Institute of Otolaryngology, Kyiv, Ukraine Purpose/Objective: Lymphoid cells are able to contact with mast cells. Materials and methods: In the investigation the methods of MLRforming and CD-determination were used. Results: Lymphoid cells of peripheral blood in patients with cancer of larynx forms increased number of so-called mast-lymphocytes rosettes (MLR) in comparison with patients with precancer diseases and healthy donors. Herewith the number of CD5+ , CD11+ , CD16+ and CD54+ blood lymphocytes was decreased in patients ofboth groups plus decreased level of CD25+ cells in larynx cancer patients and the number of CD 20+ , CD95+ and CD3+ lymphocytes was (had) at the normal level. Analysis of subpopulations of lymphocytes that forms the contacts with mast cells revealed the predominant participation in MLR-forming CD3+ and CD25+ cells in patients with larynx cancer and CD19+ and CD11+ in patients with precancer diseases. Conclusions: So the subpopulation compositions of lymphoid cells contacting with mast cells are varied in different diseases.

Purpose/Objective: Phages are viruses specific to bacteria. Their application in drug-resistant bacterial infections has become significant alternative for antibiotics. However, phage therapy still evokes discussion due to its safety. Studies of phage influence on immunological system are necessary to provide more profound understanding of their safety and effectiveness. In this work we evaluated the effect of T4 head proteins on reactive oxygen species (ROS) production by granulocytes. Materials and methods: Highly purified, by two-step affinity and sizeexclusion chromatography ( 0.05). Conclusions: Adhesion to type I collagen downregulates MMP-1 and 9 gene expression and secretion from CoMtb stimulated NHBEs. In contrast, TIMP-1 and 2 were unaffected. Therefore, MMP activity depends on ECM-cell interactions which might be important in the regulation of the inflammatory tissue destruction characteristic of TB.

P0385 Respiratory epithelial cells mediate distinct inflammatory responses to different bacterial pathogens M. Melin, A. Vuorela, H. Salo & O. Vaarala Department of Vaccination and Immune Protection, National Institute for Health and Welfare, Helsinki, Finland Purpose/Objective: Streptococcus pneumoniae (Pn), Haemophilus influenzae (Hi), and Moraxella catarrhalis (Mc) are potential respiratory pathogens, which generally colonize the nasopharynx of humans early in life. Carriage of these bacteria decreases with age, but the immunological mechanisms functioning against colonization are largely unknown. IL-17 expressing Th17 cells have been found to mediate mucosal immunity to many bacterial pathogens. In this study we aimed to characterize the cellular immune responses Pnc, Hi and Mc trigger in human cells. Materials and methods: Nasopharyngeal epithelial cells (Detroit 562) were incubated with live bacteria for 1 h. The proportion of adherent bacteria was determined by plate counting. During the next 23 h the epithelial cells were cultured with antibiotics to inhibit bacterial multiplication. Blood peripheral mononuclear cells (PBMCs) collected from healthy adults were incubated with heat-killed bacteria for 96 h. The cytokines (mRNA) expressed by the epithelial and PBM cells were measured by RT-qPCR. Results: Exposure to live bacteria induced a rapid proinflammatory response in the epithelial cells; expression of IL-1a, IL-1b, IL-6, IL-8, and TNF-a, was increased but no significant differences were seen between cells exposed to different bacteria after the first hour. However, after 24 h, epithelial cells exposed to Mc or Hi expressed increased levels of all these cytokines, whereas cells exposed to Pn did not differ significantly from unstimulated cells. The proportion of bacteria binding to the epithelial cells was highest for Mc and Hi. Stimulation of PBMCs with Pn, in contrast to Mc and Hi, induced a marked IL-17 biased immune response coupled with down-regulation of IL-10. Mc induced a stronger IFN-c mediated, Th1-type, proinflammatory response in the PBMCs than Pnc or Hi. Bacterial exposure also induced expression of Foxp3, typically considered a marker for regulatory T cells. Conclusions: The results indicate that Pnc, Hi and Mc induce distinctly different types of immune responses in respiratory epithelial cells and T cells, suggesting that the host immune mechanisms against these pathogens may differ. Airway epithelial cells have the capacity to

modulate the activation of immune cells, which means that the innate responses bacteria induce in epithelial cells can affect the adaptive immune response.

P0386 Role of epithelial E-cadherin in gut immune homeostasis H. Meinicke,* M. Brack,* A. Bremser,* P. Rauf,* H. Pircher, M. P. Stemmlerà & A. Izcue* *Centre of Chronic Immunodeficiency, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany, Immunology, Institute of Medical Microbiology and Hygiene, Freiburg, Germany, àMolecular Embryology, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Purpose/Objective: The transmembrane molecule E-cadherin is an essential component of epithelial junctions. It has emerged that it also plays a role in the immune system, as lymphocytes can express Ecadherin-specific receptors. CD4+ Foxp3+ regulatory T cells (Treg) are enriched for the E-cadherin receptors CD103 and KLRG1, suggesting a link between CD4+ Treg and E-cadherin recognition. As gut Treg are key to prevent intestinal inflammation, we decided to assess the role of intestinal epithelial E-cadherin on gut Treg homeostasis. Materials and methods: To check the effects of lack of intestinal Ecadherin on the immune system we used genetically modified mice. Complete lack of E-cadherin is not compatible with embryonic development, and specific deletion of E-cadherin from the intestinal epithelium also leads to prenatal death. To circumvent this problem, we replaced one allele of E-cadherin by the closely related molecule Ncadherin. The other allele of E-cadherin was specifically deleted on intestinal epithelial cells using a Villin-Cre E-cadherin flox system, so that E-cadherin positive cells throughout the body express both E- and N-cadherin but intestinal epithelial cells only express N-cadherin (gut E-Cad KO mice). We have then analysed the intestinal and systemic immune compartment from these mice using flow cytometry, microscopy and quantitative PCR. Results: Unlike E-cadherin-sufficient littermates, gut-E-Cad KO mice show flora-dependent intestinal inflammation. This was not due to the ectopic N-cadherin expression as N-cadherin transgenic mice harboring one wildtype copy of E-cadherin showed no phenotype. These mice also presented increased CD4+ T cell numbers in the gut. Interestingly, the frequency of Treg among the intestinal CD4+ T cell population was also increased, mostly due to the accumulation of Foxp3+ cells expressing the cadherin receptor KLRG1. The frequency of Treg expressing the E-cadherin receptor CD103 did not change significantly. As a control, we did not find increased KLRG1+ Treg in the gut from IL-10-deficient mice, which also have flora-dependent intestinal inflammation. Conclusions: Mucosal lymphocytes are controlled by local factors, and E-cadherin expressed by gut epithelial cells could be involved in the control of the intestinal Treg pool.

P0388 Soluble factors from alveolar epithelial cells increase intracellular killing using nitric oxide independent pathways D. Petursdottir, O. D. Chuquimia & C. Fernańdez Immunology, Stockholm University, Stockholm, Sweden Purpose/Objective: We have shown that factors from alveolar epithelial cells (AECs) increase macrophage intracellular killing of Bacillus Calmette-Gue´rin (BCG) through a nitric oxide-independent mechanism. We aim to identify a potential mechanism by which this increased killing is mediated. Materials and methods: Bone marrow-derived macrophages (BMM) and pulmonary macrophages (PuM) were isolated from C57BL/6 mice


Poster Sessions 315 and infected with BCG. After a 4 h infection, bacteria were removed and cells incubated further in media with or without IFN-c (20 ng/ml) or supernatants from AEC (AECsup). After culturing, intracellular bacterial killing was assessed, RNA was harvested and mRNA quantified using qPCR. Cytokines were also measured with ELISA. Results: Treating BMM with IFN-c, increased intracellular killing compared with that of cells cultured in medium alone whereas in PuM IFN-c was ineffective. In both cell-types, IFN-c treatment increased transcription of iNOS, IP-10 and IL-12 and secretion of IL-12 and IL6. Transcription of suppressor of cytokine signaling (SOCS)1 was higher in PuM than BMM. SOCS1 expression was important for mediating the ineffectiveness of IFN-c in increasing intracellular killing in PuM, as PuM from SOCS1/IFN-c-/- mice showed enhanced killing after IFN-c treatment. On the other hand, AECsup treatment did not affect iNOS, IP-10 nor IL-12 expression but increased Arg1 transcription and IL-6 secretion. Conclusions: BMM and PuM responded to IFN-c indicating that both cell types possess receptors able to recognize and transmit signals delivered by IFN-c. The only difference was that IFN-c only increased intracellular killing of BCG in BMM but not in PuM. On the other hand, treating cells with AECsup increased intracellular killing of BCG in both types of macrophages. This increased killing was not associated with increases in the pro-inflammatory effectors IL-12 or iNOS, indicating that the mechanism by which intracellular killing is increased after treatment with AECsup is not through an M1 activation pathway.

P0389 Synoviocytes change phenotype and function after Treg-depletion in arthritic mice M. Bo¨ttcher,* C. Wunrau, T. Pap & T. Kamradt* *Institute of Immunology, University Hospital Jena, Jena, Germany, Institute of Experimental Musculoskeletal Medicine, University Hospital Muenster, Muenster, Germany Purpose/Objective: Immunization with Glucose-6-phosphate isomerase (G6PI) induces arthritis in susceptible strains of mice. Depletion of regulatory T cells (Tregs) prior to immunization switches the usually acute, self-limiting course to a non-remitting, destructive arthritis. This provides a good possibility to study relevant molecular switches for the transition from acute, self-limiting to chronic, destructive arthritis within one mouse model. To examine the role of fibroblast-like synoviocytes (FLS), which can support and modulate immune responses via the production of proand anti-inflammatory mediators, the expression of a panel of surface markers characteristic of FLS was determined. Next, the phenotype and function of FLS from mice with either acute, self-limiting or nonremitting, destructive arthritis was studied. Materials and methods: FLS from DBA/1 mice that developed either the acute or the chronic form of arthritis have been isolated from the joints over a time course of 56 days.To investigate the phenotype of FLS flow cytometric methods as well as quantitative realtime-PCR and ELISA studies have been performed. For the functional clarification of those cells the matrix-associated transepithelial resistance invasion (MATRIN) assay and a cartilage attachment assay have been used. Results: It was found that murine FLS stably express several surface markers over several passages in vitro. Furthermore, FLS from Tregdepleted mice produced significantly more cytokines [e.g. Interleukin 6 (IL-6)] upon stimulation with other cytokines, growth factors and TLR ligands. This increased susceptibility to cytokine-stimulation in chronic animals compared to acute ones is observable throughout the disease course (56 days). Additional functional differences include the collagen-destructive potential and the potential to attach and eventually invade wild type cartilage. Here, FLS from Treg-depleted chronic arthritic mice showed a higher invasive and destructive potential.

Conclusions: Our results are compatible with the hypothesis that synoviocytes from Treg-depleted, arthritic mice acquire an autonomously aggressive phenotype that contributes to the switch from acute to chronic arthritis. P0390 The regulatory effect of lactobacilli on Staphylococcus aureus induced inflammatory response in intestinal epithelial cells and immune cells Y. Haileselassie,* M. A. Johansson,* C. Zimmer,* D. H. Petursdottir,* J. Dicksved, M. petersson,à J. O. Persson,à C. Fernandez,* S. Roos, U. Holmlund* & E. Sverremark-Ekstro¨m* *Department of Immunology, Stockholm University, Stockholm, Sweden, Department of Microbiology, Swedish University of Agricultural sciences, Uppsala, Sweden, àDepartment of Mathematics, Stockholm University, Stockholm, Sweden Purpose/Objective: Dysbiosis early in life has been associated with immune mediated disease, such as allergy and inflammatory bowel disease. Previously we have shown differences in the early gut microbiota between children who developed allergic disease at five years of age and children that remained non-allergic. The mechanisms how the microbiota interacts with and influences the immune maturation after birth remain unclear. Here we aimed to investigate how different species of bacteria influence the response of the intestinal epithelial cells (IEC) and the immune cells. Materials and methods: We exposed intestinal epithelial cell (IEC) lines (HT29 and SW480) to culture supernatants from seven Lactobacillus (L.) strains and three Staphylococcus (S.) aureus strains. Further peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated with bacteria conditioned IEC supernatants. The level of cytokines and chemokines in the IEC and PBMC supernatants were analyzed qualitatively and quantitatively using human proteomic array and ELISA. Results: The IEC lines produced a very limited set of cytokines and chemokines following bacterial exposure. Only S. aureus 161.2 induced an inflammatory response by the IEC characterized by the production of CXCL-1 and CXCL-8/IL-8. In PBMC, most of the tested Lactobacillus and Staphylococcus strains were able to induce IL-6 production, but only S. aureus 161.2 induced IFN-c and IL-17. The simultaneous presence of epithelial factors did not significantly alter the response. Notably, the S. aureus induced IFN-c and IL-17 production by PBMC, but not CXCL-8/IL-8 production by IEC, was down regulated by the simultaneous presence of any of the different Lactobacillus strains. Conclusions: S. aureus 161.2 induced a strong inflammatory response in both PBMC and IEC, but there was a limited influence of IEC secreted factors on the PBMC response. Interestingly, Lactobacilli attenuated the S. aureus induced inflammatory response by immune cells, but not that of IECs, indicating a specific regulatory role on immune cells, although the mechanisms need to be further investigated.


316 Poster Sessions P0391 The role of ceramide kinase in regulating LPS and TNF-alfa induced inflammation in A 549 cells V. Hinkovska-Galcheva,* A. J. Morris, S. Van Wayà & J. A. Shayman§ *Internal medicine, University of Michigan, Ann Arbor, MI, USA, Internal medicine-Cardiology Molecullar and Cellular Biochemistry, University of Kentucky, Lexington, KY, USA, àDepartment of Pediatrics, University of Michigan, Ann Arbor, MI, USA, §Department of Internal Medicine-Nephrology, University of Michigan, Ann Arbor, MI, USA Purpose/Objective: Purpose: Activation of Toll-like receptors (TLRs) by specific pathogens leads to the production of proinflammatory mediators to initiate an inflammatory process. This is followed by a counter-regulatory, anti-inflammatory response to prevent excessive damage to the host. Tumor necrosis factor (TNF-a), one of the first cytokines to be released after activation of TLRs, is a key pro-inflammatory cytokine, and was reported that is down regulated by ceramide and its metabolite ceramide-1-phosphate (C1P)(Rozenova K, et al., JBC, 2010; Lamour N, et al., JBC, 2011; Jozefowski et al, J Immun., 2010). Additionally, it has been reported that activation of TLR4 signaling by lipopolysaccharide (LPS) is dependent upon production of ceramide by acid sphingomyelinase (Cuschieri et al., Surg Infect, 2007). We reported recently ceramide-dependent PP2A regulation of TNF-a induced IL-8 production in respiratory epithelial cells (Cornell T, et al., A. J. of Physiology, 2009). Objective: We are studying the regulation of inflammatory response through the action of phosphatases such as PP2A. Specifically, we hypothesized that in respiratory epithelial cells, LPS and TNF-a-induced inflammation is dependent upon activated ceramide kinase (CERK) and the resulting production of ceramide-1phosphate (C1P). Materials and methods: Methods: We used A549 cells stably transfected with hCERK. Additionally, cells were transfected either with siRNA duplex oligonucleotides targeting, CERK, PP2A or PP1 or with

non-targeting siRNA duplex oligonucleotides (control) using Lipofectamine (Invitrogen). Results: Results: Both LPS and TNF-a stimulation of A549 and A549/ hCERK transfected cells increased CERK activity and also increased CERK phosphorylation. Mass spectrometry analysis revealed a significant increase of C1P. CERK activation impacted PP2A expression and activity, and led to changes in the regulation of Il-8 and TNF-a production. We also observed co-localization of CERK and PP2A by co-immunoprecipitation and by confocal microscopy using GFP-PP2A and RFP-CERK over-expression constructs.

Conclusions: Conclusions: CERK appears to be a key enzyme involved in regulating inflammation by increasing C1P and influencing the activity of the regulatory phosphatase, PP2A. These studies were funded by AHA 0930039N.


Poster Sessions 317

Poster session: Ageing P0395 Aged T cell subsets are characterized by a distinct microRNA signature N. Teteloshvili,* A. van den Berg,* R. J. van der Lei,* P. G. Jellema,* N. van der Geest, L. Brouwer, A. M. H. Boots & B. J. Kroesen* *Department of Pathology and Medical Biology, University Medical Center Groningen, Groningen, The Netherlands, Department of Rheumatology and Clinical Immunology, University Medical Center Groningen, Groningen, The Netherlands Purpose/Objective: Decline of immunological responsiveness in elderly is at least in part attributed to changes in the composition of the T cell compartment. Recently, microRNAs (miRNA) have emerged as novel players in the regulation of T cell function. Little is known, however, on the expression of specific miRNAs and their role in altered T cell function with age. Aim of the study was to investigate the age associated changes in miRNA expression within defined T cell subsets in young and old healthy individuals. Materials and methods: T cell subsets (naı¨ve, memory, CD4 and CD8 cells) derived from young and elderly healthy subjects were sorted based on CD3, CD4 and CD45RO expression. RNA was isolated and miRNA expression patterns were determined for pooled T cell subsets (n = 5) using the agilent human miRNA microarray platform (V2) based on Sanger miRbase (release 10.1). Results were validated by qRTPCR. A computational analysis was performed to identify miRNA putative targets and related molecular pathways. Results: Hierarchical clustering showed differential expression of miRNAs mainly between naı¨ve and memory subsets. Age related differential expression was observed predominantly within the naı¨ve CD45RO- T-cell population. Seventeen miRNAs showed at least twofold up- or downregulation in aged naı¨ve T cells. Analysis of individual samples revealed a statistically significant age related upregulation for miR-21, miR-223 and miR-451 by qRT-PCR. Computational analysis revealed putative miRNA targets associated with cell proliferation, apoptosis and insulin signaling pathways. Conclusions: Age-related changes in miRNA expression are found predominantly within the CD45RO-T cell compartment. It remains to be established if the differentially expressed miRNAs identified within the CD45RO- T cell subset in this study, converge with the accumulation of end-differentiated CD45RO- effector T cells reexpressing CD45RA.

P0396 Ageing alters the frequency and function of CD161+ CD8+ T cells K. S. M. van der Geest,* W. H. Abdulahad,* P. L. Chalan,* M. G. Huitema,* B. J. Kroesen, E. Brouwer* & A. M. H. Boots* *Rheumatology and Clinical Immunology, University Medical Center Groningen (UMCG), Groningen, The Netherlands, Medical Biology, University Medical Center Groningen (UMCG), Groningen, The Netherlands Purpose/Objective: Age-associated changes of immune function may affect recognition of pathogens, autoantigens and cancer cells. Ageing is associated with accumulation of T cells which express NK markers. Recently, circulating CD8+ T cells expressing the NK marker CD161 have been identified as potent producers of pro-inflammatory cytokines (IFN-gamma, IL-17) in young individuals. So far, little is known about the influence of ageing on CD161+ CD8+ T cells. Therefore, we determined whether ageing alters the frequency and function of circulating CD161+ CD8+ T cells. Materials and methods: We assessed the relative and absolute number of CD161high and CD161int CD8+ T cells in peripheral blood from 62

healthy donors with increasing age (20 91 years). We analyzed intracellular expression of IFN-gamma and IL-17 by CD161high and CD161int CD8+ T cells from young (20 40 years) and old donors (>60 years) after stimulation with PMA/calcium ionophore + BFA. In the same donors, we also assessed intracellular expression of perforin and granzyme B in unstimulated CD161high and CD161int CD8+ T cells. Results: In general, absolute numbers of CD8+ T cells decreased with age. Both the proportion and absolute number of circulating CD161high CD8+ T cells decreased with age. Whereas the proportion of CD161int cells increased with age, the absolute number of these cells remained stable. In contrast to CD161- CD8+ T cells, nearly all CD161high and CD161int cells produced IFN-gamma, regardless of age. CD161high cells produced IL-17 and expressed the transcription factor RORgt. Interestingly, this potency to produce IL-17 was decreased in old donors. In general, CD161int cells hardly produced IL-17. Both CD161high and CD161int cells expressed perforin. Although the majority of CD161int cells produced granzyme B in young and old donors, granzyme B expressing CD161high cells could be only detected in old donors. Conclusions: We show that the frequency and function of CD161+ CD8+ T cells is altered in non-pathological ageing. Firstly, CD161highCD8+ T cells clearly decrease with age. Secondly, CD161highCD8+ T cells produce less IL-17 in old individuals and become better equiped with cytotoxic effector molecules. How this affects the response to novel pathogens, autoantigens and cancer cells remains to be established. P0397 Ageing shifts the Treg-Th17 balance K. S. M. van der Geest,* W. H. Abdulahad,* M. G. Huitema,* B. J. Kroesen, E. Brouwer* & A. M. H. Boots* *Rheumatology and Clinical Immunology, University Medical Center Groningen (UMCG), Groningen, The Netherlands, Medical Biology, University Medical Center Groningen (UMCG), Groningen, The Netherlands Purpose/Objective: Ageing may affect various lymphocyte subsets, including regulatory T cells (Tregs) and T helper 17 (Th17) cells. Conflicting results on Treg numbers in young and old donors have been reported.Furthermore, whereas various studies in young donors have shown a remarkable plasticity between Tregs and Th17 cells, little is known about the Treg-Th17 balance in healthy elderly people. Therefore, the aim of our study was to determine whether ageing shifts the Treg-Th17 balance. Materials and methods: We assessed the number of naı¨ve (CD25intCD45RA+) and memory (CD25highCD45RA-) Tregs in donors with increasing age. Furthermore, we assessed the propensity for production of IL-17 by memory Tregs and the total CD4+ memory population in young and old donors. Results: First, we found that ageing is associated with a decrease of circulating naı¨ve Tregs. This loss of naı¨ve Tregs in the elderly could be entirely attributed to the loss of CD31+ recent thymic emigrant Tregs. Unlike naı¨ve Tregs, the number of circulating memory Tregs increased with age. Next, we found that a small fraction of memory Tregs was able to produce IL-17, but this was lower in the elderly compared to young donors. In fact, the total CD4+ memory population from older donors produced less IL-17 compared to young donors, and an increased memory Treg/Th17 ratio was observed in the elderly. Conclusions: In conclusion, the number of circulating naı¨ve Tregs gradually decreases with age, as less recent thymic emigrant Tregs are present in the elderly. In contrast, the combined observation of increased numbers of memory Tregs and decreased numbers of Th17 cells tilts the balance towards Tregs in healthy elderly people. This finding would be in line with previous studies indicating that anti-


318 Poster Sessions inflammatory immune responses are essential to preserve health in old age by neutralizing the pro-inflammatory state that tends to develop later in life (inflamm-ageing).

P0399 Altered regulation of Lck in T cells with aging T. Fulop, H. Garneau, A. Le Page, C. Fortin, A. Larbi, G. Dupuis, N. Allard & K. Tsvetkova Universite´ de Sherbrooke, Medicine, Sherbrooke, QC, Canada Purpose/Objective: Our aim was to study whther the tyrosine phosphatase SHP-1, a key regulator of T cell signal transduction machinery is, at least in part, responsible for the impaired T cell activation in aging. Materials and methods: T cells separated from young and elderly subjects. T cell stimulated via TCR. Use of WB, confocal microscopy, FACScan, biochemical enzyme assays and thymidine incorporation assay. Results: We showed that a dysregulation of the Csk/PAG loop in activated T cells from elderly individuals favored the inactive form of phosphorylated Lck (Tyr505). Dynamic movements of these regulatory proteins in lipid raft microdomains was also altered in T cells of aged individuals. We showed that SHP-1 activity was upregulated in T cells of aged donors compared to young subjects. Pharmacological inhibition of SHP-1 resulted in recovery of TCR/CD28-dependent lymphocyte proliferation and IL-2 production of aged individuals to levels not significantly different than those of young donors. Furthermore, we report differences in the active (Y394) and inactive (Y505) phosphorylated forms of Lck in response to T cell activation in elderly donors. Conclusions: Our data suggest that the regulatory role of SHP-1 in T cell activation extends to its involvement in Lck-dependent negative feedback in aging. Modulation of SHP-1 activity could restore altered T cell functions in aging, suggesting a powerful tool for improvement of immunosenescence.

P0400 CMV-specific T cells with highly effector capacity are associated with 2-years all-cause mortality in healthy elderly individuals S. Ferrando-Martinez,* E. Ruiz-Mateos, R. S. de Pablo-Bernal, J. Delgado,à R. Solana,§ M. A. Munõz-Fernandez* & M. Leal *Gregorio Maranõn University Hospital, Laboratory of Molecular Immuno-Biology, Madrid, Spain, Biomedicine Institute of Sevilla, Laboratory of Immunovirology, Sevilla, Spain, àHospital San Juan de Dios del Aljarafe, Internal Medicine, Sevilla, Spain, §Department of Immunology IMIBIC, Reina Sofia University Hospital, Cordoba, Spain Purpose/Objective: To analyze CMV-specific T cell response in healthy elderly and its relatioship with human mortality. Materials and methods: Sixty-seven subjects of the CARRERITAS cohort were selected according with the following inclusion criteria: (1) older than 50 years old, (2) without hospitalization during the last years, (3) without active infections, (4) without comorbidities or pharmacological treatment affecting the immune sistem and (5) positive IgG serology for cytomegalovirus (CMV) infection. T cell CMV-specific response was analyzed by multiparametric flow cytometry (IFNg, TNFa, IL2, MIP1a, CD107a and PRF1 production in response to the pp65 peptide pool). Results: CMV-specific T cell response polyfunctionality was decreased in non-survivors. In addition, this subjects showed increased percentages of highly effector T cells expressing CD107a and perforine (CD107a+PRF1+) without IFNg coexpression. The CD107a+PRF1a+ subset was increased in both, CD4 and CD8 T cells and was correlated with higher percentages of CD57-expressing T cells. A multivariate analysis showed that percentages of CD8 T cells expressing

CD107a+PRF1+ (but not CD4 T cells nor the age) were independently associated with time to death. Conclusions: Aberrant accumulation of the CMV-specific CD8+PRF1a+ CD107a+ T cell response is associated with lower human survival. This population could be a a predictive marker of a immune collapse.

P0402 Cytokine production by monocytes from elderly patients with Candida-associated denture stomatitis H. G. Lima,* P. Freitas-Faria,* K. H. Pinke,* M. P. P. Nascimento,* V. C. Porto & V. S. Lara* *Department of Stomatology Area Pathology, Bauru School of Dentistry University of Saõ Paulo, Bauru, Brazil, Department of Prosthodontics, Bauru School of Dentistry University of Saõ Paulo, Bauru, Brazil Purpose/Objective: This study aimed to evaluate the cytokine production by monocytes, challenged in vitro with C. albicans, obtained from peripheral blood of elderly denture wearers with denture stomatitis (DS), compared with elderly denture wearers without DS and elderly and young non-denture wearers. Materials and methods: The isolated monocytes were cultivated in 24-well flat-bottomed culture plates, in the absence or presence of lipopolysaccharide (LPS) or heat-killed C. albicans ATCC 90028. After 18 h, the supernatant was collected and submitted to the enzymelinked immunosorbent assay (ELISA) for determination of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-a), interleukin-6 (IL-6), CXCL8, IL-1b, monocyte chemotactic protein-1 (MCP-1) and anti-inflammatory cytokines IL-10 and transforming growth factor-b (TGF-b). Results: The results demonstrated, in general, changes in monocytes from the elderly with DS, as compared to other groups: lower spontaneous production of CXCL8 and MCP-1; lower levels of TNF-a, IL-6, IL-1b, CXCL8, MCP-1 and IL-10 after stimulation with LPS; and reduced production of TNF-a and IL-6 after stimulation with C. albicans. Comparing young and old, regardless of the presence of DS, the results revealed changes in the monocytes of the elderly: a lower production of TGF-b, spontaneous and after stimulation with C. albicans. Conclusions: In conclusion, the dysfunctional in vitro production of pro- and anti-inflammatory cytokines by monocytes from elderly patients with DS may represent an additional aspect associated with susceptibility to the development and to the persistence of DS. Still, the dysfunction in monocytes of elderly groups, regarding the in vitro production of TGF-b, may represent aspects related to immunosenescence.

P0403 Cytomegalovirus (CMV) dependent and independent changes in the ageing of the human immune system: a transcriptomic analysis S. Marttila,* T. Kuparinen,* J. Jylha¨va¨,* L. Tserel, P. Peterson, A. Hervonen,à M. Jylha¨à & M. Hurme* *Department of Microbiology and Immunology, The School of Medicine, University of Tampere, Tampere, Finland, Institute of General and Molecular Pathology, University of Tartu, Tartu, Estonia, àThe School of Health Sciences, University of Tampere, Tampere, Finland Purpose/Objective: Ageing is associated with profound decline of the immune capacity, which is manifested as increased morbidity and mortality. It has been proposed that chronic antigen stress, mainly from persistent CMV infection, may have a causative role in immunosenescence. The persistent CMV infection has for example been associated with accumulation of exhausted, non-proliferative CD8+ cells that lack the expression of costimulatory receptors CD27 and


Poster Sessions 319 CD28, a hallmark of decreased immune competence. However, the number of individuals affected by CMV increases with increasing age, so that the great majority of the elderly are affected by CMV. Thus it can be difficult to establish which ageing-associated changes are due to increased CMV prevalence and which are only due to increased chronological age. We now wanted to identify genes and pathways that are affected with ageing independently of CMV infection. Materials and methods: We performed a transcriptomic analysis of peripheral blood mononuclear cells (PBMCs) with Illumina Human HT12 v4 BeadChip in a cohort of CMV seropositive (CMV+, n = 140) and seronegative (CMV-, n = 6) nonagenarians using CMV seronegative young individuals (n = 11, 19 30 years of age) as controls. Data was analysed with Chipster (CSC) and the affected pathways were identified with IPA software (Ingenuity Systems). Results: Our results show that the gene expression profiles of CMV-and CMV+ nonagenarians are different from each other. When comparing to the CMV-controls we found 667 and 559 genes to be differentially expressed in CMV- and CMV+ nonagenarians, respectively; 333 of these were common to both groups. Similarly, there were differences in canonical pathways affected; 45 and 16 pathways were affected in CMV- and CMV+ nonagenarians, respectively, and 10 of the pathways were common. Interestingly, NK-cell and TCR-signalling pathways were affected only in the CMV- nonagenarians. The up regulation of pro-inflammatory genes and pathways was however common to both groups. Conclusions: Our data indicate that the CMV dependent and independent changes in the ageing of the immune system are fundamentally different. The results imply that inflamm-aging is independent from CMV infection, but that the cell-mediated immune functions, mediated by T cells and NK cells, are different depending on the CMV serostatus.

P0404 Effects of prolonged intense exercise on the adaptive immune response in elderly and young athletes M. A. Moro-Garcıá,* R. Alonso-Arias,* B. Fernańdez-Garcıá & C. Lo´pez-Larrea* *Hospital Universitario Central de Asturias, Immunology, Oviedo, Spain, Department of Morphology and Cell Biology, Universidad de Oviedo, Oviedo, Spain Purpose/Objective: Exercise induces a series of changes on the immune system depending on their intensity and duration. In fact, transient states of immunosuppression are induced after intense physical activity and yet beneficial exercise anti-inflammatory effects have been described over many diseases and longevity. Materials and methods: To study the impact of intense exercise for long periods of life on adaptive immunity at different ages we compared phenotypical and functional features of T lymphocytes of young (n = 27) and elderly athletes (n = 15) with young (n = 30) and elderly non-athletes (n = 30). We characterized leukocyte and lymphocyte subpopulations by flow cytometry and measured the T cell proliferation and activation response (CD69) against anti-CD3. We also studied the percentage of recent thymic emigrants (RTEs) by quantification of TREC by real-time PCR. Specific antibody titers against CMV were determined by ELISA. Results: Leucopenia was found in both groups of athletes, mainly explained by low levels of neutrophils and lymphocytes. Exercise induced higher frequencies of NK, B lymphocytes and CD8+ T cells, whereas CD4+ T lymphocytes showed significant lower levels in the elderly athletes. Moreover, young athletes showed significant differences in all parameters that define the immune risk profile (IRP), with characteristics of an aging immune system, but we did not find differences between elderly groups. Less differentiated subsets of T lymphocytes were more frequents in non-athletes, with the exception

of CD8+ T lymphocytes in young individuals. The analysis of TREC content in the elderly groups showed no significant difference in either the CD4+ or CD8+ T lymphocytes. In the young non-athletes group we observe an increase in the content of TREC in CD8+ T cells, but not in CD4+ T cells. Moreover, functional response of CD4+ and CD8+ T lymphocytes was significantly impaired only in young but no in elderly athletes. Conclusions: Intensive training for long periods throughout life induces important phenotypical and functional changes on the adaptive immune response. These changes are most striking in young individuals and they damped with physiologic immune aging.

P0405 Intensity of the humoral response to CMV throughout the life configure phenotypical and functional status of the immune system in elderly R. Alonso-Arias, M. A. Moro-Garcıá, A. Echeverrıá-de Carlos & C. Lo´pez-Larrea Hospital Universitario Central de Asturias, Immunology, Oviedo, Spain Purpose/Objective: CMV infection exerts an enormous impact on human immunity being associated with an immune impaired response and also with a variety of chronic diseases and the overall survival in elderly individuals. History of CMV infection, and not only infection per se, may be determining the changes induced on the immune response. Materials and methods: To study the impact of titers of anti-CMV antibodies on immune system we compared phenotypical and functional features of T lymphocytes of 92 healthy elderly donors and 70 young healthy controls. We characterized lymphocyte subpopulations by flow cytometry and measured the T cell proliferation and activation response (CD69) against CMV. We also studied the percentage of recent thymic emigrants (RTEs) by quantification of TREC by real-time PCR. Specific antibody titers against CMV were determined by ELISA. Results: Titers of anti-CMV antibodies was used as a measure of accumulated antibodies throughout the life, and in fact titers were significantly higher in elderly and correlated positively with the specific CD4+ T cells responses to CMV. In elderly, the antibody titers were associated with the differentiation degree and the TREC content in CD4+ T cells, were correlated to other parameters belonged to the IRP and emerged as a conditioning factor over the ability to respond to immunization in vivo. Lack of correlations in young subjects may be due to lower anti-CMV antibody titers that they show. However, at similar levels of antibodies differences in highly differentiated and naı¨ve T cells between young and elders were accentuated as titers increase. The reduction in absolute counts of naı¨ve CD4+ T cells, also observed in individuals with higher titers, may be acting as a strategy to compensate the expansion of differentiated cells and to avoid the increase of total T cells. Conclusions: In summary, our data show that titers of anti-CMV antibodies and not only CMV-seropositivity influence the differentiated status and the immunocompetence in the elderly, emerging as an important target in the improvement of the immune system function in elderly.


320 Poster Sessions P0406 Long term injected with D-galactose altered the immune responses in C57BL/6J mice

P0410 Renal replacement therapy enhances CD8 T cell differentiation in young end-stage renal disease patients

J. Liau* & M. L. Chen *Chang Jung Christian University, Graduate Institute of Medical Sciences, Tainan, Taiwan, Department of Nutrition and Health Sciences, Chang Jung Christian University, Tainan, Taiwan

R. W. J. Meijers,* N. H. R. Litjens,* L. E. A. de Wit,* A. W. Langerak, A. van der Spek, C. C. Baan,* W. Weimar* & M. G. H. Betjes* *Erasmus Medical Center, Internal Medicine, Rotterdam, The Netherlands, Erasmus Medical Center, Immunology, Rotterdam, The Netherlands

Purpose/Objective: Age-related changes of the immune system contribute to the increased susceptibility of elderly persons to infectious diseases. Mice chronically injected with D-galactose (D-gal) have been used as an animal aging model. However, less attention has been paid to the changes of systemic immune responses on D-gal induced aging mice. Materials and methods: To investigate the changes of cytokines on Dgal injected mice. The C57BL/6J mice of model control (MC), Gal-100, Gal-150 and Gal-500 groups were subcutaneous injected with 0, 100, 150 and 500 mg/kg/day for 8 weeks. The brain and supernatants form immune cell culture were collected for cytokines assay. Results: Our results indicated that Ab were significantly increased in Gal-500 group, opposite to the activity of the mice. Furthermore, 150 mg/kg/day D-gal injected mice had higher cytokines production, especially TNF-a level in brain homogenates, but not in Gal-500 group. Moreover, D-gal injection toward decreased the IL-2 secretion, and increased the IL-4 production by splenocytes. Conclusions: This study demonstrated that mice injected with 150 mg/kg/day D-gal could induce inflammatory cytokines production in brain, but not affect by 500 mg/kg/day injection group. There need more studies to confirm the mechanism of immune responses in D-gal induced aging mice.

P0407 Long term injected with D-galactose altered the immune responses in C57BL6J mice C. C. Liau* & M. L. Chen *Chang Jung Christian University, Graduate Institute of Medical Sciences, Tainan, Taiwan, Department of Nutrition and Health Sciences, Chang Jung Christian University, Tainan, Taiwan Purpose/Objective: Age-related changes of the immune system contribute to the increased susceptibility of elderly persons to infectious diseases. Mice chronically injected with D-galactose (D-gal) have been used as an animal aging model. However, less attention has been paid to the changes of systemic immune responses on D-gal induced aging mice. Materials and methods: To investigate the changes of cytokines on Dgal injected mice. The C57BL/6J mice of model control (MC), Gal-100, Gal-150 and Gal-500 groups were subcutaneous injected with 0, 100, 150 and 500 mg/kg/day for 8 weeks. The brain and supernatants form immune cell culture were collected for cytokines assay. Results: Our results indicated that Ab were significantly increased in Gal-500 group, opposite to the activity of the mice. Furthermore, 150 mg/kg/day D-gal injected mice had higher cytokines production, especially TNF-a level in brain homogenates, but not in Gal-500 group. Moreover, D-gal injection toward decreased the IL-2 secretion, and increased the IL-4 production by splenocytes. Conclusions: This study demonstrated that mice injected with 150 mg/kg/day D-gal could induce inflammatory cytokines production in brain, but not affect by 500 mg/kg/day injection group. There need more studies to confirm the mechanism of immune responses in D-gal induced aging mice.

Purpose/Objective: End-stage renal disease (ESRD) patients have a defective T cell mediated immune system, which is related to excessive premature immunological ageing. The aim of this study is to examine whether this premature ageing is changed by renal replacement therapy (RRT). Materials and methods: For this purpose, we studied circulating T cells of healthy individuals (n = 60), ESRD patients without RRT (n = 30, eGFR15 years ago, 19 healthy, age-matched controls (HC) and nine HC aged >60 years were included. Indicators of T-cell-immunosenescence were studied: proportions of peripheral CD45RA+ CD27+ CCR7+ naı¨ve T-cells, the T-cell-receptor-excision-circles (TRECs) as markers for recent thymic emigrants (RTE, CD45RA+ CD127+ CD31+), Ki67expression as a marker of peripheral replication, T-cell-receptor (TCR) diversity and IL-7 as a proliferative cytokine for T-cells.

Results: Naive T-cells of TP were significantly decreased compared to HC but still higher than HC > 60 years. In TP, total counts of RTE were almost equal to HC > 60 years. Proportions of intestinal derived CD127+ CD103+ naı¨ve CD8+ T-cells were significantly increased in TP compared to HC and HC > 60 years. TP demonstrated significantly lower TRECs in naı¨ve T-cells which correlated with time post thymectomy and a higher percentage of Ki67-expresssing naı¨ve T-cells. IL-7 levels of TP were similar to HC > 60 years. TP showed less polycloncal TCR distribution than HC. Conclusions: The findings indicate that changes of the peripheral naı¨ve T-cell subset in TP may resemble the findings of immunosenescent elderly persons after thymic involution. The peripheral naı¨ve Tcell-homeostasis after thymectomy is maintained mainly by extrathymic expansion of pre-existing naı¨ve T-cells to compensate the diminished thymic output. The pathophysiological role of these alterations, such as infectious complications or autoimmunity in later life, has to be determined in long-term follow-up.


Poster Sessions 323

Poster Session: Antigen Receptors: Specificities, Repertoires & Functions P0417 Characterization of three new HLA-A alleles S. I. Lozano-Ramos,* J. Gil, E. Campos, C. Ambros, P. Caro, S. Mantecon, L. Mongay, P. A. Correa,à M. J. Herrero, R. Colobran§ & E. Palou *Immunologia-LIRAD, Germans Trias I Pujol Hospital, Badalona, Spain, Immunologia-LIRAD, Blood Tissue Bank Germans Trias I Pujol Hospital, Badalona, Spain, àImmunologia-LIRAD, Blood Tissue Bank Germans Trias i Pujol Biomedical Research Institute Foundation, Badalona, Spain, § Immunologia-LIRAD, Blood and Tissue Bank Germans Trias i Pujol Biomedical Research Institute Foundation Vall D¢Hebron University Hospital Research Institute Foundation, Badalona, Spain Purpose/Objective: During the routine in the HLA laboratory is common to find some patients or donors who don«t exactly match with the databases of HLA. The characterization of new alleles is a very necessary task in the improvement on haematopoietic transplant, solid organ transplantation. In this report we describe the identification of three new HLA-A alleles found in three individuals typed in our lab. Materials and methods: The new alleles were detected during routine HLA typing by SBT, whose results showed a mismatched position with a known allele in each case. All three HLA-A alleles were cloned and nucleotide sequenced in isolation. Results: The first new allele is equal to A*68:02:01:01 but with one nucleotide change at position 127 in exon 2 resulting in a coding change at codon 19 (GAG’AAG) with the replacement of a glutamic acid (E) by a lysine (K). In the protein structure this amino acid residue is in a beta sheet in the a1 domain. The nucleotide sequence is available at Genbank, accession number JQ815887, andtheWHO Nomenclature Committee assigned the name A*68:92. The second allele is identical to A*29:02:01:01 except for one nucleotide change in the 188 position in exon 2 resulting in a coding change at codon 39 (GACˆ GGC) with the replacement of an aspartic acid (D) by a glycine (G). This position codifies a residue located in a loop between two beta sheets of the a1 domain which interacts with the peptide and with the TCR. The nucleotide sequence is available at Genbank, accession number JQ815888, and the WHO Nomenclature Committee assigned the name A*29:38. The last one is equal to A*23:01:01:01 with one nucleotide change in position 615 resulting in a silent mutation at codon 181 (CGCˆCGT) which codifies an arginine. In this case this allele was found in a LAL patient who was candidate for haematopoietic stem cell transplantation and who shares the mutation with her father. The nucleotide sequence is available at Genbank, accession number JQ815889, and the WHO Nomenclature Committee assigned the name A*23:01:09. Conclusions: We have described three new HLA-A alleles, found in different individuals: two of them carry amino acidic changes in the a1 domain, A*68:92 and A*29:38, while the last one shows a silent change, A*23:01:09.

P0419 IFI16 is an early sensor of Listeria monocytogenes DNA in the cytosol stimulating IFN-beta expression K. Hansen,* S. B. Jensen,* T. A. Decker, K. A. Horan* & S. R. Paludan* *Institute of Biomedicine, Aarhus University, Aarhus, Denmark, Department of Microbiology Immunology and Genetics, University of Vienna, Vienna, Austria Purpose/Objective: Infection with Listeria monocytogenes (L. monocytogenes) induces a potent innate immune response. Induction of the

immune response is dependent on bacterial secretion of listeriolysin O (LLO) which mediates the escape of L. monocytogenes to the cytosol after entry by phagocytosis. Previous studies have shown L. monocytogenes DNA and cyclic dinucleotide monophosphates secreted by bacteria as potent inducers of IFN-b. IFI16 is a predominantly nuclear protein which distributes to the cytosol upon viral infection and UV irradiation. Recent studies show that IFI16 directly interacts with cytosolic DNA to induce IFNb. Materials and methods: THP1, a non-adherent monocyte-like cell line differentiated into macrophage-like cells was used for all studies. L. monocytogenes homogenates were prepared by sonication of bacterial overnight cultures and treated with RNase or DNase before transfection into cells. Short-hairpin-RNA was used for knock-down of the intracellular DNA sensor IFI16. Co-localisation between bacterial DNA and IFI16 was investigated by confocal microscopy. Results: In THP-1 cells L. monocytogenes-induced a potent IFN-b expression. This induction was dependent on bacterial escape into the cytoplasm. Short-hairpin-RNA knock-down of the intracellular DNA sensor IFI16 strongly reduced L. monocytogenes-induced IFN-b expression indicating a role of IFI16 in the sensing of L. monocytogenes. Transfection with L. monocytogenes homogenates induced IFN-b expression in differentiated THP1 cells, and this was sensitive to DNase treatment of the homogenates. Furthermore, we observed L. monocytogenes DNA present in the cytoplasm during infection and this colocalized with IFI16 and the adaptor molecule STING. Conclusions: Based on these results we conclude that IFI16 is a sensor of L. monocytogenes DNA in macrophages stimulating IFN-b expression.

P0420 MHC Class I-restricted recognition of Immunodominant viral or tumoral epitopes by human CD4 T cells X. Saulquin, A. Arnold Leger, L. Gautreau, F. Legoux & M. Bonneville INSERM U892, Centre de Recherche en Cance´rologie, Nantes, France Purpose/Objective: It is generally considered that MHC class I-restricted antigens are recognized by CD8+ T cells whereas MHC-class IIrestricted antigens are recognized by CD4 T cells. Several examples of MHC class-I restricted CD4+ T cells have been reported but the functional relevance of MHC I crossreactvity of the CD4 T cell compartment has not been assessed yet. This has been addressed through the systematic analysis of human HLA-A*0201-restricted CD4 T cells directed against various immunodominant viral or tumoral antigens. Materials and methods: ex vivo frequencies of human CD4+ T cells specific for several viral or tumoral A2/peptide complexes were assessed through a pMHC multimer-based enrichment approach in immune or non-immune donors. MHC class I-restricted CD4 T cell lines and clones directed against (liste des *pitopes ELA, PGT, pp65, HCV1 E´) were generated and analyzed for specificity, functional avidity and ability to recognize various target cell lines in vitro. Results: CD4+ T cells directed against every pMHC class I complexes tested were detected in all donors. CD4 T cell lines and clones recognized target cell lines in vitro in a peptide MHC-class I dependent but CD4 coreceptor-independent manner. While these cells were seldom overrepresented in immune donors, they expressed TCR of very high affinity and showed a different cytokine production profile compared to their CD8 T cells counterparts. Conclusions: This study shows that MHC I-crossreactive CD4 T cells, though present in all individuals, are scarce and not usually engaged in adaptive immune responses against the corresponding pMHC class I complex. Nevertheless, these cells represent a natural source of high affinity TCR that could be exploited for TCR gene transfer strategies.


324 Poster Sessions P0421 Recruitment and phosphorylation of the subsynaptic pool of LAT at the immune synapse requires the SNARE protein VAMP7 C. Hivroz,* P. Larghi,* D. Williamson, K. Chemin,* J. M. Carpier,* S. Dogniaux,* K. Gaus & T. Gallià *Inserm U932, Institut Curie, Paris, France, Cellular Membrane Biology Group, Centre for Vascular Research, Kensington, Australia, àInstitut Jacques Monod, Mtnem Inserm ERL U950, Paris, France Purpose/Objective: The LAT adaptor plays a crucial role in T cell antigen receptor (TCR) induced signalling. Two pools of LAT are present in T cells, one at the plasma membrane and one in intracellular sub synaptic vesicles. Recent results have shown that TCR activation results in the recruitment and phosphorylation of LAT from these sub synaptic vesicles. This raises the question of the mechanisms involved in the recruitment and fusion of these intracellular LAT pools. The purpose of this study was to characterize the mechanisms involved in the docking of the intracellular pool of LAT. We herein investigated the role of the vesicular SNARE protein, VAMP7, in the docking of LAT containing sub synaptic vesicles at the plasma membrane and in TCR signalling. Materials and methods: The role of VAMP7 in LAT recruitment at the immune synapse was investigated using high resolution imaging techniques, including total internal reflection fluorescence microscopy (TIRFM), and photo-activatable localization microscopy (PALM). This was performed in Jurkat T cells silenced for VAMP7 expression using a lentivirus encoding two different shRNAs specific for VAMP7 and a control shRNA. Signalling in T cells was analyzed by Western blot analysis as well as by purification of signalosomes induced by TCR activation. Some of the results were reproduced in primary CD4+ T cells from VAMP7 KO mice. Results: Our results show that VAMP7 decorated vesicles are recruited and fuse at the plasma membrane of T lymphocytes upon TCR triggering. Moreover, they show that VAMP7 co-localizes with the intracellular pool of LAT and is required for LAT recruitment and phosphorylation at the immune synapse. Finally, analysis of the TCR induced signalling in T cells invalidated for VAMP7 expression reveals that the LAT containing signalosome induced by TCR triggering, does not form correctly but that yet some signalling occurs, i.e. ZAP70 is phosphorylated as well as SLP76. Conclusions: Our data show that upon TCR triggering the VAMP7 SNARE protein controls docking and fusion of pre-existing intracellular pools of LAT at the immune synapse and that these events are required to induce the formation of signalling complexes. One implication of our results is that control of recruitment and fusion of endocytic compartments plays a crucial role in TCR signalling. Another implication is that the TCR signalling cascade is not linear but rather involved recruitment at the right place of components of the signalling pathway. These results thus suggest that imbalances in signalling regulation might be due to impaired intracellular trafficking of signalling molecules.

P0422 TCR or BCR CDR3 spectratypes differ between blood and spleen and between old and young patients, and can be used to detect myelodysplastic syndrome

responding lymphocytes. There are several millions of different CDR3 sequences of the TCR Vb chain in human blood and about the same number of B cell clones. CDR3 length distributions (also called spectratypes) show variations between individuals and over time. However, the complexity of CDR3 length distribution patterns and the large amount of information included in each sample (e.g. 32 length distributions of the TCRa chain, and 24 length distributions of TCRb) calls for the use of machine learning tools for full exploration. Materials and methods: We have used supervised machine learning methods to analyze CDR3 length distributions from a variety of sources. Results: We have found that the splenic B cell CDR3 length distributions are characterized by low standard deviations and few local maxima, compared to peripheral blood distributions. In addition, we show that healthy elderly people’s B cell CDR3 length distributions can be distinguished from those of the young. Finally, we show that a machine learning model based on TCR CDR3 distribution features, mainly on TCRVb22, can detect the myelodysplastic syndrome (MDS) with approximately 93% accuracy. Conclusions: In summary, supervised learning models, based on a selection of distribution based features, may facilitate the use of CDR3 spectratyping as a monitoring tool for the health of the immune system.

P0423 The influenza nucleoprotein-specific memory B-cell pool is composed of multiple clonotypes with little clonal hypermutation S. Reiche, Y. Dwai & C. Jassoy Institute for Virology, University of Leipzig, Leipzig, Germany Purpose/Objective: Immunization against influenza virus induces a pauciclonal expansion of antigen-specific B-cells. The resulting clonal plasma cell pools contain a highly diverse spectrum of somatically mutated cells. To examine how the antigen-specific B-cell diversity is maintained, we examined the repertoire of influenza nucleoprotein (NP)-specific memory B-cells 2 weeks after vaccination. Materials and methods: Influenza NP-specific memory B-cells were isolated in a multi-step process from peripheral blood using proteincoated magnetic beads. The purity of the NP-specific B-cells was determined by ELISpot. Immunoglobulin G variable regions were amplified from single B-cells by RT-PCR, sequenced, cloned and expressed as recombinant monoclonal antibodies. Antigen specificity was determined by ELISA. Results: Influenza NP-specific B-cell preparations were highly pure. More than 60 different clonotypes belonging to IGHV subgroups 1, 3, 4, 5, and 7 were identified in three subjects. The clonotype repertoire varied significantly between the subjects. Somatically mutated variants were detected for a minority of the clonotypes. Several memory B-cells had low numbers of somatic mutations. Conclusions: Large numbers of different clonotypes and few somatically mutated variants indicate that the memory B-cell repertoire is primarily composed of different clonotypes rather than somatically mutated variants. Additionally, despite probable multiple exposures to influenza virus the vaccination induced several new influenza nucleoprotein-specific memory B-cell clonotypes in all donors.

R. Mehr,* Y. M. Pickman* & D. Dunn-Walters *The Mina & Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel, Department of Immunobiology, School of Medicine Guy’s Campus, Kings’ College London, London, UK Purpose/Objective: Complementarity determining region 3 (CDR3) is the most hyper-variable region in B cell receptor (BCR) and T cell receptor (TCR) genes, and the most critical structure in antigen recognition and thereby in determining the fates of developing and


Poster Sessions 325 P0424 Titer and avidity of IgG antibodies against Toxoplasma gondii and conventional PCR in patients with ocular diseases L. C. de Mattos,* A. I. C. Ferreira,* C. C. Brandaõ de Mattos,* F. B. Frederico, G. C. Almeida Jr, C. S. Meiraà & V. L. PereiraChioccolaà *Molecular Biology, Faculdade de Medicina de Sao Jose do Rio Preto, Sao Jose do Rio Preto, Brazil, Hospital de Base FUNFARME, Ophthalmology Outpatient Clinic, Sao Jose do Rio Preto, Brazil, àParasitology, Insituto Adolfo Lutz, Sao Jose do Rio Preto, Brazil Purpose/Objective: To evaluate titers and avidity of IgG antibodies against Toxoplasma gondii and the presence of genomic DNA of T. gondii in peripheral blood of patients with toxoplasmic retinochoroiditis versus other ocular diseases. Materials and methods: The sera of ocular disease patients were analyzed by indirect immunofluorescence, enzyme immunoassay and

avidity tests and genomic DNA was investigated by conventional PCR. Statistical analysis used Fisher’s exact test and the unpaired t test. Results: Of the total sample, 65.7% (163/248) of patients were positive by ELISA and 34.3% (85/248) were non-reactive for IgG antibodies against Toxoplasma gondii. According to indirect immunofluorescence, titers of anti-T. gondii antibodies higher than 4000 were more common in patients with toxoplasmic retinochoroiditis compared to patients with other ocular diseases (8.1% versus 1.0%). No patients had IgM antibodies. Avidity of more than 60 prevailed in both groups. PCR identified genomic DNA more commonly in patients with toxoplasmic retinochoroiditis (21/62 33.9%) than in those with other ocular diseases (1/101 0.9%). Conclusions: Toxoplasmic retinochoroiditis is common in patients with ocular diseases. Most patients with toxoplasmic retinochoroiditis have lower titers of highly avid IgG antibodies against T. gondii. PCR results suggest that T. gondii is found circulating in blood regardless of the presence of ocular lesions related to toxoplasmosis.


326 Poster Sessions

Poster Session: B Cells & Plasma Cells P0425 A crucial function of the ligand-binding domain of Siglec-G, an inhibitory receptor of B1 cells ¨ zgo¨r & L. Nitschke S. Hutzler, L. O Department of Biology, University of Erlangen-Nuremberg, Erlangen, Germany Purpose/Objective: Siglec-G is a transmembrane protein of the Siglec family, which inhibits BCR signalling specifically on B1 cells. With it first extracellular Ig-like domain Siglec-G binds a2,3- and a2,6-linked sialic acids. These ligand interactions can take place to glycoproteins either on the same cell surface (in cis) or to ligands on other cell surfaces (in trans). We wanted to analyse whether the ligand-binding domain is important for the biological function of Siglec-G. Materials and methods: We generated a Siglec-G R120E knockin mouse with a mutation in the critical amino acid involved in ligandbinding. We also developed a monoclonal anti-Siglec-G antibody, which did not exist so far, to study the Siglec-G expression pattern. Results: Siglec-G R120E mice showed a large increase of the B1 cell population in the peritoneal cavity and in the spleen. B1 cells of these mice showed a better survival in vitro and expressed a changed BCR repertoire. Furthermore, Siglec-G R120E mice showed increased levels of serum IgM and increased Ca2+ responses of their B1 cells. These phenotypes strongly resemble the phenotypes of Siglec-G-deficient mice. With our new anti-Siglec-G antibody we detected a quite uniform expression pattern on B cell subsets, therefore the B1-cell specific functions of Siglec-G cannot be attributed to a higher expression pattern on the surface of B1 cells, when compared to conventional B2 cells. Conclusions: These experiments demonstrate an essential role for the ligand-binding of Siglec-G. We presume that the binding to sialic acid containing cis-ligands on the B1-cell surface regulates its inhibitory signalling function.

P0426 A role for IgG1 in immunity to Salmonella infection Y. Zhang,* S. Essex,* N. Cumley, J. Heath,* C. MacLennan,* S. Kracker,à R. May, C. Buckley,* A. Cunningham* & K. M. Toellner* *MRC Centre for Immune Regulation College of Medical and Dental Science, The University of Birmingham, Birmingham, UK, College of Bioscience, The University of Birmingham, Birmingham, UK, àDeutsches Rheuma-Forschungszentrum (DRFZ), Berlin, Germany Purpose/Objective: The immune response to Salmonella infection initially is characterized by Th1 type cellular immunity, while germinal center and high affinity antibody development is delayed. Long-term protection from infection is mediated by humoral immunity. In mice the dominant Salmonella-specific antibody isotype is IgG2a, whereas IgG1 is only a minor component of the primary response. IgG2a dominance suggests that this isotype is most adapted to control challenge with Salmonella. Here, we have assessed whether IgG1 plays a significant role in immunity to Salmonella typhimurium (Stm) challenge. Materials and methods: IgG1-deficient or wild-type mice were vaccinated with Salmonella outer membrane proteins. Five weeks later, mice were infected with high dose of Salmonella (SL3261). Complement dependent bacterial killing assay and phagocytosis assay were used to further detect the role of IgG1 for Salmonella infection. Results: While wild type mice are protected at this stage, IgG1deficient mice had much higher bacterial burdens in livers and spleens. Protection from Salmonella infection inversely correlated with the

concentration of Salmonella-specific IgG1. This was not due to deficiencies in high affinity antibody production: Total antigen-specific IgG levels were similar to wild type mice, due to compensatory production of IgG2a and IgG3. Experiments with haptenated protein show that absence of high-affinity IgG1 is compensated by maturation of high-affinity antibody of other IgG subclasses. T cell memory and polarization of T cells in the early secondary response is also not affected, nor is the capacity to promote complement-dependent lysis. In vivo and in vitro experiments show that protection by opsonisation of Salmonella with immune serum and antibody mediatedSalmonella uptake by macrophages is severely reduced. Conclusions: IgG1 leads to compensatory Ig class switching to other affinity matured IgG classes, but these do not efficiently protect from Salmonella reinfection. IgG1 is mediating efficient opsonization and macrophage uptake, while no effect is seen from IgG1 deficiency on complement dependent lysis or on T cell responses.

P0427 A spontaneous mutation with low B cell phenotype discovered during the analysis of the function of Themis2 in B cells H. Hartweger,* M. Peirce, V. Peperzak,à D. Tarlintonà & V. Tybulewicz* *Immune Cell Biology, MRC National Institute for Medical Research, London, UK, Kennedy Institute of Rheumatology, University of Oxford, London, UK, àImmunology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Purpose/Objective: Thymocyte-expressed molecule involved in selection (Themis) is expressed in T lymphocytes and has recently been reported to have a role in thymocyte positive selection. Themis2, sharing similar domains and high sequence similarity with Themis, is expressed in B lymphocytes, macrophages and dendritic cells. We hypothesized that Themis2 might have an analogous role in B cell development or activation to Themis in T cells and therefore created a Themis2-deficient mouse strain. During the generation of this strain we serendipitously found a spontaneous mutation leading to the loss of most mature B cells. Materials and methods: We derived mice from a Themis2-targeted KOMP-CSD embryonic stem cell clone. These mice have been bred to recombinase bearing deleter-strains to obtain Themis2-deficient animals, which were analysed for Themis2 expression as well as defects in B cell development or activation. Results: We show that Themis2 is expressed in all populations of B cells analysed, with lowest levels in germinal centre B cells and recently activated B cells. Redundant expression of other Themis family members was not found in any B cell population, either in wildtype or Themis2-deficient mice. Analysis of Themis2-deficient mice has confirmed complete ablation of Themis2. Preliminary results indicate no defects in B cell development or activation or after immunisation with T-dependent antigens. During the analysis of these mice however, we observed a defect in early B cell development in the bone marrow, which did not correlate with the Themis2 genotype. Further breeding experiments indicate that this defect is segregating freely from Themis2 with an X-linked recessive, Mendelian inheritance pattern. Sanger-sequencing of cDNA from these mice of the coding sequence of Bruton’s tyrosine kinase, the most likely candidate for the spontaneous mutation, did not reveal any mutations. Conclusions: The ubiquitous expression of Themis2 in the B cell compartment and its transcriptional regulation after activation suggests a yet unknown role for Themis2 in B cell biology. Currently, we plan to further analyse these mice for defects in immune responses or B cell activation in response to various, other stimuli. We will use exome sequencing in order to pinpoint the spontaneous mutation, which we have termed atropos.


Poster Sessions 327 P0428 Accumulation of circulating autoreactive naı¨ve B cells reveal defects of early B cell tolerance checkpoints in patients with ¨gren’s syndrome Sjo E. Corsiero,* N. Sutcliffe,* H. Wardemann, C. Pitzalis* & M. Bombardieri* *EMR, William Harvey Research Institute, London, UK, Molecular Immunology Group, Max Planck Institute for Infection Biology, Berlin, Germany Purpose/Objective: Sjo¨gren’s syndrome (SS) is an autoimmune disease characterized by high affinity circulating autoantibodies and characteristic B cell disturbances with a predominance of naı¨ve and a reduction of memory B cells in the periphery. It is unknown at what stages of B cell differentiation tolerance checkpoints are defective in SS. Here we aimed to determine the frequency of self-reactive and polyreactive B cells in the circulating naı¨ve compartment of SS patients. Materials and methods: Single IgD+CD27- naı¨ve B cells were FACS sorted from 4 SS patients and RNA used to amplify Ig VH and VL genes which were then cloned and expressed as recombinant monoclonal antibodies displaying an identical specificity of the original B cells. B cells from two healthy donors (HDs) were used as control. Recombinant antibodies were tested towards different autoantigens to determine the frequency of autoreactive and polyreactive clones. Results: A total of 66 individual recombinant antibodies were generated from naı¨ve B cells of SS patients. Analysis of the VH and VL gene usage showed no significant differences between SS and HD. Our data showed increased reactivity towards ANA (43% SS clones versus 25% HD clones) and ENA (19% SS clones) but not increased polyreactivity in peripheral naı¨ve B cells from SS patients, demonstrating an accumulation of autoreactive naı¨ve B cells in the periphery of SS. Conclusions: Here using a novel strategy to express recombinant antibodies from single B cells we demonstrated an elevated frequency of autoreactive naı¨ve B cells in the circulation of SS patients strongly suggesting the existence of early defects in B cell tolerance checkpoints in SS.

P0429 Alpha-1-antitrypsin promotes B regulatory lymphocytes P. Cal, M. Mizrahi, D. Ochayon & E. C. Lewis Clinical Biochimestry, Ben Gurion University of the Negev, Beer Sheva, Israel Purpose/Objective: B lymphocytes appear to play a role in autoimmune diabetes and also during islet allograft rejection. B regulatory lymphocytes, on the other hand, protect non-obese diabetic (NOD) mice from diabetes in an IL-10-dependent mechanism and also promote T regulatory cells. Alpha-1-antitrypsin (hAAT), an acute phase anti-inflammatory protein, induces immune tolerance during islet allograft transplantation in a mechanism that involves T regulatory cells. Recent findings suggest that B cells are required for hAATmediated protection of islet allografts. Aim: Examine whether hAAT increases the proportion of B regulatory cells in culture and in various animal models. Materials and methods: Splenocytes from GFP-IL-10 transgenic mice were added stimulatory concentrations of CD40 ligand (CD40L), LPS and B cell activating factor (BAFF), in the presence or absence of hAAT (0.5 mg/ml). Cell-specific IL-10 production was evaluated by Flow Cytometry. GFP-IL-10 transgenic mice were treated with hAAT (60 mg/kg) and then grafted with allogeneic skin. The proportion of B regulatory cell population out of B cells was evaluated in draining lymph nodes (DLN) 7 days later by Flow Cytometry. Peritoneal cavity cell populations were assessed in hAAT transgenic mice (hAAT+/+) and

wild-type mice during non-sterile inflammation, as provoked by cecal puncture. All surgical procedures were SHAM-controlled. Results: In cultures stimulated with CD40L, LPS and BAFF, hAAT caused IL-10-positive B regulatory cell population size to increase 1.57 ± 0.06-, 1.5 ± 0.03- and 1.34 ± 0.04-fold, respectively (P < 0.001). In skin-grafted hAAT-treated mice, IL-10-producing B regulatory cell population size increased 3.06 ± 0.12-fold compared to skin-grafted untreated mice. hAAT+/+ mice responded to non-sterile inflammation with a 3.56 ± 0.65-fold increase in peritoneal IL-10positive B regulatory cells compared to wild-type mice. Conclusions: B regulatory cells may play a role in the immunoregulatory activity of hAAT. Further studies are required to elucidate the mechanism of B regulatory expansion by hAAT.

P0430 Analysis of CD22 knockin mice with mutated ligand-binding and signalling domains J. Mu¨ller, M. Woehner, I. Obermeier & L. Nitschke Biology Chair of Genetics, University of Erlangen-Nuremberg, Erlangen, Germany Purpose/Objective: CD22 is a transmembrane protein, which mediates the inhibiton of the calcium signal after BCR crosslinking. The extracellular domain of CD22 can bind to its ligands a2,6-linked sialic acids, while the cytoplasmic domain contains inhibitory motifs. The purpose of this study was to analyse the ligand-binding and signalling domain of CD22 independently and thereby determine their functional interplay. Materials and methods: We generated CD22-R130E knockin mice, which have a mutated binding pocket in the extracellular domain of CD22. These molecules are no longer able to bind their ligand a2,6linked sialic acid. We also generated CD22-Y5,6F and CD22-Y2,5,6F mice with mutated ITIM-domains, so that the inhibitory SHP-1 phosphatase is no longer able to bind the CD22 cytoplasmic tail. Results: In the bone marrow (BM) we found a reduction of recirculating B cells in the CD22-Y5,6F and CD22-Y2,5,6F mice similar to the phenotype of CD22-deficient mice. In contrast CD22R130E mice did not show this phenotype. In the spleen all mice showed a reduced marginal zone B cell population. Similar to CD22deficient B cells, only CD22-Y2, 5, 6F B cells showed increased turnover, as determined by BrdU incorporation in vivo. Ca2+ measurements revealed a decreased flux after BCR stimulation in CD22R130E B cells, whereas the CD22-Y5,6F and CD22-Y2,5,6F B cells showed a higher Ca2+ flux. The CD22-R130E mutation affected the CD22 association to the BCR. Conclusions: These data indicate that the maintenance of B cells in the marginal zone is dependent on the ligand-binding of CD22, as well as on the CD22 mediated signalling. However the reduction of recirculating B cells in the BM seems to be caused by the mutations in the ITIM domains of CD22. We could find a increased turnover rate, despite lower B cell numbers in the bone marrow and therefore conclude that B cells with defect signalling domains have a higher apoptose rate. These cells undergo apoptosis before they recirculate into the BM. The calcium flux after BCR crosslinking seems to be mediated by both the ligand-binding domain as well as by the signalling tail of CD22. In summary we found an important role for both functional domains of CD22 considering B cell homing, apoptosis and signalling.


328 Poster Sessions P0432 B cell response to pneumococcal RrgA and RrgB antigens and its relationship with carriage in children and adults M. S. Ahmed,* A. Kasbekar, C. Loh, S. Leong,à M. McCormick,à M. Barocchi,§ V. Masignani,§ B. Flannagan* & Q. Zhang* *Clinical Infection Microbiology and Immunology, Institute of Infection and Global Health, Liverpool, UK, ENT, Alder Hey Children’s Hospital, Liverpool, UK, àENT, Royal Liverpool and Broadgreen University Hospital, Liverpool, UK, §Vaccinology, Novartis Vaccine, Sienna, Italy Purpose/Objective: Streptococcus pneumoniae remains a leading cause of childhood morbidity and mortality around the globe, even after three decades of 23-valent polysaccharide (PPV23) and one decade of 7-valent conjugate vaccines (PCV7) have been available in the market. Pneumococcal pilus plays an important role in their adherence to host respiratory epithelium. Pilus proteins (RrgA and RrgB) were found to be associated with increased bacterial colonization and invasiveness and a heightened inflammatory response in the host. We are investigating the B cell response to pilus proteins (RrgA and RrgB), developed in consequence to natural colonisation of pneumococcus;whether this priming can sufficiently induce immunological memory to protect against pneumococcal carriage and the potential of these proteins as mucosal vaccines in children. Materials and methods: Adenotonsillar tissues, peripheral blood and nasal swab samples were collected from patients undergoing adenotonsillectomy. Nasal swab was cultured for pneumococcal carriage. Adenotonsillar mononuclear cells (MNC) were cultured with or without concentrated pneumococcal culture supernatants (CCS), to assess memory B cell responses by ELISPOT and to detect antibody responses by ELISA. Serum and salivary antibodies to pneumococcal RrgA and RrgB pilus proteins were measured by ELISA and by Western Blot. Results: Preliminary results suggest that, pneumococcal colonisation is common in young children, which tends to decline with advancing age. The natural immunity to RrgA and RrgB proteins mount with progression of age. Anti-RrgA response seemed to develop earlier than anti-RrgB. Conclusions: Serum antibody titres were higher in culture-negative children than those who were culture-positive for pneumococcus, suggesting that these antibodies are protective against pneumococcal carriage.

P0433 B-cell exposure to self-antigen induces regulatory B cells as well as IL-6- and TNF-a-producing B cell phenotypes in healthy humans B. Kristensen,* A. Langkjær,* B. E. Hansen,* H. Schultz,* L. Hegedu¨s & C. H. Nielsen* *Infection Medicine and Rheumatology Clinic, Institute for Inflammation Research, Copenhagen, Denmark, Department of Endocrinology and Metabolism, Odense University Hospital, Odense, Denmark Purpose/Objective: The ability of human B cells to secrete IL-10 after stimulation with self-antigens and to regulate self-antigen-induced Tcell responses in humans is still unclear. The present study was undertaken in order to determine whether exposure of normal human B cells to human thyroglobulin, a self-antigen with relevance to autoimmune thyroid disease, induced a regulatory phenotype or a proinflammatory phenotype in subsets of B or CD4+ T cells. Materials and methods: Thyroglobulin-pulsed B cells were isolated from healthy donors and co-cultured with autologous T cells. The production of IL-10 and TGF-b, in addition to the pro-inflammatory cytokines, TNF-a and IL-6 were observed and recorded. Results: Pulsing with foreign antigen, tetanus toxoid, induced a Th1response with minimal IL-10. Pulsing of B cells with thyroglobulin

stimulated 1.10 ± 0.50% of B cells and 1.00 ± 0.20% of CD4+ T cells for IL-10 production, compared to 0.29 ± 0.19% of B cells (P = 0.01) and 0.13 ± 0.15% of CD4+ T cells (P = 0.006) following TT-pulsing. Thyroglobulin-stimulated IL-10 secreting B cells were enriched within CD5+ and CD24high cells. While thyroglobulin-pulsed B cells induced only modest proliferation of CD4+ T cells whereas B cells pulsed with TT induced vigorous proliferation. Conclusions: Thus, B cells mediate self-antigen specific IL-10, TNF-a and IL-6 production in co-cultures with T cells and contributes actively to secretion of these cytokines.

P0434 B1 cells but not B2 cells exert regulatory activities through cellcell contact independent pathway S. Y. Lin & B. L. Chiang Graduate Institute of Immunology and Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Purpose/Objective: B1 cells which are distinguished from conventional B2 cells belong to a special peripheral B lymphocyte subset and are mainly found in peritoneal and pleural cavities. Since B1 cells constitutively produce natural IgM in the absence of exogenous antigenic stimulation, they are thought to be parts of innate immunity. The basal level of IL-10 by B1 cells increases in response to stimulation with lipopolysaccharides or CpG. On the other hand, B2 cells could not produce IL-10 actively until they interact with T cells, stimulate with Ig and CD40, or contact with Toll-like receptor ligands. Based on the different characters of these two B cell populations, we speculate that B1 cells might play a regulatory role to suppress proliferation of T cells. In this study, we like to investigate further on the regulatory activities exerted by both B1 and B2 cells. Materials and methods: We used wild type BALB/c mice and C.129P2 (B6)-Il10tm1Cgn/J mice, which are IL-10 knock-out mice, to test whether CD90.2-CD5+ B1 cells and B220+ B2 cells could suppress proliferation of CD4+ T cells which are activated by anti-CD3 and antiCD28 antibodies. Moreover, we used transwell experiment to clarify whether the immunosuppressive function of B1 cells is dependent on cell-cell contact. Naı¨ve T cells were labeled with CFSE. These T cells activated by anti-CD3 and anti-CD28 antibodies cultured with B1 cells or B2 cells with transwell study or not. After 3 days, T cells were separated from B cells and analyzed by FACScan as well as [3H] thymidine incorporation. The cultured supernatants were collected to measure the cytokine profiles by ELISA. Results: Allogeneic stimulated proliferation of T cells was significantly suppressed by wild type B1 cells cultured supernatant. Neither wild type B2 cells cultured supernatant nor B1 and B2 cells cultured supernatant from IL-10 knock-out mice could suppress T cells proliferation. In addition, B1 cells could significantly inhibit T cells proliferation even with transwell experiment. The cytokines productions of T-B2 co-culture were IL-10, IL-2 and IFN-c while the cytokines productions of T-B1 co-culture were abundant IL-10 and some IFN-c. Conclusions: These data suggested that B1 cells might play a regulatory role to suppress T cells proliferation by secretion of IL-10 and it is cell-cell contact independent. In the future, B1 cells might be studied further for their application to modulate immunological diseases.


Poster Sessions 329 P0435 Bcl3 overexpression leads to loss of marginal zone B cells N. Ho¨velmeyer, M. Wo¨rns, P. Adams & A. Waisman Institute for Moleculare Medicine, Mainz, Germany Purpose/Objective: B cell homeostasis is regulated by multiple signaling processes including BAFF- B cell receptor and NF-kB signaling. Bcl-3 is a nuclear member of the IjB family that was originally identified as an oncogene in a subset of patients with chronic lymphatic leukemia. After nuclear translocation, Bcl3 associates with the NF-jB subunits p50 and p52 thereby enhancing cell proliferation and carcinogenesis through activation of cyclin D1 expression. Thus, Bcl-3 acts mainly as an oncogenic coactivator of NF-jB, although it is also able to repress NF-jB target gene transcription. Bcl-3 is required for lymphoid organogenesis and germinal center responses since mice deficient for Bcl-3 are immunodeficient due to microarchitectural defects of the lymphoid organs. Materials and methods: We used a mouse strain allowing for the conditional and tissue specific overexpression of Bcl-3 upon Cremediated recombination. The coding sequence of Bcl-3 was inserted into the ROSA26 locus under control of the CMV early enhancer/ chicken b actin promoter. Expression of Bcl-3 is prevented by a loxP flanked transcriptional STOP cassette. The compound overexpression of Bcl-3 and eGFP is permitted only after Cre-mediated recombination of the transcriptional STOP cassette in cells and tissues in which Cre is expressed.We used FACS analysis, ELISA and different immunizations protocols to investigate the role of Bcl3 overexpressin in B cells. Results: Mice overexpressing Bcl3 appeared normal with a slight increased spleen at the age of 8 weeks. This increasement becomes more pronounced with age. FACS analysis revealed an accumulation of follicular B cells while the marginal zone B cells are absent. Further, B cells isolated from Bcl3BOE mice show reduced proliferative capacity upon a-IgM and LPS stimulation. But these B cells keep the ability to prolong survival in vitro. Moreover, T dependent immunization in Bcl3BOE mice revealed increased numbers of germinal center B cells. Conclusions: Our results show a crucial role of Bcl-3 in survival of follicular B cells and terminal B cell differentiation.

P0436 Conjugate vaccination generates isotype-switched memory B-cells which depend on bystander T-cell help for their activation E. Clarke,* N. Williams,* R. Heyderman & A. Finn* *School of Cellular and Molecular Medicine, University of Bristol, Bristol, UK, Malawi-Liverpool Wellcome Trust Clinical Research Programme, University of Malawi College of Medicine, Blantyre, Malawi Purpose/Objective: Polysaccharide (PS) conjugate vaccines generate PS-specific memory B-cells (BMEM) through the recruitment of CD4+ T-cells, upon which the induction of BMEM depends, via a conjugated protein (PT). However, in isolation the BMEM thus generated appear unable to sustain immune protection following the waning of the initial serum antibody response. We have sought to establish whether this fundamental limitation of the conjugate vaccines reflects the ineffective activation of the BMEM population as a result of the lack of cognate T-cell help subsequently available to them. Materials and methods: A group of healthy adults (n = 20) who had previously been vaccinated routinely with a Men C conjugate vaccine were boosted with a Men C * tetanus toxoid (TT) conjugate vaccine. Men C and TT specific BMEM in the circulation were enumerated using a memory ELISpot assay. The role of bystander T-cells and other noncognate stimuli in driving the differentiation of BMEM into Pc was subsequently established using antigen stimulation experiments employing both immunomagnetic cell separation and transwell experiments.

Results: Conjugate vaccination generated IgG+CD27+ BMEM but not IgM+CD27+ BMEM specific for Men C. We demonstrate that both the Men C and TT-specific BMEM, generated following the administration of the conjugate vaccine continue to require CD4+ T-cells in order to differentiate effectively into plasma cells.Nonetheless, bystander T-cells are able to provide such signals to the PS-specific BMEM with comparable effect to the cognate T-cell help available solely to the TT-specific BMEM population. Heat-killed meningococci drive the differentiation of the PS-specific BMEM through bystander T-cell activation which is further enhanced by non-cognate T-cell independent innate signals. The bystander effects of T-cells activated by the conjugated TT within the vaccine were also demonstrated on BMEM in vivo. Conclusions: These data support the hypothesis that the differentiation of PS-specific BMEM, at the time of bacterial encounter, depends on the capacity of bacterial PT to effectively activate bystander T-cells. Thus, priming such responses through including bacterial PT within conjugate vaccine preparations should be further evaluated.

P0437 CpG-ODN induces PD-L1 expression on human B cells and CpG ODN -treated B cells decreased IL-5 and IL-13 production from antigen-stimulated human CD4+ cells S. Kubo,* T. Yamada,* K. Ougi & S. Fujieda* *Otolaryngology, Fukui University, Yoshida-gun Fukui Pref., Japan, Otolaryngology, Shinseikai Toyama Hospital, Toyama Pref., Japan Purpose/Objective: Co-stimulatory molecules are important for regulating T cell activation and immune response. Programmed death ligand 1 (PD-L1) also known as CD274 or B7-H1 has emerged as an important immune modulator that can block T cell receptor signaling. We investigated the effect of CpG-ODN on the human B cells in vitro. The expression of co-stimulatory molecule of B cells and its function were analyzed. Materials and methods: B cells and T cells were separated from human peripheral blood mononuclear cells with magnetic beads. We have investigated whether PD-L1, and other costimulatory ligands could be expressed in human B cells stimulated by CpG-DNA. We sought to determine the effect of CpG-DNA-treated B cells on T helper 2 (Th2) cytokine production in Cry j 1 (Japanese pollen antigen)stimulated human CD4-positive cells from patients with seasonal allergic rhinitis caused by Japanese cedar pollen. Results: CpG-DNA strongly induced the coinhibitory molecule ligand, PD-L1 of human B cells. Results show that nuclear factorkappa B (NF-jB) signaling is involved directly in CpG-DNA- induced PD-L1 expression in human B cells. CpG-DNA-treated B cells reduced Cry j 1-induced IL-5 and IL-13 production in CD4-positive cells. When the binding of PD-1 to PD-L1 was inhibited by PD-1-Ig, this chimera-molecule reversed the previously described reductions in IL5- and IL-13-production. In contrast, the CpG-B-treated B cells increased both IFN-c and IL-12 production in the presence of Cry j 1stimulated CD4-positive cells. CpG-DNA simultaneously reduced the expression of B7RP-1 (also known as inducible costimulator ligand (ICOSL), B7-H2) and the ligand of CD30 (CD30L). Conclusions: CpG ODN increased PD-L1-expression. CpG ODN treated B cells decreased IL-5 production from antigen-stimulated human CD4+ cells. These results suggested that the treatment of CpGDNA suppressed antigen-specific IL-5 production via PD-1- PD-L1 ligation. This study reinforces the idea of CpG-DNA being a potential therapeutic modality through B cells and its signaling pathway being a target for drug interventions against allergic diseases.


330 Poster Sessions P0438 Dietary omega-3 fatty acids induce a stronger B cell response in mice with antigen-induced inflammation S. Thorleifsdottir,* V. Tomasdottir,* A. Vikingsson, I. Hardardottirà & J. Freysdottir* *Departmen of Immunology and Centre for Rheumatology Research, Faculty of Medicine University of Iceland, Landspitali The National University Hospital of Iceland, Reykjavik, Iceland, Centre for Rheumatology Research, Landspitali The National University Hospital of Iceland, Reykjavik, Iceland, àBiochemistry and Molecular Biology, Faculty of Medicine University of Iceland, Reykjavik, Iceland Purpose/Objective: Omega-3 fatty acids may, in addition to their anti-inflammatory effects, affect resolution of inflammation. They are substrates for specialized pro-resolving lipid mediators that block production of pro-inflammatory mediators, inhibit infiltration of polymorphic neutrophils and stimulate phagocytosis by macrophages. The effects of omega-3 fatty acids on the adaptive phase of the resolution of inflammation has not been much studied. The objective of this study was to determine the effects of omega-3 fatty acids on the adaptive immune response in antigen-induced peritonitis. Materials and methods: Female C57BL/6 mice were fed diets without or with 2.8% fish oil for 4 weeks. Peritonitis was induced by vaccination with mBSA in Freund«s adjuvant followed by an i.p. injection of mBSA. The mice were euthanized before and 1, 2, 5 and 10 days after mBSA injection. mBSA-specific antibodies in serum were determined by ELISA, peritoneal B cells analyzed by flow cytometry and germinal center B cells and mBSA in spleen evaluated by staining cryosectioned spleen sections. Results: Germinal centers increased in size and number following administration of mBSA. There were more germinal centers and the germinal centers were larger in spleen from mice fed the omega-3 fatty acid diet than in mice fed the control diet. Serum levels of mBSA specific IgG and IgM antibodies increased slightly following mBSA administration. Mice fed the omega-3 fatty acids had higher serum levels of mBSA specific IgM antibodies than mice fed the control diet. However, serum levels of IgG were similar in the two dietary groups. There were more peritoneal B cells in mice fed the omega-3 fatty acid diet than in mice fed the control diet. The amount of mBSA present in the spleen increased in both groups, reaching maximum 2 days after administration of mBSA. Mice fed the omega-3 fatty acid diet had less mBSA in their spleen on day 2 than mice fed the control diet. Conclusions: These data indicate that the adaptive B cell response is more intense, with increased number of B cells, increased levels of mBSA specific IgM antibodies and increased number and size of splenic germinal centers in mice fed the fish oil diet than those fed the control diet. Therefore, dietary fish oil may enhance B cell response in antigen-induced inflammation and provide better protection in future encounters with the antigen.

P0439 Differential regulation of IRF4 during extrafollicular B cell differentiation, and its role for Ig class switching L. A. George, Y. Zhang, K. Nakamura, I. MacLennan, G. Anderson, P. Lane, W. Jenkinson, J. Marshall & K. M. Toellner Immunity and Infection, University of Birmingham, Birmingham, UK Purpose/Objective: The transcription factor IRF4 is essential for immunoglobulin class switch recombination (CSR) and plasma cell differentiation. Whilst high levels of IRF4 induce terminal differentiation to plasma cells, low-level expression of IRF4 is associated with CSR. Our aim was to study the kinetics of IRF4 induction and dissect the role of high and low-level IRF4 expression in CSR.

Materials and methods: To study IRF4 induction, we immunised quasi monoclonal (QM) mice, which have a high frequency of B cells specific for the model antigen 4-hydroxy-3-nitrophenylacetyl (NP), with NP conjugated to Ficoll (NP-Ficoll). This induces B cell activation with CSR, followed by parallel differentiation of both plasma cells and T-independent germinal centre B cells. To test the role of IRF4 for Ig class switching in different phases of the antibody response, mice with NP-specific B cell receptors deficient in NFjB1 or overexpressing mir125 were produced. Results: We show that low-level expression of IRF4 is induced within minutes of B cell activation. These become B blasts that undergo CSR. Three days after immunisation, B cells differentiate into germinal centre cells that lose IRF4 expression or extrafollicular plasmablasts. Plasmablasts express high levels of IRF4 and Blimp1, and do not undergo further CSR. We show that whilst both NFjB1 and mir125b regulate IRF4 and plasma cell differentiation, they have different effects on the early phase IRF4 induction and Ig class switching in B blasts. Conclusions: The results are compatible with a role for low-level expression of IRF4 for CSR in early B blasts.

P0440 Expression and functionality of proteinase activated receptor-2 (PAR-2) in murine B cells M. Campbell,* A. Crilly,* G. McGarvie,* R. Plevin, W. R. Ferrell,à C. S. Goodyearà & J. C. Lockhart* *School of Science, Centre for Musculoskeletal Science, University of the West of Scotland, Paisley, UK, Strathclyde Institute of Pharmacy and Biomedical Science, University of Strathclyde, Glasgow, UK, àInstitute of Infection Immunity and Inflammation, University of Glasgow, Glasgow, UK Purpose/Objective: PAR-2 is a seven transmembrane G protein coupled receptor (GPCR) that when activated by proteolytic cleavage results in the production of cytokines and chemokines(1). PAR-2 is expressed on numerous immune and non immune cells(2), however, it has previously been reported to be absent on B cells(3), although this remains controversial. The current study aims to address this controversy by demonstrating both PAR-2 expression and a functional role for PAR-2 in murine B cells. Materials and methods: PAR-2 expression on murine B cells was assessed by flow cytometry. B cells were negatively enriched from C57BL/6 splenocytes using magnetic separation. Purified B cells were activated in vitro with the specific PAR-2 activating peptide SLIGRLNH2 (100 lM), supernatants collected 1, 4 and 24 h after stimulation, and analysed by ELISA for IL-6 and IL-10. Ca2+ signalling was measured by flow cytometry and pERK1/2 by western blotting. For sub-setting studies, spleens, bone marrow and peritoneal lavages were collected from female F2rl1-/- (PAR-2 deficient) and wild type C57BL/6 littermates, and cells stained with fluorescent antibodies for analysis by flow cytometry. Results: Our data reveal that PAR-2 is expressed at low levels on splenic B cells, and this can be augmented by activation with SLIGRL-NH2.The activation of B cells with SLIGRL-NH2 also induced the increased secretion of IL-6 and IL-10. Furthermore, receptor activation induced Ca2+ mobilisation and the phosphorylation of ERK1/2 in B cells, indicative of intact PAR-2 signalling. Interestingly, the absence of PAR-2 in F2rl1-/- mice resulted in alterations in the composition of the B cell compartment, namely B1a and marginal zone B cells. Conclusions: This study is the first to demonstrate the expression of functional PAR-2 on murine B cells, and implicates PAR-2 as a regulatory factor in the homeostasis of the innate-like B cell repertoire. 1. Johansson et al.2005 Journal of Leukocyte Biology;78:967 75


Poster Sessions 331 2. Shpacovitch, V. et al., 2008. Journal of leukocyte biology, 83(6), 1309 22. 3. Lim, S. Y. et al., 2006. British journal of pharmacology, 149(5), 591 9. This work was supported by The University of the West of Scotland, Arthritis Research UK (18306) and Tenovus Scotland

P0441 Expression of Toll-like receptors 7 and 9 in B cell populations of ¨gren’s syndrome patients with Sjo M. Karlsen, T. Hansen, R. Jonsson & S. Appel Medicine and Dentistry, The Gade Institute, Bergen, Norway Purpose/Objective: Sjo¨gren’s syndrome (SS) is a rheumatic autoimmune disease, with focal lymphocyte infiltrations and inflammation in exocrine glands, resulting in destruction of glandular tissue. Primarily, salivary and lachrymal glands are affected, leading to reduced production of saliva and tears. A conse-quence of this is dry eyes and dry mouth (sicca syndrome). Other organs can also be involved in SS, and patients have, as many other rheumatic patients, major problems with fatigue. The cause of SS is so far not known, and there is no cure available. Only symptomatic treatment can be offered, and even if the inflammation is halted, it is not certain that the glands can be regenerated. To generate new and more effective treatments for these patients, more information on the development and cause of this disease is needed. As SS is characterized by the production of autoantibodies, B cells have been recognized as important cells in the pathogenesis of SS and other rheumatic diseases. Toll-like receptors (TLR) are pattern recognition receptors that induce activation responses after ligand binding, and they are found on many immune cells. TLR7 and 9 are localized intracellulary in the endosomal compartment where they recognize nucleic acids. Both have been suggested to be involved in autoreactivity towards ‘self’. Materials and methods: In this study we utilized peripheral blood mononuclear cells to investigate the expression levels of TLR7 and TLR9 in various B cell subsets of SS patients and healthy controls using eight colour flow cytometry. Results: Preliminary data suggests that memory B cells from SS patients have elevated expression levels of TLR 7 and 9. Conclusions: Further analyses will be needed to evaluate possible functional differences of these cells.

P0442 FCGR3A-158V/F polymorphism may correlate with hypogammaglobulinaemia in patients after Rituximab treatment E. Villegas,* J. Iglesias,* N. Martıńez-Pomar,* A. M. Gutie´rrez & N. Matamoros* *Immunology, Son Espases Hospital, Palma de Mallorca, Spain, Hematology, Son Espases Hospital, Palma de Mallorca, Spain Purpose/Objective: Rituximab is a chimeric monoclonal antibody directed toward CD20, a B-cell surface marker that has been proven effective in depleting normal and malignant B cells in vivo. Rituximab is widely used in the treatment of B-cell malignancies and induces almost a complete depletion of normal B lymphocytes in peripheral blood for an average of various months. In some cases, Rituximab treatment causes prolonged hypogammaglobulinaemia. It has been reported that the presence of valine (V) at position 158 of FCGR3A (CD16) has a higher affinity to human IgG than the phenylalanine (F) allele. The hypogammaglobulinaemia after Rituximab treatment could be correlated with this polymorphism. Materials and methods: This study initially included 16 patients who received treatment with Rituximab. The FCGR3A gene polymorphisms

were determined by allele specific polymerase chain reaction (PCR). Genomic DNA was extracted from peripheral blood using a DNA isolation kit under the manufacturer’s instructions. Results: The patients tested for the FCGR3A-158V/F polymorphism were classified in low-affinity group (158 F/F) and high-affinity group (158 F/V and 158 V/V). Of the 16 patients initially tested for the polymorphism, 5 in Rituximab maintenance and 11 hypogammaglobulinemic subjects after Rituximab treatment, 3 patients had homozygous F/F, 11 had heterozygous V/F and 2 had homozygous V/V. Conclusions: The aim of this study is to demonstrate the possible correlation between immunoglobulin levels after Rituximab treatment and the FCGR3A-158V/F polymorphism. In theory, patients classified in low-affinity group may have lower levels of immunoglobulins in comparison with high-affinity group. The confirmation of this result may imply the introduction of these studies as a diagnostic test and provide a more accurate Rituximab treatment to avoid secondary hypogammaglobulinaemia.

P0443 Functional elimination of double-stranded DNA-specific B Lymphocytes suppresses disease activity in SCID model of mouse lupus S. Chausheva, N. Mihaylova, V. Gesheva, N. Kerekov, K. NikolovaGaneva & A. Tchorbanov Immunology, Institute of Microbiology Bulgarian Academy of Sciences, Sofia, Bulgaria Purpose/Objective: Self-specific B cells play a main role in pathogenesis of Systemic lupus erythematosus (SLE). The elimination of B and T cells involved in the pathological immune response is a reasonable approach for effective therapy of SLE. In the present study we established an autoimmune model by transferring purified B and T cells from MRL/lpr mice to SCID mice and tested the effects of the chimeric molecule that selectively targeted pathological autoreactive Blymphocytes. Materials and methods: The protein chimeric molecule was constructed by coupling an DNA- mimotope peptide DWEYSVWLSN to a monoclonal anti-CD32 (FccRIIb) antibody. This engineered molecule is able to cross-link cell surface immunoglobulin with the inhibitory FccRIIb on DNA-specific B cells. Female SCID mice were transferred with isolated B+T cells from MRL/lpr mice and the animals were treated with DNA- peptide chimera. Cytokine assays, ELISpot, Signal transduction, Proliferation assay, ELISA and FACS analyses were also performed. Results: The specific elimination of the DNA-specific B cells in MRLtransferred SCID mice restricts not only anti-dsDNA IgG production, but also autoreactive T cell activation and proliferation. In contrast, untreated MRL-transferred SCID mice experienced an increase of disease-associated antibody levels and developed glomerulonephritis similar to intact MRL/lpr mice. Conclusions: In the present study we report a possible way to restrict the communication between autoimmune B and T cells, leading to suppression of lupus syndrome in MRL/lpr cell-transferred SCID mice. The functional elimination of autoantigen-specific B cells leaves autoreactive T cells alone without potency of prolonged pathogenetic effects. In the present transferred SCID model of mouse lupus we have a possibility to study post-elimination disease processes and autoreactive T cell behavior after treatment with the protein-engineered antibody.


332 Poster Sessions P0444 Gene profiling of human interleukin-10 producing regulatory B cells W. Lin, E. W. M. Chua, N. Shadan, S. M. Mustafah, I. C. H. Low, J. Lum, K. Duan, J. J. Y. Tai, A. Larbis, F. Zolezzi, M. Poidinger, S. C. Wong & S. Calbo Immunos, Singapore Immunology Network (SIgN), Singapore, Singapore Purpose/Objective: B cells which possess regulatory function (Bregs) have been identified in mouse models of multiple sclerosis, rheumatoid arthritis and colitis. These Bregs are characterized by their ability to produce interleukin-10 (IL-10) which down-modulate inflammation. Similarly, we were able to detect IL-10 producing Bregs in human blood and spleen after ex vivo short time stimulation. Since IL-10 producing Bregs did not have a particular phenotype, a detailed profiling of this Breg subpopulation is needed in order to identify them more accurately based on unique cell surface markers and transcription factors. Materials and methods: B cells were stimulated for 48 h with CpG and anti-Ig which are the most potent stimuli for inducing IL-10 secretion in B cells. Bregs were then isolated based on IL-10 secretion. Microarray analysis was performed in order to identify differentially expressed genes between IL-10 negative (IL-10-) and IL-10 positive (IL-10+) B cell populations. Results: Both naı¨ve and memory B cells can secrete IL-10. Microarray analysis showed hierarchical clustering of B cells based on IL-10 expression. A total of 614 genes were found to be differentially expressed between the IL-10- and IL-10+ B cell populations. Conclusions: All subsets of B cells have the potential to produce IL-10 following CpG and anti-Ig stimulation. Our microarray findings may allow the identification of new subpopulations of human B cells with potential immunoregulatory function.

P0445 Generation of B-cell memory from the in-vitro-induced germinal center B cells D. Kitamura Division Molecular Biology, Research Institute for Biomedical Sciences, Tokyo University of Science, Noda, Chiba, Japan Purpose/Objective: During T-cell dependent immune responses, antigen-bound B cells proliferate extensively to form germinal centers (GC) in the peripheral lymphoid organs and then differentiate into either long-lived plasma cells (LLPCs) or memory B (Bmem) cells, which constitute a B-cell part of the immunological memory. To understand the molecular mechanisms for the development of LLPCs and Bmem cells, we sought to construct an in vitro model system that recapitulates the GC reaction. Materials and methods: Mouse naı¨ve B cells were cultured on the feeder cells expressing CD40L and BAFF (named 40LB) with cytokines, IL-4 in the primary, and IL-21 in the following secondary culture. The B cells from these conditions were transferred into syngeneic mice, and after certain days, the mice were analyzed by flow cytometry, ELISA, and ELISPOT. Results: In this culture system, the B cells extensively proliferated to generate germinal center-phenotype B (induced GC B: iGB) cells expressing switched or unswitched Ig classes. The iGB cells after primary culture with interleukin (IL)-4 develop into Bmem cells in vivo that elicit rapid immune responses in the presence of cognate helper T cells, whereas the secondary culture with IL-21 inhibits the Bmem development and allows their development into LLPCs that produce significant amount of IgG1 for more than one month in the bone marrow. The subsequent IL-21 withdrawal partially restores the Bmem development and abolishes the LLPC development. In addition, the transfer of the secondary iGB cells that are specific to a certain membrane antigen inhibited tumor formation of concomitantly

transferred melanoma cells expressing the same antigen in the lungs of the recipient mice. Furthermore, we have established a system to selectively expand antigen specific iGB cells using the 40LB cells expressing a membrane-bound antigen and Fas-ligand. Conclusions: This novel culture system has enabled in vitro differentiation from naı¨ve B cells into Bmem or LLPC precursors, which can colonize and mature in their destined sites in vivo, and therefore will be useful to elucidate molecular mechanisms for the late B-cell development in GC. It also offers a model system to develop a B-cell-mediated anti-tumor therapy, in which patients will be transferred with autologous iGB cells selected for binding to a specific tumor antigen.

P0446 HA-specific memory B cell responses to pandemic 2009 H1N1 and seasonal influenza viruses in children and adults W. Mahallawi,* A. Kasbekar, C. Loh, K. Hoschler,à M. McCormick,§ S. Leong,§ P. McNamara– & Q. Zhang* *Institution of Infection and global health, University of Liverpool, Liverpool, UK, Alder Hey Children’s Hospital, University of Liverpool, Liverpool, UK, àVirus Reference Department, Health Protection Agency, London, UK, §ENT Department, Royal Liverpool University Hospital, Liverpool, UK, –ENT Department, Alder Hey Children’s Hospital, Liverpool, UK Purpose/Objective: Backgrounds: Influenza is a highly contagious and acute respiratory infection caused by influenza virus in the mucosa of respiratory tract. Nasal-associated lymphoid tissues (NALT) are mucosal immune organs in the nasopharynx, among which adenoids and tonsils are major components of human NALT. Aims of study (i) to investigate whether prior exposure/infection of pandemic 2009 H1N1 virus (pH1N1) primes for immunological memory in human NALT and, (ii) To determine whether it cross reacts with seasonal H1N1 and H3N2 viruses. Materials and methods: Adenotonsillar tissues were obtained from children and adults undergoing adenoidectomy and /or tonsillectomy due to adenoidal hypertrophy or tonsillitis. Adenotonsillar MNC were cultured in RPMI medium with or without the addition of influenza antigens. Peripheral blood samples were also taken for immunological analysis. HA-specific memory B cell responses to pandemic 2009 H1N1, seasonal H1N1 and H3N2 viruses were analysed by Elispot assay, following stimulation by surface antigens derived from respective influenza viruses. Serum anti-HA IgG antibody and haemagglutination inhibition titres were measured by ELISA and a standard HAI assay respectively. Results: In individuals who had evidence of prior exposure to the 2009 H1N1 virus (who had anti-pH1N1 HAI titre340), there were significant numbers of pH1N1 HA-specific memory B cells which respond not only to the homologous pH1N1, but also cross-react to the sH1N1 viruses. However, there was no significant memory B cell response to the seasonal H3N2 HA after the pH1N1 antigen stimulation. Stimulation with the sH1N1 antigen induced only a moderate increase in the numbers of HA-specific ASC to the homologous sH1N1 and heterologous pH1N1, and no significant increase in numbers of specific ASC to sH3N2. In contrast, the pH1N1 antigen stimulation induced significantly higher HA-specific memory B cell responses, not only to the homologous pH1N1, but also to the heterologous sH1N1 than the sH1N1 antigen in patients with serum anti-pH1N1 HAI titre 340. There was a significant correlation between numbers of HA-specific antibody secreting cells (ASC) to pH1N1 and that to sH1N1 after stimulation by the pH1N1 surface antigens. Conclusions: Pandemic 2009 H1N1 influenza virus induces strong HA-specific memory B cell responses which cross react with the seasonal H1N1 but not seasonal H3N2 viruses.


Poster Sessions 333 P0447 Hypoactive Syk elicits B cell-mediated autoimmunity and a pre-diabetic state S. Ko¨nigsberger,* J. Prodo¨hl,* V. Weis,* M. Andreas,* M. Stehling,* T. Schumacher, R. Bo¨hmer* & F. Kiefer* *Department Vascular Cell Biology, Max Planck Institute for Molecular Biomedicine, Muenster, Germany, Department of Neurooncology, Neurology Clinic and National Center for Tumor Diseases, University Hospital of Heidelberg, Heidelberg, Germany Purpose/Objective: The closely related non-receptor protein tyrosine kinases Syk and Zap-70 are of central importance in transducing signals from antigen receptors to downstream signaling mediators, a process ensuring proper selection, maturation and activation of lymphocytes. Partially overlapping protein expression patterns and reported functional redundancies at the signal transduction level still raise the question to which degree the kinases can replace each other in the immune system. Materials and methods: To fully elucidate kinase interchangeability in vivo, we generated mice, which carry a Zap-70 cDNA ‘knock-in’ at the locus of Syk that is controlled by intrinsic Syk promoter elements and disrupts wildtype Syk expression. Results: Kinase replacement strongly reduced Erk1/2-mediated survival and proper selection of developing B cells, demonstrating critical dependence on BCR signaling quality. The observed alteration in BCR signaling quality was accompanied by a preferential development and survival of marginal zone B cells, prominent knock-in serum autoreactivity, high levels of anti-insulin antibodies, proteinuria and an age-related glomerulonephritis. Development of concomitant fasting glucose intolerance in Zap-70 knock-in mice highlights aberrant B cell selection through hypoactive Syk as a potential novel risk factor for type 1 diabetes and suggests attenuated BCR signaling output as a mechanism to cause biased cellular and Ig repertoire selection. Conclusions: Consequently, lowering Syk kinase activity in B cells through e.g. somatic mutation, alternative splicing / exon skipping or changes in promoter methylation might therefore contribute to autoimmune predisposition.

P0448 IL-2 induced MAPK-ERK pathway triggered human plasma cell differentiation through BACH2 Repression C. Delaloy, G. Caron, S. Le Gallou, C. Pastoret, K. Tarte & T. Fest Rennes University Hospital, INSERM U917, Rennes, France Purpose/Objective: Terminal differentiation of B lymphocytes into plasmocytes (PCs) involves a well-established transcription factor cascade. However, temporal dynamics of cell signaling pathways regulating transcription factor network during germinal center (GC) reaction remain poorly defined. Materials and methods: To gain insight into the molecular processes and extrinsic factors required for B cell differentiation, we set up a controlled primary culture system combining BCR signal, Toll like receptor activation and T cell help in the form of CD40L and cytokines to differentiate human naı¨ve B cells into PCs.. Results: We identify T-cell-produced IL-2 to be critically involved in ERK1/2 triggered PC differentiation. This early signaling event controlling specification to plasma cell fate operates independently of proliferation and survival functions. Compared to no cytokine activated B cells, IL-2-primed B cells display a gene expression profile of a more differentiated fate.Chemical inhibition of the MAPK-ERK pathway impairs IL-2 mediated PC differentiation and rescues the expression profile of several key genes, maintaining GC B cell fate. One of them encodes the transcription factor BACH2, which represses BLIMP1 the master regulator for PC differentiation, and whose

expression is physiologically shutdown in centrocytes. Partial inhibition of BACH2 is sufficient to drive PC differentiation in absence of IL2, and suppresses the effects of ERK1/2 inhibition on IL-2 induced PC differentiation as well. These results support the notion that the concentration of BACH2 fine-tunes the gene regulatory network involved in PC differentiation. The molecular mechanism of ERKdependent BACH2 repression is under investigation. Conclusions: Altogether we identify IL-2 as a novel early master regulator required to overcome the repressive forces that block PC differentiation through ERK activation and BACH2 inhibition, sustaining BLIMP1 expression.

P0449 IL-21 derived from human follicular helper T cells acts as a survival factor for secondary lymphoid organ, but not for bone marrow, plasma cells R. Ana Belen, B. Rodriguez-Bayona, A. Campos-Caro & J. A. Brieva-Romero Hospital universitario Puerta Del Mar, Unidad De Investigacioń, Ca´diz, Spain Purpose/Objective: OBJETIVE: IL-21 induces the differentiation of activated B lymphocytes into plasma cells (PC), but its direct effect on PC remains uncertain. This study analyzes the role of IL-21 on human in vivo -generated PC. Materials and methods: MATERIAL AND METHODS: Expression of constitutive IL-21R on human PC from different organs was examined by flow cytometry. IL-21R mRNA from PC was quantified by RT-PCR. Tonsillar and bone marrow (BM) PC were purified by a combination of magnetic-selection and FCC-sorting. PC cultures without/with IL21 or purified follicular helper T (Tfh) cells. Analysis of proliferating cells was performed by detecting BrdU incorporation. Apoptotic PC were determined by labeling active caspases with the fluorochromelabeled inhibitor FAM-VAD-FMK. Measurement of Ig secreted to culture supernatants was performed by ELISA. Results: RESULTS: IL-21R was clearly expressed on PC from human tonsil, lymph node and spleen (secondary lymphoid organs, SLO), but barely on terminally mature BM. PC. IL-21 enhanced Ig-secretion by isolated SLO PC, but not BM PC. Tonsillar Tfh lymphocytes are known to secrete IL-21. Purified Tfh-cells induced a marked increase of Ig-production by tonsillar PC, and this effect was impaired when endogenous IL-21-production was blocked. IL-21 provoked a rapid and transient phosphorylation of STAT3 in tonsillar PC. Tfh-cells or exogenous IL-21 reduced tonsillar PC apoptosis and increased PC recovery, but dit not modify their non-proliferating status. Conclusions: CONCLUSIONS: These results suggest that IL-21 derived from Tfh-cells may act as a survival factor for SLO PC in vivo.

P0450 Immunoglobulin gene usage and specificity of plasmablasts generated during secondary dengue infections R. Appanna,* M. H. Xu,* V. Hadinoto,* K. Joensson,* Y. X. Toh,* T. Balakrishnan,* Y. S. Leo, C. I. Wang* & K. Fink* *Singapore Immunology Network, Agency for Science Technology and Research A*STAR, Singapore, Singapore, Department of Infectious Diseases Communicable Disease Centre, Tan Tock Seng Hospital, Singapore, Singapore Purpose/Objective: Memory B cells generated after exposure to dengue viral (DENV) infection are a central component in shaping immune memory. Re-exposure to dengue virus of a different serotype is often associated with increased viral replication and disease pathogenesis. In repeated infection, the rapid activation of memory B cell leads to the generation of massive amounts of cross-reactive anti-


334 Poster Sessions bodies. In the acute phase of a secondary infection, this B cell memory pool probably outcompetes the newly generated virus specific B cells. Materials and methods: To characterize the specificity of plasmablasts and memory B cells generated during and after acute infection, we performed single cell RT-PCR for sequence analysis of Ig variable regions. Results: Contrary to the polyclonal plasmablast repertoire we found that memory B cells isolated one month after disease were surprisingly oligoclonal and of different genetic composition than plasmablasts. Our current data revealed highly preferential usage of the VH1 gene in DENV binding plasmablasts. In contrast, skewed cell memory VH family repertoires (VH3) were noted in long lasting circulating cells isolated over a month after infection. Whether VH gene usages reflect a specific antigen/epitope selection is currently under investigation. Conclusions: These data further suggest that plasmablasts represent a population of re-activated memory B cells with a potentially different origin than circulating steady state memory B cells.

P0451 Implication of proteins syntaxin-3 and syntaxin-4 in constitutive secretion of ig from human plasma cells M. L. Go´mez Jaramillo, L. Delgado-Pe´rez, G. Jimeńez Go´mez, E. Reales Rodrı´guez, V. Rivas Guerrero, R. M. Mateos Bernal, A. Garcıá-Poley, J. A. Brieva Romero & A. Campos-Caro Hospital Universitario Puerta Del Mar, Unidad De Investigacioń, Ca´diz, Spain Purpose/Objective: Plasma cells (PC) are B-lymphocytes terminally differentiated with the purpose of manufacturing and secreting immunoglobulins (Ig). This study explores the presence of several SNARE proteins in human PC and examines their functional roles on Ig secretion process. Materials and methods: The U266 human myeloma cell line was used as model of Ig secreting cell line. Expression of constitutive SNARE proteins on human PC was examined by Western-blot and protein localization was analysed using immunofluorenscence confocal microscopy. Experiments of loss of function were performed by nucleofection of U266 with specifics iRNA. Overexpression assays were generated by transfection of constructs for native SNAREs and deleted protein lacking the transmembrane domain, both in fusion with Ruby as a reporter gen. Transfected cells, either iRNA o plasmid, were isolated by FACS-sorting and cultured for 24 h. IgE levels into the cultured supernatants were measured by ELISA and the corresponding cell pellets were used to analyse the inhibition or overexpression of each protein by Western-blot. Results: Syntaxin-3 and Syntaxin-4 were detected in human PC and human cell line U266. The predominant location of Syntaxin-3 and Syntaxin-4 is on the cell surface but they could be also observed intracellularly. Although both Syntaxins shown to interact with SNAP23, functional studies demonstrated a different role of these proteins in Ig-secretion. Interference of Syntaxin-4 expression, but not of Syntaxin-3, significantly inhibited Ig secretion in PC. Overexpression of wild-type Syntaxin-4 induced a marked decrease of Ig-secretion whereas overexpression of the mutant form of the protein did not modify significantly this secretion. Increase of both wild-type Syntaxin3 and mutant form did not cause changes on Ig secretion levels. Conclusions: The present study demonstrates that Syntaxin-4, but no Syntaxin-3 plays a critical role in the Ig-secretion and this data suggest that Syntaxin-4 along with SNAP-23 appears to be a component of the SNARE complex in human PC Ig-secretion.

P0452 Increase of IgA+, but not IgM+ memory B cells after vaccination with Pneumovax23 A. Meyer-Bahlburg, K. Schu¨tz & U. Baumann Hannover Medical School, Pediatric Pneumology and Neonatology, Hannover, Germany Purpose/Objective: The mature B cell compartment is comprised of at least two B cell subpopulations, namely naı¨ve follicular mature and marginal zone B cells. Whereas naı¨ve follicular mature B cells are crucial during T cell dependent immune responses, marginal zone B cells are the main player during T cell independent immune responses. In humans, IgM+ memory B cells in the peripheral blood have been suggested to represent equivalents of splenic marginal zone B cells. However, this view remains very controversial. Materials and methods: We therefore analyzed the changes in the composition of the B cell compartment in peripheral blood of healthy individuals following immunizations with Pneumovax23 inducing primarily a T cell independent immune response. Results: Whereas we could not observe a significant change within the IgM+ memory B cell compartment we found a significant increase, both in relative and absolute numbers, in IgA+ memory B cells. Moreover, a ‘non-responder’ to vaccination, defined by no significant increase in Pneumovax-specific IgM or IgG, had very low, but still few, IgA+ memory B cells in peripheral blood. Conclusions: Our data show, that Pneumovax23 primarily induces an IgA+ memory B cell reponse. Although it is well known that individuals with IgA deficiency show often normal responses to pneumococcal infection and vaccinations with Pneumovax23, these results suggest that IgA is primarily induced in response to these antigens and might serve as a predictor for the immune response.

P0453 Innate CD19+ CD45Rlo cell population exhibit high proliferation rates in homeostatic conditions and is able to respond after TLR stimulation B. De Andres,* I. Cortegano,* C. Prado,* B. Palacios,* M. Alıá,* C. Ruiz,* N. Serrano, M. A. R. Marcos & M. L. Gaspar* *Centro Nacional de Microbiologıá, Unidad de Inmunobiologıá, Madrid, Spain, Centro de Biologıá Molecular, Consejo Superior de Investigaciones Cientı´ficas, Madrid, Spain Purpose/Objective: We described previously a novel splenic B cell population CD19+ CD45RloCD21lo (19+45Rlo) from embryonic origin, that preferentially home on perifollicular areas of unmanipulated follicles. These cells harbor a plasmablast phenotype (Blimp-1+Xbp1+Pax-5+) and spontaneously release IgG1 and IgA. Here, we show that these cells are different from classical follicular, transitional, marginal zone B cells, B1 cells, the innate response activator B cells (IRA), and aged-associate B cells (ABCs). Materials and methods: Multiparemetric flow cytometry analyses were performed on samples from different lymphoid organs. BrdU uptake and adoptive transfers of sorted 19+45RloLy5.1+ cells on Rag2c-/- mice were performed. PCR array experiments from cell cycle genes were performed on sorted samples. IL6, IL10, IL12 and GM-CSF were quantified by RT-qPCR, magnetic bead arrays, and intracellular flow cytometer cytokine detection. Constitutive phosphorylation of pERK was also determined by flow cytometry studies. TLRs ligand stimulation, BAFF/IL4 and T-dependent stimuli were used for in vitro activation of 19+45Rlo cells. Results: 19+45Rlo cells are present in Peyer’s Patches, peripheral blood and spleen but not in lymph nodes or thymus. In the spleen they appear from 7 days post-natal age and are minimally present in CBA/ CaHN (btk-). In homeostatic conditions, 50% of 19+45Rlo cells are labeled after in vivo 17-day continuous administration of BrdU.


Poster Sessions 335 Purified 19+45RloLy5.1+ cells transferred on Rag2c-/- mice survived up to 40 days. PCR array experiments performed on sorted 19+45Rlo cells compared to 19+45R+ and to peritoneal B1 cells, confirmed an active cell cycling signature of 19+45Rlo cells. In vitro stimulation of sorted 19+45Rlo cells with LPS, CpG, and BAFF/IL4 induced cell proliferation and IgG1/IgA secretion. IL10-transcripts were found in resting 19+45Rlo cells that also released IL10 after activation with LPS and BAFF/IL4. Finally, constitutive p-ERK1/2 was present in 19+45Rlo cells, reflecting a pre-activation stage. Conclusions: We conclude that 19+45Rlo cells represent a novel component of the innate adaptive immune system with unique features, responsible of rapid IgG- and IgA-humoral responses. Additionally, we propose that 19+45Rlo cells may play a potential immunoregulatory role in the initial phases of the encounters with common pathogens.

P0454 Interaction between B-1 cell and B16 melanoma cell: a mutual increase in cell survival M. F. L. Laurindo, F. G. Thies, A. F. Popi, R. R. Novaes e Brito, E. C. Perez & M. Mariano UNIFESP, Microbiology Immunology and Parasitology, Saõ Paulo, Brazil Purpose/Objective: B-1 cells can be differentiated from B-2 cells because they are predominantly located in the peritoneal and pleural cavities and have distinct phenotypic patterns and activation properties. The role of both cell populations in cancer progression is still controversial. Previous work in our laboratory showed that physical interaction of B-1 cells with B16 cells in vitro leads to an increase in tumor cells metastatic potential. The aim of this study is to elucidate possible effects of this interaction on B-1 cells. Materials and methods: Peritoneal B-1 cells were cultured alone or with B16 cells.B-1 cell survival and proliferation were evaluated by flow cytometry using propidium iodide and CFSE respectively. Considering that IL-10 is an autocrine growth factor that is used by B-1 cells to support their self renewal, we hypothesized that B-1/B16 cell interaction would increase the production of IL-10 by B-1 cells. The cytokine production was analysed by Th1/Th2/Th17 cytokines cytometric bead array (CBA) kit. Expression of STAT3, pSTAT3 and Bcl2 was assessed by Western Blot. Results: B-1/B16 cells contact increased B-1 cells survival and proliferation in vitro. In order to clarify if contact between these two cell populations was necessary to increase B-1 cell survival; B-1 cells were cultivated on transwell which were placed on tumor cells or with B16-conditioned medium. In both conditions, it was observed an increment in B-1 cell survival. We also observed that after direct or indirect contact of B-1/B16 cells, B-1 cells became resistant to high dose of radiation. Analysis of cytokine secretion showed a strikingly increase in IL-10 production by B-1 cells after contact with B16 cells, associated with higher levels of STAT3 phosphorylation. Conclusions: Our data clearly show that interaction of B-1 cells with melanoma cells, besides changing the tumoral lineage also affect B-1 cells, leading to both increased cell viability and rate of proliferation. Further, soluble factors that were secreted by B16 cells were sufficient to augment B-1 cell viability and IL-10 production. Impressively, after direct or indirect contact with the B16 cells, B-1 cells became resistant to radiation-induced cell death. Thus, future studies that assess the importance of concomitant immunity and other conventional therapies in cancer treatment are needed.

P0455 Interleukin-21-induced granzyme B-expressing B lymphocytes regulate T cells and infiltrate human solid tumors S. Hofmann,* K. Dahlke, K. Sontheimer, M. Hagn,à C. Kaltenmeier,* T. F. E. Barth,§ T. Beyer,* F. Reister,– D. Fabricius,** R. Lotfi,* O. Lunov, G. U. Nienhaus, T. Simmet, R. Kreienberg,– P. Mo¨ller,§ H. Schrezenmeier* & B. Jahrsdo¨rfer* *Institute of Clinical Transfusion Medicine and Immunogenetics, University of Ulm, Ulm, Germany, Institute of Clinical Pharmacology, University of Ulm, Ulm, Germany, àCancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Vic., Australia, §Institute of Pathology, University of Ulm, Ulm, Germany, –Department of Gynecology and Obstetrics, University of Ulm, Ulm, Germany, **Department of Pediatrics, University of Ulm, Ulm, Germany, Karlsruhe Institute of Technology (KIT), University of Karlsruhe, Karlsruhe, Germany Purpose/Objective: B cells can exhibit potent regulatory functions. Conflicting data exist regarding the role of B cells in lymphocytic infiltrations of tumors. Here, we demonstrate, that granzyme B (GrB)expressing B cells in adjacency to IL-21-providing T cells can be found in the tumor microenvironment of various solid tumors including breast, ovarian and cervical carcinomas. GrB-dependent regulation of T cell responses is known from both, regulatory T cells (Treg) and plasmacytoid dendritic cells. Materials and methods: Cell culture, FACS analysis, laser scanning confocal microscopy, Western blotting, histology. Results: In the current study, we find that IL-21 induces human B cells to express high levels of GrB and to develop regulatory potential towards co-cultured T cells by GrB-dependent degradation of the T cell receptor z-chain. More detailed characterization of IL-21-induced GrB+ B cells reveals a CD19+ CD38+ CD1d+ CD147+ phenotype and expression of further regulatory molecules including IL-10, IDO and CD25. Of note, CD5+ B cells exhibit a significantly higher potential to express GrB than CD5- B cells, and GrB induction is accompanied by activation of both B cell receptor- and Toll-like receptor-associated signaling pathways. Conclusions: In summary, we demonstrate for the first time, that IL21 induces GrB-expressing regulatory B cells, which can be detected in the microenvironment of solid tumors and which may contribute to the modulation of cellular adaptive immune responses by Treg-like mechanisms. Our findings may provide the basis for the development of novel diagnostic and cell therapeutic approaches to the management of malignant, autoimmune and graft-versus-host pathologies.

P0456 Interleukin-33 mediates activation and proliferation of effector and regulatory B cells S. Sattler,* L. Hussaarts, G. S. Ling,* A. Romaine,* D. Xu,à F. Y. Liewà & F. P. Huang* *Department of Medicine, Imperial College, London, UK, Department of Parasitology, Leiden University Medical Centre, Leiden, The Netherlands, à Division of Immunology Infection and Inflammation, University of Glasgow, Glasgow, UK Purpose/Objective: IL-33 is a novel IL-1 family cytokine with seemingly contrasting pro- and anti-inflammatory properties. Different types of immune cells express the IL-33 receptor ST2 and therefore can respond to IL-33 to initiate and regulate immune responses. Importantly, IL-33 has also been implicated in a wide range of inflammatory conditions such as allergic, autoimmune and cardiovascular diseases. In the past, many IL-33 related studies have been performed using recombinant human IL-33 in murine systems. We have observed previously that human IL-33 induces CD25 expression and IL-10 production in murine B cells. During this work we aimed to further


336 Poster Sessions characterize the potential pro- and anti-inflammatory properties of murine IL-33 on the activation and function of B cells. Materials and methods: In vivo and in vitro B cell activation in response to IL-33 stimulation was studied by analyzing B cell functional markers by flow cytometry. IL-33 mediated B cell proliferation was investigated by CFSE dilution and thymidine uptake experiments. Results: We report here that, although the murine cytokine is as expected significantly more potent, both human and mouse IL-33 induce B cell CD25 and IL-10 expression. The effect on B cell activation is largely independent of the genetic background of the mouse models tested. Interestingly however, mice of different backgrounds show significant differences in the IL-33 induced IL-10 production by B cells. We also found that IL-33 induces proliferation of splenic CD19+ B cells as well as upregulation of ST2 expression on their surface, suggesting that IL-33 may act on B cells directly. However, our findings from further in vitro experiments using isolated splenic B cells indicate that an indirect effect of IL-33 is also involved in driving the B cell responses. In addition, IL-33 induces upregulation of the low-affinity Fc receptor for IgE, CD23, as well as the costimulatory molecule CD86 on B cells. Conclusions: Considering the diverse and crucial roles of B cells in both Th1 and Th2 immune responses, this work adds valuable information to our understanding of the immunological effects mediated by the novel cytokine IL-33.

P0457 Long-lasting and protective IgM antibody responses against Salmonella typhi C. Perez-Shibayama,* C. Gil-Cruz,* L. Cervantes-Barragan, R. Pastelin-Palacios,à E. Hisaki,§ Q. Chai,* A. Isibasi,– C. LopezMacias– & B. Ludewig* *Kantonal Hospital St. Gallen, Institutte of Immunobiology, St Gallen, Switzerland, Deparment of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA, àDeparment of Biology, Faculty of Chemisty UNAM, Mexico City, Mexico, §National School on Biological Sciences IPN, Immunology, Mexico city, Mexico, – Mexican Social Security Institute IMSS, Medical Research Unit on Immunochemistry, Mexico city, Mexico Purpose/Objective: IgM antibodies can efficiently clear bacterial infections through the activation of the classical complement pathway. However very little is known about the mechanisms that govern the generation of long-lasting IgM. S. typhi porins are a candidate sub-unit vaccine that induces protective antibody responses. Here, we studied the mechanisms by which S. typhi porins induce a sustained IgM antibody response and the role of these antibodies in the protection against infection with S. typhi.. Materials and methods: Porins-specfic IgM antibodies were measured by ELISA and antibody-secreting cells were enumerated by ELISpot analysis. CD4 T cell responses were assessed following in vivo restimulation using intracellular cytokine staining. Porins-specific IgM hybridomas were generated and monoclonal antibodies used for in vivo protection assays. Results: Boostered immunization with porins induced long-lasting, T helper cell-dependent IgM responses. Marginal zone B cell contributed to the first wave of IgM production after primary immunization, whereas a CD4+ T cell-dependent germinal center reaction generated long-lasting memory B cells of the IgM type following booster immunization. Importantly, transfer of porins-specific IgM monoclonal antibodies protected mice from challenge with S. typhi. Conclusions: In conclusion, long-lasting IgM memory B cell responses against porins represent an important layer of antibacterial protection against S. typhi infection.

P0459 Marginal zone B cells in autoimmune arthritis A. K. E. Palm & S. Kleinau Uppsala University, Cell and molecular biology, Uppsala, Sweden Purpose/Objective: Breakage of self-tolerance and induction of autoimmune arthritis is achieved in susceptible strains of mice by immunization with bovine (B) collagen type II (CII) in Freund’s complete adjuvant (FCA). We have recently shown that naturally BCIIreactive marginal zone B cells (MZB) in the spleen expand rapidly following this immunization. Here we have investigated when and where true autoreactive B cells to murine (M) CII in relationship to BCII develop in collagen-induced arthritis (CIA), with emphasis on the MZB population. Materials and methods: Cell suspensions were prepared from the spleen and peripheral lymph nodes from DBA/1 mice at different time points after BCII immunization. Mice immunized with ovalbumin (OVA) in FCA or FCA only served as controls. The B cell response was analyzed by flow cytometry and ELISpot. Serum was analyzed for antigen specific antibodies in ELISA. Results: We show here that the initial autoimmune B cell response to MCII is induced and mediated by MZB in the spleen. The response is comparable to that towards BCII. One week later a B cell response to MCII appears in the lymph nodes. Notably, a small MZB-like population in the lymph nodes expand almost twofold after immunization with BCII but not with OVA or FCA. These cells are defined as B220posCD23lowCD1dhigh and are distinguishable from follicular B cells (FOB). The MZB-like cells are also CD80highFccRIIbhigh MHCIIlowin contrast to FOB. IgM antibodies to MCII are found in serum a few days after BCII immunization with initially less titres than to BCII. In contrast, IgG antibodies to MCII are detectable at higher titres than to BCII about 2 weeks after immunization. Conclusions: MZB trigger the autoimmune response in CIA. The autoreative B cell response in the lymph nodes develops later, involving FOB and the generation of IgG antibodies. The expansion of a MZBlike population in the lymph nodes is specific for the autoimmune reaction and may be of importance for the pathogenesis of CIA. However, the question remains whether these MZB-like cells have migrated from the spleen and whether they are CII-specific.

P0460 Oral immunization with T dependent antigen gives lifelong IgA immunity with long-lived plasma cells and memory B cells distinct the ones formed after systemic immunization M. Bemark,* P. Bergqvist,* A. Stensson,* L. Hazanov, R. Komban,* R. Mehr & N. Y. Lycke* *Microbiology and Immunology, University of Gothenburg, Gothenburg, Sweden, Bar-Ilan University, Faculty of life science, Ramat Gan, Israel Purpose/Objective: There is a discrepancy in the literature with regard to if mucosal immunization leads to long-term protection and development of memory. We reasoned that this could reflect differences between T dependent and independent responses and devised a system to address the response to a T dependent oral antigen. Materials and methods: A strictly T cell dependent oral antigen was created by conjugating the hapten NP to the T dependent mucosal antigen cholera toxin to create NP-CT that was used for oral immunizations in mice. Results: Repeated oral immunization resulted in strong antigenspecific IgA responses, with up to 15% of the gut plasma cells being NP-specific. Transfer of NP-specific GFP-labelled B cells prior to immunization demonstrated that the response was initiated in stomach-proximal Peyer’s patches (PP) through invasion of preexisting germinal centres (GC), then spread to more distal PP and finally to MLN and spleen. The presence of clonally related sequences


Poster Sessions 337 in different organs and between PP indicated that activated B cells could travel between germinal centres, a notion that was further strengthened by that transferred GC B cells could invade PP GC after iv transfer if antigen was present. Oral immunization with NP-CT resulted in secretion of specific IgA into the gut and IgA and IgG in serum for more than 2 years. A strong boost response 1 year after immunization demonstrated that memory B cells had formed. Transfer of GFP+ NP-specific B cells prior to immunization resulted in traceable long-lived class-switched memory B cells that expressed CD73, CD80 and CD273 and were situated in B cell follicles in PP, MLN and spleen. Oral priming followed by systemic challenge resulted in a strong response with rapid formation of plasma cells in the small intestinal mucosa, spleen and bone marrow, while systemic immunization followed by oral challenge did not generate any response. A difference in memory B cells formed after oral and systemic responses was further demonstrated in transfer experiment, where PP, and to lesser extent MLN, memory B cells preferentially produced IgA and spleen ones IgG in recipient mice. Conclusions: We conclude that oral immunization with a T dependent antigen generates life-long immunity with long-lived plasma cells and memory B cells, and that there are distinctions between memory B cells formed after oral and systemic immunization.

P0462 Pro- and anti-inflammatory plasma cells can be distinguished by the expression level of sialyltransferase regulating IgG sialylation M. Ehlers,* A. Winkler, C. Hess & V. Holecska *Institute for Systemic Inflammation Research, University of Luebeck, Luebeck, Germany, German Rheumatism Research Center, Laboratory of Tolerance and Autoimmunity, Berlin, Germany Purpose/Objective: The Fc glycosylation pattern determines the proor anti-inflammatory effector function of IgG antibodies, whereby sialylated IgGs are anti-inflammatory. However, how differentially glycosylated IgG antibodies develop is unknown. We sought to examine the Fc glycosylation of antigen-specific IgG molecules and plasma cells (PC) after induction of inflammation or tolerance. Materials and methods: We administered chicken ovalbumin (OVA) under inflammatory or tolerance conditions to mice and analyzed OVA-reactive IgG Fc glycosylation and the expression level of the alpha 2, 6 sialyltransferase in OVA-reactive PCs. Results: Stimulation with protein antigens under inflammatory conditions induces PCs expressing low levels of alpha 2,6-sialyltransferase and producing de-sialylated IgGs. In contrast, PCs induced upon tolerance failed to downregulate alpha 2,6-sialyltransferase expression and secreted immunosuppressive sialylated IgGs. Conclusions: Our data show a novel antigen-specific immunoregulatory mechanism mediated by PCs expressing high levels of alpha 2,6 sialyltransferase and producing anti-inflammatory sialylated IgGs that are formed upon tolerance induction. Thus, pro- and anti-inflammatory PCs can be distinguished by the expression level of alpha 2,6 sialyltransferase.

P0463 Profiling B cell responses toward bacterial pathogens in human tonsils E. Faenzi,* F. Buricchi,* E. Viciani, A. Manetti, M. Barocchi, G. Galli,* F. Castellino,* G. Del Giudice* & O. Finco* *Novartis Vaccines and Diagnostics, Research Translational Medicine, Siena, Italy, Novartis Vaccines and Diagnostics, Research Microbial Molecular biology, Siena, Italy Purpose/Objective: The first contact between microorganisms and the human host takes place at the mucosal sites, in particular the upper

respiratory tract and oro-pharyngeal mucosa. Data currently available on primary and recall B cell response in nasopharyngeal-associated mucosa are limited, due to the fact that more frequently the immune memory response to a pathogen or a vaccine is analyzed in PBMC isolated from peripheral blood. Therefore, the understanding of the mechanisms underlying the effector and memory response at the site of host-pathogen interaction is of great interest in the vaccine field. The aim of the present study was to evaluate effector and memory B cells from palatine tonsils of patients that underwent surgery due to recurrent tonsillitis (RT) or obstructive sleep apnea syndrome (OSAS) by ELISpot assay. Materials and methods: In order to avoid high background signal due to hyper-activated condition of B cells in tonsils, we have developed an ad-hoc protocol to enumerate simultaneously IgG and IgA antigenspecific B cells. The optimized protocol was applied to enumerate IgG+ and IgA+ plasmacells (PC) and memory B cells (MBC) specific for selected antigens of S.aureus, S.pneumoniae and Group A streptococcus. Results: Frequencies of plasmacells and MBC were calculated as percentages of total IgG/IgA positive B cells in tonsils of 41 subjects aged from 4 to 42. Microbiologic culture data from tonsil swabs were also available for each analyzed sample. Conclusions: We investigated if there were any differences in the antigen recognition profile between plasmacells and MBC of the same tonsil and tried to define a relationship between microbiological data and the pattern of the B cell response in tonsils.

P0464 Reassessment of immunoglobulin joining-chain expression during B cell development: an early marker of plasma cell differentiation in germinal centers F. Lechouane, Z. Oruc, V. Pascal, M. Cogne´ & C. Sirac Centre National de la Recherche Scientifique UMR 7276, Controˆle de la re´ponse immune B et des lymphoprolife´rations, Limoges, France Purpose/Objective: Immunoglobulin Joining (IgJ)-chain is a 15 kDa peptide expressed by antibody secreting cells and required for assembly of pentameric IgM and dimeric IgA. It is required for the transport of these polymeric immunoglobulins (pIg) to the mucosal surface. Despite this definite role, it still remains controversial to what extent plasma cells producing monomeric Ig isotypes also express this peptide. Likewise, the exact stage of IgJ-chain production during B cell differentiation into plasma cell is not clearly defined. Materials and methods: To address this issue, we created a trangenic murine lineage that express the GFP under the control of the welldefined murine IgJ gene enhancer/promoter. This model allows us to follow IgJ-chain expression during in vivo and in vitro differentiation of B cells into plasma cells. Results: We found that all plasma cells express a high level of GFP in every lymphoid organ observed including spleen and bone marrow. We confirmed that GFP positive cells also expressed IgJ protein. Interestingly, we detected a B cell subpopulation that express a lower level of IgJ-chain (GFPint cells) in secondary lymphoid organs. Flow cytometry, immunohistochemistry and transcriptional analyses showed that these cells consisted in a large proportion of germinal center (GC) cells. However, Pax5 appeared to be slightly decreased in these GFPint cells compared to resting B cells, while Prdm1 and Xbp1 expression are unchanged. In vitro stimulation of spleen B cells with LPS confirmed the expression of IgJ-chain in all CD138+ plasmablasts but also revealed a population of CD138-/GFP+ activated B cells. ELISPOT experiments performed on these IgJ-chain expressing B-cells indicates these cells secreted larger amount of Ig than their IgJ-negative counterpart. Conclusions: At first, this IgJ-GFP model is a reliable tool for PC tracking. Secondly, our results indicate that IgJ is already produced in


338 Poster Sessions B-cells that seem to be pre-committed in the PC differentiation. Characterizing more precisely the features of this population would allow us to better understand early events of PC differentiation.

P0465 Regulatory B cells can modulate maturation and function of human dendritic cells and are partially defective in systemic lupus erythematosus C. Jamin,* A. Morva,* A. Achour,* S. Lemoine,* A. Saraux, P. Youinou* & J. O. Pers* *Brest University Medical School Hospital, Immunology, Brest, France, Brest University Medical School Hospital, Rheumatology, Brest, France Purpose/Objective: Mature dendritic cells (DCs) are stimulators of Tcell immune response, whereas immature DCs support T-cell tolerance. It has been demonstrated that murine B cells can hinder the inflammatory response by inhibition of IL-12 production by DCs. The current study was aimed at determining the regulatory capacity of human B cells on DC maturation and function. Materials and methods: Peripheral blood monocytes from six healthy donors (HDs) and six systemic lupus erythematosus (SLE) patients were differentiated into immature DCs with GM-CSF and IL-4, and into or mature DCs with LPS and IFNc. B lymphocytes were activated by CD40 and TLR9 and cultured with DCs. Expression of HLA-DR, CD80, CD86 and IL-12 by the DCs were evaluated by flow cytometry. The DC-dependent proliferation of CFSE-labelled T cells was also evaluated in the presence of B lymphocytes. Phenotypic analyses of B cells and inhibitory experiments were performed in co-cultures. Results: Activated B lymphocytes restrained the development of monocytes into immature DCs and their differentiation into mature DCs. They decreased the density of HLA-DR from mature DCs, the expression of CD80, CD86, and also the production of IL-12 Furthermore, they inhibited the DC-induced T cell proliferation. These modulations were mediated by CD19+IgDlowCD38+ CD24lowCD27- B lymphocytes and needed CD62L for the control of CD80 and CD86, and a soluble factor for the control of IL-12. Mature SLE DCs were insensitive to the regulation of IL-12. Conclusions: Human B cells can regulate DC maturation and function and consequently T cell-dependent inflammatory response. This regulation is partially deficient in SLE due to refratory DCs. This may influence inappropriate balance in SLE between effector inflammatory response and tolerance induction.

P0466 Regulatory role of B-1 cells in streptozotocin-induced diabetes in mice A. M. Alvares, M. C. T. Novo, A. F. Popi & M. Mariano Federal University of Sao Paulo, Microbiology Immunology and Parasitology, Saõ Paulo, Brazil Purpose/Objective: Recently, an important role of B cells in promoting type 1 diabetes has been demonstrated. Concomitantly, it has also been shown that B-1a cells, a subtype of B lymphocytes, can be involved in various autoimmune diseases. Based on these data, this study aims to investigate the involvement of B-1 cells in the development of murine streptozotocin (STZ)-induced diabetes. Materials and methods: BALB/c and BALB/xid (B-1 cell-deficient mice) male mice strains were treated intra-peritoneally with STZ (40 mg/kg) for 5 days. It is important to note that BALB/c mice do not develop diabetes when this dose of STZ was used. Blood glucose levels (BGL), cellular profile in the peritoneal cavity and cellular pancreatic islet infiltration were evaluated in experimental and control animals by flow cytometry. Further, pancreas were processed

and analyzed histologically or immunohistochemically for insulin expression. Results: Our findings showed that BALB/xid mice become diabetic after STZ treatment with BGL higher than BALB/c control mice (P < 0.001). Corroborating this data, the amount of insulin labeled beta cells of STZ-treated BALB/xid mice was always lower than control groups. Surprisingly, B-2 population increase in the peritoneal cavity of STZ-treated mice 10 days after treatment (P < 0.001) while CD4+ T cells increase only in BALB/xid diabetic mice (P < 0.01). Histological analysis showed lower number of pancreatic islets in diabetic mice (BALB/xid) as compared with pancreas in BALB/c mice. Additionally, the cellular evaluation of pancreas showed infiltrating T cells in these mice. To evaluate the role of B-1 cells in diabetes induction, peritoneal B-1 cells obtained from BALB/c mice were purified based on expression of CD19+ CD23- by FACSAriaII Cell Sorter and adoptively transferred intra-peritoneally to BALB/xid mice before or after the STZ treatment. BALB/xid mice adoptively transferred were not diabetic after STZ-treatment and B-1 cells were observed infiltrating pancreatic islets 2 days after STZ treatment. Conclusions: Our data demonstrate that B-1 cell-deficient mice showed higher reactivity to STZ treatment with more severe symptoms, intensive pancreas damage, insulin deficiency and high BGL. In addition, B and T cells increase in peritoneal cavity of BALB/ xid STZ-treated mice while B-1 cells migrate to pancreatic islets of non diabetic mice.

P0467 Revealing the role of Akt E. Cox, N. Ho¨velmeyer & A. Waisman University Medical Center of the Johannes Gutenberg-University, Institut for Moleculare Medicine, Mainz, Germany Purpose/Objective: The serine/threonine kinase Akt is expressed in three isoforms (Akt1, Akt2 and Akt3) which share a similar structure and analogue functions. Using distinct downstream pathways they regulate cellular metabolism, cell survival and proliferation, being important for peripheral B-cell maturation and early stages of T cell development. In B cells Akt is a critical check point in different phases of proliferation and differentiation. Especially the generation of marginal zone and B1 B cells depends on Akt1 and Akt2. In this study we investigated in more detail the role of Akt in B cell development and maturation. Materials and methods: T. Wunderlich created a mouse allowing for the expression of an N-terminally myristoylated murine AKT carrying in addition a C-terminally attached Myc TAG (ROSA-AKT-C) in cell types that express the Cre recombinase (unpublished). The myristoylation signal recruits AKT-C to the plasma membrane where AKT becomes phospho-activated. This mouse was crossed to CD19 Cre mice leading to a B cell specific overexpression of Akt (AKTBOE). For FACS analysis of AktBOEand CD19 Cre control mice we isolated cells from bone marrow, spleen, lymphnodes, mesenteric lymphnodes, as well as cells from the peritoneal cavity and Peyer’s patches. Furthermore, we used CD19-sorted B cells for functional survival and proliferation assays. Results: AktBOE mice displayed a spleenomegaly accompanied by enlarged lymph nodes compared to control mice. FACS analysis of bone marrow showed a reduction of re-circulating B cells while immature B cells were not affected. In the spleen we found a loss of CD23 expression by B cells. Further, we found increased absolute numbers of B and T cells and more neutrophiles, monocytes and mature macrophages in the spleen. Finally, the investigation of immunoglobulin titers by ELISA revealed reduced IgM, IgG1 and IgG3 serum levels. Conclusions: The B cell specific overexpression of Akt underlines its importance in B cell maturation and homeostasis.CD23 serves as a


Poster Sessions 339 lymphoma-marker because of its differential expression on various types of lymphoma. The fact that AktBOE mice have almost no CD23 expression on B cells hinds towards some kind of lymphoma in these mice.

P0468 Ro52- and Ro60-specific B cell pattern in the salivary glands of ¨gren’s syndrome patients with primary Sjo

signaling. The PI3K/Akt pathway appears to be involved in this exacerbated terminal differentiation. Interestingly, in our models, terminal differentiation occurs independently of antigen trigerring, germinal center formation or T-cell help yet giving rise to long-lived PC in spleen. In striking contrast, strong constitutive BCR signaling, mimicking chronic contact with cognate antigen, completely prevented both in vitro and in vivo PC differentiation.

L. A. Aqrawi,* K. Skarstein, G. Øijordsbakken & K. Brokstad* *The Gade Institute, Broegelmann Research Laboratory, Bergen, Norway, The Gade Institute, Section for pathology, Bergen, Norway Purpose/Objective: Primary Sjo¨gren’s syndrome (pSS) is characterised by the presence of autoantibodies against the ribonucleoprotein (RNP) particles Ro/SSA and La/SS-B, and mononuclear cell infiltration of exocrine tissues, especially salivary and lachrymal glands. Low numbers of autoantigen-specific memory B cells and elevated levels of plasma cells have previously been detected in the peripheral blood (PB) of pSS patients compared to controls1). Since both Ro52 and Ro60specific cells have been detected in the salivary glands (SG) of pSS patients, we aimed to characterise the SSA-specific B cell pattern in SG biopsies. Materials and methods: A series of double immunohistochemical stainings were performed on paraffin-embedded tissue from 10 wellcharacterised pSS patients for each Ro52 and Ro60 along with CD19, CD20, CD27, or CD5, respectively. Results: Ro52 and Ro60-specific cells detected in SG tissue were found to be CD19+ B cells located outside the CD19+/CD20+ B cell zones (BCZ) and also interstitially. These SSA-specific cells were also quantified. No SSA-specific cells were observed within the CD20+ BCZ. Hence, no SSA-specific memory B cells were detected in these individuals. Contrary to this, SSA-specific cells were found to be CD19+/CD27++, demonstrating that they are differentiating short or long-lived plasma cells. Also, no SSA-specific cells were CD5+, indicating that they do not belong to the B-1 B cell subset. Conclusions: Together our findings suggest that these lower levels of SSA-specific memory B cells in PB and absence of SSA-specific memory B cells in SG of pSS patients could be resulting from activation of these cells into plasma cells at the site of inflammation. 1. Aqrawi LA, Skarstein K, Bredholt G, Brun JG, Brokstad KA. Autoantigen-specific memory B cells in primary Sjogren’s syndrome. Scand J Immunol 2012;75(1):61 8.

P0470 Sustained B-cell receptor signaling prevents B cell terminal differentiation into plasma cells F. Lechouane,* A. Bonaud,* L. Delpy,* S. Casola, Z. Oruc,* G. Chemin,* M. Cogne´* & C. Sirac* *CNRS UMR7276, Immunology, Limoges, France, IFOM, The FIRC Institute of Molecular Oncology Foundation, Milan, Italy Purpose/Objective: B-cell terminal differentiation into antibody secreting plasma cells (PC) features a transcriptional shift driven by the activation of plasma cell lineage determinants. Little is known about the signals inducing this change in transcriptional networks and the role of the B Cell Receptor (BCR) in terminal differentiation remains especially controversial. Materials and methods: We used immunoglobulin light chain transgenic mice expressing suboptimal surface BCR levels and LMP2A knock-in animals with defined BCR-like signal strengths to explore the influence of BCR signaling in terminal differentiation independently of cognate antigens. Results: We observed a strong increase of PC numbers both in vivo and in vitro upon weak, antigen-independent constitutive BCR

Conclusions: We demonstrated an inverse correlation between BCR signal strength and PC development. These findings provide new insights onto the role of the BCR in PC differentiation and point to the need to resolve BCR signaling to guarantee terminal differentiation.

P0471 Syk is required in B cells for effective antibody responses J. Ackermann, J. Nys, E. Schweighoffer & V. Tybulewicz MRC National Institute for Medical Research, Immune Cell Biology, London, UK Purpose/Objective: Spleen tyrosine kinase (Syk), a pivotal kinase in B cell signalling, is recruited to phosphorylated tyrosines in immunoreceptor tyrosine-based activation motifs (ITAMs) after stimulation of the B cell receptor. Binding to these motifs leads to phosphorylation of Syk and subsequent activation of important downstream signalling molecules. Syk-deficient mice have no mature B cells due to a complete block in B cell development at the immature stage. To study immune responses of Syk-deficient primary B cells, we used a mouse model, which allows inducible deletion of Syk. Materials and methods: To achieve inducible deletion of Syk in mature B cells, we used mice, carrying Cre recombinase fused to two mutated oestrogen receptors and a floxed allele of the Syk gene together with either a wildtyp or a knockout allele of the syk gene. These mice were treated with tamoxifen to induce Syk deletion. Immune responses and B cell activation in these mice were analysed in vivo and in vitro. Results: B cell numbers drop after inducible Syk deletion, but a significant proportion of Syk-deficient B cells survives on a long time scale. We showed that Syk-defcient B cells are unresponsive to BCR stimulation and less responsive to stimulation of TLR recptors. Mice with Syk-deficient B cells had reduced titers of immunoglobulin after immunization with thymus-dependent or thymus-independent antigens. In addition mice with Syk-deficient B cells show strongly impaired germinal centre formation. Conclusions: A long-lived B cell population remains after deletion of Syk. These cells appear to be impaired in responding to BCR and TLR stimulation and have impaired abilities to form germinal centre B cells.


340 Poster Sessions P0472 The deubiquitinases A20 and CYLD do not share overlapping functions during B cell differentiation and activation Y. Chu,* V. Soberon,* R. Beyaert, R. Massoumi,à G. van Loo & M. Schmidt-Supprian* *Max Planck Institute of Biochemistry, Molecular Medicine, Martinsried, Germany, Department for Molecular Biomedical Research, Ghent University, Ghent, Belgium, àDepartment of Laboratory Medicine, Lund University, Malmoe, Sweden Purpose/Objective: The deubiquitinases TNFAIP3/A20 and CYLD are critical negative regulators of signaling events leading to the activation of NF-jB transcription factors. They share similar mechanisms by removing non-degradative K63-linked polyubiquitin chains from an overlapping set of substrates. Loss of A20 in B cells results in impaired immune homeostasis, chronic inflammation and autoimmunity. In contrast, the consequences of CYLD-deficiency in B cells are controversial, ranging from an absence of effects to dramatic B cell hyperplasia. These differences could be due to varying compensation by A20. The aim of this study is to address a potential redundancy between A20 and CYLD in B cells. Materials and methods: We generated mice lacking both A20 and CYLD in B cells and studied B cell differentiation in these mice. In addition, we investigated the responses of the A20/CYLD-deficient B cells to different B cell mitogens by measuring the activation status, proliferation and cytokine production in vitro. Results: The combined loss of A20 and CYLD did not exacerbate the developmental defects of A20-deficiency in B cells. In addition, loss of both A20 and CYLD did not have additive effects on B cell activation, proliferation and the NF-jB-dependant production of the proinflammatory cytokine IL-6. Conclusions: We concluded from this study that the lack of phenotypic effects in CYLD-deficient mice is not due to compensation by A20 but that A20 and CYLD do not functionally cooperate during B cell differentiation and activation.

P0473 The in vivo role of Blimp-1 SUMOylation in B Cell Development C. Reichhold,* B. Tirosh & A. Waisman* *Institute for Molecular Medicine, University Medical Center Johannes Gutenberg University Mainz, Mainz, Germany, Faculty of Medicine Pharmacologyute for Molecular Medicine, Hebrew University, Jerusalem, Israel Purpose/Objective: Recent in vitro studies have shown the importance of SUMOylation (a ubiquitin-like posttranslational modification) on lymphocyte development, in particular early lymphocyte and Plasma cell (PC) development. PC development and differentiation is controlled by BLIMP-1, a transcriptional repressor that is necessary and sufficient to allow B cells to differentiate into antibody secreting cells. The study presented here uses a B cell specific Sentrin protease 1 (SENP1, a deSUMOylation protease) conditional knockout mouse model to investigate the influence of SUMOylation on lymphocyte development, mainly focusing on BLIMP-1 dependent PC development. Materials and methods: The role of BLIMP-1 SUMOylation was investigated in SENP1 conditional knockout mice crossed to CD19Cre and AID-Cre lines, therefore lacking SENP1 from early B cell developmental stage and following the germinal center reaction, respectively. Different B cell subsets were analyzed by flow cytometry, quantitative real time-PCR and Western blot. Sheep red blood cells were used for in vivo stimulation whereas Lipopolysaccharide and IL-4 was used for ex vivo stimulation. Results: We discovered that BLIMP-1 undergoes a reversible modification with SUMO-1, which facilitates BLIMP-1 turnover and

proteasomal degradation. Increase in SENP1 activity stabilized BLIMP-1, while a decrease promoted its degradation. This data indicates that SUMOylation of BLIMP-1 regulates its intracellular stability. In in vivo experiments we can show an impairment of B cell development due to a decreased amount of total B cells in these mice. Furthermore, in naı¨ve mice, we observe increased numbers of immature B cells in spleen and lymph nodes and of germinal center B cells in Peyer’s patches, as well as in spleens of sheep red blood cell immunized mice. We could also show ex vivo an impaired ability of B cell survival after Lipopolysaccharide (LPS) and IL-4 stimulation. Conclusions: This study shows a direct influence of posttranslational SUMOylation on B cell development in an in vivo mouse model. In our preliminary data the elevated germinal center B cell numbers underline the influence of SUMOylation on BLIMP-1 activity, as impaired SUMOylation of BLIMP-1 leads to its degradation and therefore an absence of termination of the germinal center reaction.

P0474 The kinetics of a memory B cell response to virus like particles in mice F. Zabel,* D. Mohanan,* A. Link, J. Bessa,à P. Saudan,§ T. M. Kuendig* & M. F. Bachmann* *Department of Dermatology, University Hospital Zurich, Zurich, Switzerland, Molecular Partners AG, Immunology, Zurich, Switzerland, à Roche AG, Research, Zurich, Switzerland, §Cytos Biotechnology AG, VP Research, Zurich, Switzerland Purpose/Objective: Vaccine-induced specific antibodies are usually responsible for protection against infection. The most common protective mechanism of antibodies is binding to pathogens which neutralizes their infectious potential and enhances their elimination by phagocytes. The most potent antibodies are produced by B cells which have undergone a germinal centre reaction. Within germinal centres, the BCRs of the specific B cells switch from IgM to IgG and hypermutate by the insertion of point mutations in their variable regions. Upon hypermutation, B cells are selected through iterative cycles of best fit for the antigen by follicular dendritic cells (FDC) and follicular T helper cells. While the GC reaction is usually relatively short-lived and ends a few months after elimination of the pathogen, memory B cells persist in the host for very long time-periods and may rapidly respond upon re-encounter of the antigen. In contrast to the well understood differentiation of naı¨ve B cells into memory and/or plasma cells, relatively little is known about the fate of memory B cells upon re-exposure to antigen. Since classical vaccines often only induce protective antibodies after several injections, it is important to understand how triggering of memory B cells may affect the development of high affinity and long lasting antibody responses. In this study we followed the kinetics of the specific memory B cell response in terms of antibody production and frequencies of specific B cells. Materials and methods: To address this question, we used virus-like particles derived from the bacteriophage Qb, which are highly immunogenic due to their repetitive structure. To trace memory B cells, congenic Ly5.1+ C57BL/6 mice were immunized with Qb, splenic memory B cells were isolated and transferred intravenously into naı¨ve Ly5.2+ C57BL/6. Twenty-four hours later recipient mice were challenged with Qb. Results: Transferred memory B cells homed the spleen. Although they did not proliferate as much as naı¨ve B cells after immunization they differentiated more quickly into plasma cells and secreted more antibodies. The number of plasma cells found in spleen is similar in a primary and memory response. However, a higher number of plasma cells were found in the bone marrow, the main source of humoral immunity, after transfer of memory B cells.


Poster Sessions 341 Conclusions: Memory B cells respond to antigen by rapid differentiation into plasma cells rather than extensive proliferation. The induced plasma cells preferentially home to the bone marrow and produce larger amounts of antibodies than plasma cells derived from naı¨ve cells.

P0475 The lymphoid/myeloid potential of B-1 cells is sustained by WNT/ beta catenin pathway M. C. T. Novo & A. F. Popi UNIFESP, MIcrobiology Immunology and Parasitology, Saõ Paulo, Brazil Purpose/Objective: The Wnt/beta-catenin signaling pathway has been shown to play an important role in controlling the proliferation, survival, and differentiation of hematopoietic cells. Several Wnt/bcatenin signaling components substantially influence hematopoietic cell fate. We have shown that B-1 cells are self-renewing cells and spontaneously express both myeloid and lymphoid restricted transcription factors. B-1 lymphocytes play a major role in autoimmunity and are related to CD5+ B-cell lymphomas and leukemias, such as CLL (chronic lymphatic leukemia). Previously, we have shown that this pathway influences B-1 cell proliferation in vitro. Considering that Wnt/beta-catenin signaling network as a critically important regulator of hematopoiesis, we have investigated the effect of quercetin, a classical Wnt inhibitor, on B-1 cells hematopoietic lineage-restricted genes expression. Materials and methods: Peritoneal B-1 cells were obtained and cultivated as described by Popi et al. (Immunology 2009;126(1): 114 22). Treatment of B-1 cells with the Wnt inhibitor quercetin induced an inactivation of the Wnt pathway and suppressed cell proliferation. Herein we have analyzed the effect of Wnt inhibition on the expression of lymphoid and myeloid genes by B-1 cells by real time PCR. Results: We have demonstrated that Wnt/beta-catenin signaling is inhibited by quercetin treatment of B-1 cells in vitro. Wnt pathway inhibition by quercetin induces silencing in Pax-5 and CD19 expression by B-1 cells. However, expression of myeloid associated genes by B-1 cells is still observed after quercetin treatment. Conclusions: Inhibition of Wnt pathway interferes with the commitment of B-1 cells to B-cell lineage, suggesting that Wnt pathway plays an important role during B-1 cell differentiation. Considering that quercetin could inhibit leukemic cell growth without suppressing normal hematopoiesis, the role of Wnt pathway on B-1 cell proliferation and differentiation could be closely related to their ability to generated leukemic cells. Based on this, B-1 cells could be considered an in vitro model to study the molecular networks that orchestrate the transformation of cancer stem cell.

P0476 The role of B lymphocytes in alpha-1-antitrypsin mediated allogeneic allograft protection M. Mizrahi, P. Cal, D. Ochayon, G. Shahaf & E. C. Lewis Ben-Gurion university, clinical bichemistry, Beer sheva, Israel Purpose/Objective: Type-1 diabetes (T1D) is characterized by immune-mediated islet beta cell destruction. Islet transplantation restores normoglycemia and is considered for clinical implementation in T1D patients. There is evidence for B lymphocyte involvement in T1D as well as in islet allograft rejection. However, a protective role for B cells is suggested by the occurrence of islet allograft rejection in B cellknockout mice. Alpha-1-antitrypsin (AAT) is an anti-inflammatory circulating protein that promotes tolerance towards islet allografts and prevents diabetes in non-obese diabetic (NOD) mice in a yet unknown

mechanism. Aim: To characterize the effect of AAT on B lymphocyte responses in the context of allogeneic islet transplantation. Materials and methods: Spleen-derived B lymphocytes were studied. B cell activation-related responses and expression of B cell activating factor (BAFF) receptor were determined under stimulatory conditions in the presence or absence of human AAT (hAAT, 0.5 mg/ml). In vivo, B cell proliferation response evoked by allogeneic skin transplantation was evaluated in draining lymph nodes (DLN) in transgenic mice that express constitutive hAAT (hAAT+/+). To determine whether the alloprotective role of hAAT is B cell-dependent, B cell-depleted hAAT+/+ chimeras were generated; the mice were rendered diabetic by streptozotocin and then transplanted with allogeneic islets. Results: In the presence of hAAT, B cells exhibited a decline in CD40 ligand-induced co-stimulatory molecules CD86 and CD80 (6.3 ± 0.1% and 33.7 ± 4.1%, respectively; P < 0.05). B cells stimulated with antiIgM (Fab)2 displayed 35 ± 5.2% less surface BAFF receptor in the presence of hAAT. In cultures stimulated with CD40 ligand, LPS or BAFF, hAAT diminished inducible B cell proliferation 2.79 ± 0.13fold, 4.62 ± 0.63-fold and 2.04 ± 0.21-fold, respectively (P < 0.05). Skin allograft-evoked B cell proliferation was 4.36 ± 1.1-fold lower in hAAT+/+ mice than in WT recipients (P = 0.0398). While hAAT+/+ transgenic mice accepted islet allografts, chimeric hAAT+/+ mice (300 mg/ dl). Furthermore we observed a higher titer of anti-dsDNA IgG antibodies and rheumatoid factors in Siglec-G-deficient MRL/MpJ fas/ lpr mice. Conclusions: Even though SiglecG deficient mice in the BALB/c background do not show spontaneous autoimmunity, based on our data we conclude that the loss of the inhibitory receptor SiglecG can contribute to the development of autoimmunity, both in mouse SLE and rheumatoid arthritis models.

P0480 The role of toll-like receptor 9 in B-cell activating factor (BAFF) expression and function in normal human B-cells E. Y. Abu-rish,* Y. Amrani & M. J. Browningà *Department of Infection Immunity and inflammation, University of Leicester, Leicester, UK, Department of infection immunity and inflammation, Institute of Lung Health, University of Leicester, Leicester, UK, àImmunology, University Hospitals of Leicester, Leicester, UK Purpose/Objective: B-cell autoreactivity is a characteristic abnormality in several autoimmune diseases. The inappropriate activation of toll-like receptors (TLRs) and/or the over-expression of B-cell activating factor (BAFF) have increasing importance in breaching B-cells’ self-tolerance. In systemic lupus erythematosus (SLE), activation of TLR7 and TLR9, accompanied by high serum levels of BAFF, are implicated in disease pathogenesis. In murine B-cells, TLR9 activation

resulted in up-regulating BAFF expression, while such a direct effect has not yet been established in normal human B-cells (nhB-cells). We therefore studied the effect of CpG-2006, a synthetic TLR9 ligand, on the expression of BAFF and its receptors (BAFF-R, TACI and BCMA) by nhB-cells. Materials and methods: qPCR, flow cytometry and ELISA assays were utilized to investigate the effect of 3lg/ml CpG-2006 on BAFF expression at the level of mRNA, intracellular and membrane-bound protein expression, and secreted protein expression, respectively. BAFF receptors expression, in response to 3 lg/ml CpG-2006 was explored using flow cytometry. The functional role of membrane-bound BAFF was studied in BCR co-simulation and blocking assays. Results: BAFF expression, in response to CpG-2006, was significantly upregulated at the level of mRNA, intracellular and membrane-bound protein. In contrast, we did not detect secreted BAFF in culture supernatants of CpG-2006-stimulated nhB-cells. CpG-2006, in addition, promoted the expression of TACI receptors, but not of BAFF-R or BCMA, in nhB-cells. We found that CpG-stimulated B-cells costimulated B-cell receptor-induced cellular proliferation of nhB-cells, an effect that was completely blocked by a BAFF-specific monoclonal antibody. Finally, CpG-2006 treatment of nhB-cells sensitised them to proliferate in response to exogenous BAFF, whereas exogenous BAFF had no effect on the proliferation of untreated B-cells. This effect was not mediated through TACI, as a TACI-specific blocking antibody failed to inhibit BAFF-mediated cellular proliferation. Conclusions: Taken together, these novel findings demonstrate a functional cross-talk between TLR9 and BAFF signaling in nhB-cell, resulting in autocrine B-cell activation, and have implications for the roles of TLR9 and BAFF in the pathogenesis of SLE.

P0481 TLR9, CD40 or BCR stimulated human naı¨ve and memory B cells exhibit differential responsiveness to the survival effect of IL-21 C. Antonio, J. Pons, N. Lanio, N. Matamoros & J. M. Ferrer Son Espases Hospital, Immunology Service, Palma de Mallorca, Spain Purpose/Objective: IL-21 is one of the most potent cytokines for human B cell proliferation and differentiation. IL-21 also influences B cell survival. The stimulatory or inhibitory effect of IL-21 depends on the maturation and activation status of the B cell, the co-stimulatory accompanying signals and the presence of other cytokines. The aim of this study was to evaluate the response of human naı¨ve and memory B cells to IL-21 after TLR9, CD40 or BCR engagement. Materials and methods: B cells were obtained from PBMC by negative selection using magnetic beads. Purified CFSE-free or CFSElabelled B cells were cultured 3 days in the presence of ODN, antiCD40 antibody or anti-IgM alone or in combination with human recombinant IL-21. Collected cells were stained with anti-CD19PCy7 and anti-CD27PCy5 monoclonal antibodies and analyzed by flow cytometry. Annexin V-FITC and Propidium Iodide staining protocol was used to evaluate the apoptosis of CSFE-free naı¨ve (CD19+ CD27-) and memory (CD19+ CD27+) B cells. Proliferation was measured by CSFE dilution in previously gated CSFE-labelled cells. Results: Spontaneous apoptosis was higher in memory than in naı¨ve B cells. All stimuli alone protected both B cell subsets from spontaneous apoptosis. When IL-21 was tested alone, only naı¨ve B cells were rescued from apoptosis. In contrast, IL-21 addition reverted the protective effect of all other stimuli on naı¨ve B cells. In memory B cells, IL-21 was also able to revert the protective effect of anti-IgM and CpG-ODN but not anti-CD40. Anti-IgM, anti-CD40, or IL-21 alone did not induce proliferation of naı¨ve or memory B cells. CpG-ODN alone induced proliferation only on memory B cells. The combination of IL-21 with anti-IgM did not induce proliferation. IL-21 and anti-CD40 induced greater proliferation of memory than naı¨ve B cells. The addition of IL-21 decreased


Poster Sessions 343 CpG-ODN induced proliferation of memory B cells and modestly increased proliferation of naı¨ve B cells. Conclusions: Memory B cells are more sensitive to spontaneous apoptosis than naı¨ve B cells and less prone to be rescued by individual stimuli. Although a survival factor for unstimulated naı¨ve B cells, IL-21 abrogates activation-induced survival of naı¨ve B cells. On the contrary, IL-21 selectively supports memory B cell responses to T dependent stimulus.

P0482 WASp and N-WASp regulate the germinal center response and production of auto-antibodies C. Dahlberg,* S. Petersen,* E. D. Jordo¨,* M. Baptista,* M. Karlsson,* S. Snapper & L. Westerberg* *Karolinska Institutet, Medicine Solna, Stockholm, Sweden, Harvard Medical School, Children’s Hospital, Boston, MA, USA Purpose/Objective: Wiskott-Aldrich syndrome (WAS) is a rare, potentially life threatening X-linked primary immunodeficiency disease. Forty to 70% of the patients develop any form of autoimmunity. WAS is caused by the WAS protein (WASp). We hypothesized that mice lacking WASp family members have a skewed development and activation of B cells due to intrinsic B cell dysfunction and decreased protective shield of the splenic marginal zone to blood-borne antigens.

Materials and methods: To determine the role of WASp and the homologues molecule N-WASp in B cell biology we have used WASp knock out (WKO) mice and mice lacking WASp and N-WASp in B cells (cDKO mice). To induce breakdown of tolerance with emergence of auto-antibodies, we immunized mice with apoptotic cells. Results: Compared to reduced uptake and decreased immune response after non-self antigen immunization, WKO and cDKO mice had normal uptake of apoptotic cells in the marginal zone and formed large germinal centers after apoptotic cell immunization. However, compared to germinal center B cells in wild type mice, B cells retained longer and proliferated less in germinal centers of WKO and cDKO mice, suggesting decreased capacity to undergo affinity maturation. When compared to wild type mice, WKO and cDKO mice had significantly higher DNA-specific IgM antibodies before and after immunization with apoptotic cells. After repeated apoptotic cell immunizations the immunological tolerance was broken in wild type mice that produced DNA-specific IgG antibodies, while WKO and cDKO mice failed to produce anti-DNA IgG antibodies. Conclusions: Together, our data show that mice lacking WASp family members respond with an atypical immune response to self-antigens such as apoptotic cells. WKO and cDKO B cells formed low quality GC-like structures upon self-antigen challenge and produced mainly auto-reactive IgM antibodies. Our data will increase the knowledge of why patients with primary immunodeficienies such as WAS develop autoimmune diseases.


344 Poster Sessions

Poster Session: Cytolytic T Cells P0483 An optimal pMHCI/CD8 interaction exists which affords maximum pMHCI sensitivity without loss of CD8+ T-cell specificity T. Williams,* M. Clement,* A. K. Sewel,* D. A. Price,* M. Bailey & L. Wooldridge* *Infection and Immunity, Cardiff University, Cardiff, UK, Molecular and Cellular Biology, University of Bristol, Bristol, UK Purpose/Objective: CD8+ T-cells recognize ‘foreign’ peptide fragments, in the context of ‘self’ major histocompatibility complex class I (MHCI) molecules on the surface of host cells. The detection of T-cell antigens is unique because it involves the binding of two receptors (TCR and CD8) to a single ligand (pMHCI). The pMHCI/CD8 interaction is characterized by very low solution binding affinities (KD = 137 lM). An incremental enhancement in CD8 binding (~1.5fold) has previously been shown to result in enhanced recognition of pMHCI, thereby demonstrating the huge potential of CD8 in strategies to enhance T-cell immunity. I aim to define the strength of the pMHCI/CD8 interaction which affords maximal enhancement of pMHCI recognition whilst still retaining specificity of pMHCI recognition by the TCR. Materials and methods: In this study, I used a system of MHCI mutants which vary in the strength of the pMHCI/CD8 interaction. Using tetramer technology, I constructed a panel of A*0201 pMHCI tetramers that exhibit an abrogated (DT227/8KA), weak (A245V), normal (wild-type), slightly enhanced (Q115E), intermediately enhanced (A245V/Kb) and super-enhanced (Kb) interaction with CD8. The latter two mutations were also combined with a reduced affinity b2-microglobulin mutation (b2M K58E). This tetramer panel was then used to examine how the strength of the pMHCI/CD8 interaction influences pMHCI recognition at the cell surface and the specificity of pMHCI recognition. Results: TCR recognition of pMHCI at the cell surface increases as the strength of the pMHCI/CD8 interaction is increased. Loss of pMHCI tetramer staining specificity was observed at pMHCI/CD8 strengths exceeding a threshold KD of ~30 lM. Thus, an optimal pMHCI/CD8 binding interaction exists (between a KD of 97 and 30 lM) which increases pMHCI sensitivity without the loss of specificity. Conclusions: This data defines the strength of the pMHCI/CD8 interaction which affords maximal enhancement of pMHCI recognition whilst still retaining specificity of pMHCI recognition. Due to the non-polymorphic nature of CD8, strategies to enhance the antigen specific T-cell response by targeting CD8 would be globally applicable to any system in which enhanced T-cell immunity is desirable.

P0485 CD40L induced by strong cytotoxic T cell epitopes on CD8 T cells contributes to helper-independent responses D. Llopiz, E. Huarte, M. Ruiz, J. Bezunartea, A. Zabaleta, J. J. Lasarte, J. Prieto, F. Borra´s-Cuesta & P. Sarobe CIMA, Gene Therapy and Hepatology, Pamplona, Spain Purpose/Objective: CD8 T-cell responses can be primed by strong cytotoxic T cell epitopes even in the absence of CD4 epitopes or DCactivating adjuvants. The aim of this work is to analyze the role of CD40/CD40L interaction on the induction of helper-free CD8 T cell responses. Materials and methods: Different strains of mice were immunized with different cytototoxic T cell epitopes emulsified in incomplete Freund adjuvant. CD40/CD40L interaction was blocked in vivo and in vitro by anti-CD40L antibodies. DC maturation as well as IFN-gamma/ CD40L expression by primed CD8 T cells was measured by flow

cytometry. Also, IFN-gamma production was analyzed by ELISA and ELISPOT. Results: We found that CD8 T-cell responses induced by immunization with strong cytotoxic T cell epitopes were inhibited in vivo by CD40L blockade. In vitro, peptide stimulation of splenocytes from immune mice induced CD40L on CD8 T cells and DC maturation, which was partially inhibited by anti-CD40L antibodies. Interestingly, CD40L blockade also inhibited CD8 responses, even in the presence of already mature DC, suggesting a role for CD40L not only in DC maturation but also in CD8 costimulation. These peptides had features of CD4 epitopes, since they helped the induction of responses against other less immunogenic CD8 epitopes. Finally, analysis of peptide epitopes used in human vaccination clinical trials showed that they also induced CD40L on CD8 cells. Conclusions: These results suggest that CD40L expression induced by strong CD8 peptide epitopes can facilitate activation of helper-free CD8 responses.

P0486 CD8beta ADP-ribosylation regulates CD8 coreceptor function T. Lischke,* V. Schumacher,* R. Hurwitz, F. Koch-Nolte* & H. W. Mittru¨cker* *Institute for Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, Core Facility Biochemistry/Proteinpurification, Max Planck Institute for Infection Biology, Berlin, Germany Purpose/Objective: The coreceptor of conventional CD8+ T cells consists of a CD8ab heterodimer and correct interaction of CD8a, CD8b, TCR, and MHC-I is decisive for antigen recognition and effective signal transduction. ADP-ribosyltransferase 2 (ART2) is an ectoenzyme expressed on T cells that utilizes extracellular nicotinamide adenine dinucleotide (NAD) to transfer ADP-ribose to arginine residues of membrane proteins. Here we show that CD8b on murine CD8+ T cells becomes ADP-ribosylated in vitro and in vivo in the presence of extracellular NAD. Materials and methods: ADP-ribosylation leads to loss of binding of certain anti-CD8b antibodies (YTS156.7.7 and 53 5.8). Other antiCD8b antibodies (H35-17) still recognize CD8b indicating that NAD causes only modification of certain epitopes and not a general loss of CD8b. Results: The ADP-ribosylation of CD8b is mediated by ART2, because loss of ART2 expression results in lack of CD8b ADP-ribosylation even in the presence of high doses of NAD. Furthermore, loss of ADPribosyl cyclase 1 (CD38), an ectoenzyme that degrades extracellular NAD, results in inevitable CD8b ADP-ribosylation upon any cell isolation procedure. ADP-ribosylation of CD8b is stable for several hours and re-appearance of unmodified CD8b is mainly due to the replacement of ADP-ribosylated surface CD8b by newly generated molecules, but there also appear to be mechanisms that remove ADPribose residues from surface CD8b. Finally, NAD treatment of ovalbumin-specific endogenous CD8+ T cells or of T cells from OT1 transgenic mice substantially reduces binding of ovalbumin-MHC-I tetramers. Since activated CD8+ T cells downmodulate ART2 expression, reduction of tetramer binding is less pronounced on effector CD8+ T cells. Nevertheless, an in vivo cytotoxicity assay revealed impaired CD8+ T cells function after i.v. NAD injection. Conclusions: In summary, we propose that ADP-ribosylation of CD8b can regulate the coreceptor function of CD8 on CD8+ T cells in the presence of elevated levels of extracellular NAD. Our current studies aim at elucidating the role of this mechanism for the regulation of CD8+ T cell responses.


Poster Sessions 345 P0487 Cross reactivity prediction based on hierarchical clustering of pMHC complexes D. A. Antunes, J. P. Silva, M. F. A. Mendes, M. M. Rigo, J. A. B. Chies, M. Sinigaglia & G. F. Vieira Department of Genetics, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Purpose/Objective: Cross-reactivity was initially described as being triggered by the great similarity between the amino acid sequences of virus-derived peptides presented in the context of the Major Histocompatibility Complex (MHC). These peptide:MHC (pMHC) complexes are recognized by Cytotoxic T Lymphocytes (CTLs), which are the effector agents of cellular immunity. However, immunologists have already described many events of cross-reactivity between epitopes sharing 70% of these cells are IL-17 positive. Therefore, we investigated the immune modulating effects of beta interferon (IFN-b), glatiramer acetate (GA) and vitamin D (VitD), on circulating IL17+ CD8+ T cells in MS. Materials and methods: PBMC were isolated from healthy controls (HC; n = 30) and relapsing-remitting (RR) MS patients in remission (no medication: n = 17, IFN-b: n = 18, GA: n = 13) and during a relapse (no medication: n = 12, IFN-b: n = 10). Additionally, PBMC from 15 IFN-b treated RRMS patients, receiving 20 000 IU VitD3/day for 12 weeks, were isolated at baseline and at 12 weeks. Intracellular FACS analysis was performed on PBMC to assess the IL-17+ cell fraction in the CD8+ T cell population. Data are given as median with corresponding interquartile range. Due to a correction for multiple testing, a P-value < 0.01 was considered significant. Results: Compared to HC, MS patients in remission had higher IL17+ CD8+ T cell percentages (0.3% [0.2 0.4] and 0.5% [0.3 0.5], respectively; P = 0.001), while MS patients during a relapse showed a trend towards higher percentages (0.5% [0.3 0.8]; P = 0.016). In patients in remission, the fraction IL-17+ CD8+ T cells did not differ between the three treatment groups. In MS patients during a relapse, IL-17+ CD8+ T cell percentages were similar in untreated and IFN-b treated patients (0.4% [0.2 1.4] and 0.7% [0.3 0.8], respectively). IL17+ CD8+ T cell percentages were comparable before and 12 weeks after VitD supplementation (0.3% [0.2 0.5] and 0.4% [0.3 0.6], respectively). Conclusions: These results show elevated IL-17+ CD8+ T cell percentages in MS patients. This, in combination with their presence in MS lesions, suggests a role for these cells in MS pathogenesis. However, therapy with either GA, IFN-b or vitamin D seemed unable to downregulate these cells in the circulation.


348 Poster Sessions P0497 Viral dsRNA-activated human dendritic cells produce IL-27 which selectively promotes cytotoxicity in naı¨ve CD8+ T cells R. de Groot, A. van Beelen, G. Bakdash, E. Taanman-Kueter, E. de Jong & M. Kapsenberg Cellbiology, Academic Medical Center, Amsterdam, The Netherlands Purpose/Objective: Dendritic cells (DCs) are central in shaping immune responses by activating naı¨ve T-cells, not only CD4+ helper T cells but also CD8+ cytotoxic T cells. Viral recognition programs DCs to express signal three molecules that promote the differentiation of effector CD8+ T cells. Besides IL-12, another DC-derived IL-12-family member, IL-27, has been reported to contribute herein. Therefore, we studied the relative roles of IL-12 and IL-27 in the induction of CD8+ T cell responses by human DCs. Materials and methods: FACS sorted naı¨ve CD8+ T cells were activated either by anti-CD3/CD28 stimulation in the presence of recombinant human IL-12 and IL-27 or by viral dsRNA-activated

human peripheral blood BDCA1+ DCs in the presence of neutralizing antibodies against IL-12 or the IL-27 receptor. After 3 6 days CD8+ T cell proliferation, granzyme B expression, cytokine production and cytotoxicity were determined. Results: Whereas IL-12 potently induces inflammatory cytokines (i.e. IFN-g and TNF-a, but not IL-2), IL-27 excels in inducing proliferation and a cytotoxic profile (granzyme B, cytotoxicity of target cells) in human naı¨ve CD8+ T cells. Compared to bacterial cell wall peptidoglycan, viral dsRNA-mimic poly (I:C) is superior in priming human BDCA1+ DCs to produce IL-12 and IL-27, which promote inflammatory cytokines and a cytotoxic profile in differentiating CD8+ T cells, respectively. Conclusions: This data supports the concept that viral dsRNAactivated human dendritic cells produce IL-27, which acts as a specialized pro-cytotoxic, anti-viral cytokine that promotes development of effector CD8+ T cells.


Poster Sessions 349

Poster Session: Effector Th Cell Subsets and Plasticity P0498 Activity of T helper cells in patients with primary Sjogren’s syndrome G. Sudzius,* A. Siaurys,* D. Mieliauskaite, I. Butrimiene, Z. Mackiewiczà & I. Dumalakiene* *Department of Immunology, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania, Department of Innovative Diagnostic Treatment and Health Monitoring Technology, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania, àState Research Institute Centre for Innovative Medicine, Department of Regenerative Medicine, Vilnius, Lithuania Purpose/Objective: To investigate whether T helper (Th1, Th2 and Th17) cells activity can differentiate in peripheral blood of patients with primary Sjo¨gren’s syndrome (pSS), non- Sjo¨gren’s sicca syndrome (nSS-sicca) and healthy controls. Materials and methods: We isolated peripheral blood mononuclear cells (PBMCs) from 34 pSS, 13 nSS-sicca patients and 13 healthy controls. We stimulated PBMCs using phorbol-12-myristate-13-acetate and ionomycin, labelled for CD4, IFN-c, IL-4 and IL-17A and analyzed these cells using flow cytometry. Results: According to our results no differences between nSS-sicca and healthy controls were found and we combined them in to one control group. Activity of Th1, Th2 and Th17 cells according to IFN-c, IL-4 and IL-17A expression in patients with pSS were similar to control group. We found the significantly increased percentage of both IFN-c and IL-17 producing Th17/Th1-like cells in pSS patients as compared to control group. We observed a significant correlation between all Th subsets activity in control group. Th1 correlated with Th17, with Th2 and Th17/Th1-like. Th2 correlated with Th17 and Th17/Th1-like. Th17 correlated with Th17/Th1-like. However, we observed correlation only between Th1 with Th2 and Th17 and Th17/Th1-like with Th17 in pSS group (see Table 1). Table 1. Correlation of Th subsets in primary Sjo¨gren’s syndrome patients and control group pSS group

Th subpopulation

Th1

Th1 Th17

Th17

Th2

Th17/Th1

r = 0.3654

r = 0.4657

ns (not

P = 0.0261

P = 0.0048

r = 0.3654

ns

P = 0.0261 Th2

r = 0.4657

significant) r = 0.7485 P < 0.0001

ns

ns

P = 0.0048 Th17/Th1

ns

r = 0.7485

ns

P < 0.0001 Control

Th1

r = 0.6811

r = 0.4997

r = 0.6184

P = 0.0001

P = 0.0093

P = 0.0008

r = 0.6811

r = 0.3981

r = 0.7667

P = 0.0001

P = 0.0440

P < 0.0001

group Th17 Th2 Th17/Th1

r = 0.4997

r = 0.3981

P = 0.0093

P = 0.0440

r = 0.4925

r = 0.6184

r = 0.7667

r = 0.4925

P = 0.0008

P < 0.0001

P = 0.0106

P = 0.0106

the fact that Th subsets activity levels seem to be unaltered in patients and controls. Therefore, we conclude that an imbalance of relationship between Th subsets activity plays a role in pSS pathogenesis.

P0499 Adaptor Src kinase-associated phosphoprotein-1 (SKAP1) differentially suppresses production of multiple cytokines and chemokines in CD4+ T-cells P. Riha & C. E. Rudd Department of Pathology, University of Cambridge, Cambridge, UK Purpose/Objective: While immune cell adaptor Src kinase-associated phosphoprotein-1 (SKAP1, formerly known as SKAP-55) regulates integrin-mediated adhesion of T-cells, little is known whether it plays additional roles in modulating other aspects of immune responses. In this study, we report that while SKAP1 positively regulates T-cell adhesion, it elicited a paradoxical inhibitory effect on a selected group of cytokines and chemokines. Materials and methods: Primary naı¨ve Skap1+ /+ and Skap1-/- CD4+ T-cells were stimulated by various concentrations of immobilized antiCD3, anti-CD28 or unspecific IgG antibodies and the production of CCL3, CCL4, CCL5, CXCL10, GM-CSF, IFN-c, IL-1a, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IL-21, IL-22, IL-27, TGF-b and TNF-a was simultaneously measured by FlowCytomix assay. In parallel, the dynamics of T-cell proliferation and cell viability was assessed by CFSE dilution and 7-AAD exclusion assays, respectively. The activity of mitochondrial oxidases was detected by MTT assays. Results: SKAP1 suppressed the production of cytokines and chemokines such as IFN-c, TNF-a, GM-CSF and CCL3 in the response to the Tcell receptor (TCR) stimulation. Th1 cytokines were affected more than Th2 cytokines, although the inhibition pattern did not strictly follow the division between Th1 and Th2 phenotype. SKAP1-dependent inhibition was not reversed by increasing the strength of the TCR signal, but unlike in the case of the other factors, IL-2 and CCL4 production could be restored by CD28 co-ligation. IL-17 production was relatively resistant to SKAP1-dependent inhibition. While this adaptor did not alter magnitude and dynamics of T-cell proliferation, it also limited the survival of non-dividing, but not proliferating CD4+ T-cells. Conclusions: SKAP1 represents the first example of an adaptor that can both enhance integrin-dependent T-cell adhesion and supress the production of certain cytokines and chemokines reducing polarization of the cytokine milieu towards Th1 phenotype.

Conclusions: The significantly increased percentage of Th cells producing both IFN-c and IL-17A in peripheral blood suggest a possible role of Th17/Th1-like cells in the pathogenesis of pSS. We observed different correlation between Th subsets activity in pSS and in control groups. These results are especially important considering


350 Poster Sessions P0500 Basophils control disease activity and T cell responses in experimental murine colitis M. Rodriguez Gomez,* Y. Talke,* C. Hofmann, I. Ketelsen,* F. Hermann,* B. Reich,* N. Goebel,* S. Shahzad-Nawaz* & M. Mack* *Department of Internal Medicine II, University Hospital Regensburg, Regensburg, Germany, Department of Internal Medicine I, University Hospital Regensburg, Regensburg, Germany Purpose/Objective: Basophils have been recognized as important inducers of T helper cell 2 (Th-2) responses in models of vaccination and infection. However, little is known about their role in autoimmunity. Using the colitis model of adoptive transfer of CD4+ CD62L+ T helper cells into lymphopenic hosts we analyzed whether basophils regulate T cell responses and modulate disease activity. We hypothesized that basophils are activated by proliferating T cells after transfer and influence colitogenic T cells. Materials and methods: In vivo basophils were depleted with antibodies against FceR1 and CD2003R or expanded with repeated injections of recombinant interleukin (IL-) 3. The phenotype of T cells and cytokine expression were quantified by in vivo cytokine capture assay, intracellular staining and quantitative RT-PCR. The weight of the mice, histological scores, colon mRNA levels and flowcytometric analysis of infiltrating cells were used to measure disease activity. In vitro basophils were isolated by FACS-sorting (using the markers FceR1 and CD49b) and incubated with activated T cells. Results: We show that adoptively transferred T cells rapidly proliferate, produce large amounts of IL-3 and expand the number of basophils. These basophils modify the phenotype of T cells during early expansion, counter-regulate disease inducing Th-1 responses and control the development of colitis. Depletion of basophils results in long lasting upregulation of proinflammatory Th-1 responses and exacerbation of colitis, while expansion of basophils with IL-3 almost completely blocks Th-1 cytokines and improves colitis. In vitro, basophil derived IL-4 or IL-6 is able to inhibit IFN gamma and IL-2 production in T helper cells, while only the combined release of both cytokines also suppresses TNF production. Conclusions: These data show a beneficial role of basophils in a T cell driven model of autoimmunity and identify basophils as potential target in inflammatory bowel diseases.

P0501 CD4+ T cell homeostasis in a monogenic autoimmune disease APECED N. Heikkila¨, H. Mannersto¨m, S. M. Laakso, H. Jarva & T. P. Arstila Haartman Institute, University of Helsinki, Helsinki, Finland Purpose/Objective: Autoimmune polyendocrinopathy-candidiasisectodermal dystrophy (APECED) is a recessive autoimmune syndrome caused by loss-of-function mutations in the Autoimmune Regulator (AIRE) gene. The clinical picture includes autoimmunity targeting especially endocrine organs, chronic mucocutaneous candidiasis and various ectodermal defects. AIRE is a transcriptional factor participating in negative selection of thymocytes and maintenance of peripheral deletional tolerance, but the details of the pathogenesis remain unknown. In APECED the peripheral CD8+ T cell homeostasis is imbalanced and associated with elevated levels of a homeostatic cytokine interleukin-7 (IL-7). In this study we display properties of CD4+ T cell population in APECED. Materials and methods: Peripheral blood mononuclear cells from APECED patients and healthy controls were stimulated with anti-CD3 antibody, permeabilized and stained with fluorescent dyes for flow cytometric analysis. IL-7 plasma levels were determined by ELISA.

Results: In patients the number of CD4+ CD45RA-CCR7- cells, considered effector cells, is increased while the number of CD4+ CD45RA+CCR7+ cells, considered naı¨ve cells, is diminished. Also the number of recent thymic emigrants (RTE), defined as CD45RA+CD31+ cells and thought to reflect the thymic conditions, is diminished. Patients’ CD4+ population exhibits an increased expression of interferon gamma and interleukin-4, markers of type 1 and type 2 helper T cells respectively. The expression of interleukin-17 was marginal both in patients and controls. No cells expressing two intracellular cytokines at the same time were detected. The alterations in cytokine levels are particularly marked in the CD4+ RTE population. The expression of IL-7 receptor in CD4+ cells is decreased and inversely proportional to the elevated plasma IL-7 concentration in patients. This is associated with increased proliferation rate and decreased CCR7 expression in CD4+ CD45RO- population. Conclusions: In APECED the CD4+ cells are hyperreactive. Their differentiation begins prematurely as RTE cells show characters of type 1 or type 2 helper T cells. The perturbations in CD4+ population are related to IL-7 dysregulation. This is of particular interest because IL-7 axis has been linked to other multifactorial autoimmune diseases such as multiple sclerosis.

P0502 CD40L expression identifies human and mouse CD8+ helper T cells M. Frentsch,* J. J. Listopad, R. Stark,* N. Matzmohr,* A. N. Hegazy,à S. Meier,* F. Gebhardt,§ A. Fro¨hlich,à A. R. Schulz,* J. C. Hafalla,– K. Matuschewski,– M. Lo¨hning,à D. Busch,§ T. Blankenstein & A. Thiel* *Regenerative Immunology and Aging, Berlin-Brandenburg Center for Regenerative Therapies, Berlin, Germany, MDC, Tumorimmunology, Berlin, Germany, àExperimental Immunology, Charite University Medicine, Berlin, Germany, §TUM, Institute for Medical Microbiology, Munich, Germany, –Parasitology Unit, MPI for Infection Biology, Berlin, Germany Purpose/Objective: CD40L expression on activated CD4+ T-helpercells (Th) is one of the most potent signals for T-cell depended activation of APC and is recognized generally as a hallmark for Th cells. Recently, we detected that CD40L is expressed also by a major memory subset of CD8+ T cells, which concomitantly are not cytotoxic, shown by lack of Granzyme B, Perforin and degranulation (CD107a). Materials and methods: human & mouse cells. Results: In various immune responses against pathogens as Listeria monocytogenes, LCMV or Plasmodium berghei as well as SV40 T antigen expressing tumor cells we detected up to 50% CD40L expressing CD8+ T cells among the antigen-specific CD8+ T-cell populations. In peripheral blood of healthy human donors on average 25% of memory CD8+ T cells express CD40L. These CD40L+ CD8+ T cells display plasticity with respect to their cytokine profile similar to CD4+ Th cells and accordingly express cytokines as IL-2, IL-4, IL-17 or IFNc. Furthermore, in vitro assays revealed that CD40L+ CD8+ T cells resemble also functional properties of Th cells and therefore are able to activate properly B cells or DC, shown e.g. by IgG or IL-12 secretion, respectively. To analyze the impact of CD40L expression on CD8+ T cells in vivo we challenged Rag1-/- mice with tumor cells and injected wt or CD40L-/- CD8+ T cells. Application of wt CD8+ T cells prevented the establishment of a solid tumor, whereas injection of CD40L-/- CD8+ T cells alone resulted in a non-controlled tumor progression similar to non-treated tumors demonstrating that CD8+ T-cell mediated immunity can be heavily impaired in the absence of CD8+ T-cell inherent CD40L expression. Conclusions: Our results disclose an essential functional relevance of CD40L expressed by CD8+ T cells. Especially, in situations of reduced


Poster Sessions 351 CD4+ T-cell help, MHC-II antigen presentation or in tumor-driven tolerogenic conditions with limited danger signals CD40L+ CD8+ T cells may exert essential helper responsibilities for immunity and thus are potent T cells to execute or support effective anti-tumor or antipathogen immune therapies. This work is supported by BMBF network ST-Thera and SFB TR36.

P0503 Characterisation of IL-10-producing CD4+ T cells induced by IL-27: implications in autoimmune inflammation? A. Young, T. O’Hagan & D. C. Fitzgerald Centre for Infection and Immunity, Queens University Belfast, Belfast, UK Purpose/Objective: Interleukin 27 (IL-27) is a heterodimeric cytokine that was originally believed to be pro-inflammatory by polarising naı¨ve CD4+ T cells to a Th1 cell phenotype. IL-27 has more recently been shown to exhibit a range of potent anti-inflammatory effects on T cells, including the up-regulation of Interleukin 10 (IL-10) production in Interferon-c+ (IFN-c+) T cells. However there is ambiguity regarding the phenotype and function of IL-10+ IFN-c+ T cells and the role these cells may play in autoimmune diseases such as Experimental Autoimmune Encephalomyelitis (EAE), an animal model of Multiple Sclerosis (MS). Materials and methods: CD4+ T cells were activated with anti-CD3 and anti-CD28 antibodies in vitro for up to 72hrs in non-polarising (NP), Th1, iTreg and Tr1 cell polarising conditions in the presence or absence (+/-) of IL-27 and analysed by flow cytometry and ELISA. For in vivo investigations, SJL mice were immunised with Proteolipid Protein 139 151 (PLP139 151) in Complete Freunds Adjuvant (CFA) to induce Relapsing Remitting EAE (RR-EAE), and were examined for the presence of IL-10+ IFN-c+ T cells at multiple phases of disease in the periphery and CNS. Results: IL-10 producing CD4+ T cells driven by IL-27 exhibited high expression of T-bet while lacking Foxp3 and IL-21 (associated with iTreg and Tr1 cells respectively). By ELISA, IL-27 up-regulated IFN-c and IL-10 secretion, while inhibiting IL-5, another Tr1-associated cytokine. This phenotype was enhanced under Th1 polarising stimuli. In vivo, IL-10+ IFN-c+ CD4+ T cells were detectible in low numbers in spleen, lymph node and in spinal cord at multiple disease phases of EAE with variation according to disease phase. Conclusions: In conclusion these studies show that IL-10+ CD4+ T cells induced by IL-27 in vitro express markers of Th1 cells. In vivo, IL10-producing Th1 cells were present in the periphery and CNS of mice with RR-EAE. Taken together our findings suggest that IL-27 drives the development of IL-10-producing Th1 cells which may modulate autoimmune inflammation.

P0504 Chemoimmunotherapy of MC38 mouse colon carcinoma: cyclophosphamide and dendritic cell vaccine treatment induces the differentiation of CD4+ lymphocytes J. Wojas-Turek,* E. Pajtasz-Piasecka, J. Kicielinska, J. Rossowska,à D. Dusà & E. Piasecki* *Laboratory of Virology, Institute of Immunology and Experimental Therapy, Wroclaw, Poland, Laboratory of Experimental Anticancer Therapy, Institute of Immunology and Experimental Therapy, Wroclaw, Poland, àLaboratory of Glycobiology and Cell Interactions, Institute of Immunology and Experimental Therapy, Wroclaw, Poland Purpose/Objective: The study aimed to determine the CD4+ cell differentiation induced by the administration of the chemotherapeutic agent and dendritic cell (DC)-based vaccines in MC38 colon carcinoma-bearing C57BL/6 mice.

Materials and methods: C57BL/6 mice with advanced MC38 tumor were injected with cyclophosphamide (CY). After three days, mice were injected with vaccines consisted of bone marrow-derived dendritic cells pulsed with tumor lysate (BM-DC/TAg) and/or dendritic cells of JAWS II line genetically modified for IL-2 production (JAWS II/IL-2) or with control gene JAWS II/Neo. Cell vaccines were administrated in three consecutive weeks. Tumor growth delay over control was estimated. On the 7th day after the last injection, spleens were harvested and splenocytes were stimulated with Concanavalin A (ConA). The percentage of CD4+ cells expressing transcription factors characteristic for lymphocytes Th1, Th2, Th17 and Treg in stimulated splenocytes was analyzed. The production of interferon c (IFN-c), interleukin (IL-)4 and IL-17A by cells stimulated with ConA was evaluated. Results: Administration of cyclophosphamide followed by dendritic cell vaccines caused tumor growth delay compared to CY alone. Chemoimmunotherapy increased the percentage of Th1, Th2 and Th17 and decreased the percentage of Treg cells among CD4+ splenocytes stimulated with ConA, especially when vaccines contained BM-DC/TAg+ genetically modified JAWS II cells. Additionally, this combined therapy resulted in increase in IFN-c, IL-4 and IL-17A production by stimulated spleen cells. Conclusions: Taken together, these findings suggest that administration of CY followed by DC-based vaccines induces the differentiation of Th1, Th2 and Th17 splenocytes and cause decrease in percentage of Treg cells in CD4+ spleen cells. Observed changes in the number of CD4+ T cell of each subsets during chemoimmunotherapy may be valuable prognostic factor and a basis for future improvements of anticancer therapy The financial support of the study is granted by the National Science Center of Poland (decision number DEC-2011/01/N/NZ4/01725).

P0505 Comparison of effector and regulatory cytokine expression of T cells in the target organ of experimental monophasic and relapsing autoimmune disease U. Kaufmann, M. Diedrichs-Mo¨hring & G. Wildner Ophthalmology, Klinikum der Universita¨t Mu¨nchen, Mu¨nchen, Germany Purpose/Objective: Human autoimmune diseases usually present with a chronic or relapsing course, while most animal models are monophasic. We could establish a rat model of relapsing autoimmune uveitis (EAU), an intraocular inflammatory disease, which enables us to investigate the underlying immune reactions. We compared the intraocular immune cell populations isolated during the course of relapsing with that of monophasic EAU. Materials and methods: Rats were immunized with retinal soluble antigen (S-Ag) peptide PDSAg (inducing monophasic EAU) or interphotoreceptor retinoid-binding protein (IRBP) peptide R14 (inducing relapsing EAU) in CFA. Intraocular cells were collected at various time points during ocular inflammation and stained ex vivo for TCR-ab and intracellular IFN-c, IL-17, IL-10 and Foxp3 expression. Results: During the course of monophasic uveitis intraocular T cells coexpressing IFN-c with IL-17, as well as IFN-c or IL-17 with IL-10, respectively, increased. In contrast, in relapsing EAU the number of IL17+ , IFN-c+IL-17+ and IL-17+ IL-10+ cells decreased at resolution, while IFN-c+ cell numbers remained elevated also during relapses. In general, only cell populations that express only one of the tested cytokines slightly increased or remained stable during recurrences. Foxp3+ cells increased in both, monophasic and relapsing EAU. Conclusions: The change of the intraocular T cell populations during EAU and the large numbers of T cells producing multiple cytokines point to a strong population dynamics in the eye. We observed differences between monophasic and relapsing disease with respect to IFN-c+ and IL-17+ populations. The strong increase of IL-10+ T cells in


352 Poster Sessions the eyes during monophasic EAU suggests a regulatory role of these cells with a potential to prevent relapses, rather than Foxp3 expressing cells, which increased in both monophasic and relapsing EAU. P0506 Der P 1 induces FOXP3+ GATA3+ T cells in allergic individuals L. L. Reubsaet,* R. Giezeman,* J. Meerding,* H. G. M. Arets, I. M. de Kleer,à B. J. Prakken,* F. van Wijk** & J. Beekman** *Center for Molecular and Cellular Intervention, UMC Utrecht, Utrecht, The Netherlands, Department of Pediatric Respiratory Medicine, UMC Utrecht, Utrecht, The Netherlands, àLaboratory of Immunoregulation, Department for Molecular Biomedical Research (DMBR) VIB, Ghent University, Ghent, Belgium , , , Purpose/Objective: Functionally distinct T helper cell subsets such as regulatory T cells (Treg) and T helper 2 (Th2) cells play an important role in allergy. Originally FOXP3 and GATA3 were shown to be the master transcription factors for Treg and Th2 cells, respectively. However accumulating evidence, mainly from murine models, indicates that these transcription factors are not restricted to a single T cell lineage and GATA3 expression has been implicated in controlling Treg physiology under inflammatory conditions. So far data on the differential function of these transcription factors in humans are lacking and it remains to be investigated whether these have additional functions in human disease. We studied the in vitro induction of FOXP3 and GATA3 upon antigen-specific activation. Materials and methods: We stimulated peripheral blood mononuclear cells (PBMC) of allergic children and non-sensitized healthy controls with allergens like Der P1 and Bet V1. After various days of culture cells were analyzed for transcription factors GATA3 and FOXP3, phenotypic markers and different cytokines to evaluate differences in Th2 and Treg responses. Results: In allergic individuals, besides a pronounced Th2 response with high GATA3, IL4 and IL13 expression, a select population of cells was found to co-express both GATA3 and FOXP3. These double positive cells were highly proliferative upon allergen stimulation and produced Th2 cytokines, but at lower levels than the GATA3 single positive cells. In addition, they were able to potently suppress T cell proliferation and the production of IFNgamma and TNFalpha in vitro. Conclusions: In conclusion, we identified a unique allergen-specific CD4+ T cell population that co-expresses GATA3 and Foxp3 and displays functional features of both a Th2 and Treg phenotype. These data further indicate that GATA3 and FoxP3 cannot be used to uniquely define human T helper cell subsets.

P0507 Distinct differentiation requirements and functional properties of microbe-specific human Th17 cells C. E. Zielinski,* D. Jarrossay, F. Ronchi, A. Lanzavecchia & F. Sallusto *Department of Dermatology and Allergology, Charite´ UniversityMedicine Berlin, Berlin, Germany, Cellular Immunology, Institute for Research in Biomedicine, Bellinzona, Switzerland Purpose/Objective: Th17 cells have emerged as a new T helper cell lineage involved in the clearance of extracellular bacteria and fungi. A dys-regulated Th17 response, however, can induce severe tissue destruction and autoimmunity. Therefore, mechanisms must be in place to shield the host from immune-mediated damage. In this study our aim was to analyze mechanisms by which human Th17 cell responses could be restrained in settings of infections. Materials and methods: We developed an in vitro assay for the generation of human antigen specific T helper cells using whole microbial antigens.

Results: We demonstrate that human Th17 cells transiently produce the anti-inflammatory cytokine IL-10 upon stimulation. Interestingly, IL-10 expression was accompanied by reciprocal down-regulation of IL-17, leading to a functional regulatory Th17 cell phenotype after the peak of the effector response. The ability of Th17 cells to express IL-10 was, however, restricted to certain antigen specificities. Ex vivo isolated C. albicans specific Th17 cells could not produce IL-10 in comparison to S. aureus specific Th17 cells. This was due to differential priming requirements of these Th17 cell sub-populations. IL-1beta instructed naı¨ve T cells to develop into a pro-inflammatory non-IL10 expressing Th17 cell subset. Th17 cell priming with S. aureus, however, was not IL-1beta dependent, leading instead to the generation of IL-10 producing Th17 cells with self-regulatory activities. Conclusions: Thus, using a novel approach that combines the in vitro priming of naı¨ve T cells by whole microbes with the ex vivo analysis of memory T cells we were able to unmask the existence of two types of Th17 cells that differ in priming requirements, TCR repertoire and function. This approach revealed that IL-1beta is a molecular switch for determining a functional memory for IL-10 expression. This has important consequences for the physiological termination of proinflammatory immune responses and the limitation of bystander damage in certain pathogen microenvironments. Targeting IL-1beta early in the differentiation process of Th17 cells (as we demonstrate with IL1ra therapy in CAPS patients) might therefore represent a promising therapeutic strategy to confer anti-inflammatory properties to cellular mediators of autoimmune diseases.

P0508 Distinctive features of classic and non-classic (Th17-derived) human Th1 cells M. Capone, L. Maggi, V. Santarlasci, M. C. Rossi, V. Querci, E. Maggi, F. Liotta, L. Cosmi, S. Romagnani & F. Annunziato Internal Medicine, University of Florence, Florence, Italy Purpose/Objective: T helper 17 (Th17) lymphocytes represent a third arm of the CD4+ T cell effector responses in addition to Th1 and Th2 cells. Th17 cells have been found to exhibit high plasticity because they rapidly shift into the Th1 phenotype in the inflammatory sites. In human beings, Th1 cells derived from Th17 cells express CD161, whereas classic Th1 cells do not, and they have been named as nonclassic Th1 cells. Materials and methods: In this study, we examined similarities and differences between classic and non-classic human Th1 cells by assessing a panel of T-cell clones, as well as CD161+ or CD161CD4+ T cells derived ex-vivo from the circulation of healthy subjects or the synovial fluid of patients with juvenile idiopathic arthritis. Results: The results showed that non-classic Th1 cells could be identified because of CD161 expression, as well as the consistent expression of retinoic acid orphan receptor C, IL-17 receptor E, CCR6 and IL-4-induced gene 1, which were all virtually absent in classic Th1 cells. Conclusions: The possibility to distinguish these two cell subsets by using such a panel of markers may allow the opportunity to better establish the respective pathogenic role of classic and non-classic (Th17-derived) Th1 cells in different chronic inflammatory disorders.


Poster Sessions 353 P0509 GM-CSF production by CD4+ T cells from MS patients E. Peelen,* J. Damoiseaux, S. Knippenberg,* A. H. Muris,* J. Smolders,à J. W. Cohen Tervaert,§ R. Hupperts– & M. Thewissen§ *School for Mental Health and Neuroscience, Maastricht University Medical Center, Maastricht, The Netherlands, Laboratory for Clinical Immunology, Maastricht University Medical Center, Maastricht, The Netherlands, àDepartment of Neurology, St. Antonius Ziekenhuis, Nieuwegein, The Netherlands, §Department of Internal Medicine, Maastricht University Medical Center, Maastricht, The Netherlands, – Department of Neurology, Orbis Medical Center, Sittard, The Netherlands Purpose/Objective: Th17 cells are believed to be the main pathogenic T cells in multiple sclerosis (MS). Recent studies in experimental models of MS suggest that GM-CSF producing T cells might be even more pathogenic. Data on GM-CSF in MS patients are scarce. We hypothesize that GM-CSF expression is elevated within the CD4+ T cell compartment of MS patients. Materials and methods: PBMC and CD4+ T cells were isolated from MS patients (n = 31 and n = 33, respectively) and healthy controls (HC; n = 21 and n = 20, respectively). GM-CSF mRNA expression was assessed by real-time quantitative PCR and is expressed relative to GAPDH expression levels. Intracellular flow cytometry was performed on PBMC from HC and MS patients (untreated, glatiramer acetate (GA) or interferon beta (IFN-b) treated patients) to determine the fraction of GM-CSF+ cells within the CD4+ T cell compartment. Differences between cohorts were assessed with the Mann-Whitney Utest and Bonferroni correction was used to compensate for multiple testing. A P-value 95% pure) from C57BL/6, BALB/c and DBA/2 mice were polarised in vitro using interleukin (IL)-6, IL-1b and transforming growth factor-b in medium containing a natural AhR agonist (tryptophan). Successful polarisation was determined by production of IL-17 measured by RT PCR and ELISA. To assess the contribution of AhR activation during Th17 polarisation; cells were cultured in the presence or absence of an AhR antagonist (CH-223191; 3 lM). Results: Th17 polarisation was achieved for all three mouse strains, with the highest levels of IL-17 protein and mRNA expression recorded for C57BL/6 tissue. However, levels achieved for C57BL/6 mice were lower than those reported by other authors. Following inhibition of the AhR using the antagonist, all mouse strains displayed at least a 50% reduction in Th17 polarisation but interestingly, levels of IL-17 mRNA and protein were particularly reduced (>80%) in the low affinity AhR DBA/2 strain mice. Conclusions: These data suggest that AhR activation is essential for optimal Th17 polarisation in all mouse strains investigated and that considerations of AhR affinity do not apply when considering the influence of a natural agonist on Th17 polarisation.

P0530 The Shc family protein adaptor, Rai, acts as a negative regulator of Th17 cell development C. Ulivieri,* M. T. Savino,* B. Ortensi, L. Emmi,à G. Pelicci, M. M. D’elios§ & C. T. Baldari* *Evolutionary Biology, University of Siena, Siena, Italy, Experimental Oncology, European Institute of Oncology, Milano, Italy, àLupus Clinic, Policlinico di Careggi AOUC, Firenze, Italy, §Internal Medicine, University of Florence, Firenze, Italy Purpose/Objective: Rai acts as a negative regulator of antigen receptor signaling in T and B cells. Rai-/- mice develop lupus-like autoimmunity associated to the spontaneous activation of self-reactive lymphocytes. Here we have addressed the potential role of Rai in the development of the proinflammatory Th1 and Th17 subsets. Materials and methods: Lymph node T cells or naı¨ve T cells were isolated from wild-type and Rai-/- mice and their cytokine profile wasdetermined by ELISPOT and qRT-PCR both as such and after stimulation in vitro with immobilized anti-CD3 mAb in the presence or absence of polarizing cytokines. The expression of rai was measured in PBL from SLE patients and healthy donors by qRT-PCR. Results: We show that Rai-/- mice display a spontaneous Th1/Th17 bias. In vitro polarization experiments demonstrate that rai deficiency favours the development and expansion of Th17, but not Th1, cells, indicating that Rai modulates TCR signaling to antagonize the pathways driving naı¨ve CD4+ T cell differentiation to the Th17 lineage. Th1 and Th17 cell infiltrates were found in the kidneys of Rai-/- mice, providing evidence that Rai deficiency contributes to the development of lupus nephritis not only by enhancing lymphocyte activation but also by promoting the development and expansion of proinflammatory effector T cells. Interestingly, T cells from SLE patients were found to have a defect in Rai expression, suggesting a role for Rai in disease pathogenesis.

Conclusions: In conclusion, we have identified Rai as a negative regulator of Th17 cell differentiation and expansion in the mouse and found evidence of an impairment of Rai expression in PBL from SLE patients, where a Th1/Th17 bias has been clearly documented, which suggests that it might subserve a similar function in humans. Given the causal role of Th17 cells in a number of autoimmune disorders, such as multiple sclerosis and rheumatoid arthritis, these data suggest that Rai may play a function in preventing autoimmunity beyond lupus nephritis.

P0531 The signalling strength of Phosphatidylinositol-3-kinase (PI3K) pathway regulates T helper subsets and plays a central suppressive role in autoimmunity D. Ielo, M. Zayoud, K. El Malki, A. Heinen, C. Hackenbruck, S. Reißig, F. Wanke, A. Waisman & F. C. Kurschus Molecular Medicine, University Medical Center of the Johannes Gutenberg, University of Mainz Institu, Mainz, Germany Purpose/Objective: Recent works have shown the importance of Phospohayidylinositol-3-kinase (PI3K) on T helper cell commitment. Using distinct genetic means we show that the intensity of the PI3K signal generate T helper cells type 1, T helper cells type 17 and induced regulatory T cells subsets. When thePI3Ks signaling pathwayis constitutively over-expressed on T cells; mice do not develop any signs of induced Experimental Autoimmune Encephalomyelitis (EAE) and of induced colitis.We show then, a new role of the PI3Ks signalthat plays a central role in induction of tolerance on autoimmunity via IL-22 and IFNc. Materials and methods: CD4+ CD62LT cells oniTreg, Th1 cells, and Th17 cells: LNs and SPs from WT mice, PD-1 KO, CD4creP110ind mice were harvested and naiveT cells were sorted using Magnetic Beads columns. The cells were cultured from 1 to 7 days under different conditions. Induction and assessment of EAE: Active EAE was induced by immunization with 50 lg of MOG35*55 peptide emulsified in CFA. Mice also received 200 ng of PTi.p. on the day of immunization and two days later. Clinical assessment of EAE was performed according to the standard criteria: from 0 to 6 based on the level of the sickness. Results: We demonstrate that different concentration of the CD28 stimulus and/or the lack of the PD-1 receptor inhibits the production of IL-17A under TH17 condition, impairs the generation of iTregs and enhances the production of IFNc under TH1. We further demonstrate that TGFb reduces the PI3K signalling strengthrestoring the production of cytokines under different conditions, altered by the higher PI3Ks pathway. Mice that over express PI3Ks, do not develop any signs of EAE and aretotally resistant to induced colitis transfer model on RAG1 KO model; the level of GMCSF and IL-17A is not affected but the production of IFNcand IL-22 is dramatically enhanced. Using neutralizing antibodies for IFNc and IL-22 we proved that either EAE and colitis can be both rescued in the transgenic model to a comparable level of the WT. Conclusions: Here we show the master role of PI3Ks on CD4+ T helper cells commitment; the major role of this cascade in influencing the cytokines production. An high activity of PI3Ks leads to a TH1 outcome, a middle activity to TH17 anda low activity to Tregs. The high production of IL-22 and IFNc protect the mouse from EAE and colitis unveiling a complete new role of PI3Ks cascade on induction of tolerance and identify IL-22 as a new potential target in autoimmunity


360 Poster Sessions P0532 The transcription factor Blimp-1 is a critical regulator of IL-10 expression by T helper 1 cells C. Neumann,* F. Heinrich,* J. Ahlers,* S. Rutz, N. MockelTenbrinckà & A. Scheffold* *Cellular Immunology, German Rheumatism Research Centre Berlin, Berlin, Germany, Department of Immunology, Genentech, San Francisco, CA, USA, àMiltenyi Biotec, Research and Development, BergischGladbach, Germany Purpose/Objective: Expression of the anti-inflammatory cytokine IL10 by pro-inflammatory T helper 1 cells (Th1) is a crucial mechanism for Th1 self-regulation and the containment of inflammatory immune responses. To identify potential transcription factors that are involved in the regulation of IL-10, we highly purified IL-10 secreting and nonsecreting Th1 cells and screened their gene expression profile for new candidates. Materials and methods: Sorted murine naı¨ve CD4 T cells were activated in vitro under Th1 polarizing conditions and subjected to cytokine secretion assay on day 5 following re-stimulation with PMA/ Iono. Subsequently, IL-10 secreting and non-secreting T cells were highly purified via FACS sorting and analysed using an Affymetrix GeneChip array for the expression of candidate transcription factors. Results: We found that Blimp-1 was selectively over-expressed by IL10 producing Th1 cells. Blimp-1 deficiency in in vitro as well as in vivo differentiated Th1 cells completely abolished IL-10 expression. In turn ectopic expression of Blimp-1 strongly induced IL-10 production by Th1 cells. Importantly over-expression of c-Maf, which also segregated with IL-10 secretion in our screen, could not rescue the IL-10 deficiency in Blimp-1-/- Th1 cells, although c-Maf over-expression was sufficient to induce IL-10 as well as Blimp-1 expression in wild-type T cells. Furthermore c-Maf expression levels were not altered in Blimp-1 deficient Th1 cells as well as after Blimp-1 over-expression, suggesting that Blimp-1 rather than c-Maf limits IL-10 expression. In addition, stability studies with sorted IL-10 producing Th1 cells revealed that also IL-10 re-expression highly correlated with Blimp-1 expression in contrast to c-Maf. Conclusions: Taken together our data identify Blimp-1 as a critical transcription factor for IL-10 expression by Th1 cells and introduce an attractive target for therapeutic manipulation of this pivotal immunoregulatory mechanism.

P0533 Toll-like receptor 9 ligand promotes vaccine potency by enhancing Follicular Helper T cell development S. Chakarov Centre de Physiopathologie de Toulouse Purpan, INSERM UMR1043, Toulouse, France Purpose/Objective: Protein sub-unit vaccines promote long-term immunity through the production of high-affinity B cell memory. To be effective, vaccine priming must induce antigen-specific Follicular Helper T cells (Tfh) that control plasma cell production and memory B cell development in secondary lymphoid tissue. Thus, understanding the mechanisms of Tfh cells and how to manipulate them could have a lot of impact for vaccine design. It was shown that soluble TLR9 ligand can be used as adjuvant for a protein antigen in vivo by enhancing the T-dependent antibody response. This effect require TLR signaling in dendritic cells (DC). Thus, it is clear that TLR9 ligand can promote antibody responses, there is little understanding of the rules governing how TLR9 signaling contribute to antibody responses in vivo. Moreover, whether complementation ofexisting vaccine with TLR9 ligand enhances adjuvancity is still unknown.

Materials and methods: We took advantage of the well-characterized T and B immune responses of C57BL/6 mice to I-Ea protein and its immunodominant MHC class II epitope using Ea52-68-I-Ab pMHCII tetramers and Flow cytometry approaches to monitor the dynamics of antigen-specific B and T cell resposnes after protein vaccination. Furthermore, using this antigen model, we can also track antigen presentation and the nature of presenting DC that appear in draining lymph nodes from immunized animals. Using this protein vaccination, we investigated the impact of CpG addition in the adjuvanticity and on antigen-specific immune response. Results: We show that addition to vaccine adjuvant of TLR9 ligand, CpG, enhances the antigen-specific B cell responses after protein vaccination in vivo. In particular, this effect correlates with an increase of antigen-specific Tfh cell. Furthermore, we observed that cDC recruited in the draining LN in CpG-primed condition secrete more IL-6, a cytokine necessary for Tfh cell differentiation in vivo, and are more prone to induce Tfh cell differentiation directly ex vivo when compared to DC from primed animals without CpG. In contrast, conventional DC bearing pMHCII on their surface which are recruited in the draining LN are less abundant in CpG-primed condition. In addition, we also showed that pDC do not capture and present Ag but participate in this bias towards Tfh cell differentiation by secreting IL6. Conclusions: Our results suggest that TLR9 ligand through DC imprints the specialized program of antigen-specific effector Tfh function needed to promote high affinity B cell immunity in vivo and could serve as a basis for manipulating the formulation of the next generation of protein vaccines.

P0534 Towards identification of gene regulatory network resulting in human TH17 differentiation S. Tripathi,* S. Tuomela,* V. Salo,* Z. Chen,* K. Laurila, B. Gupta,* ¨ ijo¨, H. La¨hdesma¨ki, B. Stockingerà & R. Lahesmaaà T. A *Molecular Immunology Group, Turku Centre for Biotechnology, Turku, Finland, Department of Information and Computer Science, Aalto University School of Science, Helsinki, Finland, àDivision of Molecular Immunology, Medical Research Council National Institute for Medical Research, London, UK Purpose/Objective: The aim of our study is to identify key regulatory networks of the early human Th17 cell differentiation process. This will be achieved through combination of genome wide and computational methods exploiting the strategy we recently used to report fundamentally new insights into human Th2 cell differentiation (Elo et al., Immunity, 2010). Materials and methods: CD4+ T cells isolated from human umbilical cord blood were used to generate Th17 cells. To generate Th17 cells, cells were activated via cross linking T cell receptor (anti-CD3+ antiCD28) and polarization was stimulated with combination of TGF- b, IL-6, IL1b, anti-IL4, and anti-IFN-c. Polarization of Th17 cell differentiation was confirmed by measuring the expression of several Th17 specific genes such as IL17A, IL17F, RORC, and CCR6. For transcriptional profiling, samples were collected at 0, 0.5, 1, 2, 4, 6, 12, 24, 48, and 72 h time points of culture. Total RNA was processed and hybridized on Illumina Sentrix HumanHT-12 Expression BeadChip, version 3.The microarray data analysis was done using Bioconductor package beadarray.Expression of selected genes was validated by RTPCR, western blot, and FACS analysis. Results: We, for the first time, report genome-wide gene expression profiling during early stages of human Th17 cell differentiation. We observed that gene expression pattern at the very early stage of human Th17 differentiation is highly dynamic. Further, we validated the selected genes at the protein level and analyzed their expression in Th1,


Poster Sessions 361 Th2 and iTreg T helper subsets.We found that ATP1B1, CXCR5, KDSR, and IL2RB are selectively regulated in Th17 condition during the early priming towards Th17 phenotype. On the other hand, CD52, VDR and CTSL1 were highly expressed also in response to initiation of some of the other T helper programmes. Our study provides an overview of genes and pathways regulated in response to induction of Th17 differentiation in humans and identifies several candidates which potentially play role in modulation of Th17 responses. Conclusions: This is the first transcriptional profiling study on early Th17 differentiation process, provides the starting point for constructing the gene regulatory network and identifying new candidates possibly regulating the Th17 differentiation in human.

P0535 Upstream stimulating factors mediated regulation of RORgammaT expression in human Th17 lymphocytes J. Dastych,* M. Ratajewski, A. Walczak-Drzewiecka* & A. Salkowska* *Laboratory of Cellular Immunology, Institute of Medical Biology of PAS, Lodz, Poland, Laboratory of Transcriptional Regulation, Institute of Medical Biology of PAS, Lodz, Poland Purpose/Objective: One of the two alternative products of RORC gene is RORcT, the orphan nuclear receptor that regulates the development of Th17 cells.Increased expression of RORcT is a hallmark of Th17 differentiation. We investigated the transcriptional regulation of RORcT in human lymphocytes to gain further insights into the process of Th17 differentiation.

Materials and methods: RORcT promoter analysis was performed with luciferase based constructs in Jurkat, HeLaand HepG2 cells.Screening for transcription factors regulating RORcT was performed by cotransfection ofRORcT promoter reporter constructs and expression vectors coding transcription factors into HeLa cells.Chromatin immunopreciptation and electrophoretic mobility shift assays were performed employing Jurkat T cells. Gene expression was assessed with real time PCR. Th17 cells were obtained from naı¨ve CD4+ T cells isolated from PBMCs cultured for 5 days under Th17 polarizing conditions. Concentration of IL-17 in supernatants was determined by ELISA.siRNA experiments were performed using nucleofection of lymphocytes with duplexes of Stealth siRNA. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s post hoc test. Results: While nonlymphatic human cells exclusively expressed RORc, Jurkat lymphocytes predominantly expressed RORcT. Analysis of human RORcT promoter activity with 5’ deletion and in situ mutagenesis analysis, chromatin immunoprecipitation and overexpression of selected transcription factors, revealed that USF-1 and USF-2 are critical for RORcT expression in Jurkat lymphocytes. USFs expression was upregulated upon differentiation of Th17 cells in vitro and siRNA mediated knockout of USFs expression resulted in significant decrease in expression of RORcT in Jurkat and in Th17 cells. Conclusions: We demonstrate the role of the USF-1 and USF-2 transcription factors in regulating the expression of RORcT in human lymphocytes.Thus, USFs are important for the molecular mechanisms of Th17 differentiation, and possible changes in the expression of USFs might be of interest for inflammatory conditions with a Th17 component.


362 Poster Sessions

Poster Session: Evolution and Selection

P0537 Signatures of positive selection in mammalian interleukins

P0536 Evaluation of genetic diversity of IGHG in 11 Lepus species

F. Neves, J. Abrantes & P. J. Esteves CIBIO/UP- Centro de Investigaçaõ em Biodiversidade e Recursos Gene´ticos, n.a., Vila do Conde, Portugal

A. Pinheiro,*, ,à P. C. Alves,*, C. Gorta´zarà & P. J. Esteves*,§ *Centro de Investigaçaõ em Biodiversidade e Recursos Gene´ticos, Universidade do Porto, Vairaõ, Portugal, Departamento de Biologia, Universidade do Porto, Porto, Portugal, àInstituto de Investigacioń en Recursos Cinege´ticos IREC, CSIC-UCLM-JCCM, Ciudad Real, Espanha Purpose/Objective: The European rabbit is unique in several immunological aspects, one of which being the possession of only one IGHG. Rabbit IgG has been extensively studied and its allelic variation characterized in detail since the early days of immunogenetics. However, the information available for this molecule in the Leporids is scarce, being limited to a few species hinge and CH2 sequences. In this study we sequenced the complete IGHG exons for 11 Lepus species. (382). Materials and methods: The four IgG exons, CH1, hinge region, CH2 and CH3, were PCR amplified and sequenced for 11 extant Lepus species: L. americanus, L. californicus, L. callotis, L. capensis, L. castroviejoi, L. corsicanus, L. europaeus, L. granatensis, L. timidus, L. townsendii and L. saxatilis. Six European rabbits were also sequenced. The primers were designed on European rabbit IGHG available sequences. (332). Results: 32 specific nucleotide differences were observed between European rabbit and hares (Lepus sp.) IgG genes corresponding to 19 aminoacid modifications. Within hares 50 SNP were found, that translate to 26 aminoacid changes. Specific aminoacids were detected for L. americanus, L. callotis, L. californicus, L. capensis, L. europaeus, L. saxatillis and L. townsendii. The greatest nucleotide and aminoacidic variability is found on the CH2 and CH3 domain. No variability was found among the studied 11 hare species hinge region. Conclusions: The studied hare species share a great sequence homology for IGHG. However, specific residues were observed for 7 Lepus species, mostly on the CH2 and CH3 domain but also on CH1. These could be related to resistance against specific pathogens. The hinge region is highly variable among species and IgG subclasses. Surprisingly, all 11 Lepus species studied share the same genetic hinge, suggesting that some selective pressure is maintaining the hinge motif in the Lepus taxon.

Purpose/Objective: Due to the host-pathogen co-evolution, the immune system and its genes are constantly evolving being under pressure and selection for adaptation, where advantageous mutations are highly favoured and deleterious mutations are quickly eliminated. Interleukins (ILs) have been identified as some of the genes of the immune system under positive selection in different mammals. Nevertheless, most studies do not specify the selected codons. Thus, we have searched for signatures of positive selection in mammalian ILs by using different codon-based maximum-likelihood (ML) approaches. Materials and methods: Sequences of mammalian ILs used in the analyses were retrieved from GenBank (http://www.ncbi.nlm.nih.gov/), Ensembl (http://useast.ensembl.org/index.html) and UniProt (http:// www.uniprot.org/). Each interleukin was aligned using ClustalW implemented in the software program BioEdit version 7.1.3 and adjusted manually. To detect signatures of positive selection in individual codons of mammalian ILs, dN/dS ratios were compared using two ML frameworks, the HyPhy package implemented in the Data Monkey Web Server (http://www.datamonkey.org/) and CODEML implemented in PAML version 4. Results: Signatures of positive selection were found in IL1A and B, IL2, IL4-IL10, IL12A and B, IL14-IL17A and C, IL18, IL20-IL22, IL25, IL26, IL27B, IL31, IL34, IL36A and G. Codons under positive selection identified by, at least, two ML methods varied between 1 and 15, being IL6 the interleukin with higher percentage of positive selected sites (2.28%, 15 codons) followed by IL7 (2.04%, 11 codons). No codons were detected in IL13, 17B and F, IL19, IL23, IL24, IL27A and IL29. Conclusions: Through protein-coding sequences comparison we are able to identify proteins under positive selection. Several studies have shown that genes related with immunity are among those proteins, including ILs. In ILs, codons located in regions where these proteins interact with their receptors are prone to be positively selected in order to influence signalling intensity. Also, signatures of positive selection may be related with escaping mechanisms from antagonistic parasiteencoded proteins to maintain the ILs functions in the immune response.


Poster Sessions 363

Poster Session: Ion Channels P0538 Altered calcium signalling in B and T cells from Systemic Lupus Erythematosus patients is related to STIM1 expression J. O. Pers,* T. Fali,* O. Mignen, M. Burgos, S. Jousse,* A. Saraux,* C. Jamin* & Y. Renaudineau* *Immunology, Brest University Medical School Hospital, Brest, France, Inserm U1078, Brest University Medical School Hospital, Brest, France Purpose/Objective: The calcium sensor STIM1 in lymphocytes is an essential mediator of the calcium influx that acts as a second messenger in response to activation and proliferation. Although mutations in STIM1 cause complex immunodeficiencies, the role of STIM1 in the T and B cell autoreactivity process during autoimmune diseases is currently unknown. Materials and methods: Using thapsigargin (Tg), an inhibitor of calcium pump of the endoplasmic reticulum (ER), elevations of cytosolic calcium from the ER and activation of the store-operated calcium entry (SOCE) were evaluated in T and B cells from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), primary Sjo¨gren’s syndrome (pSS), and healthy controls (HC).

Concomitantly, STIM1 expression was evaluated by flow cytometry in T and B cell subsets. Results: Higher constitutive calcium entry and increased Tg-mediated extracellular calcium entry, but not ER calcium entry, were observed in T and B cells from SLE patients compared with controls. No significant differences could be observed in SOCE/ORAI molecules by RT-qPCR. However, STIM1 expression was quantitatively different according to the disease. SLE T cells express more STIM1 than HC and pSS patients, whilst RA T cells express lower level. SLE B cells express higher STIM1 molecules than pSS B cells, whereas HC and RA B cells express similar low levels. In SLE B cells, STIM1 is overexpressed in all B cell subpopulations with highest levels detected in autoreactive transitional B cells (MFI 9.5 ± 2.3 in SLE versus 1.5 ± 0.7 in HC, P > 0.001). Interestingly, STIM1 level is strongly correlated with the constitutive calcium entry and the extracellular calcium influx (both, P < 0.01). In contrast, activity of the disease and autoantibody production were not correlated. Finally, CpG and anti-IgM co-stimulations are as efective as CpG and anti-CD40 stimulations to induce STIM1 expression in normal B cells as well as cell cycle progression in a B cell line. Conclusions: In conclusion, these findings suggest that differential STIM1 expression may be an important determinant in the SLE autoreactivity process.


364 Poster Sessions

Poster Session: Lymphocyte Development P0539 A time resolved analysis of nc/mRNA and protein expression throughout Th cell activation M. von Bergen,* K. Reiche,* N. Ho¨sler, M. Rockstroh,* F. Horn, J. Hackermu¨ller* & J. Tomm* *Proteomics, Helmholtz Centre for Environmental Research, Leipzig, Germany, Institute for Clinical Immunology, University of Leipzig, Leipzig, Germany Purpose/Objective: Activation and differentiation of immune cells are crucial for an efficient and controlled immune response. To be able to secrete cytokines, naı¨ve T helper-cells need to get in contact with antigen presenting cells (APC) to become activated and differentiate into the different T helper-cell subsets responsible for the different cytokines.These processes are controlled by a combination of transcriptional and translational regulatory mechanisms. In terms of transcriptional control non coding RNAs have been proven recently to be crucial first for epigenetic effects but also for transcription and translation. The effects can be assessed first by RNA seq and second by proteomic approaches to control for the effects of regulatory processes. Therefore we combined here RNAseq and proteomics to reveal the regulatory and effective mechanisms in T cell activation and differentiation into Th1 cells. Materials and methods: For analysis of RNAs cells were lysed in Trizol and RNA was extracted out of the aqueous phase followed by tiling arrays and transcriptome sequencing. Proteins were extracted from the phenolic phase of the Trizol extraction and analysed via GeLC-MS/MS. Results: We identified ~22.000 transcriptionally active regions (TARs) in the genome which are differentially expressed throughout this process and fall into diverse coding and non-coding transcript categories.Out of 10 buffy coats 2445 proteins were unambiguously identified in at least five of 10 buffy coats. Among the 409 proteins regulated over the activation time course were known transcription factors like STAT1, NF-kB and others, which are known to be involved in cellular differentiation of immune cells. Conclusions: The combination of transcriptome and proteome data allow identification of new ncRNAs as well as deciphering the influence of non-coding sequences on the abundance of relevant factors in T cell activation.

P0540 CD8aa TCRab intraepithelial lymphocytes: a unique distinct T cell lineage S. Mayans, D. Stepniak, S. Palida, R. Skinnakazu, B. Arlian, H. Cheroutre & F. Lambolez Developmental Immunology, La Jolla institute for Allergy and Immunology, La Jolla, USA Purpose/Objective: We aimed to define the thymic development and MHC restriction of CD8aa TCRab intraepithelial lymphocytes (IEL) of the small intestine. Materials and methods: We cloned TCR genes isolated from naturally arising CD8aa TCRab IEL and retrovirally express these TCRs in bone marrow (BM) chimera. Results: First, we successfully sequenced and cloned three TCRab isolated from single cells and then expressed them retrovirally in Rag KO BM chimera. Analysis of the chimera showed that all three TCR clones gave rise to T cells. The T cell that developed were CD4- CD8bor CD8aa TCRab cells and phenotypically identical to CD8aa TCRab IEL isolated from un-manipulated wild type mice. In addition, they were preferentially found in the gut. In order to define the MHC

restriction of these particular TCRs, BM chimeras in various MHCdeficient backgrounds were generated. Subsequent analysis of the chimera for the presence of CD8aa TCRab IEL demonstrated that they are dependent either on KbDb MHC I or on b2m-dependent MHC I molecules aside from KbDb. Conclusions: Our results show for the first time that TCRs originally cloned from CD8aa TCRab IELs can only give rise to this identical T cell lineage but not conventional CD4 and CD8ab T cells. In addition, this implies that the CDR3 from the TCR sequence will exclusively allow the selection of this unique T cell population most likely through an instructive model. Finally, CD8aa TCRab IELs can also harbor clones selected on different MHC molecules.

P0541 Characterization of the development and function of thymic Hassall’s corpuscles M. Laan,* X. Wang,* R. Bichele,* K. Kisand,* H. S. Scott & P. Peterson* *Molecular Pathology, University of Tartu, Tartu, Estonia, Department of Molecular Pathology, The Centre for Cancer Biology & The School of Medicine, The School of Molecular and Biomedical Science, The Institute of Medical and Veterinary Science & University of Adelaide, Adelaide, SA, Australia Purpose/Objective: Thymic Hassall‘s corpuscles (HCs) are enigmatic tissue structures with poorly defined origin and function. Since recent studies have indicated a role for the Autoimmune Regulator (Aire) in HC development, the aim of this study was to clarify whether HCs are derived from the Aire+ medullary thymic epithelial cells (mTECs), and whether the unique features of Aire+ mTECs to express and present tissue specific antigens (TSA) to the developing thymocytes, are preserved at the HC-stage of mTEC development. Materials and methods: Two different Aire-reporter mouse models (Aire-LacZ and Aire-GFP) as well as Aire KO mice were used to elucidate the relations between Aire and HCs. Also, FACS-based cell sorting followed by quantitative PCR was used in order to define gene expression profiles of different mTEC populations. Results: In the Aire-reporter mice the majority of HCs stained also positive for LacZ or GFP whereas in the Aire KO mice the numbers of HCs were severely reduced. The post-Aire developmental stages of mTEC development were characterized by pronounced downregulation of MHC class II and CD80, and of most of the Aire-dependent and Aire-independent TSAs, with the exception of keratinocytespecific genes. In the final stage of maturation, the mTECs lost their nuclei to become HCs and specifically expressed certain keratinocytespecific autoantigens, such as desmogleins (DGs) 1 and 3. Conclusions: HCs comprise a post-Aire mTEC population which specifically expresses keratinocyte-specific antigens. Via cross-presentation of these TSAs by APCs to the developing thymocytes, HCs may contribute to tolerance induction against these pemphigus vulgarisrelated antigens.

P0542 Contribution of secondary immunoglobulin heavy chain rearrangements to primary antibody diversification R. Kumar,* O. Kanagawa, S. Fagarsan & S. Casola* *FIRC Institute of Molecular Oncology Foundation (IFOM), Milano, Italy, Laboratory for Mucosal Immunity, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan Purpose/Objective: V(D) J recombination is a process catalyzed by Recombination Activation Genes-1 and -2 in B-lymphocyte progenitors to assemble the variable (V) region genes of the immunoglobulin heavy and light chain polypeptides. In mammals, the establishment of


Poster Sessions 365 a sufficiently complex primary Ig repertoire is essential for life-long immunity against foreign pathogens. Current knowledge indicates that IgH V-gene diversification results from single successful VDJ recombination events. Recent reports have challenged this model showing occurrence both in humans and mice of secondary IgH rearrangements. Importantly, this process has been so far described in transgenic mouse models expressing non-functional IgH chains or in the context of autoimmunity, raising the question of its physiological relevance. This project thus aims to precisely estimate the contribution of secondary IgH rearrangements to the establishment of a normal antibody repertoire. Materials and methods: A unique mouse strain generated by nuclear reprogramming of an intestinal IgA plasma cell expressing a nonautoreactive BCR was employed, which allows a rapid and easy tracking of B-cells undergoing secondary IgH rearrangements. Results: B-cell progenitors in cloned IgA mice are expected to express the same pre-rearranged IgH V gene. By phenotype analysis we observed that over 20% of peripheral mature B-cells in IgA heterozygous mice expressed IgM on the cell surface, suggesting the occurrence of secondary IgH rearrangements in a substantial number of developing B cells. Sequencing of IgA rearrangements in sorted IgM+ B cells of IgAH/+ mice revealed that both VH replacement and direct VH-to-JH joining contributed to the disruption of the original VH rearrangement, thereby triggering novel rearrangements on the second (germline) IgH chromosome. Sequence analysis of CDR3 regions suggested that secondary IgH rearrangements likely occurred in pro-B cells as they carried n nucleotides introduced by terminal deoxynucleotidyl transferase (TdT). Interestingly, V-gene analysis in B cells undergoing secondary IgH rearrangements, revealed a strong bias for V-genes adjacent to the pre-rearranged VH gene as recombination substrates. Conclusions: Altogether these results provide evidence for a major contribution of secondary IgH rearrangements to the diversification of the primary antibody repertoire under conditions of normal B-cell development.

P0543 Differential induction of TH1-17 and TH-9 cytokines in human naı¨ve t helper cells by B7h- and B7.1-mediated costimulation C. Gigliotti, R. Mesturini, M. F. Soluri, A. Woldetsadik, A. Chiocchetti & U. Dianzani Department of Health Sciences, Interdisciplinary Research Center of Autoimmune Diseases (IRCAD), ‘A. Avogadro’ University of Eastern Piedmont, Novara, Italy Purpose/Objective: ICOS and CD28 are expressed by T cells and are involved in costimulation of cytokine production in T helper (TH) cells. ICOS binds B7h expressed by several cell types, whereas CD28 binds B7.1 and B7.2 expressed by antigen presenting cells (APC). This work compared the activity of recombinant B7h-Fc and B7.1-Fc in induction of TH17 and TH9 cytokine secretion in human naı¨ve TH cells. Materials and methods: Purified naı¨ve TH cells were activated with anti-CD3 mAb in the presence of either B7h-Fc or B7.1-Fc plus different combinations of rIL-1b, rIL-6, rIL-21, rIL-23, rTGF-b1, and rIL-4. Secretion of IL-17A, IL-17F, IL-17A/F, IL-10, and IL-9 was assessed by ELISA in the supernatants. Levels of the IL-26 mRNA were evaluated by Real-time PCR in the cell lysate. Results: Results showed that, in the presence of rTGF-b1+ rIL-1b (a TH17 polarizing condition), B7h-Fc was more effective than B7.1-Fc in inducing IL-17A and IL-10, whereas B7.1-Fc was more effective in inducing IL-17F and IL-26. These different patterns were supported by further addition of rIL-6, rIL-21, and rIL-23 in the culture medium. In the presence of rTGF-b1+ rIL-4 (a TH9 polarizing condition), B7.1-Fc induced high levels of IL-9 that was instead barely detectable using

B7h-Fc. Upon dual costimulation with both B7h-Fc and B7.1-Fc, secretion of IL-17A and IL-17F was similar to that induced by B7.1 alone, with predominance of IL-17F, but secretion of high levels of IL10 mimicked that induced by B7h-Fc alone; secretion of IL-9 displayed intermediate levels between those induced by B7.1 alone and B7h-Fc alone. Conclusions: These data showed that B7h and B7.1 have different effects on secretion of TH17 and TH9 cytokines in naı¨ve TH cells, and that the effect of contemporary use of both costimuli is not the simple sum of the effects exerted by each of them.

P0544 Flipping of lipids by a putative transporter is essential for B-cell development and function M. Yabas,* C. E. Teh,* S. Frankenreiter,* D. Lal,* B. Whittle, D. T. Andrews, E. M. Kucharska,* S. Broer,à C. C. Goodnow* & A. Enders* *Immunology Department, The John Curtin School of Medical Research, The Australian National University, Canberra, SA, Australia, Australian Phenomics Facility, The John Curtin School of Medical Research, The à Australian National University, Canberra, SA, Australia, Division of Biomedical Science and Biochemistry, Research School of Biology The Australian National University, Canberra, SA, Australia Purpose/Objective: B cells with their ability to secrete a wide array of antibodies against various invading pathogens play a central role in the generation of humoral immunity. The development of B cells takes place in the bone marrow of most mammals in a highly regulated process that depends on cell intrinsic and extrinsic factors such as signaling through the precursor-B cell receptor (pre-BCR) and, at least in the mouse, the Interleukin-7 receptor (IL-7R), and the activation of transcription factors. However, the role of the composition of the cell membranes on the development of B cells is still poorly understood. Here we describe a new ENU-induced X-linked B cell deficiency syndrome in mice caused by a mutation in Atp11c, a previously uncharacterized member of the P4-ATPase family thought to serve as flippases that catalyze aminophospholipid transport from the exoplasmic to the cytoplasmic leaflet of cell membranes. Materials and methods: Analysis of mice with an ENU-induced point mutation in Atp11c by flow cytometry and genetic crosses as well as in vitro assays to determine the uptake of aminophospholipids in cells from mutant and control animals. Results: We show that ATP11C deficient mice have a near complete absence of B cells in the blood due to a severe block at the transition from the pro-B to pre-B cell stage of B cell development in the bone marrow. This block could partially be rescued by the introduction of a transgenic B cell receptor whereas elevated levels of Interleukin-7 or enforced expression of Bcl-2 showed only a minimal increase in peripheral B cell numbers. Similarly, in the spleen the number of B cell was also greatly reduced except for normal numbers of marginal zone B cells. This correlated with a reduced internalization of fluorescently labeled phosphatidylserine in developing pro-B cells but not in other developmental stages of B cells or different cell types in the bone marrow and spleen. Interestingly, despite the reduced B cell numbers the response to T-independent antigen was normal but both the primary and memory antibody response to T-dependent antigens was greatly reduced with the most severe reduction seen in the germinal centre-dependent response to the hapten azo-benzen-arsonate. Conclusions: The results demonstrate that the phospholipid transporter ATP11C is crucial for early B cell development and antibody production, and reveal an intimate and novel connection between phospholipid transport and B lymphocyte development and function. Our results also provide a new candidate gene for the many currently unclassified human primary immune deficiencies.


366 Poster Sessions P0545 HDAC7 plays a significant role in IL7 signalling during the Pro-B/ Pre-B transition of B cell development M. MacKenzie, M. Anderson & S. Matthews Immunology, University of Dundee Medical Research Institute, Dundee, UK Purpose/Objective: In many cellular systems Histone Deacetylases (HDACs) play important roles in the regulation of cell differentiation, proliferation and survival. HDACs regulate chromatin structure and thus gene transcription by reversing histone lysine acetylation. As such they been identified as key players in the development of the immune system with evidence suggesting HDAC7 acts as a gene expression switch regulating T cell development and function while HDAC9 increases regulatory T cell function by interacting with the transcription factor FOXP3. A role for HDACs in B cell development and function is poorly characterised and where our interest lies. Materials and methods: We have bred an HDAC7 floxed/floxed mouse with a vav-cre transgenic mouse in order to delete HDAC7 from all haematopoietic cells including B cells. To investigate a role for HDAC7 in B cell development we analysed the phenotype of lymphoid tissues in WT and KO mice (Spleen, Lymph Nodes and Thymus) using flow cytometry and qPCR. Results: Using flow cytometry we identified a significant reduction in size of these tissues due to a large reduction in cellular content of B cells and T cells. To investigate the B cell phenotype further we looked in the bone marrow again identifying a significant reduction of Pre-B, immature and mature B cells. A small but significant increase in Pro-B cell number suggested a partial block in development between the ProB and Pre-B stage of development. This is a critical point during B Cell development where IL7 signalling drives expression of the pre-BCR complex. The pre-BCR complex in turn induces specific intracellular signals that drive the transcription of genes relevant for the proliferation, survival and expansion of developing B cells. In addition, defects in the expression of IL-7Ra chain in HDAC7-/- pro-B cells, as well as down stream effects such as RAG1/2 expression and IgM expression may account for the observed B cell phenotype in our mice. Conclusions: These data suggest a key role for HDAC7 in controlling B cell development and further highlights the importance of HDACs within the adaptive immune system.

P0546 IL-7Ra plays a critical role in the development of B cells D. T. Patton,* A. Plumb,* J. Seo* & N. Abraham *Microbiology and Immunology, Life Sciences Insitute, Vancouver, Canada, Microbiology and Immunology and Zoology, Life Sciences Insitute, Vancouver, BC, Canada Purpose/Objective: IL-7 is a critical cytokine throughout lymphocyte biology and controls the development of T and B cells from lymphocyte precursors in the thymus and bone marrow. The molecular signalling events downstream of the IL-7Ra are not fully characterised. Y449 is critical for the development and function of T cells; the role of this motif has not been investigated in B cells, and due to the clear role of IL-7Ra in B cell development, we decided to evaluate the role of IL7RaY449 motif in B cell development. Materials and methods: We used flow cytometry to examine a knockin mouse possessing a point tyrosine to phenylalanine mutation at position 449 (IL7Ra449F mice) within the cytoplasmic YxxM SH2binding motif of IL-7Ra, as well as IL-7 over-expressing, TSLPR-/- and IL-7Ra-/- mice. Results: Signalling downstream of IL-7RaY449 is critical during early B cell development, as numbers of B cells in the bone marrow of IL7Ra-/- mice and IL-7Ra449F were reduced at pro-B and pre-B cell stages, suggesting that Y449 is the major signalling residue downstream of the

receptor in bone-marrow B cell development. IL-7Ra also controls B cell development in the spleen, as IL-7Ra449F mice have a partial and IL-7Ra-/- mice a near-total block at T1-T2 transition. However, IL7Ra449F was required for follicular, but not marginal zone B cell populations. Over-expression of IL-7 had the opposite effect on follicular B cells, increasing their numbers, but had no effect on marginal zone B cells. Any role for TSLP was ruled out, as TSLPR-/-mice had an identical B cell phenotype to wild-type mice. Experiments using bone-marrow chimera suggested that the defect seen in pre-B cells was due to defective IL-7Ra signalling within the B cell, whereas the splenic defect was due to defective IL-7Ra signalling in non-B cells, as WT and IL-7Ra449F B cells showed an identical phenotype in a mixed environment and did not express IL-7Ra nor signal through pSTAT5 in response to IL-7. Conclusions: IL-7 plays a key role in the development of B cells from the earliest stages, however signalling downstream of IL-7Ra Y449 is only required at the pro-B and pre-B cell stages in the bone marrow. However, in the spleen, follicular, but not marginal zone B cells are critically dependent on IL-7Ra signalling levels and are controlled by IL-7Ra signalling in an as-yet unidentified manner extrinsic to the B cell compartment.

P0547 Inhibition of glycogen synthase kinase-3 regulates T cell development in vitro H. Schroeder, M. Janas & M. Turner Lymphocyte Signaling and Development, Babraham Institute, Cambridge, UK Purpose/Objective: The development of functional non-autoreactive T cells requires receptor-mediated transition through multiple checkpoints in the thymus. Double negative 3 (DN3) thymocytes are selected for the presence of a rearranged TCR beta chain in a process termed beta-selection which requires signalling via the preTCR and Notch1. Pre-selection DN3 are referred to as DN3a and express low levels of CD27 and CD98, while post selection DN3 (which are TCRbeta positive) express higher levels of CD27 and CD98. Additional signalling from CXCL12 ensures optimal proliferative expansion of DN3 thymocytes. Signal integration by these receptors converges on core pathways such as the Phosphatidylinositol-3-kinase (PI3K) pathway. Glycogen Synthase Kinase 3 (GSK3) is generally thought to be negatively regulated by the PI3K pathways. Materials and methods: Murine DN3a were FACS sorted as negative for CD4/CD8/CD44/CD11b/CD19/NK1.1/Gr-1/cdTCR/Ter119 and CD25highCD98low. FACS sorted DN3a were cultured with OP9 or OP9-Delta Like 1 stromal cells or in wells that had been coated with 10 lg/ml of recombinant mouse Delta Like 4. In the stromal cell-free cultures DN3a cells were cultured in the presence or absence of recombinant murine CXCL12 and IL-7. In some experiments the GSK3 inhibitor CHIR99021 was added with the DN3a cells from the start of culture. After up to 96 h of culture numbers of gated lymphocytes were determined and analysed by FACS. Results: We have shown that a GSK3-inhibiting drug, CHIR9902,1 promotes the proliferative expansion of DN3a cultured with recombinant Delta Like 4 and CXCL12. Here we show that developmental progression of DN3a is promoted by CHIR99021. Furthermore, inclusion of CHIR99021 allowed differentiation in the absence of preTCR- or Notch1-mediated signalling. Inactivation of GSK3 using CHIR99021 appears to antagonize IL-7-mediated inhibition of development at the DN stage. In addition to the effect on T cell development, CHIR99021 increased IL-7 dependent proliferation and caused enhanced cell recovery in these experiments. Conclusions: These experiments indicate a potentially important role for inactivation of GSK3 during the process of beta-selection. A stromal free culture system that promotes beta-selection may offer a


Poster Sessions 367 new drug discovery platform for screening regulators of proliferation, differentiation and apoptosis.

P0548 Matrix attachment regions flanking the IgH intronic El enhancer are important cis- and trans-regulators of somatic hypermutations E. Pinaud, M. Marquet, S. Bender, P. Rouaud, A. Garot, Y. Denizot & M. Cogne´ CNRS Unite´ Mixte de Recherche 7276, Controle de la Re´ponse Immune B et des Lymphoproliferations, Limoges, France Purpose/Objective: The immunoglobulin heavy chain (IgH) intronic enhancer region (El) is a combination of both an enhancer core element (220 bp) and two 310 350-bp flanking scaffold/matrix attachment regions (S/MARs herein named MARsEl) that were first defined by nuclear matrix-binding assays in vitro. The El core enhancer region is critical for efficient V-D-J recombination at early stages of B cell development. Strikingly, the physiological role of MARsEl is still unclear even if several nuclear factors have been found to bind to this region: a B cell transcription factor named BRIGHT/Arid3a; AT rich binding proteins expressed in T cells and in B cell progenitors (SATB2); and also a negative regulatory factor named NF-lNR whose expression decreases at late stages of B cell development when cells express high levels of Ig heavy chains. Materials and methods: We generated a mouse model carrying an endogenous deletion of MARs and performed a detailed analysis of its consequences on B cell development. Results: We found that, unlike the core El element, the absence of MARs did not affect B cell development but led to a significant decrease in somatic hypermutations in Peyer’s patch germinal centre B cells. In the intronic region downstream IgH J segments, the mutation frequency was also significantly reduced in the corresponding region of the Ig Kappa locus. Conclusions: Our data revealed that IgH MARsEl regions are critical regulatory elements that participate in efficient recruitment of SHM machinery to Ig loci target sequences. In germinal centre B cells, we postulate that MARsEl also act in trans to recruit Ig Kappa loci and contribute spatially to the SHM process.

P0549 Progenitor deprivation provokes acute T cell lymphoblastic leukemia V. Martins* & H. R. Rodewald *Institute of Immunology, University Hospital of Ulm, Ulm, Germany, Division of Cellular Immunology, German Cancer Research Center, Heidelberg, Germany Purpose/Objective: Continuous thymus function is thought to depend on a steady supply of T cell progenitors from the bone marrow. To address the fate of a thymus in the complete absence of developmentally competent bone marrow progenitors, we have transplanted normal wild-type thymus into Rag2-/-gc-/-KitW/Wv mice. Materials and methods: Compound mutant Rag2-/-gc-/-KitW/ Wv mice lack competitive hematopoietic stem cells (HSC) and are devoid of T cell progenitors. Using this strain as recipient for wild-type thymus grafts,. Results: we unexpectedly find that thymocytes persist and that T lymphocytes continue to be produced and exported for several months. Sequencing of the expressed alpha and beta TCR loci in progenitor-deprived thymus grafts shows that the TCR repertoire is still diverse. The analyses of single mutants as recipients for the thymus transplants indicate that gc-mediated signals alone play a key role in the competition between thymus-resident and bone marrow-derived progenitors. Hence, the turnover of each generation of thymocytes is

not only based on short life span but is also driven via expulsion of resident thymocytes by fresh progenitors entering the thymus.However, a frequent consequence of progenitor-independent thymus autonomous function is the development of T cell acute lymphoblastic leukemia (T-ALL).Recipient mice developed hepatosplenomegaly and full-blown signs of T-ALL. Blast cells in bone marrow, blood and spleen had cell surface phenotypes reminiscent of T-ALL in humans, the tumors were transplantable and were clonal, based on the analysis of the TCR rearrangements. These murine T-ALLs bore mutations in the heterodimerization and PEST domains of Notch1. Rag1 and Rag2 mRNA continued to be expressed in the leukemic cells but Rag expression was not required for leukemogenesis. Conclusions: We propose that competition between old and new progenitors in the thymus provides a quality control mechanism that ensures that young outcompete old progenitors, and that abrogation of this competition results in thymus-intrinsic T cell development, followed by the development of leukemia.

P0550 Spatio-temporal regulation of Notch ligand expression defines functional microenvironments in the human thymus: implications in the TCR Alpha Beta versus TCR Gamma Delta T-cell fate decision M. J. Garcia-leon,* J. L. De La Pompa, J. C. Ferreres Pinãs,à M. Garrido Pontnouà & M. L. Toribio Garcıá* *Cellular Biology and Immunology, Centro de Biologıá Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain, Cardiovascular Developmental Biology, Centro Nacional de Investigaciones Carciovasculares, CNIC, Madrid, Spain, àHospital Universitario Vall d’Hebron, Banco de Tejidos Fetales, Barcelona, Spain Purpose/Objective: Interaction of Notch receptors with Notch ligands (DLL1, DLL4, JAG1, JAG2) expressed on the thymic microenvironment (TME) is essential to support T-cell development. DLL4 is the essential ligand that drives T-cell specification, but little is known about the ligands involved is subsequent differentiation of pre-T cells into either TCRab or TCRcd cells. As T-cell development implies the active movement of developing thymocytes throughout distinct thymic regions, we ought to determine whether a differential distribution of Notch ligands at defined anatomical regions could endow discrete intrathymic niches with specific developmental functions once T-cell specification has been induced by DLL4. Materials and methods: We analysed the spatio-temporal regulation of Notch ligand expression in the human thymus by immunohistochemistry and confocal microscopy. The characterization of particular TME niches defined by Notch ligands in vivo provided the framework to approach the specific functions of those ligands in the TCRab versus TCRcd cell fate of human pre-T cells using the OP9 co-culture system. Results: We found that distinct Notch ligands are confined to defined histological regions and stromal cell types, and this distribution changes along thymus ontogeny. The four ligands are expressed at high levels in the postnatal thymus (PNT) at the cortico-medullary junction (CMJ) and thymic medulla. In contrast, JAG1 and DLL4 are essentially undetectable in the PNT cortex, although DLL4 is expressed on cortical thymic epithelial cells (cTECs) in the human embryonic thymus (90th percentile (median 39 ng/ml). In 33.3% of those (3.72% of total sample) with the highest leptin levels (median 46 ng/ml), significantly elevated levels of serum IFN-g were also found (mean 27.11 pg/ml, range 17.5 38.5 pg/ml). In neonates with leptin levels ~50th percentile (median 12 ng/ml) or 0.01) at the peak of the inflammatory process and a drastic increase during resolution P = 0.01. Interestingly, while LEC expansion was due to increase in the number of small lymphatics, the second peak corresponded to the histological finding of a small number of lymphatic with an enlarged lumen. This change in number and shape of the lymphatic bed coincided with the infiltration of B-lymphocytes and peak of lymphotoxin signal in the gland. Accordingly, cannulation of LTbRKO, demonstrated absence of the second peak of LEC proliferation and lack of lymphatic expansion by IF, suggesting a role for this molecule in lymphatic modification during inflammation. LTbRKO mice did not show significant differences in the percentage of BEC as compared to the WT. Conclusions: Ectopic lymphoid follicles associated vasculogenesis fails to recapitulate the regulation observed in secondary lymphoid organs. The changes observed in the lymphatic bed seems to be regulated by the presence of ectopically expressed lymphotoxin, suggesting a novel therapeutic role for this molecule in inflammation.

P0739 Postnatal dynamics in small intestinal immune cell populations N. Torow,* O. Pabst & M. W. Hornef* *Hannover Medical School, Microbiology, Hannover, Germany, Hannover Medical School, Immunology, Hannover, Germany Purpose/Objective: More than half of the body’s professional immune cells are involved in the maintenance of intestinal homeostasis. Already in the fetus gut associated lymphoid tissues begin to form and the highly dynamic process of intestinal immune homeostasis is initiated. During delivery of the neonate a transition of the intestine from a sterile to an increasingly colonized environment occurs. The commensal flora is a very important modulator of the innate and adaptive immune mechanisms that contribute to the homeostasis. Therefore, we hypothesised that the postnatal adaptation and colonization of the small intestine directly influences the intestinal immune cell composition. The present study aims to investigate the postnatal dynamics in small intestinal immune cell populations.

Materials and methods: Small intestine immune cells were isolated and subjected to FACS analysis. The following markers of CD45+ cells were assayed to define subpopulations: CD4, CD8aa, CD8ab, TCRab, TCRcd, MHCII, MHCII CD11c, MHCII CD11b F480.The immune cell subsets in the small intestine of B6 mice were analysed at d6, d12, d28 and d56 after birth. Results: During the 2nd week after birth mainly the B cell population (MHCII+ CD11c-CD11b-F480-) expands and reaches a peak around day 12. TCRcd and CD8aa subsets start to expand between d12 and day 28 after birth whereas the TCRab and CD8ab positive population increases only after day 28. The relative abundance of the CD4 posititive cells decreases from day 12 onwards. The myeloid fraction of the small intestinal immune cells (MHCII+ CD11c+ and MHCII+ CD11b+ F480+) remains quite stable over time. Conclusions: The lymphocyte composition in the small intestine is highly dynamic whereas the relative abundance of the myeloid compartment remains stable during the postnatal period.

P0740 TCR triggering controls IL7 responsiveness and homeostatic expansion of CD4+ T cells during IL7 therapy D. Leboeuf,* O. Hennion-Tscheltzoff,* S. D. Gauthier,* C. L. Mackall & M. Guimond* *Maisonneuve Rosemont Hospital Research Center, Immunology and Microbiology, Montreal, QC, Canada, NCI, Pediatric Oncology Branch, Bethesda, USA Purpose/Objective: Lymphocyte regeneration following high dose chemotherapy occurs via homeostatic peripheral expansion (HPE). While HPE efficiently regenerates CD8+T cells, it is inefficient for CD4+ and induces a skewing of the lymphocyte repertoire towards cells with high affinity for self-peptide MHC complexes (p-MHC). Interleukin-7 (IL7) is essential for thymopoiesis and HPE of naı¨ve T cells. Previous studies in humans have demonstrated that supraphysiological doses of IL7 could increase T cell counts and TCR diversity. However, the exact mechanism is not completely understood. Given the critical role of thymopoiesis in CD4+ recovery, we hypothesized that the proliferative effect of IL-7 therapy must rely largely on recent thymic emigrants (RTE) expansion. Materials and methods: Adoptive transfer of single positive CD4 and CD8 thymocytes and LN T cells into wt or lymphopenic hosts was used for all experiments. IL7 treatment consists of six daily doses of 10 lg. Results: Following adoptive transfer of RTEs isolated from Rag-GFP mice, we did not find evidence that IL7 therapy induced stronger proliferation of RTEs. However, single positive CD4+ thymocytes (CD4+SPT) were more responsive to IL7 therapy according to stat5 phosphorylation and BCL-2 induction and that lower doses of IL7 were sufficient to induce proliferation in vivo. We also found that CD4+SPT were more sensitive to TCR signalling than RTEs and peripheral CD4+ T cells (CD4+PERI) according to miR181a micro-RNA quantification. Despite the requirement of TCR stimulation for IL7 to induce proliferation of CD4+PERI and CD4+SPT, residual proliferation of CD4+SPT was observed upon transfer into IL7 treated MHC II-/hosts. This proliferation was abrogated when IL7 therapy was delayed; suggesting that TCR triggering received inside the thymus was responsible for this effect. Increasing the number of dendritic cells by treating mice with FLT3 ligand augmented proliferation of CD4+ T cells by IL7. Finally, we found that cells undergoing proliferation during IL7 therapy were low affinity CD4+ cells to p-MHC. Conclusions: Together our data support a model wherein IL7 therapy affects CD4+ T cells as they egress from the thymus and once in the periphery, these cells rapidly diminish their responsiveness to IL7. Therefore a residual thymic function is likely to augment the success of IL7 therapy in lymphopenic patients.


Poster Sessions 431

Poster Session: Transcription and Epigenetics P0741 DELTA4BAFF alternative splicing is regulated by interferongamma AND SC35 protein J. O. Pers,* G. J. Tobon,* L. Corcos, G. Dujardin, P. Youinou* & L. Le Pottier* *EA 2216 ‘Immunology & Pathology’, Brest University and European University of Brittany, Brest, France, INSERM UMR1078, Brest University and European University of Brittany, Brest, France Purpose/Objective: The B-cell activating factor (BAFF) is a potent survival factor involved in the pathogenesis of autoimmune diseases. Recently, we reported the discovery of a new transcript for BAFF, D4BAFF lacking exon 4 -, which is mainly detected in autoimmune diseases and acts as a transcription factor for its own gene. However, the mechanisms implicated in D4BAFF induction and up-regulation are unknown. In this study we analyzed the induction and regulation of D4BAFF. Materials and methods: First, to study the alternative splicing of BAFF exon 4, we transfected a minigene construct, centered on exon 4, into RAMOS B cells.To determine the proteins implicated in exon 4 inclusion/exclusion, we co-transfected the minigene together with each of the plasmids coding for the main splicing proteins (SC35, SRp40, SRp55, SRp20 and hnRNPA1), and the ratios between exon4 inclusion/ exclusion were evaluated by RT-PCR. Second, we examined the effects of different cytokines on D4BAFF induction. Results: RAMOS cells presented exon 4 skipping (ratio inclusion/ exclusion: 6.8) after minigene transfection. Following co-transfection of the minigene with coding plasmids for splicing proteins, only the overexpression of SC35 showed effect in the splicing of exon 4, promoting exon 4 inclusion (ratio: >30).Incubation of different cell lines with several cytokines showed that IFN-cwas able to induce D4BAFF-transcript. Thus, after IFN-c stimulation in the minigene model, the ratio inclusion/exclusion markedly decreased (1.5). IFNcmodifies the balance between SC35 and another member of hnRNPs family (hnRNP C1/C2) favouring the alternative splicing of exon 4. Conclusions: These results demonstrated that IFN-c induces D4BAFF, modifying the function of SC35 protein and increasing the expression of hnRNPC1/C2. Our study provides an expanded conceptual view of BAFF gene regulation, and contributes to a better understanding of the mechanisms involved in BAFF up-regulation in autoimmunity.

P0742 Discovery of a new alternative splice variant of human Interleukin4 and study of its functionality as a nonsense-mediated decay C. Hauvespre,* V. Courtois,* K. De Luca,* C. Legras-Lachuer & R. Sodoyer* *Sanofi Pasteur, Research, Marcy l’Etoile, France, ProfileXpert-LCMT, Faculte´ de Me´decine et de Pharmacie, Lyon, France Purpose/Objective: Interleukin-4 (IL-4) is a pleiotropic cytokine regulator of immunity in health and disease states. IL-4 is a cytokine responsible of the Th2 response during infections with parasites, allergy or asthma. An alternatively spliced variant of human IL-4, known as IL-4d2, is deleted of the second exon. IL-4d2 can be considered as an inhibitor of IL-4 in immune response. Doing experiments on human T cells, a new alternative splice variant of IL-4 has been cloned and sequenced. In the present study, we explain the discovery of a new alternative spliced variant of IL-4 and investigate the expression of this new transcript. Materials and methods: A bioinformatics study is conducted to investigate the presence of the alternative splice site consensus sequences. Using a transfection and a non-sens mediated decay

(NMD) inhibition approach by cycloheximide (CHX), we show that this new transcript is targeted by NMD degradation. Results: 101 base-paired (bp) of the second intron are retained between the second and the third exon. Alternative splicing consensus sequences are presents in the flanking sequences of the retention. The base-pairs retained downstream the second exon generate a frameshiftinducing a premature stop codon (PTC) in the third exon. This PTC is located 53 bp upstream the junction between the exons 3 and 4, the last junction exon-exon. This is characteristic of a nonsensemediated decay (NMD) construct. Conclusions: The function of this new variant is likely to be involved in the regulation of the IL-4 expression. Thereby, we can imagine a role in regulating the Th2 response by this new mRNA.

P0743 DNA double stranded breaks enhance the transcription of AIRE target genes M. Guha, U. Halasorg, K. Kisand & P. Peterson Biomedicum, Institute of General and Molecular Pathology, Tartu, Estonia Purpose/Objective: The autoimmune regulator (AIRE) gene shows a predominant expression in thymus and is the major regulator of negative selection of autoreactive T-cells. Mutations in AIRE protein result in onset of autoimmune polyendocrinopathy-candidiasis-ectodermaldystrophy (APECED). One reported AIRE interacting parter is topoisomerase 2 and this interaction has been found to promote topoisomerase 2a initiated double stranded breaks. We aimed to study the possible involvement of double stranded DNA breaks in AIRE transcriptional activation of target genes in AIRE-expressing HEK293 cells after the treatment with etoposide or merbarone, the well known inhibitors of topoisomerase 2a. Materials and methods: HEK cell line culture AIRE inducible stable cell line was cultured in DMEM media supplemented with 10% tetracyclin negative fetal calf serum, 100 U/ml penicillin/streptomycin and 0.15 mg/ml G418. AIRE expression was induced with Doxycyline (Dox) at 2.0 lg/ml next day after plating. The protein expression was verified with western blotting. RNA isolation and real time PCR- Total RNA was isolated from cells at different time intervals using Trizol reagent following manufacturer’s protocol. Yield and purity of RNA was determined by nanodrop. Gene expression level of AIRE target gene was detected by quantitative PCR using qPCR SYBR Green Core Kit on ABI Prism 7900HT. Results: We observed an upregulation of the AIRE target genes after the treatment with etoposide that stabilizes topoisomerase 2 to DNA and prevents religation of DNA breaks. In contrast, another topoisomerase 2 inhibitor, merbarone, which blocks topoisomerase mediated DNA cleavage did not result in up-regulation of AIRE target genes. This effect was not a consequence of variability in Aire expression levels but rather indicates the specific differencies of these two topoisomerase 2 inhibitors and a possible role of double stranded breaks in up-regulation of Aire target genes. We next examined the extent of DNA double stranded breaks under our experimental settings using BrdU TUNEL assay and FACS analysis. The analysis showed that indeed there was a significant increase in DNA double stranded breaks under the etoposide treatment compared to nontreated cells or cells that were treated with merbarone. Further, in co-immunoprecipitation experiment, we found AIRE interaction with topoisomerase 2 and this interaction increased in presence of etoposide. Conclusions: With these results we conclude that etoposide enhances the up-regulation of AIRE target genes through the involvement of DNA double stranded breaks.


432 Poster Sessions P0744 Epigenetic regulation of CTLA4 in different subpopulations of T cells J. Kaurson, K. Kisand, K. Kisand & R. Uibo Immunology Group Institute of General and Molecular Pathology, University of Tartu, Tartu, Estonia Purpose/Objective: Cytotoxic T lymphocyte antigen 4 (CTLA4) transmits inhibitory signals to T cells and is critical for maintaining tolerance to self antigens. It is differentially expressed in T cell subpopulations. Regulatory T cells (Tregs) express CTLA4 constitutively, but in effector T cells CTLA4 expression is up-regulated in response to T cell activation. The mechanisms governing regulation of CTLA4 expression in T cell subpopulations are not entirely clear. Chromatin structure and histone modifications are important regulatory factors that affect the transcriptional status of genes. This study aims to determine if differential expression of CTLA4 is regulated through epigenetic mechanisms by comparing histone modification profiles of T cell subpopulations. Materials and methods: Using immunomagnetic separation, naı¨ve CD4+ and CD8+ T cells and CD4+ CD25+ Tregs were isolated from peripheral blood mononuclear cells. Purity of cells was determined by flow cytometry. Chromatin immunoprecipitation (ChIP) was performed with antibodies to various histone modifications, including histone H3 trimethylation at lysine 4 (H3K4me3) and at lysine 27 (H3K27me3). ChIP samples were analyzed by qPCR with primers covering different regions of Ctla4, including non-coding regulatory regions. Results: H3K4me3 is a marker of active promoters and enhancers. We found that the promoter, gene body and distal regulatory regions were enriched for H3K4me3 in Tregs. In naı¨ve CD4+ and CD8+ T cells, level of H3K4me3 was also high at the promoter and promoter proximal regions, although lower compared to Tregs. There was no marked difference in the level of the transcriptionally silenced euchromatin marker, H3K27me3 between Tregs and CD4+ T cells. In CD8+ T cells, however, all the studied regions were enriched for H3K27me3. Conclusions: This bivalent chromatin pattern consisting of a repressive and an activating epigenetic marker could represent a ‘transcription-ready’ state for CTLA4 in naı¨ve CD8+ T cells. However, since Tregs and naı¨ve CD4+ T cells are very similar in their epigenetic profiles, it is possible that differential expression of CTLA4 in T cell subpopulations is regulated by other mechanisms as well. RNA Pol II presence together with epigenetic changes at the promoter and potential distal regulatory regions upon activation of T cells need further studies.

P0745 Evaluation of hematopoietic differentiation by expression level of key molecules K. Polgarova, E. Fronkova, E. Mejstrikova, T. Kalina & O. Hrusak 2nd Faculty of Medicine and University Hospital Motol, Childhood Leukaemia Investigation Prague (CLIP), Charles University, Prague, Czech Republic Purpose/Objective: For a long time, hematopoiesis has been seen as a continuous restriction of cell fate potentials till the development of mature blood cells of all lineages. However, in last years several works documented unexpected plasticity of hematopoietic cells. So called lineage infidelity and/or aberrant expression of different molecules, including the key cell fate regulators, is usually observed in acute leukemia (AL). How exactly these changes affect the cell behavior in background of differentiation and whether they rise from naturally present plasticity of normal cells remains often unclear. Therefore we aimed to evaluate the simplified transcription profile of hematopoietic

progenitors of different lineages in relation to the profile of their malignant counterparts as defined by flow cytometry. Materials and methods: Using qRT-PCR we investigated mRNA expression of 90 different molecules and five control genes in sorted hematopoietic progenitors. The genes included key differentiation and proliferation regulators (e.g. EBF1, MNDA) and also molecules without known function in hematopoiesis (e.g. NFIL3, PAWR). The tested subpopulations included CD34+ Lin- cells, B-lymphoid and myeloid subpopulations from bone marrow, T-lymphoid subpopulations from thymi and blasts from different AL cases (lymphoblastic, myeloblastic, AL of ambiguous lineage). Results: The basic differences between lineages were observed in expression of well known lineage markers and regulators, e.g. CD19, PAX5, GATA3, thus forming expected clusters in clustering analysis; however, even some of these basic molecules (e.g. CD79a) presented with low aberrant expression not attributable to the contamination, observed mainly in early developmental stages. Specific transcription patterns were observed in AL of different lineages disclosing possible importance of currently poorly described genes (e.g. CCDC26). Complete changes in transcription profile towards different lineage was observed in particular stages of ambiguous lineage AL suggesting role of specific regulators (e.g. CSF3 signaling). Conclusions: The fidelity of transcription profile of different lineages was lower than expected and in early stages comparable to AL. Our results show a need to redefine so called aberrant expression present also in normal hematopoietic progenitors. Supported by GACR P301/10/1877.

P0746 Patterns of DNA methylation in the T-cells and monocytes from the elderly and the young L. Tserel,* M. Saare,* K. Kisand,* L. Milani, A. Metspalu & P. Peterson* *Molecular Pathology and Centre for Translational Genomics, University of Tartu, Tartu, Estonia, Estonian Genome Centre and Centre for Translational Genomics, University of Tartu, Tartu, Estonia Purpose/Objective: During the process of aging, several changes contribute to the suppressed immune responses in the immune system of elderly individuals. Both adaptive and innate immune systems are affected. In T-cell compartment, the changes include aberrant T cell phenotypes and reduced activity. This is mainly the result of thymic involution that leads to the reduction of naı¨ve and increase of memory and activated T cell numbers. In turn, monocytes are key regulators of the innate immune system because of their antigen presentation and phagocytic function. Although the numbers of monocytes are stable during the aging, their functional capacity to activate TLR or to express cytokines is often decreased. DNA methylation is an epigenetic modification that plays an important role in several processes, including aging. DNA methylation occurs throughout the genome including repeats, genes and intergenic regions. CpG islands are 0.5 2 kb regions of DNA with high GC content that are often associated with promoter regions. The CpG methylation is linked with transcriptional silencing, as it can block the binding of transcription factors and recruit DNA binding repressors. In this study, we have identified genome-wide DNA methylation differences in CD4 and CD8 T cells and in monocytes of young and elderly individuals. Materials and methods: CD4+ and CD8+ T cells, and CD14+ monocytes were purified from the peripheral blood of four healthy young and four aged volunteers of Estonian Genome Centre using Ficoll gradient method combined with magnetic activating cell sorting (MACS) method. To assess the whole genome methylation and


Poster Sessions 433 expression patterns, Infinium HD Methylation Assay and HumanHT12 v4 Expression BeadChips were used. Results: We identified methylated CpG sites in human peripheral blood CD4 and CD8 T-cells and in monocyte cell populations. Many CpG sites were specifically methylated in T-cell or monocyte subpopulations. Importantly, we found several CpG positions that were differentially methylated in young and elderly individuals suggesting age-related methylation changes in studied cell populations. We have also analyzed the correlation of CpG methylation with nearby gene expression in corresponding cell populations. Conclusions: Human peripheral blood CD4+ and CD8+ T-cell and monocyte populations have differential methylation patterns in young and elderly individuals. The differential methylation may contribute to the phenotypic and functional changes, and to suppressed activities of the immune responses during aging.

P0747 T helper 9 cell development under epigenetic control during T cell maturation A. Ramming, D. Druzd, J. Leipe, H. Schulze-Koops & A. Skapenko Division of Rheumatology and Clinical Immunology, Medizinische Klinik und Poliklinik IVUniversity of Munich, Mu¨nchen, Germany Purpose/Objective: Histone modification patterns are key players in T helper (Th) cell regulation. However, their impact on differences in cytokine response patterns during maturation of the adaptive immune system is less understood. Here, we analyzed histone modifications in promoter regions of T-bet, GATA-3, PU.1, IRF4 and RORC in T cells of different maturation-status. Materials and methods: Recent thymic emigrants (RTEs), matured naı¨ve and memory CD4 T cells were isolated from cord blood of neonates or peripheral blood of adults for studying histone modification patterns and related gene and cytokine expression. Results: The repressive histone H3 lysine 27 trimethylation (H3K27me3) dominated the PU.1 promoter in RTEs, whereas in matured naı¨ve and further in memory T cells H3K27me3 overrepresentation was increasingly counterbalanced by permissive methylation at H3K4 and histone H3 acetylation. Notably, naı¨ve T cells required more intense stimulation to switch the chromatin pattern in the PU.1 promoter from a repressive to a permissive state and thus to produce interleukin (IL)-9 than memory T cells. Inhibition of repressive histone methylation by the specific inhibitor, 3deazaneplanocin A, induced Th9-specific PU.1 expression even with stimulation conditions that would normally lead only to the production of Th0 cytokines. On the other hand, prevention of histone acetylation by the histone acetyltransferase inhibitor, curcumin, diminished PU.1 expression after stimulation that would normally induce production of IL-9. Whereas RTEs differentiated into Th9 cells producing exclusively IL-9, matured naı¨ve or memory CD4 T cells developed into effectors consisting of IL-9-producing cells as well as of cells capable of IL-4, IFN-c, IL-5, or IL-17 coproduction together with IL-9. Conclusions: The data indicate that Th9 cell development is under stringent control during T cell life suggesting a growing contributive role of IL-9 in immune responses initiated by matured T cells.

P0748 The influence of NF-jB signaling in the conserved regulatory region of the AIRE gene K. Koñd, V. Kont, M. Saare & P. Peterson Department of General and Molecular Pathology, University of Tartu, Tartu, Estonia Purpose/Objective: The AIRE (Autoimmune Regulator) gene is essential for the establishment of central tolerance in the thymus. In humans, mutations in the AIRE gene cause the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE regulates the promiscuous gene expression of tissue-restricted antigens (TRAs). NF-jB is essential in the transcriptional regulation of AIRE gene and the lack of NF-jB decreases Aire dependent TRA gene expression. Human AIRE and mouse Aire share a similar conserved sequence containing NF-jB binding sites 3 kb upstream of the transcription start site. The aim of the research was to study whether the NF-jB pathway affects AIRE expression through the upstream NF-jB binding site containing conserved sequence. Materials and methods: We cloned the NF-jB conserved sequence upstream of different AIRE promoter fragments in a luciferase reporter plasmid. HEK293 cells were transfected with the reporter constructs and the NF-jB pathway was activated by TNFa or PMA/ionomycine stimulation. The effect of the NF-jB conserved sequence was assessed by the luciferase activity. In addition, we overexpressed various Rel family members and stimulated the cells with TNFa or PMA/ ionomycine and the activity of the promoter constructs was measured via luminescence. Results: Our preliminary results show that the NF-jB signaling does not activate the reporter gene expression through the conserved sequence upstream of AIRE promoter region. However, analysis of the AIRE promoter region without the NF-jB conserved region showed that gene expression was activated through the AIRE promoter during TNFa or PMA/ionomycine stimulation.Overexpression of Rel family members showed no significant effect on the activation of the promoter constructs with or without NF-jB conserved region. Conclusions: In conclusion, our findings based on luciferase assays suggest that the conserved NF-jB sequence upstream of AIRE promoter region does not mediate activating signals, although, further analyses are required to reveal the asssociation between NF-jB signaling and the conserved NF-jB binding sites upstream of AIRE gene.


434 Poster Sessions P0749 Transcriptional regulation of GARP expression S. Haupt, Q. Zhou, S. Herrmann, H. Schulze-Koops & A. Skapenko Division of Rheumatology and Clinical Immunology, Medizinischen Klinik und Poliklinik IV University of Munich, Mu¨nchen, Germany Purpose/Objective: The membrane-associated molecule, glycoprotein A repetitions predominant (GARP) is highly expressed on activated regulatory CD25+ CD4 T cells (Tregs), but not on resting Tregs or resting or activated CD25- CD4 T cells. The aim of this work was to delineate the transcriptional regulation of GARP gene expression. Materials and methods: Human GARP promoter sequences were cloned into the basic pGL4.10 vector. Several CNS regions were inserted upstream of the minimal promoter of the pGL4.24 vector. Foxp3 was integrated in the mammalian expression vector pDEST12.2. The mouse T cell line, EL-4, was transfected with the promoterreporter-constructs and stimulated with anti-CD3/anti-CD28 antibodies. In addition, cells were transfected with the Foxp3 expression vector and/or treated with retinoic acid (RA). The enhancer/attenuater function of CNS on the promoter activity was analyzed after

transfection of CNS-reporter-constructs and stimulation with PMA and ionomycin. Chromatin immunoprecipitation of freshly isolated and activated human CD4+ CD25+ and CD25- T cells was performed to evaluate the trimethylation state of histone 3 at lysine 4 and at lysine 27, as well as the acetylation state of histone 3 of promoter and CNS regions. Results: Foxp3 and RA synergistically induced luciferase expression in a concentration dependent manner. Histone modifications at the promoter region changed in CD25+ Tregs towards a more accessible chromatin configuration upon T cell activation. In contrast, in CD25T cells it was rather repressively modified. A CNS region located upstream of the GARP gene exhibited an open chromatin configuration in CD25+ T cells upon TCR stimulation. When inserted upstream of a minimal promoter, this CNS region enhanced luciferase activity in response to T cell stimulation. Conclusions: Thus, GARP transcription appears to be initiated by coordinate action of Foxp3 and RA and is additionally modulated via CNS. The transcriptionally active status of the GARP gene is supported by histone modifications in the promoter and conserved regions.


Poster Sessions 435

Poster Session: Asthma/Allergy P0750 A fusion protein of flagellin and ovalbumin as novel vaccine candidate for allergies: suppressing TH2 responses and preventing murine intestinal allergy S. Schu¨lke,* M. Burggraf, Z. Waibler,à A. Wangorsch,* S. Wolfheimer,* S. Vieths,* M. Toda & S. Scheurer§ *Allergology, Paul-Ehlich-Institut, Langen, Germany, Junior Research Group ‘Allergy models’, Paul-Ehlich-Institut, Langen, Germany, àJunior Research Group ‘Novel vaccination strategies and early immune responses’, Paul-Ehlich-Institut, Langen, Germany, §Paul-Ehrlich-Institut, Allergology, Langen, Germany Purpose/Objective: The TLR5 agonist flagellin has been associated with immune modulatory functions making it an interesting adjuvant for allergy treatment. In this study we investigated the potency of fusion proteins containing Listeria monocytogenesis-derived flagellin flaA to induce DC activation and modulate ovalbumin-specific T cell responses. Moreover, we checked the fusion proteins potency for the treatment of allergies in vivo. Materials and methods: Bone marrow-derived myeloid dendritic cells (mDC) from BALB/c, C57BL/6, IL-10-/-, and TLR-signalling deficient (MyD88-/-) mice were stimulated with rOva, rflaA, rflaA plus rOva, or a recombinant fusion protein consisting of rflaA and rOva (rflaA:Ova). Immune modulating properties of rflaA plus rOva and rflaA:Ova were investigated by DC:T cell co-culture using CD4+ T cells from Ova-TCR transgenic or Ova/alum-immunized mice. A murine model of Ovainduced intestinal allergy was applied to assess the capacity of flaA:Ova as potential allergy vaccine. Results: rflaA:Ova induced up-regulation of TLR5 on mDC and, dosedependently, even higher IL-6 and IL-10 secretion than equimolar amounts of rflaA. Moreover, rflaA:Ova induced IL-10 mediated suppression of TH1 and TH2 cytokine secretion from Ova-TCR transgenic CD4+ T cells and CD4+ T cells derived from TH2-biased mice upon Ova/alum immunization. The strong activation of mDC by rflaA:Ova could be suppressed in a dose-dependent manner by application of both inflammasome (Z-VAD-FMK, glybenclamid) and proteasome (lactacysteine) inhibitors. Prophylactic vaccination with rflaA:Ova was shown to protect against intestinal allergy, reduce T cell activation and TH2 cytokine secretion. Additionally, rflaA:Ova suppressed Ova-specific IgE and induced Ova-specific IgG2a antibodies, whereas rflaA or rOva provided alone or as a mixture did not have comparable protective effects. Conclusions: The rflaA:Ova fusion protein showed enhanced TLRmediated immune modulating capacities, probably attributed to the proximity of the adjuvant and allergen. rflaA:Ova was able to suppress TH2 responses both in vitro and in vivo. Therefore, rflaA: allergen fusion proteins are promising vaccine candidates for intervention in IgE-mediated allergy.

P0751 A hypoallergenic variant of the major birch pollen allergen Bet v 1 shows distinct characteristics in antigen processing and T cell activation C. Kitzmueller*, M. Wallner, S. Deifl,* S. Mutschlechner,* G. Zlabinger,à F. Ferreira & B. Bohle* *Institute for Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria, Department of Molecular Biology, University of Salzburg, Salzburg, Austria, àInstitute of Immunology, Medical University of Vienna, Vienna, Austria Purpose/Objective: BM4 is a novel genetically engineered variant of Bet v 1, the major birch pollen allergen. BM4 differs from Bet v 1 in

only five amino acids but lacks the typical Bet v 1-like fold. As a consequence, BM4 displays negligible IgE-binding while it contains all relevant T cell epitopes of Bet v 1 except one. The aim of this study was to compare BM4 and Bet v 1 with regard to internalization, antigenprocessing and presentation by antigen-presenting cells as well as T cell activation. Materials and methods: Proliferative responses to BM4 and Bet v 1 of peripheral blood mononuclear cells and Bet v 1-specific T cell clones were analysed. Fluorescently labelled BM4 and Bet v 1 were used to study surface binding, endocytosis and intracellular degradation by monocyte-derived DC (mdDC). Both proteins were digested by endolysosomal extracts of mdDC. BM4- and Bet v 1-pulsed mdDC were employed to assess the kinetics of activation of Bet v 1-specific T cell clones and the polarization of naı¨ve T cells. Results: BM4 displayed a significantly stronger T cell-activating capacity than Bet v 1. Furthermore, BM4 bound more efficiently to the surface of mdDC. Internalisation and trafficking into acidic compartments as well as lysosomal degradation were faster for BM4 than for Bet v 1. BM4-pulsed mdDC induced enhanced proliferative responses at earlier time-points in Bet v 1-specific T cell clones. T cells primed with BM4-pulsed mdDC synthesized less IL-5 than those primed with Bet v 1-pulsed mdDC. Conclusions: The loss of the Bet v 1-fold changes the protein’s interaction with the human immune system at the level of antigenpresenting cells resulting in altered T cell responses. By combining low IgE-binding with a strong T cell-activating capacity that modulates the allergen-specific T cell response, BM4 represents a highly interesting candidate for specific immunotherapy of birch pollen allergy.

P0753 A peptide mimicking an important carbohydrate epitope crossreactive between birch proteins and celery allergen Api g 5 A. Lukschal, J. Wallmann, M. Bublin, H. Breiteneder, B. Hantusch,à I. Pali-Scho¨ll§ & E. Jensen-Jarolim4,5 *Division of Comparative Immunology and Oncology, Center for Pathophysiology Infectiology and Immunology, Institute of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria, Department for Medical Biotechnology, Research Center for Pathophysiology Infectiology and Immunology, Institute of Pathophysiology and Allergy, Medical University of Vienna, Vienna, Austria, à Institute of Pathology, Medical University of Vienna, Vienna, Austria, § Center for Pathophysiology Infectiology and Immunology Institute of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria, –Messerli Research Institute of Universtity of Veterinary Medicine, Vienna, Austria Purpose/Objective: Allergic reactions to celery are often caused by cross-reactivity to major birch pollen allergens like Bet v 1 and Bet v 2. Nevertheless, about 40% of the cross-reacting IgEs are directed towards yet unidentified allergens of 32 63 kDa in birch, and towards Api g 5 in celery. It has been demonstrated that IgE-binding to Api g 5 is strongly dependent on its carbohydrate moieties. Monoclonal antibody BIP 3 was originally raised against birch pollen extract and cross-reacts with Api g 5. For epitope definition, we aimed to generate peptides mimicking the BIP 3 epitope, so called mimotopes. Materials and methods: Phages from a random-peptide phagedisplay library were selected for specific binding to monoclonal antibody BIP 3 and for their ability to inhibit IgE-binding of allergic patients to birch pollen extract. Three phage clones were chosen for intraperitioneal immunization of BALB/c mice and induced antibodies were tested on blotted birch pollen extract. To test if the mimicked epitope was comprised of carbohydrate moieties, we compared serum reactivity of immunized mice to Api g 5 and the structurally unrelated plant glycoprotein horse radish peroxidase (HRP). As a negative


436 Poster Sessions control nonglycosylated grass pollen allergen Phl p 5 was used in ELISA. Results: After 3 rounds of biopanning 7 different sequences were derived from 14 positive phage clones. The most frequent sequence also elicited the strongest immune reaction in mice. All 3 clones selected for immunization induced antibodies crossreactive for the major 63 kDa band recognized by BIP 3 in birch pollen extract and Api g 5. The antibodies induced by the mimotope immunizations were also reactive towards natural purified Api g 5 and the glycoprotein HRP, but not to the control allergen Phl p 5. Conclusions: The peptide mimotopes characterized in this study mimic a carbohydrate epitope cross-reactive between Api g 5 and high molecular weight birch pollen proteins. As a peptide mimotope can be considered a superior immunogen in comparison to carbohydrate moieties, it may be suitable for epitope-specific immunotherapy and diagnosis. Supported by grants SFB F01808-B13and F4606-B19 of the Austrian Science Fund and Nano-Health 819721 of the Austrian Reseach Agency.

P0754 A pollen hybrid molecule as novel therapeutic for the treatment of Fagales multi-sensitization U. Pichler,* M. Hauser,* E. Hoflehner, P. Briza,à S. Mutschlechner,§ B. Bohle,§ U. Wiedermann-Schmidt, F. Ferreira* & M. Wallner* *Department of Molecular Biology, Laboratory for Allergy Diagnosis and Therapy, University of Salzburg/Christian Doppler, Salzburg, Austria, Department of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Vienna, Austria, àDepartment of Molecular Biology, University of Salzburg, Salzburg, Austria, §Department of Pathophysiology, Laboratory for Immunomodulation, Medical University of Vienna/Christian Doppler, Vienna, Austria Purpose/Objective: Allergies against birch pollen and pollen of related trees belonging to the botanical order of Fagales are the most common cause for spring pollinosis in the temperate climate zone of the Northern hemisphere. However, specific immunotherapy is mainly performed with birch pollen extracts, limiting the success of this therapeutic intervention in areas where birch is not endemic. Materials and methods: Thus, in the present study we generated a pollen hybrid molecule (PH) by PCR-based recombination of T cell epitopes of the low IgE-binding isoforms of the major allergens from birch, hazel, alder, oak, and hornbeam. In silico analyses of PH revealed that the molecule carries an epitope previously identified as critical for the Bet v 1-fold and closely connected to IgE binding, immunogenicity, and T cell polarization of Bet v 1. Therefore, the structure of PH was modified accordingly by replacing seven consecutive amino acids within the Bet v 1-part of the molecule by the homologous sequence of Mal d 1, resulting in a fold-variant of the molecule (PH4). Results: Both hybrid molecules showed reduced binding of Bet v 1specific IgE antibodies and an elevated activation of PBMCs from birch pollen allergic donors. Compared to the parental allergens, the pollen hybrid molecules could induce antigen-specific TH1 cells in a mouse immunization model. In dendritic cells the modified structures of both hybrids directly affected antigen-uptake and processing. The prophylactic potential of both proteins was tested in a mouse model of oral tolerance, indicating that the pollen hybrids could protect mice from Fagales pollen induced lung inflammation. Conclusions: Compared to wild-type allergens the pollen hybrid molecules interacted differently with dendritic cells, leading to a deviated T cell response in vivo. In combination with the low IgE binding properties our immunological data emphasizes the potential of PH and PH4, respectively, as novel tools for the treatment of Fagales multi-sensitization.

P0756 Absence of Foxp3+ regulatory T cells during allergen provocation does not exacerbate murine allergic airway inflammation A. M. Baru, V. Ganesh, C. Hesse, C. Untucht & T. Sparwasser Twincore, Institute of Infection Immunology, Hannover, Germany Purpose/Objective: Tregs have been implicated in modulating allergic immune responses. However, their influence on distinct phases of development of allergies remains unclear. In this study we aimed to comprehend the specific involvement of Foxp3+ Tregs in airway inflammation during allergen provocation. This reflects a clinically relevant situation, assuming prior allergen sensitization to have already occurred, and thus addresses a potential Treg mediated therapeutic role. Materials and methods: Standard OVA-Alum model was used for induction of allergic airway inflammation in Foxp3-eGFP-DTR transgenic (DEREG) mice. A group of sensitized mice received diphtheria toxin treatment to deplete Tregs prior to allergen challenge. Serum immunoglobulin levels were determined by ELISA to confirm comparable sensitization across groups. Inflammation was scored by differential cellular infiltration in broncheo-alveolar lavage (BAL) and cytokines secretion by in-vitro re-stimulated mediastinal lymph node cells. Lung pathology was assessed by histology and qPCR. Results: Comparable antigen-specific serum immunoglobulin levels implicate equivalent sensitization across all groups of mice. No significant alteration was observed in numbers or types of cells infiltrating BAL of Treg depleted mice. Th2 cytokines secreted by restimulated mediastinal lymph node cells were comparable. Double blinded histological analysis of lung sections revealed no enhanced pathology in lungs of Treg depleted mice. To rule out strain specific differences we followed similar regimen in DEREG mice on a comparatively allergy resistant genetic background (C57BL/6). Consequently, Treg depletion during allergen challenge did not enhance lung inflammation even in C57BL/6 mice. Conclusions: Absence of Foxp3+ Tregs during allergen challenge does not exacerbate allergic airway inflammation in mice. Differential disposition to allergies due to genetic backgrounds does not alter the inflammatory response observed in this regimen. We propose less pronounced role of Foxp3+ Tregs under physiological conditions in inhibiting allergic responses during the allergen provocation phase as compared to their influence during the sensitization phase. These results signify the temporal regulation exerted by Foxp3+ Tregs and may influence their application as potential therapeutics.

P0758 Acrolein blocks antibody-formation upon nasal sensitization with KLH in BALB/c mice F. Roth-Walter,* A. Willensdorfer,* C. Stremnitzer, C. Schultz, S. Diesner, R. Zadnikar,* J. Fazekas, E. Untersmayr, I. Pali-Scho¨ll* & E. Jensen-Jarolim* *Messerli Research Institute Comparative Medicine, University of Veterinary Medicine Vienna Medical University Vienna and University Vienna, Vienna, Austria, Division 1-Comparative Immunology and Oncology, Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria Purpose/Objective: Smokers have an increased risk for respiratory diseases. Hence, we sought to investigate the contribution of acrolein during nasal sensitization, since acrolein is generated in large amounts in cigarettes. Materials and methods: BALB/c mice were nasally sensitized 6 times in biweekly intervals with KLH alone or with KLH in conjunction with acrolein. Immune response was analyzed on the level of specific antibodies in ELISA and by cytokine determination from splenocytes after antigen-specific -stimulation.


Poster Sessions 437 Results: In the absence of adjuvant, nasal application of KLH alone was sufficient to induce KLH-specific antibody-titers of IgG1, IgG2a, IgG2b, IgA and IgE, whereas nasal sensitization with KLH in the presence of acrolein completely abrogated antibody-formation. In contrast, KLH-stimulated splenocytes of mice sensitized with KLH in conjunction with acrolein secreted higher levels of IFN-g than mice sensitized with KLH alone. Conclusions: Nasal application of acrolein impairs the induction of a proper humoral immune response by preventing antigen-specific antibody formation, but it supports innate release of IFN-c. Hence, acrolein in smoke significantly modulates the innate and adaptive immune responses in smokers, thereby likely affecting susceptibility to infections in smokers.

P0759 Allergen-specific immunotherapy induces regulatory sialylated IgGs M. Ehlers,* C. M. Oefner, A. Winkler, C. Hess, D. Petzold* & M. Bergerà *Institute for Systemic Inflammation Research, University of Luebeck, Luebeck, Germany, Laboratory of Tolerance and Autoimmunity, German Rheumatism Research Center, Berlin, Germany, àCharite´, Central Institute for Laboratory Medicine and Pathobiochemistry, Berlin, Germany Purpose/Objective: Allergen-specific immunotherapy (SIT; hyposensitization) is associated with the development of regulatory T cells and allergen-specific IgG serum antibodies. However, the function of SITinduced IgG is still unclear. Recently, it has been shown that the Fc glycosylation pattern determines the pro- or anti-inflammatory effector function of IgG antibodies, whereby sialylated IgGs are antiinflammatory. However, the IgG Fc glycosylation pattern induced by SIT is unknown. We sought to examine the Fc glycosylation and antiinflammatory quality of IgG molecules formed upon allergen-specific tolerance induction. Materials and methods: We administered chicken ovalbumin (OVA) under inflammatory or tolerance conditions to mice and analyzed OVA-reactive IgG Fc glycosylation. The anti-inflammatory function of differentially glycosylated anti-OVA IgGs was further investigated by studies with dendritic cell (DC) cultures and in an in vivo model of allergic airway disease. Additionally, we analyzed the Fc glycosylation pattern of birch pollen-reactive serum IgGs following successful SIT in patients. Results: Stimulation with protein antigens/allergens under inflammatory conditions induced de-sialylated IgGs. In contrast, tolerance induced immunosuppressive sialylated IgGs that were sufficient to block antigen-specific T and B cell responses, DC maturation and allergic airway inflammation. Importantly, successful SIT in allergic patients also induced sialylated allergen-specific IgGs. Conclusions: Our data show a novel antigen-specific immunoregulatory mechanism mediated by anti-inflammatory sialylated IgGs that are formed upon tolerance induction. These findings may help to understand und to develop novel allergen-specific therapies for the treatment of allergy.

P0760 Allergen-specific mucosal IgA induced following sublingual immunotherapy protect against allergic inflammation in a murine model of allergic inflammation C. Rask, J. Brimnes, S. Urioste, K. Lund & V. Renard-Wagtmann ALK-Abello´, Pharmacology, Hoersholm, Denmark Purpose/Objective: Sublingual immunotherapy (SLIT) with allergen extract has been shown to be efficacious in treating allergy. A hallmark

of allergen-specific immunotherapy is the induction of non-IgE allergen-specific antibodies primarily of the IgG1 and IgG4 isotypes which may, in addition to being robust markers of immunotherapy, be at least partly responsible for the clinical effect of the treatment through their capacity to compete with effector-cell bound IgE antibodies for allergen binding. It has also been hypothesized that allergen-specific IgA antibodies induced after successful SLIT could act in a similar manner and prevent allergen absorbance, and thereby subsequent allergic reactions. The objective of this study was to use a knock-out mouse strain lacking active transport mechanisms of IgA to mucosal surfaces for exploring the role of SLIT-induced mucosal IgA in protection against allergic inflammation. Materials and methods: Wild type (WT) and poly immunoglobulin receptor knock-out (pIgRKO) mice were sensitized to Phleum pratense (Phl p) by intraperitoneal injections of alum-adsorbed allergen extract. Sensitized mice were administered Phl p extract or buffer sublingually. Thereafter, mice were challenged intra-nasally with Phl p pollen in order to mimic a grass pollen season. Numbers of sneezes, allergenspecific antibody levels and total numbers of eosinophils in nasal lavage (NAL) and bronco-alveolar lavage (BAL) were used as read-outs for the efficacy of SLIT. Moreover, the induction of allergen-specific IgA on mucosal surfaces and in serum was determined following 9 weeks of sublingual administration. Results: SLIT with Phl p extract in WT mice significantly suppresses allergen-induced inflammation measured as attraction of eosinophils to BAL and induction of allergen-specific IgE antibody response in BAL and serum. This protection following Phl p SLIT towards allergen-induced inflammation could not be observed in the pIgRKO mice lacking active transport of IgA to mucosal surfaces. In addition, we could find higher allergen-specific IgA response in saliva, serum and BAL from WT mice as compared to pIgRKO mice. Conclusions: Allergen-specific mucosal IgA antibodies are induced after SLIT in WT mice and seem to play a protective role against allergic inflammation in a murine model of grass pollen induced allergic rhinitis.

P0761 Allergen specific T and B cell proliferation in pollen allergic patients measured by a CFSE dilution based assay J. Eckl-Dorna,* R. Campana, R. Valenta & V. Niederberger* *Otorhinolaryngology, Medical University of Vienna, Vienna, Austria, Division of Immunopathology, Center of Physiology and Pathophysiology, Medical University of Vienna, Vienna, Austria Purpose/Objective: In past years alternative methods to radioactive measurements for cellular proliferation, such as assessment of dilution of the dye 5, 6-carboxy-fluorescein diacetate succinimidyl ester (CFSE) in the flow cytometer, have been established. The latter method has one major advantage: It allows not only for measurement of proliferation in total peripheral blood mononuclear cells (PBMCs) but also in subpopulations e.g. T cells or B cells by multicolour flow cytometry. In the present study we sought to establish a CFSE dilution based assay to determine which cell types contribute to a proliferative response to allergen in vitro. Materials and methods: To that aim we isolated PBMCs from birch and grass pollen allergic patients and labelled them with CFSE. Cells were cultured in the presence or absence of highly purified recombinant allergens (rBet v 1, rPhl p 5) for 1 week whereupon proliferation was measured by flow cytometry. Results: T cell proliferation upon allergen stimulation was observed in PBMC cultures of all patients over a wide range of allergen concentrations using co-staining for CFSE and the pan T cell marker CD3. More importantly co-staining for the B cell marker CD20 revealed, that also a subfraction of B cells proliferated in response to allergen stimulation.


438 Poster Sessions Conclusions: Thus we demonstrated that in PBMC cultures of allergic patients both B and T cells proliferate specifically in response to allergen stimulation in vitro. Since each of the tested patients mounted allergen-specific antibody responses it is possible that the proliferating B cell sub-fractions contained allergen-specific B cells. The fact that in PBMC cultures of allergic patients not only T cells proliferate in response to allergen should be borne in mind when interpreting classical proliferation assays such as 3H thymidine incorporation.

P0763 Alpha purothionin, a new wheat food allergen, belongs to a family of plant defence proteins S. Pahr,* C. Constantin, N. G. Papadopoulus,à G. Stavroula,à M. Ma¨kela¨,§ T. Rito,§ C. Ebner,– A. Mari,** S. Scheiblhofer, J. Thalhamer, I. Mittermann,* S. Vrtala* & R. Valentaàà *Division of Immunopathology, Department of Pathophysiology and Allergy Research Center of Pathophysiology Infectiology and Immunology Christian Doppler Laboratory for the Development of Allergen Chips, Medical University of Vienna, Vienna, Austria, Division of Immunopathology, Department of Pathophysiology and Allergy Research Center of Pathophysiology Infectiology and Immunology, Medical University of Vienna, Vienna, Austria, àAllergy Department, University of Athens, Athens, Greece, §Skin and Allergy Hospital, Helsinki University Central Hospital, Helsinki, Finland, –Ambulatory for Allergy and Clinical Immunology, Ambulatory for Allergy and Clinical Immunology, Vienna, Austria, **IDI-IRCCS, Center for Molecular Allergology, Rome, Italy, Department of Molecular Biology, University of Salzburg, Salzburg, Austria, ààDivision of Immunopathology, Department of Pathophysiology and Allergy, Research Center of Pathophysiology Infectiology and Immunology Christian, Doppler Laboratory for Allergy Research, Medical University of Vienna, Vienna, Austria Purpose/Objective: Wheat is an important source for IgE-mediated food allergy. Avoidance of wheat products is currently the only therapy for wheat food allergic patients whereas allergen-specific approaches such as immunotherapy would require a detailed knowledge and availability of the disease-causing allergens. Aim of this study was the isolation, identification and characterization of new allergens recognized by wheat food allergic patients for diagnosis and treatment of wheat food allergy. Materials and methods: We screened a wheat cDNA library with serum IgE antibodies from patients suffering from wheat food allergy. The cDNA coding for novel wheat food allergen, alpha-purothionin, could be isolated and identified by sequence analysis. Recombinant alpha-purothionin was expressed, purified and characterized regarding molecular properties. The IgE-reactivity was tested in wheat food allergics, grass pollen allergic patients and non-atopic individuals. Allergen-specific rabbit antibodies were used to screen different cereal and bread extracts. To investigate the allergenic activity, we performed basophil degranulation experiments. Results: In this study we report the isolation of an IgE-reactive cDNA clone coding for a novel wheat food allergen alpha purothionin which belongs to a family of plant defence proteins. Homologue proteins to alpha purothionin could be detected by allergen specific rabbit antibodies in many other cereal and bread extracts. Serum IgE antibodies from wheat food allergic patients reacted specifically with alpha purothionin and allergenic activity was demonstrated in basophil degranulation and skin prick test experiments. Conclusions: Recombinant alpha purothionin may be useful for the diagnosis and possibly immunotherapy of IgE-mediated wheat food allergy.

P0764 Analysis of the lung inflammation induced by Alternaria alternata spores or extracts in C57BL/6, TLR-4 and MyD88 knockout mice O. Denis,* M. Vincent,* G. Treutens,* S. De Prins* & K. Huygen *Allergology Program, Scientific Institute of Public Health (WIV-ISP), Brussels, Belgium, Service Immunology, Scientific Institute of Public Health (WIV-ISP), Brussels, Belgium Purpose/Objective: The mould Alternaria alternata is an important cause of allergic rhinitis and asthma and exposure to airborne spores of Alternaria can trigger severe asthma. Our objective was to analyse the lung immune response induced in mice instilled with spores or extracts of Alternaria and to analyze the requirement of TLR4 and MyD88 signalling for its development. Materials and methods: WT C57BL6, TLR4 KO or MyD88 KO mice were instilled with Alternaria spores or extracts. Cellular recruitment in the lungs was analysed early (3 days) or later (5 weeks) during the response. Cytokine/chemokine lung expression profiles were investigated at the same time points by RT-qPCR. Results: After 3 days, C57BL/6 mice showed an accumulation of neutrophils in the airways, this recruitment was the strongest with the spores. This stronger inflammatory property was confirmed by a stronger activation of IL-17, CXCL-2, CCL-3 and IL-1a expression in the lungs of mice stimulated with the spores. After 5 weeks a mixed lung inflammatory response was induced by the spores. In contrast, only eosinophils were recruited after extract instillation. This correlated with an increased lung expression of IL-4, IL-13, CCL11 and CCL24. Of note, the expression of IL-33 was similar for spores and extracts at week 5 but only the extract induced an early expression of this cytokine at day 3. Very similar cell recruitments were observed in wild type C57BL/6 mice and in TLR-4 KO mice stimulated with the extracts. However mice genetically inactivated in the MyD88 adaptor had an almost complete inhibition of lung neutrophil recruitment at day 3 and eosinophils at week 5. This correlated with a decreased expression of inflammatory gene expression, an inhibition of IL-33 expression at day 3 and a decreased IL-4, IL-13, CCL11 and CCL24 expression at week 5. Conclusions: Alternaria spores and extracts have inflammatory and allergic properties, be it that spores have a tendency to induce inflammatory responses while extracts induce stronger allergic responses. TLR-4 signalling is dispensable but MyD88 is compulsory for these responses. Moreover, there is a correlation between an early IL-33 expression, a low inflammatory response and the accumulation of eosinophils during the chronic phase induced by the extract.

P0766 Apoptotic effect of Tamoxifen on neutrophils bronchial cells in horses with Recurrent Airway Obstruction: a new therapeutic approach G. Moran,* B. Perez,* N. Morales,* L. Vidal,* J. S. Galecio, J. S. Galecio, N. Vasquez,à H. Folch§ & C. Henriquez§ *Pharmacology, Universidad Austral de Chile, Valdivia, Chile, Veterinary Clinical Science, Universidad Austral de Chile, Valdivia, Chile, à Pathology, Valdivia Hospital, Valdivia, Chile, §Immunology, Universidad Austral de Chile, Valdivia, Chile Purpose/Objective: Recurrent airway obstruction (RAO, ‘heaves’) is an asthma-like condition that develops in mature horses following stabling and exposure to dusty hay and straw. The hallmark of this disease is that hay/straw exposure induces clinical airway obstruction, airway neutrophilia and increased airway mucous production in RAO susceptible horses. The aim of this work was to evaluate the rate of apoptosis in bronchial neutrophils in RAO-affected horses during acute crisis and remission; to observe the effect of the treatment on the


Poster Sessions 439 apoptosis rate of affected horses with tamoxifen; and to evaluate the concordance of these findings with clinical signs, levels of specific immunoglobulin and type of cells found in bronchoalveolar lavage fluid (BALF). Materials and methods: For this purpose, nine RAO susceptible horses sensitized to Aspergillus fumigatus (RAO herd) were selected for use in this study. The animals will be exposed to dusty/moldy hay, and once the signs of the disease appear were carefully evaluated recording clinical signs, result of endoscopic examination; and BALF evaluation (immunoglobulin and cellular content). Later, will be treated with 100 mg PO of tamoxifen every other day in a remission environment. During treatment and after the drug administration ended, periodical evolution of RAO signology. The sampling will include physical and endoscopic examination and collection of BALF and blood for analysis. Flow cytometry using commercial AnnexinV-FITC and propidium iodide was used to quantify early and late apoptotic leukocytes, respectively. Results: The results showed a significant increase in early apoptosis in peripheral blood and bronchial granulocytic cells in RAO-affected horse’s treatred with tamoxifen. In addition, we showed that RAOaffected horses treated with this drug displayed a significant decrease in neutrophils in BALF and a concomitant improvement in their clinical status; with a decrease in total levels of immunoglobulins against A. fumigatus. Conclusions: Our study showed that tamoxifen, a drug used for breast cancer, has the ability to induce apoptosis in granulocytic cells from peripheral blood and BALF with a concomitant improvement in their clinical status. Finally, further in vitro and in vivo studies are needed to better understand tamoxifen treatment because as an anti-carcinogen, tamoxifen could be used to treat chronic, inflammatory pathologies including those associated with granulocytes and allergic diseases, such as asthma or equine RAO

P0767 Association of HLA-DR1 with the allergic response to the major mugwort pollen allergen: molecular background B. Knapp,* G. Fischer, D. Van Hemelen,à I. Fae, B. Maillere,§ C. Ebner,– W. Schreiner,* B. Bohleà & B. Jahn-Schmidà *Department for Biomedical Computersimulation and Bioinformatics, Medical University Vienna, Vienna, Austria, Department of Blood Group Serology, Medical University Vienna, Vienna, Austria, àDepartment of Pathophysiology and Allergy Research, Medical University Vienna, Vienna, Austria, §CEA iBiTecS, Service d’Ingeńierie Molećulaire des Proteínes, Gif-sur-Yvette, France, –Allergieambulatorium, Reumannplatz, Vienna, Austria Purpose/Objective: Mugwort pollen allergens represent the main cause of pollinosis in late summer. The major allergen, Art v 1, contains only one single immunodominant, solely HLA-DR-restricted T cell epitope (Art v 125 36). The frequency of HLA-DRB1-01 is highly increased in mugwort-allergic individuals and HLA-DR1 serves as restriction element for Art v 125 36. However, Art v 125 36 also binds to HLA-DR4 with high affinity and DR1-restricted Art v 125 36 -specific T cell receptors can be activated by HLA-DR4 molecules. Materials and methods: To understand the predominance of HLADR1 in mugwort allergy in spite of the degeneracy in HLA/peptidebinding and TCR-recognition, we investigated the molecular background of Art v 125 36/MHC/TCR interactions in the context of HLADR1 compared to -DR4. Results: The majority of Art v 125 36 -specific T cell lines and clones from HLA-DR1 carrying, mugwort pollen-allergic donors reacted to synthetic and naturally processed Art v 1 peptides when presented by HLA-DR1 or HLA-DR4 expressing antigen presenting cells. However, at limiting peptide concentrations DR1 was more effective

in T cell stimulation. In addition, the minimal epitope for 50% of Art v 125 36 specific T cells was shorter for DR1 than for DR4. In vitro binding assays of Art v 125 36 mutant peptides to isolated DR1- and DR4-molecules indicated similar binding capacities and use of the same register. In silico simulation of Art v 125 36 binding to HLA-DR1 and -DR4 suggested similar binding of the central part of the peptide to either molecule, but a higher flexibility of the N- and C-terminal amino acids and detachment at the C-terminus in HLA-DR1. Conclusions: The predominance of HLA-DR1 in the response to Art v 125 36 may be explained by the tendency of superior peptide presentation by DR1 compared to DR4 found in vitro. Computer simulation supported our experimental data by demonstrating differences in peptide mobility within the HLA-DR complex which may influence TCR-binding. We suggest that the minor differences observed in vitro may be more relevant in the microenvironment in vivo, so that only presentation by HLA-DR1, but not -DR4 permits successful T cell activation.

P0768 Bone marrow-derived dendritic cells inappropriately reflect allergen sensitization with house dust mite aeroallergens in the context of complement activation Y. Laumonnier, C. Engelke, I. Schmudde, H. Stro¨ver & J. Ko¨hl Insitute for systemic inflammation research, University of Luebeck, Luebeck, Germany Purpose/Objective: Pulmonary dendritic cells play critical roles in allergen uptake and Th cell differentiation towards Th2 and Th17 effector cells. Previous studies have demon-strated a protective role for C5a and a proallergic role for C3a during allergen sensi-tization suggesting that C3a and C5a either promote or suppress DC functions during initial allergen encounter. Using bone-marrow-derived (BM) DCs as a surrogate for pulmonary APCs, we aimed at better defining the roles of C3a and C5a in DC-mediated development of maladaptive Th2 and Th17 immunity in allergic asthma. Materials and methods: GM-CSF-differentiated bone marrow BMDC from wildtype (wt), C3aR-/-, C5aR-/- and C3aR-/-/C5aR-/- mice were obtained after 9 days and were pulsed in vitro for 24 h with crude extract from house dust mite (HDM). 1 · 106unpulsed or pulsed cells were then transferred intra-tracheally into wt recipient mice. The following parameters were used to define the allergic phenotype: airway hyperresponsiveness (AHR), inflammatory cell infiltration, ex vivo cytokine production from pulmonary lung cells, histologic examination of mucus production. Results: We found that BMDCs from wt and C3aR-/- mice induced a strong asthmatic response, characterized by a marked increase in AHR, strong Th2 but minor Th17 and Th1 cytokine production and a mixed eosinophilic and neutrophilic infiltration of the lung. In contrast, AHR, Th2 cytokine production and eosinophilic inflammation were substantially decreased following adoptive transfer of C5aR-/- or C3aR-/- C5aR-/- BMDCs. However, mice treated with C3aR-/- C5aR-/BMDCs showed strong neutrophilic infiltration and an increased IFNg production. Conclusions: We found that adoptive transfer of allergen-pulsed C5aR-/- or C3aR-/- BMDCs does not recapitulate the findings of an increased allergic phenotype in C5aR-/- mice and a decreased allergic phenotype in C3aR-/- mice after intratracheal HDM administration. In the BMDC adoptive transfer setting, C5aR plays a critical role in the development of asthma whereas the role of C3aR is at best minor. Our data suggest that adoptive transfer of allergen-pulsed BMDC does not appropriately reflect the sensitization process towards aeroallergens such as HDM by lung resident DCs, at least in the context of complement activation.


440 Poster Sessions P0769 Caffeic acid decreases eosinophilic airway inflammation on blomia tropicalis murine model of allergy T. Cana Brasil Carneiro,* R. Santos Costa,* N. V. Queiroz Carneiro,* S. Assis Lima,* A. T. Cerqueira Lima,* M. Santos Serafim Machado,* K. M. Campos Dourado,* L. C. Pontes de Carvalho, N. M. Alcaˆntara Neves* & C. Alexandrina Viana Figueireˆdo* *Imunofarmacologia, Universidade Federal da Bahia, Salvador Bahia, Brazil, Imunologia, Fundaçaõ Osvaldo Cruz, Salvador Bahia, Brazil Purpose/Objective: To evaluate the anti-allergic effect of Caffeic Acid (CA) in a murine model of allergy to Blomia tropicalis mite. Materials and methods: Groups of 6 AJ mice were sensitized by subcutaneous injections with 100 lg of B. tropicalis antigen in 4 mg/ml aluminum hydroxide on days 0 and 7. Twenty-four hours (24 h) after the last sensitization the animals received four (4) intranasal challenges (10 lg per animal) at intervals of 1 day. Allergic animals were treated orally with 10, 100 or 200 mg/kg of CA or with 3 mg/kg of Dexametazone (Dex). On day 15 the animals were euthanized and some parameters were evaluated such as number of leukocytes/ eosinophils in bronchoalveolar lavage (BAL), determination of eosinophil peroxidase activity (EPO) in BAL and levels of IgE anti-Bt in the serum. Results: Treatment of animals with CA demonstrates the ability of CA in reducing allergic parameters such as the total number of cells andnumber the eosinophils in BAL (P < 0.01) in relation to the untreated BT-sensibilized and challenged mice. The oral treatment with CA also displayed a significant reduction in the levels of EPO in BAL (P < 0.001). As expected, the administration of 3 mg/kg of Dex significantly suppressed the number of eosinophils and total inflammatory cells (P < 0.001) and decreased EPO activity in BAL (P < 0.001). Bt-immunized mice produced higher levels of specific IgE antibodies than the normal control group (P < 0.001). However, treatment with CA was not able to reduce IgE antibody levels, in contrast to animals treated with Dex (P < 0.05). Conclusions: CA effectively reduced some immunological parameters related to cell migration responsible for airway inflammation in a murine model of allergy induced by B. tropicalis but did not interfere in IgE antibody production.

P0770 Can innate inflammatory factors break immunotherapy-induced T cell tolerance in patients with allergic asthma? U. C. Kucuksezer,* I. Tahrali,* S. Adin-Cinar,* B. Gemicioglu, A. C. Akdis,à M. Akdisà & G. Deniz§ *Immunology, Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey, Pulmonology, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey, àImmunology, Swiss Institute of Allergy and Asthma Research, Davos, Switzerland, §Immunology, Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey Purpose/Objective: Asthma is a disease of the respiratory system, with 3 main subgroups; the allergic group forming the biggest proportion. Allergen-specific immunotherapy cures allergic diseases, with great success. The immunologic mechanisms and contributing factors of immunotherapy is still a question to be answered. Innate inflammatory cytokines and ligands for certain Toll like receptors (TLRs) are shown to release T-cell unresponsiveness to allergens in PBMCs of healthy individuals, which may resemble convertion of healthy status to allergic. Materials and methods: This study aims to investigate roles of these inflammatory factors in immunotherapy model which provides allergen-specific and induced unresponsiveness in allergic asthmatic patients. PBMCs isolated from 3 allergic asthma patients who received

immunotherapy to known allergens were stained with CFSE in order to investigate allergen-specific CD4+ T cell proliferation, cultured with the existence and absence of immunotherapy allergen as well as control allergen, and stimulated with IL-1beta, IL-6 and ligands for TLR4 and TLR8. Results: First results of this ongoing study support the tolerancebreaking effects of IL-1beta and IL-6. Conclusions: More number of patients should be investigated in order to reveal the possible roles of these inflammatory conditions in breaking of peripheral tolerance

P0771 CD4+ CD25+ FOXP3+ T cells are Decreased in Patients with Allergic Conjunctivitis J. G. C. Jorge,* S. A. M. E. Sańchez-Alonso, P. O. J. L. PalomaresOrdon˜ez, H. E. Hong,* P. T. M. Pe´rez-Tapiaà & J. M. M. C. JimeńezMartıńez *Departamento de Farmacobiologıá, Centro de investigacioń y de Estudios Avanzados del I.P.N., Mexico City, Mexico, Unidad de Investigacioń Instituto de Oftalmologıá Fundacioń Conde de Valenciana, Departamento de Inmunologıá, Mexico City, Mexico, àEscuela Nacional de Ciencias Biolo´gicas del I.P.N., Unidad de Desarrollo e Investigacioń en Bioprocesos, Mexico City, Mexico Purpose/Objective: Allergic conjunctivitis (AC) is one of the most common eye disorders in clinical practice. It has been shown that AC is a disorder mediated by Th2 lymphocytes producing IL-4 and IL-5, where the eye damage is caused by a type I hypersensitivity. On the other hand, It has been suggested in asthma and rhinitis that T regulatory cells (Tregs) CD4+ CD25+ FOXP3+ have been involved in control allergic status, favoring an optimal microenvironment with immunosuppressive cytokines (IL-10, TGF-b). Meanwhile the immune status of the ocular microenvironment has evolutionally adapted itself to prevent the induction of excess inflammation, thereby protecting its delicate structures from the damages of inflammation. However, it is not been established whether Treg cells can modulate allergic response. Based on the above background, the present study focused to observe the role of Treg cells in human allergic conjunctivitis. Materials and methods: Peripheral blood mononuclear cells (PBMC) were isolated from blood samples of healthy donors (HD) and ACpatients, and then PBMC were labeled with mAbs against CD4, CD25 and FOXP3. Also PBMC were co-cultured with their autologous monocytes. After 24 h, the culture medium was removed and optimal dose of antigen stimulation Dermatophagoides pteronyssinus (Der p) was added. After 7 days, all cells were labeled with mAbs against CD4, CD25 and FOXP3. The cells labeled were analyzed by flow cytometry. The supernatant culture was recovered to analyze by ELISA the cytokines IL-10 and TGF-b. Results: AC-patients showed 55-times more CD4+ CD25+ cells than HD. Most of CD4+ CD25+ cells were FOXP3), when we compared mean fluorescence intensity (MFI) of FOXP3 in CD4+ CD25+ cells, we observed a decreased expression in AC-patients than HD. We note significant increase in CD25+, CD4+ CD25+ after antigen stimulation. However, no changes were detected in frequency of CD4+ CD25+ FOXP3+ and FOXP3) cells after stimulation with Der p. interestingly, we observed a decreased MFI for FOXP3 in AC-patients after culture than HC in PBMC. In supernatant of PBMC cultures from AC patients, no differences were observed in TGF-b levels after Der p stimulation. In contrast, we found increased IL-10 concentration after antigenic stimulation. Conclusions: Despite we observed higher frequency of CD4+ CD25+ in AC-patients, these cells were FOXP3), more interesting, the few cells FOXP3+ showed a diminished MFI. The increase in IL-10 but not TGF-b, may be associated with Th2 dominant environment. These


Poster Sessions 441 data suggest that allergic conjunctivitis status could be related with a regulatory dysfunction, as has been suggested in asthma and rhinitis.

P0773 Chimeras of Bet v 1 and its homologue Api g 1 show highly varying degrees of lysosomal stability B. Gepp,* N. Lengger,* P. Briza, M. Hauser,à U. Smole,* F. Ferreira,à C. Radauer* & H. Breiteneder* *Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria, Department of Molecular Biology, University of Salzburg, Vienna, Austria, àChristian Doppler Laboratory for Allergy Diagnosis and Therapy, University of Salzburg, Vienna, Austria Purpose/Objective: We have previously shown that the major birch pollen allergen Bet v 1.0101 and its homologue in celery Api g 1.0101 differed in their ability to polarise the allergen-specific immune response. In order to identify surface regions responsible for this behaviour, we produced four chimeric proteins of Bet v 1.0101 and Api g 1.0101. In each of them, roughly one fourth of the surface area of Api g 1.0101 were replaced by the corresponding residues of Bet v 1.0101. Resistance to lysosomal degradation enhances immunogenicity. Therefore, our aim was to test the lysosomal stability of these chimeric proteins. Materials and methods: Chimeric proteins of Bet v 1.0101 and Api g 1.0101 were constructed. The surface residues forming the Ploop (Api-Bet-1), the region opposite of the P-loop (Api-Bet-2), the area surrounding the C-terminus (Api-Bet-3), or the C-terminal alpha helix (Api-Bet-4) of Bet v 1.0101 were grafted onto Api g 1.0101. The resulting proteins were expressed in Escherichia coli and purified by standard chromatographic methods. Secondary structures were checked by circular dichroism spectroscopy. For the lysosomal degradome assay, Bet v 1.0101, Api g 1.0101 and the chimeras were digested with mircosomal/endo-/lysosomal enzymes. Reactions were stopped by heat denaturation followed by SDS-PAGE analysis and mass spectrometry. Results: All chimeric proteins adopted secondary structures equivalent to Api g 1.0101. Results for the degradome assay showed that one of the four chimeric proteins (Api-Bet-2) had a remarkably higher lysosomal stability compared to Bet v 1.0101 and Api g 1.0101. A 50% degradation of Bet v 1.0101 and Api g 1.0101 was observed after 12 h while 80% of Api-Bet-2 remained stable even after 96 h. In contrast, almost total degradation was observed for Api-Bet-1, -3 and -4 after 12 h. Conclusions: Altering the protein structure when constructing allergen chimeras can result in unexpected increase or decrease of the overall protein stability. In our study, which was supported by grants P22559-B11 (CR) and SFB-F4608 (HB) from the Austrian Science Fund, we produced a chimeric protein whose stability was remarkably greater than the starting molecules Bet v 1.0101 and Api g 1.0101. This change may result in a shift of the immune response polarisation as compared to the wildtype allergens.

P0775 CMRF-35-like molecule 1 (CLM-1) regulates IL-4-dependent responses and is required for allergic eosinophilic airway inflammation I. Moshkovitz,* D. Karo-Atar,* M. Itan,* A. Hershko & A. Munitz* *Clinical Microbiology and Immunology, Tel-Aviv University, Tel-Aviv, Israel, Department of Medicine, Meir Medical Center, Laboratory of Allergy and Clinical Immunology, The Herbert Center of Mast Cell Disorders, Kfar Saba, Israel Purpose/Objective: Asthma is a chronic disease of the airways, which is currently on the rise. IL-4 is a hallmark and central cytokine

orchestrating multiple Th2 immune responses/diseases including asthma. Thus, defining pathways regulating IL-4-induced responses may have significant therapeutic potential. CMRF-like-molecule-1 (CLM-1) is an immunoreceptor tyrosine-based inhibitory motif-containing receptor, which is predominantly expressed by myeloid cells. Notably, the in vivo role of CLM-1 is unknown and whether it regulates IL-4-induced responses is unclear. Materials and methods: Wild type (WT) and Clm1-/- mice were challenged with Aspergillus fumigatus (Asp) extract or IL-4. CLM-1 expression was assessed in the lungs (flow cytometry). Broncho alveolar lavage fluid (BALF) and lung draining lymph nodes were assessed for total and differential cell counts. Th2 cytokine/chemokine content and IgE levels were assessed in BALF and serum, respectively. WT and Clm1-/- bone marrow (BM) derived macrophages (MF) and eosinophils (Eos) were stimulated with IL-4 and assessed for CCL17 and Relm-a secretion. Results: CLM-1 was differentially expressed by various lung myeloid cells. Following Asp-challenge, CLM-1 was specifically upregulated by eosinophils and alveolar macrophages. Asp-challenged Clm1-/- mice displayed decreased BAL cellular infiltration as well as decreased chemokine and IgE production. Surprisingly, Asp-challenged Clm1-/mice displayed elevated levels of IL-4 suggestive of inappropriate IL-4 consumption by IL-4-responsive cells. Consistently, IL-4-challenged Clm1-/- mice displayed decreased BAL cellular infiltration as well as decreased chemokine levels. Indeed, IL-4-stimulated Clm1-/- BM-MF and BM-Eos displayed significantly decreased Relm-a and CCL17 production in comparison with IL-4-activated WT cells. IL-4R levels were indifferent between WT and Clm1-/-- cells. Finally, human CLM-1 was upregulated in peripheral blood eosinophils and monocytes obtained from allergic rhinitis patients. Conclusions: These results demonstrate a key role for CLM-1 in the regulation of allergic airway inflammation likely by regulating IL-4 mediated effects. To the best of our knowledge, this is the first line of evidence suggesting a role for CLM-1 as an activation molecule in vivo.

P0776 Contribution of sensitization phase to intensity of contact hypersensitivity reaction in rats A. Popov,* I. Mirkov,* D. Miljkovic, S. Belij,* J. Dokic,* L. Zolotarevski,à D. Kataranovski* & M. Kataranovski* *Department of Ecology, Institute for Biological Research ‘Sinisa Stankovic’, Beograd, Serbia, Department of Immunology, Institute for Biological Research ‘Sinisa Stankovic’, Beograd, Serbia, àMilitary Medical Academy, Institute for Pathology, Beograd, Serbia Purpose/Objective: Allergic contact dermatitis is a common skin inflammatory disease in humans, which pathophysiology is studied mostly on animal model referred to as contact hypersensitivity (CHS). For development of this reaction priming of hapten-specific T cells is necessary (sensitization phase), with subsequent activation of haptenspecific effector T cells, mediators of skin inflammation during elicitation phase. Relationship between sensitizing dose and the intensity of CHS expression was shown in humans, however mechanisms underlying such dose-response are virtually unexplored. In this study, the impact of sensitization phase on the intensity of CHS expression was investigated in a rat model of CHS to dinitrochlorobenzene (DNCB) using low (0.4%) and high (4%) sensitization doses at a constant challenge dose (0.13% or 1.3% DNCB) for each of the sensitizing doses. Materials and methods: Ear thickness, cell infiltration and IFN-c content in the medium conditioned by organ-cultured ear skin explants were analyzed during elicitation phase. Basic indices of draining lymph node (DLN) activity (cellularity, proliferation), CD4+/ CD8+ composition, the production of interferon-c (IFN-c) and


442 Poster Sessions interleukin-17 (IL-17), main effector cytokines in CHS, were analyzed during sensitization and elicitation phases. Results: Sensitization with high DNCB dose resulted in significantly more intensive inflammatory ear skin response (each of the parameters measured) to challenge with either 0.13% or 1.3% DNCB dose, compared to the response of animals sensitized with low DNCB dose and challenge with respective dose. Higher DLN cellularity, proliferation, CD4+ and CD8+ cell number, and in vitro hapten stimulation of IFN-c and IL-17 production following sensitization with 4% versus 0.4% might have contributed to more pronounced CHS expression in these animals. DLN (auricular) of animals sensitized with 4% and elicited with 0.13% or 1.3% expressed higher proliferation as well as inflammatory cytokine production compared to 0.4%/0.13% or 0.4%/ 1.3% sensitization/elicitation regime, respectively. Conclusions: Presented data demonstrated contribution of the strength of sensitization to the intensity of CHS response suggesting that it depends greatly on generation of effector (IFN-c and IL-17) producing cells.

P0777 Cross-reactivity profiling allows classification of allergenic pectate lyases into two distinct families M. Wolf,* M. Hauser,* M. Wallner,* M. Himly,* C. Ebner, P. Briza,à A. Mari,§ H. Behrendt– & F. Ferreira* *Department of Molecular Biology Christian Doppler Laboratory for Allergy Diagnosis and Therapy, University of Salzburg, Salzburg, Austria, Allergieambulatorium am Reumannplatz, Vienna, Austria, àMolecular Biology, University of Salzburg, Salzburg, Austria, §IDI-IRCCS, Center for Molecular Allergology, Rome, Italy, –Technische Universita¨t and Helmholtz Center, ZAUM Center for Allergy and Environment, Munich, Germany Purpose/Objective: The botanically unrelated families of Asteraceae and Cupressaceae are widely distributed over the Northern hemisphere. Especially in the USA, Europe, and East Asia their pollen represents one of the clinically most relevant allergenic sources. However, the major disease eliciting allergens all belong to the same protein family of pectate lyases. To date, recombinant allergenic pectate lyases showing native folds are not available, thus most studies elucidating pectate lyase allergies were performed with allergen extracts. Materials and methods: In the present study, the five most important natural allergenic pectate lyases from Asteraceae as well as Cupressaceae pollen were characterized. Therefore, the allergens were purified to homogeneity from crude pollen extracts by standard chromatography techniques. Direct and inhibition ELISA were performed to determine patients’ serum IgE binding to the different allergens including sera from four distinct geographical areas (Northern America, the Mediterranean Area, Central Europe, and Asia, respectively). Furthermore, cross-reactivity of purified allergens was assessed in a mouse model. Results: Purified proteins were characterized physicochemically in terms of identity, structural integrity, and proteolytic stability. The optimized methods allowed purification of structurally intact pectate lyases without detectable auto-proteolysis, degradation, or truncation products. In ELISA experiments, the different cohorts included in the study reacted strongly with the sensitizing pectate lyase, endemic in the particular geographic area. For both, human as well as murine sera, a high degree of cross-reactivity was observed within the pectate lyase families of Asteraceae and Cupressaceae, respectively, whereas between the two families the level of cross-reactivity was limited.

Conclusions: These results allow the classification of allergenic pectate lyases according to their botanical origin in two distinct families, which will provide the basis for the selection of suitable candidate molecules for molecule based allergy diagnosis and therapy.

P0778 Curation of the IUIS allergen database based on sequence similarity and protein family classification C. Radauer,* A. Pome´s, A. Nandy,à F. Ferreira,§ P. Rozynek,– M. Raulf-Heimsoth,– R. E. Goodman** & H. Breiteneder* *Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria, Indoor Biotechnologies Inc., Research and Development, Charlottesville, VA, USA, àAllergopharma Joachim Ganzer KG, Research and Development, Reinbeck, Germany, §Christian Doppler Laboratory for Allergy Diagnosis and Therapy, University of Salzburg, Salzburg, Austria, –Ruhr-University Bochum, Institute of Prevention and Occupational Medicine of the German Social Accident Insurance, Bochum, Germany, **Lincoln, Food Allergy Research & Resource Program, University of Nebraska, Lincoln, NE, USA Purpose/Objective: The IUIS Allergen Database (www.allergen.org) is the official site of the unambiguous and systematic nomenclature of allergens. Allergen names consist of an abbreviation of the scientific name of their source, an allergen and an isoallergen number. We aimed to correct existing database entries based on new sequence information. Materials and methods: The database was manually searched for entries with missing sequence data, biochemical names similar to those of other allergens from the same source, or inconsistent allergen numbers. Allergen sequences were analysed by pairwise and multiple sequence alignments and phylogenetic trees derived from these alignments. Results: Four types of incorrect allergen designations were identified. (1) Highly similar allergens from the same source with different numbers. This applies to Amb a 1 and Amb a 2 from ragweed (60 70% sequence identity between Amb a 1 and Amb a 2 isoallergens), to Ara h 3 and Ara h 4 from peanut (91% sequence identity) and to the nine Chironomus thummi thummi allergens (v2 = 1 9) from the globin family, some with sequences identities >50%. (2) Different numbers for homologous allergens from different species from the same taxonomical family. Sec c 1 from rye and Tri a 28 from wheat are dimeric a-amylase inhibitors. Hor v 21 (c-hordein) from barley has 76% sequence identity to c-secalin from Secale strictum, a homologue of Sec c 20 from rye. (3) Duplicate entries. Hor v 1 and Hor v 15 from barley refer to the same monomeric a-amylase inhibitor BMAI-1. Equ c 4 and Equ c 5, horse dander latherins, were originally identified based on peptide sequences belonging to the same complete sequence. (4) Single entries referring to a group of different proteins. Caseins from cow’s milk are collectively termed Bos d 8. However, caseins comprise four different proteins (aS1, aS2, b, and j) with sequence identities between a and b-caseins below 20% and no homology to j-caseins. Conclusions: Based on these Results: , the IUIS Allergen Nomenclature Sub-Committee renamed Amb a 2.01 to Amb a 1.05, Ara h 4.01 to Ara h 3.02, v2 = 4 8 to isoallergens of v2 = 3, Sec c 1 to Sec c 28, and Hor v 21 to Hor v 20. The entries Hor v 1 and Equ c 5 were deleted. Bos d 8 was split into four entries: Bos d 8.0101 (aS1-casein), Bos d 9.0101 (aS2-casein), Bos d 10.0101 (b-casein), and Bos d 11.0101 (jcasein). This study was supported by grant P-22559-B11 from the Austrian Science Fund.


Poster Sessions 443 P0779 Decreased NK regulatory cells in children with atopic dermatitis E. Aktas Cetin,* N. Akdeniz,* S. Baris, M. Kosker,à F. Cosan,§ I. Barlan, Y. Camcioglu– & G. Deniz* *Department of Immunology, the Institute of Experimental Medicine (DETAE), Istanbul University, Istanbul, Turkey, Division of Pediatric Allergy and Immunology, Marmara University, Istanbul, Turkey, à Department of Pediatric Infections Disease, Faculty of Medicine, Istanbul University Cerrahpasa, Istanbul, Turkey, §Division of Rheumatology Department of Internal Medicine, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey, –Department of Pediatric Infections Disease, Faculty of Medicine, Istanbul University Cerrahpasa, Istanbul, Turkey Purpose/Objective: Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease characterized by distributed eczematous skin lesions. Numerous studies demonstrated increased frequency of allergen-specific Th2 cells producing increased interleukin (IL)-4, IL-5 and IL-13 in the peripheral blood of AD patients. However little is known about the role of natural killer (NK) cells. NK cells are one component of the innate immune system and have the ability to both lyse target cells and it has been showed that human NK cells are able to polarized functionally different subsets. Recent studies showed that NK cells also display potent regulatory function. In this study, NK1 and NK regulatory cytokine profiles, the expression of activatory receptors as well as the cytotoxic activity of NK cells in AD were investigated. Materials and methods: The study group consists of children with AD (n = 8, mean age = 9.2 ± 3) and healthy subjects (n = 6, mean age = 8.6 ± 4). The patients were multisensitized to at least three aeroallergens and had high serum total IgE levels. Peripheral blood mononuclear cells (PBMC) were used as effector cells and K562 cell line was used as target cell with an effector: target (10:1) ratio. Cytotoxic activity (by using CFSE-labeled K562 as target cells), expression of CD16brightCD56dim and CD16dimCD56bright NK cell subsets, NK cell activatory receptors and intracellular IL-10 & IFN-c levels were also determined by flow cytometry. Statistical analyses were performed by Mann Whitney U-test. Results: In AD patients CD16brightCD56dim, CD16dimCD56bright NK cell subsets, percentages of CD3) CD16+ CD56+ cells and NK cell cytotoxic capacity were significantly decreasedcompared tohealthy subjects (P = 0.002, P = 0.037 and P = 0.007, respectively). In addition, C type lectin activatory receptor NKG2D/CD94 was found diminished in AD patients in comparison to healthy subjects (P = 0.05 and P = 0.012, respectively). IL-10 secreting regulatory NK cell ratio dramatically decreased in AD patients, whereas IFN-csecreting of NK1 cells was found to be similar in both groups. Conclusions: Our results suggested that decreased expression of CD16brightCD56dim, CD16dimCD56bright subsets, activatory receptor NKG2D/CD94, cytotoxic activity and IL-10 secretion of NK cells might play a role in the pathogenesis of atopic dermatitis.

P0780 Design of a hypoallergenic Alt a 1 trimer for specific immunotherapy S. Gutfreund,* T. Twaroch,* S. Spitzauer & R. Valenta* *Department of Pathophysiology and Allergy Research, Christian Doppler Laboratory for Allergy Research, Center for Pathophysiology Infektiologie and Immunology, Vienna, Austria, Department of Laboratory Medicine, Institute of Medical and Chemical Laboratory Diagnostics, Vienna, Austria

majority of Alternaria-specific IgE and exhibits high allergenic activity in sensitized patients. The objective of this study was the design, construction and characterization of a hypoallergenic Alt a 1 for specific immunotherapy. Materials and methods: In order to reduce IgE reactivity and allergenic activity of Alt a 1 the cystein residues stabilizing the proteins fold were mutated to serines. To increase the molecules immunogenicity and ability to induce allergen-specific protective IgG antibodies upon immunization, a recombinant trimeric form of the mutant was expressed in Escherichia coli. Results: The monomeric Alt a 1 serine mutant had completely lost IgE reactivity and a recombinant Alt a 1 trimeric form was expressed in E. coli and purified.. Conclusions: The recombinant hypoallergens may be used for specific immunotherapy of allergy to Alternaria.

P0781 Design of hypoallergenic derivatives of the major cat allergen Fel d 1 by rational reassembly as a mosaic protein M. Curin,* I. Swoboda,* M. Focke,* K. Blatt, P. Valent, H. Gro¨nlund,à S. Spitzauer§ & R. Valenta* *Department of Pathophysiology and Allergy, Christian Doppler Laboratory for Allergy Research, Research Medical University of Vienna, Vienna, Austria, Division of Hematology and Hemostaseology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria, àDepartment of Medicine, Clinical Immunology and Allergy Unit, Karolinska Institute and University Hospital, Stockholm, Sweden, § Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria Purpose/Objective: Due to the perennial exposure to cats and due to the high association with severe asthma manifestations the major cat allergen Fel d 1 is regarded as one of the most important respiratory allergens. The objective of this study was the rational design of recombinant Fel d 1 derivatives with reduced IgE reactivity and preserved T cell epitopes that are suitable for vaccination and tolerance induction. Materials and methods: Seven recombinant mosaic proteins were generated by reassembly of non-IgE-reactive peptides of Fel d 1 which contained the sequence elements for induction of allergen specific blocking IgG antibodies and those containing important T cell epitopes. Recombinant mosaic proteins were expressed in E. coli using codonoptimized synthetic genes. They were compared with recombinant Fel d 1 regarding their structural fold by circular dichroism, IgE-binding capacity, ability to activate allergic patients‘ basophils and ability to induce allergen-specific blocking IgG antibodies upon immunization. Results: Although each of the mosaic proteins had lost the alpha helical fold typical for Fel d 1, a strong reduction in IgE reactivity as well as allergenic activity in basophil activation assays was only obtained for three constructs, two reassembled fragments (Fel d 1 MB, Fel d 1 MC) and a fusion of the latter two in which the cysteines of Fel d 1 MC were replaced by serines. Immunisation of rabbits with the hypoallergens induced high levels of IgG antibodies that inhibited IgE reactivity of cat allergic patients to Fel d 1 in a comparable manner as IgG antibodies induced with the wildtype allergen. Conclusions: In conclusion, we report the development of hypoallergenic reassembled Fel d 1 proteins suitable for vaccination and tolerance induction in cat allergic patients.

Purpose/Objective: Alternaria alternata is one of the major elicitors of fungal allergy and plays a major role in the development of severe respiratory forms of allergy. Alt a 1, a protein of approximately 13 kDa has been identified as the major allergen in Alternaria. Alt a 1 binds the


444 Poster Sessions P0782 Development and characterization of recombinant olive pollen allergens, rOle e 5, 6 and 8 for diagnosis and therapy of olive pollen allergy T. Garmatiuk,* T. Twaroch,* S. Quirce, M. Curin,* H. Hochwallner,* S. Spitzauerà & R. Valenta* *Department for Pathophysiology and Allergy research, Christian Doppler Laboratory for Allergy research, Center for Pathophysiology Infektiologie and Immunology, Vienna, Austria, Department for Allergy, Hospital La Paz Health Research Institute, Madrid, Spain, àDepartment of Laboratory Medicine, Clinical Institute for Laboratory Medicine, Medical University of Vienna, Vienna, Austria Purpose/Objective: Olive pollen is one of the most important causes of allergy in Mediterranean countries and certain parts of America. Ole e 1 an approximately 20 kDa protein is the major allergen in olive pollen which is recognized by more than 90% of olive pollen allergic patients. The aim of our work was to investigate the prevalence of IgE recognition of three olive pollen, Ole e 5 a superoxide dismutase, Ole e 6, a 10 kDa protein with yet unknown biological function and Ole e 8, a calcium-binding protein. Materials and methods: Thirty sera from clinically well characterized patients with olive pollen allergy from Spain were tested for IgE reactivity to olive pollen extract and purified Ole e 1 by immunoCAP measurements. Recombinant olive pollen allergens, rOle e 5, rOle e 6 and rOle e 8 were expressed as C-terminally hexahistidine-tagged proteins in Escherichia coli and purified using Ni-NTA agarose. The structure of rOle e 5, rOle e 6 and rOle e 8 were studied by UV-circular dichroism. IgE reactivity with sera from olive pollen allergic patients was tested by ELISA. Results: Circular dichroism showed that the recombinant allergens were folded proteins with a predominant beta sheet structure in the case of rOle e 5 and rOle e 8 and alpha helical structure in the case of Ole e 6. Despite symptoms of olive pollen allergy, only twenty six of the patients were positive when tested for IgE reactivity with total olive pollen extract. Twenty three showed IgE reactivity to the major olive pollen allergen Olee 1 (78%), 57% of exhibited IgE reactivity with rOle e 5.20% and 27% of patients reacted with Ole e 6 and Ole e 8 respectively. Interestingly, all four patients negative for olive pollen extract could be detected with a combination ofrOle e 5, rOle e 6 and rOle e 8. Conclusions: We have produced three recombinant olive pollen allergens rOle e 5, rOle e 6 and rOle e 8 which are useful for component-based testing of olive pollen allergy and eventually for allergen-specific immunotherapy of olive pollen allergy.

P0783 Development of A Rhinovirus-Induced House Dust Mite Asthma Exacerbation Mouse Model L. Tao,* F. Goh,* D. NG,* X. Zhang,* V. Chow, N. Bartlett,à S. Johnstonà & F. Wong* *Pharmacology, National University of Singapore, Singapore, Singapore, Microbiology, National University of Singapore, Singapore, Singapore, à Respiratory Medicine & Allergy, Imperial College London, London, UK Purpose/Objective: Rhinovirus (RV) causes the majority of virus-induced asthma exacerbations. The purpose of the present study was to establish an asthma exacerbation mouse model by combing a mouse model of allergic asthma using a clinically relevant allergen house dust mite (HDM) with intra-tracheal inoculation of RV. Materials and methods: Mice were sensitized and challenged with HDM or saline, and infected with RV-1B, a minor group RV which binds to mouse airway epithelial cells or UV-inactivated RV. Bronchoalveolar lavage fluid and lung homogenates were examined for inflammatory cells infiltration, and Th2 cytokine protein and mRNA

expressions. Airway hyperresponsiveness was measured using direct airway resistance analysis. Results: Compared to HDM/UV-inactivated RV-treated mice, HDM/ RV-treated mice developed significant increases in airway infiltration of neutrophils, eosinophils and lymphocytes. The HDM/RV-treated group also produced higher protein levels of IL-4, IL-5, IL-13. Besides, the HDM/RV-treated group generated higher mRNA levels of eotaxin1 and MUC5AC. However, although we found significant difference in airway hyperresponsiveness to methacholine challenge between saline/ RV-treated group and saline/UV-inactivated RV-treated group, the difference between HDM/UV-inactivated RV-treated group and HDM/RV-treated group did not reach significant level. Conclusions: Taken together, we have developed a model of RV-1Binduced exacerbation of the Th2 responses in HDM-mediated allergic asthma. Additional works are required to illustrate enhanced airway hyperresponsivness in HDM/RV-treated mice. This new asthma exacerbation model will be useful to study asthma immunology and for novel drug discovery and development.

P0784 Different TLR agonists exert diverse effects on immune cells from allergic individuals S. Deifl,* C. Kitzmu¨ller,* P. Zlabinger, P. Steinberger & B. Bohle* *Department of Pathophysiology and Allergy Research, Christian Doppler Laboratory for Immunomodulation, Center for Pathophysiology Infectiology and Immunology, Vienna, Austria, Center for Pathophysiology Infectiology and Immunology, Institute of Immunology, Vienna, Austria Purpose/Objective: Toll-like receptor (TLR) ligands have been considered as promising adjuvants in vaccines for allergen-specific immunotherapy. We compared different TLR-ligands, namely Pam3CSK4 and FSL-1, targeting TLR2, lipopolysaccharide (LPS) and monophosphoryl lipid (MPL) A, targeting TLR 4 and flagellin, targeting TLR5, regarding their immunostimulatory effects on cells from allergic donors. Materials and methods: Peripheral blood mononuclear cells (PBMC) were stimulated with TLR-ligands in the presence or absence of autologous plasma and the levels of pro- and anti-inflammatory cytokines were assessed in supernatants. Monocyte-derived dendritic cells (mdDC) were tested for upregulation of CD40, CD80, CD83, CD86, CD58, CCR7 and PD-L1 in response to TLR ligands. Their functional activity was tested in mixed leukocyte reactions (MLR). Moreover, their expression of different members of the IL-12 family was assessed by real-time PCR. CD4+ CD45RA+ T cells were incubated with autologous TLR-activated mdDC to study their polarizing potential to induce Th1- or Th2-like responses. Finally, the influence of TLR ligands on allergen-uptake by monocytes was analysed using fluorescently-labelled Bet v 1, the major birch pollen allergen. Results: All TLR ligands induced TNF-a and IL-10 synthesis in PBMC from allergic patients whereas only TLR4 ligands induced IFN-c production. The presence of autologous plasma elevated all cytokine levels. All TLR ligands induced the functional maturation of mdDC. However, different expression patterns of surface molecules on mdDC in response to different TLR ligands were found. LPS-matured mdDC primed Th1-like cells. In contrast, MPL-A, Pam3CSK4 and FSL-1 promoted Th0-like responses, whereas flagellin-matured mdDC induced the differentiation of Th2-like cells. All TLR ligands except flagellin enhanced the uptake of allergen by APC. Conclusions: Activation of TLR4, TLR5 and the heterodimers TLR1/2 and TLR2/6 has different effects on functional maturation and allergen-uptake of antigen-presenting cells from allergic patients, thereby resulting in different T cell-polarizing capacities. Our data indicate that not all TLR-ligands are equally well suited as adjuvants in allergy vaccines.


Poster Sessions 445 P0785 Directed in vitro evolution as a tool for the identification of conformational IgE epitopes of the major birch pollen allergen Bet v 1 E. Guhsl,* G. Hofstetter,* C. Ebner, W. Hemmer,à H. Breiteneder* & C. Radauer* *Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria, Ambulatorium fu¨r Allergie und Immunologie Reumannplatz, Vienna, Austria, àFloridsdorfer Allergiezentrum, Vienna, Austria Purpose/Objective: Only little is known about the IgE-binding epitopes of the major birch pollen allergen Bet v 1, which have been shown to depend on the native structure of the allergen. In order to identify conformational epitopes, we performed directed in vitro evolution of non-IgE-binding structural homologues to proteins carrying single Bet v 1 epitopes. To this end, we chose (S)-norcoclaurine synthase (NCS) from Thalictrum flavum (meadow rue), a structural homologue of Bet v 1 with only 25% sequence identity as a template subjected to a combination of random mutagenesis and phage display. Materials and methods: NCS was expressed in Escherichia coli and purified by chromatographic methods. Secondary structure was verified by circular dichroism spectroscopy. IgE binding to birch pollen allergic patients’ sera was tested by IgE ELISA. Random mutagenesis of NCS was performed by PCR using mutagenic nucleotide analogues. Phage display libraries in the filamentous phage M13 were created by inserting PCR products into the phagemid pTP127. Bet v 1-specific antibodies were purified from a pool of Bet v 1-sensitised patients’ sera by affinity chromatography using immobilised Bet v 1. Several panning rounds were performed to select phages with binding activity to Bet v 1-specific IgE. IgE binding activities of enriched phages and of bacterial colonies representing single clones were analysed by transfer to nitrocellulose and immunostaining. Results: Purified recombinant NCS revealed a secondary structure with high similarity to that of Bet v 1, but no IgE binding in an ELISA using individual sera from 35 Bet v 1-sensitised birch pollen allergic patients. Phage libraries with diversities of 105 106 different clones were created. Sequencing of 27 randomly picked clones from the unselected libraries showed average mutation rates of 16/161 amino acids (range 8 24). IgE immunoblotting of phages enriched by two rounds of biopanning showed an increase of the amount of bound IgE, whereas the unselected library lacked IgE binding ability. Screening of single clones after the first panning round yielded 4 of 20 clones which expressed IgE binding mutant proteins. Conclusions: In vitro evolution of NCS by random modifications of surface residues is a suitable tool for defining IgE binding residues on Bet v 1. This study was supported by grant P-22559-B11 from the Austrian Science Fund.

P0786 Dry-roasting enhances peanut immunogenicity and promotes Th2biased responses in a murine model of peanut allergy W. R. Hillson, A. E. Moghaddam & Q. J. Sattentau Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Purpose/Objective: Peanut allergy is a significant public health concern in many Western developed countries, but its aetiology remains unclear. Thermal processing (including roasting) has been implicated epidemiologically and serologically in peanut allergenicity, but no direct in vivo evidence for this putative link has so far been identified. Oxidation has been linked to altered immunogenicity, and our group has previously demonstrated Th2 immunomodulatory activity of oxidation-derived DAMPs such as reactive carbonyl species (RCS).

Here we have studied the biochemical properties of DR peanut allergens and have determined their immunogenic and allergenic profiles in a murine model of food allergy. Materials and methods: Total peanut protein extracts and purified major allergens Ara h 1 and 2/6 were prepared from commercial or inhouse dry-roasted (DR) peanuts. To compare the allergenicity of DR with raw peanut allergens, BALB/c mice were s.c. primed with noncrosslinked fractions and subsequently challenged via the intra-gastric route with total peanut extract. Balb/c mice were also directly sensitized and challenged intra-gastrically with total peanut extract. Results: In line with previous data, DR peanut proteins showed significant increases in RCS content, a hallmark of oxidative stress, and a high level of protein aggregation. Mice immunized s.c. with DR antigens demonstrated a stronger adaptive immune response than those immunized with raw, as revealed by allergen specific Th2-biased T and B cell responses, including a greater induction of functional IgE production. Only mice systemically sensitized by DR-derived allergens responded robustly with Th2 cytokines such as IL-4 and IL-5 in their mesenteric lymph nodes upon gastric challenge. The enhanced allergenic profile of DR peanuts was also confirmed in a direct GI sensitization and challenge model, resulting in enhanced Th2 and IgE responses. Conclusions: Together these data provide the first direct link between roasting and enhanced peanut allergenicity in a murine model, lending support to epidemiological and serological findings in humans. The enhanced overall immunogenicity of DR peanut cannot be explained by crosslinking alone since this effect was observed even when such species were depleted. Given our preliminary data, the modulated immunogenic profile may be explained in part by the presence oxidation products (including RCS) in DR peanut.

P0787 Ectopic activation of the JAK/STAT pathway leads to loss of barrier function in Drosophila melanogaster airways K. Kallsen,* K. Uliczka,* H. Heine & T. Roeder* *Department of Zoophysiology, Institute of Zoology, Kiel, Germany, Division of Innate Immunity, Research Center Borstel, Borstel, Germany Purpose/Objective: Asthma bronchiale is a chronic inflammatory disease of the lungs that is becoming a major health issue in industrialized countries. Asthma pathogenesis is thought to depend on the interaction of genetic and environmental factors but the underlying molecular mechanisms are not well understood. Asthma susceptibility genes like STAT6 are most likely involved in the pathogenesis. For this gene, many functions have been described in asthma related adaptive immune responses. However, the major STAT6 expressing cell type is the bronchial epithelial cell. So far, asthma research has mainly focused on the adaptive immune response. Recently, the epithelium has gained more and more attention. In asthmatics, not only is the epithelium abnormal and has a disturbed barrier function, but also STAT6 expression is enhanced. Therefore it is necessary to analyze the effects of the activation of JAK/STAT signaling on the airway epithelium. Materials and methods: To address this question we used Drosophila melanogaster as a model organism. Drosophila has unique advantages for this purpose like the absence of an adaptive immune response and the structure of its airways which consist of epithelial cells only. Furthermore, Drosophila has a simplified JAK/STAT signaling system, which is composed of only one representative protein at each position of the cascade. Results: Here we show that the JAK/STAT pathway can be activated in the airways by hypoxia, a condition important in asthma pathogenesis. Larvae with constitutive JAK/STAT activation do not survive their developmental L2 stadium. A conditional activation after the L2 stadium induces phenotypical changes in the tracheae: liquid filled


446 Poster Sessions branches und melanization, an immune reaction of Drosophila. Microarray analyses of these tracheae suggest an involvement of WNT and TGF-b pathways in the induction of this phenotype. Conclusions: Our data indicate a crucial role of JAK/STAT signaling in the barrier function of the Drosophila airway epithelium. This function is likely to be conserved. Therefore, it is possible that the asthma susceptibility gene STAT6 is important to maintain the barrier function of the human airway epithelium (supported by DFG, SFB/ TR22, project A07).

P0788 Effect of boiling and autoclaving on allergenicity of Anisakis simplex N. Carballeda-Sangiao,* F. Olivares, A. I. Rodriguez-Mahillo,* I. Moneo,* M. Tejada, A. Mendizabalà & M. Gonzalez-Munõz* *Immunology, Hospital Carlos III, Madrid, Spain, Centro Superior de Investigaciones Cientificas, Instituto de Ciencia y Tecnologia de los Alimentos y Nutricion, Madrid, Spain, àMercamadrid, Instituto de Salud Publica, Madrid, Spain Purpose/Objective: Anisakis simplex is a fish parasite with a worldwide distribution. Humans are accidental hosts. The ingestion of parasitized fish with live larvae L3 can result in the development of anisakiasis and sometimes can induce IgE-mediated reactions. Some authors claim that infection is the only mechanism for allergic disease. However, others consider that allergic episodes can be elicited by infection or exposure to allergen remaining in food with dead larvae. It is believed that is due to heat stable and pepsin resistant allergens for example Ani s 4. The aim of this work was to detect the presence of biologically active heat resistant A. simplex allergens after high temperature and pressure treatment (autoclaving) of parasitized fish muscle. Materials and methods: Fish fillets from hake (Merluccius merluccius) and tuna (Thunnus thynnus) were artificially parasitized with A. simplex and subjected to boiling (10 min) followed by autoclaving during 40 min at 121C. Fish muscle extracts (FME) were obtained by grinding followed by sonication and centrifugation. A. simplex larvae were treated in the same conditions to obtain an L3 extract (ASE). A. simplex heat resistant antigens and allergens were detected and quantified in FME and ASE by immunoblot using specific rabbit antisera and/or sensitised patients’ sera pool. Autoclaving buffer of the larvae (AB) was also analysed. The FME was tested in a flow cytometry basophil activation test on A. simplex sensitized patients. A non parasitized fish muscle extract was used as a negative control. Results: We could detect several antigens between 6 and 16 kDa and above 36 kDa in the AB but not in the ASE. When revealing with patients’ sera pool we detected several allergens between 6 and 16 kDa. In the FME we mainly detected an allergen around 20 kDa and an antigen around 16 kDa. This extract activated the basophils of sensitized patients in a dose-dependent way. Conclusions: The presence of heat resistant antigens and allergens from A. simplex in artificially parasitized autoclaved fish muscle and autoclaving buffer was demonstrated.

P0790 Efficacious peptide-based immunotherapy in a model of allergic airways inflammation occurs independently of IL-10 K. J. Mackenzie, P. M. Fitch, D. Nowakowska, M. D. Leech, A. J. McFarlane, A. Ilchmann, S. E. M. Howie, J. Schwarze & S. M. Anderton MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK Purpose/Objective: Immunotherapy using short allergen-derived peptides holds promise for the treatment of allergic asthma, and also

offers potential as a preventative approach. However, greater understanding of the immunological mechanisms involved in peptide-based immunotherapy (PIT) is required to maximise translational opportunities. Materials and methods: We have developed a murine model of allergic airway inflammation driven by adoptively transferred Th2 polarised (hence antigen-experienced) ovalbumin (OVA)-reactive CD4+ T cells (OT-II cells). Robust features of allergic airway inflammation are induced in this model following OVA airway challenge, and the Th2 cells are congenically marked, enabling tracking. PIT was applied to this model using the immunodominant OVA T cell epitope pOVA 323 339 given intravenously prior to allergen challenge, and the effects of PIT on disease parameters and immunological readouts were then assessed. Results: PIT significantly reduced the severity of AAI in this model. Four days after PIT, immediately prior to OVA challenge, PIT treated mice had substantially increased numbers of OT-II cells in lymphoid organs, compared to controls. This is in marked contrast to the early deletional effects of PIT we have previously seen using antigen-naı¨ve OT-II cells. Th2 OT-II cells from PIT treated mice were also unable to induce disease upon direct transfer into the lungs and subsequent OVA airway challenge. Importantly, although Th2 OT-II cells from PIT treated, but not control, mice produced substantial quantities of IL-10 in response to OVA challenge in vitro, PIT remained efficacious even in conditions of IL-10R blockade. Furthermore, the therapeutic effects of PIT were evident even in the context of increased severity of allergic airways inflammation induced by IL-10R blockade. Conclusions: PIT is effective at combating the pathogenic effects of antigen-experienced Th2 cells in a model of allergic airway inflammation. Although PIT induces substantial IL-10 production from Th2 OT-II cells in this model, the therapeutic effects of PIT were not dependent on IL-10.

P0793 Epicutaneous sensitization induces a Der p 2-specific, TH2-biased antibody response independent of TLR4 expression C. Stremnitzer,* K. Szalai, A. Willensdorfer, P. Starkl,* S. Schrom,* U. Reichart,à S. Knapp§ & E. Jensen-Jarolim– *Department of Pathophysiology and Allergy Research, Comparative Immunology and Oncology, Medical University of Vienna Vienna, Austria, Comparative Medicine Messerli Research Institute, University of Veterinary Medicine, Vienna, Austria, àInstitute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria, §Division of Infectious Diseases and Tropical Medicine, Department of Medicine I, Center for Molecular Medicine of the Austrian, Academy of Sciences, University of Veterinary Medicine Vienna, Medical University Vienna, University Vienna, Vienna, Austria, –Comparative Medicine Messerli Research Institute, University of Veterinary Medicine Vienna, Medical University Vienna, University Vienna, Vienna, Austria Purpose/Objective: The major house dust mite allergen Der p 2 is a structural and functional homologue of MD2 (TLR4-CD14-MD2 complex). An asthma model in TLR4-deficient mice has suggested that the allergic immune response against Der p 2 is dependent on TLR4 signaling. We investigated whether similar mechanisms are important for mediating Der p 2-induced allergy of the skin. Materials and methods: C57BL/6 WT and TLR4-deficient mice were epicutaneously sensitized 4 times in 3 week intervals with recombinant Der p 2 in combination with or without LPS. Immune response was monitored for specific antibodies by ELISA. Skin sections were analyzed for histological changes and immune cell infiltration. Results: Co-application of low and high LPS doses with Der p 2 induced similar levels of allergen-specific IgG1 and IgE antibodies in C57BL/6 WT and TLR4-deficient mice. In skin sections, increased


Poster Sessions 447 infiltration of mast cells could be determined in both mouse strains, already 6 h after the final challenge. Moreover, we observed high allergenic potential of Der p 2 alone, based on high antibody titers and immune cell infiltration. Conclusions: Our results suggest that epicutaneous sensitization to Der p 2 does not depend only on activation of TLR-4 signalling but also on other, yet undefined mechanisms.

P0794 Epigenetic effects in mice following ozone exposure A. Jang, D. Bae & C. Park Internal Medicine, Soonchunhyang University, Bucheon, Korea Purpose/Objective: Ozone exposure can be inducing and inciting factor for asthma. Epigenetics is the study of inherited changes in phenotype or gene expression caused by mechanisms other than changes in the underlying DNA sequence. The epigenetic effect to ozone exposure needs to be determined. The aim of this study was to investigate the effects of ozone exposure on epigenetic change, airway hyperresponsiveness, and airway inflammation in a murine model. Materials and methods: BALB/c mice were exposed to 2 ppm ozone for 3 h, 3 days, 7 days, 14 days, and 21 days. Enhanced pause (Penh) to methacholine was measured. Cell differentials in bronchoalveolar lavage (BAL) fluid were done. Epigenetic enzyme such as DNMT1, DNMT3A, MECP2, HDAC3, and MBD1 4 were quantified in the lung tissue homogenate using real time PCR. Results: The ozone exposure group for for 3 h, 3 days, 7 days, 14 days, and 21 days. demonstrated increased Penh at methacholine concentration of 12.5, 25, 50 mg/ml, with shift in the dose-response curve to the left, compared with that of filtered air group. Neutrophilsand lymphocytes in BAL fluid were increased in ozone exposed group compared with those in filtered air group. After ozone 2ppm exposure DNMT1, MECP2 increased in 21 days and DNMT3a and HDAC3 increased in3 h, 3 days, and 21 days, and MBD1 4 variably increased by ozone exposure time. Conclusions: These findings demonstrate that ozone exposure can change enzymes related to epigenetics.

P0795 Evidence for T cell-independent boost of allergen-specific secondary IgE responses by B cell epitopes M. Narayanan,* M. Focke-Tejkl, R. Valentaà & B. Linhart§ *Department of pathophysiology and Allergy Research, Center for Pathophysiology Infectiology and Immunology, Medical University of Vienna, Vienna, Austria, Christian Doppler Laboratory for Allergy Research, Medical University of Vienna, Vienna, Austria, àDepartment of pathophysiology and Allergy Research, Doppler Laboratory for Allergy Research, Center for Pathophysiology Infectiology and Immunology Christian, Medical University of Vienna, Vienna, Austria, §Department of pathophysiology and Allergy Research, Center for Pathophysiology Infectiology and Immunology, Vienna, Austria

Materials and methods: Based on the hapten-carrier model developed by Benacerraf, a 31 amino acid peptide derived from the major grass pollen allergen Phl p 1, which was devoid of T cell epitopes recognized by BALB/c mice, was coupled to unrelated carrier molecules. Immunization of BALB/c mice with the peptide/carrier allowed the induction of peptides-specific IgE antibody responses. Sensitized mice were then boosted with peptide constructs devoid of carrier-specific T cell epitopes and for control purposes with the original immunogens, the original carrier molecules without peptide or PBS. Peptide-specific IgE responses were determined by ELISA and by using rat basophil degranulation assays. Results: Interestingly, we found that peptide-constructs devoid of the original carrier molecules and thus devoid of T cell epitopes could induce increases of secondary peptide-specific IgE responses. Application of the T cell epitope-containing carriers alone had no influence on ongoing IgE responses. Conclusions: Our results suggest that B cell targeting approaches may be needed for the treatment of established allergy.

P0797 Extracellular heat shock protein 70 inhibits impairment of airway neutrophils responses in induced allergic airway inflammation E. Servuli, N. Troyanova, E. Bolkhovitina & A. Sapozhnikov Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Immunology, Moscow, Russia Purpose/Objective: Immune responses of airway neutrophils are thought to be impaired in asthma. Stress-associated chaperon heat shock protein 70 (Hsp70) is known to be potent to alter neutrophil activation in response to bacterial infection. Here we analyze the effect of extracellular Hsp70 application on neutrophil infiltration and activity upon induced allergic airway inflammation (IAAI). Materials and methods: IAAI in BALB/c mice was induced by intraperitoneal ovalbumin (OVA)/Alum injection and subsequent OVA aerosol challenges. A group of mice received intra-pharyngeal injection of murine Hsp70 at 24 h after the last allergen challenge. Total and differential number of bronchoalveolar lavage (BAL) cells and percentage of neutrophils in bone marrow was analyzed at 48 h after the last allergen challenge. Potential of bone marrow neutrophils from Hsp70 treated mice to generate reactive oxygen species (ROS) in response to phorbol-meristil acetate was investigated. Results: Hsp70 application decreased total BAL cell number and eosinophil percentage, however maintained neutrophil infiltration at 48 h after the last allergen challenge. The percentage of neutrophils in bone marrow of mice that received Hsp70 was comparable to that exhibited allergic inflammation. Simultaneously treatment with Hsp70 restored the ability of bone marrow neutrophils to generate ROS, which was detected to be suppressed in mice with IAAI. Conclusions: Thus protective mechanism of extracellular Hsp70 in IAAI could be related to inhibition of airway neutrophil responses impairment.

Purpose/Objective: IgE-mediated allergy, a hypersensitivity disease affecting more than 25% of population, is characterized by the induction of an allergen-specific Th2 response and the production of allergen-specific IgE antibodies. Clinical studies showed that systemic allergen-specific production in allergic patients is strongly boosted by respiratory allergen contact and that patients become more sensitive to allergen contact. Despite the key role of IgE antibodies for the pathology of allergic disease, the mechanisms underlying allergenspecific secondary antibody responses are still not completely understood. The aim of this study was to investigate what allergen epitopes (B cell, T cell epitopes) are needed for the boosting of allergen-specific secondary IgE responses.


448 Poster Sessions P0798 Functional characterization of allergen-specific T cell responses in a humanised mouse model using a human mugwort-specific T-cell receptor and HLA-DR1

P0799 Granulocyte/macrophage colony-stimulating factor-dependent CD11b lung dendritic cells are required for induction of T helper 2 immunity to inhaled dust mite allergen

A. Neunkirchner,* L. F. Mager, V. M. Reichl,* K. G. Schmetterer,* D. Wojta-Stremayr,* E. Rosloniec,à R. Naumann,§ B. Jahn-Schmid,– B. Bohle** & W. Pickl* *Christian Doppler Laboratory for Immunomodulation, Institute of Immunology, Medical University of Vienna, Vienna, Austria, Institute of Immunology, Medical University of Vienna, Vienna, Austria, àMedicineRheumatology and Pathology, Veterans Affairs Medical Center, Memphis, TN, USA, §Transgenic Core Facility, Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany, –Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria, **Department of Pathophysiology and Allergy Research, Christian Doppler Laboratory for Immunomodulation, Medical University of Vienna, Vienna, Austria

Q. Zhou,* A. W. Ho, Y. Tang,à F. Ginhoux,§ B. Y. Chua,à H. S. Wongà & D. M. Kemenyà *Microbiology, National university of Singapore, Singapore, Singapore, Immunos, Singapore Immunology network, Singapore, Singapore, à Microbiology, National University of Singapore, Singapore, Singapore, § Immunos, Singapore Immunology Network, Singapore, Singapore

Purpose/Objective: Chicken Ovalbumin (Ova) is a frequently used model allergen in allergy and asthma research and most in vivo experiments are performed in T cell receptor (TCR) transgenic (tg) mice expressing a murine TCR specific for Ova in the context of a murine restriction element (I-Ad). We here aimed to generate double tg mice expressing a human TCR specific for the immuno-dominant epitope of the major mugwort (Artemisia vulgaris) pollen allergen Art v 1 in the context of the human restriction element HLA-DR1. Materials and methods: To obtain high expression levels the allergenspecific human TCR variable sequences were chimerized with murine TCR constant sequences. Resulting transgenes were cloned into the pTcass vector system and thus put under the transcriptional control of the natural TCR alpha and beta promotor/enhancer elements. Allergen-specific TCR tg founder mice were cross-bread with HLADR1+ B10.M-DR1dlAb1-Ea mice. Results: Immunophenotyping of double tg TCR/HLA-DR1 mice revealed clear-cut expression of the Art v 1-specific TRBV18 chain on peripheral blood CD3+ T cells and HLA-DR1 expression on CD14+ monocytes and B220+ B cells. In vitro, splenocytes from TCR/HLADR1 double tg mice but not of HLA-DR1 single tg mice or wt mice specifically proliferated upon incubation with the human-relevant immuno-dominant Art v 125 36 peptide or whole Art v 1 protein. No proliferation was observed upon incubation with control peptides or proteins. Allergen-specific cellular proliferation is accompanied by the production of a balanced cytokine milieu including IFN-gamma, IL-2, IL-4, IL-6, IL-13 and IL-17. The effect on T-cell phenotype, specific antibody production and lung function after in vivo challenge with specific allergen via the airways will be described and discussed. Conclusions: A humanised allergy model, in which all components of the allergen-specific synapse are well-defined enables to analyze the relevant T-cell dependent pathways by which allergic diseases can be influenced in vivo and will provide important insights into the pathophysiology of allergic diseases and their possible cure in the future. The research was funded by the Christian Doppler Society, the Austrian Science Fund (FWF): SFB 4609-B19 and SFB F1816-B13 and the Austrian Research Promotion Agency Bridge grant: 812079 & Biomay AG.

Purpose/Objective: Although it is well established that dendritic cells (DCs) are essential for allergic immune responses in several mouse models of asthma but the specific role of different lung DC subsets in initiating Th2 immunity remains poorly understood. The purpose of this study was to investigate the contribution of tissue resident dendritic cells in inducing Th2 immune response to the tropical allergen Blomia tropicalis. Materials and methods: C57BL/6 mice were intra-nasally sensitized with Blomia tropicalis extract (Blo t) and subsequently challenged via the same route. Bronchoalveolar lavage (BAL) eosinophils and neutrophils were determined by expression of Singlec-F and Ly6-G respectively. Mucus secretion was detected with periodic acid Schiff stain (PAS). T cell cytokines were determined by culture of draining lymph node cells with 20 mg/ml Blo t extract for 5 days and assay of culture supernatants by ELISA (R&D systems). Results: Intra-nasal administration of Blo t without adjuvant induced a robust allergic response characterized by high eosinophil (60%) and low neutrophil (10%) infiltration. Hematoxylin and eosin staining identified substantial mononuclear cell infiltration of the lungs and PAS staining demonstrated mucus hyper-secretion and goblet cell hyperplasia. Draining lymph node cells cultured with Blo t extract secreted large amounts of IL-4 (200 pg/ml), IL-5 (2000 pg/ml), IL-13 (4000 pg/ml) and very little IFN-g ( rDer p 2/1C) induced IgG antibodies in rabbits that inhibited IgE reactivity of HDM-allergic patients to Der p 1 and Der p 2. Conclusions: The preclinical characterization indicates that in particular rDer p 2/1S may be used as a safe hypoallergenic molecule for tolerance as well as vaccination approaches to treat HDM allergy.

P0803 Identification and characterization of pulmonary stem cells in the pathogenic mechanism and alleviation of allergic airway inflammation C. J. Chiu & B. L. Chiang Graduate Institute of Immunology, National Taiwan University, Taipei, Taiwan Purpose/Objective: Asthma is a very complex and heterogeneous disease that is characterized by airway inflammation and airway hyperresponsiveness (AHR). Recent studies have been focused on characterization and identification of lung stem cells for regenerative therapy in asthma. Based on the theory of alveolar development in the newborn lung, pulmonary stem cells are enriched in the neonatal mice


450 Poster Sessions and becoming the rare population in the well-development adult mice. Therefore, we isolated and explored the biological effect of the potential SSEA-1+ pulmonary stem cells (PSCs) derived from neonatal mice. Materials and methods: SSEA-1+ PSCs derived from neonatal mice were enriched by magnetic beads. The characteristics of SSEA-1+ PSCs were evaluated using immunofluorescence staining, real-time PCR, and FACS analysis. Further, in vivo biological function of SSEA-1+ PSCs was studied and analyzed in OVA-induced asthmatic mice. Results: Single-cell suspensions derived from neonatal mouse lung tissue were SSEA-1+ or Sca-1+ population. SSEA-1+ PSCs were highly expressed in the neonatal mice, and then were downregulated in the adult mice. Enriched SSEA-1+ PSCs have the ability to differentiate into AQP5+ type I pneumocytes and surfactant protein C-expressed type II pneumocytes. FACS and real-time PCR showed that CCSP transcript and protein level were increased in SSEA-1+ PSCs compared with SSEA-1) Sca-1) pulmonary cells. Adoptive transfer of SSEA-1+ PSCs reduced AHR and prevented airway damage through decreasing eosinophil infiltration and inhibiting IL-5, eotaxin and TSLP production in OVA-induced asthma mice. Conclusions: In conclusion, SSEA-1+ PSCs might have the potential to repair lung damage or to inhibit inflammatory effects in the pathological process of asthma. Therefore, we conclude that the potent immunomodulatory effect of PSCs might be beneficial for treating asthma disease.

P0804 Identification of a major IgE-binding epitope of beta-parvalbumins using scFv antibodies D. Ackerbauer, M. Kostadinova, M. Bublin & H. Breiteneder Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria Purpose/Objective: Fish allergy is associated with IgE-mediated hypersensitivity reactions to b-parvalbumins, which are small calciumbinding muscle proteins, and represent the major for 95% of fish allergic patients. A high degree of cross-reactivity of parvalbumins from various fish species has been described. We aimed to express and purify single chain variable fragment (scFv) antibodies specific for b-parvalbumins for their detection in fish protein extracts and for the identification of major IgE epitopes. Materials and methods: The phage clone gco9 specific for bparvalbumins was isolated from the ETH-2 phage display library by sequential panning against parvalbumins from Atlantic cod, carp and rainbow trout. The soluble scFv-gco9 was expressed in the E. coli nonsuppressor strain HB2151 and extracted from the periplasm. The purified antibody was obtained by nickel-based affinity chromatography and its specificity was tested in Western blot using fish protein extracts. The binding capacity of scFv-gco9 for the natural parvalbumins Gad m 1, Cyp c 1 and Onc m 1 of Atlantic cod, carp and rainbow trout, respectively, was tested in ELISA. Inhibition ELISAs were performed to investigate the ability of this antibody to block sIgEbinding to b-parvalbumins in sera of fish allergic patients. Results: The purified scFv-gco9 was able to detect b-parvalbumins of Atlantic cod, carp and rainbow trout as single 12 kDa bands in Western blot using fish protein extracts. The antibody also strongly recognized natural Gad m 1, Cyp c 1 and Onc m 1 in direct ELISA. The binding capacity of scFv-gco9 to nGad m 1 was independent of the presence or absence of Ca2+ ions. In contrast, a mouse monoclonal anti-parvalbumin antibody only reacted with the calcium-bound form. In competitive ELISA, the scFv antibody was able to inhibit binding of sIgE from fish allergic patients’ sera to all three b-parvalbumins by 80%. Conclusions: In this work, supported by grant SFB-F4608 from the Austrian Science Fund, we isolated and produced a scFv antibody

specific for a calcium-independent major IgE epitope of Atlantic cod b-parvalbumin.

P0805 IgE knock-in mice reveal a key role for IgE in basophil-mediated active systemic anaphylaxis P. Yu,* W. Lu¨bben,* A. Okhrimenko,* C. Sto¨berl, A. TurquetiNeves,à S. Bauer,* G. Riethmu¨ller§ & D. Voehringer– *Institute of Immunology, Philipps-Universita¨t Marburg, Marburg, Germany, Institute of Immunology, LMU Mu¨nchen, Mu¨nchen, Germany, àDepartment of Infection Biology, Institute of Medical Microbiology Immunology, Erlangen, Germany, §Institute of Immunology, LMU Mu¨nchen, Mu¨, Germany, –Department of Infection Biology, Institute for Medical Microbioloby, Erlangen, Germany Purpose/Objective: IgE production is tightly regulated at the cellular and genetic level and is believed to be central to allergy development. At least two pathways exist that lead to systemic anaphylaxis reactions in vivo: IgE-sensitized mast cell and IgG1-sensitized basophil mediated anaphylaxis. Although passive anaphylaxis, by application of allergen and allergen-specific antibodies in mice allows for the determination of the contribution of different immunoglobulin isotypes, the analysis of a dynamic, immunization mediated antibody responses is not currently possible. Materials and methods: We generated IgE knock-in mice (IgEki) which express the IgE heavy chain instead of IgG1 in order to analyze the contribution of IgG1 and IgE to active anaphylaxis in vivo. Results: IgEki mice display increased IgE production both in vitro and in vivo. In addition a defect of membrane expression of chiemric IgE in vivo was observed, which suggests that mIgE expression id fundamentally differenet from mIgG1 expression in vivo The sensitization of IgEki mice by immunization followed by antigen challenge leads to increased anaphylaxis. Homozygous IgEki mice, which lack IgG1 due to the knock-in strategy, are most susceptible to active systemic anaphylaxis. Depletion of basophils demonstrates their importance in IgE-mediated anaphylaxis. Conclusions: Therefore we propose that an enhanced-, antigenspecific, polyclonal, IgE response, as is the case in allergic patients, is the most efficient way to sensitize basophils to cause systemic anaphylaxis in vivo.

P0806 IgE reactivity profiling of allergic individuals in the Philippines C. R. Cabauatan,* C. Lupinek,* R. Weiss, M. Focke-Tejkl,* P. L. Bhalla,à M. B. Singh,à P. A. Knight,à M. van Hage,§ J. D. A. Ramos– & R. Valenta** *Department of Pathophysiology and Allergy Research, Center for Pathophysiology Infectiology and Immunology Medical University of Vienna, Vienna, Austria, Department of Molecular Biology, Christian Doppler Laboratory for Allergy Diagnostic and Therapy, University of Salzburg, Salzburg, Austria, àPlant Molecular Biology & Biotechnology Laboratory, Melbourne School of Land and Environment, The University of Melbourne, Parkville, Vic., Australia, §Department of Medicine Clinical Immunology and Allergy Unit, Karolinska Institutet and University Hospital, Stockholm, Sweden, –The Graduate School, University of Santo Tomas, Manila, Philippines, **Department of Pathophysiology and Allergy, Research Center for Pathophysiology Infectiology, Christian Doppler Laboratory for Allergy Research, Immunology Medical University of Vienna, Vienna, Austria Purpose/Objective: There is only limited information about allergy and relevant allergens in the Philippines. This study aimed to investigate the allergen profile in the Philippines.


Poster Sessions 451 Materials and methods: A questionnaire for Allergy Screening from the International Study of Asthma and Allergy in Childhood was used to identify individuals with and without allergic symptoms. Allergen specific-IgE antibodies were determined by ELISA using pollen extracts from several local grasses such as Cynodon dactylon, Chloris barbata, Imperata cylindrica, Saccharum spontaneum, Sporobulus indicus, Oryza sativa and Zea mays. In addition, chips containing 103 micro-arrayed purified allergen molecules were used to determine the molecular IgE reactivity profiles in random subgroups of 79 symptomatic and 20 asymptomatic subjects. IgE binding studies were performed in purified rOry s 1 from rice and galactose-a1,3-galactose (a-Gal); and IgE inhibition assay with glycosylated nPhl p 4 was done. Nitrocelluloseblotted extracts from local grasses were analyzed for the presence of major grass pollen allergens with specific antibody probes. Results: Interestingly, we found that not only symptomatic subjects but also asymptomatic individuals exhibited IgE reactivity to pollen extracts of local grasses. A detailed characterization of the IgE reactivity profiles with micro-arrayed allergens revealed an unusual preferential sensitization to mainly glycosylated grass pollen allergens such as nCyn d 1 and nPhl p 4 in both groups whereas the major protein allergens in grass pollen were not recognized. The specificity of the IgE reactivity to carbohydrate epitopes with poor allergenic activity was confirmed by IgE inhibition studies. No sensitization to the allergenic a-Gal was observed. Symptomatic allergic sensitizations were confined to nonglycosylated protein allergens from house dust mites and animals. Conclusions: Our study reveals that subjects from the Philippines exhibit a high prevalence of asymptomatic IgE sensitization to nonallergenic carbohydrate epitopes in grass pollen whereas symptomatic allergic sensitization seems to be confined to protein allergens from mites and animals. These results have impact on the development of allergen-specific forms of immunotherapy.

P0807 Immature dendritic cells in patients with ill-controlled asthma C. J. Chang,* Y. H. Yang, H. Y. Hsu, K. H. Chu, C. J. Chiu & B. L. Chiang *College of Medicine, Graduate Institute of Oncology, National Taiwan University, Taipei, Taiwan, Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan Purpose/Objective: Ill-controlled allergic asthma, a chronic inflammatory disease, has been increasingly observed in both developed and developing countries. Inhalant steroid has been applied to control disease that is transient used to reduce disease severity, however, which could not be used to cure disease completely. Therefore, to approach alternative pathogenic mechanisms involved asthma facilitates to develop new therapeutic targets. Previous studies revealed Th2 cells and other inflammatory cells play a major role in the initiation and pathological development of asthma, therefore, allergenic antigens specific adoptive immune responses play an important role in pathogenic mechanisms of disease. Dendritic cells (DCs) are professional antigen presenting cells that play a crucial role to establish adoptive immune responses to various inflammatory stimuli. The well accepted concept that human blood monocytes can be induced to develop immature DCs after treating GM-CSF and IL-4. Materials and methods: In our study, MoDCs were used to evaluate whether function of DCs involved pathogenicity of asthma that MoDCs derived from 6 patients with ill-controlled asthma or derived from 10 health donors. All of patients were discontinued to treat systemic or inhalant steroid before blood collection. Results: MoDCs derived from asthma patients presented lower levels of accessory molecules, including CD40, CD80, CD83, CD86 or MHC class II compared with those molecules derived from MoDCs of healthy donors. Consistently, MoDCs cultured from patients presented

potent capability to uptake fluoresce-conjugated model antigen than MoDCs derived from health donors. In addition, results of mix lymphocytes reaction revealed that MoDCs generated from patients have a weak capacity to prime naı¨ve CD4+ T cells proliferation compared with those derived from health donors. These data indicate that patients with ill-controlled asthma have less potency of MoDCs. Interestingly, both MoDCs derived from patients and health donors were activated and maturated after LPS stimulation, however, most of MoDCs cultured from asthma patients still have shown weak potency to prime naı¨ve T cells than those generated from health donors. Conclusions: These data indicate that less potent and immature DCs in patients with ill-controlled asthma might be play a role in pathogenic mechanism of disease.

P0808 Immune mechanisms mediating suppression of allergic airway inflammation upon Salmonella infection V. Ganesh, A. M. Baru, C. Untucht & T. Sparwasser Twincore, Institute of Infection Immunology, Hannover, Germany Purpose/Objective: Prevalence of asthma, a Th2 biased inflammatory airway disease has dramatically increased over the past few decades. Mere reciprocal regulation of Th1 and Th2 phenotypes failed to justify the concomitant rise in allergic and autoimmune disorders characterized by dys-regulated Th1 or Th17 immune responses. Recent studies have highlighted the role of immune-regulatory mechanisms in modulating allergic reactions. A longitudinal study by Pelosi et al. (Allergy, 2005) demonstrated an inverse correlation between Salmonella infection and susceptibility to asthma, however, the mechanism remains obscure. In this study we attempted to identify the mechanism(s) mediating this suppression. Materials and methods: Experimental allergic airway inflammation was induced by standard OVA-Alum model. A group of mice were infected with Salmonella during the regimen. Inflammatory responses were estimated by cellular infiltration in broncheo-alveolar lavage (BAL). Serum immunoglobulin levels and cytokines from in vitro restimulated mediastinal lymph node cells were measured by ELISA. Lung pathology was determined by histology and quantitative PCR. Alteration in cellular frequencies was determined by flow cytometry. In vitro differentiation and stability of Th2 cells were investigated. Results: Mice infected with Salmonella showed markedly reduced total cellular infiltration and eosinophilia in the BAL. Infected mice showed increased titers of antigen-specific IgG2a in sera, but no significant alteration in IFN-g was detected. Reduced IL-4 secretion was observed which correlated to decreased MUC5AC expression in the lungs. No demonstrable change in the frequencies of Foxp3+ Tregs was observed. Infected mice showed a significant increase in cells expressing CD11b+/ Gr1+/Ly6Cint. Using in vitro co-cultures we demonstrate that CD11b+/ Gr1+/Ly6Cint cells do not inhibit Th2 differentiation but destabilize Th2 phenotype by down-modulating GATA-3 expression. Conclusions: We observed amelioration of airway inflammation in Salmonella infected mice. This suppression was Treg independent. No substantial shunt towards a Th1 phenotype was detected. We demonstrate expansion of CD11b+/Gr1+/Ly6Cint cells upon Salmonella infection, which exert their suppressive function by influencing the Th2 stability. This could be a potential mechanism of protection from asthma.


452 Poster Sessions P0809 Immunological reactions to the carbohydrate antigen alpha-Gal D. Kollmann,* C. Ebner, W. Emminger,à A. Mangold,§ H. J. Ankersmit§ & B. Bohle* *Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria, Allergy Clinic Reumannplatz, Allergy Clinic Reumannplatz, Vienna, Austria, àAllergy Clinic Rennweg, Allergy Clinic Rennweg, Vienna, Austria, §Department of Surgery, Medical University of Vienna, Vienna, Austria Purpose/Objective: Gala1-3Galb1-4GlcNAc-R (alpha-Gal) is a carbohydrate expressed by non-primate mammalians. It is the main reason of hyperacute graft rejection in xenotransplantation. Recently, alpha-Gal has been described as an important allergenic structure in red meat allergy. Since the role of carbohydrates in allergy is still controversial, we sought to investigate the relevance of alpha-Gal in meat allergy and to identify proteins carrying the allergenic sugar. Materials and methods: Twenty patients who had experienced generalized urticaria after consuming meat were included. All patients showed IgE-reactivity to beef or pork in ImmunoCAP. IgG and IgE antibodies specific for alpha-Gal were analysed by ELISA. Protein extracts were produced from beef and pork and used in immunoblots to test IgE-reactivity to individual meat proteins. Additionally, sera from 15 patients receiving a biological heart valve and 8 patients receiving mechanical prostheses obtained preoperatively, 10 days and 3 months postoperatively were analysed. Results: All meat-allergic patients displayed alpha-Gal-specific IgG and 19/20 patients showed alpha-Gal-specific IgE. In immunoblots, 13/ 19 alpha-Gal sensitized patients showed IgE specific for a protein of approximately 160 kDa, equivalent to bovine gamma globulin (BGG). The same protein was detected to be glycosylated. In inhibition-ELISA, IgE-reactivity to BGG was completely abolished after pre-incubation with alpha-Gal. The majority of biovalve recipients displayed high levels of alpha-Gal-specific IgG which increased in 12/15 individuals 10 days post surgery. Five of these patients showed increased alphaGal-specific IgE levels already 10 days post surgery. None of the patients with mechanical bioprostheses showed a rise in alpha-Galspecific antibodies. Conclusions: Our data indicate that IgE-reactivity to alpha-Gal on BGG plays an important role in meat allergy. We currently investigate why the presence of high levels of alpha-Gal-specific IgG antibodies is incapable of blocking IgE-binding to the carbohydrate. The occurrence of alpha-Gal-specific IgE in biovalve recipients post surgery indicates, that sensitization to alpha-Gal can develop although alpha-Gal-specific IgG is present.

P0810 Immunomodulatory and bronchodilatory potential of Allium cepa L. and its flavonoid quercetin in vitro T. T. Oliveira,* K. M. C. Dourado,* T. C. B. Carneiro,* M. S. S. Machado,* R. S. Costa,* E. S. Velozo, L. C. Pontes de Carvalho,à D. F. S. A. Vasconcelos,* N. M. Alcaˆntara Neves* & C. A. Figueiredo* *Institute of Health Sciences, Biorregulation, Salvador Bahia, Brazil, Faculty of Pharmacy, Pharmacy, Salvador Bahia, Brazil, àGonçalo Moniz Research Institute, FIOCRUZ, Fiocruz, Salvador Bahia, Brazil Purpose/Objective: This study was conducted investigating the in vitro anti-inflammatory activity and bronchodilator potential of the methanolic extract of the Allium cepa L. (AcE) and its flavonoid quercetin (Qt) on the production of IL-4, IL-5 and IL-13 by spleen cell; production of nitric oxide on peritoneal macrophages cultures, and their bronchodilator potential on isolated trachea from mice. Materials and methods: To this end, the allergic process was induced in A/J mice by the administration of Blomia tropicalis antigen. The

animals were sensitized with 100 lg per animal s.c. in 4 mg/ml of [AL (OH)3] on days 0 and 7; after the last sensitization the animals received four intranasal challenged (10 lg per animal i.n) with Blomia tropicalis (BtE) mite at intervals of one day. Spleen cells isolated from allergic animals were incubated with different concentrations of AcE and Qt, stimulated or not with pokeweed mitogen (PWM, 2.5 lg/ ml); cultures were then incubated for 2 days and supernatants were collected for cytokine measurement. Peritoneal macrophages were isolated from normal mice and incubated with different concentrations of AcE and Qt for 1 h in the presence or not of LPS (concentration) for 24 h. After this period, the supernatants were collected and analyzed for nitrite by Griess reaction. The amount of nitrite in the sample was determined using a sodium nitrite standard curve. Additionally, trachea from non sensitized mice were sectioned into rings, and placed in tanks for isolated organ with Krebs-bicarbonate solution and aerated with a carbogen mixture (95%O2 and 5% CO2). The rings were contracted with carbachol to evaluate the presence of functional epithelium, and after reaching a plateau of contractile state, the rings were stimulated with bradykinin. Cumulatively increasing concentrations of AcE or Qt were added. Concentration response curve was constructed and the data were analyzed. Results: The production of inflammatory cytokines (IL-4, IL-5 and IL-13) and nitric oxide was significantly reduced by the treatment with different concentrations of AcE and Qt when compared with positive control. Also was observed a dilator effect of AcE and Qt on carbacholinduced contraction in isolated trachea. Conclusions: The results obtained in this study suggest that Allium cepa L. and Qt have potential as anti-asmatic drugs by both immunomodulatory and bronchodilatory properties. The molecular mechanisms wherebyAllium cepa L. and Qtact are under exploration by our group.

P0812 Induction of mucosal tolerance with structurally different antigens: studies on underlying mechanisms J. Akgu¨n, I. Schabussova, K. Hufnagl, C. Wild & U. Wiedermann Institute of Specific Prophylaxis and Tropical Medicine, Medical University Vienna, Vienna, Austria Purpose/Objective: In a mouse model of poly-sensitization, we have shown that intranasal administration of a linear synthetic hybrid, composed of immunodominant T-cell epitopes of the major birch and grass pollen allergens Bet v 1, Phl p 1 and Phl p 5, induced tolerance in mice sensitized with the 3 allergens. Tolerance induction was associated with reduced allergic inflammation and increased production of the regulatory cytokine IL-10 in the lungs. On the other hand, tolerance induction with the whole recombinant (r) Bet v 1 led to reduction of airway inflammation within the lung without the induction of IL-10 in the lung. Thus, we want to understand the mechanistic pathways of tolerance induction by these two structurally different antigens by investigation of their uptake and presentation by antigen presenting cells. Materials and methods: In order to follow up their internalization, hybrid and rBet v 1 were conjugated with the fluorescence dye 5 (6)Carboxyfluorescein (FAM). For in vivo studies, constructs were administered intranasally. After various time points (0, 1, 6, 24 and 48 h) the antigen uptake in the nasal-associated lymphoid tissue (NALT), lung, bronchial lymph nodes, spleen and blood was investigated by flow cytometry. Furthermore, FAM-hybrid and FAMrBet v 1 were co-cultured with isolated murine mononuclear lung cells in vitro in a time dependent manner (0, ½, 1, 6 and 24 h) and cells capturing these antigens were identified and characterized. Results: In vivo, both antigens were taken up by two main populations: CD11c(+) CD11b(+) macrophages and CD11c(+) CD11b()) dendritic cells in the lung and NALT. Interestingly, more FAM-hybrid than FAM-rBet v 1 molecules were detected within the lung cells. None


Poster Sessions 453 of the investigated antigens have been detected in the spleen or in the blood. In vitro, macrophages, dendritic cells and additionally B cells were internalising the antigens, and B cells displayed the population with the highest antigen uptake capacity. With respect of the time kinetic these B cells internalised the FAM-hybrid about 6 h earlier than the FAM-rBet v 1. Conclusions: In vivo and in vitro kinetic studies demonstrate that there are differences between the antigen uptake of the linear multipeptide (hybrid) and the conformational allergen (rBet v 1). Further studies on internalisation pathways are currently ongoing.

P0813 Inflammatory marker sTREM-1 reflects the clinical stage and respiratory tract obstruction in allergic asthma bronchiale patients and correlates with number of neutrophils M. Buc,* M. Bucova,* M. Suchankova,* M. Dzurilla,* D. Kantarova, H. Novosadova,à E. Tedlova,à S. Urban,à E. Hornakova,à M. Seligova,à V. Durmanova,* J. Javor* & E. Paulovicova§ *Department of Immunology, School of Medicine, Comenius University, Bratislava, Slovakia, Internal Department, Jesenius School of Medicine, Comenius University Martin, Slovakia, àDepartment of Pneumology and Phthisiology, School of Medicine, Comenius University, Bratislava, Slovakia, §Slovak Academy of Science, Institute of Chemistry Centre of Excellence Glycomed, Bratislava, Slovakia Purpose/Objective: TREM-1 (Triggering receptor expressed on myeloid cells), is constitutively expressed on the surface of myeloid cellsneutrophils, monocytes and macrophages. The membrane form of TREM-1 can be cleaved from the surface of activated myelocytes and its soluble form (sTREM-1) is released into microenvironment. The highest levels of sTREM-1 have been found in patients with sepsis and other inflammatory diseases caused mainly by extracellular microorganisms as well as inflammatory states of non-infectious origin. The knowledge that asthma bronchiale is an inflammatory disorder has prompted us to investigate the plasma levels of new inflammatory marker sTREM-1 that is released from the surfaces of activated neutrophils and monocytes. Materials and methods: The plasma levels of sTREM-1 were analysed by a sandwich Elisa test in the cohort of 76 patients with allergic asthma bronchiale and 39 healthy controls. Either Anova or Mann Whitney U-test were used to determine the difference and the statistical significance. The association of sTREM-1 plasma levels with clinical stage of AB was evaluated by Kruskal Wallis test. For correlation analysis of non-parametric continuals and nominal variables the Spearmans’ two-tailed test was used. Results: Our results revealed more than 3.5 times higher levels of sTREM-1 in AB patients (92.3 pg/ml ± 125.6) compared with healthy subjects (25.7 pg/ml ± 9.2; P = 0.0001). Higher levels of sTREM1were found also in patients with exacerbated AB (170.5 pg/ml ± 78.2) compared with non-exacerbated AB patients (59.1 ± 78.2; P < 0.0001), patients with respiratory tract obstruction (176.4 pg/ ml ± 177.8) than those without obstruction (51.99 pg/ml ± 64.0; P < 0.0001) and patients with anti-IgE therapy (P < 0.0001). Levels of sTREM-1 correlated with number of leucocytes (P = 0.002), and absolute number of neutrophils (P = 0.001). Conclusions: The levels of sTREM-1 in AB patients have not been studied yet, so we cannot compare our Results: . Plasma levels of sTREM-1 are highly elevated in severe forms of asthma, reflect the clinical stage of AB, state of exacerbation, respiratory tract obstruction and correlate with the number of leukocytes, mainly neutrophils. Our results highlight the potential usefulness of the assessment of the soluble form of TREM-1 in plasma of AB patients.

P0814 Inhibition of histamine H1 receptor activity modulates proinflammatory cytokine production of dendritic cells through c-Rel dependent pathway J. L. Suen & C. L. Lee Graduate Institute of Medicine, Kaohsiung Medical Universtiy, Kaohsiung, Taiwan Purpose/Objective: Histamine displays diverse effects on immune regulation through four types of histamine receptors (HRs). Among them, type 1 receptor (H1R) plays an important role in allergic inflammation. Dendritic cells (DCs), which express at least three types of HRs, are professional antigen-presenting cells controlling the development of allergic inflammation. However, the molecular mechanisms involved in H1R-mediated NF-kB signaling of DCs remain poorly defined. Materials and methods: Bone marrow-derived dendritic cells (BMDCs) were treated with H1R inverse agonist ketotifen to interrupt the basal H1R-mediated signaling. The crosstalk of H1R-mediated signaling and NF-jB pathway was examined by NF-jB subunits analysis using Western blotting and TNF-a promoter activity using chromatin immunoprecipitation assay. Results: The data showed that blockage of H1R signaling by ketotifen inhibited the proinflammatory cytokine TNF-a and IL-6 production of BM-DCs. H1R specific agonist was able to enhance the TNF-a production, but this overexpression was significantly inhibited by NF-jB inhibitor, suggesting crosstalk between H1R and NF-jB signaling in DCs. After comprehensive analysis of NF-jB subunits, cRel protein level was found to be significantly down-regulated in ketotifen-treated BM-DCs, which led to inhibition of the promoter activity of TNF-a. Conclusions: Our results suggest that c-Rel controls H1R-mediated proinflammatory cytokine production in DCs. This study provides a potential mechanism of H1R-mediated signaling and NF-jB pathway crosstalk in allergic inflammation.

P0815 Inhibition of the co-stimulatory molecule OX40 significantly inhibits inflammation in the chronic house dust mite model of allergic airway inflammation K. Burrows,* C. Dumont, K. Dixon,* M. Catley,* D. Marshall* & S. Shaw* *Pharmacology, UCB Pharma, Slough, UK, Cellular Sciences, UCB Pharma, Slough, UK Purpose/Objective: Allergic asthma is a chronic inflammatory disease of the lungs that is punctuated by exacerbations caused by infection or exposure to allergens. Using a mouse model that mimics certain aspects of allergic asthma this study investigated how blocking OX40 would effect the recruitment of inflammatory cells to the lung. Materials and methods: Male balb/c mice were treated with a murine OX40 targeting antibody twice weekly, via subcutaneous injection, from day 0 for 5 weeks. From day 0, mice were sensitized to house dust mite (HDM) extract via intranasal administration 4 times a week for 5 weeks. At the end of week 5, the study was terminated. Blood was taken from the animals and serum prepared. Animals were cannulated and brochoalveolar lavage (BAL) fluid was collected. Lungs were removed and tissue digest carried out in order to release immune cells within the lung tissue. Inflammatory cell types in the BAL and lung tissue digests were determined using flow cytometry. Results: Blockade of OX40 resulted in a significant decrease in the number of eosinophils, neutrophils and CD4+ T cells in both the BAL and lung tissue digest compared to vehicle treated animals.


454 Poster Sessions Blocking OX40 significantly reduced the number of IFN-c (Th1), IL-17A (Th17) and IL-4 (Th2) producing CD4+ T cells in both BAL and lung tissue digests. Conclusions: Blocking OX40 significantly reduces the infiltration of numerous inflammatory cell types into the lungs of mice. It is also interesting that blocking OX40 has effects on Th17 and Th1 cells. This indicates that blocking OX40 may be beneficial in the treatment of allergic asthma.

P0816 Inhibitory effects of hydrogen sulfide on lung oxidative stress in allergic mice H. H. A. Ferreira,* L. R. Benetti,* D. Campos,* S. A. Gurgueira, A. E. Vercesi, S. A. Teixeira,à J. Florenzano,à C. E. V. Guedes* & S. K. P. Costaà *Laboratory of Inflammation Research, Saõ Francisco University, Bragança Paulista SP, Brazil, Laboratory of Bioenergetics, State University of Campinas, Campinas SP, Brazil, àDepartment. of Pharmacology, University of Sao Paulo, Saõ Paulo SP, Brazil Purpose/Objective: Recent studies show that endogenous hydrogen sulfide (H2S) plays an anti-inflammatory role in the pathogenesis of airway inflammation. This study investigated whether exogenous H2S may counteract oxidative stress-mediated lung damage in allergic mice. Materials and methods: Female BALB/c mice previously sensitized with ovalbumin (OVA) were treated with sodium hydrosulfide (NaHS) 30 min before OVA challenge. Forty-Eight hours after antigenchallenge, the mice were killed and leukocyte counting as well as nitrite plus nitrate concentrations were determined in the bronchoalveolar lavage fluid, and lung tissue was analysed for nitric oxide synthase (NOS) activity, iNOS expression, superoxide dismutase (SOD), catalase, glutathione reductase (GR) and glutathione peroxidase (GPx) activities, thiobarbituric acid reactive species (TBARS) and 3-nitrotyrosine containing proteins (3-NT). Results: Pre-treatment of OVA-sensitized mice with NaHS resulted in significant reduction of both eosinophil and neutrophil migration to the lungs, and prevented the elevation of iNOS expression and activity observed in the lungs from the untreated allergic mice, although it did not affect 3-NT. NaHS treatment also abolished the increased lipid peroxidation present in the allergic mouse lungs and increased SOD, GPx and GR enzyme activities. Conclusions: These results show, for the first time, that the beneficial in vivo effects of the H2S-donor NaHS on allergic airway inflammation involve its inhibitory action on leukocyte recruitment and the prevention of lung damage by increasing endogenous antioxidant defenses, thus making of H2S donors a potential new class of therapeutical agents useful for treatment of lung diseases characterized by the presence of inflammatory cells and oxidant/antioxidant imbalance. Financial support: FAPESP and CNPq

P0817 Intratracheal administration of Fab fragments of an antigenspecific monoclonal antibody suppresses asthmatic responses in mice S. Yoshino, N. Mizutani, Y. Ammmori & H. Torii Pharmacology, Kobe Pharmaceutical University, Kobe, Japan Purpose/Objective: Fab fragments (Fabs) of antibodies maintain the ability to bind specific antigens but lack the binding site for complement (C) as well as the site for binding to receptors on effector cells including mast cells, basophils, and macrophages that play an important role in immune and allergic diseases. In the present study, we investigated whether intratracheal administration of Fabs of a

monoclonal antibody (mAb) specific for ovalbumin (OVA) was able to suppress asthmatic responses in mice. Materials and methods: Asthmatic responses were induced in Balb/c mice either by passive sensitization with anti-OVA pAbs or by active sensitization with OVA followed by intratracheal challenge with the antigen. Fabs prepared by the digestion of an anti-OVA IgG1 (O1 10) mAb with papain were intratracheally administered 30 min before the antigenic challenge. Normal IgG Fabs were used as a control. Results: Intratracheal administration of O1 10 Fabs markedly suppressed the early and late phases of asthmatic responses caused by passive sensitization with anti-OVA pAbs as well as by active sensitization with OVA when the in vivo responses were measured by specific airway resistance (sRaw). Control Fabs failed to affect the asthmatic responses. The significantly reduced number of neutrophils in bronchoalveolar lavage fluids (BALF) as well as in the lung tissue was seen in mice treated with O1 10 FabsO1 10. Fabs were also effective in suppressing asthmatic responses induced by passive sensitization wtih anti-OVA IgE mAb (OE-1). In cotrast, intratracheal injection of intact O1 10 failed to suppress the asthmatic responses. The suppression of asthmatic responses by O1 10 Fabs was associated with significantly higher and lower levels of MMCP-1 and C3a, respectively. In vitro studies revealed that the capture of OVA by O1 10 Fabs resulted in prevention of the following binding of intact anti-OVA pAbs or OE-1 to the captured OVA. Conclusions: Asthmatic responses appear to be downregulated by intratracheal administration of Fabs of an antigen-specific mAb via the mechanism of the formation of antigen and Fab complexes in the airway, resulting in the prevention of antigen and intact antibody binding essential for induction of asthmatic responses.

P0818 Isolation and characterisation of CD8 T cells with receptor specificity for house dust mite allergen, Der p 1 K. H. S. Wong, G. M. Grotenbreg & D. M. Kemeny Department of Microbiology Yong Loo Lin, Immunology Programme, School of Medicine, National University of Singapore, Singapore, Singapore Purpose/Objective: CD8 T cells have multiple functions in asthma. Tc2 CD8 T cells exacerbate airway inflammation through the secretion of type 2 cytokines, while the Tc1 CD8 T cells may inhibit type 2 airway inflammation and airway hyperresponsiveness via a number of different mechanisms such as the production of type 1 cytokines and perforin-mediated cytotoxicity. Therefore, there exists the potential to target CD8 T cells to for treatment of asthma and other allergic diseases. Our aim was to isolate CD8 T cells specific for the house dust mite protein, Der p 1, and to characterise the T cell receptor (TCR) of these allergen specific T cells. Materials and methods: DNA vaccination was employed to generate a CD8 T cell response in C57BL/6 mice. Plasmid vector encoding a MHC I epitope from Der p 1 was delivered intradermally by skin tattoo and CD8 T cell responses measured by IFN-g ELISPOT and flow cytometry with MHC I tetramers. T cells isolated from immunized mice were cultured in vitro with the antigenic peptide and screened for antigen specificity using MHC I tetramers. TCR genes were identified using 5’ RACE amplification. Results: Following DNA vaccination, 1 1.5% of splenic CD8 T cells were shown to be specific for the Der p 1 epitope. Screening following 4 cycles of re-stimulation showed that 95% the CD8 T cells in the culture were Der p 1-specific. These CD8 T cells killed peptide pulsed EL4 cells and secreted IFN-g and TNF-a. 5’ RACE amplification followed by sequencing of TCR genes showed that the cells were clonal with alpha (TRAV7-5*01F) and beta (TRBV5*01F) TCR chains. Conclusions: We have generated a CD8 T cell response by DNA vaccination and characterized the TCR usage of allergen specific T cells


Poster Sessions 455 that recognize an epitope derived from the house dust mite protein, Der p 1. This will facilitate investigation into CD8 T cell regulation of asthma by allergen specific CD8 T cells and the development of a CD8 based vaccine for asthma.

P0819 Isolation, expression and characterization of IgE reactive-portions of high molecular weight glutenin like wheat food allergens A. Baar,* S. Pahr,* C. Constantin,* S. Giavi, N. Papadopoulos, A. Pelkonen,à M. Ma¨kela¨,à C. Ebner,§ A. Mari,– S. Vrtala* & R. Valenta* *Department of Pathophysiology and Allergy Research, Center for Pathophysiology Infectiology and Immunology, Vienna, Austria, Allergy and Immunology Research Center, University of Athens, Athens, Greece, à Skin and Allergy Hospital, Helsinki University Central Hospital, Helsinki, Finland, §Ambulatory for Allergy and Clinical Immunology, Ambulatory for Allergy and Clinical Immunology, Vienna, Austria, – Center for Molecular Allergology, IDI-IRCCS, Rom, Italy Purpose/Objective: Wheat can be grown in a wide climatic and geographic range and is one of the most consumed cereals worldwide but it is also an important allergen source. In allergic patients wheat ingestion can lead to urticaria, atopic dermatitis, and gastrointestinal symptoms and to anaphylaxis. Materials and methods: We isolated IgE-reactive clones from a wheat seed cDNA library with sera from wheat food allergic patients. Two of these clones showed sequence identities with a gene coding for the High molecular weight glutenin x-type subunit Bx7 precursor (HMW Bx7). The IgE reactive sequences were cloned into the E. coli expression vectors pMal-c4x or pET17b and expressed as soluble hexaistidine- and maltose-binding-protein-tagged fusion proteins (m43, m82) comprising aa 344 618 and aa 600-795 of the complete protein and as hexahistidne-tagged recombinant protein (43). Dot blot analyses were performed with sera from wheat food allergic patients. Moreover, antibodies were raised against 43 to detect homologous proteins in other allergenic plant food. Results: The two tested wheat food allergic populations exhibited a similar allergen recognition frequency, 15% of the Greek (n = 26) and 10% of the Finnish (n = 60) patients showed IgE reactivity to the allergen portions m43 and m82. We detected homologous proteins with the rabbit anti-43 antibodies in extracts from spelt, rye, barley and sunflower seeds. Additional, we found HMW Bx7- related proteins in roll, brown bread, rye bread and in gluten free bread extracts. Conclusions: In summary, we expressed two IgE reactive parts of the novel wheat food allergen HMW Bx7 as soluble proteins in E. coli and showed that they are useful for in-vitro diagnostic tests to detect wheat food allergic patients. P0820 Low levels of serum B-cell activating factor among humans with high IgE reactivity to the nematode Ascaris A. Bornacelly, N. Acevedo & L. Caraballo Institute for Immunological Research, Immunology, Cartagena, Colombia Purpose/Objective: The gene TNFSF13B encoding the B-Cell Activating Factor (BAFF) is a putative candidate for resistance to Ascaris in the 13q33 locus. The polymorphism rs10508198 (G>C) is associated with specific IgE/IgG levels to Ascaris and the resistance marker (rABA1), suggesting that genetic variation in TNFSF13B may affect antibody response to Ascaris. Mechanisms are unknown but effects are more noticeable in asthmatics. We aim to analyze the serum BAFF levels (sBAFF) among high and low IgE/IgG producers and according to the BAFF-R expression, genotype and asthma status

Materials and Methods: We analyzed 842 subjects (375 asthmatics/ 467 controls) from Cartagena (Colombia) exposed to Ascaris. sBAFF levels were measured by ELISA (R&D Systems). Speci?c IgE/IgG against Ascaris and rABA-1 were measured by ELISA. Surface expression of BAFF (CD257) and BAFFR (CD268) was evaluated on monocytes and B cells (n = 83) by flow cytometry (Cyan ADP; Dako). Statistical analyses were done on IBM SPSS v20 and GraphPad Prism. Results: There was an inverse relationship between sBAFF levels and sIgE to Ascaris; individuals with high IgE to Ascaris had less sBAFF levels (Mean SD: 842 ± 320 versus 788 ± 306 pg/ml in high-IgE, P = 0.029). In addition, sBAFF levels were inversely correlated to the surface expression of BAFF-R on CD19+ B cells (r2 = -0.53, P = 4 · 10-5). sBAFF levels did not differ among low and high antiAscaris IgG producers. Mutant CC homozygotes had more specific IgE to Ascaris (P = 0.03); increased probability of high IgE levels to Ascaris >75th percentile (OR 2.67, 95%CI 1.15 6.18, P = 0.02) and less IgG to Ascaris (P = 0.002). However this polymorphism was not associated with sBAFF levels (Mean SD GG: 831 ± 307 versus CC: 818 ± 282 pg/ ml P = 0.53). Its effect remains to be evaluated at the transcriptional level since BAFF was not detected in the surface of CD14+ monocytes or B cells. There was no difference in sBAFF levels between asthmatics and controls Conclusions: sBAFF levels are related to the levels of specific IgE to Ascaris, supporting the role of TNFSF13Bas underlying gene for Ascaris susceptibility on the 13q33 locus. We hypothesize that individuals with high IgE to Ascaris may harbor variants decreasing gene expression or leading to a hypoactive isoform. Causative polymorphisms and mechanisms remain to be elucidated

P0821 M-cell specific targeting by neuraminidase-functionalized microparticles as a novel oral immunotherapy for food allergy C. Schultz,* S. C. Diesner, F. Roth-Walter,3,4 T. Eiwegger,– A. Pollak,– Z. Szepfalusi,– I. Pali-Scho¨ll,à E. Jensen-Jarolim,à F. Gabor** & E. Untersmayr* *Department of Pathophysiology and Allergy Research, Center of Pathophysiology Infectiology and Immunology, Medical University of Vienna, Vienna, Austria, Department of Pathophysiology and Allergy Research, Center of Pathophysiology Infectiology and Immunology Medical University of Vienna, Vienna, Austria, àDepartment of Pediatrics and Adolescent Medicine, Center of Pathophysiology Infectiology and Immunology Medical University of Vienna, Vienna, Austria, §Department of Pathophysiology and Allergy Research, Center of Pathophysiology Infectiology and Immunology, Institute of the University of Veterinary Medicine Vienna, Medical University of Vienna Messerli Research Vienna, Austria, –Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria, **Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Vienna, Austria Purpose/Objective: There is still no accepted causative treatment available for food allergic disorders. Recently, we demonstrated in a murine study that mucosal M-cell targeting with Aleuria aurantia lectin (AAL)-coated Poly (D, L-lactide-co-glycolide) (PLGA) microparticles (MPs) represents a promising oral treatment approach in IgE mediated allergy. Due to its structural similarities with AAL we aimed to analyze neuraminidase (NA) from Vibrio cholerae as a novel M-cellspecific functionalization substance and compared NA-, AAL- and wheat germ agglutinin (WGA)-coated, allergen-loaded MPs as a treatment option in food allergy. Materials and methods: Targeting substances and the functionalized MPs were characterized and tested for their suitability for oral application by Caco2-ELISA, digestion experiments and in a human M-cell like co-culture model. Next, we analyzed the therapeutic


456 Poster Sessions properties of the coated MPs in vitro by stimulation of naı¨ve splenocytes and in vivo in a BALB/c food allergy model. Results: NA-coated MPs revealed high binding specificity to a-LFucose and Monosialoganglioside 1 (GM1) in cellular ELISA as well as significant enhanced transepithelial transport when M-cells were present in the co-culture model. For investigation of immunogenicity of NA and NA-functionalized MPs we stimulated splenocytes from naı¨ve mice. Cytokine evaluations revealed a significant increase of IL10 and IFN-c. Intraperitoneal injection of the food allergen ovalbumin (OVA) followed by two oral challenges induced a strong IgE-mediated, OVA-specific response. After 6 oral treatments either with uncoated or WGA-, AAL- or NA-functionalized OVA-loaded PLGA-MPs, murine immune responses were re-evaluated. Treatment with NA-coated MPs induced increased OVA-specific IgG2a and IgA, whereas IgE and IgG1 levels decreased. In intestinal lavages, we observed a significant reduction of total and OVA-specific IgA levels compared to all other groups. Furthermore, significantly elevated IL-10 and IFN-c were measured when splenocytes from animals treated with NA-coated MPs were stimulated with OVA. Conclusions: Based on these results we propose NA to have a beneficial immunomodulatory capacity. NA represents a promising PLGA-MP functionalization substance for treatment of type I food allergy.

P0823 Molecular and immunological characterization of Der p 18, a chitinase-like house dust mite allergen Y. Resch,* K. Blatt, U. Malkus,à I. Swoboda,§ S. Seiberler,* I. Mittermann,* F. Horak,– N. Novak,** V. Krzyzanek, P. Valent, R. Valenta* & S. Vrtalaàà *Department of Pathophysiology and Allergy Research, Center for Pathophysiology Infectiology and Immunology, Vienna, Austria, Division of Hematology and Hemostaseology, Department of Internal à Medicine I, Medical University of Vienna, Vienna, Austria, Institute of Medical Physics and Biophysics, University of Mu¨nster, Mu¨nster, Germany, §Section of Molecular Biotechnology, University of Applied Science, Vienna, Austria, –Allergy Centre, Vienna West, Vienna, Austria, **Department of Dermatology and Allergy, University of Bonn, Bonn, Germany, Academy of Sciences of the Czech Republic, Institute of Scientific Instruments of the ASCR, Brno, Czech Republic, ààChristian Doppler Laboratory for the Development of Allergen Chips, Medical University of Vienna, Vienna, Austria Purpose/Objective: The house dust mite allergen Der p 18 belongs to the glycoside hydrolase family 18 chitinases, which can be found in viruses, bacteria, fungi, plants, animals and humans. It is a major allergen in HDM-allergic dogs but its relevance for house dust mite allergic patients has not been studied in detail. This study aimed to further characterize this allergen on a molecular, structural and immunological level. Materials and methods: Der p 18 was expressed in E. coli, purified to homogeneity and its secondary structure was analyzed by circular dichroism. Der p 18-specific IgG antibodies were produced in rabbits to localize the allergen in mites using immunogold electron microscopy and to search for cross-reactive allergens in other allergen sources (i.e. mites, crustacea, mollusca, insects). IgE reactivity of rDer p 18 was tested with sera from 278 HDM-allergic patients and its allergenic activity was analyzed in basophil degranulation experiments. Results: Recombinant Der p 18 was expressed as a folded protein which shows cross-reactivity with Der f 18 from D. farinae but not with proteins from the other tested allergen sources. The allergen is present in the gut wall of the mites but in contrast to most of the important HDM allergens only to a very small extent in the fecal pellets. Der p 18 reacted with IgE from 6.25% of mite allergic patients from Central

Europe, however, the IgE reactivity was considerably higher in HDMallergic patients suffering from atopic dermatitis (20%). rDer p 18 induced a dose-dependent upregulation of CD203c on basophils from Der p 18-positive patients. Conclusions: Der p 18 is a genus-specific allergen, which seems to be important for patients with severe disease manifestations (e.g. atopic dermatitis).

P0824 Monitoring of regulatory T cells in children with allergy before and one year after sublingual specific immunotherapy (SLIT) by flow cytometry: preliminary study A. Stelmaszczyk-Emmel,* A. Zawadzka-Krajewska & U. Demkow* *Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, Warsaw, Poland, Department Pediatrics Pneumonology and Allergology, Medical University of Warsaw, Warsaw, Poland Purpose/Objective: Allergen specific immunotherapy (ASIT) is being used for 100 years and is so far the only specific treatment of pollen/ dust allergies. In spite of that, objective laboratory test to monitor the results of ASIT is not available yet. Recently different studies started to highlight the link between regulatory T cells (Tregs) and the outcome of immunotherapy. Some authors demonstrated that upregulation in Tregs may be considered as response-related biomarker for ASIT. The aim of the study was to test the frequency of CD4+ CD25highFoxP3+ CD127) regulatory T cells in patients with allergy before and one year after SLIT. Materials and methods: The study involved 30 children with pollen allergy. All patients had characteristic symptoms of asthma, allergic rhinitis and allergic conjunctivitis. PBMCs were stained with monoclonal antibodies (anti-CD25 PE-Cy7, clone M-A251; anti-CD4 PECy5; anti-CD127 PE and anti-FoxP3 Alexa Fluor 488, clone 259/D, Becton Dickinson). The samples were evaluated using flow cytometer Beckman Coulter FC500. Results: Tregs in peripheral blood were identified as CD4+ CD25highFoxP3+ CD127) T cells. The number of Tregs is expressed as a percentage of all CD4+ T cells. The percentages of Tregs in both groups of patients (before n = 25 and after one year of SLIT n = 17) were similar (median 2.21; 2.81, respectively). Twelve patients were analyzed at both time points. Seven patients showed increase of Tregs after treatment and in case of 4 patients Tregs were lower. This results also did not differed significantly (before n = 12 median 2.67; one year after treatment n = 12 median 2.91). Conclusions: This preliminary study did not demonstrate that natural Tregs can serve as potential biomarker of effectiveness of SLIT. But definitive conclusions should be drawn after enlargement of study population.

P0825 Naive and memory T cell response to Der p 1 allergen presented by dendritic cells in the presence of by M. bovis BCG bacilli in allergic asthma M. Kowalewicz-Kulbat,* J. Staszek,* P. Szpakowski,* S. Kosinski, M. L. Kowalski, F. Bietà & J. Pestel§ *Department of Immunology and Infectious Biology, University of Lodz, Lodz, Poland, Department of Clinical Immunology and Microbiology, Medical University of Lodz, Lodz, Poland, àINRA Centre de Tours, UR 918, Nouzilly, France, §Universite des Sciences et Technique de Lille, UMR 8576, Lille, France Purpose/Objective: Dendritic cells (DC) are crucial in the regulation of Th1/Th2 polarization. Allergy is caused by an excessive development of Th2 profile in immune response to environmental allergens. Many


Poster Sessions 457 asthmatic patients develop allergy to Der p 1-major allergen of Dermatophagoides pteronyssinus house dust mite. Mycobacteria as strong Th1 inducers are potent candidates to diminish the harmful Th2 profile in allergic disorders. We asked a question whether in the presence of Der p 1, Mycobacterium bovis BCG vaccine and a recombinant BCG producing human IL-18 (rBCGhIL-18) were able to polarize Th2 towards Th1 lymphocyte response of naı¨ve and memory T cells. Materials and methods: Monocyte-derived dendritic cells (MoDC) were prepared from asthmatic patients who responded to Der p 1 in skin prick test and healthy BCG vaccinated donors. MoDC were stimulated for 24 h with Der p 1 in presence/absence of M. bovis BCG or rBCG-IL-18 bacilli. The response of naı¨ve and memory T cells to pulsed MoDC was evaluated by determining the IFN-gamma and IL-5 production by ELISA test. Results: In response to Der p 1-pulsed MoDC, autologous naı¨ve and memory T cells from allergic patients produced IL-5 significantly more frequently and more intensively as compared with T cells from healthy donors. However, in the group of allergic patient’s memory T cells, in response to Der p 1-MoDC released significantly more intensively IL-5 than naı¨ve T cells. BCG and rBCG-hIL-18 bacilli in the presence of Der p 1 presented by MoDC decreased the IL-5 production neither by naı¨ve nor memory T cells. In contrast to healthy donors, naı¨ve T cells from allergic patients, in response to Der p 1-stimulated MoDC produced significantly more intensively IFN-gamma than identically stimulated memory T cells. Interestingly, BCG and rBCGhIL-18 in the presence of Derp 1 significantly enhanced the intensity of IFN-gamma production by naı¨ve and memory T cells in the group of allergic patients and healthy donors. Conclusions: The BCG vaccine as well as rBCGhIL-18 was shown to display in vitro the ability to induce IFN-gamma production by naı¨ve and memory T cells without the significant IL-5 supression. Thus the potential immunomodulatory role of BCG in allergic asthma seems to be more essential for Th1 enhancement than Th2 supression. Supported by the MS grant N N401 015236

P0826 Participation of c-Jun N-terminal Kinase 2 (JNK2) in the induction of ovalbumin-induced bronchial asthma J. Mizuguchi & M. Furuhata Department of Immunology, Tokyo Medical University, Tokyo, Japan Purpose/Objective: Bronchial asthma is a chronic inflammatory airway disease characterized by airway hypersensitivity (AHR), inflammatory cell infiltration into the airways including T-helper 2 (Th2) cells, eosinophils, and macrophages. Th2 responses are regulated by several factors including GATA3 and mitogen-activated protein kinases (MAPKs). cJun N-terminal kinase (JNK)2, a member of MAPKs, plays a crucial role in the induction of cell proliferation, apoptosis, and inflammation. In the present study, we examined whether JNK2 is involved in the induction of ovalbumin (OVA)-induced bronchial asthma using JNK2-deficient mice. Materials and methods: C57BL/6J wild type (WT) and JNK2deficient mice were sensitized with OVA in PBS intraperitoneally, followed by intranasal administration of OVA. T and B cells from WT or JNK2-deficient mice cells were transferred into Rag2-deficinet mice and immunized with OVA, followed by intranasal administration of OVA. Mice were assayed for AHR, histological section, cytokine levels in bronchoalveolar lavage (BAL) fluid, and antibody production 24 h after the last OVA administration. Results: JNK2-deficient mice displayed reduced OVA-induced AHR, IgG1 and IgE antibody responses, and airway inflammatory lesion compared with WT mice. Cytokine levels including IL-4, IL-10, and

IFN-c in BAL fluid were also reduced in JNK2-deficient mice relative to WT mice. Transfer of JNK2-deficient T or B cells together with WT B or T cells into Rag2-deficinet mice resulted in diminished inflammatory responses compared with WT T cells plus WT B cells, suggesting that both T and B cells from JNK2-deficient mice are implicated in the pathogenesis of OVA-induced bronchial asthma. Conclusions: JNK2 activation is involved in the induction of OVAinduced bronchial asthma, probably through participation of both T and B cells.

P0828 Persistence of allergen-specific IgE responses in patients suffering from AIDS K. Marth,* E. Wollmann,* D. Gallerano,* R. Valenta* & E. Sibanda *Department of Pathophysiology, Center for Pathophysiology Infectiology and Immunology, Vienna, Austria, Asthma Allergy & Immune Dysfunction Clinic, Asthma Allergy & Immune Dysfunction Clinic, Harare, Zimbabwe Purpose/Objective: The infection of CD4+ cells by HIV leads to the progressive destruction of CD4+ T lymphocytes and, after massive reduction of CD4+ cells, to AIDS. To study if HIV-infected patients suffering from AIDS with a severe reduction of CD4+ cells can suffer from symptoms of IgE-mediated allergy and produce allergen-specific IgE antibody. Materials and methods: In total 69 HIV-infected allergic patients from Zimbabwe were studied. Among these patients, 27 had CD4 counts below 200. Allergy was diagnozed according to case history, physical examination and skin prick testing. Serological analysis of allergen-specific IgE antibodies was performed with an allergen chip, containing 163 purified allergen molecules or with the MAST-CLA assay, containing a panel of 36 allergen extracts. IgE antibody levels specific for seasonal allergens (Art v 1, Art v 3, Bet v 1, Cup a 1, Cyn d1, Ole e1, Phl p 1) were quantified with ImmunoCAP measurements when follow-up sera obtained at different time points were available. HIV infection was confirmed serologically and the disease was staged clinically. Determination of CD4+ and CD8+T lymphocyte subset numbers were performed by flow cytometry. Results: The predominant allergic symptoms of HIV-infected patients were IgE-mediated symptoms such as allergic rhinoconjunctivitis and urticaria whereas T cell mediated symptoms (e.g. atopic dermatitis) ceased in patients with very low CD4 counts. In accordance to the clinical symptoms IgE responses specific for house dust mite, grass pollen and moulds were most frequent. ImmunoCAP measurements of IgE levels specific for seasonal allergens indicated that even patients with CD4 counts 0.05). The body mass index was not altered after the change in diet (P = 0.660). Conclusions: Consumption of whole milk and butter for 3 months significantly decreases respiratory tract complaints in children. Wheezing, the only symptom reactive to medication (bronchodilatators), was not affected by the dietary advice. We conclude that a simple dietary change can improve respiratory tract complaints without side effects significantly.


Poster Sessions 465

Poster Session: DC, MHC, T Cells and Disease P0859 12/15-lipoxygenase-mediated lipid oxidation regulates maturation of dendritic cells T. Rothe,* E. Zinser, S. Ro¨ssner, S. Uderhardt,* V. Bochkov,à O. Oskolkova,à G. Schett,* A. Steinkasserer & G. Kro¨nke* *Department of Internal Medicine 3, Institute for Clinical Immunology, Erlangen, Germany, Department of Internal Medicine I, University Hospital Erlangen, Erlangen, Germany, àDepartment of Vascular Biology and Thrombosis Research, Medical University of Vienna, Vienna, Austria Purpose/Objective: The enzyme 12/15-lipoxygenase (12/15-LO) mediates lipid oxidation and its expression is restricted to different cells of the monocytic lineage. To further determine the physiological role of this enzyme, we studied a potential role of 12/15-LO during the regulation of DC maturation and the shaping of the consecutive T-cell response. Materials and methods: We analysed bone marrow-derived DCs of 12/15-LO-deficient mice with regard to their maturation status and cytokine profile in vitro. Moreover we examined the consequences of 12/15-LO-deficiency on autoimmune diseases like EAE. Results: Differentiated bone marrow-derived DCs highly expressed 12/15-LO. These cells were enriched in 12/15-LO-specific phosphatidylcholine-derived oxidation products (oxPAPC). Deletion of 12/15LO resulted in an increased expression of co-stimulatory molecules. Likewise, 12/15-LO-/- DCs displayed an altered cytokine profile with an elevated expression of the Th17-promoting IL-23 subunit p19. Addition of the 12/15-LO product oxPAPC, in turn, interfered with the maturation of DCs and the expression of p19. After initiation of a specific immune response 12/15-LO-deficient mice displayed an altered cytokine expression profile within their lymph nodes and showed an increased differentiation of Th17 T-cells. In accordance with these findings, the Th17-driven disease model of experimental autoimmune encephalomyelitis (EAE) exacerbated in 12/15-LO-/mice. Conclusions: Together these data identify 12/15-LO as a major source of bioactive phospholipid oxidation products in DCs and indicate a central role for enzymatic lipid oxidation during the regulation of the maturation status of DCs and the initiation of an adaptive immune response.

P0860 Activation of invariant natural killer T-cells in periodontitis lesions M. Nowak,* B. Kra¨mer, M. Haupt,à P. N. Papapanou,§ J. Kebschull,– P. Hoffmann,** S. Jepsen,à S. Perner,* I. G. H. Schmidt-Wolf & M. Kebschullà *Department of Prostate Cancer Research, Institute of Pathology, University of Bonn, Bonn, Germany, Department of Internal Medicine I, University of Bonn, Bonn, Germany, àDepartment of Periodontology Operative and Preventive Dentistry, University of Bonn, Bonn, Germany, § Division of Periodontics Section of Oral and Diagnostic Sciences, Columbia University College of Dental Medicine, New York, NY, USA, – Cold Spring Harbor Laboratory, Watson School of Biological Sciences, Cold Spring Harbor, NY, USA, **Department of Human Genetics, University of Bonn, Bonn, Germany, Department of Internal Medicine III, University of Bonn, Bonn, Germany Purpose/Objective: Periodontitis is one of the most prevalent human inflammatory diseases. The major clinical phenotypes of this polymicrobial, biofilm-mediated disease are chronic and aggressive periodontitis, the latter being characterized by a rapid course of destruction that is generally attributed to an altered immune-inflammatory response against periodontal pathogens. Whereas aggressive

periodontitis has been causally linked to infection with Aggregatibacter actinomycetemcomitans (A.a.), the major pathogen in chronic periodontitis is Porphyromonas gingivalis (P.g.). Still, the biological basis for the pathophysiological distinction of the two disease categories has not been well documented yet. Type I natural killer T (NKT) cells are a lymphocyte subset with important roles in regulating immune responses to either tolerance or immunity, including responses against bacterial pathogens. Here, we delineate the mechanisms of NKT cell activation in periodontal infections. Materials and methods: Dendritic cells (DC) were generated from murine bone marrow by 6-days-culture with GM-CSF. NKT cells were isolated from murine livers. DC were challenged with A.a. or P.g. for 24 h. before they were used as stimulator cells for NKT cells for 24 48 h. before cytokines were tested by ELISA. Inflammatory responses in DC upon challenge with A.a. and P.g. were analyzed by Illumina microarray analysis of in-vitro infected DC. De novo glycosphingolipid turnover in bacterial challenged DC was tested using a fluorescent alpha-galactosidase A substrate and flow cytometry. Results: We show a selective infiltration of type I NKT cells in aggressive, but not chronic periodontitis lesions in vivo. Murine DCs infected with A.a. triggered a type I interferon response followed by type I NKT cell activation. This stimulation was dependent on the expression of CD1d molecules and signaling via Toll-like receptors in DCs. In contrast, infection with P.g. did not induce NKT cell activation. The stimulatory capacity of DCs on NKT cells required DC self-activation through binding of type I interferon receptors and betainterferon secretion. Addition of exogenous beta-interferon to P.g.infected DCs restored the ability to activate NKT cells. Conclusions: Our study provides a conceivable biological distinction between the two periodontitis subforms and identifies factors required for the activation of the immune system in response to periodontal bacteria.

P0861 Activation of Th17 pathway in potentially prediabetic first degree relatives and type 1 diabetic patients after exposure of diabetesassociated autoantigens J. Vcelakova,* L. Petruzelkova,* V. Stavikova,* S. Kolouskova,* Z. Vesely & K. Stechova* *2nd School of Medicine Charles University and University Hospital Motol, Paediatrics, Prague, Czech Republic, 2nd School of Medicine Charles University and University Hospital Motol, Bioinformatics Centre, Prague, Czech Republic Purpose/Objective: Type 1 Diabetes (T1D) appears to be mainly a Th mediated autoimmune process however its complexity is still not fully understood. New discovery of molecule or pathways converge in betacell destruction is a potential target for future therapeutic avenues. In our study we analysed the effect of diabetes-associated autoantigens on peripheral blood mononuclear cells (PBMC) by gene expression microarray and quantitative RT-PCR with the aim to identify prediabetes-associated cell processes. Materials and methods: PBMC were sampled from first degree relatives of T1D patients (FDR), T1D patients and healthy controls (HC). Isolated PBMC were cultivated with and without mixture of diabetogenic autoantigens (GAD65, IA2 and proinsulin derived peptides) for 72 h. Then co-culture total RNA was isolated and gene expression arrays were done on 43 probands by high density Phalanx array. We saw the significant changes in Th-17 pathway. In the next step we analysed 53 probands by qRT-PCR TaqMan Gene Expression assays (Life Technologies) focusing on known players of this pathway (RORC, STAT3, IL-17A and TGFb). Results: We observed higher expression of IL-17A at T1D patients compared to FDR autoantibody negative (P = 0.035). Interestingly we saw significantly higher gene expression of TGFb (P = 0.01) and


466 Poster Sessions STAT3 (P = 0.007) comparing T1D patients and FDR autoantibody positive to HC. Gene expression of RORC had similar trend as the TGFb and STAT3 but the difference was not significant (P > 0.05). Conclusions: TGFb cytokine is responsible for Th17 and Treg lymphocytes balance. In our study higher expression of TGFb is associated with Th17 pathway change which is in contrast to previous study. Better understanding of this step can contribute developing immune-based intervention therapy. Supported by the project (Ministry of Health, Czech Republic) for conceptual development of research organization 00064203 (University Hospital Motol, Prague, Czech Republic)

P0862 Aggravation of atherosclerosis by MHC class II antigen deficiency is associated loss of regulatory T cells and expansion of proinflammatory Ly-6hi monocytes M. Wigren, S. Rattik, H. Bjo¨rkbacka, G. Nordin Fredrikson & J. Nilsson Lund University, Clinical Sciences, Malmo¨, Sweden Purpose/Objective: Adaptive immunity in atherosclerosis includes both pro- and anti-inflammatory immune responses. It has been assumed that atherosclerosis involves a loss of tolerance against modified self-antigens generated in response to hypercholesterolemia and that presentation of such antigens on MHC class II molecules lead to activation of pro-inflammatory Th1 cells. Furthermore, anti-inflammatory Tregs have been shown to reduce atherosclerosis development indicating that it might be possible to modulate the balance of proand anti-inflammatory responses. We have previously shown that immunizations of hypercholesterolemic mice with an Apo B-100 peptide based vaccine have the ability to reduce atherosclerosis and at the same time increase Treg frequency. Moreover, in hypercholesterolemic mice immunizations with the adjuvant Alum alone also reduce atherosclerosis and increase Tregs which is associated with a capture of oxLDL antigens. Materials and methods: To further study the role of adaptive immune responses in atherosclerosis we have used hypercholesterolemic mice lacking MHC class II (ApoE-/- MHCII-/-). Results: As expected ApoE-/- MHCII-/- mice had reducedlevels of CD4+ cells, including Tregs as well as low levels IgG and IgM and Th1 and Th2 cytokines in plasma. CD115+ monocytes were reduced in spleen as well as the plasma levels of TNF-a, IL-1b and IL-6 indicating reduced systemic inflammation. In spite of this, ApoE-/- MHCII-/mice had significantly more atherosclerosis as assessed both by en face Oil Red O staining of the aorta (4.7 ± 2.9% versus 1.9 ± 1.3%; P < 0.01) and cross-sectional area of subvalvular lesions (7.7 ± 2.2 · 105 versus 4.6 ± 2.8 · 105 mm2; P < 0.05). Moreover, macrophage accumulation in lesions was significantly increased (44.8 ± 8.0% versus 24.8 ± 7.8% MOMA-2 stained area; P < 0.001). Furthermore, pro-inflammatory Ly-6chi monocytes were increased in the spleen. Conclusions: The present observations unexpectedly show that the net effect of MHC class II-dependent antigen presentation in atherosclerosis is athero-protective. The combination of reduced frequency of protective Tregs together with the increase in disease promoting Ly-6chi monocytes can be the cause of the increased atherosclerosis in this animal model.

P0863 Allergic diseases exhibit various TREC number in CD4+ and CD8+ T-cell subpopulations E. A. Blinova,* V. S. Kozhevnikov* & V. A. Kozlov *Research Institute of Clinical Immunology RAMS, laboratory of clinical immunopathology, Novosibirsk, Russia, Research Institute of Clinical Immunology RAMS, laboratory of immunopoiesis regulation, Novosibirsk, Russia Purpose/Objective: We used T-cell receptor excision circles (TREC) to investigate the mechanisms for replenishment of the peripheral T cell poll in allergic diseases. The study included 26 patients with atopic dermatitis (AD), mean age 26 ± 9.7 years and 50 patients with different form of bronchial asthma (BA), mean age 43 ± 13.8 years. Since the number of TREC decreased with age, for each group of patients was selected a group of 22 healthy people. The average age of donors was 27 ± 4.6 years for patients with AD, and 43 ± 12.3 years for patients with BA, respectively. Materials and methods: TREC number was measured by quantitative Real-time PCR technique in pure populations of CD4+ and CD8+ lymphocytes using specific primers, probe and standard. CD4+ and CD8+ T cells were sorted by flow cytometer FACSAria (Beckton Dickinson) using fluorescent-conjugated anti-CD3 and anti-CD4 antibodies. Reanalysis of the isolated subsets showed that the purities were at least 95%. Results: Patients with AD had reduced TREC levels in CD4+ and CD8+ cells, but absolute TREC content in this populations was the same as in normal controls. Also absolute number of CD4+ and CD8+ cells was increased in AD. In contrast, patients with BA had no dissimilarities in the TREC number within both CD4+ and CD8+ Tcell subpopulations from healthy volunteers, as there were no significant differences in TREC content depending on the form of asthma and IgE levels. But a large variation was observed in TREC level according to the duration of the disease. Adults suffering from asthma about 1 year had increased absolute and relative numbers of TREC regarding to healthy controls, whereas in patients with disease duration >1 year they were comparable with donor values. Both groups of patients with the different disease duration had enlarged absolute number of CD4+ T cells and slightly extended absolute number of CD8+ T cells. Conclusions: Thus, men suffering from AD have a normal thymic function, and TREC ‘dilution’ caused by a high rate of the peripheral T cell proliferation under conditions of the chronical antigen stimulation. In the onset of BA peripheral expansion and thymopoiesis are imbalance, and thymic emigrants make a grater contribution to the replenishment of the T cell pool. Patients suffering from asthma more than 1 year received additional corticosteroids therapy, that may be related to the turning of TREC level to the normal range in these people.

P0864 Altered co-stimulatory phenotype of Nrf2 knockout dendritic cells is not a result of elevated ROS levels L. Abbas Al-Huseini, S. Sethu, J. Hamdam, Y. Tingle, C. Goldring, K. Park & J. Sathish Translatinal Medicine, Molecular and Clinical Pharmacology, Liverpool, UK Purpose/Objective: Dendritic cells (DCs) are antigen-presenting cells which play a pivotal role in adaptive immune responses. Cellular redox state has an important impact in DC immune function. The redox homeostasis in DCs is mainly controlled through the activity of the transcription factor, Nrf2. Our previous findings revealed that loss of Nrf2 resulted in enhanced co-stimulatory molecule expression and altered immune functions in dendritic cells. In the current study, we


Poster Sessions 467 investigated whether the loss of Nrf2 alters basal ROS levels in DCs. Additionally; we tested whether the increase in the co-stimulatory molecule expression is directly due to any changes in ROS levels in Nrf2 knockout DCs. Materials and methods: Bone marrow-derived immature DCs from Nrf2 knockout and Nrf2 wild type mice were assayed for ROS levels by flow cytometry. ROS levels were lowered by treatment with vitamins C and E (ROS scavengers) followed by evaluation of co-stimulatory molecule expression and ability of DC to stimulate T cell proliferation. Results: Nrf2 knockout DCs have higher levels of basal ROS compared to Nrf2 wild DCs. Vitamin C & E treatment reduced the levels of ROS in Nrf2 knockout DCs to levels comparable to that of the wild type. However, elevated co-stimulatory molecule (MHCII and CD86) expression of Nrf2 knockout DCs could not be reversed by vitamins treatment. Conclusions: Our results indicate that the increase in ROS levels in Nrf2 knockout DCs does not contribute to the increase in the costimulatory molecule expression.

P0865 Amelioration of experimental autoimmune encephalomyelitis by quinoline-3-carboxamide ABR-215757 is due to target of T cell activation S. Helmersson,* A. Sundstedt, P. Andersson,* T. Leanderson* & F. Ivars* *Lund University, Immunology, Lund, Sweden, Active Biotech AB, Active Biotech AB, Lund, Sweden Purpose/Objective: ABR-215757 (5757) is a quinoline-3-carboxamide (Q-compound) currently in clinical development for systemic lupus erythematosus (SLE). Q-compounds have shown efficacy in different autoimmune diseases where they ameliorate the disease. However, the mechanism of action is still unknown. Materials and methods: Experimental autoimmune encephalomyelitis (EAE) is a T cell dependent mouse model of multiple sclerosis. We have used this model to try to understand the mechanism underlying the amelioration by 5757. Results: Of the 5757 effectively reduces induction of EAE. This mechanism is an early effect since treatment with 5757 during the first 5 days of the disease also reduces EAE development. Influx of T cells and myeloid cells to the brain is significantly reduced by 5757. EAE is a T cell dependent disease and the activation of T cells occurs early in the peripheral lymphoid organs. T cells show a reduced proliferation in the presence of 5757. The effector function of the T cells is affected since T cells from treated animals show a reduced production of IFN-c and IL17. The ex vivo antigen specific recall response is also heavily reduced in cells from 5757 treated animals. Conclusions: Taken together, this suggests that 5757 treatment affects T cell activation and maturation of effector cells, thereby reducing EAE development.

P0866 Analysis of in vitro T-cell responses of PBMCs from patients with pemphigus vulgaris and healthy controls using desmoglein 3 peptides and peptide conjugates as antigens H. Szabados,* K. Uray,* P. Sillo´, F. Hudecz,à S. Ka´rpa´ti & S. Bo¨sze* *Eo¨tvo¨s Lorańd University, Research Group of Peptide Chemistry Hungarian Academy of Sciences, Budapest, Hungary, Department of Dermato-Venereology and Skin Oncology, Semmelweis University, Budapest, Hungary, àDepartment of Organic Chemistry, Eo¨tvo¨s Lorańd University, Budapest, Hungary Purpose/Objective: Pemphigus vulgaris (PV) is a rare, life-threatening autoimmune bullous skin disorder. The presence of autoreactive serum

antibodies (IgG1 and IgG4) against desmosomal adhesion proteins, desmoglein 1 (Dsg1) and desmoglein 3 (Dsg3), results in mucosal lesions, mucocutaneous blisters and erosions. Autoantigen-specific Tcells also play a crucial role in the initiation and perpetuation of Dsg3/ Dsg1-specific T-cell responses. In PV the CD4+ autoreactive T-cells recognize specific immunodominant regions from proteins Dsg3 and/ or Dsg1. Patients show autoimmunity only to protein Dsg3 in the early stage of disease, while at the later stage, non-crossreactive immunity appears to both proteins Dsg3 and Dsg1. Protein Dsg3 is the primary target antigen in PV. Materials and methods: In our study T-cell epitope regions have been selected and represented by synthetic oligopeptides. Synthetic peptides with distinct in vitro T-cell response on the peripheral blood monomorphonuclear cells (PBMC) isolated from PV patients have been chosen for conjugation. Conjugating the T-cell peptides to carrier molecules can increase their in vitro stimulating activity on PBMC. The selected peptides were conjugated to oligotuftsin [(TKPKG)4] carrier. These peptides and peptide conjugates were used for in vitro stimulation of the PBMC from PV patients and healthy controls. After 24 and 48 h of incubation the produced IFN-c was determined from the supernatants by ELISA. Results: The synthetic Dsg3 peptides and peptide-oligotuftsin conjugates induced different in vitro IFN-c production rate on PBMC obtained from PV patients and healthy controls determined by ELISA. The in vitro stimulatory activity of oligopeptides and peptideconjugates will be discussed. Conclusions: Our approach identified promising oligopeptide conjugate candidates as synthetic antigens.

P0867 Analysis of peripheral blood CD56+ and CD57+ NK and NKT cells subsets in pregnant women K. Halacheva,* S. Buzalov, M. Angelova, B. Popov* & E. Slavov* *Trakia University Medical faculty, Molecular biology immunology and medical genetics, Stara Zagora, Bulgaria, Trakia University Medical faculty, Obstetrics and Gynecology, Stara Zagora, Bulgaria Purpose/Objective: Increased peripheral blood natural killer (NK) cells have been associated with unexplained reproductive failures. Data about the functional role of different NK cells and natural killer T cells (NKT) subsets during pregnancy are contradictory. The aim of this study was to investigate the differences in the relative distribution and the phenotype characteristic of peripheral blood subpopulations of NK and NKT cells in pregnant women with unexplained pregnancy loss compared to healthy pregnant women. Materials and methods: Fifteen women in the first trimester of pregnancy with a history ofpregnancy loss (Group I), 12 healthy women in the first trimester of pregnancy (Group II), and 27 nonpregnant fertile women with a history of successful pregnancies (Controls) were included in this study. Five-color flow cytometry was used to assess the percentage of peripheral blood CD3- CD56+ cells (CD56-NK), CD3- CD57+ cells (CD57-NK), CD3+ CD56 + (CD56-NKT, CD3+ CD57+ cells (CD57-NKT). Results: CD56-NK and CD57-NK as a mean percentage of total peripheral lymphocytes were significantly decreased in the healthy pregnant women (8.97 ± 3.09 and 3.42 ± 1.75, respectively) compared to controls (12.64 ± 4.4 and 5.73 ± 2.63) and to Group I (13.32 ± 4.92 and 6.22 ± 2.81). Pregnant women with a history of pregnancy loss had significantly higher percentage of CD56-NK and CD57-NK (13.32 ± 4.92 and 6.22 ± 2.81) than those of Group II (8.97 ± 3.09 and 3.42 ± 1.75), but no differences were found when compared to controls (12.64 ± 4.4 and 5.73 ± 2.63). CD56-NKT percentage was nearly equal in all study groups (Group I 5.66 ± 3.08, Group II 4.64 ± 2.97, Controls 4.50 ± 2.71). CD-57-NKT was significantly decreased in healthy pregnant women (6.02 ± 4.35) compared to


468 Poster Sessions Group I (10.30 ± 5.67) and decreased without reaching statistical significance compared to Controls (9.00 ± 5.33). Conclusions: On the basis of these results we may suppose that CD57 positive NKT and NK subsets may be involved in the immunological phenomenon of pregnancy and in the immune response disturbances in risk pregnancy.

P0868 Antigen presenting cells are important to modulate CD4+ T cells in renal ischemia and reperfusion injury M. T. Amano, M. Correa-Costa, T. T. Braga, V. Andrade-Oliveira, A. Castoldi, M. Y. Miyagi, M. I. Hiyane & N. O. S. Camara Institute of Biomedical Sciences University of Sao Paulo, Immunology, Saõ Paulo, Brazil Purpose/Objective: Ischemia and reperfusion injury (IRI) is an acute inflammatory response considered to be the main cause of acute renal injury in kidneys. Antigen presenting cells (APC), as dendritic cells (DC) and macrophages, have an important role in IRI, although a few is known about their functionality and their possible role in T cell activation in this model. In this way, we investigated whether APC were involved in IRI and their participation in T cell modulation in this model. Materials and methods: We used C57Bl/6 and C57Bl/6- CD11c-DTR mice to perform the ischemia and sham group as control. In order to deplete, we used a clodronate protocol to deplete phagocytes or the CD11c-DTR mice diphtheria toxin (4 ng/g of mice) to deplete CD11c+ cells. Results: We observed that after 24 h of reperfusion, urea (>250 mg/ dl) and creatinine (>1 mg/dl) levels were increased in ischemic group, indicating injury. No difference of DC numbers were observed in the kidney and draining lymph nodes, however, DC from ischemic group were more activated, presenting more pronounced expression of CD86. We then used two models of depletion of APC, as mentioned above. Both protocols resulted in increased levels of urea (>300 mg/dl) and creatinine (>2 mg/dl) in the serum of APC depleted mice. In order to investigate the influence of DC in CD4+ T cell modulation in IRI, we analyzed the phenotype of CD4+ T cells in DC depleted mice after ischemia and reperfusion induction and we observed that the population of CD4+ CD69+ (activated) T cells was increased, while no differences were observed in CD4+ CD25+FOXP3+ T cells (Tregs), indicating that DC might be important for the regulation of T cell response in IRI. Conclusions: We concluded that APC are involved in IRI and they modulate CD4+ T cells in order to control the injury. Support: FAPESP, CNPq.

P0870 Assessment of CD8 positive NK cells and T cells subsets in pregnant women E. Slavov,* S. Buzalov, M. Angelova, B. Popov* & K. Halacheva* *Trakia University Medical faculty, Molecular biology immunology and medical genetics, Stara Zagora, Bulgaria, Trakia University Medical faculty, Obstetrics and Gynecology, Stara Zagora, Bulgaria Purpose/Objective: The purpose of the study was to estimate the phenotype alterations in the peripheral blood NK and T lymphocyte subsets of women with unexplained pregnancy loss compared to healthy pregnant women. Materials and methods: Fifteen women in the first trimester of pregnancy with a history of previous pregnancy loss (Group I), 11 nonpregnant women with a history of previous pregnancy loss (Group II), 12 women in the first trimester of pregnancy with no history of previous pregnancy loss (Group III), and 27 healthy fertile women with

a history of successful pregnancies (controls) were included in the study. Five-color flow cytometric analysis was used to assess the percentage of CD3- CD56+ cells (NK), CD3+ CD56+ T cells (NKT), CD8+ CD11b+ cells (CD8/CD11b) and their subsets: CD3CD56+ CD8+ CD11b+ NK cells (CD8+NK), CD3- CD56+ CD8bright CD11b+ NK cells (CD8brNK), CD3- CD56+ CD8- CD11b+ NK cells (CD8-NK), CD3+ CD56+ CD8+ CD11b+ NKT cells (CD8+NKT), CD3+ CD56+ CD8bright CD11b+ NKT cells (CD8brNKT), CD3+ CD56+ CD8- CD11b+ NKT cells (CD8-NKT), and CD3+ CD56- CD8+ CD11b+ T cells (CD8+T). Results: We found that the percentage of NK (8.9 ± 3.0) and CD8/ CD11b (9.4 ± 4.7) in Group III was significantly lower compared to controls (12.6 ± 4.4; 13.4 ± 4.8 respectively). The percentage of these populations in Group I and Group II did not differ compared to controls. The percentage of NK in Group I (13.3 ± 4.9) was significantly higher, compared to Group III. The percentage of NKT in all the groups was nearly equal. A significantly lower percentage of CD8+T was detected in Group III compared to controls (3.2 ± 2.4 versus 5.0 ± 1.8) and to Group II (3.2 ± 2.4 versus 6.6 ± 4.8). The percentage of CD8-NK and CD8-NKT cells was lower in Group III compared to Group I and Group II. The percentage of CD8+NK and CD8+NKT in all the groups was nearly equal. A significantly lower percentage of CD8brNK and CD8brNKT cells was detected in Group III compared to Group II. Conclusions: Our results can suggest that, in addition to NK cells, T cells with a phenotype CD3+ CD56- CD8+ CD11b+ could be involved in the immune response during normal pregnancy. Further studies are needed to clarify the role of CD8+ CD11b+ T cells, CD8+NK and CD8+NKT cell populations in complications of pregnancy.

P0871 Astrocytic FasL induces apoptosis of autoimmune T cells and is crucial for recovery from EAE X. Wang,* F. Haroon,* S. Karray, M. Deckertà & D. Schlu¨ter* *Institute of Medical Microbiology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany, INSERM U753, Institut Gustave Roussy, Villejuif, France, àInstitute of Neuropathology, University of Cologne, Cologne, Germany Purpose/Objective: Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease characterized by accumulation of T cells in the CNS and its recovery requires the termination of inflammation and apoptosis of infiltrating T cells in the CNS. However, the T cell apoptosis inducing cell population still remains to be identified. To address the role of astrocytic FasL in the regulation of T cell apoptosis in EAE, astrocyte-specific FasL deficient mice were generated and investigated for EAE. Materials and methods: Astrocyte-specific FasL knockout mice were generated by utilizing the Cre/loxP system under control of the human glial fibrillary acid protein (GFAP) promoter. EAE was induced by immunizing mice with MOG35 55 peptide and assessed daily by clinical score. Demyelination was detected by histology. Infiltrating leukocytes were isolated from spinal cord and characterized by flow cytometry. Expression of cytokines in spinal cord was analyzed by qRT-PCR. In vitro co-cultures of FasL+ and FasL- astrocytes, respectively, with T cells were used to study the induction of T cell apoptosis as determined by annexin V and caspase-3 staining. Results: FasL was efficiently and specifically deleted in astrocytes of GFAP-cre FasLfl/fl (KO) mice, while its expression was not affected in FasLfl/fl (control) mice. Compared with FasLfl/fl littermates, GFAP-cre FasLfl/fl mice developed significantly more severe EAE accompanied with widespread demyelination and more T cell infiltration. At day 22 post immunization, mRNA levels of IL-17, IFN-g, TNF and GM-CSF were also significantly elevated in the spinal cord of GFAP-cre FasLfl/f


Poster Sessions 469 mice. Consistently, astrocytes isolated from GFAP-cre FasLfl/fl mice failed to induce T cell apoptosis in vitro. Conclusions: FasL expression of astrocytes is required for the induction of T cell apoptosis and the elimination of infiltrating T cells in the CNS, which contributes to the recovery of EAE.

P0872 Calcitriol modulates the CD46 pathway by controlling its expression and function on T cells K. Kickler,* S. Ni Choileain,* A. Williams, A. Richards* & A. L. Astier* *MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK, MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK Purpose/Objective: The complement regulator CD46 is a costimulatory molecule for human T cells that induces a regulatory Tr1-like phenotype, characterized by large amounts of IL-10 secretion and low levels of IFNg. Secretion of IL-10 upon CD46 costimulation is largely impaired in T cells from patients with multiple sclerosis (MS). Vitamin D can exert a direct effect on T cells, and may be beneficial in several pathologies, including MS. Herein, we examined whether active vitamin D (1,25(OH)2D3 or calcitriol) could modulate the CD46 pathway and restore IL-10 production by CD46-costimulated T cells from patients with MS. Materials and methods: CD4+ T cells were purified from the blood of 15 healthy donors and 11 patients with relapsing-remitting MS (7 IFNbeta treated, 4 untreated). We compared T cell responses to CD3/ CD46 costimulation in presence or absence of calcitriol. We assessed the levels of expression of CD46, CD25 and CTLA-4 on activated T cells, and we measured proliferation and quantified IL-10 and IFNg production. Results: In healthy T cells, calcitriol increased expression of CD25 and CTLA-4 expression on CD46-costimulated T cells, while it concomitantly decreased CD46 expression. Similar changes were observed in MS T cells except for CD25: while CD25 was normally induced by CD46 costimulation in MS T cells, addition of calcitriol consistently inhibited its induction. Despite the aberrant effect on CD25 expression, calcitriol increased the IL-10:IFNg ratio, characteristic of the CD46-induced Tr1 phenotype, in both T cells from healthy donors and patients with MS. Moreover, bystander CD4+ T cells activated in presence of supernatants from CD46-costimulated T cells with calcitriol exhibited a lower proliferation rate. Conclusions: We show that calcitriol affects CD46 expression and functions, and that it promotes anti-inflammatory responses mediated by CD46. Moreover, it might be beneficial for T cell responses in MS, although further studies should be performed to fully understand the effects of calcitriol on the CD46 pathway in T cells.

P0873 CCL17-producing dendritic cells at the crossroads of intestinal inflammation A. Heiseke,* I. Fo¨rster, W. Reindl* & A. Krug* *Klinikum rechts der Isar TUM, 2. Medizinische Klinik, Mu¨nchen, Germany, Life & Medical Sciences (LIMES) Institute Universita¨t Bonn, Immunology and Environment, Bonn, Germany Purpose/Objective: The incidence of intestinal inflammations such as Crohn’s disease and ulcerative colitis is constantly rising in Western countries. As current treatment options are not effective in all patients the need to find new drug targets is urgent. CCL17, a chemokine predominantly produced by dendritic cells (DCs) has been implicated to have proinflammatory roles in several atopic diseases as well as atherosclerosis. As central player of the innate and adaptive immune

system, DCs play a fundamental role in the development of intestinal inflammation. Recent publications provided insights in the diversity of DC subsets involved in this process. In the work presented here, we assessed the role of CCL17 and CCL17-expressing DCs in particular, in murine experimental colitis. Materials and methods: By challenging CCL17-deficient mice with chemically and immunologically induced colitis, the role of CCL17 was investigated. To characterize the different subsets of DCs cells within the secondary lymphatic tissues during colitis development multicolour FACS analysis and functional ex vivo assays were performed. Results: CCL17-deficient mice displayed markedly reduced signs of inflammation after disease induction in two models of experimental colitis. Reduced disease development was associated with diminished levels of TH1/TH17 cells, as well as higher levels of Foxp3+ regulatory T cells. Characterization of cell infiltrates into the lamina propria revealed that inhibition of colitis induction in CCL17-deficient mice was accompanied by increased numbers of CD103+ DCs, as well as reduced expression of CCR7, crucial for recruitment to the mesenteric lymph nodes, on DCs within the colon. Further, the majority of CCL17-expressing DCs in the inflamed colon are found in the MHCIIhighCD11b+ CD103+/- CD8a- intestinal DC subset, which was shown to have proinflammatory activity. Conclusions: The work presented here clearly demonstrates a central role for CCL17 and CCL17-producing DCs in the development of intestinal inflammation. The in-depth characterization of these DCs will provide further essential insights in the processes shaping the intestinal immune response.

P0874 CD8 T cell Dendritic Cell crosstalk up-regulates CD40L expression and IL-12p70 production Q. W. Tay, L. C. Ho, P. Y. Low & D. M. Kemeny National university of Singapore, Microbiology, Singapore, Singapore Purpose/Objective: CD8 T cells have been shown to skew the immune response by stimulating dendritic cells (DC) to produce IL-12p70. The purpose of our study was to investigate the effect on CD8 T cell expression of CD40L. Materials and methods: Ovalbumin (OVA)-specific T cell receptor transgenic mouse CD8 T cells (OT-I) were isolated by positive selection using anti-CD8 coated MACS beads and co-cultured splenic DC isolated using Optiprep and by positive selection with anti-CD11c coated MACS beads, and cultured in complete medium (RPMI 1640, 10% Fetal calf serum (FCS), antibiotics and non-essential amino acids and 0.5% 2 mercaptoethanol). CD40L expression was measured by Flow cytometry, IL-12p70 was determined by ELISA (R&D systems). CD8 T cells were activated with PMA (10 ng/ml) and Ionomycin (400 ng/ml) to induce CD44high effector memory cells. CD11c+ splenic DCs were pulsed with 1 lg/ml of peptide (typically SIINFEKL) for 1 h before co-culture with pre-activated CD8 T cells at a ratio of 1 DC to 3 T cells. Altered SIINFEKL peptides (SAINFEKL, EIINFEKL, SIIRFEKL, SIINYEKL) were purchased from AnaSpec Inc (USA). Results: Co-culture of effector memory CD8 T cells with DC up regulated CD8 T cell expression of CD40L that reached a maximum after 6 h. IL-12p70 levels in the culture reached 80% of its maximum value after 8 h. Altered peptide ligands SAINFEKL and SIINYEKL induced intermediate levels of CD40L while EINFEKYL and SIIRFEKL failed to induce CD40L on CD8 T cells. There was a corresponding reduction in IL-12p70 in the medium. Using CD8 T cells stimulated with anti-CD3 and CD28 addition of IL-12p70 enhanced CD40L expression on CD8s and addition of IL-12p70 neutralising antibody reduced CD40L expression. Conclusions: CD8 T cell receptor dependent signals stimulate DCs to secrete IL-12p70 that up-regulates CD40L expression on CD8 T cells.


470 Poster Sessions P0875 Characterization of swine skin and lung dendritic cells and their response to influenza virus N. Bertho,* P. Maisonnasse,* E. Bouguyon,* F. Marquet,* B. Da Costa,* G. Simon & I. Schwartz-Cornil* *INRA, Animal Health, JOUY-EN-JOSAS, France, ANSES, Porcin Virology and Immunology Unit, Ploufragan, France Purpose/Objective: Swine is a natural host of influenza virus. Swine influenza strains can contaminate humans. Once infected, swine present identical symptoms as human, such as anorexia, pyrexia, cough, fever and nasal discharge. It has been recently shown in the mouse that dendritic cells (DC), and among them inflammatory TNF/ iNOS-producing DC (Tip DC), are partly responsible both for the virus clearance and for the inflammatory pathology (Aldridge, PNAS 2009). Moreover, lung DC can be infected by influenza virus, although, interestingly, only the cross-priming CD103pos DC subpopulation actually releases viral particles (Moltedo, PlosPathogen 2011). To establish if these DC/influenza interactions are idiosyncratic in the mouse model or can be generalized to natural hosts of influenza virus, we wanted: 1 To characterize the swine DC subpopulations network, first in the skin and then in the lung. 2 To describe their susceptibility to influenza virus infection and their capacity to produce new virions. Materials and methods: Primary skin and lung cells were extracted and Facs-phenotyped (CadM1, CD206, CD209, CD14). Putative DC subpopulations were cell-sorted and the expression of genes specific for DC subpopulations (Flt3, ZBTB46, MAFB, BatF3, CSF1-R, XCR1, CX3CR1, CCR2, CLEC10A) were tested by q-PCR. Lung DC were infected with swine influenza virus and effective infection and virus production were tested by NP and NS1 immunofluorescence staining and by titration of the virus release. Results: In a previous study, we described the different DC subpopulations present in pig skin (Marquet, PlosONE 2011). The deepening of this last study allowed us to revisit the classification of human skin DC subpopulations according to phenotypic and transcriptomic similarities between swine, human and mouse subsets, by reclassifying CD14pos dermal DC as monocyte-derived DC and by identifying unambiguously the CD172aneg/CadM1pos/XCR1pos cDC as equivalent to the murine CD103pos cross-priming dermal DC. We then identified the lung counterparts of these skin DC subpopulations and described their capacities to replicate influenza virus. Conclusions: All together these data establish the swine as a model of choice for the study of normal and pathologic immune responses against influenza infection.

P0876 Circulating dendritic cells of multiple sclerosis patients are dysregulated and their frequency is correlated with MS-associated genetic risk factors N. Hellings,* K. Thewissen,* A. H. Nuyts, N. Deckx, V. F. I. Van Tendeloo, P. Stinissen,* Z. N. Berneman & N. Cools *Hasselt University, Biomedical Research Institute (BIOMED), Diepenbeek, Belgium, Antwerp University, Experimental Hematology, Antwerp, Belgium Purpose/Objective: Dendritic cells (DC) are widely known as professional antigen-presenting cells and provide an important link to the adaptive immune system where they regulate the balance between immunity and tolerance. Alternations in the DC compartment can ultimately lead to the induction or perpetuation of autoimmune diseases such as multiple sclerosis (MS). This study aims to identify alterations in DC phenotype and functionality in MS. Moreover, the contribution of genetic risk factors to DC alterations was determined.

Materials and methods: An ex vivo analysis of myeloid (mDC) and plasmacytoid DC (pDC) was carried out on peripheral blood of MS patients (n = 104) and age- and gender-matched healthy controls (HC, n = 112). Frequencies and expression of costimulatory (CD80 and CD86) and migratory molecules (CD62L, CCR5 and CCR7) were investigated. Interleukin (IL)-12p70 and interferon (IFN)-a secretion was measured following Toll-like receptor (TLR) challenge. Study subjects were genotyped for HLA-DRB1*1501 and IL-7R a. Results: A significant decrease of circulating pDC was found in peripheral blood of patients with chronic progressive MS (CPMS) compared to relapsing-remitting (RR) MS and HC. No differences in blood frequencies of mDC were found between different study groups. Both mDC and pDC of MS patients show shifts in the expression of CD86, CCR5 and CCR7 indicating that activation and migratory patterns of DC change during MS. Moreover, RRMS patients showed a reduced upregulation of CD86 on pDC and enhanced IL-12 production by mDC after TLR ligation, indicative of altered DC responsiveness. Treatment of MS is associated with a decrease of CD62L-positive mDC and pDC. HLA-DRB1*1501 carriers have reduced frequencies of circulating mDC as compared to non-HLA-DRB1*1501 carriers. Moreover, patients not carrying the protective IL-7Ra haplotype 2 have lower frequencies of pDC in the peripheral blood, indicating that genetic risk factors may impact the DC compartment of MS patients. Conclusions: Our data indicate that circulating DC subsets undergo changes in phenotype and functionality during MS disease. This study further provides evidence that MS-associated genetic risk factors such as HLA-DRB1-1501 and absence of IL-7Ra haplotype 2 have an impact on the DC compartment and thereby may contribute to the induction and/or maintenance of autoimmune responses.

P0878 Contrasting responses of DC and NK cells to IFN-I contribute to host resistance to viral infection T. P. Vu Manh,* T. Baranek,* Y. Alexandre,* N. Zucchini,* S. H. Robbins,* K. Crozat,* U. Kalinke & M. Dalod* *CIML, Immunology, Marseille, France, TWINCORE, MHH/HZI, Hannover, Germany Purpose/Objective: Type I interferons (IFN-I) play a major role in immune defense against viral infections in mammals. Depending on the target cells, IFN-I either promote or inhibit cell proliferation and survival. How IFN-I mediate such opposite effects is not well understood. On dendritic cells (DC), IFN-I promote the up-regulation of major histocompatibility class I and co-stimulatory molecules, and the trans-presentation of IL-15. On natural killer (NK) cells, IFN-I promote entry into cell cycle, survival and cytotoxicity. However, the mechanism of action of IFN-I on NK cells, either directly by IFNAR triggering or indirectly through IL-15 presentation by DC, is still controversial. Materials and methods: We combined the use of mutant mice, functional genomics, and flow cytometry analysis of transduction molecule phosphorylation to investigate how murine cytomegalovirus (MCMV) infection modulates the transcriptome and the antiviral activity of DC subsets and NK cells, in particular the role of IFN-I. Results: We showed that splenic DC subsets undergo similar transcriptomic changes early after infection, with the induction of many IFN-Istimulated genes, including genes involved in the recognition of viral infection, and inhibition of genes involved in proliferation. We showed that this occurs at least in part under cell-intrinsic instruction by IFN-I. NK cells induced genes involved in proliferation, which can be accounted for by stimulation through IL-15. We demonstrated that MCMV infection in vivo or IL-15 stimulation in vitro increase E2F expression and phosphorylation in NK cells, in a phosphatidylinositol 3-kinase dependent manner. Finally, we showed that the ability of DC, but not of NK


Poster Sessions 471 cells, to respond to IFN-I contributed to promote health over disease during MCMV infection. Conclusions: In conclusion, we showed that DC and NK cells respond differently to IFN-I during a viral infection in vivo. DC are highly sensitive to IFN-I stimulation, due in part to their higher expression of STAT1/2. NK cells respond weakly to direct IFN-I stimulation but strongly to IFN-I-induced IL-15. This study demonstrates how contrasting responses of primary cell types to IFN-I can emerge as a result of combined quantitative differences in the signaling module downstream of IFNAR and qualitative differences in sensitivity to secondary cytokine mediators induced by IFN-I.

P0879 DCs expressing IDO controls the fungal loads, activation costimulatory molecules and T cells proliferation in PCM developed by B10.A and A/J mice E. F. Araujo & V. L. G. Calich Institute of Biomedical Sciences, Immunology, Sao Paulo, Brazil Purpose/Objective: PURPOSE/OBJECTIVES: Paracoccidioides brasiliensis is a pathogenic fungus restricted to Latin America and its natural route of infection is the inhalation of fungal particles. In paracoccidioidomycosis (PCM), the regulatory mechanisms mediated by innate and cellular immunity are still unclear. Indoleamine, 23-dioxygenase (IDO) is an IFN-gamma induced enzyme which catalyzes the tryptophan metabolism along the kynurenine pathway. It is known that IDO can control host-pathogen interaction by inhibiting the proliferation of intracellular microorganisms due to tryptophan starvation and by its immunosuppressive effect on T cell immunity. The aim of our work was to investigate the influence of IDO on the behavior of dendritic cells (DCs) in the course of the infection of susceptible (B10.A) and resistant (A/J) mice clarifying some important aspects on the immunosuppression associated with the severe forms of PCM. Materials and methods: We worked with B10.A and A/J mice using control and 1-methyl-DL-tryptophan (1MT, an IDO inhibitor)-treated mice. Control and 1MT-treated B10.A and A/J mice were infected i.t. with one million yeasts and analyzed at 96 h post infection. Dendritic cells of the lung were purified through magnetic beads and the parameters of influence of treatment with 1MT in IDO were analyzed such as mRNA expression; CFU counts; NO production; levels of kynurenine and cytokines; characterization of subpopulations of DCs present in the lungs; lymphocyte proliferation assay. Results: In 96 h post infection of B10. A and A/J mice, 1MT was shown to decrease the frequency of DC cells expressing IDO and also the activation of co-stimulatory molecules CD40, MHC II and CD86. In both mouse strains IDO mRNA expression was augmented, driving tryptophan catabolism, kynurenine production and decreased fungal loads. On the other hand, 1MT restore proliferation of TCD4+ and TCD8+ cells in B10. A mice and TCD8+ in A/J mice, showing a known effect on cells expressing IDO. In total lung cells (before magnetic beads) was observed the same effect of decreased co-stimulatory molecules, CD40, MHC II and CD86 in 1MT-treated mice. Conclusions: CONCLUSION: DCs cells of resistant and susceptible mice use the enzyme IDO to control mechanisms of immunosuppression in infected mice, diminishing fungal burdens but also suppressing T cell proliferation.

P0880 DEF6, a Rho-GEF with a unique domain organisation involved in TCR mediated signal transduction aggregates via a coiled-coil domain to form cytoplasmic foci E. Mollett,* F. Hey, F. Sablitzky,à M. Searle* & P. Jones§ *Chemistry, University of Nottingham, Nottingham, UK, Biochemistry, University of Leicester, Leicester, UK, àGenetics, University of Nottingham, Nottingham, UK, §Biomedical Sciences, University of Nottingham, Nottingham, UK Purpose/Objective: Itk, a Tec family tyrosine kinase, regulates signalling molecules at the immune synapse including DEF6 (also known as SLAT or IBP), a Rho-GEF with a poorly characterised role in TCR mediated signal transduction (Gupta et al. J Biol Chem 2003, 278, 23541). Itk- and DEF6- deficient T cells both fail to activate ERK (Chen et al. Immunity 2008, 29, 899; Schaeffer et al. Science 1999, 284, 638), have decreased susceptibility to CD3-induced apoptosis (Schaeffer et al. Science 1999, 284, 638), and exhibit aberrant IL-17 expression (Gomez-Rodriguez et al. Immunity 2009, 31, 587). DEF6deficient mice develop a systemic autoimmune disease similar to SLE (Fanzo et al. J Clin Invest 2006, 116, 703), and rheumatoid arthritis (Biswas et al. Immunol Rev 2010, 233, 79). Mice primed to develop experimental autoimmune encephalomyelitis lacking DEF6 showed resistance to its development (Canonigo-Balancio et al. J Immunol 2009, 183, 7259). Our study aims to investigate the link between Itk and DEF6, and to explore the molecular mechanisms of DEF6 function. Materials and methods: Phosphorylation assay. Transient transfection with GFP tagged constructs. Structural prediction. Results: We have found that Itk phosphorylates the PH domain of DEF6 (submitted). This lays N-terminal to the DH-like domain (DHL), a feature unique to DEF6 and its B cell homolog, SWAP70. Transient transfection of a DEF6 mutant mimicking Itk phosphorylation forms cytoplasmic foci in Cos7 cells that co-localize with DCP1, a marker of P-bodies; structures containing machinery to degrade RNA (Kedersha et al. Methods Enzymol 2007, 431, 61). The DHL domain alone can form foci that do not co-localize with DCP1. Whilst the DHL domain exhibits GEF activity, structural prediction suggests it does not form the usual Rho-GEF DH domain structure. Additionally, the amino acid composition of the DHL domain is characteristic of a group of prion proteins (Fiumara et al. Cell 2010, 143, 1121) and some P-body proteins. In DEF6 this region is likely to form coiled-coil rather than beta-sheet based aggregation to drive the formation of the foci. Conclusions: Our data suggest that the atypical domain structure of DEF6 is responsible for maintaining a meta-stable conformation, easily changed by phosphorylation to promote GEF activity and/or cytoplasmic granule formation potentially involved in the regulation of translation. P0881 Dendritic cell mediated immune reactions to mycobacterial antigens in young healthy BCG vaccinated individuals P. Szpakowski, W. Rudnicka & M. Kowalewicz-Kulbat Department of Immunology and Infectious Biology, University of Lodz, Lodz, Poland Purpose/Objective: Dendritic cells (DC) are indispensible in initiation of adaptive responses to infective agents. The aim of study was to analyze the relationship between mycobacterial antigen induced responses of monocyte derived dendritic cells (DC) manifested on the level of surface receptors and cytokine production and capacity of mycobacterial antigen pulsed DC to stimulate autologous CD4, CD8 and CD56 cells. The study was performed in healthy subjects


472 Poster Sessions (17 30 years old) who were given 2 or 3 doses of BCG vaccine at their childhood. Materials and methods: Blood CD14+ monocytes separated with immunomagnetic method, cultured with IL-4 and GM-CSF gave DC. The receptors on DC stimulated with PPD or BCG bacilli and the phenotype and intracellular IFN-gamma in CD14-cell fractions responding to mycobacteria-pulsed DC were estimated by flow cytometry. ELISA was used for released cytokine detection. Results: An individual variation was noticed in the responses of DC to PPD and whole BCG bacilli manifested on the level of co-receptors CD86, CD80, CD40, HLA-DR and DC-SIGN as well as IL-10 and IL-23 releasing. However, PPD and BCG caused a significant decrease in DCSIGN on DC from almost all volunteers. As opposed to PPD, BCG enhanced a CD86 density on and IL-10 releasing by DC in the majority of donors. A high frequency of IL-10 production characterized DC from tuberculin-negative volunteers. An antigen presentation function of PPD or BCG pulsed DC towards autologous CD4+, CD8+ and CD56+ cells was estimated as an intracellular IFN-gamma. A great individual variation in the profile of responding T and NK cells was observed. There was a positive correlation between HLA-DR expression and IFN-gamma response of CD56+ cells stimulated with PPDpulsed DC and a negative correlation between HLA-DR density and intracellular IFN-gamma in total lymphocytes. Conclusions: The complexity of DC mediated immune reactions to mycobacteria requires regarding in a designation of better vaccines than BCG. Supported by the MS grant N N401 015236

P0882 Dendritic cells orchestrate innate immunity against bacterial kidney infection A. Tittel, C. Heuser, N. Garbi & C. Kurts Institutes of Molecular Medicine and Experimental Immunology, University of Bonn, Bonn, Germany Purpose/Objective: Urinary tract infections, such as pyelonephritis (PN), affect a large proportion of the population worldwide and are mostly caused by the uropathogenic gram-negative bacteria Escherichia coli (UPEC). Dendritic cells (DCs) form an abundant network in the kidney tubulointerstitium. Their role in bacterial pyelonephritis is unknown. Here we studied that role in a murine pyelonephritis (PN) model induced by transurethral instillation of UPEC. Materials and methods: Instillation of UPEC twice at a 3 h interval increased infection rates from 16% after a single instillation to 84%. To investigate the role of DCs in early PN we used CD11c transgenic deleter mice to deplete DCs with 8 ng/gbw diphtheria toxin. After kidney digestion renal phagocytes were intracellularly stained for CXCL2 and TNFa and were analyzed by flow cytometry. Results: Already 3 h after the second instillation, resident kidney DCs produced most of the intrarenal CXCL2 and TNFa. These cells recruited and activated neutrophilic granulocytes, which are critical in the defense against PN. When we depleted DCs using CD11c. DTR mice during the first bacterial instillation, neutrophil recruitment as well as bacterial clearance was markedly delayed. However, DC depletion also caused infection-independent granulocyte release from the bone marrow commencing after 24 h. The resulting neutrophilia paradoxically improved bacterial clearance when DCs were depleted 1 day before infection. This side effect was also seen in CD11c. DOG mice, another transgenic line allowing conditional DC depletion. Here we introduce a new transgenic line, CD11c. LuciDTR mice, which is unaffected by such early neutrophilia. However, both CD11c. LuciDTR mice and CD11c. DTR mice, but not CD11c. DOG mice, showed neutrophilia after 72 h, which probably resulted from increased granulopoiesis. Conclusions: All three lines feature time-windows, during which neutrophilia is negligible. Studies within these time windows allowed

us to demonstrate that the tubulointerstitial DC network serves an innate immune sentinel function against bacterial pyelonephritis, by rapidly recruiting neutrophils into the infected kidney.

P0884 E2-2-mediated down-regulation of plasmacytoid dendritic cell function limits type I interferon, T cell responses and virus control in early life E. Belnoue, P. Fontannaz, A. F. Rochat, C. Tougne, A. Bergthaler, P. H. Lambert, D. D. Pinschewer & C. A. Siegrist Pathology-Immunology, University of Geneva, Geneva, Switzerland Purpose/Objective: Cytopathic viruses causing acute infections in immunologically mature hosts often follow a prolonged course in early life, characterized by high viral titers maintained for several weeks. Accordingly, lymphocytic choriomeningitis virus (LCMV, WE strain) runs an acute course in adults but a protracted course in infant BALB/c mice, in which LCMV-specific T cells fail to expand and control infection. To identify whether these defects were due to a dysfunction of innate immunity in infant mice, we analyzed early events of viral infection. Materials and methods: Type I IFN production was assessed early after LCMV infection of infant and adult mice by ELISA and real-time PCR. We compared viral titers (by plaque assay) and T cell responses (by FACS) of LCMV-infected infant/adult wild-type and infant/adult IFNAR -/- mice. Activation and function of plasmacytoid and conventional dendritic cells were analyzed by FACS and real-time PCR. The effect of supplementing recombinant IFN-alpha on viral control and T cell responses was investigated in infant mice. Results: Our data showed an insufficient immediate-early IFN-alpha production in infant mice, which fails to support early viral control and the expansion of LCMV-specific T cells: disrupting IFNAR signaling in adult mice mimicked a protracted LCMV infection. Plasmacytoid dendritic cells (pDCs) which are the main source of type I IFNs in LCMV infection failed to acquire an activated phenotype in infant mice and displayed defective function in vivo upon LCMV infection reflected by the low expression of the central pDC transcription factor E2-2 and related genes (TLR7/9, IRF-7 and IRF8), MyD88 and NF-KappaB. In contrast, the in vitro function of early life pDCs was normal suggesting an in vivo negative regulation of infant pDCs. Direct evidence of the contribution of type I IFNs for early life infection control was demonstrated by given exogenous IFNalpha which restored virus control and CTL functionality in infant mice. Conclusions: In this study, we identify an age-specific down-regulation of multiple factors which are critical for the activation and function of pDCs and demonstrate that it is orchestrated by the E2-2 pDC regulator. We show that this down-regulation prevents the immediate-early burst of type I IFN and the early viral control and permanently impairs T cell responses. This defect was overcome by giving exogenous IFN-alpha which restored virus control and T cell responses in infant mice. The results suggest that early life pDC responses are tightly regulated in vivo, possibly to avoid potentially harmful inflammatory or autoimmune reactions.

P0885 Effect of Th17/Th1/Treg balance and microRNA expression in A. actinomyctemcomitans accelerated atherosclerosis R. Jia, T. Ochiai & M. Yamamoto Nihon University School of Dentistry at Matsudo, Oral Immunology, Matsudo Chiba, Japan Purpose/Objective: Recent studies have shown that there is an association between periodontal disease and cardiovascular disease. We


Poster Sessions 473 previously reported that the TLR signaling pathway plays an important role in Aggregatibacter actinomyctemcomitans (A. a.) mediated atherosclerosis in apolipoprotein E-deficient spontaneously hyperlipidemic (Apoeshl) mice. However, it is not known whether the causal or primary immune mechanism is involved in periodontopathic bacteriaassociated atherosclerosis. Atherosclerosis is a chronic inflammatory disease regulated by T lymphocyte subsets. In this study, we investigated whether the functional imbalance between Th17, Th1, and regulatory T (Treg) cells, and Th17-related microRNA expression existed in A. a.-challenged Apoeshl mice. Materials and methods: The mice were intravenously treated with live A. a. HK1651 or vehicles. Histomorphometric features of atheromatous lesions, serum IFN-gamma, IL-17 and IL-10 levels, and gene expression of Th17-related molecules and microRNA (miR) were examined. Results: We observed that A. a. challenge induced a Th17/Th1 shift in Apoeshl mice. A. a.-challenged splenic Th17 cells greatly increased during early stages of bacteria-challenge, compared to Th1 cell increase accompanied with Th17 cell reduction during later stage of challenge. In contrast, splenic Treg cell-changes in the A. a.-challenged group were almost similar with the control group. Similarly, differentiation factors (TGF-bplus IL-6, TL-17RA and IL-21), growth and stabilization factor (IL-23), and transcription factor (STAT3) involved in the development of Th17 cells, as well as Th1-related IFN-c were also detected in A. a.challenged mice. Furthermore, we found that miR-326 expression was upregulated in the hearts of A. a. challenged mice.

Conclusions: These results suggest that Th17/Th1/Treg imbalance and Th17 cell-associated miR-326 expression exists during early atherosclerosis in A. a.-challenged Apoeshl mice, suggesting a potential role of Th17/Th1/Treg imbalance in the progression of A. a.- accelerated atherosclerosis.

P0888 Enhanced CD8 T cell priming in DAP12 and FcR-gamma deficient mice G. B. Gmyrek, D. B. Graham, H. M. Akilesh, G. S. Blaufuss & W. Swat Washington University School of Medicine, Pathology and Immunology, St. Louis, MO, USA Purpose/Objective: The disruption of the immunoreceptor tyrosinebased activation motif (ITAM) signaling in mice with combined deficiency in DAP12 and FcR-gamma (DF mice) was shown to have either positive or negative effects on dendritic cell function. However, the exact role of dendritic cell ITAM (DC ITAM) signaling in CD8 T cell priming was not explored so far. Materials and methods: To determine the requirement of DC ITAMs in CD8 T cell priming, we performed a series of immunization experiments with MHC class I restricted peptide (OVA257 264) to evaluate CD8 T cell priming followed by analysis of dendritic cell subsets and evaluation of IFN type I and cytokine responses. Results: We found sharply increased CD8 T cell priming in DF mice, as indicated by increased frequency of IFN-gamma producing cells after re-call response with OVA257 264 peptide (SIINFEKL) in vitro. Given that many DC subsets contribute to modulation of T cell

responses in vivo we hypothesized that the difference in CD8 T cell priming could be related to activation of specific DC subsets resulting in polarization of the T cell response. In this line of investigation we found an increased number of monocyte-derived- and plasmacytoid dendritic cells in the draining lymph nodes of DF mice after immunization with SIINFEKL. We tested if there was a cell autonomous defect due to ITAM signaling deficiency, and noted no difference in the ability of sorted CD8a DCs from DF and WT mice to prime exogenous CD8 T cells ex vivo. Subsequently, given that IL-12 significantly contributes to increased IFN-gamma production, we noted that monocyte-derived DCs (moDC), from bone-marrow of DF mice, expanded after GM-CSF culture produced increased amounts of IL-12, along with up-regulation of transcription factor IRF-8. Consistent with this scenario the treatment of DF mice with IFNAR1-specific MAR1-5A3 mAb followed by the immunization with SIINFEKL, showed dramatically decreased IFN-gamma production. Conclusions: Collectively, we showed that DC ITAMs negatively regulate IFN type I responses and IL-12 production controlling CD8 T cell priming.

P0889 Enteropathogenic Escherichia coli engages in temporal regulation of the inflammasome by interaction with NLRC4 and NALP3 N. Pramanik,* M. Lucas,* C. Bryant, M. Bajaj Elliott* & N. Klein* *UCL Institute of Child Health, immunology and infectious disease, London, UK, Department of Veterinary Medicine, Cambridge University, London, UK Purpose/Objective: Enteropathogenic Escherichia coli (EPEC) is a leading cause of acute and chronic diarrhoea in developing nations, principally affecting children T; SNP12: rs2066845, 2722G>C; SNP13: rs2066847, insC), IL17A (rs2275913, 197G>A) and IL-23R (rs11209026, 1142G>A) encoding genes, which products promote Th17 response. Materials and methods: Two hundred and thirty one individuals were studied, including 48 Polish patients with sarcoidosis, 13 of which presented with Lo¨fgren’s syndrome (LS) and 83 control subjects. NOD2/CARD15 genotyping was performed by PCR-RFLP technique. Real-time PCR amplification with LightSNiP or TaqMan SNP Genotyping Assay was used for detection of IL-17A and IL-23R alleles, respectively. Results: There was no significant difference in the distribution of NOD2/CAR15, IL-17A and IL-23R alleles and genotypes in sarcoidosis patients and controls. However, a tendency was observed towards the lower representation of IL-23R mutation among patients when compared to controls (1/47 versus 9/83, RR = 0.25, P = 0.091). LS patients more frequently presented with the SNP13 variant (insC) when compared to healthy individuals (4/13 versus 6/80, RR = 5.43, P = 0.031). The prevalence of the SNP13 variant (insC) was also seen when LS patients were compared to those lacking LS symptoms (4/13 versus 3/34, P = 0.080). It is postulated that the presence of any of 3 NOD2/CARD15 SNPs influence the transcription potential of this gene. Also in the present study patients with the presence of any studied NOD2/CARD15 mutations significantly prevailed among LS as compared to non-LS cases (7/13 versus 6/34, P = 0.026) and controls (7/13 versus 14/80, RR = 5.29, P = 0.008). In the studied group, majority of LS patients were carrying DRB1*03 (8/13 versus 8/34, P = 0.020) as compared with non-LS cases. Interestingly, HLADRB1*03-positive LS patients were more frequently carrying NOD2/ CARD15 variants (5 out of 8; 62.5%) and the concomitant presence of these 2 genetic factors was more frequent among LS as compared to non-LS patients (5/13 versus 1/34, P = 0.004). Conclusions: Therefore, the contribution of the factors associated with DRB1*03 and NOD2/CARD15 SNPs contribute to LS highly inflammatory variant of sarcoidosis.


Poster Sessions 493 P0963 p110gamma PI3K deletion in LDL receptor-deficient mice reduces macrophage proliferation but not M1/M2 polarization in atherosclerotic lesions T. M. Zotes,* C. F. Arias,* J. J. Fuster, S. Pe´rez-Yaguë,* R. Spada,* E. Hirsch,à M. Wymann,§ A. C. Carrera,* V. Andre´s & D. F. Barber* *Department of Immunology and Oncology (DIO), Centro Nacional de Biotecnologıá (CNB-CSIC), Madrid, Spain, Department of Epidemiology Atherothrombosis and Imaging, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain, àDepartment of Genetics Biology and Biochemistry, Center for Molecular Biotechnology University of Torino, Torino, Italy, §Department of Clinical and Biological Sciences, Institute of Biochemistry and Genetics, University of Basel, Basel, Switzerland Purpose/Objective: Atherosclerosis is an inflammatory disease driven by atherogenic lipids that accumulate in arterial walls. It is regulated by the immune system and shares characteristics with other inflammatory diseases, such as tissue infiltration of activated monocyte/macrophages and T cells. Macrophage number in atherosclerotic lesions is controlled by monocyte migration to plaque and by in situ macrophage proliferation. Differentiation to an inflammatory (M1) or anti-inflammatory phenotype (M2) is also implicated in atherosclerosis progression. We studied the role of phosphoinositol-3-kinase (PI3K) p110c in the regulation of macrophage proliferation and polarization towards proinflammatory (M1) or anti-inflammatory (M2) phenotypes in atherosclerotic lesions. Materials and methods: We analyzed atherosclerosis development in LDLR-/- p110c+/- and LDLR-/- p110c-/- mice, and performed expression and functional assays in tissues and primary cells from these and from p110c+/- and p110c-/- mice. Results: Atherosclerotic plaques in fat-fed LDLR-/- p110c-/- were smaller and had reduced immune cell infiltration compared with LDLR-/- p110c+/- controls. This coincided with decreased macrophage proliferation in atherosclerotic lesions of LDLR-/- p110c-/- mice. This proliferation defect was also observed in p110c-/- bone marrow-derived macrophages (BMM) stimulated with macrophage colony-stimulating factor (M-CSF), and was associated with accumulation of the growth suppressor p27Kip1. In contrast, proliferation of infiltrating T cells was unaffected in LDLR-/- p110c-/- mice. p110c deficiency did not affect macrophage polarization towards the M1 or M2 phenotypes in atherosclerotic plaques and cultured BMM. Conclusions: Our results suggest that p27Kip1 accumulation and the ensuing inhibition of macrophage proliferation contribute to atheroprotection in LDLR-null mice lacking p110c. Nonetheless, p110c deletion does not appear to be involved in infiltrating macrophage polarization or in infiltrating T cell proliferation.

P0964 Paracrine Interactions in neuroinflammation N. Patel, M. R. H. White & P. Paszek Faculty of Life Sciences, University of Manchester, Manchester, UK Purpose/Objective: Neural progenitor cells (NPCs) are potent immunomodulatory agents, capable of promoting tissue repair in cases of neurodegenerative disease. As yet, the mechanisms by which they aid regeneration in disease conditions are unknown. Here, we investigate the interactions between NPCs and the main drivers of neuroinflammation, microglia. By studying the dynamics of a gene transcription factor, and major neuroinflammatory mediator, Nuclear Factor kappaB (NF-kB), we aim to elucidate paracrine interactions that may provide therapeutic effects in the diseased brain. Materials and methods: We have adopted a systems level approach to study NF-kB dynamics in a mouse model of neuroinflammation. Modified murine microglial (BV.2) and NPC (C17.2) lines were used

to make single cell- and dual cell-cultures, which were stimulated with different doses of a potent inflammatory agent [either tumor necrosis factor-a (TNF-a) or lipopolysaccharide (LPS)]. Population-level NFkB activation was observed, using cells expressing NF-kB promoterdriven luciferase and live cell luminometry. This was complimented with quantitative single cell analysis of NF-kB dynamics, using cells expressing fluorescently- tagged NF-kB and confocal microscopy. Results: Our results demonstrate interactions between microglia and NPCs in a neuroinflammatory environment. NF-kB response in these cell lines show distinct stimulus-dependant and dose-time activation profiles. Interestingly, some cells within an NPC population demonstrate NF-kB unresponsiveness to inflammatory stimuli, even at saturating doses. Conclusions: Quantitative, multi-scale analysis of NF-kB dynamics reveals complex NF-kB-mediated interactions between microglia and NPCs. These interactions require further investigation to determine whether they serve to orchestrate a regenerative phenotype in the inflamed brain.

P0965 Required contribution of IL-6, IL-17 AND IL-23 to control the paracoccidioidomycosis F. A. Rocha, J. S. Silva & M. A. Rossi Imunology, School of Medicine of Ribeiraõ Preto, University of Saõ Paulo, Ribeiraõ Preto, Brazil Purpose/Objective: Paracoccidioides brasiliensis (Pb), a thermally dimorphic fungus, is the causative agent of paracoccidioidomycosis (PCM), one of the most frequent systemic mycosis that affects the rural population in Latin America. T helper cells type 17 (Th17) are described as an arm of the immune system that enhances host protection against several infections, including mycosis. Considering the better understanding of the mechanisms involved in resistance to P. brasiliensis infection is necessary, our aim was to evaluate the Th17 and related cytokines participation during the PCM. Materials and methods: We worked with male C57BL/6 (WT) and IL-6, IL-17 receptor (R) and IL-23 knockout (KO) mice that were intravenously inoculated with 1 · 106 yeast forms of Pb18, a highly virulent P. brasiliensis strain. We measured cytokines mRNA and production from lung homogenate, besides, followed the fungal growth by recovering of colony forming units (CFU) and verified the inflammatory process and granulomas formation during de Pb experimental infection by cytometry and histopathology analysis. Results: Our results show that Pb-infection induces increased IL-6, IL17 and IL-23 mRNA lung expression and production compared with uninfected mice. Evaluating the susceptibility to the infection, the IL6KO mice showed the highest mortality rate. At 15 and 30 dpi, the CFU recoveries from the lung, liver and spleen of all KO mice were increased in relation to WT group. Histopathological analysis showed that IL-6 and IL-17 deficiency assisted absence of compact granulomas due to impaired formation of reticulin fibers accompanied of disorganized CD4+ T cell infiltration at lung tissue, favoring an increased fungal load. The absence of IL-6, IL-17R or IL-23 induced lower production of IFN-g and IL-10 compared with WT lung. Additionally, the frequency of CD4+IL-17+ T cells also this cytokine production were decreased in the absence of IL-6 or IL-23. Conclusions: Taken together, these results demonstrate that IL-6, IL17 and IL-23 contribute to control of experimental Pb-infection through an efficient granulomatous organization.


494 Poster Sessions P0966 Role of microglia and macrophages on retinal pigment epithelial cells degeneration and the factors influencing these in age related macular degeneration G. Devarajan, J. Forrester & I. Crane Division of Applied Medicine, University of Aberdeen, Aberdeen, UK Purpose/Objective: Age related macular degeneration (AMD) is one of the common causes of blindness in elderly population. Retinal pigment epithelial (RPE) degeneration is one of the early events in AMD and these RPE cells mainly die through apoptosis. Maintaining a normal and functional RPE layer is vital for healthy vision. Therefore understanding the factors that induce RPE cell death in AMD would help identify a therapeutic approach. Inflammation is one of the key factors that influence the development of AMD. Infiltrating macrophages and resident microglia are associated with AMD and may influence RPE degeneration. Therefore we sought to identify the effects of cytokines, microglia and macrophages on RPE degeneration. Materials and methods: An in vitro system to model in vivo conditions was set up by co-culturing murine macrophages derived from bone marrow cells or microglia from central nerves system on a confluent murine RPE cell layer under different conditions for 48 h at a ratio of 1:2 respectively. These conditions include RPE cells and macrophages or microglia pre-treated for 24 and 18 h respectively before the co-culture in the presence of pro inflammatory mediators, oxLDL, IL-1b, TNF-a, IL-6, and IFN-c that are present in the microenvironment in which AMD develops. After co-culture the apoptotic cells were detected using annexin V staining by flow cytometry. Results: Co-culture of un-treated RPE cells and un-treated BMDM or microglia does not increase apoptosis in RPE cells. However coculture of un-treated RPE cells with pre-treated macrophages increased apoptosis in RPE cells but pre-treated brain microglia failed to increase apoptosis in RPE cells. Co-culture of pre-treated microglia and macrophages were able to increase apoptosis in treated RPE cells. These macrophages/microglia increase apoptosis in RPE cells through cell contact. In addition to macrophages and microglia, combination of cytokines and oxLDL also increases apoptosis in RPE cells. Conclusions: The above findings provide a greater understanding of the pathogenesis of AMD, specifically the role played by microglia/ macrophages in RPE degeneration and how oxidative stress and cytokines act together to bring about RPE degeneration. Better understanding of the pathogenesis of AMD will provide better therapeutic strategies.

P0967 S100B in the ocular inflammatory response; cytokine and chemokine modulation J. Niven, J. Hoare, P. Teismann & I. Crane Division of Applied Medicine, Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK Purpose/Objective: S100B is a Ca2+ binding protein which can potentially act as a pro-inflammatory mediator via the receptor for advanced glycation end products (RAGE); however its involvement in inflammation is still unclear. In the human condition, Endogenous Posterior Uveitis, retinal inflammation with autoimmune aetiology is associated with loss of sight. Both S100B and RAGE are reported to be present in the retina and the purpose of the study was to determine whether S100B is involved in retinal inflammation. Materials and methods: Using the mouse model, experimental autoimmune uveitis, Topical Endoscope Fundal Imaging allowed disease progression to be followed post-immunization (pi) with uveitogenic peptide, in S100B-deficient and wild type mice. Eyes were

snapping frozen for histological grading and immunohistochemistry. Real-time PCR array analysis was used to confirm changes in cytokine and chemokine expression in diseased matched retinas from S100Bdeficient and wild type mice. This method was also used to investigate the response of a murine macrophage cell line RAW 264 to treatment with S100B, with cytokine production confirmed by ELISA. Results: We have shown using clinical grading, that disease is significantly reduced in S100B deficient mice compared to wild type, at days 15 and 21. At day 24 pi this disease reduction was also shown by histological grading of the infiltrate, although the composition of the infiltrate remained unchanged. In S100B knockout mice compared to wild-type, at day 24 pi there was an overall reduction in expression of proinflammatory cytokines and chemokines in diseased matched retinas. We confirmedthat the macrophage cell line express RAGE and that this expression increases upon activation with PMA. Treatment of the macrophages with S100B resulted in an increase in proinflammatory cytokines and chemokines, in particular IL-1b and CCL22. ELISA analysis confirmed a significant increase in CCL22 with 2 and 5 lM S100B compared to un-treated cells. Conclusions: These results suggest that S100B may be involved in augmenting the inflammatory response in uveitis and this may be via a direct effect on macrophages.

P0968 The conformational significance of hinge region glycosylation on the solution structure of monomeric IgA1 in IgA nephropathy O. Vennard,* K. Molyneux,* L. Rayner, S. J. Perkins & J. Barratt* *Infection Immunity and Inflammation, University of Leicester, Leicester, UK, Structural Immunology Group, University College London, London, UK Purpose/Objective: Intrinsically characterised by the deposition of IgA1 in the renal mesangium, IgA nephropathy (IgAN) remains the most common form of glomerular inflammation worldwide. Within 20 years of diagnosis, up to 30% of patients progress to end stage renal disease. It is widely accepted that IgAN is caused by an inherent abnormality in the IgA1 molecule. Aberrant IgA1 hinge O-glycosylation, presenting as undergalactosylation, is observed in patients and is strongly implicated in the pathogenesis of IgAN. Analytical ultracentrifugation (AUC) represents a powerful technique in the study of macromolecular size and shape in biologically relevant conditions. This study focuses upon the complex relationship between structurally induced changes in the IgA1 molecule through hinge region glycosylation, and its potential effects in the causation and prognosis of IgAN. Materials and methods: Serum IgA1was isolated using jacalin affinity chromatography from a healthy control and 2 progressive IgAN patients. Monomeric IgA1 was separated by FPLC. The relative hinge region O-galactosylation was determined using a helix aspersa (HA) lectin binding assay including a standard curve containing serum samples with decreasing lectin binding. High lectin binding relates to low galactosylation. AUC sedimentation velocity experiments were performed at 20C with rotor speeds of 20 000 and 30 000 rpm for 16 h in PBS. Data was analysed using size-distribution plots c(s) using SEDFIT to determine monodispersity and establish sedimentation coefficients (S). Results: The HA lectin binding values of the healthy subject and the patients 1 and 2, expressed as arbitrary units from a standard curve, were 22, 32 and 31, the sedimentation coefficients were 6.85 S, 6.7 S and 6.55 S respectively. Conclusions: Sedimentation coefficient, a measure of molecular elongation increases when a molecule becomes more compact. The decrease in s value with increased HA binding and therefore decreased galactosylation, suggests a change in shape associated with the undergalactosylation of serum IgA1 in IgAN patients.


Poster Sessions 495 These results show that the conformational state of monomeric IgA may be directly altered via hinge glycosylation, potentially providing an implication to the increased deposition of IgA in IgAN.

P0969 The effect of interleukin-15 on inflammatory bone metabolism A. Mitani, H. Takeda, T. Kikuchi, K. Soboku, S. Fujita, Y. Ishihara & T. Noguchi Aichi Gakuin University, Periodontology Sch Dentistry, Nagoya, Japan Purpose/Objective: Interleukin-15 (IL-15), mainly induced by macrophage or epithelium cell, is important for early response to compensate the time difference between innate immunity and adaptive immunity and deeply involved the initial immune response at local inflammatory site. However, it is still unclear about the effect on inflammatory bone metablism like periodontitis and peri-implantitis. Thus, we examine the effect of IL-15 on osteoblast and osteoclast differentiation. Materials and methods: Bone marrow cells (BMCs) were collected from femurs of 4-week-old ddy mice. Osteoblasts from 2-day-old ddy mice were isolated from the calvaria. Osteoclast formation using primary cells was determined in co-cultures of osteoblasts (1.6 · 104 cells/well) and BMCs (4 · 106 cells/well). They were cocultured for 7 days in a 24-well plate in the presence or absence of 10-6 M prostaglandin E2 (PGE2) and/or IL-15 (10, 100 ng/ml). To detect osteoclasts, the cells were stained for Tartrate-Resistant Acid Phosphatase (TRAP). TRAP-positive multinucleated cells containing three or more nuclei were counted as osteoclasts. Osteoclast formation using cell line was determined in RAW 264 cells (6 · 103 cells/well). They were cultured for 5 days in a 48-well plate in the presence or absence of receptor activator of nuclear factor-jB ligand (RANKL) at 10, 25, 50 ng/ml. Osteoclast detection was carried out as described above. Results: The number of osteoclasts in co-culture was decreased by IL15 in a dose-dependent manner. The osteoblasts in co-culture seemed to disappear and be apoptotic cell death. This phenomenon was attenuated when T-cells and NK-cells were removed from BMCs. The number of osteoclasts in RAW 264 cells was increased by IL-15 in a dose-dependent manner. Conclusions: IL-15 might promote inflammatory bone destruction by both promoting osteoclast differentiation and osteoblast apoptotic cell death. It is possible for IL-15 to be immunological therapeutic target for local inflammatory bone resorption like periodontitis and periimplantitis.

P0970 The genes of AP-1 transcription complex as the potential factors which have an influencing on atherosclerosis development M. Sautin Faculty of Medicine, Peoples’ Friendship University of Russia, Moscow, Russia Purpose/Objective: Searching and research of the expression of the genes, which are connected with the risks of atherosclerosis development. Materials and methods: It was used material of 34 autopsies taken from patients who were suffered from the ischemic attacks of the brain and acute heart failure. Mean age of the patients was 77.6 years old. It was used PubMed and UniGene bases during preparing the list of the genes. According to this list maps gene interactions were prepared. We looked throw the genes whose expression was changed more than 2 times. According to the maps key gene complexes are transcription factors AP-1 and NF-kB. NF-kB factor is not specific for the atherosclerosis process; therefore we have excluded it from research. Transcription factor AP-1 (Activating protein 1) is including proteins

of Fos-, Jun- and ATF- families. Expression level of the components of AP-1 transcription factor at the blood vessel intimae was determined by Real-time PCR. Expression of the JunB, c-Jun, JunD, cFos genes was studied. Expression of the genes was normalized on the housekeeper gene GAPDH. Results: cFos expression at the affected intimae of the blood vessel in comparison with the not affected intimae was increased in more than two times. JunB expression at the affected intimae was increased too. JunD expression in two patients was increased less than 2 times. In the case of 3 patients gene expression was increased more than 10. c-Jun expression decreased in 1 case. In the case of 2 patients’ it was increased in more than 10 times. Thus, expression of the cFos, JunB, JunD, c-Jun genes in the in the most cases was increased. Conclusions: Our data relate to those of other authors that the expression of the certain genes of the AP-1 transcription factor may vary in different pathological processes. Increasing of the level of cFos gene expression in 3 or more times is observed in patients, who were developed complications of atherosclerotic damage of the arteries which are supplying the tissue of the brain. Our data suggest that the genes of AP-1 transcription factor are playing key role at the atherosclerosis processes.

P0971 The right dose determines a poison influence of benzo(a)pyrene on the infection with Salmonella enterica C. Fueldner, S. Riemschneider, K. Zoldan, J. Knauer & J. Lehmann Cell Engineering/GLP unit, Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany Purpose/Objective: Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BaP) are environmental contaminants exerting deleterious effects towards the immune system, ranging from immunosuppression to fatal inflammatory disease. Since exposure to BaP is ubiquitary, risk assessment was conducted to determine upper limits for tolerable daily intake doses (subtoxic concentrations). However, most studies demonstrating hazardous effects of BaP used concentrations in the toxic range. Therefore, the aim of this study was to investigate the influence of subtoxic BaP-concentrations on the outcome of an infection with Salmonella enterica (S. e) in a murine model. Materials and methods: Mice were infected with Salmonella enterica and simultaneously exposed to subtoxic doses of BaP.. Results: Importantly, exposure to BaP in subtoxic concentrations markedly increased survival of mice after infection with S. e. However, BaP-treated animals developed a long-term infection with higher bacterial burden in spleen and liver when compared to vehicle controls. The number of B lymphocytes was significantly increased at day 90 post infection in BaP-exposed mice, which was accompanied by higher serum titers of S. e.-specific IgG1 and IgG2c antibodies. Furthermore, IL-17 and IL-22 mRNA levels in splenocytes were significantly upregulated after long-term infection in BaP-treated mice. Conclusions: Although BaP-treatment in subtoxic concentrations increased survival of mice during S. e. infection, it also promoted the survival of bacteria with a consequent increase in inflammatory responses. Our data suggest that constant BaP-exposure in subtoxic concentrations during an infection favours a bacteria-driven chronic inflammation.


496 Poster Sessions P0973 Unravelling the causes of inflammatory steroid resistance via a systems biology approach N. Valeyev* & G. Welsh *College of Engineering Mathematics and Physical Sciences, University of Exeter, Exeter, UK, School of Clinical Sciences, University of Bristol, Bristol, UK Purpose/Objective: Steroid resistance is a generic problem which occurs in a number of autoimmune diseases. Treatment failure occurs in up to 30% of patients treated with steroids for inflammatory diseases. While it has been shown that there are significant homeostatic differences in cytokine and growth factor production profiles of interacting cell populations between steroid resistant and steroid sensitive cases, the mechanism of the steroid resistance is not understood. Recent evidence has implicated a steroid resistant CD4+ T cell subpopulation in the perpetration of steroid resistant disease. Materials and methods: In order to investigate the inflammatory causes of steroid resistance we combined available experimental information into an extended systems model of our earlier work on

interdependent cellular interactions. This project applies a systems based approach to the problem of steroid resistance using Steroid Resistant Nephrotic Syndrome (SRNS) as an example and is applicable to other steroid resistant cases. Results: Using our unique cultured human glomerular cells and glomerular cell models we have measured cytokine and growth factor production in reponse to steroids using ELISA and mass spectrometry. These results allowed us to develop a model of the response of the kidney glomerulus to steroids. The model allowed the generation of a new hypothesis by predicting the appearance of pathological conditions as a result of altered cytokine and growth factor production rates and/or degradation rates in the glomerulus. Conclusions: Our model predicts that glucocorticoids can modulate the cytokine concentrations and as a result of feedback loop interactions between cytokines and chemokines in a number of case may lead to the shift from pathologic conditions to healthy homeostasis. At the same time, we show that there can be cases when steroids can aggravate the disease and even introduce new disease etiologies.


Abstracts 497

Poster session: Hepatitis & Liver Immunology P0974 Beneficial effects of Ocimum gratissimum aqueous extract on rats with CCl4-induced acute liver injury T. C. Hsu,* B. S. Tzang, C. C. Chiu* & Y. W. Wang * Institute of Microbiology and Immunology, Chung Shan Medical University, Taichung, Taiwan, Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan Purpose/Objective: Ocimum gratissimum (OG) is known as a food spice and traditional herb, which has been recommended for the treatment of various diseases. Herein we investigated the effects of OG leaf aqueous extract (OGAE) on reducing hepatic injuries in rats after CCl4 challenging. Materials and methods: To investigate the hepatoprotective effect of OG aqueous extract (OGAE), male Wistar rats challenged by carbon tetrachloride (CCl4) were used as the animal model of chronic hepatic injury. Catalase assay (CAT) and DPPH assay was assessed in CCl4administrated rats. Expression and phosphorylation of protein was determined and quantitated by immunoblotting using specific antibodies and densitometric analysis. Results: Significantly increased serum catalase and DPPH levels were detected in CCl4-administrated rats that were treated with OGAE or sylimarin as compared to those rats that were treated with saline or CCl4. In contrast, significantly decreased stress proteins including HSP70 and iNOS were observed in livers of CCl4-administrated rats that were treated with OGAE or sylimarin as compared to those rats that were treated with saline or CCl4. Moreover, significant decreases of MMP-9/MMP-2 ratio, uPA, phosphorylated ERK (p-ERK) and NFjB (p-P65) were detected in livers of CCl4-administrated rats that were treated with OGAE or sylimarin as compared to those rats that were treated with saline or CCl4. Conclusions: These findings imply that OGAE can efficiently inhibit CCl4-induced liver injuries in rats and may therefore be a potential food or herb for preventing liver injuries.

P0975 CCR5-32 and UGT1A1 28

mutations in HCV patients

L. Sadovska,* L. Piekuse,* M. Kreile,* J. Keiss & A. Kruminaà * Riga Stradins University, Laboratory of Molecular Genetics, Riga, Latvia, Latvian Infectology Center, Liver disease department, Riga, Latvia, à Latvian Biomedical Research and Study Center, Genetics, Riga, Latvia Purpose/Objective: The role of CCR5-D32 and UGT1A1 28* mutations in HCV patients is not entirely clear, but it is known that CCR5D32 contributes to immunology and UGTA1A 28* mutations cause Gilbert’s syndrome which affects the liver. Presumably, a patient homozygous for both of these mutations is more likely to be at risk group for HCV infection. The objective to this study was to determine whether the occurrence of mutations CCR5-D32 and UGT1A1 28* is higher in HCV patients than in normal healthy group. Materials and methods: To achieve our goal, we performed PCR for the genes in question and visualized the results in 6% polyacrylamide gel electrophoresis and by sequencing the PCR products. In this study we analyzed 262 individuals infected with hepatitis C and 187 healthy donors from general population, in total*243 male and 206 female individuals. For statistical analysis we used PLINK software, for biomedical association we used linear regression using additive model. Results: Allelic frequency for CCR5-D32 mutation was 0.239 in HCV patient group and 0.176 in control group (P = 0.0399); for (TA)7 allele it was 0.546 in HCV patient group and 0.355 in general population (P = 0.00000005.

For 79 patients there was more detailed information about their infection viral RNA levels before and after therapy, viral genotype, iron, ferritin, ALT before and after therapy, viral clearance right after and 3 months after therapy. We found no significant association between patient’s genotypes and RNA levels, ALT and iron levels (P > 0.05). Almost significant association was found between CCR5D32 mutation and ferritin level by additive model (BETA -120.4, P = 0.077). Moreover, there was no association with viral clearance right after therapy, but 3 months after therapy for positive RNA CCR5-D32 allele frequency was 0.297, for negative 0.0714 (P = 0.0363). Conclusions: Patients with mutations in CCR5 and UGT1A1 are more likely to be infected with HCV, as their immune response is altered and their liver is weakened. It seems that UGT1A1 28* can affect liver function also in heterozygous state, but that way it does not cause Gilbert’s syndrome. Presumably, the CCR5-D32 mutation correlates with unresponsiveness to therapy.

P0976 CD39+ CD4+ T cells display a pro-inflammatory signature in patients with autoimmune hepatitis C. Grant, R. Liberal, B. S. Holder, Y. Ma, G. Mieli-Vergani, D. Vergani & M. S. Longhi King’s College London, Institute of Liver Studies, London, UK Purpose/Objective: Autoimmune hepatitis (AIH) is associated with numerical and functional regulatory T cell (Treg) defects. A recently described Treg subset expresses the ectonucleotidase CD39, which renders Tregs capable of suppression by initiating an ATP/ADP hydrolysis cascade culminating in the production of immunomodulatory adenosine. CD39+Tregs exert preferential control over Th17 cells, an effector subset involved in AIH liver damage. CD39 is also present on a proportion of CD4 memory lymphocytes with effector function. Regulatory and pro-inflammatory features of CD39+ CD4+ cells in AIH are untested. We therefore sought to provide a phenotypic signature of CD39+ CD4+ cells in AIH. Materials and methods: Of the 14 AIH patients and 6 healthy subjects (HS) were studied. Circulating CD39+ CD4+ cells were phenotyped by flow cytometry using monoclonal antibodies to CD4, CD39, CD25, FOXP3, Granzyme B, CD62L, CTLA-4 and RORC. The frequency of TGFb and IL17A-producing cells was assessed by intracellular cytokine staining after incubation with Leukocyte Activation Cocktail (LAC). Results: Compared to CD39- CD4+ cells, a higher proportion of CD39+ CD4+ cells expressed Treg and Th17 markers in AIH and health. Compared to HS, CD39+ CD4+ cells in AIH patients contained fewer cells positive for the Treg-associated markers CD25, FOXP3, and Granzyme B, while CD62L or CTLA-4 positive cell frequencies were similar. After LAC stimulation, the frequency of TGFb-producing cells among CD39+ CD4+ cells was lower in AIH patients compared to HS. In contrast to Treg markers, CD39+ CD4+ cells from AIH patients contained a higher proportion of cells positive for the Th17 transcription factor RORC than those from HS. There was no difference in the proportion of IL17A-producing cells within CD39+ CD4+ cells between AIH and HS. Conclusions: The CD4+ population expressing the ectoenzyme CD39 contains a high proportion of cells expressing Treg and Th17 markers. In AIH the lower frequency of cells positive for conventional Treg markers and the higher number of cells positive for RORC indicates that the CD39++ CD4+ population is skewed towards a pro-inflammatory phenotype. Whether the effector potential of Th17 committed lymphocytes is mitigated by the presence of CD39 awaits investigation.


498 Poster Session: Myeloid Cell Development P0977 CD8ßlow a novel differentiation marker and a prominent population in chronic hepatitis B infection L. Walker, E. Marrinan, E. Barnes & P. Klennerman Nuffield Department of Medicine, University of Oxford, Oxford, UK Purpose/Objective: Failure of antigen-specific CD8 T cells is a recognised feature of chronic hepatitis B (HBV) infection, however the bulk CD8 T cell population is also characterised by low expression of CD28 and poor IL-2 production and proliferation capacity (Hoare D et al. 2008) in keeping the phenotype of late differentiated CD8 T cells. We have previously observed a prominent CD8 T cell population in chronic HBV to be CD8a+blow (Kang W et al. 2009) this is most obvious in the cells which either lack (CD161-) or have low expression of CD161 (CD161+) (a molecule associated with liver homing). In this study we aimed to explore the relationship between these two observations in chronic HBV, chronic Hepatitis C (HCV) and healthy controls (HC). Materials and methods: Peripheral blood mononucleocytes were obtained from patients with chronic HBV (n = 37), chronic genotype 1HCV (n = 24) and healthy controls (n = 15). All patients were treatment naı¨ve. FACS analysis was performed on both cell surface antibody staining using a panel of activation/exhaustion/differentiation markers (CD25, CD38, CD69, HLA-DR, PD-1, CD8a, CCR7, CD62L, CD45RA, CD45RO, CD28, CD27, CD57) and intracellular cytokine staining following PMA/ionomycin stimulation. Results: Prominent populations of CD161- and CD161+ CD8a+blow T cells can be identified in healthy individuals as well as those with chronic viral hepatitis, however a significantly greater population of CD161-/CD161+ CD8a+ CD8blow T cells is seen overall in chronic HBV compared to healthy controls (mean 39.5% HBV, mean 24.74% HC, P = 0.05). The CD8a+blow T cells have the CD28- CD27- CD57+ CD62L- CCR7- CD45RA- phenotype of late differentiation in both healthy controls and patients with chronic HBV and HCV and express high levels of the late activation marker HLA-DR. CD161- CD8a+blow CD8 T cells produce significantly more IFN-c and TNF-a on stimulation with PMA/ionomycin than their CD161- CD8a+bhigh counterparts and express greater levels of Ki67 and perforin. Conclusions: CD8blow status represents a novel marker for late differentiated CD8+ T cells and may alter the ability of CD8+ T cells within this population to respond to CD8ab dependent epitopes. The prominence of CD8a+blow T cells in chronic HBV infection is likely to profoundly influence the immune environment, with important implications for the development of immunotherapy and treatment.

P0978 Cell-targeting gold nanorods critically affect the outcome of liver inflammation in vivo by modulating macrophage polarization M. Bartneck,* T. Ritz,* H. A. Keul, M. Wambach, J. Bornemann,à U. Gbureck,§ J. Ehling,– T. Lammers,– F. Heymann,* N. Gassler,** T. Lu¨dde,* C. Trautwein,* J. Groll§ & F. Tacke* * Department of Medicine III, Medical Faculty RWTH Aachen, Aachen, Germany, RWTH Aachen, DWI e.V. and Institute of Technical and Macromolecular Chemistry, Aachen, Germany, àMedical Faculty RWTH Aachen, Electron Microscopic Facility (EMF), Aachen, Germany, § Department and Chair of Functional Materials in Medicine and Dentistry, University Hospital Wu¨rzburg, Wu¨rzburg, Germany, – Department of Experimental Molecular Imaging (ExMI), Helmholtz Institute for Biomedical Engineering, Aachen, Germany, **Medical Faculty RWTH Aachen, Institute of Pathology, Aachen, Germany Purpose/Objective: Hepatic macrophages critically promote liver inflammation and fibrogenesis. Thus, hepatic macrophage-specific immunomodulatory nanoparticles represent a promising therapeutic

option in liver diseases. We reported recently that human primary macrophages are strongly polarized by nanoparticle surface chemistry in vitro. Here, we investigated the toxicity, distribution, and therapeutic effects of gold nanorods (AuNR) in acute and chronic liver injury in vivo, aiming to develop novel therapeutic strategies for the treatment of liver disease. Materials and methods: We studied the concentration-dependent organ distribution of AuNR coated with either CTAB, PEG, or with the tripeptides GLF, or RGD in vivo in C57BL/6J mice using inductively coupled plasma-mass spectrometry, electron microscopy, and in vivo imaging (micro-CT). The therapeutic effects of the AuNR were studied in Concanavalin A (ConA)-mediated acute hepatitis as well as in carbon tetrachloride (CCl4)-induced fibrosis, using biochemistry (ALT, AST, hydroxyproline), histology, immunohistochemistry, and flow cytometry. Macrophages were isolated from liver and analyzed for polarization by qPCR-based assays. Results: We found that the AuNR predominantly accumulate in liver in vivo, specifically in hepatic macrophages. At high concentrations (1200 lg/kg), CTAB-coated AuNR slightly increased infiltrating inflammatory CD11b+F4/80+Gr1high macrophages in the liver without evidence of hepatotoxicity in vivo. Interestingly, upon ConA-induced hepatitis, RGD-peptide-modified AuNR significantly deteriorated liver damage, decreased the frequency of resident CD11b- F4/80+Gr1low Kupffer cells accompanied by strong changes in the expression of surface markers and function-related genes expressed by classically or alternatively activated macrophages, compared to untreated controls. Similar observations were found in chronic liver injury, with a trend towards increased liver fibrosis development by RGD-coupled AuNR. Conclusions: AuNR efficiently target hepatic macrophages, and modifications of nanoparticle surface chemistry affect the phenotype and functionality of both liver infiltrating and resident macrophages, thereby impacting the hepatic response towards acute or chronic injury.

P0979 Characterization of two human monoclonal anti-E2/HCV antibodies for potential application in prevention and treatment of HCV infection R. A. Diotti, G. Sautto, E. Criscuolo, N. Mancini, L. Solforosi, M. Clementi & R. Burioni Universita` Vita-Salute San Raffaele, Microbiology, Milano, Italy Purpose/Objective: HCV is a major cause of chronic liver disease worldwide and the current antiviral therapies results successful in approximately half of treated patients. Liver disease caused by HCV infection is a common indication forliver transplantation but graft re-infection is universal and often results in graft loss. Immunotherapies employing neutralizing antibodies, able to inhibit HCV infection, directed against the HCV viral glycoproteins E1 and E2, involved in cell viral entry, have the potential to control or prevent graft reinfection. Moreover, anti-HCV neutralizing antibody can find a potential application for the treatment of chronically infected patient. Here we characterized two potent neutralizing anti-E2/HCV human monoclonal antibodies (mAbs). Materials and methods: We previously characterized two anti-HCV E2 human mAbs as Fab fragments, e20 and e137. To obtain molecules more suitable for therapeutic purposes, we generated whole IgG e20 and e137. Both mAbs were tested in ELISA and immunofluorescence assay (IF) to study the affinity and the reactivity on different genotypes. To determine the capacity of these antibodies to interfere with E2CD81 binding, we performed an inhibition of binding (IOB) of CD81 to recombinant E2 glycoprotein by ELISA. For studying biological activity of e20 and e137, neutralization assay against HCVpseudo-


Abstracts 499 particles of five genotypes and cell culture infectious HCV system based on genotype 2a was performed. Results: The antibodies showed high affinityfor E2 recombinant glycoprotein (genotype 1a) and were able to recognize HEK293T cells expressing HCV E1-E2 from the principal HCV genotypes. Both IgGs showed dose dependent IOB activity and at the maximum tested concentration the inhibition was about 97%. Neutralization assay showed that these antibodies are strong neutralizers of the tested genotypes with IC50 ranging from 0.02 to 2 lg/ml. Conclusions: e20 and e137 are potent cross-reactive and crossneutralizing mAbs able to inhibit E2-CD81 binding. These data suggests that both antibodies are directed against conserved and critical residues for viral infectivity on E2 glycoproteins. Based on their features, both IgGs may assist in the development of an effective passive immunotherapy for the prevention of viral reinfection of the liver graft and treatment of chronically infected patients.

P0980 Chemokine presentation by liver sinusoidal endothelial cells as therapeutic target in murine T cell-mediated hepatitis K. Neumann,* N. Kruse,* K. Wechsung,* M. Schumann,* A. Ku¨hl, U. Erben,* M. Zeitz* & K. Klugewitz* * Charite´ Universita¨tsmedizin Berlin, Medizinische Klinik I, Berlin, Germany, Charite´ Universita¨tsmedizin Berlin, Institut fu¨r Pathologie, Berlin, Germany Purpose/Objective: Leukocyte adhesion and transmigration is one of the central paradigms of inflammation. Within the liver sinusoids, chemokines initiate the first crucial step of lymphocyte migration into the parenchyma. Especially liver sinusoidal endothelial cells (LSEC) present chemokines to T cells increasing their biological activity. We here investigated mechanisms of chemokine transport by LSEC and whether inhibition of endothelial chemokine presentation influences autoimmune hepatitis. Materials and methods: In the murine model of Concanavalin (Con) A-induced T-cell mediated hepatitis chemokine expression was determined. Chemokine uptake, transcytosis and presentation by LSEC were visualized by confocal microscopy and functionally investigated in transmigration assays. Effects of reduced endothelial chemokine presentation on the development of hepatic inflammation were monitored by alanine transaminase and histology. Results: During Con A-induced hepatitis, liver mRNA expression of the pro-inflammatory CXC chemokine ligand (CXCL)9 and CXCL10 was significantly increased. Furthermore, CXCL9 was particularly shown within LSEC. LSEC internalized basolateral CXCL9, CXCL10 and CXCL12 via clathrin-coated vesicles and provided these chemokines immobilized on the glycosaminoglycans heparan sulphate and chondroitin sulphate to CD4+ T cells, thereby increasing transmigration in vitro. Blockage of the clathrin-dependent cellular transport pathway significantly reduced endothelial chemokine internalization and consequently chemokine-dependent CD4+ T-cell transmigration across LSEC. Administration of a clathrin inhibitor in vivo suppressed Con A-induced autoimmune hepatitis and decreased accumulation of activated CD4+ T cells expressing the CXC chemokine receptor 3. Conclusions: Intervention in endothelial chemokine provision in vivo affects chemokine-dependent migration of pro-inflammatory CD4+ T cells into the liver, thereby counteracting development of autoimmune hepatitis. Thus, chemokine presentation by LSEC during liver inflammation might be a promising therapeutic target in hepatitis.

P0981 Corticosteroids affect hepatitis C infection by modulating plasmacytoid dendritic cells but not interferon-a signaling J. Kwekkeboom,* P. E. de Ruiter, P. P. C. Boor,* Q. Pan, J. de Jonge, H. J. M. Metselaar,* H. W. Tilanus & L. J. W. van der Laan * Erasmus MC University Medical Centre, Gastroenterology and Hepatology, Rotterdam, The Netherlands, Erasmus MC University Medical Centre, Surgery, Rotterdam, The Netherlands Purpose/Objective: Chronic hepatitis C virus (HCV) infection is one of the leading indications for liver transplantation, but outcomes are often compromised by re-infection of the graft. Several studies have indicated that the use of corticosteroid-based immunosuppression is a risk factor for severe HCV recurrence. The mechanism for the steroidmediated effect on HCV is not fully elucidated, but recent studies using in vitro HCV models found no direct effect on viral replication. The success rate of interferon-a (IFN-a) based antiviral therapy is significantly lower post-transplantation than in the non-transplant HCV population; however the impact of steroids on the antiviral activity of IFN-a is unknown. Therefore, the aim of this study is to investigate the effect of steroids on the antiviral activity of IFN-a and the impact on the primary IFN-a-producing cells, the plasmacytoid dendritic cells (PDCs). Materials and methods: As a model for HCV replication we used the Huh7 hepatoma cell line, stably transfected with the non-structural coding sequence of HCV directly coupled to a luciferase reporter gene (Huh7-ET), and treated with IFN-a in the presence or absence of different doses of prednisolone or dexamethasone. A Huh7 cell line stably transfected with a luciferase gene under the control of an interferon response element (Huh7-ISRE-Luc) was used to investigate effects on IFN-a signal transduction. To investigate the effects of steroids on PDCs, Huh7-ET cells were co-cultured with human PDCs in the presence or absence of steroids. Results: HCV replication was inhibited by 10 IU/ml IFN-a by more than 99% of control levels. Treatment with increasing doses of dexamethasone or prednisolone did not significantly affect HCV replication. When combining IFN-a with dexamethasone or prednisolone, no interference with the inhibition of HCV replication by IFN-a was observed. Moreover, dexamethasone and prednisolone had no effect on IFN-a signal transduction as measured in Huh7-ISRE-Luc cells. However, when Huh7-ET cells were co-cultured with PDCs, a significant reduction of HCV replication was observed, which was almost completely reversed by treatment with steroids. Conclusions: We found no evidence that corticosteroids interfere with signal transduction and antiviral action of IFN-a. However, steroids may affect HCV replication post-transplantation by reducing the antiviral capacity of PDCs.

P0982 Differential migration patterns of CD8 T cells primed in the liver and gut I. Eickmeier,* D. Seidel,* J. R. Gru¨n, A. A. Ku¨hlà & E. Schott* * Charite´ Universita¨tsmedizin, Hepatology and Gastroenterology, Berlin, Germany, German Arthritis Research Center (DRFZ), Bioinformatics, Berlin, Germany, àCharite´ Universita¨tsmedizin, Immunopathology/RCIS, Berlin, Germany Purpose/Objective: Patients with inflammatory bowel disease (IBD) are frequently affected by autoimmune liver diseases, suggesting that these anatomical distinct diseases may share pathogenic features. Since IBD is T-cell mediated, it is possible that T cells activated in the gut migrate to the liver where they cause inflammation. In earlier experiments we have shown that gut- as well as liver-activated T cells


500 Poster Session: Myeloid Cell Development accumulate in the liver, but only gut-activated CD8 T cells are licensed to migrate to the small intestine. In contrast liver-activatedCD8 T cells retain the capacity to migrate through lymph nodes. Here we analysed the expression of activation and adhesion molecules of these differentially activated T cells, which may explain their migration behaviour. Materials and methods: Antigen-specific naı¨ve OT-I CD8 T cells were adoptively transferred into mice, in which ovalbumin is expressed in the liver (TF-OVA mice) or in the small intestine (iFABP-OVA mice). Effector T cells were isolated from liver and mesenteric lymph nodes after three days. Activation assays as well as microarray analysis and flow cytometry were used to examine distinct or shared imprinted patterns of these cells. Results: Transcriptome analysis of naı¨ve, liver- and gut-activated CD8 T cells identified 10 326 differentially regulated IDs. Hierarchical clustering clearly discriminated naı¨ve, liver- and gut-activated T cells. Activation in the gut induced the typical gut-specific CCR9/a4b7 T cells. In contrast liver-activated T cells displayed increased expression of a4b1, a6b1, Ly6C, PD-1 and CD62L, whereas activation markers as well as aLb2 were upregulated in both cell types. The adhesion molecules ICAM-1 and VCAM-1 are constitutively expressed in the liver and were upregulated upon inflammation, whereas MAdCAM-1 expression could not be detected in the liver. Conclusions: Liver-activated CD8 T cells display a unique phenotype compared to naı¨ve and gut-activated T cells. High expression of CD62L and Ly6C may be responsible for their migration through lymph nodes. Only gut-activated T cells show gut-specific migration, but they also accumulate in the liver. This accumulation probably results from their adhesion to VCAM-1 and ICAM-1 in the liver. The migration of activated CD8 T cells is a one way route from the gut to the liver and may be responsible for the development of liver disease in patients with IBD.

P0983 Estudy of LDL influence in the interaction between HCV E2 protein and the surface receptors of ECV304 cell A. C. Urbaczek,* W. C. Generoso, A. Tansini,* L. M. Fonseca,* F. Henrique-Silva & P. I. Costa* * UNESP, Clinical Analysis, Araraquara, Brazil, UFSCAR, Genetic and Evolution, Saõ Carlos, Brazil Purpose/Objective: The HCV presents a world-wide prevalence of 3%, being the Hepatitis C one of the most important public health problems. The virus is enveloped and presents 10 different structural and non-structural proteins, amongst them the envelope 2 glycoprotein (E2). The E2 protein presents a strong association with the LDL receptor, suggesting that the HCV may use it to invade cells. An association between HCV and LDL from human serum was demonstrated in preliminary studies which report that lipoproteins may increase HCV infectivity. The aim was to produce a recombinant protein similar to HCV E2 protein, codified by partial E2 protein gene (without TM domain) and not glycosylated, to analyze its binding to the surface receptors of ECV304 cells, in the presence and absence of human LDL, in order to verify the influence of LDL in this binding process. Materials and methods: The coding gene of the E2-like protein fused to the GST was cloned in the pET-42a vector and transformed into E. coli bacteria strain Rosetta, which was induced to protein expression with IPTG, at 37C, 300 rpm for 3 h. The proteins were purified by glutathione column. ECV304 cells were incubated at 4C for 90 min with 20 lg of E2 protein only and with 20 lg of E2 protein added of 40 lg of human LDL. The cells were washed with PBS pH7.2 and incubated at 4C for 30 min with anti-his antibodies conjugated with APC. The cells were washed with PBS pH7.2 and submitted to analysis in FACS Canto BD cytometer, software Diva FACS..

Results: The flow cytometry results showed that the E2 recombinant protein bound to 20% of the cells and the proteins with human LDL bound to 35% of the tested cells. Conclusions: The flow cytometry revealed that the addiction of human LDL to E2 recombinant proteins provided an increase of 75% in the binding to surface receptors of ECV304 cells, phenomenon that may increases the HCV infectivity. Financial support: FAPESP (2008/58957-0) and FUNDECIF.

P0984 Expression and function of Toll-like receptors in human hepatic stellate cells J. Zeromski,* A. Mania, M. Kaczmarek,* I. Mozer Lisewska,à B. Brzezicha,* M. Frydrychowicz,* R. Safadi§ & A. Kowala Piaskowskaà * Clinical Immunology, University of Medical Sciences, Poznan, Poland, Pediatrics, University of Medical Sciences, Poznan, Poland, àInfectious Diseases, University of Medical Sciences, Poznan, Poland, §Hadassah Medical Center, Liver & Gastroenterology Unit, Jerusalem, Israel Purpose/Objective: Human hepatic stellate cells (HHSC) play pivotal role in chronic liver diseases. They are involved in the induction and progression of hepatic fibrosis leading to liver cirrhosis. Toll-like receptors (TLRs) are known to be expressed on/in HHSC and some of them, such as TLR4 have been claimed to promote liver fibrosis. Presence and function of other TLRs in HHSC remains totally inknown. The aim of the current study was to to get some information about the expression and function of these structures in the cells in question. Materials and methods: Established cell line of HHSC, LX2 was used. Cell cytospins were searched by immunocytochemistry (ICH) and cell suspensions by flow cytometry (FC) for TLR1-10 expression, as a per cent of positive cells and mean fluorescent intensity (MFI). RNA was isolated from cells and tested for mRNA specific for respective TLRs. Cells were subjected to short time culture with appropriate TLR ligands and afterwards assessed by FC for TLR expression. Culture supernatants were searched for a number of released cytokines by Flexnet (BD) cytometric assay. Results: LX2 cells were shown to express all TLR1-10, both, tested by ICH and FC, but the per cent of positive cells varied. TLR mRNA was evidenced also for all TLRs tested. Culture with ligands resulted usually in lower MFI expression than in medium alone. SSpolyU, TLR8 ligand was the only one, that provided slightly higher MFI value of LX2 cells as compared to medium alone. The only cytokine, or rather chemokine, produced by LX2 cells in reasonable amounts (up to 800 pg/ml) was stromal cell-derived factor-1 (SDF-1a also known as CXCL12). Its production however appeared to be constitutive feature of LX2 cells, because it was also secreted by cells cultured without any TLR ligands. Another cytokine, being also a chemokine, that was secreted by cells in tiny but measurable range (53 pg/ml) was IL-8. It was the case, when cells were cultured in medium supplemented with FLA-BS, ligand of TLR5. Besides, cells cultured in medium (without any ligand) secreted TGF b1, in amounts of 96 pg/ml. Conclusions: Expression of TLRs (1 10) appears to be common phenomenon on/in LX2 cells. Their activation by TLR ligands has relatively modest effect on TLR expression and function. Presumably, in the case of HSC in vivo, these cells are boosted by other, hitherto unknown receptors or agents to manifest their profibrotic activity.


Abstracts 501 P0985 HCV Core protein induced immune disordre via oxidative stress and thiol redox alteration K. Ben Larbi Zarki, M. Rodri¢guez Iglesias, M. C. Duran Ruiz & F. F. Garcia Cozar Biomedicine Biotechnology and Public Health, Universidad de Ca´diz, Ca´diz, Spain Purpose/Objective: The network linking HCV infection, inflammation, free radical production, and carcinogenesis applies very well to HCV-mediated chronic liver damage, just as it applies to any chronic inflammatory condition. Research into the role of structural and nonstructural proteins of HCV and the changes induced in cytokine expression oncogenes, antioncogenes, and intracellular kinases shows that HCV is by itself and not only through inflammation able to induce ROS, an effect specific to this virus. This free radical production, accompanied by oxidative genomic injury, constitutes the first step of a cascade of genomic and postgenomic events that play an important role in HCC. More information is necessary from recently introduced technologies for proteomics that will hopefully close the gap between hypothesis and understanding. In the present study, we provide further evidence in support of this postulate. The technique used in our search, combining proteomics and proteine redox modification by S-glutathionylation, can help identifying proteins specifically modified under HCV Core pathological conditions. Materials and methods: In the present work, we have engineered lentiviral vectors expressing the HCV core to determine how the pattern of protein oxidation is affected by disrupting thiol reduction pathways in CD4 Jurkat cells expressing HCV core protein. As a step towards characterizing relative susceptibilities of cell proteins to oxidation and identifying what changes can altere signalling or metabolic regulation, we have taken a proteomic approach (mono and two-dimensional electrophoresis; and MALDI-TOF MS) to identifying thiol proteins that become oxidized in both CD4 Jurkat cells expressing HCV Core and JKT cells exposed to oxidative stress (H2O2); This model was previously reported to cause extensive S-thiolation of cellular proteins. we also monitored oxidized thiols in situ by a technique that involves labelling with the fluorescent probe and analyzed by flow cytometry in a CyanADP-MLE (DakoCytomation) using an UV enterprise laser set at 30 Mw. Results: Core protein increased S-glutathionylation in CD4 Jurkat cells. These results suggest that expression of core protein and subsequent oxidation of the glutathione pool and oxidative cysteine modifications inducing glutathiolation may be an important cause of the Alteration of immune cell function and the progression from chronic hepatitis C to HCC. Conclusions: This new insight into the mechanisms for HCV mediated immune evasion and lymphocyte homeostasis alteration may offer novel therapeutic targets for one of the most devastating human malignancies in the world today

P0987 Hepatocytes mediate Notch dependent immune regulation in response to inflammation in the regenerating mouse liver S. Burghardt, A. Erhardt & G. Tiegs Institute of Experimental Immunology and Hepatology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Purpose/Objective: A single injection of concanavalin A (ConA) to mice induces acute Th1-mediated hepatitis. Tolerance against ConA rechallenge develops within 8 days and is mediated by IL-10 predominantly produced by CD4+ CD25+Foxp3+ regulatory T cells (Tregs) and Kupffer Cells. This study was intended to identify the role of hepatocytes (HC) in ConA-induced immunoregulation.

Materials and methods: HC or splenic DC were isolated from salineor ConA-pretreated wt, IL-10-, IFNc-, or interferon regulatory factor (IRF)-1-deficient mice. Subsequently, HC or DC were co-cultured with splenic CD4+ T cells from wt, DEREG or IL-10 KO mice and stimulated with anti-CD3 for 60 h. Cytokine release was measured by ELISA. Frequencies of CD4+Foxp3+ Tregs and Notch1+ T cells were quantified by FACS analysis. Results: Naive CD4+ wt T cells co-cultured with wt HC from ConAtolerant mice showed significantly increased IL-10 levels indicating a tolerant phenotype. T cells were identified as major source of IL-10. In contrast, splenic DC from ConA-tolerant mice failed to induce IL-10 release in naı¨ve wt T cells. CD4+ wt T cells co-cultured with ConAprimed IFNc- or IRF-1-deficient HC released significantly lower levels of IL-10 compared to co-cultivation with wt HC from ConA-tolerant mice. Moreover, the c-secretase inhibitor DAPT, which blocked Notch activation, prevented IL-10 secretion. Interestingly, HC from ConAtolerant mice increased the frequency of CD4+Notch1+ T cells as well as receptor density of Notch1 on CD4+ T cells. Furthermore, Foxp3 expression was elevated in co-cultures containing T cells from DEREG mice and ConA-tolerant HC compared to non-primed HC. Moreover, HC from ConA-tolerant mice promoted the TGFb-driven conversion of naı¨ve wt T cells to Foxp3+ Tregs which was again abrogated by DAPT. Conclusions: We showed that HC from ConA-tolerant mice induce an IL-10-secreting regulatory phenotype in naı¨ve T cells and convert these cells into Foxp3+ Tregs in the presence of TGFb. The conversion depends on an intact IFNc-dependent Th1 response and on Notch signalling. The failure of splenic DC to induce IL-10 expression indicates that the generation of an IL-10 producing T cell subset is restricted to liver-resident non-professional APCs favouring the ‘liver tolerance effect’ as well as regeneration in response to inflammation.

P0989 Human cytomegalovirus infection of human hepatic sinusoidal endothelial cells promotes CD4 T cell recruitment and posttransmigrational activation T. Bruns,* H. Zimmermann,* A. Pachnio, P. A. Moss & D. H. Adams* * NIHR Biomedical Research Unit and Centre for Liver Research, University of Birmingham, Birmingham, UK, School of Cancer Sciences, University of Birmingham, Birmingham, UK Purpose/Objective: Animal studies suggest that endothelial cells and not hepatocytes are the site of cytomegalovirus (CMV) latency and reactivation in the liver and the source of secondary viral spread. Furthermore, murine CMV infection of sinusoidal endothelium is able to break immunotolerance and induce a strong CD8 T cell effector response. The aim of this study was to investigate, whether CMV infection of human hepatic sinusoidal endothelial cells (HSEC) modulates the ability of the liver to recruit and activate CD4 lymphocytes. Materials and methods: Recombinant endotheliotropic eGFP-labeled CMV was propagated in RPE-1 cells and purified by ultracentrifugation in tartrate/glycerol gradients. Primary HSEC were isolated from explanted livers, grown to confluence and infected with CMV over 2 h. Infection was confirmed by fluorescence microscopy and plaque assay on fibroblasts. Expression of adhesion molecules and costimulatory on infected HSEC were analyzed by flow cytometry and cell-based elisa. Resting CD4 T cells, regulatory T cells and CMV-specific CD4 clones were perfused over HSEC monolayers 24 h after infection under constant flow simulating physiological shear stress and adhesion and transmigration recorded using phase contrast microscopy. Static transmigration assays through HSEC into collagen were used to study phenotype and cytokine production of transmigrating lymphocytes using flow cytometry.


502 Poster Session: Myeloid Cell Development Results: Human sinusoidal endothelial cells were permissive to CMV infection. CMV infection of HSEC resulted in an increase of ICAM-1 and a decrease of ICAM-2, PD-L1 and CD40 and secretion of CXCL10 and CCL5. Under flow, transendothelial migration of CD4 T cells was increased through CMV-infected endothelium and predominantly mediated by ICAM-1. Transmigrated allogeneic CD4 effector memory T cells from CMV-seropositive and seronegative donors displayed a strongly increased expression of CD69 at 24 h and CD25 after 48 h after transendothelial migration through CMV-infected HSEC into collagen and demonstrated a Th1 phenotype (T-bet+, IFNg+, TNFa+) independently from HSEC MHC class II expression. Conclusions: CMV infection of HSEC facilitates the up-regulation of cell-adhesion molecules and chemokines resulting in increased adhesion, transmigration and activation of CD4 T cells. This may explain how human CMV infection not only provokes significant hepatitis but also increases hepatic immune activation in graft rejection.

P0990 IL-33 exacerbates liver ischemia-reperfusion injury in mice P. Zhu,* L. Xie, L. Yang,* L. Duan,à M. Fang,à F. Gongà & J. Yang* * Department of Internal Medicine, Institution of Immunology, the First college of Clinical Medical Science, Yichang city, China, Department of Surgery the First college of Clinical Medical Science, Institution of Immunology, China Three Gorges University Yichang China, Yichang city, China, àDepartment of Immunology, Institution of Immunology, Tongji Medical College Huazhong University of Science and Technology Wuhan China, Yichang city, China Purpose/Objective: The aim of this study was to examine the role of IL-33 on liver I/R injury in mice. Materials and methods: A partial lobar liver warm ischemia model was performed. The expression of IL-33 and ST2 were analysed in sham mice and liver I/R injury mice, using real-time polymerase chain reaction, western blotting, immunohistochemisry staining and confocal microscope approaches. The liver function, the level of cytokines and TLR4 and the infiltration of neutrophils were measured in sham, anti-IL-33 antibody, rIL-33 protein and saline group by ELISA, flow cytometry, immunohistochemisry staining, respectively. Results: Our results show that both mRNA and protein levels of IL-33 were overproduced in I/R injury mice but not sham mice (P < 0.05). The major sources of IL-33 during liver I/R injury were injury hepatocytes and the vascular endothelial cells. Those mice pre-treated with anti-IL-33 antibody 1 h before ischemia showed decreased serum alanine aminotransferase levels, inhibited production of proinfiammatory cytokines such as IL-1b, IL-18, TNF-a, and IL-6 as well as decreased of infiammatory cell infiltration by down regulation of TLR4 on kupffer cell, leading to the prevention of liver I/R injury, when compared with controls (P < 0.05). Histology revealed that pre-treated with anti-IL-33 antibody significantly ameliorated hepatocellular damage (P < 0.05). At the same time, those mice pre-treated with rIL-33 protein revealed more serious liver injury than control mice (P < 0.05). Conclusions: our study confirms that IL-33/ST2 signalling is involved in liver I/R and that inhibition of IL-33 can protect the liver from I/R injury by reducing IL-1b, IL-18, TNF-a, IL-6 release and infiammatory cell infiltration through down regulation of TLR4 expression on kupffer cell.

P0991 Immune cell CD39 and CD73 expression in autoimmune hepatitis and health C. Grant, R. Liberal, B. S. Holder, Y. Ma, G. Mieli-Vergani, D. Vergani & M. S. Longhi Institute of Liver Studies, King’s College London, London, UK Purpose/Objective: CD39 is an ectoenzyme that works in tandem with CD73 to degrade ATP and ADP into AMP and immunosuppressive adenosine. In mice, CD4+ CD25high regulatory T cells (Tregs) express both CD39 and CD73. In humans CD39+ Tregs have potent immunomodulatory capabilities, despite variable CD73 expression. Autoimmune hepatitis (AIH) is an inflammatory liver disorder characterised by reduced Treg frequency and function. CD39 and CD73 expression by different immune cell subsets has not been fully characterised. Our objective was to determine the frequency of circulating CD39+ and CD73+ immune cells in AIH and health. Materials and methods: Twenty AIH patients and 10 healthy subjects were studied. The frequency of CD19+ (B cells), CD11c+ (dendritic cells), CD8+, CD56+ (natural killer cells), CD4+ CD25- and Treg cells expressing CD39 and CD73 was measured by flow cytometry. Results: CD19+ and CD11c+ populations contained the highest frequency of CD39+ cells in both AIH patients and HS. CD39 was expressed to a lesser extent by Tregs, and the frequency of CD39+ Tregs was lower in AIH than in HS. The frequency of CD39+ cells within the CD4+ CD25-, CD56+ and CD8+ subsets was low in both groups, but CD39+ CD8+ lymphocytes were fewer in AIH than in HS. The CD19+ and the CD8+ subsets contained the highest frequencies of CD73+ cells in both groups, with CD73+ CD8+ lymphocytes being more numerous in AIH than in health. A lower proportion of Tregs from both groups were positive for CD73. The frequency of CD73+ cells within CD11c+, CD56+ and CD4+ CD25- populations was low in both AIH and health, but AIH patients displayed higher frequencies of CD73+ CD56+ and CD73+ CD4+ CD25- lymphocytes than HS. Conclusions: Circulating immune cells display differential CD39 and CD73 expression patterns. In AIH, the low proportion of CD39+ cells within Tregs and CD8+ lymphocytes may account for impaired immune-regulation. Both in AIH and health, only a small proportion of Tregs are positive for CD73, suggesting that completion of the ectonucleotidase cascade is performed by CD19+ and CD8+ cells, the higher frequency of CD73+ cells in AIH possibly reflecting an inflammatory state. Since CD19+ cells express the highest frequency of both CD39 and CD73, their possible role as regulatory B cells needs to be investigated in future studies.

P0992 Immunohistochemical study of Toxocara-induced hepatic inflammation: characterizing the immune response players A. Othman, D. Ashour & D. A. Mohamed Parasitology Faculty of Medicine, Tanta University, Tanta, Egypt Purpose/Objective: The aim of this study was to characterize the key immune cells and the rate of apoptosis in hepatic inflammation during the course of experimental infection by Toxocara canis. Materials and methods: Mice experimentally infected with Toxocara canis were divided into two groups: mice with primary infection by Toxocara, and those infected after sensitization by Toxocara excretorysecretory antigen. CD4+, CD8+, andBcl-2-expressing T lymphocytes were identified in the liver by immunohistochemistry at different durations post-infection. Results: We observed recruitment in both CD4+ and CD8+ T lymphocytes with difference in count and localization within the liver. These cells were detected within and around Toxocara-induced granulomas as well as in isolated inflammatory foci in the portal tracts


Abstracts 503 or within the hepatic parenchyma. The antiapoptotic protein Bcl-2 showed no significant change at different periods post-infection. On the other hand, immunization of mice with Toxocara excretorysecretory antigen prior to experimental infection caused earlier and more pronounced recruitment of CD8+ T cells to the liver and enhanced expression of Bcl-2. Conclusions: These results suggest a dynamic change in key immune cells according to duration of infection as well as the immune status of the host.

P0993 Induction and functionality of hepatic regulatory CD4+ T cells C. Rudolph,* N. Kruse,* K. Neumann,* U. Erben,* A. Ku¨hl,* A. Derkow, E. Schott, M. Zeitz,* A. Hamannà & K. Klugewitz* * Charite´ University-Medicine Berlin, Medical Clinic I, Berlin, Germany, Charite´ University-Medicine Berlin, Medical Clinic Hepatology and Gastroenterology, Berlin, Germany, àDeutsches Rheuma-Forschungszentrum, Berlin, Germany Purpose/Objective: Liver sinusoidal endothelial cells (LSEC) play an important role in shifting local immune responses to tolerance in major histocompatibility complex (MHC) I-restricted models of antigen presentation. Their impact in MHCII-mediated antigen presentation in the context of tolerance and immunity is still under investigation. Recently, in a bone marrow chimeric mouse model expressing MHCII exclusively on non-hematopoietic cells like LSEC, the induction of CD4+ CD25low forkhead box protein (FoxP)3- regulatory T cells by LSEC has been described. Materials and methods: In a model of T cell-mediated autoimmune hepatitis, we adoptively transferred OVA-specific CD8+ and CD4+ T cells alone or in combination with LSEC-primed CD4+ T cells (TLSEC) into TF-OVA mouse, expressing OVA exclusively in the liver. The suppressive capacity of TLSEC was analyzed by CD8 T cell suppression assay in vitro. We further investigated the mechanisms involved in induction of TLSEC phenotype. Transfer colitis model was used to gain insights into the ability of regulatory TLSEC to suppress the development and progression of intestinal inflammation. Results: TLSEC suppress a T cell-mediated autoimmune hepatitis in vivo. In vitro TLSEC have the capacity to suppress proliferation, activation and development of effector molecules of CD8+ T cells. We investigate mechanisms involved in the induction and function of regulatory CD4+ T cells by LSEC. Especially retinoic acid seems to play an important role during induction of TLSEC phenotype. Surprisingly, TLSEC do not only home into the liver but also migrate into the gut, enabled by expression a4b7 integrin and CC chemokine receptor (CCR)9. Initial experiments showed an inhibitory effect of TLSEC on intestinal inflammation by reduced lamina propria lymhocyte cell counts and a slightly reduced clinical colitis score. Conclusions: The suppressive capacity and gut-homing properties of TLSEC open for the intriguing possibility that recruitment of liverprimed CD4+ T cells into the intestine might modulate the immunological balance of the gut.

P0994 Induction of human CD14+HLA-DR- Myeloid Derived Suppressor Cells by Hepatic Stellate Cells B. Ho¨chst,* F. A. Schildberg, M. Ballmaier,à P. Sauerborn,* J. Schilling,* I. Mu¨ller,§ F. Gieseke,§ P. Knolle & L. Diehl* * Institutes of molecular Medicine and experimental Immunology, AG Diehl, Bonn, Germany, Institutes of molecular Medicine and experimental Immunology, AG Knolle, Bonn, Germany, àDepartment of Pediatric Hematology, Medizinische Hochschule Hannover, Bonn, Germany, §Forschungsinstitut Kinderkrebs-Zentrum, AG Mu¨ller, Hamburg, Germany Purpose/Objective: Tumors have evolved different mechanisms to evade the host immune response and generate a suppressive network. In addition to regulatory T cells (Tregs) and tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs) are of great importance and are becoming a focus of interest. The only possibility to overcome this problem will be a careful phenotypical and functional analysis of all potential MDSC subsets in different clinical settings and different immunological compartments. Identification of better markers will facilitate these studies. More indepth analysis of the interaction of MDSC with other cell types will help understanding the biological function and finally, the specific targeting of human MDSCs will enhance the effect of immune-based therapies in cancer. Materials and methods: MDSCs have gained a lot of attention in recent years, mainly in mouse models. However, the results from murine studies indicate that human MDSCs will need to be analyzed in more detail in cancer patients in order to understand the induction and function of these cells in patients. One major hurdle remains the heterogeneity of these cells. humans, MDSC are inadequately characterized because of the lack of uniform markers. The described phenotype includes CD33+, CD11b HLA-DR- cells (immature phenotype) CD14+, HLA-DR- cells (monocytic like) and CD15+, HLADR- cells (granulocytic like). They negatively regulate the immune response by effecting NK and T cells. Potential mechanisms, which underlie this inhibitory activity range from those requiring direct cellcellcontact. The only possibility to overcome this problem will be a careful phenotypical and functional analysis of all potential MDSC subsets in different clinical settings and different immunological compartments. Identification of better markers will facilitate these studies. More in-depth analysis of the interaction of MDSC with other cell types will help understanding the biological function and finally, the specific targeting of human MDSCs will enhance the effect of immune-based therapies in cancer. Results: Here we show that human Hepatic Stellate Cells have the capability to induce Myeloid derived Suppressor Cells from circulation Monocytes in a cell-cell contact dependent mechanism. Conclusions: Further investigation of the exact mechanism will provide opportunities to prevent the induction of Myeloid Derived Suppressor Cells. This represents a potential target for immunotherapy.

P0995 Induction of sustained tolerance towards experimental ConA hepatitis depends on CD4+ T and NKT cells B. Claass, A. Erhardt & G. Tiegs Institute for Experimental Immunology and Hepatology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Purpose/Objective: In mice, a single i.v. injection of the T cell mitogen Concanavalin A (ConA) leads to severe acute T cell and Kupffer Cell (KC) dependent hepatitis. Following a sublethal treatment of ConA, mice develop resistance towards hepatitis after additional


504 Poster Session: Myeloid Cell Development ConA injections within 8 days. This resistance is characterized by regulatory T cell and KC derived IL-10 dependent immunoregulation. However, IL-10 production was less regulated during sustained tolerance observed between 14 and 42 days. Here we investigated the role for B cells, CD4+ T and NKT cells, Tregs and KCs in the induction of long-lasting liver tolerance during the first ConA treatment. Materials and methods: Liver damage was quantified by plasma transaminase activities and/or by histology 8 h after a single ConA injection or after ConA restimulation 14 days after the first ConA treatment. Tregs were depleted from DEREG (DEpletion of REGulatory T cells) mice by diphtheria toxin i.p. treatment 1 day before first ConA injection. KCs were depleted by i.v. injection of Cl2MDP liposomes 2 days before the first ConA stimulation. A role for antibody mediated protection was investigated by ConA restimulation in Jh/Jk double knock-out mice 42 days after the first ConA treatment. To investigate if induction of liver resistance depends on CD4+ T cells, RAG1-/- mice were reconstituted with liver CD4+ T cells and treated with ConA. Results: Liver injury was detectable 8 h after a single ConA injection but was completely absent 14 days after ConA restimulation. However, neither absence of Tregs nor absence of KCs during the first ConA treatment abrogated liver tolerance in response to ConA restimulation on day 14. Also, B cell deficiency did not abrogate liver tolerance towards ConA. ConA treatment of Rag1-/- mice reconstituted with CD4+ T cells induced hepatic injury. ConA tolerance upon restimulation was inducible in these reconstituted mice. However, ConA pretreatment of RAG-/- mice 14 days prior to reconstitution failed to induce tolerance. Conclusions: CD4+ T and NKT cells seem to be involved in induction of sustained liver tolerance towards ConA observed from day 14 onwards. Involvement of Tregs and KCs during the first ConA treatment might play a minor role in the induction of long-lasting liver tolerance towards ConA.

P0997 Innate and adaptive control of immunity to Salmonella in the liver J. R. Hitchcock, E. A. Ross, A. Flores-Langarica, J. Marshall, C. Cook, M. Khan, S. Bobat, R. Coughlan, G. Reynolds, D. H. Adams & A. F. Cunningham Immunity and Infection, University of Birmingham, Birmingham, UK Purpose/Objective: Non-typhoidal Salmonella e (NTS) such as Salmonella Typhimurium (STm) can cause invasive and often fatal disease in children and HIV-infected adults in developing countries. The liver is a major target of this infection yet how immunity is regulated in this site is incompletely understood. Whilst important for understanding how STm can kill, examining hepatic immune function also allows the study of how innate and adaptive responses co-ordinate to control infection in non-lymphoid sites. Materials and methods: Microscopy, FACS and biochemical techniques were used to assess the impact of systemic STm infection on the murine liver in a resolving model of NTS infection. Results: There was a profound and rapid impact of infection on the liver. Inflammatory lesions formed from day 2 of infection, peaked as T cells effected STm clearance and had resolved when bacteria had been cleared. Despite the striking inflammatory response and substantial necrosis, liver function was largely normal. Lesions mostly contained multiple myeloid CD11c+ and F4/80+ subsets, with small numbers of CD4 and CD8 T cells, but not B cells. Before infection, Kupffer cells were the dominant monocyte population but this changed rapidly after infection. T cells orchestrated foci development. In particular, T-bet in T cells was required for optimal T cell migration and T cell IFNc, but not TNFa, expression. In addition, loss of T-bet increased numbers of hepatic FoxP3+ T cells but did not alter the early inflammatory

response. In contrast, infected IFNc KO livers resembled those of noninfected mice despite having substantial bacterial burdens. Conclusions: This work details the complex phenotype and organization of hepatic lesions after STm infection and the discrete roles of IFNc in this response. Moreover, it identifies a central role for T-bet in balancing T cell phenotype in the liver post-infection. Exploiting these findings will help identify how best to promote bacterial clearance whilst minimizing the collateral cost of immune responses to the host.

P0998 LLT1 engages CD161 at the immunological synapse between T cells and hepatocytes M. Purbhoo, N. Kumar & S. Ashraf Imperial College London, Section of Hepatology, London, UK Purpose/Objective: CD161+ CD8+ T cells are liver resident and associated with chronic hepatitis C virus infection. However, little is known about how CD161+ T cells engage target cells at a molecular level. CD161 is a co-stimulatory molecule that engages Lectin Like Transcript 1 (LLT1) on target cells and potentiates IFNc production by T cells. It may thus be important in generating an effective anti-viral immune response. We have investigated the expression of LLT1 by hepatocytes, and the partitioning of CD161 and LLT1 at the immunological synapse between T cells and hepatocytes. Materials and methods: LLT1 expression was investigated by flow cytometry and semi-quantitative RT-PCR in (1) Huh7 cells, (2) Huh7 cells containing the JFH-1 subgenomic replicon, and (3) Huh7/ replicon cells cured with IFNa. CD161 and LLT-1 were fluorescently tagged and expressed in immortalized T cells (Jurkat) or Huh7 cells, and their distribution at the T cell/hepatocyte immune synapse determined by confocal microscopy. Results: Huh7 cells constitutively expressed low levels of LLT1. The JFH-1 subgenomic replicon cells upregulated LLT1 at both surface protein and mRNA levels. Huh7 cells stimulated with TLR3 and TLR 7/8 agonists known to upregulate LLT1 on hematopoietic cells, did not show increased levels of LLT1. CD161 and LLT1 colocalised at T cell/ hepatocyte immune synapses. These molecules formed microdomains which gradually coalesced into extended, perforated accumulations which represent a novel, atypical spatial arrangement for costimulatory molecules in T cells. Conclusions: This study demonstrates that HCV may upregulate LLT1 on infected hepatocytes, independent of TLR-mediated recognition of virus. Hepatocytes expressing LLT1 form a stable immunological synapse with T cells expressing CD161, which forms independent of the presence of antigen. Within the synapse, CD161/ LLT1 show a unique spatial distribution, and such patterning may underpin the molecular mechanism of how CD161 enhances T cell activation.

P0999 Loss of chemokine receptor CCR6 diminishes recruitment of IL-17producing gamma/delta T cells to the liver leading to increased liver inflammation and fibrosis L. Hammerich,* F. Heymann,* H. W. Zimmermann,* S. Huss, S. A. Lira,à C. Trautwein* & F. Tacke* * Medical Clinic III, University Hospital Aachen, Aachen, Germany, Institute of Pathology, University Hospital Cologne, Cologne, Germany, à Immunology Institute, Mount Sinai School of Medicine, New York, NY, USA Purpose/Objective: The chemokine receptor CCR6 is known to be expressed on some T helper cells and cd T cells, monocyte derived dendritic cells and B cell subsets. It plays an important role in mucosal


Abstracts 505 immunity, but its role in liver disease is largely unknown. In this study we investigated its functional relevance in chronic liver disease and hepatic fibrosis. Materials and methods: Wildtype (wt) and CCR6-/- mice were treated with the hepatotoxic agent carbon tetrachloride (CCl4) thrice weekly over a period of 4 weeks. Liver damage, inflammation and fibrosis development were assessed by biochemical methods, histology, immunostaining, flow cytometry and qPCR. Human liver samples from patients with chronic liver disease (n = 50) were analysed by qPCR. Results: CCR6 and its ligand CCL20 are strongly upregulated upon chronic liver injury in mice and in human patients with cirrhosis. CCR6-/- mice develop more severe hepatic fibrosis compared to wt mice when treated with CCl4. They also show higher liver inflammation compared to wt mice, as indicated by increased overall infiltration of immune cells to the liver. We could not detect changes in infiltrating macrophages or composition of T helper cell subtypes between wt and CCR6-deficient mice, but expression of interleukin-17 (IL-17) was significantly reduced in livers of CCR6-/- mice. While wt mice showed an accumulation of IL-17-producing cd T cells in the liver upon CCl4 treatment, this cell type was markedly reduced in livers of CCR6-/mice. The adoptive transfer of wt cd T cells into CCR6-/- mice restored hepatic inflammation and fibrosis in the chronic injury model. Conclusions: We propose a CCR6-dependent pathway for recruitment of IL-17-producing cd T cells in chronic liver injury, that protects the liver from excessive inflammation and fibrogenesis.

P1000 Metformin aggravates Con A induced liver injury V. Volarevic,* M. Milovanovic,* B. Simovic Markovic,* M. Stojanovic,* M. Misirkic, L. J. Vucicevic, V. Trajkovic, N. Arsenijevic* & M. L. Lukic* * Center for Molecular and Stem Cell Research, Immunology, Kragujevac, Serbia, Faculty of Medicine, Immunology, Belgrade, Serbia Purpose/Objective: Several cases of liver injury related to hepatotoxicity of metformin, the most commonly prescribed oral antidiabetic medication, have been reported recently but mechanism of metformin induced liver injury remains unclear. We tested metformin hepatotoxicity and its effects in Con A induced hepatitis in mice. Materials and methods: Con A hepatitis was induced in susceptible C57BL/6 and CBA mice, relatively resistant BALB/c mice as well as in CBA iNOS-/- mice. Liver enzymes, histology, mononuclear cell (MNC) infiltration, cytokine production, expression of Akt, NF-kB, p38, AMPK, apoptosis of MNCs and autophagy were analyzed. Results: Single injection (400 mg/kg dissolved in saline, i.p) metformin did not induce liver damage, but the same dose of metformin significantly enhanced Con A (12 mg/kg dissolved in saline i.v) induced liver injury in BALB/c C57Bl/6 and CBA mice as evaluated by liver enzymes and histology. We found an increased level of TNF-a, IFN-c in the sera and an increased number of TNF-a, IFN-c and IL-17 producing CD4+ T cells, IFN-c producing NK and IL-4 producing NKT cells, activated CD80+ CD86+ IL-12 producing F4/80+ macrophages and CD11c+ dendritic cells (DCs) and aggressive B2 cells all of which are involved in disease process. Liver specific (CD4+ CXCR3+ Tbet+IL-10+ and CD4+ CD69+ CD25-) T regulatory cells were downregulated by metformin. Metformin significantly increased expression of both Akt and NF-kB in the liver as well as influx of activated CD4+ CD27+ cells, CD4+ and CD8+ CD62L- CCR7- effector memory cells. We also noticed significantly increase of iNOS expression both in the liver and spleen by metformin. Deletion of iNOS attenuated both Con A induced disease and the effects of metformin: there was no difference in percentage of liver specific T regulatory cells, total number of liver infiltrated effector T cells, macrophages and DCs.

Conclusions: Metformin aggravates Con A induced liver injury by enhancing activation of immune cells in Akt/NF-kB dependent and p38 and AMPK independent manner affecting influx of effector and liver specific regulatory cells and NO production. These data suggest that in the inflamed liver metformin have deleterious effects.

P1001 MHC class I transfer from stellate cells to LSEC allows for enhanced immune surveillance in the liver K. Scho¨lzel, F. A. Schildberg, P. A. Knolle & D. Wohlleber Insitute of Molecular Medicine and Experimental Immunology, University of Bonn, Bonn, Germany Purpose/Objective: The liver is known for its unique immunological properties. Besides liver parenchymal cells, the hepatocytes, also nonparenchymal cells like dendritic cells (DC), liver-resident macrophages, the Kupffer cells, and liver sinusoidal endothelial cells (LSEC) interact with passenger leukocytes and modulate immune responses. However, little is known about the contribution of stellate cells, known to be responsible for contraction of the vessel diameter and storage of vitamin A. Here, we addressed the question, if stellate cells contribute to immune surveillance by (cross-) presenting exogenous antigen to circulating naı¨ve or effector CD8 T cells and the consequences of such antigen presentation during viral infection of the liver. Materials and methods: We employed a transgenic mouse model with stellate cell-specific expression of the MHC class I molecule H2-Kb under the control of the GFAP-promoter (GFAP-Kb mouse). Mice were infected with 5 · 108 pfu of Adenovirus expressing the model antigen ovalbumin (AdOVA) followed by adoptive transfer of activated OT-I T cells which recognize SIINFEKL, a H2-Kb restricted peptide of ovalbumin. Stellate cells and liver sinusoidal endothelial cells were isolated as described previously. Results: Using this model system, we found that H2-Kb-restricted CD8 T cells were stimulated in vivo to proliferate and express cytokines suggesting that stellate cells could directly interact with circulating naı¨ve CD8 T cells. Further, we found that stellate cells play a role in generating antiviral CD8 T cell response to viral infection of the liver. However, a detailed characterization of the GFAP-Kb mouse revealed that stellate cells transferred MHC-I molecules to other hepatic cell populations like LSEC, DCs and Kupffer cells. We excluded erratic gene expression driven by GFAP-promoter in LSEC by RTPCR. Rather, we directly demonstrate transfer of H2-Kb molecules from stellate cells to H2-Kb-negative LSEC. Such molecule-transfer rendered LSEC capable of stimulating H2-Kb-restricted CD8 T cells in an antigen-specific fashion. Conclusions: Our results provide insight into new mechanisms of how local immune regulation in the liver can be achieved, i.e. by transfer of MHC-I molecules from stellate cells to LSEC. This phenomenon may contribute to the unique immune functions of the liver.


506 Poster Session: Myeloid Cell Development P1002 Modification of a single lysine in a CYP2E1 epitope induces immune-mediated DILI in BALB/c mice D. Njoku,* J. Cho, L. Kim, L. Strouss, E. McCarthy, K. Gilbert, M. Amzelà & N. Rose§ * Johns Hopkins University, Anesthesiology Pathology Pediatrics, Baltimore, MD, USA, Johns Hopkins University, Anesthesiology, Baltimore, MD, USA, àJohns Hopkins University, Biochemistry, Baltimore, MD, USA, §Johns Hopkins University, Pathology Molecular Microbiology and Immunology, Baltimore, MD, USA Purpose/Objective: Key steps in the pathogenesis of immune-mediated drug-induced liver injury (Im-DILI) have not been identified. After receiving halogenated anesthetics, anti-seizure medications, antibiotics or non-steroidal anti-inflammatory drugs, susceptible patients develop Im-DILI thereby increasing their morbidity and often their mortality. In anesthetic Im-DILI patients, granulocytic hepatitis, trifluoroacetyl chloride (TFA) and IL-4-mediated cytochrome P4502E1 (CYP2E1) IgG4 antibodies support the diagnosis, while CYP2E1 epitopes responsible for the pathogenesis of Im-DILI are unknown. We previously demonstrated a CYP2E1 epitope [Gly113-Leu133 (JHDN5)] containing a single lysine that was recognized by sera from anesthetic DILI patients with specific MHC II haplotypes. We showed that JHDN5 was recognized by splenocytes from mice with experimental Im-DILI induced by immunizations with liver proteins covalently altered by TFA, a drug hapten formed during metabolism of halogenated anesthetics. We hypothesize that covalent modification of a single lysine in JHDN5 induces IL-4-mediated, Im-DILI in BALB/c mice. Materials and methods: JDN5 was modified by TFA (TFA-JHDN5) using the methods of Goldberger and Anfinisen. We confirmed 81.5% modification of JHDN5 using the method of Habeeb. BALB/c mice were immunized with 100 lg of an unrelated CYP2E1 epitope or JHDN5 ± TFA emulsified in CFA or CFA alone on days 0 and 7 and killed on day 21. IL-4 deficient (KO) mice were similarly treated with CFA ± TFA-JHDN5. Histology scores, antibodies and cytokine levels were analyzed using Mann Whitney U-test. A P value 5 years (CHB/r); (5) 12 with non alcoholic fatty liver disease (NAFLD). Three liver samples with a mild increase of aminotransferases but without histological changes, served as controls. Histological activity index and staging of fibrosis were also assessed. RNA was extracted and cDNA was synthesized using standard protocols. mRNA expression of TGFb isoforms (TGFB1, 2, 3), activins (A, B, C, E), ALK4, ALK5, SMAD molecules (SMAD2, 3, 4, 7), and CTGFwas examined using quantitative real time PCR. Statistical analysis was performed using SPSS and P values < 0.05 were considered significant. Results: Patients with CHB/r exhibited a significant increase of SMAD7 and ALK4 mRNA expression compared to CHB/d patients, and reduced levels of TGFB1, SMAD2, SMAD3, and CTGF. A significant increase of SMAD7 was also found in NAFLD patients compared to untreated viral hepatitis patients and those who did not respond to any treatment. Moreover, NAFLD patients were presented with elevated levels of TGFB1, TGFB3, INHbC, ALK5, and SMAD4. Considering the intensity of inflammation, SMAD7, ALK5, and INHbC exhibited a significant increased expression from absent to minimal inflammation with a gradual reduction as inflammation exacerbates. Conclusions: Our data indicate that in cases with low grade fibrosis, as NAFLD (characterized by a lower incidence of severe liver complications and fibrosis progression) and CHB/r, SMAD7 overexpression might be a mechanism limiting the fibrogenic effect of TGFb suggesting that its induction may provide a target for novel therapeutic approaches. This research has been co-financed by the ESF and Greek national funds through the Operational Program ‘‘Education and Lifelong Learning’’ of the NSRF Research Funding Program: Heracleitus II. Investing in knowledge society through the European Social Fund.

P1005 Peritoneal macrophage inflammatory profile in cirrhosis is dependent on the etiology and is related to ERK phosphorylation level M. Martıńez-Esparza,* A. Tapia-Abellań,* A. J. Ruiz-Alcaraz,* T. Hernańdez-Caselles,* C. Martıńez-Pascual, M. Miras-Lo´pez, J. Such,à R. France´sà & P. Garcıá-Penãrrubiaà *Biochemistry and Molecular Biology (B) and Immunology, University of Murcia, Murcia, Spain, Hospital Universitario Virgen de la Arrixaca, Unidad de Trasplante Hepa´tico, Murcia, Spain, àHospital Universitario, Unidad Hepa´tica, Alicante, Spain Purpose/Objective: The aim of this work is to identify functional differences in the inflammatory profile of monocyte-derived macrophages (M-DM) from ascites in cirrhotic patients of different etiologies, alcohol- and hepatitis C virus (HCV)-related cirrhosis, trying to extrapolate studies from liver biopsies to immune cells in ascites. Materials and methods: We studied 45 patients with cirrhosis and non-infected ascites, distributed according to disease etiology, HCV (n = 15) or alcohol (n = 30). Cytokines and cellular content in ascites were assessed by ELISA and flow cytometry, respectively. Cytokines and ERK phosphorylation level from peritoneal monocyte-derived macrophages isolated and stimulated in vitro were also determined. Results: A different pattern of leukocyte migration to peritoneal cavity and primed status of macrophages in cirrhosis is observed depending on the viral or alcoholic etiology. Whereas no differences in peripheral


Abstracts 507 blood cell subpopulations could be achieved, T lymphocyte, monocyte and polymorphonuclear cell populations in ascites were more abundant in HCV versus alcohol etiology. Cirrhosis of HCV etiology is associated to a decreased inflammatory profile in ascites compared with alcoholic etiology. Higher levels of IL-10 and lower levels of IL-6 and IL-12 were present in ascitic fluid from HCV group. Isolated peritoneal monocyte-derived macrophages kept their primed status in vitro for the extent of 24 h culture. Increased phosphorylation of ERK1/2 was observed in ALC peritoneal macrophages at baseline compared with those from HCV patients, although addition of LPS induced higher phosphorylation increases of ERK1/2 in macrophages from HCV than from ALC patients. Conclusions;An increased macrophage inflammatory status is present in ascites of alcohol-related cirrhotic patients compared with that of HCV-related. This fact could be related to differences in bacterial translocation episodies or regulatory T cell populations. These findings would contribute to identify potential prognostic and/or therapeutic targets for chronic liver diseases of different etiology.

P1006 Positive M2-ELISAs with negative anti-mitochondrial antibodies (AMA) on IFT: a diagnostic dilemma? C. Weiler-Normann,* A. W. Lohse* & F. Haag *1st Medical Clinic, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Purpose/Objective: Immune fluorescence testing (IFT) combined with ELISA techniques to determine the specificity of autoantibodies have refined and facilitated the diagnosis of primary biliary cirrhosis (PBC) over the past years. Positive AMA with specificity for the M2 antigen have become an established diagnostic criterion. However, in our liver clinic we encountered a substantial number of patients with negative AMA who nevertheless showed positive ELISA results for M2 antibodies. The aim of the present study was to evaluate the significance of positive M2-ELISA- results with discordant negative IFT Results. Materials and methods: Patient sera were screened using Hep2-cells (Inova), rat kidney, liver, and stomach sections (Menarini) and the M2 EP (MIT3)-ELISA (Inova). All sera screened for M2 antibodies in 2011 were included. Results: Of 278 patients tested for M2 antibodies in 2011, 112 (40.3%) were positive. Surprisingly, 26 of these (23.2%) were judged negative for AMA on tissue sections. In nine of these cases (34.6%) PBC was diagnosed on the basis of clinical/histological criteria. In AMA/M2discordant patients, ELISA Titers were significantly higher in patients suffering from PBC according to the guidelines (69.68 ± 41.22 units) than in añon-PBC’ patients (31.85 ± 8.332 units; P = 0.0011). Nine AMA/M2-discordant patients showed untypical cytoplasmic staining patterns in Hep2 cells, of whom 4 (44.4%) were diagnosed as having PBC. Of the 17 patients without cytoplasmic staining, 5 (29.4%) were diagnosed with PBC. Conclusions: Some patients with PBC can present with negative testing for AMA, but react positive in the M2-ELISA. Conversely, patients with conditions other than PBC can exhibit positive M2ELISAs. Most of these latter patients have elevated immunoglobulins on analysis, and M2-ELISA-levels in these patients are usually low. In conclusion, if clinical suspicion of PBC exists, M2 testing should be performed even in the absence of positive AMA.

P1008 Recruitment mechanisms for primary and malignant B cells to the human liver S. Shetty,* T. Bruns,* C. J. Weston,* Z. Stamataki,* G. M. Reynolds,* H. M. Long, S. Jalkanen,à S. G. Hubscher, P. F. Lalor* & D. H. Adams* *Centre for Liver Research, University of Birmingham, Birmingham, UK, School of Cancer Studies, University of Birmingham, Birmingham, UK, à Department of Microbiology and Inflammation, University of Turku, Turku, Finland Purpose/Objective: B cells are present within chronically inflamed liver tissue and recent evidence implicates them in the progression of liver disease. In addition a large proportion of hepatic lymphomas are of B cell origin. The molecular signals that regulate normal and malignant B cell recruitment into peripheral tissue from blood are poorly understood leading us to study human B cell migration through hepatic sinusoidal endothelial cells (HSEC) in flow-based adhesion assays. Materials and methods: We used isolated human HSEC in flow assays with purified peripheral blood B cells to elucidate the molecular mechanisms of B cell recruitment via HSEC. The contribution of conventional adhesion molecules, ICAM-1 and VCAM-1 and unconventional molecules VAP-1 and CLEVER-1/stabilin-1 was assessed by using function blocking antibodies. We repeated our experiments with two B cell lymphoma cell lines, CRL-2261 and Karpas 422, and primary malignant B cells. We assessed the contribution of chemokines by performing transwell assays and adding chemokines to our flow assays. We also tracked the motility of B cells and lymphoma cell lines on HSEC using tracking software. Results: In flow assays B cells were captured from shear flow without a prior rolling phase and underwent firm adhesion mediated by VCAM1. Unlike T cells, which displayed vigorous crawling behaviour on the endothelium, B cells remained static before a proportion underwent transendothelial migration mediated by a combination of ICAM-1, VAP-1, CLEVER-1/stabilin-1 and the chemokine receptor CXCR3 and CXCR4. B cell lymphoma cell lines and primary malignant B cells from patients with chronic lymphocytic leukaemia and marginal zone B cell lymphoma also underwent integrin-mediated firm adhesion involving ICAM-1 and/or VCAM-1 and demonstrated ICAM-1 dependent shape-change and crawling behaviour. Unlike primary lymphocytes the malignant cells did not undergo transendothelial migration which could explain why lymphomas are frequently characterised by intravascular accumulation of malignant cells in the hepatic sinusoids. Conclusions: Our findings demonstrate that distinct combinations of signals promote B cell recruitment to the liver suggesting the possibility of novel targets to modulate liver inflammation in disease. Certain features of lymphocyte homing are maintained in lymphoma recruitment to the liver suggesting that therapeutic targets for lymphocyte recruitment may also prevent hepatic lymphoma dissemination.

P1009 Relationship between synthesis of alpha2-macroglobulin and changes in serum cytokine in rats T. Kuribayashi,* T. Seita,* E. Momotani & S. Yamamoto* *Azabu University, Immunology, Sagamihara, Japan, National Institute of Animal Health, Research Team for Paratuberculosis, Tsukuba, Japan Purpose/Objective: The a2-macroglobulin (a2M) is a typical acute phase protein in rats. We have investigated the kinetics of a2M after inflammatory stimulation. Furthermore, we estimated that interleukin (IL)-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1) were contributed the synthesis of a2M. However, little is available on the time-dependent changes on synthesis of a2M in hepatocytes. In


508 Poster Session: Myeloid Cell Development this study, synthesis of a2M in hepatocytes was estimated by immunohistochemistry and correlations between synthesis of a2M and cytokines (IL-6, CINC-1) were investigated. Materials and methods: Sprague-Dawley rats (age, 9 weeks) were used. Turpentine oil was intramuscularly injected at 0.4 ml/rat to induce acute inflammation. Three rats at each time point were scarified under anesthesia with pentobarbital before treatment and at 6, 12, 18, 24, 36, 48 or 72 h after injection of turpentine oil. Blood was collected from the aorta and the liver was removed. The presence of a2M in the liver was investigated by immunohistochemistry, and serum levels of a2M, IL-6 and CINC-1 were measured by ELISA. Results: a2M was not detected in the liver before injection of turpentine oil. a2M was detected in whole lobules of liver after 12 h after injection of turpentine oil, when high serum levels of IL-6 or CINC-1 were observed. a2M was distributed around the central vein at 36 h. However, small amounts of a2M were detected in the liver at 48 h, when peak serum levels of a2M were observed. a2M was apparently synthesized in response to stimulation by IL-6 and CINC-1, and synthesis of a2M in hepatocytes peaked long before peak a2M serum levels were seen. Conclusions: In conclusion, synthesis of a2M appears to be correlated closely with serum levels of IL-6 and CINC-1.

P1010 Role of mitogen activated protein kinases and PI3K-Akt on the cytokine inflammatory profile of peritoneal macrophages from ascites of cirrhotic patients A. Tapia-Abellań,* A. J. Ruiz-Alcaraz,* T. Hernańdez-Caselles,* R. France´s, P. Garıá-Penãrrubia* & M. Martıńez-Esparza* *Biochemistry and Molecular Biology (B) and Immunology, University of Murcia, Murcia, Spain, Hepatic Unit, Hospital General Universitario, Alicante, Spain Purpose/Objective: To compare the role played by several Mitogen Activated Protein Kinases (MAPKs) and PI3K-Akt pathways on the release of cytokines in monocyte-derived macrophages (M-DM) obtained from the ascites of cirrhotic patients to identify novel targets for pharmaceutical intervention to prevent hepatic damage. Materials and methods: M-DM were isolated from ascites of cirrhotic patients and stimulated in vitro with LPS and heat killed Candida albicans in the presence or absence of the inhibitors for MEK1, p38 MAPK, JNK and PI3K. Ascites and cell culture supernatants were assayed by ELISA for TNF-a, IL-6 and IL-10. MAPK phosphorylation levels were determined by Western blot. Results: We found that release of the pro-inflammatory cytokines, IL6 and TNF-alpha at baseline was more effectively reduced by the MAPK inhibitors, while basal IL-10 anti-inflammatory cytokine secretion, was only, but strongly (91.6%) affected by inhibition of PI3K. The incubation of peritoneal M-DMs in the presence of LPS and heat killed C. albicans increased the release of IL-6, TNF-alpha and IL10. LPS-induced pro-inflammatory secretion was more sensitive to MAK inhibitors, while that induced by C. albicans was more susceptible to inhibition of PI3K. Finally, inhibition of PI3K almost completely suppressed secretion of IL-10 in stimulated M-DM. Conclusions: These results demonstrate pro-inflammatory cytokines release depends on MAPK signalling pathways and differ depending of microbial stimulus in this clinical setting, and confirm the prominent role of PI3K-Akt pathway in the modulation of IL-10 mediated antiinflammatory function.

P1011 Salmonella infection promotes cross reaction of gut activated t cells to liver antigen leading to immune-mediated cholangitis in mice D. Seidel,* I. Eickmeier,* A. Ku¨hl, B. Wiedenmann* & E. Schott* *Medizinische Klinik m. S. Hepatologie und Gastroenterologie, Charite´ Universita¨tsmedizin Berlin, Berlin, Germany, Medizinische Klinik m. S. Rheumatologie und Klinische Immunologie, Charite´ Universita¨tsmedizin Berlin, Berlin, Germany Purpose/Objective: A dysregulated immune response against components of the gut flora may be involved in the pathogenesis of inflammatory bowel diseases (IBD). Infections with pathogens could further trigger cross reactions against extraintestinal autoantigens in genetically susceptible individuals. Primary sclerosing cholangitis (PSC) is strongly associated with IBD. T cells infiltrating the livers of patients with PSC display an a4b7+ CCR9+ phenotype, indicating their provenance from the gut-associated lymphatic tissues (GALT). This finding led to the hypothesis that activation of the adaptive immune system in the GALT could initiate an aberrant reaction against antigens in the liver. Materials and methods: Naive antigen-specific CD8 OT-I T cells were transferred i.v. into transgenic mice, which express their nominal antigen ovalbumin (OVA) in enterocytes of the small intestine (iFABPtOVA) or in the bile duct epithelia (ASBT-OVA), or into double transgenic mice (iFABPxASBT-OVA). To evaluate whether Salmonella promotes immune-mediated liver disease, mice were infected orally with S. typhimurium SL7207 (Sm) or S. typhimurium SL7207 expressing the MHC-I epitope OVA257 264 (SmOVA). The effector function of OT-I T cells was evaluated by measuring IFN-g production and by an in vivo cytotoxicity assay. Inflammation in the liver was assessed by measuring plasma ALT and by histologic analysis. Results: Adoptively transferred OT-I T cells acquired effector function characterized by production of IFN-c and in vivo cytotoxicity in iFABP-tOVA as well as in double transgenic iFABPxASBT-OVA mice, while only a minority of OT-I T cells became activated in ASBT-OVA mice. Activated OT-I T cells migrated into the liver, but caused cholangitis only in the presence of OVA in the biliary epithelia. ALTlevels were elevated in the plasma of double transgenic iFABPxASBTOVA mice, but not in the single transgenic lines. Infection with Sm or SmOVA enhanced the activation of OT-I T cells in the GALT and exacerbated cholangitis in iFABPxASBT-OVA mice. Conclusions: The activation of T cells in the gut led to cross reactivity in the liver when the same antigen was expressed in both compartments. The severity of cholangitis was increased by oral Salmonella infection. Our findings suggest that cross reactivity of gut-derived T cells to endogenous antigens may be involved in the pathogenesis of PSC.

P1012 SepSecS-induced TH17-associated autoimmune hepatitis in mice J. Rauch, F. Schulte, M. Sebode, A. Carambia, A. W. Lohse, C. Weiler-Normann & J. Herkel Departmend of Medicine I, University Medical Center HamburgEppendorf, Hamburg, Germany Purpose/Objective: Autoimmune Hepatitis (AIH) is a chronic inflammatory liver disease of unknown pathogenesis; its characteristics include intrahepatic periportal lymphocytic infiltrates and circulating autoantibodies. Autoimmunity to the SepSecS molecule may be of pathogenetic relevance, since SLA/LP autoantibodies that recognize the SepSecS molecule are highly specific for AIH. Here we explored the role of SepSecS immunity in AIH. Materials and methods: Mice were immunised with recombinant murine SepSecS protein in complete Freund‘s adjuvant (CFA).


Abstracts 509 Primary immune responses were analysed by in vitro re-stimulation of draining lymph node cells. Liver inflammation and cytokine response of liver infiltrating lymphocytes were determined. Results: SepSecS-immunised C57BL/6 mice and IL-10-/- mice on C57BL/6 background developed SLA/LP autoantibodies. However, SepSecS-immunised C57BL/6 mice did not develop liver inflammation; in contrast, IL-10-/- mice manifested histological liver inflammation with periportal lymphocytic infiltrates, reminiscent of human AIH. In response to saline/CFA, IL-10-/- mice showed mild parenchymal liver inflammation of a granulomatous type, but not the characteristic periportal inflammation seen in response to SepSecS/ CFA. Lymphocytes from SepSecS-immunised IL-10-/- mice, but not from C57BL/6 mice could transfer histological hepatitis both to C57BL/6 or IL-10-/- mice. In response to CD3 antibody stimulation in vitro, lymph node cells of IL10-/- mice secreted more IL-17 than those of C57BL/6 mice (1230 versus 460 pg/ml), indicating a general tendency of IL10-/- mice for TH17 differentiation. In response to SepSecS re-stimulation, lymph node cells of IL-10-/- mice secreted considerably more IL-6 (480 versus 65 pg/ml) and IL-17 (550 versus 15 pg/ml) than lymph node cells of C57BL/6 mice. Accordingly, liverinfiltrating lymphocytes, notably CD4 T cells, isolated from SepSecS/ CFA immunised IL-10-/- mice secreted significantly higher amounts of IL-17 (524 versus 72 pg/ml) and IFNc (7590 versus 1234 pg/ml) in response to SepSecS re-stimulation, than IL-10-/- mice immunised with saline/CFA. Conclusions: Immunity to SepSecS can cause hepatitis in susceptible IL-10-/- mice. Periportal liver inflammation was associated with a TH17 response of SepSecS-primed lymphocytes.

P1013 Serum HLA-G is associated with liver damage rather than with liver graft acceptance J. Kwekkeboom,* V. Moroso,* B. van Cranenbroek, F. Fai-A-Fat,* L. J. W. van der Laan,à H. J. M. Metselaar* & I. Joosten *Gastroenterology and Hepatology, Erasmus MC University Medical Centre, Rotterdam, Netherlands, Laboratory Medicine, Radboud University Medical Centre, Nijmegen, Netherlands, àSurgery, Erasmus MC University Medical Centre, Rotterdam, Netherlands Purpose/Objective: HLA-G is a non-classical HLA class I molecule which expression in healthy individuals is restricted to invading trophoblasts in placenta, which is thought to contribute to protection of the fetus from immunological attack by the mother. On basis of cross-sectional studies it has been suggested that increased serum HLAG levels after liver transplantation (LTX) are associated with graft acceptance. The aim of this study was to determine whether longitudinal variations in serum HLA-G concentrations after LTx are associated with signs of graft acceptance. Materials and methods: Serum HLA-G levels were quantified by ELISA in a cohort of 32 patients with end-stage liver diseases, both before transplantation and at several time points during the first year after transplantation, and for comparison in 24 age- and gendermatched healthy subjects. In addition, HLA-G was quantified in T-tube bile collected during the first few weeks after LTx. Results: Pre-LTX serum HLA-G levels in the patients were significantly higher compared to those in healthy individuals, suggesting that soluble HLA-G is released from diseased livers. Pre- and post-LTx serum HLA-G levels were positively correlated with serum transaminases and bilirubin, indicating release during liver damage. Patients with an early acute rejection episode displayed significantly elevated serum HLA-G concentrations compared to non-rejectors during the first 2 weeks after LTx. Starting at 1 month after LTX, serum HLA-G levels gradually decreased. Interestingly, bile secreted by the liver graft during the first few weeks after LTx contained HLA-G, suggesting that HLA-G is produced in the liver graft early after LTx.

Conclusions: Our data do not support the hypothesis of a tolerogenic role for HLA-G after LTX, but rather suggest that serum HLA-G levels are associated with liver (graft) damage.

P1014 Synergistic effect of HLA-class II DRB1*1301 and activating full length KIR2DS4 in the susceptibility to type I autoimmune hepatitis L. Fainboim,* N. Paladino,* A. Podhorzer,* G. Theiler,* S. Paz, H. A. Fainboim & M. Cuartiroloà *Inmunogene´tica, INIGEM, Ciudad Autońoma de Buenos Aire, Argentina, Hepatologıá, Hospital F. J. Muniz, Ciudad Autońoma de Buenos Aire, Argentina, àHepatologıá, Hospital Nacional de Pediatrıá J. P. Garrahan, Ciudad Autońoma de Buenos Aire, Argentina Purpose/Objective: A previous study from our center revealed a difference in the genetic predisposition to type I autoimmune hepatitis (AH) between children (PAH) and adults (AAH). The haplotype HLADRB1*1301-DQB1*603 is strongly associated with PAH in most pediatric populations studied worldwide. Because some activating KIR genes were associated with autoimmune diseases, in the present study we investigated the possibility of a combined effect of these two highly polymorphic systems. Materials and methods: The study included 233 healthy controls, 81 type I PAH and 44 AAH patients. Genomic DNA was used to identify the presence or absence of each 16 KIR genes, by two PCR amplifications: PCR-1 for domains D1and D2 combined and the PCR-2 for the TM/cytoplasmic region. Nineteen 5«-digoxigeninlabeled probes were used in a sequence-specific oligo-nucleotide probing (SSOP) approach. The KIR2DS4 gene was amplified by PCR with primers to analize the full-length and the deleted version of KIR2DS4. HLA class I and class II alleles were typed by PCR amplification and hybridization with SSOP for the analysis of exon 2 and 3 (class I), or exon 2 polymorphisms (class II). Results: The frequency of all 16 tested KIR genes were similar when we compared controls with AAH or PAH patients. In Argentinian Caucasoid healthy population, the frequency of KIR2DS4-alleles containing the 22 bp deletion in exon 5 account for 81% versus a frequency of the full-length gene of 39%. In contrast, in PAH patients the full-length 2DS4*001 allotype was present in 68% (P < 0.0001) and in 53% of AAH. We followed the method of Svejgaard and Ryder to find out whether class II allele or KIR full-length allele represent the primary association. The combined presence of both factors provided an OR value of 40.3, higher than the product of the RR of the two independent factors. The presence of the truncated form of KIR2DS4, showed a protector effect with an OR of 0.22. In AAH, only the combined presence of HLA-DRB1*1301 and functional KIR2DS4 showed an increased susceptibility (O r = 5.8) not observed by these two factors independently. Conclusions: These results represent the first evidence of a synergistic effect between a class II allele and an activator KIR gene in the susceptibility to develop autoimmune hepatitis.


510 Poster Session: Myeloid Cell Development P1015 The ABCs of viral hepatitis acute viral hepatitis

defining biomarker signatures for

D. Duffy,* R. Saleh, M. Laird,* L. Le Fouler,à M. El-Daly,§ A. Casrouge,* M. Abdel Hamid,§ M. Mohamed,§ M. Rafik, A. Fontanetà & M. Albert* *Institut Pasteur, ICD, Paris, France, Faculty of Medicine, Aim Shams University, Cairo, Egypt, àEmerging Disease Epidemiology, Institut Pasteur, Paris, France, §Liver Disease Research Unit, National Hepatology & Tropical Medicine Research Institute, Cairo, Egypt Purpose/Objective: Viral hepatitis is the leading cause of liver disease worldwide and can be caused by several agents, including: Hepatitis A, B and C. The host response to liver infection has been an area of intense study. We exploited recent advances in the assessment of biomarker signatures in order to define unique and common responses during three acute infections, all sharing the same tissue tropism. Patients were recruited as part of a hospital based surveillance program in two ‘fever hospitals’ specialized in infectious diseases in Cairo, Egypt. Materials and methods: We performed multi-analyte profiling (MAP) measuring the concentrations of 182 molecules in the serum of acute Hepatitis A, Hepatitis B, and Hepatitis C infected individuals, as well as healthy controls. Patients with negative anti-HCV Ab and positive HCV-RNA were considered as acute hepatitis C cases. Acute Hepatitis B was defined by a positive IgM anti-hepatitis B virus core and circulating levels of hepatitis B surface antigen. Acute Hepatitis A was defined as positive anti-HAV IgM Ab. Results: Statistical analysis revealed an analyte signature based upon eight proteins, which applied to Principle Component Analysis distinguished HCV patients from HAV/HBV-infected individuals and healthy controls. Notably, the signatures of HAV and HBV host response were indistinguishable in a hierarchical cluster analysis of all samples; suggesting that these RNA and DNA viruses share a similar mode of pathology in contrast with Hepatitis C. However when HAV and HBV patients were directly compared, six differentially expressed serum proteins were identified. Within the Hepatitis C cohort we could separate cleared and non-cleared patients based on just five molecules. One of these was IP-10, which we and others have previously highlighted as a predictive biomarker of Hepatitis C clearance, thus validating the approach used here. Conclusions: This medium throughput discovery approach has revealed previously unrecognized virus host interactions. The identification of hepatitis virus specific biomarkers will lead to novel functional insights of disease mechanisms.

P1016 The lymphotoxin-ß receptor and its role in hepatocyte-mediated liver regeneration K. Behnke, U. R. Sorg & K. Pfeffer Institute for Medical Microbiology & Hospital Hygiene, Heinrich-HeineUniversity Du¨sseldorf, Du¨sseldorf, Germany Purpose/Objective: The liver retains a capacity for regeneration in response to injury. Loss of at least 30% of liver mass leads to synchronized proliferation of mature hepatocytes (compensatory hyperplasia). It has previously been shown that mice deficient in LTbR (LTbR-/-) exhibit reduced survival after partial (70%) hepatectomy (PHx). Therefore, liver regeneration was analyzed in LTbR-/- mice compared to wild-type (WT) mice. Materials and methods: 70% PHx was performed in WT and LTbR-/(KO) mice. Using microarray (MA) analysis, the gene expression profile of liver tissue was analyzed 12 h post PHx and, where appropriate, verified by realtime RT-PCR. H/E-staining of liver sections and cytokine ELISAs were performed 0, 12, 24, and 48 h

post PHx. Serum protein levels (GPT, GOT, Bilirubin, pancreatic amylase, alkaline phosphatase, glucose) were analyzed 0, 12, 24, and 48 h post PHx. At similar time points H/E-staining of liver sections and cytokine ELISAs were performed. Results: It was confirmed that LTbR-deficient mice have a decreased survival rate compared to WT mice (62% versus 90%, respectively). Interestingly, surviving LTbR-/- animals show no delay in liver regeneration compared to WT animals. MA analysis identified a panel of differentially expressed genes; most prominent among these was a markedly decreased expression (in LTbR-/- mice) of murinoglobulin-2, a proteinase inhibitor of the a2-macroglobulin family. Also, TNF expression was increased twofold in the LTbR-/- cohort (confirmed by ELISA). Levels of the liver transaminases GPT and GOT were similar in both cohorts, while levels of pancreatic amylase were significantly decreased in LTbR-/- animals 24 h post PHx. In contrast, alkaline phosphatase was significantly increased in KO animals 24 and 48 h post PHx, and surprisingly, still elevated 10 days after PHx. Liver sections of LTbR-/- animals showed a higher number of hemorrhagic/ necrotic areas and more vacuolisation of hepatocytes 24 and 48 h post PHx. Conclusions: After PHx, LTbR-/- mice show a massive change in their gene expression profile compared to WT animals. In addition, their cytokine expression profile is altered and several serum proteins appear to be deregulated. This clearly demonstrates the importance of LTbR signaling in liver regeneration and its exact role in will be elucidated in further studies.

P1017 The role of Foxp3+ regulatory T cells in the development of an adaptive immune response in a persistent HBV mouse model Y. A. Ga¨bel,* L. R. Huang,* N. Garbi,* U. Protzer & P. A. Knolle* *Molecular Medicine, Institutes of Molecular Medicine and Experimental Immunology, Bonn, Germany, Virology, Institute of Virology Technische Universita¨t Mu¨nchen/Helmholtz Zentrum Mu¨nchen, Munich, Germany Purpose/Objective: Worldwide around 2 billion people are infected with the Hepatitis B Virus (HBV), from whom approximately 350 million people are chronically infected. They are at high risk of developing liver cirrhosis and hepatocellular carcinoma (HCC). To facilitate the development of therapeutic strategies against chronic Hepatitis B infection, it is mandatory to have a better understanding of the detailed mechanism responsible for the induction of HBV persistence. The forkhead box P3 transcription factor (Foxp3) has been shown to influence several adaptive immune responses, for example in the context of LCMV, where protective roles for regulatory T cells (Treg) have been uncovered (Rowe et al., Immunology 2012). Here we analyze the role of Foxp3+ Treg on the induction of HBV-specific CTL responses upon HBV infection in a novel mouse model. Materials and methods: As in vivo studies of HBV are hampered by the lack of a suitable animal model, we here take advantage of our recently developed mouse model for the induction of HBV persistence in mice (Huang et al., Gastroenterology 2012). These mice were infected with a low-dose (1 · 108 i.u./mouse) adenoviral vector carrying a 1.3-fold overlength human HBV genome (AdHBV) to cross the species barrier. To deplete Foxp3+ Treg, we used Foxp3.LuciDTR-4, which display 95% Treg depletion following injection of diphtheria toxin (DT) (Suffner et al., J. Immunology 2009). Results: In AdHBV infected Foxp3.LuciDTR-4 mice we observed a dramatic decline of the serum HBsAg but not HBeAg level, which dropped below detection limit at d10, whereas the control groups remained positive for HBsAg. Additionally, the serum ALT level of those mice peaked at day 7 by fourfold. Further, we analyzed the HBVspecific CTL response among total CD8+ T cells. We found that after


Abstracts 511 depleting Foxp3+ cells there is an increase in HBs190 97 -specific, but not HBc93 100 -specific CD8+ CTLs in the liver and spleen. Conclusions: During persistent HBV infection, Foxp3+ Treg plays a role in the suppression of HBV-specific CTL response, especially for the development of an HBs-specific adaptive immunity. Further analysis for revealing the detailed mechanism of tolerance induction towards HBV has to be investigated.

P1018 The secretion of cytokines has a greater influence on the severity of acute hepatitis than the survival of infiltrating CD8 T cells M. Vo,* L. E. Holz,* V. Benseler,* S. S. Tay,* C. McGuffog,* A. Andreas,* D. Hilton, G. McCaughan,à D. G. Bowen* & P. Bertolino* *Liver Immunology, Centenary Institute, Sydney, Australia, WEHI, Molecular Medicine, Melbourne, Australia, àLiver Immuobiology, Centenary Institute, Sydney, Australia Purpose/Objective: Acute hepatitis is often mediated by CD8 T cells that kill target cells or secrete hepatotoxic cytokines. Although it is known how cytotoxic T cells mediate liver damage, the parameters that regulate acute hepatitis are not well understood. To determine whether some intrinsic T cell parameters might play a role in this regulation, we used the well-characterized Met-KbTg mouse model of autoimmune mediated hepatitis (AIH). Materials and methods: Acute liver injury in Met-Kb mice is induced by the adoptive transfer of TCR Tg CD8 T cells activated in lymph nodes and recognizing their cognate antigen in the liver. Tg CD8 T cells transferred into Met-Kb typically induced a severe but selflimiting autoimmune hepatitis (AIH). To investigate whether cytokine regulation and apoptosis of CD8 T cells were critical in limiting the acute damage induced by CTLs, we performed transfer experiments using TCR transgenic T cells deficient for either suppressor of cytokine signaling (SOCS-1) or the pro-apoptotic molecule Bim. Results: Bim-/- Tg cells accumulated in liver of recipient mice at sixfold higher levels compared to wt Tg cells. Despite this substantial accumulation, the outcome of hepatitis remained unchanged, neither prolonged nor more severe. This data suggest that although T cells died in a Bim-dependent process, T cell survival was not a critical parameter in limiting liver damage. In contrast, SOCS-1-deficient Tg T cells induced a more severe hepatitis than their wt counterparts. SOCS-1-/- Tg isolated from the liver displayed upregulated IL-2Ra chain, required to form the high affinity IL-2R. Upregulated IL-2Rawas associated with higher IFNg, CTL activity, and proliferation rates, consistent with enhanced effector function and supporting a critical role for cytokines in CTL function. Conclusions: These data demonstrate that the propensity of CD8 T cells to mediate acute hepatitis is determined by the quality, rather than the quantity of CTLs infiltrating the liver. In the long term, both Bim and SOCS deficiency led to the accumulation of CD8 T cells that were unable to cause chronic liver damage. These findings reveal the existence of mechanisms that are able to silence potentially autoreactive T cells surviving acute hepatitis. Such mechanisms might explain why immune infiltrates do not always correlate with ALT levels in patients chronically infected with HCV or with autoimmune hepatitis.

P1019 TREM-1 expression is elevated on monocytes in cirrhotic patients S. Gurney,* C. Graham, R. Metcalf, L. Greathead, M. Foxton,à S. Singh,* N. Soni* & P. Kelleher *Chelsea and Westminster Hospital, Intensive Care Unit, London, United Kingdom, Imperial College London, Immunology, London, United Kingdom, àChelsea and Westminster Hospital, Hepatology, London, United Kingdom Purpose/Objective: Sepsis and spontaneous bacterial peritonitis (SBP) are common sequelae in patients with cirrhosis. There is increasing interest in the role of monocytes in bacterial infection in cirrhotics. Triggering Receptor Expressed on Myelocytes 1 (TREM-1) modulates the immune response via an intracellular signalling cascade with resultant production of pro-inflammatory cytokines. It has been used as a biomarker in the diagnosis of bacterial infection but its role in patients with cirrhosis is unknown. We aim to evaluate TREM-1 as a biomarker in the diagnosis of SBP. Materials and methods: Venous blood samples were obtained from 11 healthy controls (HC) and 14 patients with advanced cirrhosis (CA), as defined by clinico-radiological criteria. Simultaneous ascitic fluid samples were taken from seven patients in the CA group. Cirrhosis severity was graded using Child-Pugh score (median: 10) and Model for End-Stage Liver Disease score (MELD) (median: 14). The CA group had no clinical or laboratory evidence of sepsis at the time of sampling. Flow cytometry was used to quantify the expression of TREM-1 on three monocyte subsets: CD14+CD16-, CD14+CD16+, CD14dimCD16+ and CD16+CD15+ neutrophils in blood and CD14+ monocytes and CD16+CD15+ neutrophils in ascitic fluid. Results: Results to date show that TREM-1 expression is significantly higher in the CA group compared to HC in the monocyte subsets CD14+ CD16- [median geometric mean fluorescent intensity (GMFI): 6250 versus 2663 (P = 0.0067)] and CD14+CD16+ [GMFI: 4666 versus 9280 (P = 0.0148)] but not CD14dimCD16+ or CD16+ CD15+ neutrophils. There is no correlation between TREM-1 expression and severity of cirrhosis by Child-Pugh or MELD score. There is no significant difference in TREM-1 expression on monocytes in ascitic fluid compared with CD14+ CD16+ monocytes in blood. Conclusions: Blood monocyte TREM-1 levels are elevated in the CA group compared to the HC group in the absence of infection. There was no difference in TREM-1 expression between blood and ascitic fluid monocytes in culture negative and non-neutrocytic ascites (PMN count 450 cells/ mm3 were randomized (2:1) to receive three immunizations every 2 weeks with DC pulsed with autologous heat-inactivated HIV-1 (Cases, n = 24) or with non-pulsed DC (Controls, n = 12). Changes in viral load (VL) as well as changes in CD4 cell counts have been evaluated. Additionally HIV specific responses were measured in PBMC samples from different time-points by LPR and by IFN-gElispot against gag, nef and gp41 HIV overlapping peptide pools, respectively. Results: VL rebounded to detectable level in all the patients. At week 12 and 24, a decrease of VL ‡ 1 log was observed in 12/22 (55%) versus 1/11 (9%) and in 7/20 (35%) versus 0/10 (0%) in cases and controls, respectively (P = 0.02, P = 0.03). CD4 drop to baseline value before any cART without differences between groups. Although only transient positive responses to HIV p24 were observed, the median change in LPR to HIV p24 at week 24 from baseline was 0.96 versus -0.50 (P = 0.02) in cases versus controls, respectively. Baseline median values of the total sum of HIV specific responses against HIV peptide pools were similar in both arms (2625 versus 2283 SFC/106 PBMC, P = 0.462). After vaccination, the median change of total sum of SFC/106 PBMC at week 24 was 3567 versus 838 SFC/106 PBMC (P = 0.0459) in cases and controls, respectively. This difference was more evident when analyzing responses against gag p17 and Nef peptide pools (P = 0.0288 and P = 0.03615, respectively). No statistically significant correlations between immune responses and VL were found. Conclusions: These results indicate that HIV-1 specific immune responses elicited by therapeutic DC vaccines could significantly change pVL set-point after cART interruption in chronic HIV-1 infected patients.

P1027 Chemokines CCL3, CCL4 and chemokine receptor CCR5 in HIV-1infected women with complicated anamnesis V. P. Chernyshov,* V. V. Podolsky, B. V. Dons’koi,* A. A. Kostyuchyk* & V. V. Podolsky * Laboratory of Immunology, Institute of Pediatrics Obstetrics& Gynecology National Academy of Medical Sci, Kiev, Ukraine, Department of Gynecology, Institute of Pediatrics Obstetrics& Gynecology National Academy of Medical Sci, Kiev, Ukraine Purpose/Objective: Chemokine receptor CCR5 plays a key role inhuman immunodeficiency virus (HIV) entry into CD4+ T lymphocytes. Chemokines CCL3 and CCL4 have been shown to possess antiviral effects by binding to the HIV-1 co-receptor CCR5. We evaluated activity of these mechanisms in HIV-infected women that used drugs, alcohol and additionally had sexual transmitted diseases. Materials and methods: Eighty three HIV-1-infected women were divided in four groups: drug edicts (DE) 20, prostitutes (P) 21, sexual transmitted diseases (STD) 23, chronic alcoholism (CA) 19. Non-infected 30 women with chronic alcoholism and 30 women with acute alcohol intoxication served as control groups. Lymphocyte subsets and expression of CCR5 on CD4+ T lymphocytes were examined by flow cytometry. CCL3, CCL4, IL-8 in blood serum were detected by ELISA. Results: Levels of CD4+ T lymphocytes, especially CD4/CD8 ratios were decreased dramatically in four groups of HIV+ women. Expression of CCR5 inCD4+ lymphocytes was decreased in all groups of HIV+ women too. Levels of CCL4 in groups DE, P and STD were decreased, but not in CA group. Changes in CCL3 were not significant in all groups of HIV+ women. Levels of IL-8 were elevated in all groups ofHIV+ women. In non-infected women with acute alcohol intoxication levels of CCL4 were decreased. Conclusions: Drugs using and sexual transmitted diseases in HIV-1infected women are related with suppressed CCR5 expression in CD4+ lymphocytes and levels of CCL4 in blood serum. Chronic alcoholism has not so great suppressive influence. Acute alcohol intoxication relates with lower levels of CCL4 in non-infected women and one should think about weaker resistance of organism against HIV in such women.

P1028 Co-infection of human immunodeficiency virus and hepatitis B surface antigen among pregnant women in Kaduna, Nigeria H. O. ABA, C. M. Z. Whong & M. Aminu Department of Microbiology, Faculty of Science, Ahmadu Bello University, Zaria, Nigeria Purpose/Objective: Human immunodeficiency virus (HIV) and Hepatitis B virus (HBV) share overlapping transmission routes and some risk factors. The HIV-infected pregnant cohort represents a unique population and infection with HBV is considered a public health problem worldwide. This study was conducted to determine the sero-prevalence of HIV and Hepatitis B Surface Antigen (HBsAg) and co-infection in pregnant women in Kaduna, Nigeria. Materials and methods: Eight hundred pregnant women attending ante-natal care in four selected hospitals were recruited for the study. Blood samples were collected and examined for the presence of HIV and HBsAg using test kits. The positive HIV blood samples were analyzed for CD4+ counts, while the positive HBsAg plasma samples were confirmed using ELISA and tested for various markers of HBV. Results: Human immunodeficiency virus was detected in 5.9% (47/ 800) and HBsAg was detected in 3.9% (31/800) of the blood samples of the pregnant women. Four (0.5%) of the women were co-infected with both viruses. Mean CD4+ count in the HIV positive pregnant women


Abstracts 515 was 396 cells/ll of blood while the mean CD4+ count in HIV and HBsAg co-infected women was 299 cells/ll of blood. Test for markers of HBV indicated Anti-HBc as the most predominant (58.1%:18/31) while Anti-HBs was the least (3.2%:1/31). The highest prevalence of 13% (7/54) and 4.4% (10/229) were recorded for HIV and HBsAg among women in age groups 36 40 years and 21 25 years respectively (P > 0.05). There was a statistically significant association between the presence of both viruses and women in polygamy and those who have undergone blood transfusion and surgery (P < 0.05). Conclusions: The results obtained in this study showed a considerably higher sero-prevalence rate of HIV and HBsAg amongst the pregnant women. Hepatitis B virus infection is a dynamic disease and coinfection with HIV impacts directly on the outcome of HBV infection, considerably complicating its natural history, diagnosis, and management, Therefore screening for HIV and HBsAg co-infection during pregnancy is essential to improve ante-natal care and inform clinical management.

P1030 Different prognostic value of cell-associated total HIV-1 DNA and integrated HIV-1 DNA for virological outcome in ART naı¨ve and treated HIV-1 chronically infected patients I. Martıńez Rıó, J. Modrego-Ruiz, E. Fernańdez-Cruz & C. Rodrı´guez-Saínz Clinical Immunology, Hospital Gregorio Maranõń, Madrid, Spain Purpose/Objective: It has been demonstrated that total HIV-1 DNA levels associated to peripheral blood mononuclear cells (HIV-1 DNAT) has prognostic value for virological outcome in HIV-1 infected patients. HIV-1 DNAT includes both integrated and unintegrated forms of HIV-1 DNA. During natural infection the majority of HIV-1 DNA exists in an unintegrated state, compared with treated patients where the majority DNA is integrated. We evaluated whether the integrated forms of HIV-1 DNA (HIV-1 DNAI) could provide a different prognostic value compared to HIV-1 DNAT for virological outcome in ART naı¨ve chronically HIV-1+ individuals. Materials and methods: In 107 ART naı¨ve HIV-1+ patients who have been previously analyzed for HIV-1 DNAT we have retrospectively measured integrated DNAI using real time PCR and relative quantification by DDCt. Patients had participated in a multicenter, randomised, double-blinded, placebo-controlled phase II clinical trial of ART combined with an HIV-1 immunogen (STIR-2102). We analyzed the prognostic value of HIV-1 DNAT and HIV-1 DNAI as categorical variables using bivariate Cox regression models and stratifying by the medians at baseline PRE-ART and POST-ART (6 weeks after initiation ART). In STIR-2102 trial the endpoint was defined as time to the first increase of HIV-1 RNA > 5000 copies/ml. Results: HR for virological failure in PRE-ART patients, introducing in the Cox model HIV-1 DNAT and HIV-1 DNAI were: HR 1.69 (95% CI, 1.02 2.79, P = 0.04) and HR 1.83 (95% CI, 1.11 3.03, P = 0.019), respectively. In POST-ART HR for HIV-1 DNAT and HIV-1 DNAIwere: HR 2.36 (95% CI, 1.39 4.02, P = 0.001) and HR 1.68 (95% CI, 1.01 2.80, P = 0.046), respectively. Conclusions: Both HIV-1 DNAT and HIV-1 DNAIshowed different independent prognostic value. In ART naı¨ve patients integrated HIV-1 DNAIwas the strongest variable associated with virological failure independently of HIV-1 DNAT. In treated patients, HIV-1 DNAT showed the strongest prognostic value for virological outcome, probably reflecting residual replication.

P1031 Dissecting HIV-1 clade C

specific antibody responses

D. Gallerano,* M. Focke, I. Makupe,à E. N. Sibanda§ & R. Valenta * Division of Immunopathology, Inst. of Pathophysiology and Allergy Research, Vienna, Austria, Christian Doppler Laboratory for Allergy Research, Inst. of Pathophysiology and Allergy Research, Vienna, Austria, à Allergy Asthma and Immunology Clinic, Gamma City Laboratory, Harare, Zimbabwe, §Asthma Allergy and Immune Dysfunction Clinic, Parirenyatwa University Teaching Hospital, Harare, Zimbabwe Purpose/Objective: Out of the different HIV strains, HIV-1 clade C is the one which is expanding more rapidly, above all in the southern states of Africa. Though, little is known about the immune response elicited by this strain in HIV infected individuals. In order to dissect the clade C HIV-1-specific antibody response in HIV-infected individuals we produced recombinant proteins and synthetic peptides. Materials and methods: For a precise mapping of epitopes lying on the HIV envelope proteins (gp120 and gp41) overlapping peptides covering the entire amino acid sequence of the two proteins of a South African HIV-1 clade C strain were synthesized by solid phase synthesis. In order to indentify epitopes on non-surface antigens the HIV-1 clade C structural, functional and accessory proteins have been expressed in Escherichia coli. In preliminary experiments 85 sera from Zimbabwe were tested for HIV-specific IgG reactivity to the produced antigens in a dot blot immunoassay. Results: The envelope epitope mapping allowed identifying immunogenic regions of gp120 and gp41. Interestingly HIV-specific IgG antibodies have been found to be elicited by both the domains which are conserved among HIV-1 strains as well as by variable domains (i.e. C1, V3 and V4 regions). On the other hand, we detected strong IgG reactivity also to proteins and peptides which have not been reported to be exposed on the surface of the virion (i.e. gp41 C-terminal region, matrix protein, Vif). Conclusions: This suggests that also proteins contained within viral particles are exposed to the immune system in an immunogenic form.

P1032 Effectiveness of herbal remedy in 6 HIV patients in Nigeria A. Jewell,* B. Okesina, T. Ajadi,à S. Rahamon§ & O. Ogunrin– * Health & Social Care Sciences, Kingston University, London, UK, Chemical Pathology, University of Ilorin, Ilorin, Nigeria, àRadiology, LAUTECH Teaching Hospital, Osogbo, Nigeria, §Chemical Pathology, College of Medicine University of Ibadan, Ibadan, Nigeria, –NAFDAC, Drug R & R, Lagos, Nigeria Purpose/Objective: The uncurable nature of HIV infection compelled many people living with the virus to seek alternative therapy. This pilot study determined effectiveness (clinical and laboratory responses) of an herbal remedy (A-Zam) used by patients seeking herbal remedy for HIV infection in Nigeria. Materials and methods: Six patients taking herbal concoction as alternative therapy for HIV infection were recruited into the study and monitored for 4 months. All the (6) patients were confirmed for presence of HIV infection using Western blot technique in the nearest teaching hospital before commencing preliminary clinical and laboratory examinations using WHO and CDC criteria. The patients were contacted daily and visited regularly after commencement of herbal medications to assess side effects, drug toxicity, compliance and effectiveness. Results: The symptoms and signs associated with HIV infection disappeared within 20 days of commencement of herbal therapy. The body weight increased from average 52.8kg to 62.7kg (9.9 ± 3.2), viral (HIV-RNA) load decreased from average 42 300 copies/ml to undetectable level (£50 copies/ml) and CD4 T cell count increased


516 Poster Session: Myeloid Cell Development from average 226 680 mm3/ll (454 ± 106 mm3/ll) at 4-month post therapy. Conclusions: This pilot study concluded that the herbal remedy (AZam) is effective in HIV infection based on dramatic improvement in both clinical features and laboratory results of HIV infection. However, a longer period is suggested to ensure that the observed improvement is sustained. Also, a large population study is needed to confirm our observation in this cohort of people

P1033 Evaluation of the presence of the compound KIR:HLA-C genotypes in the virologic outcome of individuals with HIV-1 chronic infection C. Rodrı´guez-Sainz, L. Herraiz, D. C. Hernańdez, L. Valor, I. Martıńez & E. Fernańdez-Cruz Servicio de Inmunologıá Clıńica, Hospital General Universitario Gregorio Maranõń, Madrid, Spain Purpose/Objective: An effect of certain HLA-C genotypes in combination with KIR2DL3 has been implicated with HCV clearance previously. However, to date neither individual HLA-C alleles nor combinations or HLA-C groups with KIR have shown a protective effect against HIV-1 infection. The objective of this work was to study whether the presence of different compound KIR:HLA-C genotypes (KIR2DL1 and its ligand the HLA-C2 allotype; KIR2DS1:HLA-C2; KIR2DL2:HLA-C1; KIR2DL3:HLA-C1; and KIR2DS2:HLA-C1) could influence the virological outcome of chronically HIV-1 infected individuals initiating antiviral-therapy. Materials and methods: Of the 187 patients with asymptomatic HIV1 chronic infection who had participated in a multicenter, randomised, double-blinded, placebo-controlled phase II clinical trial of ART combined with an HIV-1 immunogen (STIR-2102), were retrospectively genotyped for HLA-C allotypes and KIR genes using sequencespecific primer PCR (Olerup SSP KIR Genotyping kit). Kaplan Meier curves and Cox proportional-hazard models were used for survival analysis. Results: Kaplan Meier analyses, stratifying by the different genotypes showed that HLA-C1 allotype and KIR2DL3 were strongly associated with virological failure (v2 value for the Log-Rank test was 8.46; P = 0.004): The mean time to virological failure (first increase of HIV1 RNA above 5000 copies/ml) was 31.60 months (95% CI, 29.29 33.92) for the group KIR2DL3:HLA-C1 and 20.74 months (95% CI, 19.07 27.06M) for the group of patients lacking the KIR2DL3:HLA-C1 genotype. In a Cox regression model, the HR for virological failure was significantly higher for individuals without the compound KIR2DL3:HLA-C1 genotype [HR, 1.90 (95% CI, 1.20 2.97, P = 0.006)]. Conclusions: The compound KIR2DL3:HLA-C1 genotype has been associated to a protective effect in a cohort of chronically HIV-1 infected individuals initiating antiviral-therapy.

P1035 High numbers of M-DC8+ monocytes and TNFa+ cells within the marginal zone of spleens from HIV-1 infected patients S. A. Degrelle,* C. A. Dutertre,* S. Amraoui,* J. P. Jourdain,* E. Oksenhendler, J. P. Viard,à L. Garderet,§ Y. Richard,* R. Cheynier* & A. Hosmalin* * Inserm U1016 Institut Cochin Paris France. CNRS UMR8104 Paris France. Univ Paris Descartes Paris France., Immunology Department, Paris, France, Assistance Publique-Hoˆpitaux de Paris (AP-HP)/Hoˆpital Saint Louis, Service d’Immunologie Clinique, Paris, France, àCentre de Diagnostic et de The´rapeutique/Hoˆpital Hotel Dieu, Unite´ fonctionnelle de The´rapeutique en Immuno-infectiologie (T2i), Paris, France, §Assistance Publique-Hoˆpitaux de Paris (AP-HP)/Hoˆpital Saint-Antoine, He´matologie Anatomie Pathologique, Paris, France Purpose/Objective: We found recently that circulating M-DC8+ monocyte numbers were significantly higher in viremic, HIV-1 infected patients than in non-viremic patients or in healthy controls. In vitro, peripheral blood mononuclear cells from viremic patients produced more TNFa in response to LPS than those from non-viremic patients or from controls, and M-DC8+ monocytes were mostly responsible for this overproduction. We quantified and localized these monocytes in spleens from HIV-1 infected patients with idiopathic thrombopenic purpura (ITP). Materials and methods: Samples were obtained after therapeutic splenectomy from HIV-infected or uninfected patients. Numbers of M-DC8+ monocytes were evaluated by 11-color flow cytometry in spleen mononuclear cells from 10 patients (six HIV-infected, four uninfected). Localization and quantification of M-DC8+ and TNFa+ cells were performed by immunohistofluorescence on spleen cryosections from 17 patients (eight uninfected, nine HIV-infected including four untreated by antiretrovirals). Results: Spleens from HIV-infected patients displayed significantly higher numbers of M-DC8+ monocytes than those from uninfected patients. M-DC8+ cells were localized in the red pulps from all patients, but were present within the marginal zone only in HIVinfected, untreated patients. Numbers of TNFa+ cells were also significantly higher in HIV-infected patients than in controls, irrespective of treatment. Some were found within the marginal zone, whereas in uninfected patients, they localized strictly in the red pulp. TNFa did not colocalize with M-DC8+ cells, but with other cell types currently being characterized. Conclusions: High numbers of M-DC8+ monocytes were not only found in the blood from HIV-infected patients, but also in the spleen. In untreated patients, these cells localized abnormally within the marginal zone. TNFaexpression was higher in spleens from HIVinfected patients than in uninfected patients, and was found within the marginal zone, but did not colocalize with MDC8 labeling. We will test further the kinetics of proinflammatory monocyte homing in a simian model over different stages of SIV infection.

P1036 Highly Active Antiretroviral Therapy (HAART) can restore the decrease of CD20+ T cell numbers in HIV-1 patients F. Fo¨rster,* A. Singla, S. K. Arora, R. E. Schmidt* & R. Jacobs* Department of Clinical Immunology & Rheumatology, Hannover Medical School, Hannover, Germany, Department of Immunopathology, PGIMER, Chandigarh, India *

Purpose/Objective: CD38 expression on CD8+ T cells is a valuable prognostic marker for disease progression in HIV infection. Like CD38, CD20 is functionally involved in calcium mobilization and coexpressed by a subpopulation of human T cells. Here we wanted to elucidate if CD20+ T cells are affected by HIV-1 infection and may have a prognostic value for the course of disease.


Abstracts 517 Materials and methods: Numbers of CD20+ T cells were determined in healthy controls, untreated and HAART-treated HIV-1 patients. Coexpression patterns of CD4, CD8, and CD38 as well as IFN-c production were analysed in CD3+ CD20+ and CD3+ CD20- T cells by multi color flow cytometry. Results: We found a significant decrease of CD20+ T cell numbers in untreated HIV-1 patients (1.4%) as compared to healthy controls (2.5%) which recovered under HAART (1.9%). Particularly, the CD8+ T cell compartment was affected revealing significant differences between healthy controls (3.4%) and both treated (1.7%) and untreated (1.1%) patients. CD38 was expressed on a few CD20+ T cells but preferentially on CD20- cells in all three groups. IFN-c production was measured upon cell activation using PMA alone or in combination with ionomycin in order to assess functional capacities of the cells. PMA alone was much more effective in CD20+ cells regardless of CD38 coexpression, indicating a supportive role of CD20 but not CD38 in T cell activation. Conclusions: Here we present data showing that CD3+ CD20+ T cells are decreased in untreated HIV-1 patients and normal numbers are restored under HAART. Expression of CD20 and CD38 is independently regulated on T cells and it is not clear by now if CD20 expression is of prognostic value. Contrary to CD38, CD20 can substitute ionophores for Ca2+ flux in early T cell activation and also strongly amplifies cell stimulation in the presence of Ca2+ ionophores, indicating that CD20 contributes to T cell activation. The study was supported by the Federal Ministry of Education and Research (BMBF, Germany) and by the Indian Council of Medical Research (Ministry of Health and Family Welfare, Govt. of India).

P0137 HIV impairs avidity maturation to EPI vaccines in children D. M. Muema,* E. Nduati,* J. Berkley* & B. Urban * KEMRI-Wellcome Trust Research Programme, Immunology, Kilifi, Kenya, Liverpool School of Tropical Medicine, Molecular and Biomedical parasitology, Liverpool, UK Purpose/Objective: HIV-infected adults and children have reduced quantities of antibodies and memory B cells to non-HIV antigens. On the other hand, avidity of antibodies to vaccine antigens is not affected by HIV in adults, probably because they have already experienced avidity maturation to most routine vaccine antigens prior to infection. Little is known regarding avidity maturation in children infected with HIV. Here, we analysed the avidity of antibodies against tetanus toxoid and diphtheria toxoid in children who acquired HIV vertically. We also measured the quantities of circulating antibodies and frequencies of memory B cells to vaccine antigens. Materials and methods: Memory B-cell frequencies were determined using cultured B-cell ELISpot. ELISA was used to determine the antibody levels while a modified ELISA was used to determine the antibody avidities. In the modified ELISA, Guanidine Hydrochloride was used to elute the antibodies after incubating the plasma (sample) with the antigen-coated plates. The ratio of the remaining antibody levels in the eluted wells to that in the control (PBS eluted) wells was taken as the avidity index. The children were then classified based on their viral loads and compared with community controls. Results: HIV-infected children, regardless of level of viremia, had significantly lower avidity indices when compared with the community controls. As previously reported, they also had lower titres of circulating antibodies as well as lower frequencies of antigen specific memory B cells. Conclusions: HIV affects not only the magnitude but also the quality of antibody response to vaccine antigens in vertically infected children.

P0138 HIV triggers interleukin 21-mediated induction of granzyme Bsecreting B cells with antiviral properties C. Kaltenmeier,* A. Gawanbacht, S. Hofmann,* K. Dahlke,à T. Beyer,* G. Ha¨rter,§ B. Gru¨ner,§ P. Kern,§ F. Kirchhoff, H. Schrezenmeier* & B. Jahrsdo¨rfer* * Institute of Clinical Transfusion Medicine and Immunogenetics, University of Ulm, Ulm, Germany, Institute of Molecular Virology, University of Ulm, Ulm, Germany, àInstitute of Clinical Pharmacology, University of Ulm, Ulm, Germany, §Comprehensive Infectious Diseases Center, University of Ulm, Ulm, Germany Purpose/Objective: Certain lymphocyte subsets including plasmacytoid dendritic cells and regulatory T cells can secrete granzyme B (GrB), thereby suppressing T cell expansion. Recently, we found that B cells can also produce GrB in response to interleukin (IL-) 21. Since HIV has been shown to be associated with elevated serum IL-21 levels, we hypothesized that GrB-expressing B cells may be induced during HIV infection. Materials and methods: Cell culture, HIV infection of cells in cell culture, FACS, laser-scanning confocal microscopy, Wetern Blotting. Results: Here, we demonstrate for the first time, that infection of CD4+ T cells with HIV 1 (NL4-3), but not mock infection, induces strong expression of IL-21. We further demonstrate that such T cells induce GrB in co-cultured B cells in an IL-21-dependent fashion. In support of these data, serum levels of both IL-21 and GrB are significantly higher in HIV-infected patients before HAART as compared to healthy controls. Up to 60% of B cells (36.2 ± 12.9%) from patients infected with HIV, but not normal B cells, express GrB. Importantly, co-culture of HIV-infected CD4+ T cells with GrB+ B cells resulted in GrB transfer, and strongly suppressed both, proliferation of T cells and virus replication as indicated by significantly reduced p24 levels in the supernatants. The observed effects were enhanced by IL21, and reduced by GrB inhibition. Conclusions: In summary, we demonstrate that HIV infection induces IL-21 in CD4+ T cells, thereby indirectly triggering the development of GrB-secreting B cells with antiretroviral properties. GrB-secreting B cells may play a so far unappreciated role in decelerating HIV expansion, particularly in the early phase of infection. On the other hand, induction of GrB in B cells may interfere with their terminal differentiation into plasma cells, which may explain the lack of an efficient anti-HIV humoral immune response in HIV-infected patients.

P1041 Immunogenicity of a universal HIV-1 vaccine vectored by DNA, MVA and ChAdV-63 in a phase I/IIa clinical trial T. Hanke,* N. Borthwick,* T. Ahmed,* E. J. Hayton,* U. Ebrahimsa,* A. Rose,* A. Black,* H. Yang,* G. Hancock,* S. Colloca, A. Nicosia, A. J. McMichaelà & L. Dorrellà * The Jenner Institute, University of Oxford, Oxford, UK, Okairos, company, Rome, Italy, àWeatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK Purpose/Objective: The develop a safe and effective vaccine against HIV-1/AIDS. Materials and methods: The major challenge that both antibody and T cell-eliciting vaccines against HIV-1 face is the extreme variability of the HIV-1 genome: a successful vaccine has to effectively target diverse HIV-1 strains circulating in the population and then must deal with ongoing virus escape in infected individuals. To address these issues, we assembled vaccine immunogen HIVconsv from the functionally most conserved regions (not epitopes) of the HIV-1 proteome with the underlying working hypothesis that early focus of vaccine-elicited immune responses on these regions will lead to a better recognition


518 Poster Session: Myeloid Cell Development and control of transmitting viruses. A gene coding for the HIVconsv immunogen was inserted into plasmid DNA (D), modified vaccinia virus Ankara (MVA; M) and non-replicating adenovirus of a chimpanzee origin ChAdV-63 (C). Currently, combined heterologous prime-boost regimens of these vaccines, namely CM, DDDCM and DDDMC, are being evaluated in a phase I/IIa trial HIV-CORE002 in healthy HIV-1/2-negative volunteers in Oxford. Results: Preliminary data indicate that the vaccines are very well tolerated and show high immunogenicity. Following the CM regimen, vaccine-induced T cell frequencies reached a median of 5150 (range 1475 16495) SFU/106 PMBC ex vivo specific for multiple, conserved, therefore in natural infection mostly subdominant HIV-1 epitopes (in contrast to 136 and 686 SFU/106 PMBC in the STEP study). Similar immunogenicity was observed for the DDDCM regimen. Results from DDDMC, epitope mapping, and phenotypic and functional characterization of these responses will be presented. Conclusions: Thus so far, the unique HIVconcv immunogen and unique vector delivery have induced T cell responses superior to other HIV-1 vaccine candidates tested in humans to date, HIVconsv is the first of the second-generation immunogens addressing the HIV-1 diversity that reached clinic and ChAdV-63 is the first adenovirus of chimp origin delivering an HIV-1-derived immunogen that reached clinic.

P1042 Immunomodulation of antigen-specific T cells by HIV gp41 transmembrane envelope protein

A. Ashkenazi,* O. Faingold,* N. Kaushansky, A. Ben-Nun & Y. Shai* * The Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel, The Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel Purpose/Objective: Modulation of T-cell responses by human immunodeficiency virus (HIV) occurs via distinct mechanisms, one of which involves down-regulation of T cells already at the stage of viruscell fusion. The membrane-bound T-cell receptor (TCR) complex is located in the vicinity of the HIV fusion site. Thus, it is conceivable that hydrophobic portions of the gp41 protein of the viral envelope that contributes to membrane fusion may interact with membraneembedded portions of the TCR and modulate T-cell responsiveness. Materials and methods: To address this hypothesis, we investigate the ability of HIV gp41 derived peptides to modulate T-cell proliferation in-vitro by examining their effect on T cells, which were stimulated at different steps of the TCR complex signal transduction. Furthermore, imaging, biochemical and biophysical approaches are implemented to identify the target protein of the HIV peptides. In-vivo studies are carried out in mice models of autoimmune disease. Results: We demonstrate inhibition of antigen-specific T-cell proliferation by peptides derived from membranotropic regions of HIV gp41. We will address the mode of action of the peptides on the transmembrane domains of the TCR complex. Furthermore, administering the peptides to an animal model of autoimmune disease mediated by pathogenic T-cells significantly reduces the severity of the disease. This seems to be associated with down-regulation of Th1 proinflammatory cytokines. Conclusions: Thus, our data imply that T-cell inactivation during HIV-cell fusion lie in part in hydrophobic portions of gp41. Disassociated from HIV, however, these HIV peptides may act as novel reagent for down-regulating undesirable immune responses.

P1044 Inhibition of HIV-replication by cell-membrane crossing oligomers (CMCOs) W. Posch,* T. Lindhorst, B. Werner, H. Bock, C. Lass-Floerl* & D. Wilflingseder* * Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austria, Ugichem GmbH, Innsbruck, Austria Purpose/Objective: Although rapidly becoming a valuable tool for gene silencing, regulation or editing in vitro, the direct transfer of siRNAs into cells is still an unsolved problem for in vivo applications. Materials and methods: For the first time, we show that specific modifications of antisense oligomers allow autonomous passage into cell lines and primary cells without further adjuvant or coupling to a cell-penetrating peptide. For this reason, we termed the specifically modified oligonucleotides ‘cell-membrane crossing oligomers’ (CMCOs). Results: CMCOs targeted to various conserved regions of HIV-1 were tested and compared to non-targeting CMCOs. Analyses of noninfected and infected cells incubated with labeled CMCOs revealed that the compounds were enriched in infected cells and some of the tested CMCOs exhibited a potent antiviral effect. Finally, the CMCOs did not exert any cytotoxicity and did not inhibit proliferation of the cells. Conclusions: In vitro, our CMCOs are promising candidates as biologically active anti-HIV reagents for future in vivo applications.

P1045 Interferon alpha induced acceleration of thymic production during acute SIV infection J. Dutrieux,* V. Fabre-Mersseman,* B. Charmeteu-De Muylder,* M. Rancez,* R. Ponte,* S. Rozlan, A. Bernard,* S. MorgadoFigueiredo,* A. Coue¨del-Courteille* & R. Cheynier* * Cochin Institute Inserm U1016, Immunology, Paris, France, Cytheris SA, Immunology, Paris, France Purpose/Objective: The processes involved in the inhibition of thymic function usually observed during primary HIV-infection are poorly understood. In humans, the acute phase of HIV-infection, that certainly drives pathogenesis, remains barely studied. The aim of this study was to define the mechanisms impacting thymopoiesis during the acute phase of SIVmac251-infection in rhesus macaques. Materials and methods: Blood samples were taken every other day for 2 weeks in 15 SIV-infected macaques. T-cell subsets were followed by flow cytometry. Plasma interferon alpha (IFNa) levels were measured by ELISA. Thymic function was evaluated by qPCR quantification of T-cell Receptor Excision Circles (TRECs) and estimation of intrathymic precursor T-cell proliferation (sj/§TREC ratio). Eight animals were autopsied at day 3, 7, 10 and 14 post-infection. Thymic expression of IFNa was evaluated by qRT-PCR and the proportion of the 12 IFNa subtypes estimated by heteroduplex tracking assay. The expression of chemokines involved in thymopoiesis was studied by qRT-PCR on thymus samples. Finally, the effect of IFNa subtypes on thymocyte differentiation was tested on simian DN cells cultured on OP9-hDL1 cells. Results: Combined analysis of the evolution of recent thymic emigrants (CD31+ naı¨ve CD4+ T-cells) in blood and TRECs pointed to the fact that, by day 7 of infection, the RTE compartment mostly contains newly exported cells with a reduced proliferation history. This change in thymocyte behavior coincided with both increased plasma IFNa levels and production of IFNa 1, 2, 3, 5 and 7 in the thymus. In culture, 6 IFNa subtypes, including IFNa3 and 7, induced strong inhibition of thymocyte proliferation, as demonstrated by lower cell counts, DP frequencies and sj/bTREC ratio at day 14. Finally, in the SIV-infected thymuses CXCL12 transcription was significantly increased while CCL19 and CCL25 expression was reduced.


Abstracts 519 Conclusions: Altogether, our data demonstrate that, during acute SIV-infection, modified chemokine expression patterns in the thymus and inhibition of thymocyte proliferation by locally produced IFNa subtypes lead to an acceleration of thymocyte differentiation thus to massive export of newly maturated T-cells. In the long term, such modification of thymopoiesis may lead to the observed reduction of naı¨ve T-cell counts and diversity.

P1046 M-DC8+ monocyte expansion and TNFa overproduction in response to LPS during HIV-1 infection A. Hosmalin,*, ,à,§ S. Amraoui,*, ,à,§ A. L. DeRosa,*, ,à,§ J. P. Jourdain,*, ,à,§ L. Vimeux,*, ,à,§ C. Goujard,– P. Loulergue,** O. Launay,** Y. Richard*, ,à,§ & C. A. Dutertre*, ,à,§ * Inserm U1016 Institut Cochin Paris France, CNRS UMR8104, Paris, France, àUniv Paris Descartes, Paris, France, §Immunology, Paris, France, –Hoˆpital de Biceˆtre AP-HP INSERM U1018, Univ Paris Sud, Le Kremlin-Biceˆtre, France, **Inserm CIC BT505 CIC de Vaccinologie Cochin Pasteur Paris France, Vaccinology, Paris, France Purpose/Objective: HIV infects activated CD4+ T cells and induces their depletion. Progressive HIV infection is fueled by chronic immune hyperactivation, mediated by inflammatory cytokines like TNFa, and related to intestinal epithelial damage and microbial LPS translocation into the circulation. Materials and methods: Using 11-color flow cytometry and cell culture after sorting, we investigated the numbers and TNFa production of fully defined myeloid populations during HIV-1 infection. Results: In 15 viremic patients, compared to 8 virologically suppressed patients or to 13 controls, circulating CD141 (BDCA-3)+ and CD1c (BDCA-1)+ dendritic cell counts were reduced. Conversely, nonclassical CD14dimCD16+ monocyte counts were increased, particularly those expressing M-DC8. These M-DC8+ monocytes were mostly responsible for the LPS-induced TNFa overproduction found in viremic patients. In vitro, CD16+M-DC8+ monocytes differenciated from classical, CD16- M-DC8- monocytes using M-CSF and GM-CSF, which is increased in viremic patient’s plasma. Conclusions: This M-DC8+ population, which is involved in the pathogenesis of chronic inflammatory diseases, might thus be considered as a major actor in the vicious circle of immune hyperactivation fueling HIV infection progression.

P1047 mCMV infection can be beneficial or harmful in retrovirus infection dependent on the order and time-point of infections J. Duppach, S. Francois, J. Peng, U. Dittmer & A. Kraft Virology, University Hospital Essen, Essen, Germany Purpose/Objective: Suppression of the immune system e.g. transplantation or HIV infection can result in reactivation of cytomegalovirus (CMV). In general, CMV reactivation can be life-threatening and therefore it should be avoided especially in transplantation patients. However, recently it was shown that CMV reactivation early after bone marrow transplantation in human adult myeloid leukemia can also be beneficial by reducing tumor relapse, indicating that CMV infection can be beneficial for the host. Different to CMV reactivation studies in AIDS patients, only limited studies are done to investigate if a primary or persistent CMV might be beneficial or harmful in HIV patients. Materials and methods: In this study we used the two well established infection models in mice, the Friend leukemia Virus (FV), as a model for retroviral infections and the murine cytomegalovirus (mCMV), as a model for human cytomegalovirus infections. Results: A primary mCMV infection resulted in enhanced FV replication in mice persistently infected with FV and enhanced

numbers of functional FV-specific CD8 T cells. However these cells become dysfunctional suggesting that expanded Treg cells might dampen the ‘newly’ generated FV-specific T cell response and inhibit the clearance of persistent FV infection. In contrast to these findings, we found that persistent mCMV limited a primary FV infection. Significantly reduced FV titers were found during a primary FV infection in persistently mCMV infected mice compared to primary FV infected naı¨ve mice. The reduced FV titers were mediated by an augmented FV-specific CD8 T cell response. In persistent mCMV infection we found that FV-specific CD8 T cells had a significant earlier and stronger in vivo killing capacity during primary FV infection compared to only primary FV infected mice. Conclusions: In conclusion, our data showed that a primary mCMV infection is harmful in persistently retrovirally infected mouse, but a persistent mCMV infection is beneficial by reducing viral titers of a primary retrovirus infection. Our data demonstrated that the mCMV mediated effect onto a retroviral infection is dependent on the sequence order and the time-point of mCMV infection.

P1048 Modifications of the redox intracellular environment during the Human Immunodeficiency Virus type I (HIV-1) infection associated with the Src kinase activation M. Curcio,* H. P. Monteiro, A. Sartori, S. Strumilo,à W. Maia,* E. Castro, S. Andrade,§ L. Simaõ,* M. Soaneà & M. Janini* * Federal University of Saõ Paulo, Microbiology Immunology and Parasitology, Saõ Paulo, Brazil, Federal University of Saõ Paulo, Biochemistry, Saõ Paulo, Brazil, àFederal University of Saõ Paulo, Infectology, Saõ Paulo, Brazi, §Federal University of Saõ Paulo, Ginecology and Charitable Association of Blood Collection COLSAN, Saõ Paulo, Brazil Purpose/Objective: Human Immunodeficiency Virus (HIV) and other structurally simple retroviruses developed, as a defense mechanism, numerous replication strategies to escape from apoptotic mechanism triggered by host cells. HIV maintains a persistent infection by stimulating intracellular (host) production of cytokines and of reactive oxygen and nitrogen species (ROS and RNS) (1). Levels of ROS and RNS and of their antioxidant counterparts determine the redox environment. Alterations on the redox environment are sensed by raising levels of the antioxidant enzyme Cu/Zn and Mn Superoxide dismutase (SOD). Such alterations are potentially associated with the regulation ofHIV-replication signaling pathways (2). Materials and methods: In the present study we used isolated human CD4 T lymphocytes submitted to a protocol of in vitro infection with purified HIV viral particles. Levels of the antioxidant enzymes, Cu/Zn and Mn-SOD, Glutathione peroxidase (GPx), Glutathione reductase (GR), and of the tri-peptide Glutathione (GSH) were measured in infected and non-infected CD4 T lymphocytes. Results: There was an increase on the activities of the antioxidant enzymes followed by a decrease on intracellular GSH levels. In addition we followed the temporal pattern of activation of Src kinase. Src is a cytoplasmic protein tyrosine kinase which has been shown to play an important role in HIV-1 replication (3). Conclusions: Our findings suggest a possible relationship between the alterations in intracellular redox environment and the activation pattern of Src kinase in HIV-infected human CD4 T lymphocytes. These results also emphasize the importance of the intracellular redox environment in regulating the Src-mediated signaling pathway associated with HIV infection.


520 Poster Session: Myeloid Cell Development P1050 Natural T regs recently emigrated from the thymus; consist of two subsets, differently impaired during HIV infection V. Terzieva,* J. Dutrieux, V. Fabre-Mersseman, M. Rancez, B. Charmeteau-de Muylder, S. Logerot, M. Andrieu,à K. Labroque`re§ & R. Cheynier * INSERM U567 CNRS UMR 8104 Paris France Universite´ Paris Descartes Faculte´ de Me´decine Rene´ Descartes UMR-S 8104 Paris France BAS IBIR Sofia Bulgaria, Immunology, Paris, France, INSERM U567 CNRS UMR 8104 Paris France Universite´ Paris Descartes Faculte´ de Me´decine Rene´ Descartes UMR-S 8104 Paris France, Immunology, Paris, France, àINSERM U1016 CNRS UMR 8104, Immunology, Paris, France, § INSERM U1016 Paris France, Immunology, Paris, France Purpose/Objective: The hallmark of HIV infection is the sustained deterioration of the CD4 T cell compartment accompanied by the exhausted thymic function. The role of different Treg populations in the course of HIV infection remains imprecisely determined with nTregs being less characterized than iTregs due to the lack of a clearly defined specific markers. However, it can be expected that similarly to non-Treg naı¨ve T-cells, HIV infection impacts on de novo production of nTregs. Materials and methods: Of the 26 healthy control (HC) individuals and 16 age/sex matched HIV-1+ subjects, ART-treated or therapy-free, were included. PBMCs were simultaneously stained for extra and intracellular markers. The determination of nTregs-RTE cells was done by multi-colors flowcytometry using combinations of anti-CD3, -CD4, -CD45RA, -CD31, -CCR7, -CD25 and -CD127. TREC quantification was performed by real-time PCR on FACS-sorted lymphocytes and expressed as TRECs/105 cells. Statistical analysis was performed using GraphPad software. Results: In the compartment of naı¨ve CD4+T cells (CD45RA+ CCR7+) we identified two subsets of recent thymic emigrants (RTE) nTreg cells (CD31+FOXP3+ naı¨ve CD4+ T-cells) differentially expressing CD25. These 2 subsets contained high concentrations of TRECs, confirming thymic proximity. In contrast, no difference in TREC levels was observed between CD25+ and CD25nTregs-RTE. In HCs CD25+ nTregs-RTE were twice as numerous as CD25- nTregs-RTE (P = 0.027). In patients’ groups an increase of RTE nTregs was observed, mostly as a consequence of CD25- nTregs-RTE expansion (P < 0.02). In ART-treated patients, they essentially consisted of CD127 cells (P < 0.05). This was not the case in the group of untreated patients (P > 0.05). Conclusions: Our results show that nTregs recently emigrated from the thymus consist of two subsets differentially expressing CD25. These subsets are differently impaired during HIV-infection. Further studies are needed to clarify their respective functions and behaviors in the course of HIV infection. Acknowledgements: This study is supported by Grant No PIEF-GA2009-253004, FP7.

P1051 Selection of sero-conversion predictive marker by serological analysis of HIV-diagnosed cases in Korea S. H. Kim,1 B. S. Choi,1 H. J. Sim,1 J. S. Lee2 & S. S. Kim1 1 Division of AIDS, Korea National Institute of Health, Cheonwon-gun, Korea, 2Korea National Institute of Health, Center for Immunology and Pathology, Cheonwon-gun, Korea Purpose/Objective: Even though the latest serological or molecular methods are used to diagnose HIV infection, it is still difficult to determine the HIV status of indeterminate cases. In this study, we identified HIV status of Korean sero-converter in HIV low prevalence area and chose the most relevant markers to predict the status of sero-

conversion by analyzing serological characteristics of HIV sero-converters. Materials and methods: We analyzed the results of the serological tests for HIV diagnosis in Korea from 2009 to 2011. The results of HIV diagnosis were classified as positive, indeterminate, and negative. Especially, we evaluated the predictive value of each antibody profile with presence of p24 antigen based on serological data of seroconverters who were assessed as indeterminate at first, retrospectively. Results: From 2009 to 2011, 2429 people were diagnosed as HIV infected individuals in Korea, and 199 of those cases (8.2%) were determined as sero-converters. Among 199 sero-converters, up to 72.2% (60/83) showed Ab-/Ag+ in screening test were sero-converted. In the case of sero-conversion, the use of antigen detection and western blot analysis, such as the group of Ag+/gp160 (OR; 88.3) orAg+/p24 (OR; 17.9), had higher positive predictive value than the Ag+ group (OR; 9.3) of indeterminate cases. Conclusions: According to our study, the HIV antigen detection and appearance of western blot profile to gp160 protein could be used as a high predictive diagnostic marker to determine the HIV status of indeterminate at first. These results would be helpful to increase the specificity of the HIV positive criteria in HIV low prevalence region without delayed interpretation of HIV status.

P1052 Polymorphisms in the HCP5 and HLA-C genes associate with time to undetectable plasma HIV RNA during highly active antiretroviral therapy (HAART) L. W. Thørner,* C. Erikstrup, L. H. Harritshøj,* G. Kronborg,à C. Pedersen,§ C. S. Larsen,– G. Pedersen,** J. Gerstoft, N. Obel & H. Ullum* * Department of Clinical Immunology, Copenhagen University Hospital, Copenhagen, Denmark, Department of Clinical Immunology, Aarhus University Hospital, Aarhus, Denmark, àDepartment of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark, § Department of Infectious Diseases, Odense University Hospital, Odense, Denmark, –Department of Infectious Diseases, Aarhus University Hospital, Aarhus, Denmark, **Department of Infectious Diseases, Aarhus University Hospital, Aalborg, Denmark, Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark Purpose/Objective: Single nucleotide polymorphisms (SNPs) in the HLA complex P5 gene (HCP5), HLA-C, and near the zinc ribbon domain containing 1 gene (ZNRD1) have been shown to influence viral load (VL) set point in HIV-infected individuals with known time of seroconversion. We aimed to determine the influence of the HCP5 rs2395029, the HLA-C rs9264942, and the ZNRD1 rs3869068 on mean VL before HAART, on time to first VL < 51 copies/ml, and on CD4 Tcell recovery during HAART. Materials and methods: The Danish HIV Cohort Study is a prospective, nationwide, population-based study of all HIV-infected individuals treated in Danish HIV clinics since 1 January 1995. From this cohort we genotyped 1382 Caucasian individuals for the rs2395029 (A>C), rs9264942 (T>C), and rs3869068 (C>T). We calculated the mean viral load before HAART for each individual with a CD4 T-cell count >200 cells/ll and calculated the CD4 T-cell recovery as the weighted average of the last CD4 T-cell count before 1 year with HAART and the first CD4 T-cell count after 1 year with HAART minus the last CD4 T-cell count before HAART. General linear models were applied to evaluate the effect of SNPs on mean VL before HAART and on CD4 cell recovery during HAART. Cox proportional hazard regression analysis was applied to assess the association with time to first VL < 51 copies/ml. All models were assuming additive or dominant genetic effects and were adjusted for


Abstracts 521 sex, age, calendar period, and start of HAART (where appropriate). VL was log-transformed before analysis. Results: The C-allele of rs2395029 was associated with lower mean VL before HAART [(mean ± SD), CC/CT: 4.1 ± 0.95 versus TT: 4.6 ± 0.83, P = 0.0003]. We found no significant associations with mean VL before HAART for the rs9264942 and rs3869068. The Calleles of rs2395029 and rs9264942 were associated with a shorter time to VL < 51 copies/ml [HR (95% confidence interval) = 1.36 (1.10 1.69), P = 0.005; HR = 1.17 (1.07 1.28), P = 0.0004, respectively]. None of the SNPs predicted CD4 T-cell recovery during HAART. Conclusions: The C-allele of rs2395029 associates with mean VL before HAART and the C-alleles of rs2395029 and rs9264942 associate with time to first VL < 51 copies/ml during HAART further emphasizing the impact of these SNPs on viral replication.

P1053 Precursor frequency of HLA-B27-restricted HIV KK10-specific CD8+ T-cells is not related to their immunodominance O. Bricenõ,* A. Moris,* V. Appay,* E. Gostick, D. Price, A. SaenzCirion,à R. Mallone§ & C. Iglesias– * Hoˆpital Pitie´-Salpetriere, U945 Infections and Immunity, Paris, France, Cardiff University, Institute of Infection and Immunity, Wales, UK, à Institut Pasteur, Unite´ de Re´gulation des infections Re´trovirales, Paris, France, §Universite´ Paris Descartes, UINSERM U986, Paris, France, – Hoˆpital Pitie´-Salpetriere, UINSERM U945, Paris, France Purpose/Objective: It has previously been demonstrated that HLAB27 allele is associated with the control of HIV replication and that HIV specific CD8+ T cell responses restricted by this HLA molecule are immunodominant and present superior functional attributes. Studies performed in mice have shown that the frequency of naı¨ve precursors impacts on the quantitative and qualitative attributes of CD8+ T cells during immune responses. We therefore hypothesized that advantageous properties of HLA-B27 restricted HIV specific CD8+ T cells may be linked to the initial frequency of naı¨ve precursors. The objective of this study was to calculate the frequency and priming capacity of naı¨ve CD8 T cells specific for HIV epitopes restricted by the protective (HLA-B27, KK10) and the non-protective HLA alleles (HLA-A2, S9L or HLA-B7 RL9). Materials and methods: Our data was generated using blood samples derived from healthy HIV-seronegative subjects. HIV reactive CD8+ Tcell precursors were enriched from 108 peripheral blood mononuclear cells (PBMCs) using cognate peptide-HLA class I tetramers and magnetic beads, and their frequency was calculated. We also expanded naı¨ve CD8+ T-cells by performing ex vivo priming with peptide-pulsed autologous dendritic cells, which were differentiated using GM-CSF and IL-4 and matured with a cytokine cocktail for 20 days in culture. For comparison, we performed the same assays for HLA-A2 restricted Melan-A (EV10) reactive CD8+ T-cells, known for their high frequency of naı¨ve precursors. Results: The frequency of Melan-A reactive CD8+ T-cell precursors was >100 cells per million CD8+ T-lymphocytes, In contrast, the frequency of KK10 reactive precursors was approximately 1 cell per million CD8+ T-lymphocytes and was not different from those measured for other HIV specificities. These findings were confirmed using the in vitroexpansion assay. Conclusions: Our results suggest that the frequency of naı¨ve precursors per se, cannot explain the acquisition of superior functional attributes or the immundominance of KK10/HLA-B27-specific CD8+ T-cell populations in individuals infected with HIV-1.

P1054 Rapid broadening of the CTL responses retards the evolution of immune escapes of HIV H. W. M. van Deutekom, G. Wijnker & R. J. de Boer Utrecht University, Theoretical Biology/Bioinformatics, Utrecht, The Netherlands Purpose/Objective: During the first months of infection with HIV the virus typically evolves immune escape mutations. These escape mutations are found in epitopes from the virus and reduce the selection pressure of CD8+ T cell responses specific for these epitopes. Recent data suggest that most of these immune escape mutations evolve early and rapidly. By using a mathematical model, we try to understand why the evolution of immune escape slows down over the time of infection, and how immune escapes depend on the breadth of the immune response to the virus. Materials and methods: We used a conventional mathematical model with the main distinguishing feature that several immune responses can together control the virus at steady state (e.g. at the viral set-point). Individual CTL responses appear at different time points during infection. Results: We find that most escapes occur early, when the diversity of the immune response is still small. A generic feature of the model is that the contribution of each immune response at steady state declines when the number of immune responses increases. Escaping one immune response provides little advantage when the breadth of immune responses is high. The impact of the breadth of the CTL response is even stronger if some of the viral epitopes are difficult to escape and/or the virus has a poor replicative capacity. Conclusions: The contribution of a single CTL clone decreases when the breadth of the CTL response increases. Escapes therefore tend to happen during early infection because the breadth of the CTL response is still small.

P1055 Single amino-acid change in a highly conserved motif of the gp41 elicits viral neutralization and protects against CD4 depletion C. Petitdemange,* A. Achour,* I. Malet, A. Sennepin,* R. Ho Tsong Fang,à J. Crouzet,à V. Calvez,à P. Debre´* & V. Vieillard* * hoˆpital Pitie´-Salpetriere, immunite´ et infection, Paris, France, hoˆpital Pitie´-Salpetriere, Virologie, Paris, France, àInnavirvax, innavirvax, Paris, France Purpose/Objective: The hallmark of most successful vaccine is the ability to induce cross-reactive neutralizing antibodies (Nabs). For HIV-1, only a handful of broadly Nabs (bNAbs) have thus far been identified. Over the ensuing years, we have shown that a highly specific and conserved motif of the gp41, called 3S, induces expression of NKp44L, the cellular ligand for an activating NK receptor on CD4 T cells render them more sensitive to autologous NK lysis. The aim of this study was to explore the possibility that substitutions inside this 3S motif could permit the development of bNAbs while preserving the capacity to block CD4 depletion. Materials and methods: To further characterized the 3S peptide, point mutations were introduced inside the 3S motif of the gp41. We tested such effect on the capacities to infect cells and the ability of the corresponding peptides to induce production of Ab that elicit viral neutralization and/or inhibit NKp44L expression on CD4T cells and NK cell function. Results: In this study, alanine-scanning allowed us to identify specific positions in the 3S motif that inhibit HIV entry and expose it to a broad spectrum of neutralizing antibodies. Importantly, for the first time, we show that specific amino-acid substitutions within a highly linear motif of the gp41 elicit strong neutralization capacity with


522 Poster Session: Myeloid Cell Development impressive magnitude, breadth and ability to durability over cross clade viruses. Furthermore, our data also show that this acquisition of neutralization capacities preserves the unique ability of anti-3S Ab to inhibit NKp44L expression on CD4+T cells and their sensitivity to NK lysis. Conclusions: Our finding suggest that specific substations into the 3S based immunogens may lead to the generation of bNAbs directed against the 3S conserved motif of gp41 and will provide foundation for a vaccine design based on ‘bi-functionals’ Abs allowing both viral neutralization and CD4 protection.

P1056 Study on the Functional role of Immunoglobulin E as surrogate marker for HIV/AIDS infection V. Baghyanathan Molecular Virology, King Institute of Preventive Medicine & Research, Chennai, India Purpose/Objective: IgE class of antibodies has been found in mammals and plays an important role in allergic and hypersensitivity reactions. Certain viral infections are known to produce specific IgE antibodies, to the extent that significant changes in the level of total serum IgE may occur. Study attempts to associate the Level of Ig E in HIV progression. Materials and methods: The study involves fifty HIV seropositive patients attending Anti-Retroviral Therapy Centre, Department of Sexually Transmitted Disease, Rajaji Government Hospital, and Madurai, India subjected for the present study. The individual involves 27 HIV/AIDS Male patients, 23 HIV/AIDS Female patients. The control sample comprises 15 HIV sero negatives. The samples were collected at the informed consent of the patients. Serum sample were collected and IgE was quantified using MAGIWELL IgE quantitative solid phase Enzyme linked Immunosorbent assay (ELISA). Results: The study documents highest percentage of deviation from the control observed in Male HIV seropositives (43.7%) and age-wise influence documents highest percentage of deviation in the age group 15 29 years (56%). Conclusions: Serum IgE level in the present study found to be elevated from the normal range documents the existence of imbalance between Th 1 and Th 2 and associated with T-cell dysfunction and a hypergammaglobulinemia. The present results suggest that elevation of circulating IgE levels may be due, at least in part, to specific IgE directed to the HIV virus rather than as a result of a nonspecific phenomenon. HIV infected adults indicate that total IgE is also increased during the early stages of disease, and this elevation appears to be independent of CD4 counts and is not correlated with the levels of other immunoglobulins, suggesting an important role for IgE as a surrogate marker of disease progression Further research need to be exploited to bring out the exact role of IgE in HIV pathogenesis.

P1057 TB-IRIS is marked by acute phase response related elevation of LBP and IL-6 O. Goovaerts,* W. Jennes,* M. Massinga-Loembe,* A. Ceulemans,* W. Worodria, H. Mayanja-Kizza, R. Colebundersà & L. Kestens* * Institute of Tropical Medicine, Microbiology, Antwerp, Belgium, Makerere University College of Health Sciences, Medicine, Kampala, Uganda, àInstitute of Tropical Medicine, Clinical Sciences, Antwerp, Belgium Purpose/Objective: The immunopathogenesis of tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is not well understood. The presence of elevated levels of IL-6 and C-reactive protein has been reported in TB-IRIS and are indicative of an acute

phase response. Stimulation of the immune system by pathogen associated molecular patterns like lipopolysaccharide (LPS) leads to the production of IL-6 and acute phase proteins. Here we aimed to explore the acute phase response in TB-IRIS, in association with the cytokine storm and plasma markers related to LPS. Materials and methods: We followed up 254 HIV+TB+ patients at Mulago Hospital in Kampala, Uganda of whom 53 developed TB-IRIS during antiretroviral therapy (ART). We compared 40 of these TB-IRIS patients with 45 HIV+TB+ patients from the same cohort who did not develop TB-IRIS (matched for age, gender and absolute CD4 T-cells). We analysed plasma levels of LPS, LPS-binding protein (LBP), sCD14, endotoxin core antibody (EndoCAb), intestinal-fatty acid binding protein (I-FABP, a marker of enterocyte damage), and 18 different cytokines before and after initiation of ART. Results: LBP levels in TB-IRIS patients were significantly lower preART (P = 0.008) and were sharply increased during TB-IRIS cases compared to HIV+TB+ controls (P = 0.003). No differences in sCD14, LPS, EndoCAb and cytokine levels were observed pre-ART between HIV+TB+ patients that did or did not develop TB-IRIS. During TBIRIS however, higher levels of IL-1ra, IL-6, IL-7, IL-8, IL-10, G-CSF and IP-10 were detected compared to HIV+TB+ controls at comparable time points (p£0.027). Surprisingly, I-FABP levels were significantly decreased during TB-IRIS and remained lower than in HIV+TB+ controls during follow-up (P = 0.005). No such differences were observed for LPS, sCD14 and EndoCAb. Multivariate analysis showed a central role for IL-6 in the acute phase response during TBIRIS (P = 0.001). Conclusions: Our data confirm a central role for IL-6 in the inflammation that develops during IRIS, while levels of LPS and EndoCAb suggested no additional effect of bacterial translocation from the gut. TB-IRIS is characterised by lower LBP levels pre-ART, followed by elevated levels during IRIS as part of the acute phase response. These findings suggest that LBP might be a useful marker for TB-IRIS.

P1058 The human leukocyte antigen-G 3’UTR 14-bp deletion is associated with poor survival in an HIV-1-infected Zimbabwean population M. Larsen,* R. Zinyama, P. Kallestrup,à J. Gerstoft,§ E. Gomo,– L. Thørner,** T. Berg,** C. Erikstrup & H. Ullum** * Copenhagen University Hospital, Clinical immunology/Blood bank, Copenhagen, Denmark, Medical research council, Ministry of health and child welfare, Zimbabwe, Zimbabwe, àCentre for Global Health, Public Health Aarhus University, Aarhus, Denmark, §Copenhagen University Hospital, Infectious diseases, Copenhagen, Denmark, –Medical Laboratory Sciences, University of Zimbabwe, Zimbabwe, Zimbabwe, **Copenhagen University Hospital, Clinical Immunology/Blood bank, Copenhagen, Denmark, Aarhus University, Clinical Immunology, Aarhus, Denmark Purpose/Objective: To examine the human leukocyte antigen G (HLA-G) 14-bp deletion polymorphism (rs16375) in relation to progression and survival among HIV-1-infected Zimbabwean individuals. Materials and methods: A treatment-naı¨ve cohort, including 150 HIV-infected and 158-HIV uninfected individuals, was genotyped for rs16375 using a competitive allele-specific PCR system. Survival among HIV-1-infected individuals followed for up to 4.3 years was compared between carriers of the +14-bp allele and -14-bp homozygous carriers by a log-rank test allowing for trend and by Cox proportional hazards regression analysis with adjustment for age and sex. Results: The HLA-G homozygous -14/-14 genotype was associated with lower CD4 cell count (P = 0.017) and higher HIV-1 RNA (P = 0.005). Furthermore, the HLA-G homozygous -14/-14 carriers


Abstracts 523 had a higher mortality rate compared with non-carriers (hazard ratio = 1.9; P = 0.04, CI: 1.033 3.522); however, this difference was not statistically significant after adjustment for CD4 cell count and HIV-1 RNA (hazard ratio = 1.4; P = 0.29, CI: 0.753 2.578). Conclusions: The HLA-G 14-bp deletion polymorphism is associated with higher viral load, more advanced progression, and increased mortality. The data suggest that high sHLA-G expression impairs the cytotoxic control of HIV.

P1059 The MHC-II transactivator CIITA is a viral restriction factor against HIV-1 replication G. Forlani,* E. Vicenzi, G. Poli,à R. Accolla* & G. Tosi* * General Pathology and Immunology, Surgical and Morphological Sciences, Varese, Italy, Viral Pathogens and Biosafety, San Raffaele Scientific Institute, Milano, Italy, àAIDS Immunopathogenesis Units, San Raffaele Scientific Institute, Milano, Italy Purpose/Objective: The MHC-II transactivator CIITA inhibits HIV-1 replication in human T cells by competing with the viral transactivator Tat for the binding to Cyclin T1 of PTEF-b. Here we analyzed the anti-viral function of CIITA in a monocytemacrophage model of HIV-1 infection, the U937 promonocytic Plus and Minus clones characterized by efficient or inefficient capacity to support HIV-1 replication, respectively. Recently, the Plus and the Minus phenotypes have been correlated to the absence or presence of the host factor TRIM22, respectively. Our purpose was to assess the functional relationships between CIITA and TRIM22.

Materials and methods: MHC-II/CIITA expression was assessed by FACS and QRT-PCR analyses. U937 Plus cells were stably transfected with CIITA vector by electroporation. Tat-dependent HIV-1 LTR transactivation was assessed by gene reporter assay. Exogenous CIITA and TRIM22 were co-immunoprecipitated in 293Tcells. Plus, Minus and Plus-CIITA cells infected with HIV-1 IIIB were monitored for RT activity over-time. Results: U937 Minus cells express MHC-II molecules on the cell surface whereas Plus cells do not. This phenotype correlates with the expression of CIITA protein restricted to Minus cells. Importantly, we show that Tat-dependent HIV-1 LTR transactivation is reduced in Minus cells compared with Plus cells. The exogenous expression of CIITA did not induce TRIM22 transcription in Plus cells, whereas it was sufficient to inhibit Tat activity and to change the HIV-1 permissive phenotype of Plus cells to a non-permissive Minus ‘like’ phenotype. Conclusions: We uncoupled the role of TRIM22 and CIITA in the inhibition of HIV-1 replication in monocytes cells. The transcriptional activity of Tat and HIV-1 productive infection were inhibited not only in TRIM22/CIITA-expressing Minus cells but also in Plus cells expressing exogenous CIITA. Thus, CIITA inhibits Tat-activity independently of TRIM22. These findings demonstrate that CIITA has a dual function against HIV-1: it triggers the adaptive immune response by promoting viral antigen presentation and it acts as an endogenous restriction factor against viral transcription.


524 Poster Session: Myeloid Cell Development

Poster Session: Inflammatory & Atopic Skin Disease P1060 AGF inhibited a LPS-induced inflammatory response by blocking NF-kappaB activity in raw 264.7 cells T. J. Kim, Y. J. Kim, S. J. Park & H. H. Kim Division of Biological Science and Technology, Yonsei University, Wonju, Korea Purpose/Objective: Inflammation is a system used by a host to defend against the presence of bacteria, viruses, or yeasts. Toll-like receptors (TLRs) in the plasma membranes of macrophages are activated when they recognize the molecular structure of a virus or bacterium. Lipopolysaccharide (LPS), an outer cell-wall component of Gram-negative bacteria, initiates an inflammatory process via TLR4. We investigated the effect of the extract of Anethum graveloens flowers (AGFs) on LPSmediated inflammation in RAW 264.7 cells. Materials and methods: The amount of NO production in LPSinduced macrophages was measured using Griess reagent. If AGF has a toxic effect on cells, one must distinguish such an effect from that of NO production, which also reduces the viability of LPS-induced cells. Hence, we compared cell cytotoxicity in RAW 264.7 cells incubated with AGF for 24 h using an EZ-Cytox kit. In addition, we investigated to determine whether the suppression of NO production was due to downregulation of iNOS expression. LPS-induced mRNA expression and the protein levels of iNOS were investigated in this experiment. In order to determine the effects of AGF on cytokines, we analyzed cytokine mRNA levels by RT-PCR. We further confirmed that the capability of AGF to activate the LPS-induced signal pathway involves downstream signal molecules such as MAPKs and Akt. Results: The extract markedly suppressed nitric oxide generation in a concentration-dependent manner in LPS-stimulated RAW 264.7 cells. It inhibited inducible nitric oxide synthase (iNOS) and the mRNA expression of cytokines such as interleukin-1 beta and interleukin-6 in LPS-stimulated RAW 264.7 cells. It also inhibited iNOS protein levels in LPS-stimulated RAW 264.7 cells. In addition, AGF decreased the LPS-induced phosphorylation of mitogen-activated protein kinases in LPS-stimulated RAW 264.7 cells. AGF inhibited the phosphorylation of Akt, an upstream molecule of the nuclear factor kappa B (NF-kB) pathway, and thus inhibited NF-kB activity in LPS-stimulated RAW 264.7 cells. Conclusions: These results suggest that AGF exerts an anti-inflammatory effect in LPS-stimulated RAW 264.7 cells by inhibiting iNOS expression and blocking the NF-kB pathway.

P1062 Cellular phenotypes implicated in reversal reaction in co-infected HIV/leprosy patients A. L. Oliveira, T. P. Amadeu, V. M. Menezes, A. C. F. Gomes, J. A. Nery, R. O. Pinheiro & E. N. Sarno FIOCRUZ, Leprosy, Rio de Janeiro, Brazil Purpose/Objective: The expanded availability of antiretroviral therapy (HAART) has added another layer of complexity to the understanding of HIV/leprosy pathogenesis. It has been reported that the initiation of HAART is associated with the development of reversal reaction (RR) in co-infected HIV/leprosy patients. However, the impact of HIV infection and HAART on the cellular immune response to M. leprae (ML) remains unknown. This study investigated the immunological profile of HIV/leprosy patients giving special attention to the cellular activation status and memory profile of CD4+ and CD8+ T cells. Materials and methods: Twenty-five individuals were assessed: coinfected HIV/leprosy patients with RR (RR/HIV); leprosy patients with

RR without HIV (RR); and healthy controls (HC). IFNg production in PBMC culture was analyzed by ELISPOT. T cell subsets were evaluated by flow cytometry for immune differentiation/activation markers. Skin biopsies were also evaluated for T cell subsets by immunofluorescence. Results: IFNg production in non-stimulated (NS) cells from RR/HIV group was higher than in RR and HC groups. RR patients also presented high IFNg production in response to ML independently of HIV infection. Cellular activation in RR/HIV patients was increased in both CD4+ and CD8+ T cells in comparison to RR and HC group as reflected by the expression of CD38 and CD69. These activation markers were also increased in PBMC from RR/HIV patients after ML stimulus. Analysis of skin biopsies from RR/HIV patients also showed augmented expression of CD38 and CD69 co-localizing with CD4 and CD8 T cells. A higher frequency in central and effector memory CD8+ T cells in response to ML in RR/HIV patients was also observed. The production of granzyme B and perforin by effector memory CD8 T cells in response to ML was associated with an increase in the death of ML-infected monocyte cells. Conclusions: These data suggest that CD38 expression in CD8 T cells might be utilized as a tool to identifying HIV/leprosy individuals at risk for RR. Besides this, an increase percentage of cytotoxic CD8 effector memory T cells could be an additional mechanism in mediating the appearance of RR in co-infected patients. Furthermore, the immune response to ML in RR/HIV patients appears to be restored during HAART therapy as indicate by the increase in IFNg production.

P1063 CINCA syndrome in an infant presenting with hydrocephalus O. Turel,* S. Uzunel,* O. Keskin, B. Akovali,* U. Erenberk* & B. Tatli* *Paediatrics, Bezmialem Vakif University, Istanbul, Turkey, Dermatology, Bezmialem Vakif University, Istanbul, Turkey Purpose/Objective: Chronic infantile neurological cutaneous and articular syndrome (CINCA) is a very rarecongenital autoinflammatory disease. It is characterized by neonatal onset urticarial-like rash, recurrent fever, central nervous system (CNS) involvement, and chronic arthropathy, peculiar facial and morphological features. Local and systemic manifestations of disease develop as a result of elevated IL-1b production which may be related to missense mutations within the gene encoding cryopyrin. Materials and methods: We describe an infant with CINCA syndrome who developed hydrocephalus. Results: A seven months old boy admitted with chronic urticaria and hydrocephalus. He was born by normal vaginal delivery following an unremarkable pregnancy. The parents were healthy and nonconsanguineous. He had attacks of urticarial rash beginning from birth, recurrent episodes of fever and progressive enlargement of head after 4 months. Blood tests showed leukocytosis, anemia, elevated acute phase reactants. Investigations for intrauterine infections and metabolic diseases were negative. Immunological workup did not reveal any congenital immunodefficiency. Cerebrospinal fluid (CSF) examination was compatible with chronic meningitis. Antibiotic treatment failed to alleviate the clinical symptoms. He wasdiagnosed as having CINCA on clinical grounds. Conclusions: CINCA syndrome is a well-defined clinical condition but its early identification is often missed. The IL-1Ra (Anakinra) treatment has given good results in CINCA subjects. Pediatricians should be aware of this rare condition in order to detect it quickly and start the appropriate treatment as soon as possible, allowing to stop the disease progression to the severe degrees such as deafness and mental retardation.


Abstracts 525 P1064 Effects of topically applied rapamycin and/or mycophenolic acid on TNCB-induced atopic dermatitis-like skin lesions of NC/Nga mice C. J. Park,* Y. J. Lee,* Y. H. Ryu,* K. E. Jung,* H. S. Kim,* B. J. Kim, H. Kang* & Y. M. Park* *Dermatology, College of Medicine, The Catholic University of Korea, Seoul, Korea, Dermatology, Chung-Ang University Hospital, Seoul, Korea Purpose/Objective: Our aim was to determine whether topically applied rapamycin and/or MPA is effective in AD-liked skin lesions of NC/Nga mice. Materials and methods: Four per cent rapamycin and/or 1% MPA were applied to the 2-chloro-1,3,5-trinitrobenzene (TNCB)-induced AD-like skin lesions in NC/Nga mice 5 days a week for 2 weeks. The mice were divided into nine groups: non-treated, TNCB only, vehicle, 4% rapamycin, 1% MPA, 4% rapamycin and 1% MPA mixtures (Rapamycin: MPA = 2:8, 5:5 and 8:2 ratio) and 0.03% protopic. The clinical efficacy of drugs was evaluated by ear thickness and severity scores of skin lesions. Histological inflammatory changes and mast cell infiltration were evaluated by H&E and toluidine blue stain, respectively. The mRNA and protein expression level of IL-4 and IFN-c in skin lesions was determined by quantitative RT-PCR and immunohistochemistry. Results: Topical application of 4% rapamycin and/or 1% MPA significantly reduced the clinical skin severity and mast cell infiltration in the TNCB-induced AD-like skin lesions, compared with vehicle (P < 0.05). One percent MPA markedly reduced both IFN-c and IL-4 mRNA expression level compared with vehicle (P < 0.05), whereas 4% rapamycin significantly reduced IFN-c but not IL-4 mRNA expression level, compared with vehicle (P < 0.05). Our results demonstrate that topical rapamcyin and/or MPA suppress TNCB-induced AD-like skin lesions of NC/Nga mice by suppressing the local Th2 and Th1 response. Conclusions: These findings suggest that rapamycin and/or MPA may be one of the promising topical therapeutic candidates for AD.

P1065 Eriodictyol inhibits mast cell degranulation through the inhibition of ceramide kinase T. J. Kim, J. M. Yoo, H. H. Kim & S. J. Park Division of Biological Science and Technology, Yonsei University, Wonju, Korea Purpose/Objective: Mast cells are the principal effector cells involved in allergic response, through the release of histamine. Eriodictyol is a unique constituent of the painted maple (Acer mono) and yerba santa (Eriodictyon californicum). Pharmacological activities attributed to eriodictyol include the control of blood vessel permeability in arthralgia and fracture, as well as antioxidant and antimicrobial effects. However, the anti-allergenic activity of eriodictyol has not been evaluate We investigated the effect of eriodictyol on mast cell degranulation and on an allergic response in an animal model. Materials and methods: Passive cutaneous anaphylaxis (PCA) analysis. The PCA animal model is widely used to evaluate the localized mast cell-mediated allergic reaction in vivo. We tested the effect of eriodictyol on allergic response in the passive cutaneous anaphylaxis (PCA) reaction. b-hexosaminidase release assay. The RBL-2H3 mast cell line was established from a rat basophilic leukemia. We evaluated the release of preformed allergic mediators using b-hexosaminidase as a biomarker of degranulation in the mast cells and then measured cell viability. The release of b-hexosaminidase was first measured following stimulation of the IgE-Ag complex.

Measurement of ceramide. We measured the ceramide levels in mast cells. Ceramide is a sphingolipid and an effector in proinflammatory processes. Accordingly, we tested the effect of eriodictyol on ceramide levels in IgE/Agstimulated mast cells using HPLC. Results: We also investigated the effect of eriodictyol on expression of the CERK involved in calcium-dependent degranulation, and on ceramide activation by multiple cytokines. Eriodictyol suppressed the release of beta-hexosaminidase, a marker of degranulation, and the expression of interleukin IL-4 mRNA. Eriodictyol inhibited the expression of CERK mRNA, reduced ceramide concentration in antigen-stimulated mast cells and suppressed the passive cutaneous anaphylaxis reaction in mice in a dose-dependent manner. Conclusions: These results suggest that eriodictyol can inhibit mast cell degranulation through the inhibition of ceramide kinase, and that eriodictyol may potentially serve as an anti-allergic agent.

P1066 Evaluation of platelet functions using impedance aggregometer N. Isiksacan,* M. Koser,* F. Cemsitoglu, U. C. Kucuksezer,à I. Bakir§ & G. Denizà *Biochemistry, Mehmet Akif Ersoy Thoracic & Cardiovascular Surgery Training and Research Hospital, Istanbul, Turkey, Dermatology, Mehmet Akif Ersoy Thoracic & Cardiovascular Surgery Training and Research Hospi, Istanbul, Turkey, àImmunology, Istanbul University DETAE, Istanbul, Turkey, §Mehmet Akif Ersoy Thoracic & Cardiovascular Surgery Training and Research Hospital, Istanbul, Turkey Purpose/Objective: Platelets are playing a major role in primary haemostasis. Their main functions are adhesion, secretion, aggregation and procoagulant activity. Formation of a hemostatic plug in response to vessel wall injury requires functional platelets and defects platelet functionsdefined as ‘Platelet Dysfunctions’. Chronic urticaria (CU) is a relapsing disease of skin, associated with itching of swelling skin and erythema, which lasts for 6 weeks or longer. Several studies indicate coagulopathy may be involved in CU etiology. The aim of this study was to investigate the platelet functions using impedance aggregometer method in CU patients. Materials and methods: Patients diagnosed as ‘CU’ from dermatology outpatient clinic were enrolled to the study. Venous blood samples from 32 patients with CU as well as 26 healthy controls were collected. Platelet counts and functions were measured. Patients with hereditary/acquired angioedema, anaphylaxis, as well as urticaria-related or systemic vasculitis- patients were excluded from the study. It was confirmed that none of the patients received oral anticoagulant drug treatment, had any infection, thromboembolism, hepatic or cardiac disease or underwent any surgical treatment. Healthy subjects had similar age and gender distribution as well as the patient population. Statistical analysis was performed by using student’s t-test. Platelet function analysis was performed as in vitro platelet activation and aggregation using impedance aggregometer method. Platelets were stimulated with ADP, ASP, TRAP and ristocetin agonists. Platelet aggregations were measured as ‘Area Under Curve’ (AUC). Results: Response of platelets from CU patients to ADP (P < 0.017), TRAP (P < 0.042), ristocetin (P < 0.028) were decreased in comparison to healthy controls. Platelet counts (P = 0.587) and response to ASP (P = 0.178) did not differ in both groups. Conclusions: Impedance aggregometer method is frequently used in determining prehaemostatic effects of antithrombotic therapies in clinical studies and in clinical practice. In this study platelet functions of chronic urticaria patients and healthy controls weremeasured with impedance aggregometer and different responses after stimulations of agonists were observed. The use of this methodology seems to be accurate and valuable in clinical practice.


526 Poster Session: Myeloid Cell Development P1067 Expression of histamine H4 receptor in human skin and amelioration of experimental acute pruritus using H4 receptor antagonist K. Yamaura,* E. Suwa,* M. Suzuki & K. Ueno* *Department of Geriatric Pharmacology and Therapeutics Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan, Research Center for Frontier Medical Engineering, Chiba University, Chiba, Japan Purpose/Objective: Histamine is a potent mediator of itch in humans, yet histamine H1 receptor (H1R) antagonists have been shown to be of limited use in the treatment of pruritic diseases. Recently described histamine H4 receptors (H4R) are expressed in hematopoietic cells and have been linked to the pathology of atopic dermatitis and asthma. Recent studies have raised the possibility that H4R may be involved in pruritic responses. Here we investigated the expression of H4R in human skin and efficacy of the H4R antagonist on pruritus using experimental mice model of acute pruritus. Materials and methods: Dermal tissue specimens were obtained from osteoarthritis patients, and immunofluoresence staining was performed to ascertain whether H4R is expressed in human epidermal tissues or dermal fibroblast cultures. Scratching behavior was induced by histamine (300 nmol), substance P (100 nmol) or serotonin (100 nmol) injected intradermally into the rostral part of the back of each mouse. Mice model of dry skin pruritus was created by topical application of distilled water following acetone/ diethylether (1:1) mixture twice daily upon the shaved area for 5 days. Fexofenadine (30/60/150 mg/kg), a selective H1R antagonist and JNJ7777120 (3/10/30 mg/kg), a selective H4R antagonist were administrated orally. Results: Immunohistochemical staining showed that K10-positive differentiated keratinocytes in the prickle cell layer and granular layer of epidermis strongly expressed H4R. In contrast, H4R expression was less in K14-positive proliferating keratinocytes in the basal layer. Cultured human dermal fibroblasts also express the H4R. The H4R antagonist JNJ7777120 significantly reduced histamine- and substance P-induced scratching behavior in a dose-dependent manner. Moreover, JNJ7777120 reduced dry skin-induced scratching behavior. However, the inhibitory effects of fexofenadine, a H1R antagonist, on pruritus of these models were much smaller than that of JNJ7777120. Neither fexofenadine nor JNJ7777120 showed reduction in serotonin-induced scratching. Conclusions: Results of this study suggest that keratinocytes increase expression of H4R following differentiation. Moreover, as suggested in this study, histamine may have an involvement via the H4 rather than the H1R in histamine- and substance P-induced pruritus and dry skininduced pruritus. The H4R antagonist may be useful for the treatment of H1R antagonist-resistant pruritus.

P1068 Hidradenitis suppurativa is characterized by a defect of Th17/Th22 CD4+ T cells and an infiltration of regulatory T cells in the skin C. Hotz,* A. Guguin,* M. Surenaud,* N. Ortonne, R. Bosc,à P. Wolkenstein,§ S. Huë* & Y. Le´vy* *Faculte de me´decine de Cre´teil, INSERM U955 team 16 UPEC, Cre´teil, France, Hoˆpital Henri-Mondor, Pathology, Cre´teil, France, àHoˆpital Henri-Mondor, Plastic surgery, Cre´teil, France, §Hoˆpital Henri-Mondor, Dermatology, Cre´teil, France Purpose/Objective: Hidradenitis suppurativa (HS) is a chronic, inflammatory skin disease. Medical treatments in use for HS (antibiotics and immunosuppressive agents) point out to a deregulated immune response against microflora. In situ antimicrobial defense requires a balance between inflammatory and protective T cell re-

sponses and their regulation through a Th17/IL-22/Treg axis. We investigated this pathway in the blood and the skin of HS patients. Materials and methods: Analyses of the phenotype and functional profile of T cell populations in PBMCs from HS (n = 17) and healthy donors (HDs; n = 11) were performed ex vivo and following in vitro stimulation. Production of cytokines (IL-17, IL-22, IFN-g) and expression of transcription factors (T-bet, RORgt, Foxp3) were assessed using qRT-PCR, multiparametric flow cytometry and functional assays using ELISA and luminex technologies. In situ, T cell populations were characterized by Histo-immunochemistry (HIC). Results: HS patients exhibited a higher frequency of CD4+IL17A+(median 2%) and CD4+IL-22+(1.32%) in the blood as compared to HDs (0.85% and 0.53%, respectively) (P < 0.05). Frequency of CD4+ IL22+ expressing CCR10, a skin homing receptor, was significantly increased in HS (15.3% as compared to 8.1% in HDs) (P = 0.033). There was no difference between groups in the frequency of CD4+ CD25highFoxP3highCD127low Treg. Stimulation of PBMC with either flagelline, staphylococcus aureus or candida albicans led to a higher production of IL-17, IL-22 and IFN-g in HS patients as compared to HDs. Expression of CD3, IFN-g, and FoxP3 mRNAs was increased in the skin of HS patients (n = 4) as compared to HDs (n = 5). In contrast IL-22 and RORgt RNA expression was decreased in HS patients. HIC analyses confirmed an infiltration of Treg, but not IL-17+ T cells in the skin of HS. Conclusions: HS patients are characterized by a high frequency of proinflammatory functional memory Th17/Th22 T cells in the blood capable to respond to bacterial products. Despite expressing homing receptors, a lower frequency of these cells was noted in the skin of patients, in contrast, to a higher infiltration of Treg. These results raise several hypotheses such as a defect of local production of antimicrobial peptides, key triggers of T cell homing, leading to the chronic perpetuation of inflammation in skin lesions.

P1071 Relationship of interleukin-13 and interleukin-33 serum concentration and selected clinical and immunological parameters in patients with atopic dermatitis A. Pszonak,* W. Owczarek,* E. Paluchowska* & J. OlkowskaTruchanowicz *Department of Dermatology, Military Institute of Health Services, Warsaw, Poland, Department of Transplantology and Central Tissue Bank, Center of Biostructure Research Medical, University of Warsaw, Warsaw, Poland Purpose/Objective: Interleukin-13 (IL-13) coordinates the allergic inflammation process. It stimulates lymphocytes B to IgE synthesis, affects the differentiation’s process and survival time of mastocytes and eosinophils. Interleukin-33 (IL-33) stimulates mastocytes, eosinophils, basophils and Th2 lymphocytes to secrete IL-13. The possible role of these cytokines in the pathogenesis of AD is indicated. To assess the relationship between serum level of IL-13 and IL-33 and severity of disease, extension of skin lesions and immunological parameters as total serum concentration of immunoglobulin E (total IgE), the amount of white blood cells and elements of leucogram in AD patients. Materials and methods: The study involved 60 patients with AD (32 women, 28 men) aged of 18 54 years and 20 healthy volunteers of control group. AD was diagnosed according to criteria of Hanifin and Rajka. Serum concentration of interleukins was evaluated by immunosorbent assay (R&D, USA). The extension of skin lesions and SCORAD were rated in patients, total IgE in serum and the number of white blood cells with the elements of leucogram was determined. Results: The mean concentration of IL-13 in AD patients’ serum was 154.4 pg/ml (95% CI = 91 217) and IL-33 14.1 pg/ml (95% CI = 12.3 16). The mean concentration of IL-13 in healthy subjects


Abstracts 527 was 149.5 pg/ml (95% CI = 132.1 167) and was significantly lower (P = 0.01) and IL-33 18.4 pg/ml (95% CI = 16.2 20.7) and was significantly higher (P = 0.01) than in AD patients. The average extension of skin lesions was 33.4% (95% CI = 25.4 41.4) and the average severity of disease was 53.7 (95% CI = 49 58.3). The study found a statistically significant correlation between serum concentration of IL-33 and the extension of skin lesions (P = 0.03) and severity of disease (P = 0.03). The mean concentration of total IgE in serum was 2500IU/ml (95% CI = 843 4157). Relationship between serum concentration of interleukins and total IgE in serum did not show significant dependency. The evaluation of dependencies between the number of white blood cells, elements of leucogram and serum concentration of interleukins shows a statistically significant correlation with IL-13 and the percentage of lymphocytes (P = 0.03) and basophils (P = 0.03). Conclusions: The mean concentration of IL-13 in serum is significantly higher and IL-33 is significantly lower in AD patients compared to healthy subjects. Serum concentration of IL-33 has a positive correlation with the extension of skin lesions and the severity of disease.

P1072 Role of the Aryl hydrocarbon receptor in skin inflammation J. H. Duarte,* P. Di Meglio,* K. Hirota,* F. O. Nestle & B. Stockinger* *Molecular Immunology, MRC National Institute for Medical Research, London, UK, King’s College London, St. John’s Institute of Dermatology, London, UK Purpose/Objective: The Aryl hydrocarbon receptor (AhR) recognizes polycyclic aromatic hydrocarbons (PAHs), a family of structurally related environmental contaminants. Recently, AhR ligation was shown to amplify the developmental program of Th17 cells and to induce IL-22. By being in constant contact with the environment, the skin is exposed to multiple pollutants that are potential AhR agonists. Exposure to the best known AhR ligand, dioxin, for instance, leads to a severe skin disorder in humans. Taking into consideration the broad expression of AhR in the skin, which includes lymphocytes, dendritic cells and epithelial cells, we aim at defining skin immune responses in wild-type and AhR-deficient mice in order to assess the impact and integration of AhR activation on different cells in the skin. Materials and methods: For this end we are making use of in vivo models of sterile injury (mechanical skin wounding), infection (Candida albicans skin infection) and immune-pathology (Imiquimod-induced psoriasiform skin inflammation). Parameters analysed included histological analysis of the skin and assessment of transcriptional changes, immune cell infiltration and ex vivo measurement of pro-inflammatory mediator secreted by resident and recruited skin cells. Additionally, the effect of dietary, endogenous and environmental AhR ligands on different cell types and its consequences for the development of skin inflammation are being assessed. Results: The inflammatory reaction following simple mechanical skin injury was found to be stronger in AhR-deficient mice, which displayed an increased influx of neutrophils to the injury site. Similar results were obtained in the Candida albicans skin infection model, where the higher inflammatory milieu of the infected skin appeared to be detrimental for the infection clearance, as mice lacking AhR had higher fungal burden. Moreover, lack of AhR resulted in a more severe phenotype induced by the TLR7/8 agonist imiquimod, with increased thickness of the epidermis, neutrophils and inflammatory macrophage infiltration, and expression of key pro-inflammatory cytokines and chemokines. Conclusions: Taken together, our results suggest that AhR signalling has a critical role in dampening immune processes in the skin. Future work will dissect the mechanisms underlying the stronger inflammatory response observed in AhR-deficient mice using cell type-specific

AhR-deficient mice, to address what effect the lack of AhR in different cell types present in the skin has in different models of skin inflammation.

P1074 Th17 cells favor inflammatory responses while inhibiting collagen production by SSc fibroblasts N. C. Brembilla,* E. Montanari,* M. E. Truchetet,* E. Raschi, C. B. Chighizola,à P. L. Meronià & C. Chizzolini* *Immunology and Allergy, University of Geneva Medical School, Geneva, Switzerland, Immuno-Rheumatology, IRCSS Istituto Auxologico Italiano, Milan, Italy, àImmuno-Rheumatology, IRCSS Istituto Auxologico Italiano and Istituto G. Pini University of Milan, Milan, Italy Purpose/Objective: Th17 cells are augmented in Systemic Sclerosis (SSc), an autoimmune disease characterized by fibrosis of the skin and internal organs due to uncontrolled fibroblast activation. Our aim was to assess whether Th17 cells and IL-17A could modulate inflammatory and fibrotic responses in dermal fibroblasts from SSc and healthy individuals (HD). Materials and methods: Fibroblasts were obtained from eight SSc skin biopsies and abdominoplasty pieces of 8 age/sex matched HD. Th17 cell clones were generated from the peripheral blood of HD upon enrichment of cells expressing CCR4/CCR6/CD161 and their cytokine production assessed by FACS analysis and multiplex beads immunoassay. MCP-1, IL-8, MMP-1 and type I collagen production was quantified in fibroblast supernatants by ELISA and RIA, and relative change in their transcription levels assessed by real-time PCR. IL-17 neutralizing antibody was used to confirm the specificity of the effects. Signaling events induced by IL-17A were investigated by western blotting and pharmacological inhibitors used to dissect the pathways involved. Results: IL-17A increased MCP-1 (P < 0.01), IL-8 (P < 0.01) and MMP-1 (P < 0.01) production in a dose-dependent manner, while having no effect on type I collagen synthesis in 8 HD and 8 SSc fibroblasts at both protein and mRNA level. IL-17A induced the production of pro-inflammatory chemokines MCP-1 and IL-8 by triggering NF-kB and p38 signaling, while enhancing MMP-1 via activation of the JNK pathway. Supernatants of activated Th17 clones strongly enhanced MCP-1, IL-8 and MMP-1 production while inhibiting collagen synthesis. IL-17A neutralization proved the role of IL-17A in mediating Th17 effects. In clone supernatants, IL-17A had an additive/synergistic activity with TNF, as shown by IL-17A/TNF blockade. Consistently, IL-17A synergized with TNF in enhancing MCP-1, IL-8 and MMP-1 production when added to fibroblast cultures. Finally, TNF/IL-17 blockade in Th17 clone supernatants resulted in enhanced collagen production specifically in SSc fibroblasts. Conclusions: Th17 cells elicit in vitro pro-inflammatory responses while limiting collagen production by fibroblasts. The increased Th17 cell number observed in SSc may impact on the inflammatory component of the disease and have rather a protective role against fibrosis.

P1075 The levels of cytokines, serum IgE and melatonin in children with atopic dermatitis with sleep disturbance C. Yung-Sen*, L. Jyh-Hong,* S. Chi,* L. Pei-Lin, L. Yu-Tsan,* W. Li-Chieh,* Y. Hsin-Hui,* Y. Yao-Hsu* & C. Bor-Luen* *Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan Purpose/Objective: Patients with atopic dermatitis (AD) frequently reported disturbed sleep leading to impaired quality of life. The aims of


528 Poster Session: Myeloid Cell Development this study are to objectively measure the characteristics of their sleep disturbance, to determine the potential roles of scratching, cytokines, and melatonin, and to evaluate the impact of sleep disturbance on behavior. Materials and methods: Forty-six AD patients and 32 healthy controls between 1 and 18 years old were enrolled. Subjective sleep quality is evaluated by questionnaire. Objective sleep parameters were determined by polysomnography and actigraphy. Serum levels of cytokines related to sleep (IL-1b, IL-4, IL-10, IL-6) and itch (IL-31 and INF-c), serum total immunoglobulin E (IgE) levels, and urine melatonin sulfate levels were measured on the subsequent morning. The SNAP-IV and the Strength and Difficulties (SDQ) questionnaires were used to evaluate behavior. Results: Subjective recognition of poor sleep quality was present in 60.9% of AD patients, compared with only 6.3% in controls. Objective measurements showed that sleep efficiency was lower in AD patients (72.2 ± 10.2% versus 81.2 ± 7.6%, P < 0.001). Sleep onset latency and wake time after sleep onset are longer in AD patients. Disease severity of AD was related to sleep efficiency (r = )0.52, P < 0.001) and movements in sleep (r = 0.69, P < 0.001). Total serum IgE levels were correlated with sleep efficiency (r = )0.46, P = 0.001). Morning urine melatonin sulfate level was higher in AD patients (87.7 ± 42.9 versus 71.1 ± 55.2 ng/ml, P < 0.001), and was correlated with sleep efficiency (r = 0.41, P = 0.01). Serum IL-1b, IL-4, IL-10, IL-6, IL-31 and INF-c levels were not associated with sleep parameters. Lower sleep efficiency in AD patients is associated with higher oppositional defiant disorder score of SNAP-IV (r = )0.47, P = 0.002), and higher conduct problems score and total difficulties score in SDQ (r = )0.42, P = 0.009 and r = )0.35, P = 0.027, respectively). Conclusions: Objective evaluation with polysomnography and actigraphy showed that poor sleep efficiency is common in AD patients, is associated with disease severity, and may have impact on behavior. Scratching movements, serum IgE, and melatonin might play a role in their sleep disturbance, and further studies are required to explore the mechanisms.

in complex with IL-1RAcP. Levels of IL-36a are increased in psoriatic skin, suggesting that the cytokine may play a role in the disease, and in support of this over-expression of IL-36a in keratinocytes causes skin inflammation in mice. Until recently, it has been difficult to study the IL-36 cytokines in vitro since the full length proteins are only active at high concentrations. It is now known, however, that N-terminal processing increases their potency a thousand-fold. It seems likely that the IL-36 cytokines are processed in vivo in a similar manner to IL-1a and b, producing more potent agonists. The truncated proteins have been shown to activate T cells and dendritic cells to produce cytokines and upregulate co-stimulatory molecules. Since mast cells are found at high numbers in the skin and are implicated in psoriasis and skin inflammation, we investigated whether IL-36a, b and c can activate mast cells. Materials and methods: Experiments were carried out in vitro using bone marrow derived mast cells (BMMCs). Results: We find that BMMCs express the specific receptor for IL-36, IL-1Rrp2, and that the IL-36 cytokines cause bone-marrow derived mast cells to secrete IL-6, with IL-36b and c being the most potent of the three. IL-36 increases the response of mast cells to stimulation via FceR1, in a similar manner to that observed to occur with IL-33. Interestingly, BMMCs deficient in another IL-1 cytokine family receptor, ST2, are significantly less able to respond to IL-36a, b and c. It is possible that ST2 is required for the expression of IL-1Rrp2 or IL-1RAcP in mast cells, or is involved in signal transduction after stimulation with IL-36a, b and c. Conclusions: These findings may be of particular relevance in disease settings where IL-36 is expressed, for example in psoriatic legions. The cytokine could act to cause mast cell pro-inflammatory cytokine secretion in addition to enhancing the response of mast cells to antigen, therefore exasperating inflammation.

P1076 Truncated forms of IL-36alpha, beta and gamma cause mast cell activation and enhance the sensitivity of mast cells to stimulation via FcepsilonR1 H. Sandig, C. E. Jobbings & S. Bulfone-Paus School of Translational Medicine, University of Manchester, Manchester, UK Purpose/Objective: IL-36a, b and c (previously IL-1F6, IL-1F8 and IL-1F9) are IL-1 family members that signal via the receptor IL-1Rrp2


Abstracts 529

Poster Session: Inflammatory Bowel Diseases P1079 Allogeneic mesenchymal stromal cells (MSC) transplant in experimental inflammatory bowel disease: modulation of gut inflammation and IL-17 dependent responses H. Ogata,* B. C. Sousa,* V. B. F. Alves,* K. C. R. M. Farias, J. C. Voltarelli, V. J. D. Silva,* V. Rodrigues Junior,* J. E. L. Chica,* J. S. Silva & C. R. B. Cardosoà *Department of Biological Sciences, Federal University of Triaˆngulo Mineiro, Uberaba, Brazil, Faculty of Medicine of Ribeiraõ Preto, University of Saõ Paulo, Saõ Paulo, Brazil, àFaculty of Pharmaceutical Sciences of Ribeiraõ Preto, University of Saõ Paulo, Saõ Paulo, Brazil Purpose/Objective: Inflammatory Bowel Diseases (IBD) is chronic inflammation of the intestinal mucosa, with uncontrolled Th1 and Th17 responses. Although there are current therapies, no treatment is at present fully effective. Since Mesenchymal Stromal Cells (MSC) is multipotent, regulatory and immunosuppressive cells, they emerge as a therapeutic option for various immune disorders. The objective was to evaluate the role of allogeneic MSC transplant in the treatment of experimentally induced IBD. Materials and methods: IBD was induced in BALB/c mice with intrarectal injection of Trinitrobenzene Sulfonic Acid (TNBS). MSC were isolated from bone marrow of C57BL/6 mice, cultured and phenotyped for characterization. IBD mice received intraperitoneal injection of MSC 24 h after disease induction and were euthanized 3, 7 and 14 days later for sample collection, besides clinical evaluation of gut inflammation. Results: MSC cultures in 4th passage showed characteristic markers, such as CD105, CD73, CD44, CD90, CD29, although CD45, CD31, CD34 and CD11b were also found, indicating some contamination of the MSC culture with cells of hematopoietic lineages. Even though, IBD mice treated with MSC presented gain of weight and reduced clinical score compared to not treat mice. There was an apparent increase in eosinophil and reduction in neutrophil influx in treated animals’ colon. Regarding intestinal cytokines, we found an increase of IL-10 and IL-12 on day 3 together with a strong reduction in IL-17 after MSC treatment. In histological analysis, the percentage of goblet cells was higher in treated group than in not treated mice, showing restoration of intestinal morphology by MSC transplant. Conclusions: MSC are able to modulate gut inflammation and IL-17 responses in experimental IBD model. Thus, MSC treatment of IBD may be a novel therapeutic tool aimed at modulating mucosal immune responses without apparent or undesirable adverse effects.

P1080 Anti-TNF therapy response in patients with inflammatory bowel disease is associated with T cell expression of CD25 and TNF receptor 2 M. K. Magnusson,* H. Strid, R. Dahleń,* A. Bajor, M. Simreń ¨ hman & L. O *Department for Microbiology and Immunology, University of Gothenburg Inst for Biomedicine, Gothenburg, Sweden, Department for Internal Medicine and Clinical Nutrition, University of Gothenburg Inst for Medicine, Gothenburg, Sweden Purpose/Objective: Anti-tumor necrosis factor (anti-TNF) agents are effective treatment options for certain patients with corticosteroid dependent or refractory inflammatory bowel disease (IBD). However, the cellular mechanisms behind anti-TNF treatment leading to therapy response are still incompletely known. Materials and methods: Blood samples were obtained before first treatment (visit 1) and 2 weeks post treatment (visit 2) from patients

who commenced anti-TNF treatment. The disease activity was assessed by the validated Mayo score and Harvey-Bradshaw Index (HBI). Response was defined as a decrease in Mayo score or HBI with ‡3. The immunological effects of anti-TNF therapy were studied on freshly isolated cells and cells stimulated ex vivo with influenza vaccine, using flow cytometry. Results: We have included 23 IBD patients (6 CD and 17 UC) into the study. Sixteen patients responded to the anti-TNF therapy, whereas seven patients did not respond. A reduction in the proportion of circulating CD25+ CD4+ T cells [23.9 (5.3 49.6) versus 20.7 (4.4 38.5), P = 0.003] among freshly isolated cells was detected in responders when comparing the first visit (pre treatment) and the second visit (2 weeks post treatment). In contrast, non-responders showed an increased frequency of CD25+ CD4+ T cells [14.3 (6.4 33.4) versus 25.3 (6.6 45.2), P = 0.03] when comparing visit 1 and 2. Levels of FoxP3+ CD4+ T cells and AnnexinV+ CD3+ T cells were similar between both patient groups. Also, there was no difference in expression levels of TNF receptor 1, TNF receptor 2 (TNFR 2) or membrane bound TNF on circulating T cells or monocytes. To compare the T cell phenotype of therapy responders and nonresponders before anti-TNF treatment, cells from visit 1 (pre treatment) were stimulated with influenza vaccine in the presence or absence of anti-TNF antibodies. The effect of anti-TNF is shown as the reduction of CD25 and TNFR2 expression relative to cells cultured without anti-TNF. Results showed that anti-TNF induced a greater reduction of both CD4+ CD25+ (35.1% (14.3 87.4) versus 21.2% (29.6 39.4), P = 0.03) and CD3+TNFR2+ (51.6% (30.2 79.7) versus 20.3% (4.1 29.4), P = 0.002) T cells in therapy responders as compared to non-responders. Conclusions: This study indicates that successful anti-TNF therapy induces a reduced activation of T cells in vivo. Moreover, the expression of CD25 and TNFR2 on in vitro stimulated T cells in the presence of anti-TNF antibodies before treatment start may predict the therapeutic response.

P1081 Colonic Ileus after intestinal surgery depends on CCR2 S. Gutweiler, T. Toepfer, J. Maurer, M. Schiwon, A. Job, C. Kurts & D. R. Engel Institute of experimental Immunology, University Clinic of Bonn, Bonn, Germany Purpose/Objective: Dendritic cells (DCs) are very potent in inducing adaptive immune responses against pathogens, but also cause intestinal autoimmune diseases like colitis. It has been shown previously that the most severe complication after abdominal operation, the post operative Ileus (POI), depends on macrophages (MPs) and memory T helper type 1 (mTh1) T cells, which are initially activated by DCs. However, the specific POI-inducing DC-subset and the role of chemokine receptors in POI-induced DC-migration is still unclear. Materials and methods: After opening the peritoneal cavity POI was induced in CCR2-/-, CX3CR1+/) and C57BL6 mice by manipulating the small bowel with moist cotton applicators once from the oral to aboral direction. After 24 h small bowel function was assessed through feeding FITC labeled Dextran and measuring its bowel progression 1.5 h later. Large bowel function was examined by inoculation of a bead into the colon and measurement of the excretion time. Cell numbers were determined using flow cytometry. Results: We found that the presence of CX3CR1-expressing DCs after abdominal surgery was dependent on CCR2, but lack of such DCs did not improve POI in manipulated small bowel segments. However, CCR2-deficiency improved gut motility in the non-manipulated large bowel. Such improved gut motility was not due to reduced dissemination of POI by mTh1-cells, because recirculation of mTh1 cells was


530 Poster Session: Myeloid Cell Development not affected. Notably, F4/80+ CD11c) MPs were significantly reduced in the large bowel of manipulated CCR2-deficient mice. Conclusions: These findings indicate that CCR2-dependent DCs are dispensable for local POI-development and suggests that CCR2dependent MPs might contribute to POI within the non-manipulated large bowel.

P1082 Deficient production of reactive oxygen species leads to a chronic DSS-induced colitis in Ncf1-deficient B10.Q mice T. Sousa*, H. Carvalheiro,* A. F. Ladeirinha, A. Alarcao, A. S. Cabrita,à L. Carvalho & M. Souto-Carneiro* *Imunology, Centre for Neuroscience and Cell Biology, Coimbra, Portugal, Pathology, Faculty of Medecine, University of Coimbra, Coimbra, Portugal, àExperimental Pathology, Faculty of Medecine, University of Coimbra, Coimbra, Portugal Purpose/Objective: Chronic Granulomatous Disease (CGD) is a genetically heterogeneous immunodeficiency disorder caused by deficiency in oxidative burst, resulting in increased susceptibility tobacterial and fungal infections. CGD is caused by mutations in the phagocyte NADPH oxidase complex, the enzyme which generatesoxygen radicals. A common clinical complication in CGD is chronic intestinal inflammation. As in humans, Ncf1 mutation leads to the lack of reactive oxygen species (ROS) in B10.Q mice, increasing their susceptibility to autoimmunity and infection. We used these mutant mice to study how the lack of ROS influences DSS-induced colitis, its transitional recovery and a second colitis induction. Materials and methods: In the study were usedhomozygous Ncf1 mutatedand B10.Q (Wt) mice. Colitis was induced by oral administration of 3% (w/v) DSS. The induction protocol consisted of 7 days of treatment with DSS followed by 7 days of resting on normal water and finally a second cycle with DSS. Micewere sacrificed at the end of each time point. During the experiment we monitored clinical scores of colitis, collected blood for serum cytokine quantification using CBAs and phenotiping by flow cytometry. We also collected colons at each time point for HE and immunohistochemical (B220, MAC1, CD3) evaluation. Results: The clinical and histological analyses revealed that Ncf1 mice had a more severe disease with a weaker recovery and signs of a chronic colitis. Cytokines quantification showed that both groups had similar Th1/Th17 behavior in the acute phase, but with earlier higher levels of IL-2, IL-6, IL-17, IL-21, IFNc and IL-10 in Ncf1. In the 2nd induction Ncf1 reduced IL-21 compared to Wt. Phenotipe data show a marked Th1/Th17 response in both groups. Ncf1 had more CD4 and CD8 T cells expressing CXCR5 and CD69, NK cells expressing CD107a, Tregs expressing CTLA4 and CXCR5 and B cells expressing CXCR4. In the acute phase and at the 2nd induction Ncf1 had more central memory andeffector CD8 T cells and less Treg compared to Wt. Compared to Wt, Ncf1 circulating monocyte pool evolved from having fewer mature CD11b+Ly6clow at T0 towards an accumulation of CD11b+Ly6chi at T1 and T2, and finally more CD11b+Ly6clow at T3. Conclusions: The clinical scores, the immunohistopathology of colon biopsies, the quantification of serum cytokines and the flow cytometric analysis of peripheral blood mononuclear cells subsets suggest that ROS absent in Ncf1-deficient B10.Q mice leads to an aberrant inflammatory state leading to the development of a chronic colitis similar to seen in CGD humans.

P1084 Dissecting the disease associated functions of S1PR/SPHK axis in DSS-induced colitis in mice P. N. Pushparaj,* M. Jayapal,* K. Narasimhan, S. Kumarà & M. H. Al-Qahtani* *Center of Excellence in Genomic Medicine Research, Faculty of Applied Medical Sciences King Abdulaziz University, Jeddah, Saudi Arabia, Center of Excellence in Genomic Medicine Research, Faculty of Applied Medical Sciences Kingabdulaziz University, Jeddah, Saudi Arabia, àLee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore Purpose/Objective: Ulcerative colitis (UC) is the most common type of inflammatory bowel disease (IBD) afflicting humans. It causes chronic inflammation and the development of ulcers of the large intestine. The aetiology of colitis is not clearly well defined. Dextran Sulphate Sodium (DSS)-induced colitis in mice is a well studied model for human UC. In the present study, we have specifically dissected the role of Sphingosine1-Phosphate Receptor (S1PR)/Sphingosine Kinase (SPHK) axis in the development of DSS colitis using a metanalysis approach and DSS-colitis model using both SPHK1-/- and SPHK2-/- mice. Materials and methods: Meta-analysis of high-throughput genomics data to decipher the role of S1PR/SPHK axis in DSS colitis in mice. The raw Affymetrix CEL files downloaded from the Gene Expression Omnibus (GSE22307) were analysed using the Genespring GX11.5 (Agilent, USA). The statistically significant gene list was filtered based on the standard twofold cut-off compared with the Day 0 expression values. DSS colitis. C57BL/6j mice were purchased from Harlan Olac. SPHK1-/- and SPHK2-/- mice were all on the C57BL/6j background. Mice were housed in specific-pathogen-free conditions in the Central Research Facility and the experiments were conducted in accordance with the respective animal experiment guidelines. For acute colitis induction, Wild-Type (WT), SPHK1-/- and SPHK2 -/- male mice were given 3.5% (weight/ volume) DSS (molecular weight 36 50 kDa; ICN Biomedicals, Aurora, OH, USA) in their drinking water from day 0 for consecutive 7 days. The control mice were given only normal drinking water. The body weight, stool consistency, and rectal bleeding were monitored daily using the modified method of Cooper and colleagues. All the mice were sacrificed on day 7. The colons were dissected and properly cleaned. Sections were taken for histology and serum for multiplex cytokine assay. Results: The meta-analysis of the high throughput genomics data showed that S1PR3 and SPHK1 were the early induced genes in the colonic epithelia of the DSS-treated WT mice. However, both SPHK1-/- and SPHK2-/- mice were protected from the DSS-induced body weight loss, pathological changes in the colon as evidenced by colon length and histological score and attenuated proinflammatory cytokine profiles when compared with DSS-treated WT mice. Conclusions: The S1PR/SPHK axis in the colonic epithelium plays a key role in the development of DSS-colitis. Moreover, both SPHK1 and SPHK2 are important for the effective induction and exacerbation of DSS-colitis.

P1085 Elevated expression of TH17-associated cytokines in the colon of active ulcerative colitis L. Greathead,* H. Cheeseman,* A. Steel, R. Goldin,* M. Nelson,à B. Gazzardà & P. Kelleher* *Medicine, Imperial College, London, UK, Gastroenterology, Chelsea & Westminster NHS Trust, London, UK, àHIV Sexual Health Directorate, Chelsea & Westminster NHS Trust, London, UK Purpose/Objective: The functions of TH17 cytokines in IBD remain controversial. Genes involved in the downstream signaling of IL-23,


Abstracts 531 the cytokine that promotes and sustains TH17 differentiation, have been shown to mediate susceptibility to UC. However mouse models of IBD have demonstrated both protective and pathogenic effects of TH17 associated cytokines. This study measured TH17 and TH1 cytokines in matched peripheral blood and colonic biopsies to determine whether there is a detectable alteration in cytokine patterns in active UC compared to those in remission and healthy controls. Materials and methods: Matched peripheral blood and colonic biopsies were studied in 8 patients with active UC, 8 patients with inactive UC and 10 healthy controls using multi parametric flow cytometry. Disease activity was defined by standard clinical criteria (Mayo score). Mucosal mononuclear cells (MMCs) were isolated by collagenase II digestion followed by mechanical disruption then cell straining. Cytokine staining performed on PBMCs and MMCs after stimulation with SEB. We measured the proportion of CD4 that expressed TH1 and TH17 cytokines as well as amount of cytokine secreted by CD4 T cells using the median fluorescence intensity (MFI). Results: UC patients in remission had significantly higher CD4 percentages in the peripheral blood compared to those with active disease (81%, 55%, P = 0.01). The proportion of CD4+ CD161+ was significantly reduced in PBMCs (active UC = 7%, control = 9%) and MMCs (active UC = 54%, control = 63%) of patients with active UC compared to controls. The MFI of IFNc and TNF on mucosal CD4 T cells was significantly raised in active UC compared to controls (P = 0.04, P = 0.01). The MFI of IL-17a on mucosal CD4 T cells was also significantly raised in patients with active UC compared to controls (P = 0.03). A significantly increased number of MMC CD4 T cells were found to express IL-22 in active UC compared to controls (1.00%, 0.75%, P = 0.01). Conclusions: Active UC is associated with increased expression of inflammatory TH1 and TH17 cytokines. Biological agents to inhibit inflammatory pathways may have a role in the therapy of UC.

P1086 Evaluation of the piroxicam-accelerated interleukin-10 knocks out mouse as a model of human inflammatory bowel disease K. Holgersen,1,2 A. K. Hansenà & T. L. Holm§ *Department of Immunopharmacology, Novo Nordisk, LIFE In vivo Pharmacology Centre, Frederiksberg, Denmark, Department of Veterinary Disease Biology, Novo Nordisk, LIFE In vivo Pharmacology Centre, Frederiksberg, Denmark, àDepartment of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark, §Department of Immunopharmacology, Novo Nordisk A/S, Ma˚løv, Denmark Purpose/Objective: The pathogenesis of inflammatory bowel disease (IBD) is still poorly understood, but it is believed to result from a multifactorial condition, where genetic and environmental factors play an interrelated role, leading to a breakdown of the intestinal homeostasis and an excessive immune response against the commensal microflora. The aim of this project is to evaluate whether the piroxicamaccelerated colitis interleukin-10 knock out mouse (PAC IL-10 k.o.) could function as a tool in the preclinical research and development of new therapeutics against IBD. Materials and methods: The PAC IL-10 k.o. model was evaluated by clinical manifestations and the immune response was characterised by haematology, histopathology, colonoscopy, ELISA and FACS analysis. Qualification was performed by examining the efficacy of treatment with biological therapies for IBD (anti-TNFa and anti-IL-12/23p40). Also, the mice were treated with an antibiotic to determine the role of commensal bacteria in disease progression. Results: The PAC IL-10 k.o. model developed a pronounced colitis immediately after the start-up of piroxicam administration, with synchronised weight loss, diarrhea and blood in stools. Clear signs of chronic colitis were present even 2 weeks after termination of

piroxicam. Acute phase proteins and granulocytes were significantly elevated in the blood compared to IL-10 k.o. controls and histological evaluation revealed hyperplasia and marked infiltration of mononuclear cells and neutrophil granulocytes in the mucosa and submucosa. Disease progression was significantly reduced when treated prophylactically with neutralising monoclonal antibodies against IL-12/23p40 and TNFa, as well as ampicillin. Conclusions: The presented data show that the PAC IL-10 k.o. mouse model may be useful as an in vivo model of human IBD, since the model resembles the corresponding human condition in many aspects.

P1087 GM-CSF modifies monocytes to develop anti-inflammatory properties that are beneficial in Crohn’s disease T. Weinhage, J. Da¨britz, G. Varga, M. Belz & D. Foell Institute of Immunology, University Hospital Muenster, Muenster, Germany Purpose/Objective: Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) showed clinical response and remission in patients with active Crohn’s disease (CD). Since GM-CSF has pleiotropic effects on monocytes, which represent the exclusive source of macrophages in inflamed intestinal mucosa we characterized GM-CSF-treated mouse monocytes in vitro and analyzed their function in vivo in a mouse model of chronic DSS induced colitis. Materials and methods: Mouse bone marrow-derived monocyte precursors were treated for 48 h with GM-CSF in vitro. Phenotypical changes were assessed by qRT-PCR and flow cytometry. Various functional properties were evaluated: mixed lymphocyte reactions, phagocytosis, adherence, cytokine production and reactive oxygen species (ROS) production. Therapeutic effects of GM-CSF-treated monocytes were assessed in a model of chronic colitis that was induced by repeated oral administration of DSS (2%). Monocytes were administered i.v. prior to start of final DSS treatment cycle and their subsequent immunomodulatory functions were evaluated in vivo by clinical monitoring (e.g. body weight), histology, immunohistochemistry and expression of inflammatory markers by qRT-PCR. The distribution of injected monocytes in the intestine was measured by in vivo imaging. Results: GM-CSF-treated monocytes expressed significantly higher levels of anti-inflammatory molecules on mRNA and protein levels (e.g. IL1-Ra, IL4-Ra, CD121b and ARG1) ex vivo. GM-CSF induces ROS production but reduces phagocytosis and adherence in monocytes. Mice treated with GM-CSF stimulated monocytes showed resistance in chronic colitis with diminished weight loss compared to control mice. (Histo-) Pathology showed less inflammatory infiltration, ulceration, and colon shrinkage. Additionally, proinflammatory mediators IL-1, IL-6 and TNF-a were decreased during the course of colitis. GM-CSF-treated monocytes enter the intestine in significantly higher number and persisted longer compared to control monocytes in DSS treated mice. Conclusions: Our results indicate that the beneficial effects of GMCSF in CD may be in part due to the induction of monocytes with anti-inflammatory properties.

P1088 HDAC dependent regulation of the IL-6/STAT3 pathway during T helper cells activation R. Glauben,* M. Wetzel,* P. Mascagni, M. Zeitz* & B. Siegmund* *Charite´ Universita¨tsmedizin Berlin, Research Center ImmunoSciences, Berlin, Germany, Italfarmaco, R&D, Cinisello, Italy Purpose/Objective: Histone modifications represent a promising new approach in cases where cell functions are to be modulated as in


532 Poster Session: Myeloid Cell Development autoimmune diseases or cancer. While several histone deacetylase (HDAC) inhibitors are currently in clinical cancer studies, we demonstrated an additional anti-inflammatory potency in murine colitis models. Here we describe a possible cellular mechanism for this effect. Materials and methods: Murine naı¨ve T helper cells were isolated via magnetic cell sorting and macrophages were derived from bone marrow (BMMF). T cells were stimulated using coated anti-CD3/CD4 antibodies, macrophages via LPS. Cells were analysed using flow cytometry, cytometric bead array or western blot. Acute DSS colitis was performed. Results: In the presence of ITF2357, the generation of FoxP3+ cells from naı¨ve T helper cells could be enhanced, the polarization to the pro-inflammatory Th17 cells suppressed. In parallel, we demonstrated a dose-dependent downregulation of the IL-6 receptor on naı¨ve CD4 T cells treated with ITF2357. This effect could be observed on the mRNA expression level and on the protein level via flow cytometry. These results were confirmed in murine colitis models, where the IL-6R expression was diminished on naı¨ve T cells within the lymphnodes, paralleled by a significant reduction of Th17 cells in the lamina propria of ITF2357-treated animals. Consequently, HDAC inhibition resulted in a reduced amount of activated/phosphorylated STAT3 in T cells identifying the IL-6/STAT3/IL-17 pathway as an important target of HDAC inhibitors. In parallel, ITF2357 treatment of BMMF leads to a dose-dependent down regulation of TNFa, IL-6 and IL-12p70 secretion by BMMF. TLR4-dependent IL-6R upregulation was significantly impaired by ITF2357, while expression of the signaling transducer CD130 was unchanged. ITF2357 reduced the ability of antigen-specific, MHC-IIdependent T cell activation. Conclusions: The present study demonstrates that inhibition of HDAC exerts an anti-inflammatory potency by modulation in T cell polarization directly, but also via affecting macrophage differentiation, leading to impaired IL-6 signalling, reduced T cell activation, thus representing a novel therapeutic approach for chronic (intestinal) inflammation.

P1089 Identification, charactarization and epitope mapping of gamma gliadin, a major wheat antigen in celiac disease B. Srinivasan,* M. F. Tejkl, I. Swoboda, C. Constantin,* I. Mittermann,* S. Pahr,* H. Vogelsang,à W. D. Huber§ & R. Valenta *Division of Immunopathology, Department of Pathophysiology and Allergy Research, Centre for Pathophysiology Infectiology and Immunology Medical University of Vienna, Vienna, Austria, Division of Immunopathology, Department of Pathophysiology and Allergy Research, Christian Doppler Laboratory for Allergy Research, Centre for Pathophysiology Infectiology and Immunology Medical University of Vienna, Vienna, Austria, àDepartment of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria, §Department of Gasteroenterology and Hepatology, Medical University of Vienna, Vienna, Austria Purpose/Objective: Approximately 1% of the population suffers from celiac disease (CD), an inflammatory disease of the small intestine elicited by wheat ingestion. Affected patients mount T cell responses and IgA antibody responses to several antigens belonging to the gliadin-containing fraction of wheat but so far no recombinant wheat antigen specifically recognized by CD patients has been produced. Aim of this study was the biochemical, molecular and immunological characterization of recombinant wheat antigens specific for CD. Materials and methods: Gliadins were fractionated into sub-fractions using ion-exchange chromatography and their antibody reactivity to patients sera was analysed by western blot and ELISA. The disease

specific antigens in the sub-fractions were identified by mass spectrometry and N-terminal sequencing of the individual protein bands. Recombinant antigens were generated in bacterial expression system. Overlapping peptides covering the entire sequence of the recombinant antigen was synthesised and IgA/IgG epitope mapping was performed by ELISA. The secondary structure of the recombinant antigen was performed using circular dicroism. Results: We developed a method for separation of gliadins by ionexchange chromatography and identified disease specific antigens in the gliadin sub-fractions by studying their reactivity to serum IgA from clinically well defined CD and non-CD patients. Through mass spectrometry and N-terminal sequencing we identified the proteins in the sub-fractions and showed that the most relevant IgA-reactive CDspecific antigens belong to the class of gamma gliadins. Based on the sequences identified by mass spectrometry we cloned the complete cDNA sequence of a gamma gliadin (GG1) for recombinant expression and purification. Secondary structure of recombinant GG1 was analysed by circular dichroism. Recombinant GG1 was successfully purified and showed highly specific IgA reactivity with sera from CD patients. IgA and IgG epitope mapping studies with synthetic peptides revealed a major immunodominant regions located at the N-terminus of the protein. Circular dichroism analysis showed that the antigens were folded. Conclusions: Recombinant GG1 should be useful for characterizing the immune response to wheat antigens and to develop diagnostic and therapeutic strategies for CD. This study was supported by the DK program IAI of the Austrian Science Fund (FWF) and by a research grant from Thermofisher/ Phadia, Uppsala, Sweden.

P1090 IL-1 mediates intestinal inflammation by promoting the accumulation of IL-17A secreting innate lymphoid cells and CD4+ Th17 cells M. Coccia,* O. Harrison,* C. Schiering, M. Asquith,* B. Becher,à F. Powrie & K. Maloy* *Sir William Dunn School of Pathology, University of Oxford, Oxford, UK, Translational Gastroenterology Unit, University of Oxford, Oxford, UK, àInstitute of Experimental Immunology, University of Zurich, Zurich, Switzerland Purpose/Objective: Although very high levels of Interleukin (IL)-1b are present in the intestines of patients suffering from Inflammatory Bowel Diseases (IBD), little is known about the contribution of IL-1b to intestinal pathology. We aimed to define the role of IL-1b in driving innate and adaptive pathology in the intestine. Materials and methods: We assessed the roles of IL-1b in complementary mouse models of inflammatory bowel disease, mediated by either innate or adaptive immune activation. Results: We show that IL-1b promotes innate immune pathology in Helicobacter hepaticus-triggered intestinal inflammation by augmenting the recruitment of granulocytes and the accumulation and activation of innate lymphoid cells (ILC). Using a T cell transfer colitis model, we demonstrate a key role for T cell-specific IL-1 receptor (IL-1R) signals in the accumulation and survival of pathogenic CD4+ T cells in the colon. Furthermore, we show that IL-1b promotes ‘type-17’ responses from CD4+ T cells and ILC in the intestine and we describe synergistic interactions between IL-1b and IL-23 signals that sustain innate and adaptive inflammatory responses in the gut. Conclusions: Our data identify multiple mechanisms through which IL-1b promotes intestinal pathology and suggest that targeting IL1b may represent a useful therapeutic approach in IBD.


Abstracts 533 P1093 Interaction between Trichinella spiralis infection and inflammatory colitis: novel immunological concepts D. Ashour,* A. Othman,* M. Shareef, H. Gaballahà & W. Mayah§ *Parasitology Faculty of Medicine, Tanta University, Tanta, Egypt, Pathology Faculty of Medicine, Tanta University, Tanta, Egypt, à Biochemistry Faculty of Medicine, Tanta University, Tanta, Egypt, § Tropical Medicine Faculty of Medicine, Tanta University, Tanta, Egypt Purpose/Objective: The aim of this study was to gain insight about time-related interaction between intestinal nematode infection and inflammatory colitis in an effort to ameliorate experimentally induced colitis using a model for ulcerative colitis, and to explore the underlying immunoregulatory mechanism of Trichinella spiralis infection. Materials and methods: Mice used were divided into four groups: group I: infected with Trichinella spiralis; group II: infected with Trichinella spiralis then subjected to induction of colitis; group III: subjected to induction of colitis and group IV: subjected to induction of colitis followed by Trichinella spiralis infection. Mice were sacrificed at 2th and 4th weeks post-colitis. Assessment of colitis was done by histopathological examination, and determination of pentraxin 3 level in the colon. Immunohistochemistry was done for identification of T regulatory Foxp3-expressing cells. Results: It was evident that T. spiralis infection ameliorated the severe inflammation induced by acetic acid. The amelioration was more pronounced when T. spiralis infection preceded the induction of colitis. Mean pentraxin 3 values were significantly lower in case of colitis with Trichinella infection as compared to negative control or colitis group at different experimental periods. Regarding the immunohistochemical staining of T regulatory cells, the highest score of positivity was detected in group I (T. spiralis alone) and the least score was in group III (acetic acid induced colitis) with the other two groups inbetween. Conclusions: T. spiralis regulatory mechanism can improve the inflammation of colon through the ‘inflammatory regulatory’ axis. Finally, it would be of great importance to apply these results in the development of new therapeutic approaches for treatment of ulcerative colitis.

P1094 KIR-mediated NK education mediates KIR-associated Crohn’s disease susceptibility L. Lin,* C. Ma, N. Aziz,* R. Rajalingam,* S. Targan,à D. McGovernà & J. Braun* *UCLA, Pathology and Lab medicine, Los Angeles, USA, NanoSystems Biology Cancer Center, California Institute of Technology, Pasadena, CA, USA, àCedars Sinai Medical Center, Medical Genetics Institute and IBD Center, Los Angeles, CA, USA Purpose/Objective: Natural Killer (NK) cells are innate effector lymphocytes in the host response to infections and tumors through a variety of activating and inhibitory receptors. NK cells require education by self-human leukocyte antigen (HLA) class I molecules through Killer cell Immunoglobulin-like receptors (KIRs) to gain proficient responses. Yet, it remains unknown whether and how KIRmediated NK education contributes to chronic inflammation in humans. Materials and methods: First, in healthy subjects bearing the simplified AA KIR haplotype, NK subsets expressing KIR2DL3 and 3DL1 were analyzed for cytokine production by single cell barcode chip [SCBC, Ma, C, et al. Nat Med (2011)]. Second, genetic distribution of HLA-C1 and HLA-Bw4, the respective ligands for KIR2DL3 and KIR3DL1, in AA haplotype Crohn’s Disease (CD) patients were analyzed by Chi square test. Third, CD patient NK culture media was profiled for cytokine production at the bulk level using multiplex ELISA system, and at the single cell level using SCBC. Lastly, an NK

coculture assay was performed to test their effect on antigenic CD4+ T cell activation and mechanism of action. Results: In this study of subjects bearing the simplified AA KIR haplotype, we show that KIR education enables NK cells to promote CD4+ T cell proliferation by eliciting expression of multiple proinflammatory cytokines and chemokines. Using the SCBC microfluidic platform, we show that the genetically educated NK subset was highly polarized towards robust production of cytokines at the single-cell level. The strongly-educating KIR-ligand pair (KIR2DL3 and homozygote HLA-C1) was enriched in CD patients, and NK cells from patients with this genotype had distinct secretion profiles compared to NK cells from weakly educated patients. Autologous NK-T cell coculture demonstrated that strong KIR education permitted NK augmentation of CD4+ T cell proliferation via secretion of proinflammatory cytokines. Conclusions: Collectively, these results extend our understanding of the functional consequences of NK education, and reveal the unappreciated capacity of NK cells to produce a wide spectrum of immune mediators that modulate the CD4+ T cell activation threshold. These findings offer a biologic basis for the correlation between NK education and KIR-associated susceptibility to CD and other chronic inflammatory syndromes.

P1095 Mononuclear phagocytes in steady-state and colitis I. C. Arnold, S. Mathisen & F. Powrie Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK Purpose/Objective: There is increasing evidence that antigen-presenting cells are key determinants in shaping both innate and adaptive immune responses in the gut, thereby contributing to intestinal homeostasis. Recently, two major populations of intestinal mononuclear phagocytes have been identified on the basis of the differential expression of the integrin subunit CD103 and the fractalkine receptor CX3CR1. The aim of this study is therefore to understand how these specific cellular subsets contribute to the pathogenesis of inflammatory bowel diseases. Materials and methods: To address the function of colonic LP mononuclear phagocytes during intestinal inflammation, we utilized a mouse model of Helicobacter hepaticus-induced colitis combined with anti-IL10R treatment in CX3CR1-GFP knock-in reporter mice. Results: Upon infection, large numbers of antigen-presenting myeloid cells were rapidly detected in the colonic lamina propria and mesenteric lymph nodes, and persisted during the course inflammation. Detailed analysis of these cells revealed the primary accumulation of an activated inflammatory monocyte population expressing MHCII+ CD11chet Ly6Chi CX3CR1int as well as the FCgReceptor IV (CD64), and producing TNFa, IL12p40 and iNos. Conclusions: We therefore suggest that CD64+ marks an activated subset of infiltrating CX3CR1int mononuclear phagocytes that produce colitogenic cytokines and perpetuate intestinal inflammation.

P1096 Neuronal CGRP and TNF alpha co-regulate each other in a mouse model of parasitic infection in the gut M. Bakri,* J. Pennock* & J. Miyan *Immunology, University of Manchester, Manchester, UK, Neuroscience, University of Manchester, Manchester, UK Purpose/Objective: Background: Neuropeptides have been associated with immune functions in many inflammatory models. In this study we examined the relationship between the sensory nociceptive neuropeptide calcitonin gene related peptide (CGRP) and the potent


534 Poster Session: Myeloid Cell Development inflammatory cytokine tumor necrosis factor alpha (TNFa) during a chronic helminth infection. CGRP has been shown to down-regulate TNFa production from macrophages in vitro whilst TNFa attenuates CGRP release from sensory neurons in vitro. Given the role for CGRP in mediating neuroinflammation, we wanted to study this interaction in vivo during an infectious challenge in the gut. Materials and methods: Methods: CGRP and its receptor antagonist, hCGRP 8 37, were administered in vivo between days 14 and 21 post Trichuris muris infection in BALB/c and AKR mice. Additionally, a TNFa inhibitor, Infliximab, was administered in vivo in chronically infected AKR mice at day 35 p.i. Results: Results: : During infection, an inverse correlation between CGRP and TNFa was observed. Furthermore, CGRP treatment in vivo reduced TNFa expression in both AKR and BALB/c mice during infection, implying a negative regulation. However, when TNFa was inhibited in vivo with infliximab, CGRP levels reduced in the colon, suggesting that CGRP may respond to local TNFa downregulation. Conclusions: Conclusion: Our study shows for the first time that CGRP and TNFa inversely correlate with each other during gut inflammation in vivo. This demonstrates a role for CGRP in the immune response during parasite infection. Whether this happens directly or indirectly is the subject of further work.

P1097 Phagocytosis of Escherichia coli by human gamma delta T cells: a possible role in the pathogenesis of Crohn’s disease S. Banthiya,* N. B. Rayment, P. Skoutari,* S. Tempest-Roe,à B. N. Hudspith, J. Hermon-Taylor & J. D. Sanderson§ *Immunology, Kings College London, London, UK, Diabetes and Nutritional Sciences Division, Kings College London, London, UK, à Biomedical Sciences, Kings College London, London, UK, §Gastroenterology, Guy’s & St. Thomas’ Hospital, London, UK Purpose/Objective: The aetiology of Crohn’s disease (CD), although unknown, is generally accepted as being multifactorial. One of the factors that appear to be instrumental in disease initiation/progression is the immunological response to the gut enteric microbiota. We have previously shown that in cases of CD, mucosal associated E. coli are able to penetrate into the lamina propria and survive within macrophages. In this present study we have not only confirmed this observation but in addition, using immunofluorescence staining (IF), identified E. coli associated with a population of cd T cells in biopsies of patients with Crohn’s disease. This would be consistent with previous literature that has indicated the role of cd T cells as APC’s. Materials and methods: Patients with CD were identified using the Montreal criteria, none of whom were on immunosuppressive or biologic therapy at the time of study. Controls were selected from a colorectal cancer screening population who had a normal bowel habit, no abdominal pain, no rectal bleeding or bloating. Rectal biopsies were taken at colonoscopy and snap frozen in liquid nitrogen. Six micrometer sections of the biopsies were taken and fixed in acetone. Polyclonal antibodies recognising cd T cells, CD68 +ve macrophages and E. coli were used to stain sections by indirect immunofluorescence (IF). Spectrally distinct fluorophores were used in order to achieve co-localisation. Sections were viewed using an epi-fluorescence microscope and were quantified by counting five randomly selected high power fields. Results: Samples were collected from 11 CD patients and 5 controls. Co-localisation of E. coli with cd T cells was seen in 9/11 CD patients but none in controls.cd/E. coli/+ve T cells were observed in both colonic epithelium as well as lamina propria and where present, accounted for 40%of the total cd T cell count. Conclusions: This is the first study to demonstrate the uptake of E. coli in a population of cd T cells within the colonic tissues of CD patients but not controls. The results above suggest that the presence of E. coli within two immunologically distinct populations of cells may

impair the host response and therefore be critical in the resolution of the chronic inflammation seen in Crohn’s disease. 1. Wu, Y., et al. (2009). Human gamma delta T cells: a lymphoid lineage cell capable of professional phagocytosis. Journal of immunology 183(9), 5622 9.

P1098 Regulatory role of the hypothalamic-pituitary-adrenal axis and adrenal glands in experimental colitis P. Reis de Souza,* H. Salles-Campos,* J. S. Silva & C. R. B. Cardoso* *Dactb, University of Saõ Paulo, Ribeiraõ Preto, Brazil, Dpmi, School of Medicine of Ribeiraõ Preto, Ribeiraõ Preto, Brazil Purpose/Objective: Inflammatory immune responses may be modulated by the hypothalamic-pituitary-adrenal axis (HPA) through neuroimmunoendocrine interactions and cortisol secretion. However, even in the presence of intact adrenal glands patients may develop chronic diseases such as Inflammatory Bowel Disease (IBD), which may be caused by an imbalance between regulatory and effector responses in the intestinal mucosa. On the other hand, adrenal glands are also involved in stress response, which may predispose to uncontrolled inflammatory diseases. Then, our objective was to investigate the role of the HPA axis and adrenal glands in experimentally induced IBD. Materials and methods: C57BL/6 mice were subjected to bilateral adrenalectomy and after a 15 day-surgery recovery period the colitis was induced by oral intake of water containing 3% (w/v) Dextran Sulfate Sodium (DSS) for 6 consecutive days. Animals were daily assessed for weight loss and clinical signs of disease. Mice were sacrificed at 6th day of colitis induction and colon samples were collected to assess cytokine production by ELISA and eosinophil peroxidase activity (EPO) by enzymatic assay. Blood samples were also obtained for evaluation of circulating leukocytes. Results: Our results showed that colitis was more severe in animals subjected to adrenalectomy, which showed greater weight loss, increased disease clinical score and early mortality when compared to colitis group. The absence of adrenal glands was also related to an increase in pro-inflammatory cytokines such as TNF-a, IFN-c and IL17 in the gut, along with an augmentation of IL-10, probably in an attempt to compensate for the exacerbated inflammatory response. Moreover, these local alterations were accompanied by reduced EPO in the intestine and diminished circulating eosinophils in the blood, indicating that adrenal produced hormones and neuroimmune interactions may be involved in the maintenance of the peripheral leukocyte pool and control of exacerbated responses in the gut mucosa. Conclusions: Taken together, our results showed HPA axis and adrenal glands play an important role in the regulation of systemic leukocytes and local inflammatory response in the gut.

P1100 Role of dendritics cells during IBD J. Schulthess, C. Arancibia, T. Steveels & F. Powrie NDM, University of Oxford, Oxford, UK Purpose/Objective: Dendritic cells (DC) participate in the fine control of immune system by promoting effective immunity against invading pathogens and in the same time by preventing excessive inflammation. Even the phenotype and the function of subsets of intestinal DC are well-characterized in mice, few are known in human. Better knowledge concerning human intestinal DC at steady state allows us to understand the role of these cells during IBD. Materials and methods: A complex panel was set up to characterize and define intestinal human DC by flow cytometry. Because of the difficulty to isolate significant number of intestinal dendritic cells from


Abstracts 535 controls and IBD patients, an in vitro system of intestinal-like DC was develop from monocytes-derived DC (MODC) which could provide sufficient numbers of DC to assess their function. Results: Our human data indicate that 20% of intestinal DC expresses CD103 while this molecule is not expressed in circulating DC in the blood, suggesting that human CD103+ DC as mice CD103+ DC might be important in maintaining intestinal homeostasis. Moreover gene expression in CD103+ and CD103) DC from human colon indicates that CD103+ DC express high level of mRNA involved in TGF-b pathway or in enzyme producing retinoic acid (ALDH1A2) compared to CD103- DC. Furthermore CD103+ DC coming from IBD patient’s loss the capacity to up regulate TGF-b pathway gens and ALDH1A2. Besides the culture and the differentiation of CD14+ monocytes from blood of control donor into intestinal-like DC with in presence or in absence of RA or FLT3 ligand or TGF-b indicate that only RA is a potent inducer of CD103 on MODC. Quantitative PCR analysis shows that CD103+ MODC up regulate mRNA related to TGF-b pathway as well as ALDH1A2 compared to CD103- monocytes-derived dendritic cells. Conclusions: Preliminary data on human intestine suggest that intestinal DC express CD103 and gene related to tolerance. When cultured with RA, MODC expressed CD103 which provide intestinallike DC phenotype. This CD103+ DC are enriched for genes related to TGF-b activation.

P1101 Th17 related genes and celiac disease susceptibility L. M. Medrano,* B. Dema,* C. Maluenda, M. A. Figueredo,* M. Fernańdez-Arquero* & C. Nuń˜ez* *Clinical Immunology, Hospital Clıńico San Carlos, Instituto de Investigacioń Sanitaria HCSC (IdISSC), Madrid, Spain, Pediatrics, Hospital Clıńico San Carlos, Instituto de Investigacioń Sanitaria HCSC (IdISSC), Madrid, Spain Purpose/Objective: Celiac disease (CD) is an inflammatory intestinal disorder caused by gluten ingestion in genetically predisposed individuals. Th17 immune response has been related to different autoimmune diseases such as Crohn’s disease, psoriasis or ankylosing spondylitis. Polymorphisms in genes involved in the Th17 pathway, such as IL23R, have been associated with susceptibility to those diseases. We aimed at exploring the role of Th17 cells in CD by studying the association of numerous single nucleotide polymorphisms (SNPs) located in Th17 genes with CD susceptibility. Materials and methods: We initially studied 735 CD patients and 549 healthy individuals, and we used a replication sample set consisting of 294 CD patients and 475 healthy individuals. All included individuals were Spaniards and Caucasian. We selected 101 SNPs in 15 Th17 genes (IL23R, RORC, IL6R, IL17A, IL17F, CCR6, IL6, JAK2, TNFSF15, IL23A, IL22, TBX21, SOCS3, IL12RB1 and IL17RA) by performing an aggressive tagging using the Haploview program. Genotyping was performed by ‘Veracode’ technology at the National Genotyping Center (CEGEN) or by Taqman technology in the case of the IL6, IL6R and TBX21 genes. Genetic frecuencies were compared between cases and controls using the chi-square test. Interactions between genes were evaluated following four different approaches: logistic regression, random forests (RF), classification and regression trees (CART) and multifactor dimensionality reduction (MDR). Results: In the case-control study, significant results were not obtained for any SNP with the excepcion of rs4969170 located in the SOCS3 gene [P = 0.0018, OR (95% CI) = 0.59 (0.42 0.84)] and one haplotype conformed by the SNPs located in the IL23R locus. However, these results were not replicated in our validation sample set. No significant interactions between the studied genes were found. Conclusions: Genetic polymorphisms in Th17-related genes do not seem to be crucial for CD development.

P1102 The Gut Microbiota and Inflammatory Immune Diseases C. MacDonald,* D. Kelly,* I. Mulder* & K. Scott *Gut Immunology, Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK, Gut Health, Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK Purpose/Objective: The current work will investigate the effect of the microbiota in autoimmune disease in two disease models: inflammatory bowel disease, a local autoimmune disease within the gut, and multiple sclerosis, a systemic autoimmune disease. We aim to identify key bacterial species present in healthy animals and to investigate the presence or absence of these microbes in diseased states. The effect of the microbial composition of the gut on the function of the cells of the immune system in healthy and colitic mice will be assessed through FACS analysis. In addition, the importance of close association of the bacteria to the mucosa to elicit their effect will be studied. Materials and methods: Denaturing Gel Gradient Electrophoresis (DGGE) has been utilised to expand our knowledge of the microbial composition of the gut. Fluorescence In Situ Hybridisation (FISH) was also used to provide information about the location of the bacteria within the gut tissue. A protocol to retrieve the mucosa-associated bacterial population from the gut was developed. The DNA collected from scraping the mouse gut mucosa was run on DGGE gels to visualise the differences between tissues, and between bacteria that are loosely and tightly associated to the mucosa. Further work will involve a mouse study in which wild-type and IL10 KO mice are treated with antibiotics. The mice are then treated with bacteria (either a single strain or a cocktail) to assess the effect of the known bacteria on microbiota composition and the host immune system. FACS analysis will investigate the effect on the differentiation of the cells in the immune system. Of the 454 Pyrosequencing and qPCR will identify the exact species present and qPCR enables the quantification of the species. Results: Thus far, DGGE profiles have been obtained of both faecal and gut mucosa bacterial profiles. Additionally, FISH has been carried out to visualise the location of the bacteria within the gut tissue, while the protocol to retrieve bacteria associated with the gut mucosa is under-going final optimisation. Conclusions: Our initial work shows a large difference between the bacterial DGGE profiles from faecal samples of wild type mice and IL10 KO mice. The profiles of loosely and tightly mucosa-associated bacterial communities show a clear difference. FISH results show the technique to retrieve the bacteria from the mucosa was effective.

P1103 The impact of NOD2 3020insC mutation in microbial-driven host innate immunity E. Milioris,* V. Iebba,* N. J. Klein,* T. Steevels, A. Simmons & M. Bajaj-Elliott* *Infectious Diseases & Microbiology, UCL Institute of Child Health, London, UK, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK Purpose/Objective: NOD2 is an intracellular pattern recognition receptor of the NOD-like receptor family. Activation of NOD2 by the muramyl dipeptide (MDP) motif of the bacterial peptidoglycan drives NF-jB signaling. The NOD2 3020insC polymorphism is a well-established genetic risk factor for Crohn’s disease. At present the impact of this mutation on enteropathogen-mediated host innate immune responses is unknown. Materials and methods: THP-1 cells transduced with WT and 3020insC NOD2 under both an intermediate and high expression promoter were utilized. PMA-treated cells were stimulated with TLR


536 Poster Session: Myeloid Cell Development (LPS & PGN) and NLR (ieDAP & MDP) ligands and infected with Campylobacter jejuni, Clostridium difficille, Enteropathogenic E. coli and Salmonella enterica. Host innate immunity was quantified by gene and protein analysis, flow cytometry and confocal microscopy. Results: MDP reduced PGN-mediated TNFa levels in cells overexpressing wild-type NOD2 by 40%, indicating that NOD2 is a negative regulator of TLR2-driven pro-inflammatory responses. Enteropathogens led to a marked increase in TNFa, while inhibiting IL-10, during co-infection with macrophages expressing the 3020insC mutant. Conclusions: Our observations suggest that 3020insC NOD2 mutant receptor responds differentially to exogenous stimuli when compared to its WT counterpart. The observed disequilibrium between the proand anti-inflammatory cytokine axes may contribute to bacterial-driven IBD pathogenesis in individuals carrying the 3020insC NOD2 mutation.

P1104 The influence of cell free probiotic supernatant on bacterial macrophage interactions Y. Seenappanahalli Nanjundaiah, Z. Ali, D. A. Wright & M. Sarker School of Science and Engineering, Teesside University, Middlesbrough, UK Purpose/Objective: Probiotics have been shown to be beneficial on patients suffering from inflammatory bowel diseases such as ulcerative colitis. However, the mode of action is unclear with many papers reporting contrary stimulatory or suppressive effects on immune cell activity. Materials and methods: This study utilised a gentamicin protection assay (GPA) to assess the influence of probiotics on both the ingestion and digestion phases of phagocytosis by immune cells. The GPA was performed with E. coli and a murine macrophage (J774) at a multiplicity of infection of 50:1 in DMEM (Dulbecco’s modified Eagle’s medium) alone or DMEM supplemented with either 20 lg/ml lipopolysaccharide (LPS), 10% cell-free Lactobacillus rhamnosus GG (LGG) probiotic bacterial supernatant or a combination of LPS-LGG. Results: Studies monitoring bacterial ingestion, demonstrated a significant reduction in bacterial uptake by macrophages when treated with LGG and the LPS-LGG combination (P < 0.05). The LPS alone had no significant effect on bacterial ingestion. In studies monitoring bacterial digestion, the LPS and the LPS-LGG combination, brought about significant increase in the digestion rate when compared to control (P < 0.05). LGG alone had no significant effect on bacterial digestion. The data suggest both LGG and LPS modulate immune cells but in a contrary manner. The LGG inhibits bacterial ingestion, but does not influence digestion, whereas LPS does not influence ingestion but enhances bacterial digestion. Conclusions: By interfering with macrophage ingestion, LGG may suppress the total microbial load associated with macrophages, and, hence, the extent to which pro-inflammatory molecules such as nitric oxide and oxygen free radicals are generated. The suppression of inflammatory promoting signals may be beneficial to the host, since overproduction of these signals may induce host damage. Future work will focus on whether the LGG driven suppression of ingestion is also associated with a reduction of pro-inflammatory molecules and can maintain this suppression in the presence of LPS, a molecule known to stimulate inflammation.

P1105 The TGF-ß activating gut-associated integrin avß8 is upregulated during DC maturation T. Fenton,* M. Lehtinen & M. Travis* *Manchester, University of Manchester, UK, Kantvik Active Nutrition, DuPont Nutrition and Health, Kantvik, Finland Purpose/Objective: Maintenance of homeostasis within the gut requires constitutive immune suppression to prevent inappropriate inflammation, punctuated by effective antigen-specific responses against pathogens. Breakdown of suppression, or failure to deal with infection, can result in inflammatory or allergic disease. Thus, understanding the cellular and molecular mechanisms that regulate intestinal immunity will be important in identifying potential therapies to improve immune-mediated disorders of the gut. We have previously found that high expression of integrin avb8 on lamina propria dendritic cells (DC) results in activation of the key cytokine TGF-b, which induces either Treg or Th17 cells, depending on the immune context. This pathway is critical to gut homeostasis, as shown by CD11c-specific avb8 knockout mice which develop severe colitis. The aim of this current study is to understand how expression of integrin avb8 on DC is controlled, and how this affects downstream immune responses. Materials and methods: Human monocyte derived DC was treated with intestine-associated molecules and the effect on DC maturation and avb8 expression was measured by flow cytometry. Results: The TLR ligand LPS upregulated expression of the TGF-b activating integrin avb8 on human DC in a dose-dependent manner. This upregulation appeared to correlate with the activation status of the DC; however, if DC were treated with gut-associated molecules that dampen activation, LPS was still able to cause upregulation of the integrin. Conclusions: Integrin avb8 upregulation on DCs by danger signals in the gut may contribute to constitutive suppression of non-specific immune responses, or enhancement of Th17 responses depending on the immune milieu. We are currently determining the functional significance of this integrin upregulation in controlling gut immune responses, which may prove to be a potential pathway for modulation of intestinal immunity.

P1106 Tumour necrosis factor alpha in non-inflammatory bowel disease enterocutaneous fistulas prospective study G. Rahbour, H. O. Al-Hassi, A. L. Hart, M. R. Ullah, S. M. Gabe, S. C. Knight, J. Warusavitarne & C. J. Vaizey Antigen Presentation Research Group, Imperial College, St. Mark’s Hospital and Academic Institute, London, UK Purpose/Objective: The aim of this study is to assess the inflammatory activity, with a particular emphasis on tumour necrosis factor alpha (TNF-a) of non-inflammatory bowel disease enterocutaneous fistula (non-IBD ECF) when compared with IBD ECF and control small bowel terminal ileum tissue. If this study can show the presence of TNF-a in the fistula tract then there would be a potential for a novel therapy for patients with persistent ECF not associated with IBD. This would be an alternative option and benefit an already surgically challenging group of patients associated with a high morbidity and mortality where it is deemed conservative medical management has failed. Materials and methods: Research ethics approval was granted for this study. Tissue biopsies were obtained from ECF at operation from nonIBD patients and from the terminal ileum in normal colonoscopy control patients. After 24 h incubation, intra cellular staining was performed using monensin to assess the on-going intra cellular production of cytokines. Data was acquired using FACS Canto II.


Abstracts 537 Further compensation and analysis of list-mode data were carried out subsequently using Win List software. Results: Results are available on ten non-IBD ECF and four control patients. The student t-test for non-paired data was used. Production of TNF- a by dendritic cells from non-IBD ECF tissue was significantly higher than that from control terminal ileum tissue (P = 0.0008). All viable cells from non-IBD ECF tissue also produced significantly higher level of TNF- a compared with control tissue (P = 0.01). There were no significant differences in the production of TNF alpha, IL17a and IFN-g by T cells in non-IBD ECF compared with cells from control terminal ileum tissue. Ongoing production of IFN-g gamma was not significantly different in all viable cells form non-IBD ECF compared with all viable cells from control terminal ileum tissue. However, the on-going production of IL17a in all viable cells from non-IBD ECF was significantly higher compared with all viable cells from control terminal ileum tissue (P = 0.04). Conclusions: Although this work is still in progress, a trend has been found in the on-going production of cytokines in non-IBD ECF compared with control tissue. Addition of a greater number of samples may show significant differences between the two groups of patients and provide validity to this trend. This data is encouraging and may provide evidence for the potential use of anti-TNF- a agent in the treatment of non-IBD ECF.

P1107 Vagal anti-inflammatory reflex in dextran sodium sulfate (DSS)induced colitis C. Cailotto,* B. J. Olivier,* L. M. M. Costes,* J. van der Vliet,* F. Hilbers,* W. J. de Jonge* & G. E. Boeckxstaens *Academic Medical Center, Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands, Department of Gastroenterology, University Hospital Leuven, Leuven, Belgium

anism modulating the immune system. We recently established that the CAIP is endogenously activated during subtle inflammation of the small intestine in a model of postoperative ileus (POI). To what extent this neuronal pathtway is also activated during colitis remains however unknown. Therefore, we evaluated vagal activation upon DSS-induced colitis using cFos expression, a marker for neuronal activation, in the dorsal motor nucleus of the vagus nerve (DMV) at different time points of DSS exposure. Moreover, selective vagal denervation of the proximal part of the colon was performed to investigate the antiinflammatory role of the vagal innervation of the colon. Materials and methods: C57/Bl6 mice were exposed to DSS in drinking water for 7 consecutive days. Mice were sacrificed at day 1, 3 and 7. The group of mice exposed to 7 days of DSS was divided into 2 experimental groups: vagal denervated (Cx) and sham-operated. Brain was collected for cFos expression and colonic tissue was collected to determine the expression of inflammatory cytokines. Results: Mice exposed to 7 days of DSS exhibited a significant increase in the disease index activity and a decreased body weight. The inflamed colons showed an increased expression of IL-6, TNFa, and IL1b compared to control mice. Colonic inflammation did not significantly increase cFos expression in the DMV. Denervation of the proximal colon induced an increase of IL6 and IL1b transcript levels in the entire colon. Strikingly, the enhanced colonic inflammation was observed only in the distal part (non-denervated) of the colon. Conclusions: In the present study, DSS-induced colonic inflammation did not trigger endogenous activation of CAIP as described during intestinal muscularis inflammation in POI. Denervation of the vagus nerve (innervating the proximal part of the colon) enhanced colonic inflammation, confirming the role of the cholinergic anti-inflammatory pathway in mucosal immune homeostasis. Strikingly, removal of this neuronal pathway affects the innate immune response of the distal colon rather than the proximal colon. Our data demonstrated that neuronal circuitry underlying the cholinergic anti-inflammatory mechanism in colitis differs from the CAIP activated during POI.

Purpose/Objective: The cholinergic anti-inflammatory pathway (CAIP) is a recently identified endogenous anti-inflammatory mech-



Poster Session: Malaria P1108 BTLA expression dampens innate as well as adaptive immunity against experimental malaria T. Jacobs,* C. Steeg,* K. Pfeffer, T. L. Murphy,à K. M. Murphy,à J. Langhorne§ & G. Adler* *Immunology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany, Institute of Medical Microbiology, Heinrich Heine University, Du¨sseldorf, Germany, àDepartment of Pathology and Immunology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO, USA, §Division of Parasitology, Medical Research Council National Institute for Medical Research, London, UK Purpose/Objective: The activation of T cells during priming but also their later function is modulated by numerous surface receptors. Activation requires signals from the TCR and costimulatory signals, which determine whether antigen recognition leads to full activation or anergy. In contrast coinhibitory receptors expressed by T cells mediate the regulation of immune responses and play a pivotal role in the maintenance of peripheral tolerance. By integrating positive and negative signals excessive activation of T cells is prevented. During malaria T cells express different coinhibitory receptors, which might protect the host from an overreaction of the immune response but might contribute to an ineffective clearance of the pathogen. Recently, BTLA (B and T lymphocyte attenuator, CD272) was described as a novel negative costimulatory receptor. BTLA is predominantly expressed on T and B cells and dampens T cell activation. In this study, we analyzed the function of BTLA during experimental malaria infection. Materials and methods: To study the function of BTLA we employed a mouse model of blood stage malaria using P. yoelii NL. This infection causes a high parasitemia in infected mice that is cleared within 3 weeks. By using BTLA-deficient and HVEM-deficient mice the respective function of these molecules were analysed. Using mixed bone-marrow chimera the function of BTLA on different immune cell populations was further elucidated. Results: BTLA expression restricts the protective immune response on different levels. BTLA::HVEM interaction regulates the number and function of the responding T cells but in addition BTLA expression also dampens B cells and phagocytic cells. However, in contrast to the manipulation of the CTLA 4 pathway, where already a transient blockade is accompanied by a massive inflammation, the lower parasitemia of BTLA- and HVEM-deficient mice is not accompanied by any signs of pathology. Conclusions: In contrast to other negative costimulatory pathways, the HVEM::BTLA pathway dampens the function of the innate as well as of the adaptive immune system during experimental malaria and thus manipulation of this pathway is an attractive target for therapeutic interventions.

P1109 CTLA-4 expression on T effector and T regulatory cells in experimental malaria C. Steeg,* T. Sparwasser & T. Jacobs* *Immunology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany, TWINCORE/Centre for Experimental and Clinical Infection Research, Institute of Infection Immunology, Hannover, Germany Purpose/Objective: In the course of an infection with Plasmodium ssp. the parasites develop via two stages: at first in the liver followed by replication in the blood. In the first phase only few hepatocytes are infected and the antigenic load is low, but in the subsequent blood

stage many erythrocytes are infected which leads to a vast amount of antigenic material delivered to the immune system. This induces a strong activation of T cells followed by the production of proinflammatory cytokines that contribute to the severe complication of malaria, the cerebral Malaria (CM). Thus the immune response has to be tightly regulated to achieve on the one hand protection against the parasite but on the other hand prevent immunopathology. CTLA-4 is one of the most effective regulators of T cells and is expressed on Treg and T effector cells. We investigated CTLA-4 expression on T cells in a mouse model for the blood stage of protection against malaria and in a model for CM. Materials and methods: We infected C57Bl/6 mice and DEREG mice (DEPletion of REGulatory T cells) with Plasmodium yoelii or Plasmodium berghei ANKA and analysed spleen cells during the development of the disease. Results: In the course of infection with P. yoelii the ratio of Treg to CD4+ T cells declines but the number of CTLA-4 positive T cells increases. These CTLA-4+ cells produce IFN-g whereas the Foxp3+ cells produce IL-10. After depletion of Treg in DEREG mice with Diphtheria toxin the number of CTLA-4+ Foxp3- T cells increases rapidly and IFN-g production of CD4+ T cells is also increased whereas TNF-a production is unchanged. However this has no influence on the parasitemia. When we infected mice with P. berghei ANKA we observed a massive induction of CTLA-4 on CD4+ as well as on CD8+ with up to 30% CTLA-4 positive cells. The CD4+ CTLA-4+ cells are still functional and produce IFN-g but no TNF. Conclusions: Our data suggest that the rapid induction of CTLA-4 on T effector cells during an infection with Plamodium is a highly dynamic counterregulatory pathway that protects the host from pathology.

P1110 P. berghei sporozoite challenge of vaccinated BALB/c mice lead to induction of humoral immunity and improved CD8+ T cell memory S. Tartz,* C. Deschermeier, V. Heussler,à P. Sebo,§ B. Fleischer* & T. Jacobs* *Immunology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany, Parasitology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany, àInstitute of Cell Biology, University of Bern, Bern, Switzerland, §Cell and Molecular Microbiology Division, Czech Academy of Sciences, Prague, Czech Republic Purpose/Objective: Protection against malaria can be achieved by activation of a strong CD8+ T cell response against the Plasmodium circumsporozoite protein (CSP). However, most subunit vaccines fail to induce a sufficient memory response. In the present study we analysed the impact of a sporozoite infection after immunization on the development of long-lasting protective immunity. Materials and methods: BALB/c mice were immunized by a heterologous prime/boost regimen against P. berghei CSP. A recombinant Salmonella typhimurium strain and a Bordetella pertussis adenylate cyclase toxoid fusion molecule were used as vaccine carriers. This immunization induces a strong CD8+ T cell response and complete protection upon sporozoite challenge, which is however, only short lived. Results: A sporozoite challenge after immunization led to prolonged protective immunity. Repeated challenge infections induced sporozoite specific antibodies that showed protective capacity. On the other hand the CSP-specific CD8+ T cell response was not substantially boosted by sporozoite infections and the magnitude of the CD8+ T cell memory seemed not to correlate with protection. The phenotype of these memory T cells was comparable; however, CSP-specific memory CD8+ T cells of challenged mice displayed stronger cytotoxic activity than


Abstracts 539 memory T cells of only immunized mice. Sterile protection was abrogated when CD8+ T cells were depleted whereas CD4+ T cells played a minor role. Conclusions: Based on these data we suggest that the increase in protective immunity observed after immunization and subsequent challenge infection is the sum of different antiparasitic mechanisms including CD8+ and CD4+ T cells as well as neutralizing antibodies, with CD8+ effector-memory T cells playing the major role. Our results indicate that a vaccine which induces a short-lived liver-stage specific immunity that prevents disease within a certain time span would allow the induction of naturally acquired immunity upon repeated sporozoite infections.

P1111 Pathogenesis and therapy of malaria-associated acute respiratory distress syndrome P. Van den Steen, K. Deroost, N. Geurts, N. Lays, S. Verhenne, H. Heremans, J. Van Damme & G. Opdenakker Rega Institute, Katholieke Universiteit Leuven, Leuven, Belgium Purpose/Objective: Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a lethal complication of malaria. No efficient treatment is available for this complication and its pathogenesis remains poorly understood. We studied the disease mechanisms and evaluated candidate treatments in a new mouse model. Materials and methods: We established a novel mouse model of MAARDS by infection with Plasmodium berghei NK65 (PbNK65). Histology, FACS analysis, RT-PCR, bronchoalveolar lavages, cell depletions and anti-inflammatory treatment with dexamethasone were performed. A new method for the extraction and quantification of hemozoin (malaria pigment) in tissues was optimized. Results: PbNK65 infection resulted in leukocyte accumulation, extensive edema and hemorrhage in the lungs. The pulmonary expression of several cytokines and chemokines was increased to a higher level than in mice infected with P. chabaudi AS, a parasite strain which does not cause MA-ARDS. CD8+ T lymphocytes were shown to be pathogenic and high doses of dexamethasone (DEX) blocked MA-ARDS through inhibition of lymphocyte proliferation and expression of chemoattractants for monocytes/macrophages, even when given after appearance of the pathology. On tissue sections, we noticed the presence of hemozoin or malaria pigment in the lungs. In view of the important inflammatory potential of this hemoglobin degradation product, we optimized a novel method to quantify hemozoin in tissues and measured the Hz content in different organs of mice infected with parasites of different pathogenicity. Significantly higher amounts of hemozoin were found in the lungs of mice with MA-ARDS. Furthermore, total Hz contents (including liver and spleen levels) were significantly higher in mice infected with P. berghei NK65 than in mice infected with P. chabaudi AS, despite of similar peripheral parasitemia levels. Further investigations to clarify the role of hemozoin in MA-ARDS are currently underway. Conclusions: Our new mouse model for MA-ARDS has many similarities to human MA-ARDS. Inflammation has a prominent role in the disease, and anti-inflammatory treatment appears highly

effective. High amounts of hemozoin were detected in the lungs and may contribute to the pathogenesis.

P1112 Abstract withdrawn.

P1113 The immuno-stimulatory and protective effect of the novel nanoparticle-coated Plasmodium yoelii merozoite surface protein (PyMSP-1) M. S. Cherif,* M. N. Shuaibu, T. Kurosaki,à Y. Kodama,à H. Sasaki,à K. Yui,§ G. K. Helegbe, M. Kikuchi, T. Yanagi– & K. Hirayama *Immunogenetics, Institut National de Sante´ Publique, Universite´ de Conakry (Guinea) & Institute of Tropical Medicine, Nagasaki, Japan, Immunogenetics, Institute of Tropical Medicine, Nagasaki, Japan, à Hopital Pharmacy Department, Nagasaki University, Nagasaki, Japan, § Division of Immunology, Department of Molecular Microbiology and Immunology, Nagasaki University, Nagasaki, Japan, –Institute of Tropical Medicine, Animal Research Center for Tropical Medicine, Nagasaki, Japan Purpose/Objective: In malaria DNA vaccine development, for induction of an efficient and protective immunity against parasite, there exists a critical need for additional delivery vehicles. We analyzed the immuno-stimulatory effect of PEI/c-PGA nanoparticle (NP)coated PyMSP-1 plasmid and investigated its in vivo stimulatory effect on dendritic cells. Materials and methods: Groups of C57BL/6 mice were immunized either with 100 lg/mouse of nanoparticle-coated plasmid (pVR1020MSP-1/PEI/c-PGA), naked (pVR1020-MSP-1) or coated control group (pVR1020/PEI/c-PGA) using different routes of administration. Mice were prime-immunized at day 0 and subsequently, two boosters given with 3 weeks intervals. Two weeks after the last boost, specific IgG and their subtype’s titres measured by ELISA. Cytokine (IL-12 and IFN-c) levels were measured in the supernatants of antigen stimulated spleen cells and sera from immunized mice using procarta-immunoassays kit. Flow cytometric analysis of various activated DC markers was also carried out. Results: Protection against P. yoelii lethal challenge, specific IgG and its subtypes and INF-c producing cell number were observed to be significantly higher in the coated-MSP-1 group than the naked group. Also, there were a significantly increased proportion of activated DCs and their elevated CD40 expression in the NP-coated immunized group as compared to naked plasmid. In the coated group, the costimulatory molecule CD80 was significantly increased when immunized subcutaneously, while CD86 molecule was increased when immunized intraperitoneally. In vivo and ex-vivo production of IL-12 were observed in the sera and the spleen cells stimulated with recombinant MSP-1. Conclusions: These data indicates that nanoparticle-coated PyMSP-1 DNA vaccine protected mice and induced activated DCs either with CD80 or CD86, and the activated DCs produced IL-12 when stimulated by rMSP-1.



Poster Session: Metabolic Disease and Diabetes P1115 B cell over-expression of Fc gamma receptor IIb inhibits the development of atherosclerosis A. P. Sage,* O. Herbin, D. Murphy,* L. Masters,* L. Baker,* J. Harrison,* M. R. Clatworthy,à K. Smithà & Z. Mallat* *Division of Cardiovascular Medicine, University of Cambridge, Cambridge, UK, Inserm, Centre de Recherche Cardiovasculaire, Paris, France, àMedicine, University of Cambridge, Cambridge, UK Purpose/Objective: The development of atherosclerosis is significantly regulated by autoimmune and inflammatory processes involving both innate and adaptive immune systems, and is accelerated in the presence of other autoimmune diseases. The inhibitory FcyRIIb has previously been associated with regulation of atherosclerosis development, but the cells types mediating these effects are unknown. Thus, we investigated the effect of B cell-specific FcyRIIbover-expression on the development of atherosclerosis in mice. Materials and methods: Atherosclerosis-susceptible, low density lipoprotein receptor -/- (ldlr) mice were irradiated and transplanted with bone marrow from B cell FcyRIIb-transgenic (BTG) mice or nontransgenic littermate controls, then fed a high-fat diet for 6 weeks. Atherosclerosis burden and plaque immune cell content was analysed histologically. Immune cell phenotypes were analysed by flow cytometry and cytokine production by intracellular flow cytometry and ELISA. Results: Compared to normal chow, a 6 week high-fat diet, known to induce CD4+ T cell activation and increased anti-oxidized lipid antibodies, significantly increased MHC class II levels in splenic and lymph node B cells as well as increased levels of BAFF in serum and spleen, suggesting B cell activation in response to high fat feeding. In ldlr chimeras, there were no differences between control and BTG groups in total cholesterol levels, body weight or total blood cell counts. FcyRIIb overexpression suppressed purified B cell proliferation in response to anti-IgM IgG but not F’ab or LPS, and did not affect splenic B cell MHCII or CD40 expression. The level of atherosclerosis at the aortic root was significantly reduced in ldlr/BTG mice compared to littermate controls. The reduced atherosclerosis in ldlr/BTG mice was associated with reduced CD3+ T cell levels within plaques, reduced ex-vivo production of IFN-y by both CD4+ and CD8+ T cells, and reduced levels of CD62Llo CD44hi CD4+ T cells in the spleen, consistent with the previously proposed role of B cells in promoting T cell activation and atheroprogression. Conclusions: FcyRIIb expression on B cells leads to reduced atherosclerosis, potentially via suppression of pro-atherogenic T cell activation and cytokine production.

P1118 Galectin-3 deficiency preserves pancreatic islets function in basal conditions and under cytotoxic stimuli I. Nikolic,* T. Saksida,* I. Stojanovic,* M. L. Lukic & S. StosicGrujicic* *Immunology, Institute for Biological Research ‘Sinisa Stankovic’, University of Belgrade, Belgrade, Serbia, Center for Molecular Medicine, Faculty of Medicine, University of Kragujevac, Kragujevac, Serbia Purpose/Objective: Galectin-3 (Gal-3) is b-galactoside-binding lectin expressed in variety of tissues and cell types and possesses diverse functions, including promotion of inflammatory response and triggering apoptosis. Since it was established that Gal-3-deficient (Gal-3-/-) mice are resistant to streptozotocin-induced diabetes and the precise role of Gal-3 in pancreatic islets activity has not been examined so far, our aim was to explore the influence of Gal-3 absence on insulin secretion and apoptosis of pancreatic islets.

Materials and methods: We exposed mouse pancreatic islets (isolated from Gal-3-/- and WT (C57BL/6) mice by collagenase digestion) to proinflammatory cytokines (IL-1b+IFN-c+TNF-a, 10 ng/ml each) or left them untreated for 24 or 96 h and analysed their survival and function. After incubation, islet apoptosis was measured by histoneDNA ELISA. In order to assess effect of Gal-3 deficiency on insulin secretion, we performed in vitro insulin release assay. Isolated islets were first treated with low glucose (1.67 mM) for 1 h and then with high glucose (16.7 mM) and released insulin was measured by ELISA. Results: Deficiency of Gal-3 promoted better survival of pancreatic islets when they were cultured in basal conditions for 96 h. In line with that, Gal-3-/- pancreatic islets preserved intact insulin secretion compared to WT islets. What is more, Gal-3 deficiency protected pancreatic islets from cytokine-induced apoptosis. Also, after 24 hlong cytotoxic stimulation, Gal-3-/- pancreatic islets retained normal insulin secretion while WT islets had lower secretion. Similarly, Gal3-/- islets had better response to glucose-stimulated insulin release in basal as well in inflammatory conditions after 24 h of culture. Conclusions: This study demonstrates that Gal-3 deficiency protects islets from cytokine-induced apoptosis and preserves their function therefore implicating the role of Gal-3 in pancreatic islets apoptosis during inflammation that occurs in diabetes pathogenesis. This study was supported by the Serbian Ministry of Education and Science (Grants No.: 173013 and 175069).

P1120 Glucose tolerance status and hsCRP in a large population-based survey Y. Tutuncu,* I. Satman,* S. Gedik,* B. Canbaz,* F. Turker,* N. Dinccag,* K. Karsidag,* A. Telci, S. Genc, B. Omer & On behalf of TURDEP-II Study Group *Faculty of Medicine Internal Medicine Endocrinology & Metabolism, Istanbul University, Istanul, Turkey, Faculty of Medicine Clinical Biochemistry, Istanbul University, Istanul, Turkey Purpose/Objective: To determine whether high sensitive C-reactive protein (hsCRP) levels differ according to glucose tolerance status. Materials and methods: Data derived from recently completed population-based survey of diabetes, obesity, hypertension and endocrine disease epidemiology in Turkish adult population (n = 26 499), TURDEP-II. Results: In female hsCRP was significantly higher than in male (mean ± SEM 3.95 ± 0.05 versus 3.53 ± 0.09 mg/dl, p Univariate analysis of variance corrected for age, gender, urban/rural, region, BMI, waist, sBP, dBP, HDL-c, non-HDL-c, 25(OH)D Vit, PTH, creatinine, TSH and FT4 revealed that hsCRP significantly differ across glucose tolerance status pmellitus (DM) and known DM groups [mean ± SEM (mg/dl)]: 4.13 ± 0.25, P = 0.023; 4.49 ± 0.33, P = 0.006; 5.65 ± 0.35, P. Conclusions: Based on our study results, mean levels of hsCRP increase from NGT through new DM categories. The fact may confirm that any abnormality of the glucose tolerance (pre-DM and DM) is associated with a low-grade inflammatory process. However, in established DM the inflammatory process may not be as important as in the earlier metabolic derangements. Turkish Scientific and Technical Research Council (TUBITAK; Project No: 106S166).


Abstracts 541 P1121 High sensitive C-Reactive protein (hs-CRP) and its correlation with angiographic severity of coronary artery disease: an Indian cohort study S. Das,* S. K. Gupta,* P. C. Ray* & G. M. P. *Department of Biochemistry, Maulana Azad Medical College, New Delhi, India, Department of Cardiology, G. B. Pant Hospital, New Delhi, India Purpose/Objective: The association between high sensitive C-reactive protein (hs-CRP) levels and the severity of coronary artery disease (CAD) is highly debated. There are very few studies assessing the role of hs-CRP levels with increasing severity of CAD. The aim of our study was to study the correlation of hs-CRP levels with angiographic clinical vessel score in an Indian population. Materials and methods: Hundred patients of angiographically proven CAD were studied of which 50 patients were of stable angina (Group I), 50 patients of acute coronary syndrome (Group II) (including 35 patients of unstable angina and 15 patients of MI) from a tertiary health center, New Delhi and a third group comprising of 50 age and sex matched healthy controls were studied over a period of 1 year. The hs-CRP levels were measured by ELISA technique and angiographic clinical vessel scoring was done for all patients. Results: The mean age of the patients was 49 ± 8.8 years (84% men, 16% women). THe mean hs-CRP levels for stable angina (Group I) (6.04 ± 2.51 mg/l) and acute coronary syndrome (Group II) (8.11 ± 2.84 mg/l) were significantly higher in CAD patients than in controls (1.54 ± 1.27 mg/l, P < 0.001). High hs-CRP levels were correlated with higher vessel scores indicating a more severe CAD (r = 0.735, P < 0.001). Conclusions: Significant correlations were found between the hs-CRP levels and the angiographic clinical vessel score. High hs-CRP levels have a correlation with the increasing disease severity in CAD patients.

P1122 Human a1-antitrypsin Blocks Heat Shock Protein 70 (HSP70)Induced Inflammation in Pancreatic Islets D. O. Ochayon, M. M. Mizrahi, P. C. Cal, B. B. Baranovski, G. S. Shahaf & E. L. Lewis Clinical Biochemistry, Ben Gurion University of the Negev, Beer-Sheva, Israel Purpose/Objective: Introduction. Heat shock protein 70 (HSP70) is an abundant chaperone and an endogenous mediator of cell damage. HSP70 enhances inflammation and promotes antigen presentation and subsequent T cell proliferation via membrane CD91. These activities may take part in islet destruction during autoimmune diabetes and during islet transplant rejection. Indeed, circulating HSP70 has been observed in patients with type-1 diabetes. The anti-inflammatory acute phase protein a1-antitrypsin (AAT) promotes islet survival in a transplantation model and in an autoimmune diabetes model. Its protective mechanism of action in these processes is unknown. Aim. Examine whether the protective activity of AAT is related to binding and inactivation of HSP70. Materials and methods: Peritoneal macrophages, bone marrow derived dendritic cells and pancreatic islets were evaluated for inflammatory responses under stimulation with polymyxin B treated recombinant HSP70 (rHSP70) in the presence or absence of clinicalgrade human AAT. Islet function was evaluated by insulin secretion. OT-I spleen cells were incubated with OVA peptide and HSP70 in the presence or absence of AAT. The effect of AAT on IL-1b/IFNc-induced surface levels of CD91 was evaluated. Binding between HSP70 to AAT was determined by direct ELISA.

Results: In all tested cell cultures, AAT diminished rHSP70-induced inflammatory responses. In HSP70-stimulated islets, IL-1b release was reduced 2.43 ± 2.01-fold, IFNc 1.73 ± 1.06-fold and IL-6 1.27 ± 1.02fold. AAT restored rHSP70-mediated disrupted islet insulin secretion. OVA/HSP70 introduced to OT-I spleen cells elevated the number of CD3+ CD8+ T-cells 2.28-fold, compared to cells incubated with OVA alone, and 2.79-fold compared to OT-I spleen cells incubated with HSP70 plus AAT. Under inflammatory conditions, surface CD91 levels were reduced in AAT-treated peritoneal macrophages 2.05 ± 0.33-fold compared to untreated macrophages. Finally, we demonstrate that rHSP70 directly binds to human AAT in a concentration-dependent manner. Conclusions: Our findings suggest that AAT is a natural regulator of inflammatory responses mediated by HSP70, and a possible target during clinical AAT treatment.

P1123 Interleukin-1 and leptin synergistically enhance matrix metalloproteinase expression in human gingival fibroblasts R. C. Williams,* A. D. Rowan, P. M. Preshaw* & J. J. Taylor* *Centre for Oral Health Research, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK Purpose/Objective: The adipokine leptin is elevated in obesity and type 2 diabetes, conditions which are associated with increased susceptibility to chronic inflammatory diseases such as periodontitis. Loss of tissue integrity in inflammatory diseases is caused by extracellular matrix (ECM) degradation by proteases, such as the matrix metalloproteinases (MMPs). Gingival fibroblasts are critical in regulating tissue homeostasis in the periodontium and synthesise MMPs in response to cytokines but the effect of leptin on these cells is unknown. Our aim was to investigate the effect of leptin in regulating MMP expression in primary human gingival fibroblasts (HGFs). Materials and methods: HGFs were isolated from gingival tissue obtained during canine exposure surgery. HGFs (between passage 5 9) were stimulated under serum-free conditions with leptin (10 lg/ ml), interleukin (IL)-1a (0.05 ng/ml) and oncostatin M (OSM) (5 ng/ ml) for 24 h. MMP mRNA expression was assessed by real-time and conventional RT-PCR. HGF proliferation was determined using a MTS-based assay. Results: Leptin significantly increased the expression of the collagenase MMP-1 in a dose-dependent manner, but did not affect the expression of the gelatinase MMP-2, compared to unstimulated HGFs. MMP-3, a stromelysin known to activate MMP-1, was expressed in both leptin-stimulated and unstimulated HGFs. Interestingly, leptin+IL-1 synergistically increased both MMP-1 and MMP-3 expression in HGF cultures above that observed after IL-1 or leptin stimulation alone. Leptin+IL-1 had no effect on MMP-2 expression. It is unlikely that the synergistic upregulation of MMP expression was due to increased HGF proliferation as leptin+IL-1 stimulation did not increase HGF proliferation above that of cells stimulated with leptin alone. Leptin+OSM did not synergistically upregulate MMP-1, MMP2 or MMP-3 expression in HGFs. Conclusions: We demonstrate that HGFs respond to leptin by upregulating MMP-1 expression. The synergy demonstrated between leptin and IL-1 highlights the importance of combinations of cytokines in regulating MMP expression. This pathway may be of mechanistic relevance to inflammatory tissue damage as occurs in periodontitis, and underpin the cross-susceptibility between inflammatory diseases associated with obesity.


542 Poster Session: Myeloid Cell Development P1124 Investigation of possible relations of SDF-1 and CXCR-4 polymorphisms and CD55 and CD59 markers with etiopathogenesis and prognosis in Type 2 diabetes B. Aydin,* E. Coskunpinar, S. Gazioglu,* B. Agachan, G. Deniz,* M. T. Yilmazà & A. O. Gurol* *Immunology, Institute of Experimental Medicine (Detae), Istanbul, Turkey, Molecular Medicine, Institute of Experimental Medicine (Detae), Istanbul, Turkey, àInternal Medicine, Istanbul Faculty of Medicine, Istanbul, Turkey Purpose/Objective: Diabetes is a metabolic hyperglycemic disease resulting from impaired insulin production, secretion or insulin resistance. Complement regulators and chemokines are pivotal in pathogenesis. In the context of complications related to T2DM, CD55, CD59 expressions, and SDF-1, CXCR-4 polymorphisms were investigated. Materials and methods: Seventy-five patients with T2DM and 73 healthy subjects were enrolled in the study. CD55 and CD59 expressions were evaluated with flow cytometry. DNA was isolated from heperinized periferic blood samples. Real-time PCR assay was carried out using LightSnip (rs17881575 Roche-Germany) for SDF-1 and LightSnip (rs2680880 Roche-Germany) for CXCR-4. Results: CD55 and CD59 expressions in patients with T2DM nephropathy, retinopathy and cardiovascular disease were significantly lower than healthy subjects. SDF-1 genotype and allele distributions between groups were not different. CXCR-4 genotype distribution wasn’t different between groups, while a low significance was observed in allel distributions. CXCR-4 T allele was increased in patients, with 1.6-fold risk in terms of disease. Although SDF-1 genotypes in nephropathics did not show any difference, a significant difference was detected for CXCR-4 genotypes. CXCR-4 A allele carriers had decreased nephropathy, while 2-fold high nephropathy frequency was observed in the carriers of CXCR-4 T allele. The nephropathy risk increases 10-fold in CXCR-4 TT genotype carriers. A significant difference was observed in SDF-1 genotypes associated with retinopathy presence. Our results show that all SDF-1 CC genotype carriers have retinopathy, and CC genotype is effective in retinopathy development, however no significance was found for CXCR-4 genotypes. For the presence of cardiovascular disease, a significant difference was observed for SDF-1 genotypes. Increased cardiovascular risk of 5- and 1.9-fold in SDF-1 T and CXCR-4 T allele carriers respectively was observed. Conclusions: This is the first study investigating together CD55 and CD59 expressions, and SDF-1 and CXCR-4 polymorphisms in T2DM. In conclusion we suggest that CD55 and CD59 have a predictive importance in the process of the disease, and the polymorphism of CXCR-4 gene promoter site (rs2680880) plays a role in the susceptibility to T2DM. Further studies with an increased number of subjects are needed in this field.

P1125 Low frequency of GITR+ cells in ex vivo and in vitro expanded Tregs from type-1 diabetic patients C. Xufre´,* M. Costa,* C. Roura-Mir,* E. Codina-Busqueta,* L. Usero,* E. Pizarro, G. Obiols,à D. Jaraquemada* & M. Martı´* *Institut de Biotecnologia i Biomedicina, Biologıá Celular Fisiologıá e Inmunologıá, Barcelona, Spain, Hospital de Mataro´, Servei d¢Endocrinologia, Mataro´, Spain, àHospital de la Vall Hebron, Servei de Nutricio´ i Endocrinologia, Barcelona, Spain Purpose/Objective: Reported alterations in regulatory T cells (Tregs) in patients with several autoimmune disorders led us to a revision of the phenotypical features of type-1 diabetic patients’ peripheral CD4+ CD25hi Tregs compared to controls.

Materials and methods: A fine cytometric analysis was designed to phenotypically characterise Tregs from type-1 diabetic (T1D) patients and controls using a staining panel including FOXP3, CTLA-4, GITR and CD127. PBMCs and sorted Tregs were cultured with different stimulus: media alone, soluble OKT3 and anti-CD3/anti-CD28 coated beads. Suppression assays of in vitro expanded Treg cells were performed. Results: The frequency of peripheral Treg cells was similar between T1D patients and controls. However, the yield of sorted Tregs was significantly lower in patients than in controls (P < 0.003). When comparing Treg phenotype between samples, the only difference concerned GITR. A significant decrease of GITR+ cells within the Treg population (P < 0.0009) and to a lesser extent in effector (P < 0.02) populations, was observed for T1D compared to controls. Since GITR is involved in costimulation, its expression was analyzed in different conditions of T cell activation. Differences were only observed for T1D Tregs versus controls when faced to suboptimal stimulation i.e, soluble anti-CD3 (P < 0.05) or medium alone (P < 0.05), but not in the presence of anti-CD3/anti-CD28 coated beads. Despite this reduced expression, expanded T1D Tregs -mediated suppression was as efficient as that mediated by their control counterparts. Conclusions: Our results suggested that GITR is a Treg-marker that would be primarily involved in Treg maintenance rather than in its suppressor function.

P1126 Mice deficient for the autoimmune regulator, Aire, display altered T cell responses against 21-hydroxylase, the major autoantigen in autoimmune Addison’s disease E. Bratland, A. Hellesen & E. S. Husebye Institute of Medicine, University of Bergen, Bergen, Norway Purpose/Objective: The autoimmune regulator, Aire, is a transcriptional regulator playing a critical role in central tolerance by promoting the display of tissue specific self-antigens. In humans mutations in the gene encoding Aire result in the unique disorder autoimmune polyendocrine syndrome type 1 (APS-1). One of the hallmarks of APS-1 is autoimmune Addison’s disease (primary adrenocortical failure) with corresponding immune responses against 21hydroxylase (21OH), an enzyme crucial for the synthesis of steroid hormones. Mice deficient for Aire also develop spontaneous autoimmunity in several organs, but adrenal autoimmunity or anti-21OH immune responses have not yet been described. We therefore wanted to investigate the influence of Aire on immune responses in mice immunized with 21OH protein. Materials and methods: C57Bl6 mice deficient for Aire and littermate controls were immunized with 21-hydroxylase (21OH) on day 0 and boosted on day 7. All mice were sacrificed on day 14, and spleens, lymph nodes, adrenals and whole blood were collected. CD4+ and CD8+ T cells were stimulated in vitro with recombinant 21OH and a panel of 21OH-derived peptides. Cell culture supernatants were assessed for interferon-c content by ELISA. T cell mediated cytotoxicity were determined using peptide pulsed or 21OH-transfected EL4 cells as targets. Results: Broad T cell responses against multiple epitopes of 21OH could be induced in C57Bl6 mice, regardless on the presence or absence of Aire. However, in the absence of Aire, T cell responses were enhanced against certain epitopes. Furthermore, some epitopes were uniquely targeted in Aire deficient mice. Conclusions: Our findings suggest that T cell responses against 21OH can be readily induced in C57Bl6 mice, and that Aire may modulate these T cell responses.


Abstracts 543 P1128 Obesity-associated autoantibody production requires AIM for retaining the immune complex on follicular dendritic cells S. Arai & T. Miyazaki Lab of Molecular Biomedicine for Pathogenesis, Center for Disiease Biology and Integrative medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan Purpose/Objective: Recent evidence has suggested a significant correlation between obesity and autoantibody production, although its mechanism remains unknown. The aim of this study is to address a functional involvement of the apoptosis inhibitor of macrophage (AIM) in this autoimmune process. AIM is a secreted protein produced by macrophages and circulates in blood at around 10 lg/ml in both human and mouse. Interestingly, AIM is associated with pentamers of natural IgM in blood. Natural IgM is polyreactive, recognizing self-antigens, and thus is believed to be associated with autoimmunity. In addition, AIM blood levels increase with progression of obesity in mice when fed with a high-fat diet (HFD). All these observations strongly suggest that AIM/IgM complex might be involved in the development of obesity-associated autoimmunity. Materials and methods: Following experiments were performed: (1) serum IgM and IgG levels were assessed in AIM+/+, AIM-/- and TLR4-/mice during the progression of obesity, (2) we analyzed autoantibodies in obese AIM+/+ and AIM-/- mice using autoantibody array, (3) influence of AIM to the binding of IgM to Fca/mR, an IgM-specific Fc receptor expressed in the splenic follicular dendritic cells (FDCs), was tested. In addition, effect of AIM on the holding of IgM-immune complex (IgM-IC) on the FDCs was analyzed. Results: In mice fed a HFD, splenic B cells were activated through stimulation of TLR4. This response increased natural IgM production, followed by expansion of IgG autoantibodies. AIM associated with IgM, and inhibited its binding to the Fca/mR such an effect protected IgM-IC from internalization mediated by Fca/mR, and thus retained the ICs on FDCs. This supported IgM-dependent antigen-presentation to B cells leading to plasma cell development. In AIM-/- mice, although elevation of IgM in response to HFD was comparable to AIM+/+ mice, the increase of IgG autoantibodies was abrogated. Conclusions: Our results show that association of AIM with IgM interferes with the internalization of IgM-ICs via Fca/mR, resulting in prolonged retention of the ICs on the surface of FDCs. This contributes to the efficient presentation of autoantigens to germinal center B cells, leading to development of IgG autoantibody producing plasma cells. Thus, suppression of AIM may have therapeutic potential for preventing obesity-associated autoimmune diseases.

P1129 Perivascular adipocytes and signaling through toll-like receptors: role in the pathophysiology of atherosclerosis C. Benaiges,* X. Garcia-Moll, E. Mateus,* C. Munõz,à E. Canto´,* E. Pe´rez,* R. Leta, E. Moga,* S. Vidal* & C. Juarez* *Immunology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, Cardiology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, à Cardiac Surgery, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Purpose/Objective: Perivascular adipose tissue has emerged as a critical regulator of vascular function implicated in the pathophysiology of atherosclerosis. Arteries are surrounded by a significant amount of perivascular adipose tissue that is an active endocrine and paracrine source of inflammatory cytokines and adipokines. Moreover, adipocytes express toll-like receptors (TLRs) to respond to lipids and other self and nonself molecules activating proinflammatory pathways. We analyzed TLR/JAK-STAT transduction pathways in adipose tissue from patients with atherosclerosis to unravel the mechanisms implicated in the pathophysiology of atherosclerosis.

Materials and methods: Perivascular and subcutaneous adipose tissues were obtained from control and atherosclerosis patients. Explants of adipose tissue were cultured in presence of TLR ligands and Affymetrix Expression Arrays were performed to analyze changes in gene expression. Western blot was carried out to analyze expression and activation levels of STATs. Immunohistochemistry was used to identify cell types and STATs’ activation in adipose tissue. Secretion of adipokines (Adiponectin, Resistin and Leptin) and cytokines (IL-6, IL10, TNF and MCP-1) in supernatants were quantified by ELISA. Results: Perivascular adipose tissue from atherosclerosis patients showed a higher expression of genes implicated in immune processes and lower expression of genes implicated in metabolism. Accordingly, perivascular adipose tissue from atherosclerosis patients showed B and T lymphocytes infiltrates. TLR stimulation of adipose tissue upregulated the expression and activation levels of STATs. While STAT3 was activated at the basal level, STAT1 was activated in tissues previously stimulated with TLR ligands. Cytokine secretion, especially IL-6, increased in supernatants from explants stimulated with TLR ligands. No significant changes in adipokine secretion were observed after TLR stimulation. Conclusions: We identified alterations in the TLR/JAK-STAT signaling pathways in adipose tissue that would help to explain the deleterious effects of lipids and other TLR ligands in the pathophysiology of atherosclerosis. The results will be very useful to understand the role played by adipose tissue in atherosclerosis and to design therapeutic approaches to control this inflammatory process.

P1130 Proprotein convertase FURIN is overexpressed in human atherosclerotic plaque leukocytes and associates with vascular stenosis H. Turpeinen,* E. Raitoharju, A. Oksanen,* N. Oksala,à M. Levula, L. P. Lyytikaïnen, M. Ka¨ho¨nen,§ M. Pelto-Huikko,– T. Lehtima¨ki & M. Pesu* *Institute of Biomedical Technology, University of Tampere, Tampere, Finland, Department of Clinical Chemistry, University of Tampere, Tampere, Finland, àDivision of Vascular Surgery, Department of Surgery, Tampere University Hospital, Tampere, Finland, §Department of Clinical Physiology, Tampere University Hospital, University of Tampere, Tampere, Finland, –Department for Developmental Biology, University of Tampere, Tampere, Finland Purpose/Objective: Atherosclerosis is a chronic, multifactorial process, where both environmental and genetic factors have critical roles. Proprotein convertase (PCSKs) proteases cleave and convert immature target proteins into a biologically active form. Many PCSK target proteins control various aspects of atherosclerosis thus making also PCSKs as key determinants in atherogenesis. Previously, 4 PCSKs (PCSK5, PCSK9, FURIN, MBTPS1) have been linked to regulation of cholesterol metabolism and plaque inflammation in experimental models. Materials and methods: We analyzed the expression of PCSK genes in human atherosclerotic plaques and pinpointed the cell types involved. Immunohistochemistry, microarray and Q-PCR were used. To further dissect the role of FURIN in atherosclerosis we genotyped 7 haplotype tagging SNPs (htSNPs) in the FURIN gene region in three independent collections of atherosclerosis patients and controls. We also addressed whether the FURIN expression in the artery wall is genetically regulated by analyzing more than 500 000 SNPs along the genome and reciprocally how the FURIN htSNPs regulate the expression of all the other genes in the artery wall. Results: FURIN, but not other PCSK genes, was shown to be universally statistically significantly over-expressed in the plaques of different arterial beds. FURIN dysregulation concentrates in the inflammatory plaque cells, such as CD3+ and CD20+ lymphocytes


544 Poster Session: Myeloid Cell Development and CD68+ macrophages. FURIN expression analysis in peripheral blood cells revealed that FURIN was induced in TCR activated CD4+ T cells, as well as upon TLR4 mediated activation of CD14+ myeloid cells. Analyzing the htSNPs we found that the FURIN SNP rs4932370 associates with the severity of atherosclerosis. SNP genotype expression correlation analyses revealed that the expression of the ITGBP3 gene is regulated by the FURIN SNP rs12903530. However, after correcting for multiple comparisons no SNPs were found to associate statistically significantly with the FURIN expression in atherosclerotic plaques. Conclusions: Taken this all together makes FURIN as one of the most interesting candidate genes important in regulation of atherogenesis and the plaque inflammation. However, the FURIN expression is not directly regulated by the polymorphisms and further investigations are needed to decode the factors that contribute to the FURIN regulation in the human atherosclerotic plaques

P1131 Resistin is a risk factor for myocardial infarction and acute coronary syndrome in the general population A. Cabrera,* D. Almeida, J. J. Alemań,à A. Gonza´lez,à B. Brito,à S. Domıńguezà & M. C. Rodrı´guezà *Hospital Universitario NS Candelaria, Universidad de La Laguna, Unidad de investigacioń, Santa Cruz de Tenerife, Spain, Hospital Universitario NS Candelaria, Unidad de Inmunologıá, Santa Cruz de Tenerife, Spain, àHospital Universitario NS Candelaria, Unidad de investigacioń, Santa Cruz de Tenerife, Spain Purpose/Objective: In mice, resistin is an adipokine that plays a role in insulin resistance (IR). On the other hand human resistin is a cytokine whose main source are macrophages and is not relevant in the RI, but is related to atherosclerosis. The few prospective studies have been conducted in patients with acute coronary syndrome (ACS) and resistin has been associated with mortality. It has not been prospectively studied whether resistin modifies the risk of myocardial infarction (AMI) in the general population; that was our goal. Materials and methods: We measured serum resistin in which so far is the largest general population sample (n = 6630). We followed this cohort for 3.5 years (cohort study ‘CDC de Canarias’), recording the incident cases of AMI and ACS and calculated the relative risk (RR) of those who occupied the top quintile of resistin (Q5) compared to the rest of the population. We adjusted RR for age, sex and tobacco in Cox models. Results: Resistin showed higher values in women (5.9 ± 0.5 versus 5.5 ± 0.5 ng/ml; P < 0.001). On average Q5 was younger (41 versus 43 years, P < 0.001) and had lower prevalence of obesity (26% versus 29%; P = 0.006), diabetes (8% versus 11%; P = 0.013), hypertension (29% versus 36%; P < 0.001) and LDL-cholesterol >160 mg/dl (16% versus 19%; P = 0.018), but showed higher smoking prevalence (34% versus 24%; P < 0.001). Q5 showed no differences in RI (HOMA2: 0.5 versus 0.51; P = 0.502) but had a higher incidence of MI [RR = 2.1 (1.1 3.9); P = 0.019] and ACS [RR = 1.7 (1.0 2.7); P = 0.044]. After

multivariate adjustment, Q5 showed higher incidence of MI [RR = 2.2 (1.2 4.1); P = 0.012] and ACS [RR = 1.8 (1.1 2.9); P = 0.027]. Conclusions: Elevated resistin acts as a risk factor for AMI and ACS in general population even though Q5 individuals are younger and have lower frequency of known cardiovascular risk factors.

P1132 Serum concentrations and mRNA expression of chemokine CCL17 in patients with type 2 diabetes mellitus and obesity: the influence of caloric restriction and sleeve gastrectomy M. Mraz,* Z. Lacinova,* P. Kavalkova,* J. Drapalova,* M. Kindlova,* D. Haluzikova,* M. Urbanova,* M. Kasalicky, S. Svacina* & M. Haluzik* *3rd Department of Medicine, General University Hospital, 1st School of Medicine Charles University, Prague, Czech Republic, Department of Surgery, Central Military Hospital, Prague, Czech Republic Purpose/Objective: CCL17 (thymus activation-regulated chemokine TARC) is a CC chemokine with chemotactic effects on Th2 lymphocytes, which are involved in the switch from proinflammatory M1 to antiinflammatory M2 macrophage phenotype in adipose tissue. The aim of our study was to assess the influence of short-term caloric restriction and laparoscopic sleeve gastrectomy (LSG) on serum concentrations and mRNA expression of CCL17 in subcutaneous adipose tissue (SCAT) of patients with obesity and type 2 diabetes mellitus (T2DM). Materials and methods: Twelve obese females with T2DM undergoing 2 weeks of very-low-calorie diet (VLCD energy content 2500 kJ/ day) and 17 non-diabetic women with 3rd grade obesity (BMI > 40 kg/m2) scheduled for LSG were included into this prospective study. Serum concentrations and mRNA expression of CCL17 together with other investigated parameters were assessed at the beginning and at the end of VLCD and before and 6, 12 and 24 months after LSG. Results: Both study procedures markedly reduced body weight and improved metabolic parameters including glucose control, insulin resistance and systemic low-grade inflammation. Two weeks of VLCD lead in our obese diabetic patients to a significant increase in serum levels of CCL17 (P < 0.001) accompanied by a 7-fold elevation of its mRNA expression in subcutaneous adipose tissue. On the contrary, extremely obese non-diabetic women showed a 30% decrease of systemic CCL17 concentrations at 24 months after LSG, while no significant change could be seen at months 6 and 12. mRNA expression of CCL17 in SCAT exerted a continuous, albeit nonsignificant, downward trend throughout the whole study period. Conclusions: Our pilot data suggest the existence of different immunological mechanisms participating in improved metabolic state after short-term and long-term interventions in patients with T2DM and obesity. A more precise definition of the role of CCL17 in these processes might offer novel insights into the mutual link between immunity, inflammation and the pathogenesis of obesity, type 2 diabetes mellitus and their complications.


Abstracts 545

Poster Session: Neurological and Neuromuscular Diseases P1134 Ab-proteases and their potential applications in MS diagnostics and treatment M. Bocharova,* M. Levitan, D. Kostyushev,* S. Krynsky* & S. Suchkov* *Pathology, I. M.Sechenov The First Moscow State Medical University, Moscow, Russia, Neurology, Astrakhan State Medical University, Astrakhan, Russia Purpose/Objective: Here, we aim at elucidating the role of autoantibodies with proteolytic activity (Ab-proteases) in MS pathogenesis and discuss their possible applications in MS diagnostics and treatment. Materials and methods: A comparative mass-spectrometric and kinetic analysis of the activity of Ab-proteases in serum samples from MS patients, their relatives and healthy donor’s war performed. Results: Ab-mediated proteolytic activity showed a marked difference between MS patients and healthy controls and a strong correlation with EDSS scores and thus with different degrees of disability in MS patients. Besides, 12 18% of the patients’ relatives were also found seropositive for MBP-specific Ab-proteases, but those endowed with activity 8 15 times lower (yet significantly higher than that of individuals seropositive for canonical anti-MBP autoAbs). In course of 3 5 years of observation,7 of 12 relatives suspicious for pre-MS showed a gradual increase in the activity of Ab-proteases, and after it reached mid-level, primary clinical and MRI-manifestations could be identified in those relatives. Another significant feature described for Ab-proteases is their sequence specificity. Anti-MBP Ab-proteases that are able to rec-ognize 48 70 and 85 170 amino acid sequences within the MBP molecule are predominantly more typical of MS patients, but not of patients with neurodegenerative diseases other than MS, while Ab-proteases for 43 68 and 146 170 amino acid sequences were found to be occurring only in MS patients. The proteolytic activity of anti-MBP autoantibodies reached its peak at 82 98 amino acid sequence, which had previously been described as having encephalitogenic and immunodominant properties. The experimental in vitro application of 82 98 fragment itself (or its mutant form Copaxone), proved able to inhibit MBP-specific at-mediated catalysis. Conclusions: Ab-proteases appear to be prospective both as potential MS biomarkers and as therapeautic tools. e.g. by changing the sequence specificity of the Ab-mediated proteolysis of the myelin sheath one may reach a reduction in the density of points of the proteolytic effects within the sheath and decrease lesion load, thus minimizing the proteolytic susceptibility of myelin-associated proteins and lowering scales of demyelination. The possibility of designing abzymes for the development of principally new catalysts with no natural counterparts is of great interest too.

P1135 Absence of IGF1R from oligodendrocytes ameliorates EAE T. Buch,* G. Locatelli, O. Prazeres da Costa,à B. Ingold,§ L. Koch,– J. Bru¨ning– & B. Becher *TU Mu¨nchen, Institute for Medical Microbiology Immunology and Hygiene, Mu¨nchen, Germany, Institute of Experimental Immunology, University of Zurich, Zu¨rich, Switzerland, àTU Mu¨nchen, Institute for Medical Microbiology Immunology and Hygiene, Munich, Germany, § Charite´, Universita¨tsmedizin Berlin, Institute of Pathology, Berlin, Germany, –Institute for Genetics, University of Cologne, Cologne, Germany Purpose/Objective: Signaling of Insulin-like Growth Factor 1 (IGF-1) through the insulin-like growth factor receptor 1 (IGF1R) mediates

anti-apoptotic mechanisms in several cell types and regulates differentiation and myelination of oligodendrocyte (ODC) precursor cells (OPCs) during development and following injury. Interestingly, IGF-1 expression is increased in the CNS parenchyma during cuprizone intoxication and surrounding sclerotic lesions in Multiple Sclerosis and in its inflammatory animal model, experimental autoimmune encephalomyelitis (EAE). However, the role of IGF1R signaling in OPCs and ODCs in the context of neuroinflammation remains unclear as published studies show contradictory Results: . Materials and methods: We used oligodendrocyte-specific deletion of IGF1R to investigate the role of IGF-1 signalling for disease course and recovery in the oligodendrocyte-ablating paradigms EAE and cuprizone intoxication. Results: The use of a late-acting myelin-specific Cre strain (MOGicre) resulted in efficient ablation of the IGF1R gene without any clinical and histological abnormalities developing in the respective animals. We observed, however, upon cuprizone-mediated oligodendrocyte death impaired remyelination. EAE was, surprisingly, ameliorated. We found that microglia was less activated as shown by CD44 and MHC class II surface expression analysis. Conclusions: Taken together we show that specific deletion of IGF1R from mature oligodendrocytes increases oligodendrocyte susceptibility to death. Yet in EAE absence of IGF1R from oligodendrocytes results in significant amelioration of disease.

P1138 Chronic gastro-intestinal worm infection enhances prion-mediated CNS-pathology and disease incubation time D. Donaldson*, B. M. Bradford,* K. J. Else & N. A. Mabbott* *Neurobiology, The Roslin Institute, Edinburgh, UK, Faculty of Life Sciences, University of Manchester, Manchester, UK Purpose/Objective: Prion (transmissible spongiform encephalopathy) infections are neurodegenerative diseases that affect both humans and animals. Factors which enhance neurodegeneration may be important determinates of the onset of clinical symptoms and disease progression. Pathogen infection is a potential risk factor in the development of other neurodegenerative diseases such as Alzheimer’s. Previous studies have shown that the gastro-intestinal worm Trichuris muris, which is restricted to the gut lumen, is capable of adversely altering CNS pathology in murine model of stroke through the induction of systemic immune responses. Therefore, the aim of this study was to assess if T. muris infection altered CNS pathology and clinical incubation time of ME7 prion disease. Materials and methods: Groups of mice were infected with ME7 scrapie prions intra-cerebrally (i.c.). At various time-points following ME7 scrapie infection, mice were co-infected with either an acute TH2inducing or chronic TH1-inducing dose of T. muris by oral gavage. Mice were culled at the clinical endpoint, after which brains were collected and pathology assessed. Results: After the onset of TSE pathology, chronic TH1-inducing T. muris infection significantly reduced the time until onset of clinical symptoms and thus the incubation of time of the i.c. scrapie disease. Chronic TH1-inducing T. muris infection prior to the development of TSE pathology enhanced vacuolation in the thalamus and septum but did not alter disease incubation. Infection with an acute TH2-inducing dose of T. muris did not alter either the incubation time or pathology. Conclusions: Co-infection with TH1-inducing and not TH2-inducing doses of T. muris is capable of enhancing neurodegeneration in ME7 scrapie-infected mice and accelerating the onset of clinical signs. Determining the basis for this enhancement may help understand how pathogenic infections alter the progression of neurodegenerative diseases and point to novel treatments.


546 Poster Session: Myeloid Cell Development P1140 Donor dependent responsiveness dictates effect of CB2-receptor drugs on human leukocyte migration in response to the chemokine SDF-1 E. S. Graham,* E. Burns,* A. Lim,* K. Moodley,* M. Glass* & C. E. Angel *Centre for Brain Research, University of Auckland, Auckland, New Zealand, School of Biological Sciences, University of Auckland, Auckland, New Zealand Purpose/Objective: The aim of this study is to better understand the function of the cannabinoid type 2 receptor (CB2) expressed by certain human immune cells circulating in blood. Various rodent experimental data suggests that CB2 drugs (CB2 receptor selective agonists) have anti-inflammatory properties in models of stroke and multiple sclerosis. The CB2 drugs appear to improve the neurological deficits, by suppressing the detrimental neuroinflammation in these debilitating diseases (often fatal). A proposed mechanism of action is blockade of leukocyte infiltration into the damage brain. Materials and methods: We assessed whether a variety of cannabinoid ligands had any cytotoxic properties towards human peripheral blood mononuclear cells (PBMC) from blood of healthy volunteers. In addition, we assessed whether cannabinoid ligands had any prochemotactic activity as previously suggested by other authors. Furthermore, we have assessed the ability of selective CB2 agonists to abrogate human leukocyte chemotaxis towards well defined proinflammatory chemokines, such as SDF-1. Migration assays were performed using 96-well plate HTS Transwell assay coupled with ATPlite measurement of migrated cells. Results: The cannabinoid drugs we tested were not cytotoxic towards human leukocytes (n = 6 donors). Nor did we observe any basal chemotactic or chemokinetic activity of the cannabinoid drugs (n = 4 donors). However, several CB2 agonists, (namely HU308 and JWH015), significantly suppressed the chemotactic responses of human leukocytes from some donors towards SDF-1 (n = 6). Interestingly, this response was donor-dependent as the CB2 agonists had no effect at all or potentiated the SDF-1 mediated migratory response (n = 3). Conclusions: This donor heterogeneity is clinically very relevant for the application of these drugs in human for neurological diseases or other inflammatory conditions where CB2 receptors are a potential target. The variability clealry demands more detailed studies to understand the donor variability and target cells.

P1141 Elevated IL-10 in Dark Agouti rats suffering from experimental autoimmune encephalomyelitis J. Blazevski,* F. Petkovic,* M. Momcilovic,* D. Miljkovic* & M. Mostarica Stojkovic *Department of Immunology, Institute for Biological Research ‘Sini a Stankovi’, University of Belgrade Serbia, Belgrade, Serbia, Department of Immunology, School of Immunology, Institute of Microbiology and Immunology, University of Belgrade Serbia, Belgrade, Serbia Purpose/Objective: Experimental autoimmune encephalomyelitis (EAE) is an animal model of neuroinflamatory and demyelinating disease multiple sclerosis. EAE in Dark Agouti (DA) rats is characterized by strong inflammation with intense infiltration of immune cells into the central nervous system (CNS) leading to neurological impairments. On the contrary, EAE in Albino Oxford (AO) rats is mild, often asymptomatic, while infiltration of immune cells into spinal cord (SC) is limited, yet present. Cytokines produced by immune cells in lymphoid tissues and within the CNS largely contribute to EAE pathogenesis. So, objective of this work was comparison of various cytokine gene expressions in SC of DA and AO rats.

Materials and methods: Rats were immunized with myelin basic protein (MBP) or SC homogenate and complete Freund‘s adjuvant. SC were isolated from rats at different stages of the disease, and homogenates and SC immune cells (SCIC) were assessed for cytokine gene expression by real-time RT-PCR and protein production by ELISA assay. Cells were isolated from lymph nodes draining the injection site (DLNC) of the animals at the induction phase of EAE and from cervical lymph nodes (CLNC) of unimmunized rats. Also, CD4+ cells were obtained from DLNC by magnetic beads separation. Results: We found higher expression of pro-inflammatory cytokines IL-17 and IFN-gamma, but also of anti-inflammatory IL-10 at the peak of EAE in SC homogenates and SCIC in DA rats comparing to AO rats. On the contrary, lower expression of anti-inflammatory TGF-beta was detected in DA rats. IL-4 gene expression was extremely low in both strains. Increased gene expression of IL-10 was accompanied with greater IL-10 production in SCIC. Higher IL-10 expression and generation were also recorded in DLNC and CD4+ DLNC, but not in mitogen-stimulated CLNC of DA rats in comparison to AO rats. Conclusions: Although IL-10 is an anti-inflammatory cytokine with a prominent role in immunoregulation and its role in the course of EAE is thought to be protective, our results showing its higher expression and production in DA rats at the peak of disease imply that IL-10 might not have such a unilateral role in EAE pathogenesis.

P1142 Expression of IL-22 in the central nervous system during development of experimental autoimmune encephalomyelitis (EAE) A. Noofeli, M. Barbour, D. Alan, O. Millington & H. Jiang Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK Purpose/Objective: Interleukin (IL)-22 is a member of the IL-10 family, which are important regulators of the inflammatory response. Interestingly, IL-22 is also produced by Th17 cells, which play a key immunopathogenic role in many autoimmune diseases. However, the function of IL-22 in the development of central nervous system (CNS) inflammation is less clear. IL-22 deficient mice are fully susceptible to the development of experimental autoimmune encephalomyelitis (EAE), while IL-22 receptor (IL-22R) is expressed on blood-brain barrier (BBB) endothelial cells and IL-22 disrupts BBB tight junctions in vitro and in vivo. Therefore, the aim of this study is to examine the expression of IL-22 in the CNS tissues and to investigate whether the expression level correlates with the development of EAE. Materials and methods: EAE was induced by immunising mice with MOG35 55 peptide emulsified in Complete Freunds Adjuvant (CFA), together with intraperitoneal injection of pertussis toxin (PTX). Naive mice or mice immunised with PBS in CFA together with PTX were used as controls. EAE clinical score and mouse weight were examined and recorded daily. At day 9, 17 and 28 post-immunisation, mice were sacrificed and spleen and spinal cord tissues harvested. Single cell suspension of spleen cells were cultured with or without MOG35 55 and supernatant were collected for cytokine detection using ELISA. Spinal cord tissues were visualised by histological and immunohistochemical staining. Results: Our data demonstrate that MOG35 55 immunised mice developed EAE around day 9 and reached peak at day 15 while PBS immunised mice remained unaffected. ELISA of the spleen cell cultures show that cells from EAE mice produced more antigen-specific IL-17, IL-22 and IFN-c at day 9 and day 17. Furthermore, immunohistological staining data show that whilst IL-22 was expressed by naı¨ve/PBS spinal cord tissues, expression was highly up-regulated in the spinal cord of EAE mice. Conclusions: Our data therefore suggest that IL-22 may play a pathogenic role in autoimmune disease in the CNS, possibly through divergent roles in both the peripheral immune and CNS systems.


Abstracts 547 P1143 FoxP3+ T-regulatory cells are reduced in peripheral blood in ischemic stroke patients J. Ruhnau,* M. Heinrich,* C. Kessler,* B. M. Bro¨ker, A. Dressel* & A. Vogelgesang *Neurology, University of Greifswald, Greifswald, Germany, Immunology, University of Greifswald, Greifswald, Germany Purpose/Objective: Stroke is a leading cause of disability and death. Recently it became evident that cerebral ischemia leads to a defect of the innate and the adaptive immune response which is associated with an increased risk of secondary infections. To date, only scarce information is available on the underlying immunopathology. T-regulatory cells (Treg) are an important inhibitor of immune responses. In experimental stroke conflicting data have been reported regarding the survivial and importance of Treg. This study aimed to quantify and phenotype Treg in the peripheral blood of stroke patients and to assess their function. Materials and methods: Stroke patients (n = 38) and age matched non-stroke controls (n = 15) were recruited. Blood samples of patients were obtained on stroke unit admission and on days 1, 3, 5 and 7 thereafter. Treg were phenotyped by staining CD4+ CD49d) FoxP3+ cells using FACS analysis. Additionally we determined CD39 and CD45RA expression to identify active and naı¨ve Treg. In a functional assay the suppressive capacity of CD4+ CD25+ CD127dim/) Treg on T-effector (Teff) was evaluated by measuring the expression of CD69 and CD154 on anti-CD3/anti-CD28 stimulated Teff. Results: In stroke patients the percentage of CD4+ T-cells expressing Treg markers was reduced on day (d) 0 (P < 0.01), d1 (P < 0.01), d3 (P < 0.05), and d5 (P < 0.01) compared to healthy controls. This effect was also seen in CD39+ Treg. Functional testing revealed an impaired inhibition of CD154 expression (P < 0.01) in stroke derived samples but no difference in CD69 expression. Conclusions: Our data show that in peripheral blood of stroke patients CD4+ T-cells contain less Treg than in controls. In addition, in stroke patients the suppressive efficacy of Treg appears partly impaired. This could either be due to impaired Treg function or due to altered Teff function. We conclude that stroke induced immune suppression is unlikely to be caused by a selective survival and extensive suppressive activity of Treg. Since we have only access to peripheral blood, we cannot exclude that Treg have migrated to other compartments.

P1144 High levels of free kappa light chains incerebrospinal fluid predict conversion to multiple sclerosis M. Espinõ,* L. Costa-Frossard, A. Muriel,à J. C. Alvarez-Cermenõ & L. M. Villar *Immunology, Hospital Ramon y Cajal, Madrid, Spain, Neurology, Hospital Ramon y Cajal, Madrid, Spain, àBiostatistics, Hospital Ramon y Cajal, Madrid, Spain Purpose/Objective: A clinically isolated syndrome (CIS) may be the initial presentation of multiple sclerosis (MS). However, some CIS never develop MS. The identification of patients at risk of MS conversion is crucial as early treatment may improve their outcome. Free kappa chains (FKC) are increased in cerebrospinal fluid (CSF) of MS patients. We studied the accuracy of CSF FKC level measurement, using a new nephelometric test, to predict conversion of CIS patients to MS. Materials and methods: We quantified this protein in CSF from 25 patients with non-inflammatory neurological diseases (NIND) and 78 consecutive CIS patients. We assessed whether high CSF FKC levels associate with CIS conversion to clinically definite MS, defined as the onset of new relapses during follow-up.

Results: A cut-off value of 0.53 mg/l (mean+2SD of NIND group CSF FKC values) was calculated. CIS patients with CSF FKC above this value showed earlier conversion to MS in univariate and multivariate Cox analysis (HR = 6.41; 95% CI = 1.88 21.78, P = 0.003). Conclusions: High CSF FKC levels accurately predict CIS patient conversion to MS.

P1146 High serum LPS values associate with lower disability in multiple sclerosis R. Alenda-Asensi,* I. Toboso,* L. Costa-Frossard, E. RodriguezMartin,* M. Espinõ,* J. C. Alvarez-Cermenõ & L. M. Villar* *Inmunology, MS unit, Hospital Universitario Ramon y Cajal, Madrid, Spain, Neurology, MS unit, Hospital Universitario Ramon y Cajal, Madrid, Spain Purpose/Objective: High LPS values may induce endotoxin tolerance in monocytes, which is characterized by a decreased production of cytokines in response to proinflammatory stimuli. We aimed to explore this phenomenon in multiple sclerosis (MS) and to investigate if increased LPS values associate with any clinical or immunological variables in the disease. Materials and methods: The study included 55 with MS patients and for comparison, 11 patientswhith other inflammatory neurological diseases (OIND), 13 patients with non inflammatory neurological diseases (NIND) and 10 healthy individuals (HC). No differences were found in age or gender between the four groups. We also monitored in MS group disease duration, time to a relapse and disability measured by the EDSS score. Serum samples were stored at )80C until assayed. LPS was measured under sterile conditions by the endopoint chromogenic Limulus Amebocyte Lysate test (Lonza). Serum levels of activin A were measured by ELISA (R&D). Peripheral blood T and B cell subsets were studiedby by flow cytometry on a FACS-Canto II instrument. Immunoglobulin and albumin indexes were assessed by nephelometry on a Beckmann nephelometer. Results: We did not find differences in the LPS levels between MS patients and the other patient groups. We classified MS patients in two groups according to serum LPS values and did not find differences between both groups in the percentages of Tor B cell subsets, immunoglobulin indexes or in activin A concentration. However, we found a striking difference in patient disability. Those with high LPS values showed a significantly lower EDSS score (P = 0.004). Conclusions: This data suggest that endotoxin-tolerance induced by high LPS serum values may contribute to regulate the abnormal inflammatory response taking place in MS and ameliorate disease course.

P1147 IL-33 protects mice from experimental autoimmune uveoretinitis D. Allan,* H. Xu, C. Pei,* M. Chen, W. Niedbala,à S. Y. Fukada,à A. G. Besnard,à J. C. Alves-Filho,à J. V. Forrester,§ F. Y. Liewà & H. R. Jiang* *Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK, Centre for Vision and Vascular Science, Queen’s University Belfast, Belfast, UK, àInstitute of Infection Immunity and Inflammation, University of Glasgow, Glasgow, UK, §Ophthalmology Department, University of Aberdeen, Glasgow, UK Purpose/Objective: IL-33 is a recently discovered IL-1 cytokine family member. Previously, we have reported that IL-33 is an important modulator of the immune system and is associated with several immune-mediated disorders. The aim of this study is to evaluate the role of IL-33 cytokine in the development of experimental autoimmune uveoretinitis (EAU).


548 Poster Session: Myeloid Cell Development Materials and methods: The expression of IL-33 and its receptor ST2 on retinal pigment epithelial (RPE) cell line was examined by immunohistochemical staining. Next the severity of IRBP peptide induced-EAU was assessed in C57BL/6 mice treated with recombinant IL-33 or PBS. Cytokine secretion and production by the draining lymph nodes (DLNs) or spleen cells were measured at day 26 after immunization. Results: We demonstrate that RPE cells expressed high levels of both IL-33 and ST2. Administration of IL-33 cytokine to EAU mice led to reduced disease severity. In line with the reduced inflammation in the retina of IL-33 treated mice, the percentage of IFN-c+ or IL-17+ cells in the DLNs and spleen was markedly lower, while IL-5+ or IL-4+ cell percentage was increased. Furthermore, antigen specific production of IFN-c, IL-17 and IL-6 by the DLN cells from IL-33 treated mice was also significantly reduced. Conclusions: Our results suggest that IL-33 may play a protective role in the development of EAU possibly via its known role in promoting the function of alternatively activated macrophages. P1148 Is Parkinson’s disease the result of autoimmunity arising from Influenza A infection of the brain? K. Bryson,* Y. Ligertwood,* M. Quigg-Nicol,* R. Mellanby,* D. Brown,* S. Anderton & A. Nash* *The Roslin Institute and R(D) SVS, University of Edinburgh, Edinburgh, UK, The Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK Purpose/Objective: The mechanism of dopaminergic neuronal cell death remains a mystery in Parkinson’s disease. Compelling epidemiological evidence links Parkinson’s disease with Influenza A infection, with 5 million people developing the Parkinson’s associated disease Encephalitis lethargica following the 1918 influenza pandemic. However, as Influenza induces an acute infection, where as Parkinson’s disease is a chronic condition, and in the majority of cases the virus is absent from the lesion, this suggests that the virus has an indirect mode of action. An autoimmune reaction may thus be the effector mechanism linking the infection to neurological disease. Using a murine model we set out to investigate the relationship between influenza A and Parkinson’s disease, and in particular, investigate the hypothesis that autoimmunity arising from infection results in dopaminergic neuronal death in the substantia nigra. Materials and methods: A murine model was established with intranasal delivery of the neurotropic H1N1 A/WSN/33 Influenza strain. Following infection, brains were harvested and examined for the presence of the virus, T cell infiltrate and dopaminergic neuronal loss by immunohistochemistry and flow cytometry. Murine behaviour was also examined for Parkinsonian symptoms. A potential autoantigen, alpha synuclein; a protein central to the pathology in Parkinson’s disease, was also examined. Mice were primed against alpha synuclein in CFA and T cell behaviour examined. Results: Preliminary data using our murine model has shown that the Influenza virus was detected in the midbrain as late as 21 days post infection. T cell subsets were also detected in the brain following infection. In addition to this, we were able to generate alpha synuclein reactive T cells, and these cells were able to traffic to the brain. Conclusions: Identifying autoimmune mediated dopaminergic neuronal loss would radically change therapeutic approaches and may thus provide new targets to prevent the disease or preserve the quality of life of the patients. We thus need to further examine the aberrant immune response in Parkinson’s disease.

P1149 Is there a functional role for KCNMA1 in the multiple sclerosis? A. Vanheel, B. Broˆne, R. Daniels, P. Stinissen, J. P. Noben & N. Hellings Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium Purpose/Objective: A more detailed insight into disease mechanisms of multiple sclerosis (MS) is crucial for the development of new and more effective therapies. MS is a chronic inflammatory autoimmune disease of the central nervous system. The aim of this study is to identify novel disease associated proteins that are functionally involved in the MS brain pathology. Materials and methods: In a previous proteomics study, brainstem proteins were obtained from Lewis rats with MBP induced acute experimental autoimmune encephalomyelitis (EAE), a well characterized disease model of MS. Samples were collected at different time points: just before onset of symptoms, at the top of the disease and following recovery. To analyze changes in the brainstem proteome during the disease course, a quantitative proteomics study was performed using two-dimensional difference in-gel electrophoresis (2D-DIGE) followed by mass spectrometry. Results: We identified 75 proteins with a significant abundance difference between the different disease stages. Regulated proteins were mapped to existing biological networks by Ingenuity Pathway Analysis (IPA). Post-synaptic density protein 95 (DLG4), a key player in neuronal signalling and calcium-activated potassium channel alpha 1 (KCNMA1), involved in neurotransmitter release, are 2 putative regulators connecting 64% of the proteins identified. The involvement of KCNMA1 in macrophage functionality was studied in vitro by using a specific functional blocker for KCNMA1, paxillin. We show that blocking of KCNMA1 altered myelin phagocytosis and proinflammatory cytokine release, disease mechanisms which are highly involved in EAE and MS pathology. We are currently investigating possible influences of this blocker on functionality of other disease relevant cells and processes using in vitro and in vivo models. Conclusions: This study will elucidate to what extent modulation via this ion channel affects disease processes in the context of EAE/MS.

P1150 Mast cells protect from post-traumatic spinal cord inflammation in mice by degrading inflammation-associated cytokines via mouse mast cell protease 4 S. Hendrix,* S. Nelissen,* E. Lemmens,* T. Vangansewinkel,* P. Vidal Vera,* L. Willems,* F. Boato,* D. Dooley,* G. Pejler, M. Maurerà & M. Metzà *Morphology, BIOMED Institute Hasselt University, Diepenbeek, Belgium, Swedish University of Agricultural Sciences, Anatomy, Uppsala, Sweden, àDermatology and Allergy, Charite´- Universita¨tsmedizin, Berlin, Germany Purpose/Objective: It becomes increasingly clear that mast cells (MCs) are not only key players in allergic diseases (e.g. asthma), but seem to play a complex role in neuroinflammatory diseases such as multiple sclerosis and stroke. However, their role during and after mechanical CNS trauma is not clear. In the present study, we have investigated the effects of MC-deficiency on the histological and clinical outcome after spinal cord injury (SCI) in mice. Materials and methods: MC-deficient W-sash c-kit mutant knockout mice (kitW-sh/W-sh), mMCP-4 deficient (mMCP4-/-) mice and control mice underwent spinal cord hemisection at thoracic level T8 resulting in a complete transection of the dorsomedial and ventral corticospinal tract. Functional recovery in SCI mice was tested with the Basso Mouse Scale. Spinal cord sections were analyzed by immunofluorescence. RTPCR and Western blotting were used to analyze cytokine/chemokine mRNA and protein levels. In degradation assays murine recombinant


Abstracts 549 IL-1b, IL-4, IL-6, IL-10, IL-13, TNF-a and MCP-1 were incubated with supernatant from BMCMC derived from either C57BL/6 or from mMCP4-/- mice. Cleaved fragments were identified using tris-tricine SDS-PAGE and analyzed by intensity analysis. Results: We show that MC-deficient kitW-sh/W-sh mice display significantly increased astrogliosis and T cell infiltration as well as significantly reduced clinical outcome after SCI compared to wildtype mice. In addition, MC-deficient mice show significantly increased levels of MCP-1, TNF-a, IL-10 and IL-13 protein levels in the spinal cord after SCI. Mice deficient in mMCP-4, a MC-specific chymase, also showed increased MCP-1, IL-6 and IL-13 protein levels in spinal cord samples and a decreased functional outcome after SCI. A degradation assay using supernatant from MCs derived from either mMCP4-/- or wildtype mice revealed that mMCP-4 cleaves MCP-1, IL-6 and IL-13, suggesting a protective role for MC proteases in neuroinflammation. These data indicate that MCs may be protective after SCI and that they may reduce CNS inflammation by degrading inflammation-associated cytokines via the mast cell-specific chymase mMCP-4. Conclusions: In summary, our results suggest a new and complex mechanism how MCs and their proteases may protect the CNS from exacerbated and/or chronic inflammation after damage.

P1151 Peripheral blood CD16+56) cells may contribute to the early diagnosis of multiple sclerosis E. Rodriguez-Martin,* E. Roldan,* L. Costa-Frossard, M. Espinõ,* R. Alenda-Asensi,* J. C. Alvarez-Cermenõ & L. M. Villar* *Servicio de Inmunologia MS Unit, Hospital Universitario Ramon y Cajal, Madrid, Spain, Servicio de Neurologia MS Unit, Hospital Universitario Ramon y Cajal, Madrid, Spain Purpose/Objective: Alterations in peripheral blood natural killer (NK) cell subset numbers and functions have been linked to multiple sclerosis (MS). NK cells are classified in two major subsets, CD56 dim and CD56 bright, which differ in their functional and homing properties. In early HIV infection, a CD16+ CD56) NK cells subset are expanded are correlated with a higher plasma viral load set point, suggesting its utility as a predictive marker for disease progression. The aim of this study was to investigate this recently described NK subset in MS and in patients with other neurological diseases. Materials and methods: We studied 63 patients with relapsing remitting MS, 16 with other inflammatory neurological diseases of the central nervous system (OIND) and 17 patients with noninflammatory neurological diseases (NIND). Peripheral blood cells were labelled with conjugated monoclonal antibodies and analyzed on a standard FACSCanto-II cytometer (BD). Results were analyzed with the Mann Whitney U-test for comparison between groups. Results: NK bright and the NK dim cells subsets did not change in MS patients when compared to the other groups. In addition, we did not find differences in CD56+ CD3) or CD56+ CD3+ NK cells subsets between the three groups of patients. However, CD16+ CD56) cells were clearly increased in OIND patients when compared to MS (P = 0.01) and NIND (P = 0.0084) patients. Conclusions: These results may be of clinical relevance in the early differential diagnosis of MS.

P1152 Protein profiling of the cerebrospinal fluid in diagnostics, prognosis and treatment management of multiple sclerosis S. Krynskiy,* M. Bocharova, D. Gnatenko,* M. Levitanà & S. Suchkov§ *Department of Pathology, I.M.Sechenov The First Moscow State Medical University, Moscow, Russia, Department of Neurological Diseases, I.M.Sechenov The First Moscow State Medical University, Moscow, Russia, àDepartment of Neurological Diseases, Astrakhan State Medical Academy, Astrakhan, Russia, §Department of Pathology, I. M. Sechenov The First Moscow State Medical University, Moscow, Russia Purpose/Objective: When dealing with diseases of the central nervous system (CNS), the cerebrospinal fluid (CSF) seems to be the most appropriate body fluid for early and accurate diagnosing. Specifity of any changes found in the protein profile of the CSF is higher, and there is higher possibility for detection of low-abundance proteins. With the CSF it is also possible to skip separation by means of electrophoresis, chromatography etc. Materials and methods: To access possible diagnostic and prognostic criteria for MS, protein profiling of the CSF was used, using patients with inflammatory and non-inflammatory diseases of the CNS as controls. Protein profiling of the CSF was performed by means of MALDI-TOF mass-spectrometry. Results: Biomarkers of multiple sclerosis (MS) are devided into 2 groups: markers of immune inflammation and markers of neurodegenerative processes. Markers of immune inflammation include oligoclonal IgG bands, light Ig chains, chromogranin A, clusrerin, CC3. Markers specific to autoimmune processes taking place in MS areautoantibodies to myelin basic protein (anti-MBP) and to myelin olygodendrocyte protein (anti-MOG). Antiganglioside antibodies (anti-GM) are more of prognostic value, as there is a correlation between anti-GM antibody type and clinical type of the disease (primary progressive, back-and-remitting, and secondary progressive). Markers of neurodegenereation are components of structures altered during the clinical attack of MS. The best studied marker is the main target of autoimmune reactions in MS myelin basic protein (MBP). High levels of MBP have been shown to correlate with upcoming relapses of MS, as well as with the disease attaining the progressive clinical type. Another prognostic marker is acidic calcium-binding protein a component of axons damaged during the course of the disease. Another important task is predicting transformation of clinically isolated syndrome (CIS) into manifest MS. Some protein markers that have shown diagnostic value are 14-3-3 protein and chemokin CXCL13.. Conclusions: Proteomics of liquor gives a possibility of diagnosing the disease and planning the protocols of treatment conveniently and fast, without excessive time and resource consumation. Hovewer, there is a number of methodological problems. One of these is the fact that it is impossible to form control groups of healthy individuals. Another reason is lack of standarts for sample collecting and storing, and lack of international bases including protein profiles of the CSF. Overcoming this obstacle is obligatory for further progress in this field of predictive medicine.

P1153 Regulation of class II MHC antigen expression in microglial cells M. Kalognomou & I. Athanassakis Biology Department, University of Crete, Heraklion, Greece Purpose/Objective: Deregulation of microglial activation mechanisms plays a critical role in the development of neurodegenerative conditions like ageing, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and schizophrenia. Studies have shown that the


550 Poster Session: Myeloid Cell Development neurodegenerative role of microglia has been highly correlated with the expression of MHC-II molecules. The aim of the present study was to define the membrane and intracellular expression of H-2A as well as the intracellular expression of H-2M, H-2O and CD74 in different activation states of microglial cells, using the BV-2 cell line. Materials and methods: Flow cytometry and confocal microscopy analysis were used to define the expression of H2-A, -O, and -M molecules in BV-2 cells. Isolated mRNAs were submitted to RT-PCR experiments using CD74 specific primers. LPS, IFN-c and IL-4 were used to explore microglial activation mechanisms. Secretion of H2-A molecules in different activation states was evaluated by ELISA experiments. Results: BV-2 cells expressed all necessary components for posttranslational regulation of MHC class II molecules and their transport to the cell surface. Regarding microglial activation mechanisms, LPS did not affect MHC-II expression but induced TNF-a secretion while promoting the cytophagic properties of microglia. The proinflammatory cytokine IFN-c reduced of MHC-II expression but increased secretion of H2-A molecules as tested by ELISA experiments. IL-4 activation reduced membrane and intracellular H2-A while inducing H2-O expression. Conclusions: Stimulation of BV-2 cells with LPS, IFN-c and IL-4 demonstrated that different activation stimuli could lead to different activation pathways of microglial cells promoting a variety of events ranging from neuroprotection to neurodegeneration. Studying the regulatory pathways of microglial MHC II expression could delineate mechanisms of stimulation versus suppression, surface expression versus secretion of MHC-II molecules during pathological conditions in neurodegenerative diseases and dictate new strategies of therapeutic approaches.

P1154 The acute-phase protein Hemopexin regulates Th17 cells and experimental allergic encephalomyelitis development S. Rolla,* G. Ingoglia, V. Bardina,* L. Silengo, F. Altruda, F. Novelli* & E. Tolosano *Department of Medicine and Experimental Oncology, University of Turin, Torino, Italy, Molecular Biotechnology Center, University of Turin, Torino, Italy Purpose/Objective: Hemopexin (Hx) is an acute phase protein synthesized by hepatocytes in response to the pro-inflammatory cytokines IL-6, IL-1b and TNF-a. Hx is the plasma protein with the highest binding affinity to heme and controls heme-iron availability in peripheral cells and also in T lymphocytes, where it modulates their responsiveness to IFNc. Recent data have questioned about an antiinflammatory role of Hx, a role that seems heme-binding independent. The aim of this study was to investigate the role of Hx in the development of a T-cell mediated autoimmune response. Materials and methods: Experimental autoimmune encephalomyelitis (EAE), the animal model of MS, was induced in WT and Hx knockout (Hx-/-) mice. The development of the disease, the levels of Hx, the activation of Th1 and Th17cells and the production of inflammatory cytokines by macrophages were evaluated. Results: During EAE Hx level increased and remained high. Hx-/mice developed a clinically earlier and more severe and pathologically exacerbated expression of EAE compared to WT controls. Hx-/- mice displayed a higher amount of CD4+ infiltrating T cells and a more severe demyelization in the CNS compared to WT. The severe EAE developed by Hx-/- mice could be ascribed to the higher amount of Th17 cells infiltrating the CNS and circulating in the periphery of Hx-/mice compared to WT mice. In vitro, T cells from Hx-/- mice polarized more efficiently towards IL-17 producing cells compared to WT lymphocytes. Moreover, the lack of Hx was associated with enhanced

production of inflammatory cytokines in the antigen presenting cells, in particular IL-6 and IL-23, the major Th17 differentiating factors. Conclusions: Over all our observations indicate that Hx could influence the generation of Th17 response during EAE and account for the anti-inflammatory role of Hx in T cell-mediated autoimmune disease.

P1155 The different role of NKT and NK cells functional activity in demyelinating polyneuropathies R. Sepiashvili,* I. Balmasova, O. Timchenkoà & N. Yushchukà *Immunology, National Institute of Allergology and Clinical Immunology, Tskhaltubo, Georgia, Immunology, Peoples’ Friendship University of Russia, Moscow, Russia, àLaboratory of Pathogenesis and Treatment Methods of Infectious Diseases, Moscow State University of Medicine and Dentistry, Moscow, Russia Purpose/Objective: Acquired autoimmune demyelinating polyneuropathies include Guillain-Barr’ Syndrome (GBS) and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), which differ in the etiology (GBS usually develops as a complication of infectious processes, immunopathogenesis, course duration, methods of treatment and therefore call for differential diagnosis. Materials and methods: Were observed 68 patients with demyelinating polyneuropathiesby flow cytometry who were divided into two groups: 42 GBS patients and 26 with CIDP. Expression of cell-surface markers such as CD3+/CD56+/CD4+, CD3+/CD56+/CD8+, CD3+/ CD56+/DN, CD3)/CD16-/CD56+, CD16+/CD56+/CD158a, h+, CD16+/CD56+/CD94+, CD16+/CD56+/NKG2D+, CD16+/CD56+/ NKp46+ were assessed by multiparametric FACS. Results: It was found that in GBS unlike in the CIDP the fall of NKT number was not present but reliably reduced the NK cell number. In all patients D4+ NKT increased 10 and more times, subpopulation CD8+ NKT significantly decreased. The changes in NK cell in GBS patients were accompanied by a decline in their expression of KIR (D158a, h), NKG2D, NKp46. The fall of natural cytotoxicity of receptor content (NKp46) in 1.8-fold on NK membrane is observed only in GBS while negative shift in receptor expression of antibody dependent cytotoxicity is observed in all manifestations ofdemyelinating polyneuropathies. Conclusions: It is expected that the observed immunopathogenetic features with NKT and NK cells participation can help in differentiation of GBS from CIDP.

P1156 The pro-inflammatory cytokines IL-1ß and TNFa both have deleterious effects on human astrocytes E. S. Graham,* J. Costa,* K. Moodley* & C. E. Angel *Centre for Brain Research, University of Auckland, Auckland, New Zealand, School of Biological Sciences, University of Auckland, Auckland, New Zealand Purpose/Objective: Astrocytes play important roles within the human central nervous system through trophic support of neurons and are inextricably involved in the brain’s immune status. Astrocytes are part of the blood-brain barrier vasculature, which maintains cellular and soluble traffic into this immune specialised tissue. Any event (immune response, infections, and drug treatments) that is detrimental to astrocyte survival or function will have a negative effect neurologically. Our aim was to investigate acute and long-term influence of neuroinflammatory events on astrocyte survival and fate. Materials and methods: We used xCELLigence technology to track astrocyte responses to various inflammatory mediators, in conjunction


Abstracts 551 with classical assays including cell counts and caspase-3 expression as markers of cell loss/death.xCELLigence is a real-time biosensor technology, which directly measures the level of adhesion of living cells. Theoretically, any treatment that significantly or acutely affects adhesion, (e.g. changes cell morphology, cell proliferation or cell death), can be investigated using xCELLigence, with appropriate validation of the responses. Specific cellular responses produce signature changes in growth curve dynamics and we reveal and explain several of these. All responses were verified and corroborated using conventional assays. Results: We show that the pro-inflammatory cytokines IL-1b and TNFa are both cytotoxic towards astrocytes. IL-1b is more potent than TNFa, with cytotoxic effects induced in the high fempto-molar range (>250 fM), whereas TNFa induced astrocyte death occurred in the pico-molar range (>250 pM). xCELLigence technology elegantly revealed the differential responsiveness of astrocytes to IL-1b and TNFa, and other cytokines including IFNc, CXCL8 and IP10, which had no influence on astrocyte viability but have important roles in neuroinflammation. Conclusions: Certain pro-inflammatory environments clearly have a detrimental effect on these very important brain immune cells. These findings have important implications for understanding the adverse events occurring during neuroinflammation that exacerbate or perpetuate the primary insult. Similar events may occur in brain diseases such as stroke or MS, where vascular inflammation and damage are hallmarks of the disease.

P1157 The role of iron in inflammatory demyelinating diseases: development of experimental in vitro and in vivo models C. Schuh, S. Hametner, M. Bradl & H. Lassmann Neuroimmunology, Center for Brain Research, Vienna, Austria Purpose/Objective: Inflammatory processes play a key role in various neurodegenerative diseases such as Multiple Sclerosis (MS), Alzheimer’s Disease and Parkinson’s Disease. Increasing evidence suggests that iron accumulation in the brain might contribute to neurodegeneration. Iron is a potential source of free radicals as it can catalyze the production of hydroxyl radicals under oxidative conditions. This can lead to amplification of tissue injury caused by oxidative damage. In preliminary experiments we found evidence that the presence of iron exacerbates H2O2 induced cell death in glial cells in vitro. Materials and methods: We thus characterized iron storage within the central nervous system (CNS) of animal models of different neurodegenerative diseases. We examined animals with acute inflammation mediated by CD8 or CD4 positive T cells and animals suffering from T cell and antibody mediated chronic inflammation due to active immunization. Further, we studied LPS induced lesions which represent CNS disease caused by the innate immune system. Similarly, we characterized oxidative damage in the different models for inflammation. Results: We did not find evidence for the presence of oxidized phospholipids or oxidized DNA in the experimental lesions, which is in contrast to our results of MS lesions. None of these models showed iron accumulation in glial cells comparable to what is seen in MS tissue. However, some iron positive microglia and perivascular macrophages were observed in acute and chronic models. As iron accumulates progressively with aging, we analyzed iron storage in aged rats. We observed that iron is stored in oligodendrocytes and myelin in certain areas of the brain such as the deep cerebellar nuclei and the

basal ganglia. To address the question, if iron could influence CNS inflammation, we compared acute young and old animals. We found a similar disease course in both groups whereas in the pathological analysis we detected higher levels of inflammatory infiltrates in young animals. In contrast, we found a higher degree of neurodegeneration in the spinal cords of old animals. Nevertheless, we did not find any evidence for iron deposition in glial cells. Conclusions: These observations necessitate the search for additional animal models mimicking the human situation more closely with respect to age dependent iron accumulation and neurodegeneration.

P1158 The Vagus nerve, importance in immunodepression following stroke L. Akyu¨z,* C. Dames,* O. Engel, K. Winek, H. D. Volk,à A. Meisel & C. Meiselà *Institute for Medical Immunologie, Charite´ Universita¨tsmedizin Berlin, Berlin, Germany, Department of Experimental Neurology, Charite´ Universita¨tsmedizin Berlin, Berlin, Germany, àInstitute of Medical Immunology, Charite´ Universita¨tsmedizin Berlin, Berlin, Germany Purpose/Objective: Impaired immune function due to overactivation of neuro-humoral stress pathways, in particular of the sympathetic nervous system, has been proposed as risk factor for the high incidence of pneumonia after stroke. However, changes in pulmonary immunity and the role of the parasympathetic vagus nerve (VN) in stroke-induced suppression of immune responses are poorly understood so far. We hypothesize that after stroke pulmonary macrophage function is impaired due to release of acetylcholine by the VN resulting in reduced lung anti-bacterial responses. This anti-inflammatory effect mediated by the VN is dependent on alpha7 nicotinic acetylcholine receptor (a7nAChR). Materials and methods: To investigate the role of the VA in strokeassociated pneumonia we analyzed effects of vagotomy (Vtx) on cellular immune responses to bacterial pneumonia in an experimental model of cerebral ischemia (MCAo) in WT C57/BL6 mice. In addition, a7nAChR-/- mice and littermate controls were used to address the role of the a7nAChR in stroke-induced immunosuppression. To determine the role of the a7nAChR expression on pulmonary immune versus parenchymal lung cells we used bone marrow chimeras reconstituted with a7nAChR-/- and WT cells. The immune responses in lung and spleen were analyzed by flow cytometry, by multiplex cytokine analysis of BAL fluid and ex vivo TLR-ligand induced cytokine secretion, and assessment of macrophage phagocytic activity. Results: Inhibition of VN activity by Vtx significantly ameliorated spontaneous bacterial infections after stroke in wt mice. Release of proinflammatory cytokines such as TNF-a by alveolar macrophages upon ex vivo stimulation with TLR-ligands was significantly increased in Vtx mice. Interestingly, our mixed bone marrow chimera experiments indicate that a7nAChR expression on lung parenchymal cells is also important for impaired pulmonary anti-bacterial responses mediated by increased VN activity after stroke. Conclusions: We could show that the VN and the a7AchR play a crucial role in suppression of lung immunity after stroke. Inhibition of parasympathetic activity by Vtx or a7AchR deficiency reduces susceptibility to bacterial lung infection after acute CNS damage. Expression of a7AchR on both lung macrophages and parenchymal cells seem to be important to the immunosuppressive effect of increased VN activity after stroke.



Poster session: Parasitic Diseases P1160 A role for plasmacytoid dendritic cells in promoting Th2 immunity against helminths R. J. Lundie,* L. M. Webb, P. C. Cook, A. T. Phythian-Adams, L. H. Jones, L. Boonà & A. S. MacDonald *Centre for Immunology, Burnet Institute, Melbourne, Australia, Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, UK, àDepartment of Cell Biology, Bioceros B.V., Utrecht, Netherlands Purpose/Objective: Plasmacytoid dendritic cells (pDC) are a distinct subset of dendritic cells that are found in the blood as well as in peripheral lymphoid tissues. While pDC are best known for their role in antiviral immunity (and their potent ability to produce type I interferons), they also appear to be involved in regulation of Th2 responses in allergy. However, it is not known how pDC respond to Th2associated pathogens such as the parasitic helminth Schistosoma mansoni, and whether they play an immunogenic or tolerogenic role in Th2 infection settings. Materials and methods: In this study, we have characterized the response of pDC in the liver effector site during S. mansoni infection, both in terms of their phenotypic activation and their ability to present antigen to naı¨ve and effector/memory CD4+ T cells. The mAb 120G8 was also used to determine the impact of pDC depletion on the development of Th2 immunity in vivo. Results: Our results demonstrate that liver pDC recognise and respond to S. mansoni antigens by up-regulating surface expression of MHC class II and co-stimulatory molecules. Liver pDC also acquire the capacity to support the proliferation of antigen-experienced IL-4/ IL-13 producing CD4+ T cells during infection. Furthermore, in the absence of pDC, S. mansoni-specific CD4+ T cell production of IL-4 and IL-13 was impaired in the liver. Conclusions: Together our data provide strong evidence that liver pDC promote Th2 immunity against S. mansoni parasites.

P1161 A SAG2A protein-based algorithm to analyze the production of IgG1 and IgG3 subclasses in sequential serum samples from patients with acute toxoplasmosis J. Mineo,* S. S. Silva,* D. A. Silva,* L. D. Vaz,* C. P. Pirovani, G. B. Barros,à E. M. Lemos,§ R. Dietze§ & J. P. Cunha-Junior– *Immunology Microbiology and Parasitology, Institute for Biomedical Sciences, Uberlandia, Brazil, Institute of Biotecnology, Universidade Estadual de Santa Cruz, Ilheús, Brazil, àFaculdade de Medicina, Universidade Federal do Espı´rito Santo, Vito´ria, Brazil, §Nućleo de Doenças Infecciosas, Universidade Federal do Espı´rito Santo, Vito´ria, Brazil, –Immunology Microbiology and Parasitology, Institute for Biomedical Sciences, Universidade Federal de Uberlaˆndia, Uberlandia, Brazil Purpose/Objective: The major aim of the present study was to evaluate the kinetics of the humoral immune response to the recombinant SAG2A antigen in comparison with T. gondii soluble antigen (STAg) through the detection of specific IgG, IgG1 and IgG3 antibodies in serum samples of patients with toxoplasmosis in different time points of infection. Also, we investigated the association of these serological markers based on SAG2A reactivity as potential tools to distinguish early acute from convalescent phase of toxoplasmosis. Materials and methods: It was carried out a prospective longitudinal study that evaluated a total of 130 serum samples obtained from 19 patients with acute toxoplasmosis after different time points of illness onset. The patients enrolled in the study were attended at the

Infectious Disease Center of the Federal University of Espirito Santo, Vitoria, ES, Brazil, after an initial screening by physicians of the region. Serological evidence of recent infection was characterized by the presence of IgM and/or IgG antibodies to T. gondii in conventional serological assays using a commercial kit (ELFA, VIDAS Toxo IgM and IgG II, Biom rieux SA, Lyon, France). Exclusion criteria were pregnancy and/or human immunodeficiency virus (HIV)-positive patients. Indirect ELISAs for the detection of IgG antibodies and their subclasses (IgG1, IgG2, IgG3, and IgG4) to SAG2A and STAg were also performed. Results: The follow up of IgM and IgA levels to STAg showed a gradual decrease, with the majority of patients (88%) seropositive for IgM up to 12 months of infection, whereas IgA seropositivity was relatively low (78%) compared to IgM (100%) in the first 3 months of infection. The follow up of IgG and IgG1 antibodies showed a similar increasing profile for both SAG2A and STAg, with slightly higher seropositivity for STAg. The kinetics of IgG3 to STAg was similar to that of IgG1, contrasting with the kinetics of IgG3 to SAG2A that showed high levels up to 6 months of infection, with continuous decreasing over the time. Higher IgG3 seropositivity to SAG2A than STAg was also observed in the initial phases of infection. A higher IgG3/IgG1 ratio for SAG2A than STAg was detected in the first 3 months of infection, with decreasing profile over the time. Conclusions: Altogether, our results demonstrate a differential kinetics of IgG3 antibodies to SAG2A and STAg in patients with toxoplasmosis up to 12 months of infection. Also, the IgG3/IgG1 ratio to SAG2A in association with classical serological markers of acute phase could constitute valuable tools to distinguish early acute from convalescent phases of Toxoplasma gondii infection.

P1162 Activation of PPAR-alpha decreases in vitro nitric oxide production and increases mice susceptibility to Toxoplasma gondii infection G. Bonfa´,* L. Benevides,* M. T. C. Fonseca,* F. R. Guimara˜es, D. M. Fonseca,* N. M. Silva,à J. S. Silva* & C. R. B. Cardoso *Bioquı´mica e Imunologia, Faculdade de Medicina de Ribeiraõ Preto, Universidade de Saõ Paulo FMRP/USP, Ribeiraõ Preto, SP, Brazil, Ana´lises Clıńicas Bromatolo´gicas e Toxicolo´gicas, Faculdade Cieˆncias Farmaceûticas de Ribeiraõ Preto, Universidade de Saõ Paulo FCFRP/ USP, Ribeiraõ Preto, SP, Brazil, àInstituto de Cieˆncias Biome´dicas, Universidade de Uberlaˆndia UFU, Uberlaˆndia, MG, Brazil Purpose/Objective: Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors, consisting of three known isoforms (a, b and c), of which PPARa is the most studied. PPARs function as lipid sensors, regulating metabolism and immune response by acting in the inhibition of pro-inflammatory cytokines. Toxoplasma gondii infection induces a robust Th1 inflammatory response with excessive nitric oxide (NO) production. The role of PPARs in the gut inflammation caused by T. gondii in different susceptible mice lineages is still unclear and the aim of this work was to evaluate the expression and activation of PPAR-a and its role in the susceptibility to T. gondii infection. Materials and methods: C57BL/6 mice were orally infected with cysts of T. gondii, ME-49 strain. The transcripts of PPAR-a were evaluated at 0, 4, 6 and 8 days post-infection (p.i.) by real time PCR (qPCR) of ileum and liver on mice inoculated with 40 cysts. One group of animals infected with five cysts was treated with PPAR-a agonist (Gemfibrozil) for mortality evaluation. NO production was assessed in the supernatant of splenocytes cultured for 48 and 72 h after T. gondii infection and agonist treatment in vitro. Results: PPAR-a expression was downregulated in ileum and liver at day 8 p.i. when compared to the previous periods evaluated. Interestingly, when mice were treated with PPARa agonist at 10 mg/


Abstracts 553 kg/day during 7 days p.i. there was an increase on mortality. In vitro infection and agonist treatment showed that PPAR-a activation reduced NO production of C57BL/6 mice infected spleen cells. Conclusions: These data indicate that PPAR-a is modulated by T. gondii infection with equals patterns of expression in ileum and liver in susceptible mice. Moreover, activation of PPAR-a induces an increase of mortality and susceptibility to infection besides a reduction in NO production caused by T. gondii. These results lead to a better understanding of the susceptibility to this infection and provide basis for future approaches aimed at controlling exacerbated gut inflammation.

P1164 ArtinM, a D-mannose-binding lectin extracted from Artocarpus integrifolia, plays a potent adjuvant role in immunization process against Neospora caninum

P1163 Apoptosis as mechanism of supression of regulatory T cells in Chagas disease

Purpose/Objective: Neospora caninum is a coccidian parasite, closely related to Toxoplasma gondii, with wide host range and worldwide distribution, causing neuromuscular disease in dogs and abortion or reproductive disorders in cattle. Neosporosis seriously impacts the dairy and beef industries leading to substantial economic losses. Cattle acquire the infection by horizontal transmission, through ingestion of food and drinking water contaminated with oocysts eliminated in faeces from canine definitive hosts; or more often, by transplacental vertical transmission, endogenous or exogenous, from an infected dam to her offspring during pregnancy. Vaccination is an important approach to control neosporosis, however, so far there is no effective vaccine to control this parasitic disease. ArtinM and Jacalin (JAC) are lectins from the jackfruit (Artocarpus integrifolia) that have important role in modulation of immune responses to pathogens. The major aim of the presente study was to evaluate the adjuvant effect of ArtinM and JAC in immunization of mice against neosporosis. Materials and methods: Six C57BL/6 mouse groups were subcutaneously immunized three times at 2-week intervals with Neospora lysate antigen (NLA) associated with lectins (NLA+ArtinM and NLA+JAC), NLA, ArtinM and JAC alone, and PBS (infection control). Animals were challenged with lethal dose of Nc-1 isolate and evaluated for morbidity, mortality, specific antibody response, cytokine production by spleen cells, brain parasite burden and inflammation. Results: Our results demonstrated that ArtinM was able to increase NLA immunogenicity, inducing the highest levels of specific total IgG and IgG2a/IgG1 ratio, ex vivo Th1 cytokine production, increased survival, the lowest brain parasite burden, along with the highest inflammation scores. In contrast, NLA+JAC immunized group showed intermediate survival, the highest brain parasite burden and the lowest inflammation scores. Conclusions: In conclusion, ArtinM presents stronger immunostimulatory and adjuvant effect than Jacalin in immunization of mice against neosporosis, by inducing a protective Th1-biased pro-inflammatory immune response and higher protection after parasite challenge.

M. P. S. Damasio,* M. O. C. Rocha, K. S. Ferreira,* V. A. A. Valente,* G. R. Sousa,à R. C. G. Fares,§ R. Correa-Oliveira§ & J. A. S. Gomes* *Morphology, Federal University of Minas Gerais, Belo Horizonte, Brazil, Clinical Medicine, Federal University of Minas Gerais, Belo Horizonte, Brazil, àTropical Medicine and Infectology, Federal University of Minas Gerais, Belo Horizonte, Brazil, §Cellular and Molecular Immunology Laboratory, Rene Rachou Research Center, Belo Horizonte, Brazil Purpose/Objective: The Chagas disease is the fourth most important tropical disease, which affects approximately 15 million people in Latin America. The mechanisms involved in the development of the severe forms of this illness are not well known yet. Recently, it has been observed an important protective role of regulatory Tcells in this disease, although its mechanism remains unclear. Thus, the aims of this study is to evaluate apoptosis as possible mechanism used by regulatory Tcells (CD4+ CD25+Foxp3+) and characterize the profile cytokines in Chagas disease. Materials and methods: To this end, the peripheral blood of 11 patients were collected and the analyzed by flow cytometry. The patients were grouped in the indeterminate (IND) and cardiac (CARD) clinical forms of Chagas disease. To evaluate the apoptotic profile, it was used monoclonal antibodies reactive to PD1, PD1L, CD39, CD95, CD95L, CTLA-4, GITR, CD107 and granzyme B conjugated with PERCP, APC, PE and FITC fluorochromes. The profile of cytokines was also evaluated through the staining of IL-1b, IL-17, TGF-b and IL-10 conjugated with PE fluorochrome. Results: The results show no significative difference in the expression of CD95, CD95L, CTLA-4, GITR and granzyme B after comparison between patients of the different groups in the presence or absence of stimulus. However, it may be observed a higher expression of PD1 after stimulus in the IND group (P = 1.000) and CARD group (P = 1.000). In addition to a reduction in the expression of CD39 after stimulus in the IND group (P = 0.312) and CARD group (P = 0.437). CARD patients present higher expression of PD-1L (P = 0.0173) than IND patients in the absence of stimulus. Moreover, the regulatory Tcells of CARD patients present higher expression of CD107 after comparison with and without stimulus (P = 0.0313). No significative difference was observed in the expression of IL-1b and IL-10. However, it may be observed a reduction in the expression of IL-17 after stimulus in both groups IND (P = 0.2087) and CARD (P = 0.3125). Furthermore, IND patients present higher production of TGF-b by regulatory Tcells, If compared to CARD patients (P = 0.1576) in the absence of stimulus. Conclusions: In conclusion, many mechanisms are involved in the apoptosis caused during the infection by T. cruzi and the presence of severe forms may be related to the fratricide of regulatory Tcells or the apoptosis of effector cells.

J. Mineo,* M. R. Cardoso,* C. M. Mota,* D. P. Ribeiro,* F. M. Santiago,* E. C. Arau´jo,* N. M. Silva,* T. W. Mineo,* M. C. Roque-Barreira & D. A. Silva* *Immunology Microbiology and Parasitology, Institute for Biomedical Sciences, Universidade Federal de Uberlaˆndia, Uberlandia, Brazil, Cellular and Molecular Biology and Pathogenic Bioagents, Faculdade de Medicina de Ribeiraõ Preto, Universidade de Saõ Paulo, Ribeiraõ Preto, Brazil

P1165 B cell epitope screening on Clonorchis sinensis through in silcoprediction and peptide microarray analysis M. R. Lee, D. W. Kim, Y. J. Kim, S. H. Lee, S. H. Cho, W. J. Lee & J. W. Ju Divison of Malaria & Parasitic Disease, Korea National Institute of Health, Cheonwon-gun, Korea Purpose/Objective: Serological diagnosis of clonorchiasis with crude antigens of C. sinensis is hampered by the cross reactivity with other trematode. Therefore identification and the use of highly sensitive and specific antigens are essential for the improvement and standardization of the serological diagnosis of clonorchiasis. In this study, we have studied to find strong antigen of C. sinensis via B cell epitope screening.


554 Poster Session: Myeloid Cell Development Materials and methods: We selected 22 peptides on 52 antigenic proteins which were reported and identification in C. sinensis utilizing five epitope prediction tools (BepiPred, BCPred, FBCPred, AAPPred and Antigenic). These peptides ranged between 13 and 29 amino acids were chemically synthesized and conjugated with biotin respectively. The peptides were spotted onto microarrays and screening the reactivity against sera from clonorchiasis patients. Seropositive human serum samples (n = 50) were collected from egg positive cases and seronegative human serum samples (n = 20) were volunteer healthy human. Results: From a total of 22 peptides contained in the microarray, nine peptides showed the antigenic reactivity against pooled sera from clonorchiasis patients. The positive peptides that reacted above the cutoff determined from volunteer healthy human. The highest of antigenic peptides were ProR1 and Cys7 derived from Proline rich protein and Cysteine proteinase, respectively. When the antigenicity of both peptides was analyzed with individual serum of clonorchiasis patients, the sensitivity showed different pattern. Conclusions: In the current study, we have identified two antigenic peptides for effective serological diagnosis could useful for diagnostic development. Our study provides the proof that the bioinformatic prediction and peptide microarray analysis are valuable tools for the discovery of C. sinensis-derived epitopes as serodiagnostic antigens with sufficient sensitivity and specificity.

P1166 B-1 cells increase susceptibility to murine visceral leishmaniasis V. Xavier,* B. C. Vivanco, W. F. K. M. Gonzaga,* C. L. Barbieri, J. D. Lopes & P. Xander* *Cieˆncias Biolo´gicas, Universidade Federal de Saõ Paulo campus Diadema, Saõ Paulo, Brazil, Microbiologia Imunologia e Parasitologia, Universidade Federal de Saõ Paulo, Saõ Paulo, Brazil Purpose/Objective: Leishmaniasis belongs to a group of diseases caused by protozoan parasites from Leishmania genus. The immune response to leishmaniasis is complex and the result of infection depends both on the genetic composition of different species of Leishmania and the host immunity. Clinical and experimental evidences suggest that activation of B cells leads to exacerbation of visceral leishmaniasis and contributes to susceptibility of cutaneous leishmaniasis. B-1 cells are a subtype of B lymphocytes whose role in the physiology of the immune system as well as in the pathogenesis of various diseases is still poorly understood. These cells produce large amounts of IL-10 cytokine which plays a key role in immunosuppression in several diseases such as leishmaniasis. In this study we investigated the role of B-1 cells in the pathogenesis of visceral leishmaniasis. Materials and methods: Experiments were performed with BALB/c, BALB/Xid or BALB/Xid mice that receive adoptively peritoneal B-1 lymphocytes. These groups were infected or uninfected with L. (L.) chagasi. Results: Our results showed that BALB/Xid mice, a mouse strain deprived of B-1 cells, infected for 45 days with L. (L.) chagasi had a significant reduction in parasite load in the spleen (0.63 · 107 ± 0.4 parasites/mg tissue) when compared to control animals BALB/c (1.87 · 107 ± 0.6 parasites/mg of tissue) or BALB/ Xid that received adoptive transfer of B-1 cells (1.44 · 107 ± 0.5 parasites/mg tissue). These data were also confirmed by qPCR. Flow cytometry analysis demonstrated changes in peritoneal B-1 cell population. Infected BALB/c mice showed a significant increase in the percentage of peritoneal B-1 cells (70.0% ± 1.50) compared to control (63.3% ± 1.88). On the other hand, BALB/Xid that received adoptive transfer of B-1 cells and were infected with L. (L.) chagasi showed a significant decrease in the percentage of peritoneal B-1 cells

(6.14 ± 3.01%) when compared to uninfected group (25.48 ± 7.60%). The IL-10 production was evaluated in supernatants from spleen homogenates of uninfected and infected mice. No differences were detected between BALB/c uninfected and infected group. However, BALB/Xid that received adoptive transfer of B-1 cells and were infected with parasite had higher levels of IL-10 when compared with unifected mice. Conclusions: Our results suggest a possible role of B-1 cells in susceptibility to L. (L.) chagasi.

P1167 CD8+ T cell epitopes: a new source for vaccination against cutaneous Leishmania major infections K. Schwonberg,* S. Brosch,* G. van Zandbergen, B. Grewe,* M. N. Harndahl,à S. Buus,à H. Schild,§ S. Tenzer§ & E. von Stebut* *Dermatology, University Medical Center of the Johannes Gutenberg, University of Mainz, Mainz, Germany, Divsion of Immunology, PaulEhrlich-Institut, Langen, Germany, àPanum Institute 18.3, University of Copenhagen, Copenhagen, Denmark, §Institute of Immunology, University Medical Center of the Johannes Gutenberg, University of Mainz, Mainz, Germany Purpose/Objective: Infections with the parasite Leishmania (L.) major are caused by the bite of a sandfly. Healing is eventually based on IFNg secretion by CD8+ and CD4+ T cells. However, it is still unclear, which peptides finally promote protection and long-lasting immunity against infections with L. major. Up to date, no vaccine against this human pathogen exists. Thus, we are highly interested in identifying peptides that mediate healing of L. major infections. Materials and methods: The most abundant proteins of both lifeforms were identified by mass spectrometry. Subsequently, from these proteins, epitopes were predicted using the computer-based algorithm SYFPEITHI. 300 Kb/Db peptides were chosen based on their predicted immunoreactivity. Additionally, all peptides were analysed for their binding-affinity towards MHC class-I molecules. A correlation between the predicted and meassured binding affinity of the peptides towards MHC class-I molecules was observed. Further, all 300 peptides were tested in in vitro assays. To this aim, peptide-loaded dendritic cells of C57BL/6 mice were cocultured with primed CD8+ T cells for 48 h. Results: Supernatants were analysed for their IFN-g, IL-4 and IL-10 content. Interestingly, we identified ~20 peptides with induction of high amounts of IFN-g and low levels of IL-4 and IL-10. Based on their cytokine profile, we randomly chose 16 peptides for further in vivo experiments. Here, C57BL/6 mice were immunized either in a prime/ boost (P/B) (10 lg peptide twice) or a prime/boost/boost (P/B/B) (20 lg/2 · 10 lg peptide) approach in one ear. Three weeks later, mice were infected with 1 · 103 metacyclic promastigotes in the alternate ear. Lesions volumes were assessed weekly. Surprisingly, five peptides partially protected mice against challenge using the P/B approach compared to PBS-control mice. This effect even increased, when mice were immunized in the P/B/B approach. Conclusions: In summary, identifying peptides which protect mice against challenge with L. major would finally lead to a long desired vaccine against this human pathogen. Further, tetramer development with L. major specific peptides would aid the understanding of T cell priming during parasitic infection.


Abstracts 555 P1168 Characterization of the virulence of Leishmania infantum isolates from human patients J. Cunha,* E. Carrillo, C. Sanchez, J. Tavares,* J. Moreno & A. Cordeiro-da-Silva* *Parasite Disease Group, Institute for Molecular and Cell Biology, Porto, Portugal, WHO Collaborating Center for Leishmaniasis National Center of Microbiology, Institute of Health Carlos III, Madrid, Spain Purpose/Objective: Leishmaniasis is a group of diseases with a variety of clinical manifestations. The severity of the disease is highly dependent on the immunological status and genetics of the host. The virulence of the species/strain also plays an important role in the progression of the infection. In this study we have characterized some strains of L. infantum in terms of in vitro cultivation and differentiation through the life cycle of promastigotes and compared their virulence and infectivity in vivo. Materials and methods: L. infantum strains used in this study were isolated from 2 HIV/Leishmania-coinfected patients and 1 immunocompetent patient with cutaneous disease. We defined the best culture conditions to grow the parasites, having tested five well-established culture media that differ in their complexity and nutrients availability. Growth curves, viability analysis, cell cycle progression and metacyclogenesis-specific Meta-1, SHERP and Histone H4 relative expression were evaluated. In adition, Leishmania infections in bone marrowderived macrophages were executed and final assays of virulence were performed in Balb/c mice. Results: From the culture media tested, NNN medium demonstrated to maintain viability and to generate stationary promastigotes, allowing the differentiation into metacyclic forms for all strains. In vitro infections showed one of the tested strains to be more infective and resistant to the hostile intracellular environment of the macrophages. We have confirmed this result with Balb/c mice infections, where this differential virulence was maintained in acute and chronic infections. In addition, other assays were carried out using mice previously infected with each strain and challenged with the most virulent strain. Mice infected with least virulent strains showed high parasite loads in spleen, liver and bone marrow; however animals inoculated and reinfected with the most virulent strain induced lower parasite loads and a protective response. Conclusions: In conclusion, this work has shown an efficient approach to characterize the virulence of Leishmania infantum strains and to study some of the underlying mechanisms that makes a host to be capable of control Leishmania infection. Acknowledgements: We thank Dr Maria da Luz Duarte from S‘o Marcos Hospital, Braga, Portugal, for kindly provide us with the virulent strain of L. infantum.

P1169 Chronic infection drives co-expression of the inhibitory receptor CD200R, and its ligand CD200, in mouse and human T cells S. Caserta, N. Nausch, A. Sawtell, R. Drummond, A. PhythianAdams, A. S. MacDonald, F. Mutapi & R. Zamoyska Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, UK Purpose/Objective: Certain parasites evade the immune response and establish chronic infections that may persist for years. Under these conditions, upregulation of surface receptors with inhibitory properties regulate immune cellular activation and associated pathology. The negative regulator, CD200 receptor, and its ligand, CD200, regulate macrophage activation and reduce pathology in viral infections. Little is known about the modulation of CD200:CD200R in T cells and during chronic helminth infection.

Materials and methods: T cells were either transiently or chronically stimulated with anti-CD3/CD28 and peptide, in vitro, or recovered from different organs (liver, spleen and mesenteric lymph nodes) of chronically infected mice with the trematode, Schistosoma mansoni (80 Cercariae, 8 weeks post-infection). T cell expression of CD200: CD200R was investigated alongside expression of activation markers (CD44, CD25 and CD69) and secretion of cytokines (IL-2, TNFa, IFNg and IL-4) following 5 h-re-stimulation with PdbU and Ionomycin by flow cytometry. Additionally, expression of CD200R in T cells in PBMC from individuals endemically exposed to Schistosoma heamatobium was correlated to cytokine secretion and parasite egg counts in the urine. Results: T cells co-express CD200 and CD200R after TCR stimulation. Sustained expression of CD200R required chronic rather than transient TCR stimulation and coincided with loss of multifunctional potential (IL-2 and TNFa) and terminal effector differentiation in T cells. Terminally differentiated CD4 T cells increased expression of CD200R in response to chronic helminth infection, with Schistosoma mansoni, particularly in organs of pathogen persistence, where Th-2 effectors accumulate in infection. Similarly, in humans infected with Schistosoma haematobium, we detected an association between IL-4 production and CD200R expression on T effector cells that correlated effectively with egg burden and, thus infection intensity. Conclusions: CD200:CD200R in T cell responses to helminths has diagnostic and prognostic relevance as a marker of infection for chronic schistosomiasis in mouse and man. CD200R efficiently marks terminally differentiated effectors. We suggest that interfering with CD200:CD200R may recover functional properties of Th2 effectors.

P1170 Evaluation of IL-17 expression in human Chagas disease G. R. Sousa,* R. Correa-Oliveira, M. C. P. Nunes,à R. C. G. Fares, A. T. Chaves, M. P. S. Damasio,§ K. S. Ferreira,§ V. A. A. Valente,§ J. A. S. Gomes§ & M. O. C. Rochaà *Tropical Medicine and Infectology, Federal University of Minas Gerais, Belo Horizonte, Brazil, Molecular and Cellular Immunology Laboratory, Rene Rachou Research Center, Belo Horizonte, Brazil, àClinical Medicine, Federal University of Minas Gerais, Belo Horizonte, Brazil, §Morphology, Federal University of Minas Gerais, Belo Horizonte, Brazil Purpose/Objective: This study was designed to investigate whether the expression of IL-17 is associated with the no apparent heart disease or cardiomyopathy clinical forms of Chagas disease. Materials and methods: It was employedacross-sectionaldesigninvolvingpatientsfromendemicareaswithin MinasGerais, Brazil, underthe medicalcareofone of us (MOCR). Positive serology for Chagas disease was determined by two or more different tests. The patients infected with T. cruzi were grouped as no apparent heart disease (NAHD) and cardiomyopathy (CARD) patients ranging in age from 18 to 65 years old. The NAHD group included 82 individuals, with no significant alterations in electrocardiography, chest X-ray and echocardiogram. All 94 CARD patients presented with dilated cardiomyopathy, characterized by the echocardiographic finding of a dilated left ventricle with impaired ventricular systolic function. A total of 24 Normal healthy individuals, ranging in age from 29 to 55 years old, from a non-endemic area for Chagas disease and showing negative serological tests for the infection were included as a control group (NI). A cytometric bead array (CBA) immunoassay kit (BD Biosciences, USA) was used for the analysis of plasmatic cytokines, including IL17. A second fluorescently labeled PE-anti-cytokine antibody was added and the concentration of the individual cytokines was indicated by their Mean Fluorescence Intensity (MFI). Data were acquired in a FACScalibur flow cytometer (Becton Dickinson-BD) and the analyses were performed using BD CBA software.


556 Poster Session: Myeloid Cell Development Results: It was compared the three groups with relation to the expression of IL-17. The Kruskal-Wallis test was used with the significance of 5%. The hypothesis of equality between the groups was rejected. The comparison was performed between groups of two. It was used the Wilcoxon test with Bonferroni correction (significance level = 0.05/3 = 0.0167). It was observed difference between the groups CARD and NAHD (P-value < 0.001) and between NI and NAHD (P-value = 0.008). There was not significative difference between the groups CARD and NI (P-value = 0.376). The expression of IL-17 was higher in patients of the NAHD group, If compared with patients in the other groups, median of 11.81 (5.79 22.86) versus 5.38 (3.87 10.55) of CARD group and 5.715 (4.25 11.31) of the NI group. Conclusions: Therefore, IL-17 expression seems to be associated with a protective cardiac function in human Chagas disease.

P1171 Evaluation of serum cytokine concentrations of patients in different clinical stages of Chagas’ disease M. C. Chambela,* P. E. A. A. do Brasil,* S. S. Xavier,* A. M. Hasslocher-Moreno,* R. M. Saraiva,* L. H. C. Sangenis,* A. S. Souza,* J. de Meis, L. DeCastro* & G. M. Sperandio da Silva* *Institute of Clinical Research Evandro Chagas, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil Purpose/Objective: The aim of this study was to evaluate serum concentrations of cytokines IL-4, IL-10, IL-12, TNF-a and IFN-c in patients in different clinical forms of Chagas’ disease. Materials and methods: We conducted a case-control study of 115 individuals. Infected individuals were divided into four stages as the Brazilian consensus of Chagas’ disease: indeterminate patients or without apparent heart disease (normal electrocardiogram and echocardiogram) cardiac patients on the stage A (electrocardiogram changes and normal echocardiogram), cardiac patients on the stage C [electrocardiogram and echocardiogram altered with compensable congestive heart failure (CHF)] and cardiac patients on the stage D (electrocardiogram and echocardiogram changed with refractory CHF). Also were included uninfected individuals with T. cruzi. We included 30 indeterminate patients, 31 cardiac patients on the stage A, 14 cardiac patients on the stage C, 11 cardiac patients on the stage D and 29 uninfected individuals. Results: Among the pro-inflammatory cytokines, IFN-c showed higher serum levels in relation to IL-12 and TNF-a. The cardiac patients on the stage A had higher concentrations of TNF-a, however, there was a significant decrease in the concentrations of this cytokine in the same time that we observed the later stages of the chronic Chagas cardiomyopathy (CCC). Both indeterminate and cardiac patients showed high levels of TFN-a and IFN-c and low levels of IL-4 and IL-10, demonstrating a dominant Th1 profile with an imbalanced immune response. Conclusions: This study demonstrated a direct proportionality in concentrations of pro-inflammatory and anti-inflammatory cytokines with respect to the left ventricular ejection fraction in all groups of patients, suggesting that this correlation could be used as a marker of progression to CCC.

P1172 IgG and IgM anti-toxoplasma gondii antibodies class in high risk pregnant women in Brazil C. C. B. de Mattos,* F. Nakashima,* M. B. Vono,* T. Pandossio,* A. C. F. Rodrigues,* S. Uezato,* L. C. J. F. Spegiorin, A. H. Oliani, D. C. M. Vaz Oliani & L. C. de Mattos* *Immunogenetics Laboratory, Department of Molecular Biology, Faculdade de Medicina de Saõ Jose´ do Rio Preto FAMERP, Saõ Jose´ do Rio Preto, SP, Brazil, Gynecology and Obstetrics Department Faculdade de Medicina de Saõ Jose´ do Rio Preto FAMERP and Hospital de Base FUNFARME, Faculdade de Medicina de Saõ Jose´ do Rio Preto FAMERP, Saõ Jose´ do Rio Preto, SP, Brazil Purpose/Objective: Toxoplasmosis is a zoonosis caused by Toxoplasma gondii, an apicomplexa obligate intracellular protozoan parasite, which infects different species including humans. Fetuses can be infected by transplacental transmission, a condition that may cause significant sequelae in babies. The knowledge of the antigenic profile of IgM and IgG anti-T. gondii antibodies during the gestational period gives clinical support towards preventing the fetal infection by this parasite. This study aimed to verify the serological profile of the pregnant women from S‘o Jos* do Rio Preto, S‘o Paulo State, Brazil, which received medical attention in a public health service from a tertiary school hospital. Materials and methods: The medical records of 793 pregnant women, attended in the High Risk Antenatal Care and Fetal Medicine, Gynecology and Obstetrics Outpatient Clinic of the Hospital de Base in S‘o Jos* do Rio Preto, S‘o Paulo State, Brazil, between 2001 and 2004, were analyzed. The serology tests performed to determine specific IgG and IgM anti-T. gondii antibodies were based in a commercial immunoenzymatic assay kit (ETI TOXOK * IgG, ETI TOXOK * IgM DiaSorin, Italy). Results: From the overall data, 503 (63.4%) were reagent and 290 (26.6%), non reagent. Among the reagent pregnant women, 32 (4.0%) presented a serology profile with both anti-T. gondii antibodies IgG and IgM positive and 471 (59.4%), with anti-T. gondii antibodiesIgG positive and IgM negative. The profile of anti-T. gondii antibodies IgG negative and IgM positive was not found. Conclusions: The prevalence of T. gondii infection is high in the region where this study was carried out and the majority of the pregnant women with positive serology tests are not under risk of gestational transmission of this parasite. However, considering that 4.0% of the pregnant women are under risk of gestational transmission, these results attract the attention for the necessity to implement a protocol for serological and molecular screening of mother and newborn babies, to contribute with the clinical guidance and to prevent babies sequelae.

P1173 IL-10 secreting, type 1 regulatory T cells and naturally occurring regulatory T cells differently modulate IgG secretion by B cells in human hypo-responsive onchocerciasis T. Adjobimey,* C. von Horn,* J. Satoguina,* J. Oldenburg, L. E. Layland* & A. Hoerauf* *Parasitology, Institute for Medical Microbiology Immunology and Parasitology (IMMIP), Bonn, Germany, Hematology, Institute of Experimental Hematology and Transfusion Medicine, Bonn, Germany Purpose/Objective: Onchocerciasis or river blindness is the second leading infectious cause of blindness after trachoma. The disease is caused by infections with the filarial nematode Onchocerca volvulus and usually present two distinct pathological forms: the hyper-reactive or Sowda form and the hypo-reactive or generalized form. The hyporeactive form is characterized by anergy and tolerance to the parasite,


Abstracts 557 whereas the Sowda form is associated with strong local immune reactions leading to immunopathology. The cellular and molecular patterns associated with this polarization of the disease are still not fully clarified. The present study has focused on the capacity of Tr1 and Foxp3 expressing regulatory T cells Tregs isolated according to their expression of CD25 alone, CD25 and CD127, and CD25 and CD49d, to modulate B cells antibody secretion in the presence or absence of additional B cell activating signals. Materials and methods: The different cell subsets were isolated from filarial-infected individuals or healthy blood spenders, using magnetic cell isolation. Purity and cell phenotype were assessed by FACS analysis. Cytokines and antibody secretion were analyzed by ELISA and cytometric bead array. Results: We could show that, the secretion of the non-cytolytic immunoglobulin IgG4 constitutes a humoral signature, associated with hyporesponsiveness and tolerance in human onchocerciasis. Using a sequential depletion-reconstitution strategy on peripheral blood mononuclear cells from patients with the generalized form of onchocerciasis, we could also demonstrate that, both Tr1 and Treg cells are involved in this IgG4 secretion. However, direct co-cultures of Tr1 and Tregs with autologous B cells reveal significant differences in the modulation of B cells antibody secretion by the two regulatory T cell types. While Tr1 are capable of activating B cells and preferentially induce IgG4 secretion, purified Foxp3 expressing CD4+ CD25+ regulatory T cells only weakly induced IgG4 secretion. Remarkable differences were also observable depending on the methods used for Treg purification. While CD4+ CD25+ Tregs slightly induced IgG4 secretion, CD4+CD25+CD127dim and CD4+CD25+CD49d- Tregs inhibited B cells activation and antibody secretion by reducing B cell proliferation, survival and maturation into plasma cells. Conclusions: These findings confirm the implication of both IL-10 secreting regulatory T cells and CD25+ Tregs in IgG4 induction during generalized onchocerciasis. Our results also suggest a direct role for Tr1 cells in IgG4 secretion by B cells, whereas Foxp3+ Tregs inhibit IgG secretion and only indirectly promote IgG4 secretion.

P1174 IL-7 reverses T cell hypo-responsiveness in chronic schistosomiasis S. Caserta, R. J. Lundie, A. Phythian-Adams, A. S. MacDonald & R. Zamoyska Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, UK Purpose/Objective: Schistosomiasis is a major cause of morbidity worldwide. Infected individuals are susceptible to re-infection and lack effective T cell memory. The T cell response against schistosomes is a combination of activation, hypo-responsiveness/anergy and strong immunoregulation. IL-7 plays non-redundant roles in both B and T cell biology, crucially regulating thymopoiesis, naı¨ve T cell survival/ homeostasis, generation/maintenance of memory T cells, transition from effector to memory, and tumor-associated T cell exhaustion. We thus investigated whether IL-7 reverses T cell hypo-responsiveness in chronic schistosomiasis. Materials and methods: T cells were recovered from different organs (liver, spleen, mesenteric and subcutaneous lymph nodes) of naı¨ve and chronically infected mice with increasing dose of Schistosoma mansoni cercariae (8 weeks post-infection) and cultured with IL-7. Applications of IL-7 were tested during schistosomiais (7 8 weeks). T cell activation, polarization, exhaustion and hypo-responsiveness were evaluated by surface marker expression and secretion of cytokines (IL2, TNFa, IFNc, IL-5 and IL-4) following 5 h-re-stimulation with PdbU and Ionomycin by flow cytometry or in ELISA assays, upon SEA and anti-CD3-restimulation.

Results: In chronic schistosomiasis, depending on pathogen burden, T cells are widely activated leading to thymic atrophy accompanied by severe reduction in T cell numbers in lymphoid organs distal from the site of persistent infection. Activated cells localizing in the site of antigen persistence differentiate into effector/effector memory cells, with a reduction in less differentiated central memory CD4 T cells. These effector T cells show typical aspects of exhaustion (PD-1, CD200R), apoptosis (low Bcl-2) and hypo-responsiveness, while maintaining IL-7Ra expression. Application of IL-7 led to an increase in effector T cell cytokine responsiveness to schistosome antigens. Conclusions: Chronic schistosomiasis leads to T cell polarization and exhaustion in the sites of parasite antigen persistence. We suggest that IL-7 may be useful to recover functional properties of Th2 effector cells and may be involved in regulating immunopathology.

P1175 Acute and chronic immune response in Leishmania infantuminfected Rhesus macaques. A novel model for visceral leishmaniasis V. Rodrigues,1 M. Laforge,1 A. Cordeiro-da-Silva,2 R. Silvestre2 & J. Estaquier1 1 UFR Biome´dicale, CNRS FRE 3235 R.T.M.G., Paris, France, 2Intituto de Biologia Molecular e Celular, Parasite Disease Group, Porto, Portugal Purpose/Objective: Visceral leishmaniasis (VL) is a chronic infectious disease caused by the protozoan Leishmania infantum or L. donovani. Rodent models have been widely used to dissect the immune response developed during VL. However, significant variations between these and human disease claim a shift towards new models capable of accurately mimicking human VL. We developed a non-human primate model for VL in Rhesus monkeys (RM) to dissect parasite dynamics and counteractive immune responses developed during acute and chronic infection. Materials and methods: RMs of Chinese origin were intravenously inoculated with L. infantum (2 · 107 parasites/kg of body weight). Infected animals were subdivided in three groups that were sacrificed at day 11 post-infection (p.i.) (group 1), day 28 p.i. (group 2) and day 250 p.i. (group 3) for internal organ removal and analysis. Additionally, blood was sampled from remaining alive animals at days 7, 14, 21, 60 and 180 p.i. Results: Quantification of parasite loads revealed the presence of parasites in the spleen, blood, liver and bone marrow early after infection. Parasite dissemination to lymph nodes (LN) was observed at the chronic phase concomitant with an increase in parasite loads in the blood and visceral organs. Early infection was characterized by splenic CD4 and CD8 T cell differentiation towards effector-memory phenotypes accompanied by increased sensibility to FasL-mediated cell death. Increased transcript levels of splenic IL-21 and IFN-c were noticed at the acute phase. We demonstrated by intracellular cytokine staining the transient expansion of IL-21+ expressing CD4+ T cells associated with the production of specific IgM and IgG antibodies towards L infantum antigens. Consistent with the absence of parasite detection in peripheral LNs during acute phase, CD4 T cell differentiation to effector cells was modest without significant modifications on the cytokine microenvironment. In clear contrast, at the chronic phase, we observed decreased transcript levels of IFNc and TNFa and increased IL-10 in both spleen and LNs. Conclusions: Collectively, our findings point out the existence of a complex response to L. infantum infection in RMs with an early reaction of the host to infection, that includes expansion of IL21+ CD4 T cells, severely restricting parasite survival and an ability of the pathogen to colonize additional organs such as the LNs at chronic stages.


558 Poster Session: Myeloid Cell Development P1176 In Leishmania major infections the presence of leukocytes on C57BL/6 or BALB/c background is sufficient to induce a C57BL/6 or BALB/c phenotype, whereas stromal cells are not M. R. Fischer,* B. Lorenz,* K. Griewank & E. von Stebut* *Department of Dermatology, University Medical Centre Johannes Gutenberg University, Mainz, Germany, Department of Dermatology, University Clinic Essen, Essen, Germany Purpose/Objective: Susceptibility to Leishmania major in BALB/c mice the result of predominant Th2 development, whereas C57BL/6 mice show a protective Th1 response. Previously, we reported that this is * in part a result of differences in DC cytokine production, e.g. IL1a/b and IL-12p80 homodimer. Others demonstrated the importance of cytokine production by stromal cells, e.g. keratinocytes. Applying a bone marrow chimera model we aimed to determine the key cell population(s) responsible for this strain-dependent dichotomy. Materials and methods: Irradiated mice were injected with 5 · 106 bone marrow (BM) cells. Six to 10 weeks later, hematopoietic engraftment was analyzed by flow cytometry of blood samples. Reconstituted mice were infected with 2 · 105 L. major promastigotes intradermally into both ears and lesion development was measured weekly. Results: To investigate this we used BALB/cxC57BL/6 F1 mice reconstituted with either BALB/c or C57BL/6 BM. Interestingly, the course of disease in infected mice reflected the origin of the BM: Mice reconstituted with BALB/c BM showed rapid disease progression, whereas mice reconstituted with C57BL/6 BM developed self-healing lesions. To further narrow down the key cell population(s) causing this effect, we applied a mixed bone marrow chimera model with mice differing only in T and B cells being either of BALB/c or C57BL/6 origin. Everything else, including the hematopoietic compartment, was uniformly BALB/cRag1-/- · C57BL/6Rag1-/- F1. These mice exhibit a disease phenotype according to the lymphocyte origin. Preliminary data shows that this holds also true in mice with C57BL/6 lymphocytes and the rest of the hematopoietic cells being of BALB/cRag1-/- origin (and vice versa). Conclusions: These experiments showed clearly, that the presence of BALB/c-derived lymphocytes is not protective, while the presence of C57BL/6 lymphocytes is sufficient for protection. To further investigate the relevance of the genetic background of individual cell populations in this important disease, we have reconstituted mice leading to the concomitant presence of C57BL/6 T and BALB/c B cells and vice versa. Overall, the method of mixed bone marrow chimeras offers various opportunities to test the effects of individual cell populations in L. major infections as well as several other diseases.

P1178 Induction of IL-10-producing CD1dhighCD5+ regulatory B cells following Babesia microti-infection Y. I. Jeong, S. H. Hong, S. H. Cho, W. J. Lee & S. E. Lee KNIH, maralia & parasitic Disease, Cheonwon-gun, Korea Purpose/Objective: Understanding the induction of immune regulatory cells upon helminth infection is important for understanding the control of autoimmunity and allergic inflammation in helminth infection. Babesia microti, an intraerythrocytic protozoan of the genus Babesia, is a major cause of the emerging human disease babesiosis, an asymptomatic malaria-like disease. We examined the influence of acute infection by B. microti on the development of regulatory B cells together with regulatory T cells. Materials and methods: To establish a well-defined animal model for B. microti infection, we monitored Babesiosis during the acute phase of infection. To confirm successful infection, spleens were weighed and

parasitemia was calculated. Next, to determine whether B. microti infection selectively induces Bregs, we analyzed the expression of cell surface markers on various splenic B cell subpopulations in uninfected and infected mice. Flow cytometry was performed to examine the frequency of IL-10-secreting CD1dhighCD5+ B cells and CD4+ CD25+ FoxP3+ Tregs in the spleen of B. microti-infected mice. Results: B. microti infection induced interleukin-10-producing CD1dhighCD5+ regulatory B cells. Moreover, the CD4+ CD25+FoxP3+ T cell frequency was increased significantly during the course of B. microti infection. After infection with B. microti, a high level of IL-10 was detected in the serum throughout the experiment. Conclusions: This study is the first demonstration of the expansion of immune regulatory cells such as regulatory B cells following infection by an intraerythrocytic protozoan parasite. These data suggest that B. microti infection in mice provides an excellent model for studying Breg-mediated immune responses, as well as indirectly elucidating the mechanism by which helminth infection regulates autoimmunity and allergic inflammation.

P1179 Insight in the intestinal immune response during a Giardia muris infection in BALB/c mice L. Dreesen, E. Claerebout & P. Geldhof Laboratory of Parasitology, Ghent University, Merelbeke, Belgium Purpose/Objective: The protozoan parasite Giardia duodenalis is one of the most commonly found intestinal pathogens in humans and animals. Previous work on Giardia infected calves revealed an upregulated expression of the peroxisome proliferator-activated receptors (PPAR) in infected animals. The upregulation of these transcription factors and their anti-inflammatory capabilities could possibly explain the chronic nature of a Giardia infection. The aim of this study was to further unravel the role of these transcription factors in the host response using a Giardia muris mice infection model. Materials and methods: In a first trial, the intestinal immune response in BALB/c mice was investigated 1, 2 and 3 weeks after an infection with G. muris. Cyst secretion in the faeces was counted daily and intestinal gene expression levels for a panel of cytokines and PPARs were examined using qRT-PCR. In a second trial, the effect of a PPARa agonist (fenofibrate) on the intestinal immune response during a Giardia infection was analysed and compared to infected control mice and mice just receiving the agonist. Identical as in experiment 1, cyst secretion was monitored daily and changes in gene expression levels were measured by qRT-PCR. Results: Analysis of the cytokine response in trial 1 showed a major upregulation of IL17A from 1 week p.i. onwards. The highest upregulation was observed at week 3, coinciding with a drop in cyst secretion. In contrast to the situation in calves, no transcriptional changes were observed for the PPARs. Identical as in trial 1, IL17 expression was also significantly upregulated in all the infected animals of trial 2, with the highest levels found in the infected animals treated with the fenofibrate. Analysis of the PPARs showed that, in contrast to trial 1, a modest upregulation of PPARa expression was observed in the Giardia infected control mice. Although this upregulation was not further induced by the fenofibrate treatment, animals receiving the fenofibrate shedded significantly less cysts compared to control animals during the first 3 weeks of an infection. Conclusions: The outcome of this study suggests that IL17 plays an important role in the protective immune reponse against G. muris in mice. The role of PPARa in the immune response is still unclear and needs further research.


Abstracts 559 P1180 Investigation into the metabolic profile and transcriptional regulation of alternatively activated macrophages during helminth infection G. Thomas,* D. Ruckerl,* B. Maskrey, P. Whitfield, M. Blaxterà & J. Allen* *Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, UK, Lipidomics Research Facility, University of the Highlands and Islands, Inverness, UK, àInstitute of Evolutionary Biology, University of Edinburgh, Edinburgh, UK Purpose/Objective: Chronic inflammation is the hallmark of many diseases, in particular those associated with the metabolic syndrome. A growing body of evidence links alterations in cellular metabolism with inflammation, in particular classical macrophage activation with glycolysis. In vitro studies have defined a role for mitochondrial lipid metabolism, and the transcription factor PPARc, in maintaining antiinflammatory alternatively activated macrophages (AAMF). Using the helminth infection model Brugia malayi, a potent inducer of AAMF, we sought to investigate the role of lipid metabolism in AAMF in vivo. Materials and methods: We obtained MF from wild type or IL4Ra-/mice 21 days after B. malayi (NeMF) infection, or 3 days post thioglycollate injection (ThioMF). MF were FACS purified and RNASeq was performed. After gene expression quantification and differential expression analysis, coordinately regulated metabolic pathways were identified using GSEA. Transcription start sites were identified using a novel method, and over-represented transcription factor binding sites (TFBSs) were assessed using Clover. Mass spectroscopy was used to identify MF-derived eicosanoids elicited in the same manner as for the RNA-Seq experiment. Results: NeMF up-regulated mitochondrial gene expression in vivo. Surprisingly NeMF expressed very low levels of PPARc, but did express PPARd. Analysis of cis-regulatory features in AAMF promoters revealed an over-represented PPAR response element. Finally, lipidomic profiling of macrophage-derived eicosanoids showed that AAMF abundantly produce the PPARd ligand prostacyclin (PGI2). Conclusions: We show that NeMF demonstrate enhanced mitochondrial metabolism, in particular the TCA cycle. Furthermore, the overrepresented PPAR motif in AAMF promoters provides evidence for PPAR-dependent gene expression in vivo. Although PPARc expression levels are low in NeMF, we show that NeMF abundantly produce the PPARd ligand PGI2 providing a mechanism for PPAR-dependent transcriptional regulation in NeMF in vivo.

P1182 LL37 specific killing of Leishmania parasites inside human macrophages E. Bank,* P. Crauwels,* N. Reiling, & G. van Zandbergen* *Paul-Ehrlich-Institut, Immunology, Langen, Germany, Forschungszentrum Borstel, Laborgruppe Mikrobielle Grenzfla¨chenbiologie, Borstel, Germany Purpose/Objective: Leishmaniasis is induced by promastigote parasites. Inside macrophages (MF) parasites change into multiplying amastigotes, propagating disease. There are different phenotypes of human MF, such as inflammatory type I (MF I) and anti-inflammatory type II (MF II) and it is not yet clear which phenotype of MF is involved in parasite propagation or killing. In a murine model for Leishmania infection it was demonstrated that parasites can be killed in an iNOS dependent manner. Materials and methods: For human MF it is unclear which antiparasitic mechanisms are used to control Leishmania. In this study we search for MF phenotype specific control mechanisms of both Leishmania major (L. major) promastigotes and amastigotes.

Results: We found that MF II are more susceptible to infection as compared to MF I. Characterizing both phenotypes in more detail revealed the antimicrobial peptide LL37 is up-regulated in MF I as compared to MF II. LL37 is shown to be involved in the innate host defence against various pathogens. Consistent with this we found that recombinant human LL37 can kill L. major parasites, extracellularly. To demonstrate its intracellular activity against Leishmania, we established a siRNA knockdown for LL37 in primary human MF. Subsequently, we found that a LL37 knockdown in MF I resulted in a significant higher promastigote survival, as compared to control MF I. Conclusions: In conclusion, we demonstrate that LL37 is able to specifically kill L. major promastigotes, not amastigotes, in human MF I. Interestingly, the lack of anti-leishmania LL37 activity in MF II suggests them to be more suitable host cells.

P1183 Lymphoid organs behavior and dinamic of lymphocyte populations in Trypanosoma cruzi infected mice by oral or intraperitoneal route J. Barreto de Albuquerque,* D. A. Farias-de-Oliveira,* J. Juberg,* V. Cunha,* C. E. Carvalho-Pinto, D. Silva-dos-Santos,* L. R. Berbert,* W. Savino,* & J. de Meis* *FIOCRUZ, Oswaldo Cruz Institute, Rio de Janeiro, Brazil, Fluminense Federal University, Immunobiology, Niteroí, Brazil Purpose/Objective: To analyze subcutaneous lymph nodes, mesenteric lymph nodes, spleen and Peyer patches behavior, dynamic of lymphocyte populations and cytokine production in experimental Chagas‘ disease infected by oral/intragastric (IG) or intraperitoneal (IP) route. Materials and methods: BALB/c mice were infected with 5 · 104 Trypanosoma cruzi trypomastigotes IP or IG and parasitemia was followed. After 9, 12, 16, 27 days, subcutaneous (SCLN) and mesenteric lymph nodes (MLN), spleen (Sp) and Peyer patches (PP) were harvested for cell counting and flow cytometry. Blood samples were collected for cytokine detection in 3, 9 and 12 days post-infection (dpi). Hearts were harvested at 15 dpi and histological analysis performed. Results: IG infected mice presented lower parasitemia and mortality than IP infected mice. Parasites appeared within the blood after 3 days in IP mice and 9 dpi, in IG mice. In IP infection, 95% of mice died before 18 dpi, while in IG group, 90% were still alive 27 dpi.SCLN and Sp showed increased number of cells in both groups; however, hypertrophy of these tissues as well as T and CD19+ cells expansion was less evident IG mice than IP. In mucosal-associated lymphoid tissues, such as PP, IP infection promoted atrophy after 12 dpi, due to decrease in CD19+ and CD4+ cells. In IG, PP cell number and T and CD19+ cell depletion occurred in later stages of infection. MLN atrophy was only observed in IG group after 16 dpi, due to depletion of CD19+ cells. Serum analysis of IP infected mice showed a Th1 bias, with increased production IFN-c and TNF-a compared to IG and controls. Interestingly, IG group seems to be Th2 biased, with higher IL4 production than IP. Heart histological sections stained with Hematoxilin and Eosin showed alterations in 15 dpi (parasite nests, inflammatory infiltrate and tissue damage) were similar in both groups, even with the lower number of circulating parasites in IG mice. Conclusions: Our results indicate that the route of T. cruzi infection is a key factor to stimulate the immune response against the parasite in the host.


560 Poster Session: Myeloid Cell Development P1184 Motor behavior dysfunction, neuronal death and glial activation in IL-12p40KO mice that were infected with Trypanosoma cruzi J. Alvarez Mosig,* A. L. Bombeiro,* L. A. Gonçalves, C. R. F. Marinho,à C. Penha-Gonçalves, M. R. D¢Impe´rio Lima,* & G. Chadi§ *Instituto de Cieˆncias Biome´dicas University of Saõ Paulo, Immunology, Saõ Paulo, Brazil, Instituto Gulbenkian de Cieˆncia, Immunology, Oeiras, Portugal, àInstituto de Cieˆncias Biome´dicas University of Saõ Paulo, Parasitology, Saõ Paulo, Brazil, §Neuroregeneration Center FMUSP, Neurology, Saõ Paulo, Brazil Purpose/Objective: Neurological disorders have been described in children and in immunosuppressed hosts with Chagas’ disease, a Latin America protozoosis caused by Trypanosoma cruzi. Recently we showed that IL-12p40KO mice that were infected by T. cruzi, but not infected wild-type (WT) mice, develop a progressive paralysis that culminates in death. Objective: To analyze the elements involved in the neurodegenerative process presented by T. cruzi-infected IL-12p40KO (KO) mice. Materials and methods: KO and WT mice were infected by T. cruzi Sylvio X10/4 and submitted to behavioral analyses. Spinal cords (SCs) of both groups were analyzed every week by RT-PCR to estimate the kinetics of parasitism and immune transcripts, or analyzed by immunohistochemistry when KO mice presented complete paralysis to quantify neurons, astrocytes, macrophages/microglias, T. cruzi and other parameters. Results: Eye-seen and computerized behavior evaluation revealed that KO mice developed a progressive paralysis, from the tail to the forelimbs (p T. cruzi amastigotes, most of them inside macrophages/ microglias. No lesions were observed in WT SCs. Compared to WT, KO SCs showed increased immunoreactive area for nitrotyrosine (239%, p T. cruzi 18S rRNA transcripts revealed the parasite was controlled in WT SC but kept increasing in KO SC (p T. cruzi transcripts were significantly lower in WT SCs. Conclusions: IL-12 and IL-23 efficiently act constraining T. cruzi parasitism in the CNS, occurring exacerbated inflammation and neurodegeneration in their absence.

P1185 Mucosal barrier attenuates exaggerated inflammatory responses to toxoplasma but does not restrict pathogen dissemination to the brain N. Kobets,* E. Domonova, D. Goncharov,* M. Mezentseva,à O. Silveistrova, E. Gubareva,* I. Shapoval,à & E. Ievleva* *Gamaleya Research Institute for Epidemiology and Microbiology, Lab of Protozoan Infection, Moscow, Russia, Central Institute for Epidemiology, Lab. of Molecular Diagnostics, Moscow, Russia, àGamaleya Research Institute for Epidemiology and Microbiology, Lab of Latent Infections, Moscow, Russia Purpose/Objective: Toxoplasma enters the host mostly through the oral route, overcomes local and systemic host resistance and passes mucosal and blood-brain barriers in order to reside in brain. As the mechanisms restricting pathogen entrance to the brain are not clearly understood the aim of this study was to compare the dissemination of toxoplasma in intraperitoneally (ip) and orally infected mice and local immune responses that accompany systemic and mucosal route of infection. Materials and methods: A/Snell mice were infected with 105 tachyzoites of T. gondii RH strain either ip or per os (intraduodenally). The levels of T. gondii DNA were assessed in blood, spleen, liver, lungs, heart, brain, eyes, lamina propria and Payer’s patches at different time points post infection (PI) by RT-PCR. The recruitment of neutrophils,

macrophages, dendritic cells, B cells and T cells to the peritoneum was studied by flow cytometry and the levels of cytokines (IFN-g, TNF-a, IL-2, IL-4, IL-6, IL-18) in spleen were assessed by PCR and flow cytometry. Results: We found that the levels of toxoplasma DNA in orally infected mice were lower in the majority of the organs under the study (for example, spleen or liver) compared to ip infected mice. However they were higher in the brain and Payer’s patches of orally infected mice. Earlier and more pronounced recruitment of inflammatory cells such as neutrophils, dendritic cells and macrophages to the peritoneum was observed in ip infected mice, while higher amounts of B cells (both CD5- and CD5+) were found in orally infected mice starting from day 5 PI. Higher levels of inflammatory cytokines such as IL-1b, TNF-a and IL-18 were found in ip infected mice compared to per os infection. Conclusions: Our results suggest that systemic toxoplasma infection directs the immune response towards better control of toxoplasma dissemination to the brain. Precise mechanisms of this control are currently under investigation.

P1186 NKT cell expansion in absence of B cells may be related to an increased regulatory NKT cell function during Trypanosoma cruzi infection F. Cardillo,* J. Nihei, & J. Mengelà *FIOCRUZ, Gonçalo Moniz Research Center ZIP 40.296-710., SalvadorBahia, Brazil, Universidade Federal do Recoˆncavo-UFRB, Centro de Cieˆncias da Sau´de-CCS ZIP 44.570-000, Cajueiro Santo Antoˆnio de Jesus-Bahia, Brazil, àFIOCRUZ and Faculty of Medicine of Petro´polis FMP-FASE (ZIP 25.680-120), Oswaldo Cruz Institute ZIP 21.040-360, Rio de Janeiro-RJ, Brazil Purpose/Objective: Chagas disease is a tropical disease caused by the protozoan parasite Trypanosoma cruzi (T. cruzi). We have previously demonstrated that Natural Killer (NK) cells were related to resistance to T. cruzi infection in C57Bl/6 mice. Depletion of total NK1.1+ cells in C57Bl/6 infected mice resulted in diminished convertion of recently activated peripheral T cells in effector/memory lymphocytes, thus resulting in T cell hyperactivation without maturation to effector functions. Additionally, we have studied the role of B cells in the activation/differentiation of peripheral T cells during T. cruzi infection. We have found that muMT knockout (KO) mice were more susceptible to T. cruzi infection and had fewer central and effector memory T cells when compared with wild type mice. Taken together, these results might suggest a relationship between B cells and NK1.1+ cells. The present study was designed to evaluate NKT cell-numbers in both muMT and WT infected mice. Materials and methods: Mice were kept in microisolators and were manipulated according to institutional ethical guidelines. Spleen cells were isolated from C57Bl/6 and C57Bl/6 muMT KO mice (1 2 months old). B cells were obtained by magnetic separation and were adoptively transferred to muMT C57Bl/6. After infection with Tulahuen strain of T. cruzi, flow cytometry analysis of the spleens from muMT or from B-cell-sufficient C57Bl/6 mice were performed. Fluorochrome-conjugated anti-alpha beta and anti-NK1.1 monoclonal antibodies were used. Results: During the acute phase of infection, NKT cells were decreased in B cell-transferred muMT, when compared to control muMT infected mice, suggesting that B cells may down-regulate NKT-cell numbers during the infection. As a result, decreased of effector/ memory T cells and increased NKT cell number may be related to diminished capacity to mobilize inflammatory cells to infected tissues, resulting in the increase in parasite load.


Abstracts 561 Conclusions: NKT cell expansion in absence of B cells might be related to an increased negative regulatory NKT cell function latter on early chronic infection, helping to inhibit the generation of effector/ memory T cells.

P1188 Pharmaceutical sodium chlorite (DAC-N-055) acts as anti-parasitic and immunomodulating compound in the defense against Leishmania parasites

P1187 Nucleosides from phlebotomus papatasi salivary gland exacerbate leishmaniasis by generating tolerogenic dendritic cellsdependent treg

P. Wentker,* K. W. Stahl, H. C. Stahl,à C. Bogdan* & U. Schleicher* *Institute of Clinical Microbiology Immunology and Hygiene, Universita¨tsklinikum Erlangen, Erlangen, Germany, Waisenmedizin Freiburg e. V., Freiburg, Germany, àInstitute of Public Health, Universita¨tsklinikum Heidelberg, Heidelberg, Germany

V. Carregaro,* D. Lima-Juńior,* D. Costa,* C. Oliveira, C. Milanezi,à J. Valenzuela,§ J. M. Ribeiro,§ F. Cunha,– & J. Silva* *Faculdade de Medicina de Ribeiraõ Preto University of Saõ Paulo, Biochemistry and Immunology, Ribeiraõ Preto, Brazil, Institute of Biological and Natural Sciences Federal University of Triangulo Mineiro, Patology, Uberaba, Brazil, àFaculdade de Medicina de Ribeiraõ PretoUniversity of Saõ Paulo, Biochemistry and immunology, Ribeiraõ Preto, Brazil, §NIAID/NIH, Section of Vector Biology and Vector Molecular Biology Unit Laboratory of Malaria and Vector Research, Bethesda, MD, USA, –Faculdade de Medicina de Ribeiraõ Preto University of Saõ Paulo, Biochemistry and Immunology Pharmacology, Ribeiraõ Preto, Brazil Purpose/Objective: Phlebotomines saliva plays a crucial role in the establishment of Leishmania infection. Among several potent pharmacologic substances, we recently purified and identified adenosine(ADO) and adenosine-monophosphate-(AMP) as saliva’s active pharmacologic compounds presents on P. papatasii that inhibits dendritic cells-(DC) functions through PGE2/IL-10-dependent mechanism. AIM: We evaluate whether ADO and 5¢AMP are compounds present into Phlebotomus papatasii saliva responsible for leishmania establishment into vertebrate host and such immunomodulatory mechanism. Materials and methods: C57BL/6WT or C57BL/6IL-10-/- mice were coinoculated with L. amazonensis promastigotes forms (1 · 105parasites/ear-i.d. route) in the presence of ADO+AMP or vehicle (PBS). The ear lesion size, parasites burden, cytokines production and inflammatory infiltrated were analyzed at 12nd week post infection. Results: ADO+AMP mimicked the exacerbative effect of saliva in leishmaniasis, increasing parasites numbers and ear lesion. Enzymatic catabolism of salivary nucleosides reversed the SGE-induced immunosuppressive effect. Such effect was associated with pro-inflammatory cytokines reduction (IFN-c, TNF-a) and IL-10 enhancement. Moreover, ADO+AMP failed to enhance ear lesion and parasite burden in IL-10-/- infected mice. Interestingly, nucleosides increased Tregs makers (GITR; CTLA-4; CD39; CD73 expression) on CD4+ CD25population, suggesting the induction of Tregs on T effector cells. Treg induction was associated with nucleosides-induced tolerogenic dendritic cells (tDC) expressing higher levels of COX2 and IL-10. Furthermore, nucleosides-induced tDC displayed a semi-mature phenotype and produced lower levels of proinflammatory cytokines in vitro and in vivo. Conclusions: We demonstrated that ADO and 5¢AMP are constituents present in P. papatasi saliva that exacerbated leishmania infection. The exacerbative effect is associated with Tregs enrichment generated by nucleosides-induced tDC in the inflammatory foci.

Purpose/Objective: Cutaneous leishmaniasis, caused by Leishmania parasites, is characterized by chronic, frequently ulcerating and slowly healing skin lesions. An efficient local therapy is not available. The widespread use of intralesional injection of antimony is limited by emerging resistant parasites and high costs. In clinical trials we obtained evidence that local application of pharmaceutical sodium chlorite (DAC-N-055) after removal of necrotic tissue accelerated the wound healing. Based on these observations we investigated possible anti-parasitic, immunomodulating and wound healing effects of DACN-055. Materials and methods: Anti-parasitic effects of DAC-N-055 on extra- and intracellular Leishmania were evaluated by microscopic and photometric analyses. The influence of DAC-N-055 on several mouse and human immune cells involved in skin wound healing [bone marrow-derived macrophages (BMM) and dendritic cells (BMDC), monocytes, fibroblasts, endothelial cells, keratinocytes, peripheral blood mononuclear cells (PBMC)] and their effector responses were investigated by mRNA (real-time PCR) and protein expression analyses (ELISA, Western blot, proteome array) as well as by an in vitro wound healing scratch assay. Results: In vitro DAC-N-055 exerted a cytotoxic effect on extracellular Leishmania promastigotes, but did not influence intracellular Leishmania amastigotes in BMM. In IFN-gamma-stimulated BMM the expression of iNOS, production of leishmanicidal NO and release of TNF and IL-6 were enhanced by DAC-N-055. In BMDC and in BMM production of IFN-alpha/beta, which is required for the early iNOS expression in murine L. major infections, was increased by DAC-N-055 upon stimulation with Leishmania, IFN-gamma or LPS. DAC-N-055 also augmented the expression of TNF and IL-6 in human MonoMac6 monocytes and HaCaT keratinocytes. Moreover, it modulated the expression of several chemokines and cytokines in human PBMC. Unexpectedly, DAC-N-055 did not show effects on the proliferation and/or migration of fibroblasts, endothelial cells and keratinocytes in wound healing scratch assays. Conclusions: Together, our data indicate that DAC-N-055 is a leishmanicidal and immunomodulating compound, which in vitro influences the production of several effector molecules and cytokines that are protective in leishmaniasis and involved in wound healing.

P1189 Proportions of CD4+ but not CD8+ memory T cells are altered in older individuals chronically infected with Schistosoma haematobium N. Nausch,* C. D. Bourke, L. J. Appleby,* N. Rujeni,* O. Lantz,à F. Trottein,§ T. Mduluza,– N. Midzi** & F. Mutapi* *Institute of Immunology & Infection Research, University of Edinburgh, Edinburgh, UK, Biology Department, University of York, York, UK, à Laboratoire d’Immunologie et Unite´ Inserm 932, Institut Curie, Paris, France, §Center for Infection and Immunity of Lille INSERM U 1019, Institut Pasteur de Lille Univ Lille Nord de France, Lille, France, – Biochemistry, Harare, University of Zimbabwe, Zimbabwe, **Schistosomiasis, National Institute of Health Research, Harare, Zimbabwe Purpose/Objective: There are indications that the efficacy of vaccines can be reduced in helminth infected individuals but so far there are no


562 Poster Session: Myeloid Cell Development studies on how helminths may mediate this effect in human or experimental studies. The efficacy of vaccines relies on the effective generation and maintenance of CD4+ and CD8+ T cell memory immune responses. Therefore the aim of this study was to characterise human CD4+ and CD8+ memory T cell accumulation in people exposed to Schistosoma haematobium infection and relate this to host infection status and schistosome-specific immune responses before and after anti-helminthic treatment. Materials and methods: Proportions of CD4+ and CD8+ memory T cells were analysed in a cohort of 105 participants who are lifelong residents in an area endemic for Schistosoma haematobium. Fifty-one participants were analysed following treatment with an anti-helminthic drug. In addition schistosome-specific effector antibody responses and the overall function of CD4+ effector/memory cells were determined. Results: Our results show that helminth infection is associated with alteration of CD4+ memory T cell proportions. This alteration was not observed in CD8+ memory T cells. The changes in CD4+ memory T cells are associated with an impaired TH1 response and changes in activation markers. Treatment with an anti-helminthic drug leads to alterations of the CD4+ memory T cell pool. Furthermore, reduced CD4+ memory T cell proportions were associated with lower schistosome-specific effector antibody responses. Conclusions: In summary, individuals infected with S. haematobium who did not to develop protective immunity with accumulative exposure show alteration in the overall CD4 T cell memory. These findings have implications for the development of the natural or the vaccine induced immune response against schistosome-specific as well as against unrelated pathogens.

P1190 The effect of vaccination with recombinant L. donovani and L. mexicana gamma glutamyl cysteine synthetase by different routes of administration on host immune responses B. Doro, M. Wiese, A. B. Mullen & K. C. Carter Immunology, University of Strathclyde, Glasgow, UK Purpose/Objective: Human leishmaniasis is a spectrum of diseases caused by protozoan parasites of the genus Leishmania. Drugs existing for the treatment of leishmaniasis have been unsatisfactory. Ideally a vaccine could prevent infection and would be a feasible control method as infected individuals are resistant to clinical re-infection. In this study the ability of recombinant L. donovani and L. mexicana gamma glutamyl cysteine synthetase (cGCS) to induce a protective immune response in BALB/c mice was determined. Materials and methods: Animals were immunised by different routes with the recombinant proteins and the effect of vaccination on production of parasite-specific IgG1 and IgG2a was determined using an ELISA assay. In addition the effect of immunisation on cytokine production by in vitro stimulated splenocytes from immunised mice was determined. Results: Immunization with cGCS inducing significantly higher titers (P < 0.05) comparing to control group. Conclusions: Vaccination induced significant Th1 and Th2 immune responses after single dose treatment.

P1191 The expression of B blood group carbohydrate under control of FUT2 gene (19q13.3) increases the risk for digestive Chagas disease L. de Mattos,* C. R. Bernardo,* A. P. Oliveira,* A. V. S. Camargo,* C. C. Brandaõ de Mattos,* C. E. Cavasini, L. S. Ronchi,à J. G. Netinho,à R. B. Bestetti,à O. Ricci Jrà & A. A. Borimà *Molecular Biology, Faculdade de Medicina de Sao Jose do Rio Preto, Sao Jose do Rio Preto, Brazil, Dermatology and Infectious Diseases, Faculdade de Medicina de Sao Jose do Rio Preto, Sao Jose do Rio Preto, Brazil, àFaculdade de Medicina de Sao Jose do Rio Preto, Hospital de Base FUNFARME, Sao Jose do Rio Preto, Brazil Purpose/Objective: The infection by Trypanosoma cruzi can result in distinct clinical manifestations of Chagas Disease (CD) including heart (cardiomyopathy) and gastro-intestinal disease (megaesophagus and megacolon). Different factors contribute for this disease but there are no genetic markers indicating what form of the disease will occur. Previous reports evaluated the ABO blood group carbohydrates in relation to CD but they did not explore the influence of FUT2 gene which controls the expression of these carbohydrates in the gastrointestinal tract. This study aimed to verify the combined effect of the FUT2 gene and the ABO blood group carbohydrates in heart and gastro-intestinal CD. Materials and methods: Two hundred and forty patients were enrolled (cardiomyopathy: n = 120; gastro-intestinal: n = 120). The FUT2 genotyping and the ABO blood group carbohydrates were identified by PCR-RFLP and hemagglutination methods, respectively. The qui-square test, the exact Fisher’s test, and the Odds Ratio were used to compare the proportions. Results: The differences between heart and gastro-intestinal CD were not statistically significant in relation to the FUT2 gene (v2: 1.141; DF: 1; P = 0.2854) and the ABO blood group carbohydrates (v2: 1.855; DF: 3; P = 0.6031) when analysed in isolation. However, when these two markers were analysed in combination, the differences were statistically significant for gastro-intestinal CD (v2: 9.961; DF: 3; P = 0.0189) and associated with the expression B (B and AB phenotypes) carbohydrates (OR: 10.969; CI 95%: 1.415 85.022; P = 0.0114). Conclusions: The role of the FUT2 gene and the ABO blood group carbohydrates in CD remains unclear and there are no evidences that these carbohydrates act as receptors for T. cruzi. Since the ABO carbohydrates expressed in the gastro-intestinal tract under control of the FUT2 gene are structurally distinct from those expressed in the heart tissues, maybe these differences increase the risk for CD in the gastro-intestinal tract, especially among those carrying the B carbohydrate. In conclusion the expression of B carbohydrate from ABO blood group system in the gastro-intestinal tract is associated with this form of CD.

P1192 The role of mast cells in protection against Leishmania donovani infection K. Carter,* K. Ross,* F. Tacchini-Cottier & C. Lawrence* *Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK, Department of Biochemistry, University of Lausanne, Lausanne, Switzerland Purpose/Objective: Visceral leishmaniasis is a major health concern in many parts of the world and designing suitable interventions to control the disease relies on understanding the role of host immunity in controlling susceptibility/resistance to infection. Materials and methods: The role of mast cells in Leishmania donovani infection was determined by comparing the outcome infection in Kit (Wsh) mast cell deficient mice and their wild-type counterparts.


Abstracts 563 Specific antibody responses and cytokine production by in vitro stimulated splenocytes from infected mice were assessed. The role of mast cells in neutrophil recruitment was determined by monitoring their levels during infection, studying the effect of depletion on the outcome of infection, and determining myeloperoxidase levels in tissues of WT and Wsh infected mice. Results: Mast cell deficient mice had significantly lower liver and splenic parasite burdens compared to wild-type mice and this reduction was associated with significant inflammation in the spleen, based on spleen weight. Conclusions: Results of studies showed that mast cells have an important role in controlling the outcome of infection and that neutrophils are involved in protection in both mouse strains.

P1193 Two unique IL-10 producing B cell subsets contribute to the host cytokine balance during Trypanosoma brucei infections F. Kauffmann, V. Bockstal, J. Cnops, C. Haynes, C. De Trez & S. Magez VIB Structural Biology, Vrije Universiteit Brussel (VUB), Brussels, Belgium Purpose/Objective: IL-10 producing B cells have been described as a new regulatory cell type important not only in balancing auto-immune diseases, but also in the establishment of tolerance during parasitic infections. Infection with Trypanosoma brucei elicits a strong proinflammatory response and although it has been established that the anti-inflammatory cytokine IL-10 is crucial to prevent death of the host from hyper-inflammation, the cellular source of IL-10 has not been clarified so far. Materials and methods: Blood serum cytokine levels and cytokine production by total spleen and liver cells was measured by ELISA. The production of IL-10 by individual cells was assessed through intracellular flowcytometric staining for IL-10 and the use of GFP IL-10 transcriptional reporter mice. Results: After control of the first parasitemia wave there is a peak of IL-10 production in the spleen. The majority of the IL-10 producing cells has a B cell phenotype and can be divided into a B220intCD19hiTIM-1+ CD23intCD11b+ CD5lowand a B220lowCD19int TIM-1- CD23- CD11blowCD5- B cell population. Both cells populations are IgM+ CD21lowLY6C+BST2+ CD11clowIgDlowCD1d- AA4.1CD138-. Two IL-10 producing B cell populations, a B220loCD19int and a B220intCD19hi can be found in the liver as well, exhibiting a similar phenotype as the splenic populations. Conclusions: Using both intracellular staining for IL-10 and GFP IL10 transcriptional reporter mice, our results suggest that two distinct regulatory B cell subsets with ‘DC-like’ features are the major producers of IL-10 in the spleen during Trypanosoma brucei infection and may be involved in activating regulatory immune responses.

P1194 Unravelling the events leading to inflammation-induced trypanosomiasis-associated acute anemia J. Cnops, V. Bockstal, C. Haynes, C. De Trez & S. Magez Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium Purpose/Objective: African trypanosomes are extracellular parasites that cause infections in human and livestock in sub-Saharan Africa. The common hallmark of T. brucei infections is hyper-inflammation due to an ill-controlled immune reaction. During the acute phase of experimental murine trypanosomiasis, there is a 50% reduction in red blood cell counts. It has previously been shown that during several microbial infections in mice, acute anemia is caused by erythrophagocytosis, and IFNc is the critical driver in this immunopathology. Our purpose is to unravel the mechanism(s) implicated in the acute inflammation-associated anemia observed following control of the first parasitemia peak. Materials and methods: 8-week-old female C57Bl/6 mice were infected by intraperitoneal injection of 5000 T. b. brucei AnTat1.1E clone parasites. Parasitemia and red blood cell counts were performed at 2-day intervals using a hematocytometer. Lymphocyte cell populations were analyzed by flowcytometry. Results: We previously observed that serum IFNg levels are highest during the first days post infection. Here, we report that IFNc-/- mice display reduced acute anemia. Moreover, this reduced pathology is also observed in beta-2-microglobulin (b2m) deficient mice but not in transporter associated with antigen processing 1 (TAP1) deficient mice, suggesting that the cell population which plays a major role in the induction of acute anemia is independent of TAP transport for surface expression of the relevant MHC I molecule. Future analysis of anemia profiles of KbDb-/- mice will determine whether the cell population is dependent on MHC Ia or MHC Ib. Flow cytometric analysis during the first days post infection reveals that at day 4 (right before the appearance of the anemic phenotype) there is a significant increase in the number and percentage of NKT cells, CD8+ T cells and NK1.1+ CD8+ cells in the liver and blood of wild type C57Bl/6 mice. Conclusions: Further flow cytometric experiments will determine the presence or absence of these cell populations in TAP1-/- versus b2m-/mice. In addition, it will be investigated which cell population is the major IFNg producer during the first days post infection. Together, these data will help to identify the cell population(s) responsible for the acute anemia during the initial phase of T. brucei infection.


564 Poster Session: Myeloid Cell Development P1195 Vitamin E as an adjuvant in an Taenia crassiceps mouse immunization E. De Gaspari Immunology, Adolfo Lutz, Saõ Paulo, Brazil Purpose/Objective: In this study we evaluates the efficacy of vitamin E as an adjuvant, using Taenia crassiceps antigens against murine cysticercosis. The efficacy of vitamin E was compared with three well known experimental adjuvants (complete Freund’s adjuvant, aluminum salts, and saponin), and assessed with respect to the humoral immune response elicited and the reduction in parasite load. Vitamin E is a well-known anti-oxidant and has been shown to exert immunostimulatory activity by dietary supplementation . The beneficial use of a-tocopherol formulated in an oil emulsion as an adjuvant has been utilized for the development of various veterinary vaccines . Materials and methods: Groups of 5 BALB/c mice were immunized intramuscularly, subcutaneously, or intraperitoneally with different doses (10 lg/mouse) of T. crassiceps antigens emulsified in mineral oil emulsions that was prepared as previously described (Valdez et al.1994). Saponin (Sigma) was prepared at a concentration of 50 lg/ mouse as reported previously (McColm et al.1982). Aluminum salts and vitamin E were prepared as described by Ito et al., 2009 and

Tengerdy and Lacerda.1991, respectively. The solution containing vitamin E and VF-Tcra was emulsified using (Ultrasonic Devices for Liquid Processing, 2 kw). Thirty days later, the mice were given a booster with the same immunizing dose of the same peptide in the same adjuvant as had been done before. Animals were bled from the retro-orbital plexus at 60 days after immunization. The sera were pooled and kept frozen in aliquots at -20C. Results: Anti-VF-Tcra IgG antibodies that were produced were detected by ELISA on day 60 in all groups independently of the immunization method and of the type of adjuvant used. The compartive study of the different adjuvants showed that vitamin E produced the high titre of antibodies and was a good adjuvant P £ 0.05. SDS*PAGE shows the pattern of VF-Tcra and VF-tso. VF-tso pathogens presented various peptides of 8 158 kDa, and the VF-Tcra pathogen was rich in G transversion at the nucleotide 713 (c.713T>G) resulting in the substitution of the serine in position 238 by a arginine: p.Ser238Arg. Conclusions: X-SCID is caused by mutations in the IL2RG gene, coding for the cytokine receptor gammasubunit common to IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 receptors, known as the common gamma chain (cC). Cytokine receptors of the cC group belong to the class I cytokine receptor family, which includes the receptors of other interleukins and hormones. They are heterodimeric or heterotrimeric transmembrane complexes expressed on the surface of cells that share a highly conserved Trp-Ser-X-Trp-Ser motif (WSXWS motif). This motif is essential in receptor function, acting as a molecular switch involved in receptor activation. The sequence variant found in the patient has not been previously reported, however, we consider it is a pathogenic mutation, as it affects the WSXWS motif.

P1198 Autoantibody specificity from T cell receptor alpha chain deficient mice A. Maceiras Oliveira, A. Agua-Doce, A. Varela & L. Graca Instituto de Medicina Molecular, University of Lisbon, Lisbon, Portugal Purpose/Objective: Mice deficient for the alpha chain of the T cell receptor (TCR a-/- mice) spontaneously develop autoimmunity. Al-

though these mice have a developmental defect of TCR a/b T cells, they are able to develop germinal center reactions (GCR) and produce all immunoglobulin (Ig) classes with help from TCR c/d cells and from a small population of TCR a-/b+ T cells. Given the fact that GCR and antibody production is impaired in TCR b-/- mice, we believe that the small population of T cells with a receptor consisting of a pre-T a and a b chain are essential for GCR. Materials and methods: In order to determine the age of disease onset, as well as, the specificity of the autoantibodies produced, we have followed TCR a-/- mice for several months to check the presence, by immunofluorescence, of autoantibodies that recognize cellular structures. Results: We found that, although at 3 months of age autoantibodies are still absent, at 5 months autoantibodies are present in significant amounts in the serum. Moreover, these autoantibodies have preferential specificity to nuclear cell components even though in some individuals anti-cytoplasmatic antibodies are also identified. Conclusions: We, therefore, conclude that a/b T cell lymphopenia in TCR a-/- mice is sufficient to drive GCR and antibody production, but with a bias towards auto-reactivity.

P1199 CD107a expression-based NK degranulation assays for classification of familial hemophagocytic lymphohistiocytosis (FHL) L. Vargas Henny,* I. Astigarraga, U. Zur Stadt,à M. AlonsoMartıńez,§ P. Francos,* I. Martıńez-Rıó,* S. Garcıá-Obregoń, D. G. Viedma§ & J. Gil* *Hospital General Universitario Gregorio Maranõń, Immunology, Madrid, Spain, Hospital de Cruces, Pediatrics, Bilbao, Spain, à Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, §Hospital General Universitario Gregorio Maranõń, Molecular Analysis Central Unit, Madrid, Spain Purpose/Objective: HLH is an uncommon, serious disease, lethal whithout treatment, characterized by a fail on the granules dependent cytotoxic activity and therefore on the immune system regulation, manifests as an uncontrolled and fatal systemic inflammatory syndrome. NK cells degranulation assays constitute a recent laboratory tool that allows an early diagnosis of familial hemophagocytic lymphohistiocytosis (FHL). In order to validate this assay, we intended to study differences between healthy individuals (HC), FHL and secondary HLH patients and establish local references values of NK cells’ CD107a expression. Materials and methods: Within December 2011 and April 2012, 20 samples from suspected HLH patients and 24 HC samples were analyzed. NK lymphocytes were incubated in complete medium (resting NK cells) or IL-2 conditioned medium (IL-2/NK cells) and posteriorly co-cultured with K-562 cells. The quantification of the CD107a percentage expression on the NK lymphocytes surface (DCD107a%) and mean fluorescence intensity (MFI) were obtained by multi-parametric flow cytometry (BD Biosciences). Descriptive statistics, Mann Whitney U-test and ROC curves analyses were performed. Results: Genetic diagnosis of FHL was confirmed in 3/4 patients with abnormal degranulation activity through sequencing of UNC13D, STX11, STXBP2 and RAB27A genes; while nine patients who fulfilled HLH criteria were diagnosed as secondary HLH. HLH diagnosis was dismissed in seven patients.



DCD107a% resting NK cells

HC

FHL

Secondary

(n = 24)

(n = 4)

P

HLH (n = 9)

P

32 ± 12

4.7 ± 3.8

0.002

28 ± 19

0.94

18.8 ± 11.7

0.003

43 ± 24

0.49

48 ± 14.7

0.015

108.6 ± 45.8

0.15

72.8 ± 54.8

0.057

177 ± 66.4

0.057

(13 55)

X ± SD (p5-p95) DCD107a% IL-2/NK cells

52 ± 13 (28 72)

X ± SD (p5-p95) MFI resting NK cells X ± SD (p5-p95) MFI IL-2/NK cells X ± SD (p5-p95)

89 ± 38.6 (43 191) 134 ± 71.5 (48 324)

A resting NK cells DCD107a% cutoff value at 10.9 (p5:13%) was identified for classification of FHL forms.

Conclusions: Normal DCD107a% ranges, sensitivity and specificity are similar to those described by other authors. Therefore we confirm and validate the NK lymphocytes degranulation assays as a very useful technique for HLH immunologic diagnosis. Further studies are needed to assess whether higher values of MFI may be helpful for secondary HLH diagnosis.

P1200 Common variable immunodeficiency (CVID) and T immunoregulatory cells F. Cinetto, A. Parolo, N. Compagno, V. Ferri, M. Gazzola, P. Tartaro, S. Carraro, M. Facco, G. Semenzato & C. Agostini Department of Medicine, University of Padua, Padova, Italy Purpose/Objective: CVID is a common immune defect in the adulthood. Besides the infective risk, CVID is characterized by an increased incidence of autoimmune diseases, mainly autoimmune cytopenias. Unbalance in the ratio between T regulatory cells (Treg, decreased) and Th17 cells (increased) has been reported in several autoimmune conditions. These cells represent targets for different immunomodulatory therapy, included intravenous immunoglobulin (IVIg). Both IVIg and subcutaneous immunoglobulin (SCIg) are currently used as Ig replacement therapy in CVID. In this study we decided to investigate the possible immunomodulatory role of SCIg therapy and, specifically, its impact on Treg and Th17 levels. Materials and methods: Ten CVID patients were subdivided according to EUROclass classification and cytofluorimetric analysis of peripheral blood Treg and Th17 cells levels was performed before and after SCIg administration. As a comparation, 10 healthy volunteers were analyzed for Treg and Th17 cells levels. Results: No significant correlation was demonstrated between B phenotype and T immunoregulatory cells levels. A significant reduction of Th17 level was observed 48 60 h after SCIG infusion (post versus pre: 1.12 ± 0.87% versus 1.71 ± 0.70%; P < 0.05). By contrast, no significant results were obtained comparing Treg levels before and after SCIg. As a result, Treg/Th17 ratio significantly increased after SCIg infusion (pre versus post: 1.60 ± 1.18 versus 2.68 ± 1.65; P < 0.05). Conclusions: In CVID, SCIg seem not to play the only role of replacement therapy, since they significantly modified Treg/Th17 balance in our group of patients. This suggest the possibility of using SCIg as immunomodulatory treatment in different settings.

P1201 Disseminated BCG-infection in patient with autosomal recessive chronic granulematosis disease L. I. Chernyshova & A. V. Bondarenko Department of Pediatric Infectious Diseases and Clinical Immunology, National Medical Academy for Post-graduate Education, Kiev, Ukraine Purpose/Objective: Mycobacterial infection is not common cause of infectious syndrome in children with chronic granulomatosis disease (CGD). Materials and methods: Observation of case with CGD with generalized BCG infection. Immunologic and genetic examinations were used for diagnosis of CGD. Results: The child was born from III pregnancy, II delivery on 37th week of gestation with body mass 2750 g. She received BCG vaccination at 4th day of birth. At 3 months the local inflammation with regional lymphadenitis developed which was treated with isoniazid for 3 months. Then bilateral purulent cervical lymphadenitis developed at 6, 9 and 11 months treated with wide spectrum antibiotics. Culture from pus was negative. PCR for Mycobacterium tuberculosis complex was negative. Since 1 year old causeless periodic fevers occurred with progressive hepatosplenomegaly, loss of weight, progressive anemia, inflammatory changes in blood for almost 6 months. Bacteriological cultures were negative. Treatment with wide spectrum antibiotics had insufficient effect. Antitubercular treatment with amycacinum, rifampin and izoniazid was started ex juvantibus for 6 months and positive effect had been received. Three months after antitubercular treatment was stopped the child had pneumonia which did not give in to traditional treatment during 2 months. Then antitubercular treatment was renewed with positive effect. At the age of 7 during the unexplained fever multiple formations were identified in the liver, biopsy showed caseous mass, histologically confirming the mycobacterial nature of lesions. Immunological findings revealed normal serum CH50, Ig A, IgM, IgG, IgE and lymphocyte subpopulations levels, NBT-test 2 14%, phagocytic activity 4 28%. At the age of five the blood samples were tested at INSERM and autosomal recessive CGD was confirmed (p22phox). Conclusions: Universal BCG vaccination of newborns may cause problems in patients with CGD. Difficulties in diagnostics of BCG infection require administration of anti-tuberculosis drugs in suspicious cases.

P1203 Enhanced formation of giant cells in common variable immunodeficiency T. H. Scott-Taylor,* K. Whiting & D. A. Websterà *London Metropolitan University, Life Sciences, London, UK, School of Life Sciences, Kingston University, Kingston, UK, àDepartment of Immunology, University College London, London, UK Purpose/Objective: To examine the propensity of peripheral blood monocytes in Common Variable Immunodeficiency to fuse to form multinucleate giant cells in relation to the tendency to form granuloma. Materials and methods: The fusion index of cells from individuals with Common Variable Immunodeficiency (CVID) and normal controls were compared in a range of cytokines, supernatants and mitogens over a week in culture. Cultured monocytes at different of differentiation were centrifuged onto coverslips and examined under electron and confocal microscopes. Results: Cell fusion of CVID peripheral blood monocytes was variable but on average was quicker, approximately twofold more frequent and formed giant cells with higher numbers of nuclei in culture medium without cytokines than normal. Addition of IL4, GMCSF, IFNg, TNFa and T cell conditioned media further induced normal and particularly CVID giant cell formation and combinations of cytokines and


Abstracts 567 monokines acted synergistically in promoting monocyte fusion. Treatment with anti INFg antibody reduced normal giant cell formation particularly, indicating a greater predisposition of peripheral CVID cells to fuse, while a greater tendency of CVID cells to fuse with immunoglobulin conditioned media my indicate the contribution of IVIG treatment in granuloma formation. CVID and normal giant cells expressed similar levels of MHC class II and costimulatory molecules and FC receptors and demonstrated metabolic and phagocytic activity with bacteria, yeast and fluorescent carboxilated beads.

CMV. The girl is Ig-substituted, has hypothyroidism, anti-mitochondria- (AMA, M2), islet-cell, insulin- and glutamate decarboxylase- but no other autoantibodies. Family history including a 3-years-old healthy brother is unremarkable. Materials and methods: Standard immune phenotypical assays including TCR-Vb-spectratying did not reveal a known type of cellular immunodeficiency. CD4/CD8 double-negative T cells were borderline increased (2 10%), but serologic parameters (VitB12, Apolipoprotein), apoptosis, killing, and lymphocyte proliferation assays were normal, lymph node histology from the older girl was ‘compatible’ with ALPS, but gene sequence of CD95, CD95L, Caspase-8, and -10 were normal. Bowel histology of the younger patient showed lymphocytic enteritis. Results: In summary, one sibling shows an iatrogenous CVID-like syndrome and multiple viral infections after anti-CD20-treated AIHA, and the other girl partial remission of atypical ALPS post-HSCT. Conclusions: In search for an unrecognized immune dysregulation syndrome, the currently ongoing steps of diagnostic work up are homozygosity mapping and exome sequencing.

P1205 Humoral immunity in patients with diGeorge syndrome

Conclusions: A 2- to 5-fold greater tendency to form giant cells was induced in peripheral CVID monocytes by an extensive range of monokines, inflammatory lymphokines and T cell supernatants. CVID and normal cell giant cells were metabolically active and phenotypically similar.

P1204 Familial multiorgan-autoimmunity syndrome with cytopenia, liver-, and islet cell autoantibodies a novel inherited immune dysregulation syndrome? M. Seidel,* W. Schwinger,* H. Lackner,* A. Deutschmann,* G. Gorkiewicz, M. R. Speicher,à K. Boztug§ & E. C. Urban* *Pediatric Hematology Oncology, Medical University, Graz, Austria, Institute of Pathology, Medical University, Graz, Austria, àInstitute of Human Genetics, Medical University, Graz, Austria, §Austrian Academy of Sciences, Research Center for Molecular Medicine, Vienna, Austria Purpose/Objective: A now 16-years-old girl from a Kurdish consanguineous family underwent stem cell transplantation (SCT) from her HLA-identical mother for suspected type 3 (non-classic) autoimmune lymphoproliferative syndrome (ALPS) after suffering from chronic immune thrombocytopenia (ITP) starting from her 2nd year of life and recurrent bacterial infections, developing into generalized massive lymphadenopathy and splenomegaly without evidence of EBV infection. Soon after successful SCT lymphoproliferation resolved, but chronic ITP recurred. Both mother and daughter have normal immunoglobulin (Ig) levels and antibodies against viral and vaccine antigens, but were found to be positive for liver/mitochondrial autoantibodies (AMA, M2) without liver/biliary disease or cryptosporidia in stool. A now 8-years-old sister was diagnosed with autoimmune hemolytic anemia (AIHA) at the age of five years. AIHA resolved under immunosuppression with rituximab, FK506 and MMF; total B cell numbers and Ig levels were normal prior to anti-CD20 therapy. Later, the girl developed a persistent CVID-like syndrome with low B cell numbers (40% of the clones were specific for the epitope within this peptide. Conclusions: The results of this study indicate that, as shown previously for CD8 T cells, CD4 T cell responses to T. parva-infected cells in individual animals are focused on a limited number of antigens/epitopes. Further studies are underway to investigate processing of the Tp9 antigen in infected cells for CD4 T cell response.

P1421 Identification of new antigens from Rhipicephalus microplus ticks associated to infestation phenotypes of susceptibility or resistance in bovine hosts G. R. Garcia,* J. M. Ribeiro, S. R. Maruyama,* L. G. Gardinassi,* K. Nelson,à D. Rossi,§ B. R. Ferreira,– F. C. Galvao,§ C. F. Zanelli,§ S. R. Valentini,§ A. M. Maia** & I. K. F. de Miranda Santos* *Biochemistry and Immunology, Medical School of Ribeiraõ Preto, University of Saõ Paulo, Ribeiraõ Preto, Brazil, National Institutes of Health-USA, National Institute of Allergy and Infectious Diseases, Rockville, MD, USA, àCenter for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, VA, USA, §School of Pharmaceutical Sciences, Saõ Paulo State University, Araraquara, Brazil, – Ribeiraõ Preto School of Nursing, University of Sao Paulo, Ribeiraõ Preto, Brazil, **School of Animal Science and Food Technology, University of Sao Paulo, Pirassununga, Brazil Purpose/Objective: Rhipicephalus microplus cattle tick causes great economic losses to livestock. Current vaccines have partial and transient effects against infestations. To develop effective anti-tick vaccines, new targets must be identified. Bovines express breed-specific, heritable, contrasting phenotypes during infestation. Composition of tick saliva proteins may be affected by these different levels of host immunity and may be crucial to hematophagy, i.e., potential antigens. Materials and methods: With DIGE, MudPIT and 454-based RNASeq we investigated the protein expression profile of salivary glands from nymphs (NSG), males (MSG) and females (FSG) from R. microplus ticks fed on resistant or susceptible bovines as well as unfed larvae (UFL) from eggs of females fed on these hosts. Results: We identified 321 different proteins: 68 in samples derived from the ticks fed on susceptible hosts, 17 only on resistant and 236 shared by both groups. DIGE results showed 20 differentially expressed

proteins in UFL, 27 in NSG and 35 in FSG. In addition, the sialotranscriptome revealed 11 676 coding sequences (CDS), with 3590 CDS for putative secreted proteins. Many differentially expressed proteins in the global analyses show similarity with proteases, nucleases, protease inhibitors, antimicrobial peptides and pathogen recognition proteins among others and are associated with the immunity raised in susceptible or resistant hosts. Conclusions: This study represents the first attempt to identify protein profiles in developmental stages of R. microplus ticks that are affected by the different host immune responses developed by susceptible or resistant bovines. It offers an opportunity to identify new protective antigens for an effective anti-tick vaccine.

P1422 Ig-free colostral whey-supplemented formula: effects on adherent intestinal microflora and immune state of normal and low birth weight piglets B. R. Tambuyzer,* L. Che, M. De Vos,* V. Huygelen,* S. Willemen,* S. Van Cruchten,* C. Casteleyn* & C. Van Ginneken* *Applied Veterinary Morphology, University of Antwerp, Wilrijk, Antwerp, Belgium, Swine Nutrition Group Institute of Animal Nutrition, Sichuan Agricultural University, Sichuan, China Purpose/Objective: The use of hyperprolific hybrid sows in pig industry has led to increased litter sizes and consequently more low birth weight (LBW) piglets. At birth these animals are less competitive, which results in lower colostrum intake, making them more prone to infectious diseases. Pig farmers often transfer piglets to brooders where they are artificially fed with formula ad libitum until weaning. This research aims to elucidate the benefit of colostral whey fraction provided in conjunction with formula by its effect on the piglet’s gut immune system and intestinal flora. Materials and methods: LBW and normal birth weight (NBW) piglets, from day 3 onwards, were fed formula or formula supplemented with colostral whey fraction (Ig-free, 1), except for NBW piglets fed supplemented formula (0.6). However, the optimal ratio of a sow-fed animal (LBW: 1.875, NBW: 2.5) was never reached. The expression of virtually all immune proteins dropped with age, except for NF-jB in animals fed supplemented formula (P = 0.03), which is consistent with the maturation of the immune system towards adaptive immunity. Conclusions: In conclusion, a more beneficial microflora is preponderant in piglets fed formula with colostral whey fraction at day 10, which seems to be related to an altered immune state of the gut. However, this effect did not prevail in the long term, possibly due to supplementation restricted to the first week.


628 Poster Session: Myeloid Cell Development P1423 Immunoproteomics analysis of Brucella abortus antigen reveals differential antibody profiles between S19-vaccinated and naturally infected cattle A. C. Pajuaba,* D. A. Silva,* K. C. Almeida,* J. P. Cunha-Junior,* C. P. Pirovani, L. R. Camillo & J. R. Mineo* *Immunology Microbiology and Parasitology, Institute for Biomedical Sciences, Universidade Federal de Uberlaˆndia, Uberlandia, Brazil, Universidade Estadual de Santa Cruz, Institute of Biotechnology, Ilheús, Brazil Purpose/Objective: Brucellosis is an infectious disease caused by bacteria of the genus Brucella and it is one of the most widespread zoonotic diseases, including infectious abortion in domestic animals and potentially debilitating infection in humans. The food-producing herds, such as cattle, sheep, goats, and pigs are highly susceptible to disease. In some areas, however, the complete control of disease is complicated by the presence of wildlife reservoirs. The diagnosis of brucellosis is mainly based on serological methods, because of the requirement for faster and more reliable diagnostic tests. The majority of the conventional serological tests use whole cell preparations, sonicated cell extracts, or lipopolysaccharide (LPS)-enriched fractions obtained from smooth (S) B. abortus strains, called the S-LPS antigen. Although LPS induces a strong antibody response and classical serological tests are mainly based on the detection of antibodies to LPS, there are some limitations in the use of these S-LPS antigen preparations. This study aimed to characterize a B. abortus S19 antigen preparation obtained by Triton X-114 (TX-114) extraction through immunoproteomics to differentiate infected from vaccinated cattle. Materials and methods: Three groups of bovine were studied: GI, 30 naturally infected cows; GII, 30 S19-vaccinated heifers; and GIII, 30 nonvaccinated seronegative cows. Serum samples from these animals were analyzed and one-dimensional (1D) and two-dimensional (2D) electrophoretic were obtained from the TX-114 hydrophilic phase antigen of the bacteria, followed by MS/MS analysis, after immunoblotting assays. Results: The results revealed a broad spectrum of polypeptides, varying from 10 to 79 kDa. 1D immunoblot showed widespread seroreactivity profile in GI compared with restricted profile in GII. Three antigenic components (10, 12, 17 kDa) were recognized exclusively by GI sera, representing potential markers of infection and excluding vaccinal response. The proteomic characterization revealed 56 protein spots, 27 of which were antigenic spots showing differential seroreactivity profile between GI and GII, especially polypeptides 0.05). Conclusions: Unlike what has been recently reported that increases in serum levels of S100A8/A9 correlated well with some clinical forms of a few inflammatory diseases such as rheumatoid arthritis, our findings demonstrated the lack of correlation between this serum biomarker and the activity of ANCA-associated vasculitis, pathology in which polynuclear neutrophils are known to be involved in mechanism injury.

P1569 Automated imaging system for the analysis of antinuclear antibodies by indirect immunofluorescence V. P. Rodriguez,* C. G. Rodrı´guez,* A. F. Hermida, D. B. Roldań,* I. R. Losquinõ* & F. F. Romero* * Clinical Biochemistry, Virgen Macarena University Hospital, Sevilla, Spain, Menarini Diagnostics, Sevilla, Spain Purpose/Objective: Antinuclear antibodies (ANA) are essential biomarkers in the diagnosis of several autoimmune diseases. Although immunoassays in solid phase as ELISA are economic and easier to standardize, they are not able to describe ANA patterns and have less sensitivity than indirect immunofluorescence (IIF) in human epithelial cells HEP2, for this reason the American School of Rheumatology recommends IIF in HEP2 as the technique of reference for the determination of ANA. Following this tendency, technologies for the reading of ANA by IIF are being developed in order to avoid the main disadvantage of the IIF; the variability of results based on the experience of the analyst. G-Sight Zenit is an automated system of Menarini Diagnostics for the capture of images and the interpretation of IIF tests that allows a semiquantitative analysis and the recognition of ANA patterns in HEP2. The use of these systems could help reduce the great variability in the determination of these autoantibodies. Objective: Analyze the degree of agreement between the results obtained with the automated system (Zenith G-Sight, Menarini Diagnostics) and the microscope observation for the analysis of the ANA by IIF. Materials and methods: We analyzed 107 serums from patients being followed by specialized consults. The samples were analyzed in parallel with the automated system Zenith G-Sight from Menarini Diagnostics (Florence, Italy) and with a fluorescence microscope Olympus BX41 (Tokyo, Japan). We used as substrate, slides of HEP2 cells, ImmunoConcepts. (Sacramento, USA) We determined the degree of agreement for pattern and titer of ANA between the images obtained by the GSight and the microscopic analysis, by the calculation of Kappa indices using the statistical program SPSSv19. Results: In the study of the patterns we obtained a kappa index of 0.78, indicating a good degree of agreement between both techniques. In the study of the titer we obtained a kappa index of 0.53, thus the agreement between both techniques was moderate.


Abstracts 669 Of the 48 samples interpreted as negative/doubtful by the G-Sight, 32 were reported as positive and 16 as negative aftermicroscope observation. Conclusions: Both methods show a good degree of agreement as far as pattern and titer. Nevertheless, it would be necessary to adjust the rank of negatives/doubtful given by the G-Sight to use this equipment in ANA screening.

P1572 Comparative study between ELISA and chemiluminescence methods for the analysis of specific ENA V. P. Rodriguez,* B. F. Pe´rez,* C. G. Rodrı´guez,* J. M. M. Bada, E. M. Madrid,* R. F. Seda* & F. F. Romero* *Clinical Biochemistry, Virgen Macarena University Hospital, Sevilla, Spain, Menarini Diagnostics, Sevilla, Spain Purpose/Objective: The autoimmunity laboratory plays an essential role in the diagnosis, classification, prognosis and follow up of autoimmune diseases through the detection of serum autoantibodies. Autoantibodies to extractable nuclear antigens (ENA) anti-Ro/SSA, anti-La/SSB, anti-Sm, anti-RNP/U1RNP, anti-Scl-70/topoisomerase I and anti-Jo-1/histidyl-tRNA synthetase are clinically important in patients with autoimmune diseases such as Sjo¨gren’s syndrome, systemic lupus erythematosus (SLE), scleroderma, dermatomyositis and polymyositis among others. Their analysis is made preferably by enzyme linked immunosorbent assay (ELISA). This method has very good sensitivity and can detect small concentrations of antibodies. Nevertheless, in the last years the development of new chemiluminescent immunoassays is changing the methodology of the autoimmunity laboratories. Objective: Analyze the degree of agreement between chemiluminescence Zenith-RA method from Menarini Diagnostics (Florence, Italy) and the habitual ELISA from Inova Diagnostics (San Diego, USA) for the analysis of specific anti-ENA, anti-Ro/SSA, anti-La/SSB, anti-Sm, anti-RNP/U1RNP, anti-Scl-70 and anti-Jo-1. Materials and methods: We analized 85 samples from rheumatology patients using both instruments Zenit-SP+ (ELISA, Inova Diagnostics) and Zenit-RA (chemiluminescence immunoassay, Menarini Diagnostics). Once we obtained the results of the specific anti-ENA we classified our patients in positive or negative according to the cut-off values recommended by the manufacturer and we determine the degree of agreement using the statistical program SPSSv19. Results: The Kappa indices show a good degree of agreement forantiSm, anti-Scl70 and anti-Ro/SSA and moderate degree of agreement for anti-La/SSB, anti-RNP/U1RNP and anti- Jo-1. Conclusions: Both methods show good degree of agreement in the analysis of specific anti-ENA. Given the advantages of CLIA techniques in front of ELISA (master curve for each lot of calibrators and controls, linearity and continuous access of samples) it could be a valid option for the analysis of specific anti-ENA in the clinical laboratory.

P1574 Development of a conformation-dependent immunoassay for the diagnosis of systemic sclerosis K. Pozniak,* G. Nacci, A. Grieco,* C. Paolini,* S. Mori,* C. Tonnini,* M. Cuccioloni,à M. Mozzicafreddo,à M. Angeletti,à A. Funaro, A. Gabrielli* & G. Moroncini* *Clinica Medica, Dipartimento di Science Cliniche e Molecolari, Ancona, Italy, Lab of Immunogenetics, Department of Genetics Biology and Biochemistry, Torino, Italy, àBiochemistry, Dipartimento di Biologia Molecolare Cellulare e Animale, Camerino, Italy Purpose/Objective: The presence of conformational, stimulatory auto-antibodies (abs) against the PDGF receptor (PDGFR) in patients

affected by systemic sclerosis (SSc) has been questioned. IgG purification procedures and technical problems in cell-based biological assays and in solid phase binding assays can disrupt agonistic conformational abs and generate artifacts. To solve these issues and to validate the presence of anti-PDGFR abs in SSc patients, we aimed at developing a conformation-dependent immunoassay to detect agonistic anti-PDGFR abs in serum samples. Materials and methods: (1) Human monoclonal anti-PDGFR autoabs were cloned from memory B cells of SSc patients; (2) different recombinant PDGFR conformers were tested to set up a capture ELISA able to detect serum anti-PDGFR auto-abs; (3) competitive binding assays using anti-PDGFR monoclonal auto-abs and recombinant human PDGF were performed to define the PDGFR epitopes bound by these different ligands; (4) a conformational peptide library spanning PDGFR extracellular domains was generated to validate epitope mapping data; (5) peptides bound by monoclonal auto-abs were synthesized and pre-incubated with agonistic auto-abs to inhibit antibody biological activity. Results: (1) Monoclonal anti-PDGFR auto-abs generated from SSc B cell repertoire exhibited different PDGFR binding and agonistic properties; (2) a map of extracellular PDGFR functional domains was obtained: PDGFR epitopes bound by stimulatory auto-abs differed from those of non-biologically active auto-abs; (3) pre-incubation of agonistic auto-abs with peptides corresponding to their epitopes inhibited collagen stimulation in fibroblasts; (4) a competitive ELISA based on the inhibitory peptides was established to detect conformational PDGFR auto-abs in serum: this assay allowed discrimination between SSc and control sera with remarkable specificity and sensitivity. Conclusions: The identification of the epitopes of the stimulatory, SSc-specific, anti-PDGFR auto-antibodies was crucial to develop a conformation-dependent immunoassay discriminating between SSc and control sera. The solid phase binding assay based on these epitopes might be used as a novel tool for diagnosis of SSc and classification of the clinical subsets of disease and related conditions such as Raynaud’s Phenomenon.

P1577 Diagnostic value of serum high mobility group box protein 1 (HMGB1) and soluble CD163 (sCD163) levels in brucellosis A. O. Ayarci,* E. Yilmaz,* D. Sigirli, F. Budak,à G. Go¨ralà & H. B. Oralà *Infectious Diseases & Clinical Microbiology, Faculty of Medicine, Uudag University, Bursa, Turkey, Biostatistics, Faculty of Medicine, Uudag University, Bursa, Turkey, àImmunology, Faculty of Medicine, Uudag University, Bursa, Turkey Purpose/Objective: Both CD4+ and CD8+ T lymphocytes play critical roles in immunity to Brucella, in part because they secrete IFN-c and activate the bactericidal functions in macrophages. Therefore, use of markers which can evaluate macrophage activation may have diagnostic and prognostic importance. High mobility group-box 1 protein (HMGB1) is a late-onset proinflammatory cytokine secreted from activated macrophages. Soluble hemoglobin scavenger receptor (sCD163) is a specific marker of antiinflammatory macrophages. The aim of this study was to test potential associations of serum levels of HMGB1 and sCD163 with Brucellosis and its acute, subacute and chronic forms. Materials and methods: Serum HMGB1 and sCD163 levels in 49 Brucellosis patients (23 acute, 15 subacute, 11 chronic) were compared with 52 healthy control subjects. Brucellosis was diagnosed by a positive blood culture and/or increased Brucella antibodies in serological tests in addition to compatible clinical symptoms. Con-


670 Poster Session: Myeloid Cell Development centrations of serum HMFB1 and sCD163 were determined by ELISA according to the protocol of manufacturer. Results: Both serum HMGB1 and sCD163 levels were significantly higher in Brucellosis patients compared with healthy controls. The median serum HMGB1 level was 77.09 (0.00 182.84) ng/ml in healthy controls, whereas it was 170.65 (13.19 188.23) ng/ml in Brucellosis patients (P < 0.001). The median serum sCD163 level was 0.57 (0.21 1.52) mg/l in healthy controls, whereas it was 1.27 (0.37 2.13) mg/l in brucellosis patients (P < 0.001). There was no statistically significant difference in serum levels of HMGB1 and sCD163 among acute, subacute and chronic cases with Brucellosis. Additionally, serum HMGB1 levels were positively correlated with sCD163 levels, whilst neither HMGB1 nor sCD163 levels were correlated with CRP, WBC and sedimentation values. Conclusions: Our study demonstrated that the levels of HMGB1 and sCD163 may be diagnostic markers for Brucellosis, but none of them can be used for differentiating three different forms of disease (acute, subacute, chronic). On the other hand, studies with larger series of patient populations are necessary to confirm the biological significance of our results. Also, further studies are needed to assess the alteration of these mediators in response to treatment.

P1578 Effector and regulatory T cell subsets in hereditary hemorrhagic telangiectasia M. De Carvalho,* M. Vaucelle,* S. Mohamed, S. Amdouni,* C. Kohler,* M. C. Bene,* P. Kaminsky & G. C. Faure* *Laboratory of Immunology, Faculty of Medicine, Vandoeuvre-les-Nancy, France, Department Internal Medicine, Universitary Hospital of Nancy, Vandoeuvre-les-Nancy, France Purpose/Objective: Hereditary hemorrhagic telangiectasia (HHT) is a disorder characterized by recurrent epistaxis, cutaneous telangiectasia, and visceral arteriovenous malformations caused by mutations in the TGF-b receptor complex. Most cases are due to mutations in the ENG (endoglin) or ACRLV1 genes. HHT patients have higher rates of severe extracellular bacterial infections, yet few studies have evaluated their immune functions. TGF-b plasmatic levels have been described as either increased or decreased in HHT patients and one study found a deficit on IFN-c, TNF-a and IL-2 Th1 cytokines. As TGF-b has an important role on either regulatory T cells (Treg) and Th17 development, we investigated the levels of these two CD4 T subsets as well as Th1-polarized T cells in the peripheral blood of HHT patients. Materials and methods: Sixteen HHT patients and 15 healthy donors (HD) were included. Treg on whole blood were identified by flow cytometry on a lymphocyte gate as CD3+CD4+CD25+CD127low/neg cells. Th1 and Th17-polarized T cells were identified after overnight culture of PBMC in serum-free AIM-V medium in the presence of PMA, Ionomycin and Brefeldine-A. Unstimulated cells were used as negative control. Cells were labeled with a viability amine-reactive dye, with antibodies to CD3, CD4 (surface) and to IFN-c, TNF-a, IL-2 and IL-17 (after permeabilisation) to identify cytokine-producing T cells. Data was acquired on a Beckman Coulter flow-cytometer. Results: Median numbers of Treg in HHT patients (9.1% of CD3+CD4+ cells, 5.3 13.7) were similar to Treg numbers in HD (7.9% of CD3+CD4+ cells, 6.7 10.9, P = ns). Also, no statistical differences were found for IL-17+ CD3 producing cells between HHT patients (0.65% of lymphocytes, 0.17 2.5) and HD (0.8% of lymphocytes, 0.2 3, P = ns). Also, there was no difference between HHT patients and controls concerning IFN-c+ or TNF-a+: IFNc+CD3+ (HHT 23% of lymphocytes, 8 58 versus HD 15% of lymphocytes, 8 42, P = ns); TNF-a+ CD3+ (HHT 43% of lymphocytes, 20 73 versus HD 44% of lymphocytes, 29 58, P = ns). However, HHT patients had a significantly lower number of IL2+

CD3+ cells (35% of lymphocytes, 12 51) compared to HD (46% of lymphocytes, 18 54, P < 0.05, t-test). Conclusions;Our results suggest that there are no major changes on effector or regulatory CD4 T cell subsets in HHT patients. Deficits on innate immunity (phagocytes) could explain increased rates of bacterial infections in this condition. Further studies with a greater number of patients could potentially favor the identification of eventual subtle changes on T cell function as well as segregation of data according to genetic mutations.

P1579 ELISpot but not ELISA detects human IL-21 secreted by antigenstimulated human PBMC J. Huang, T. Bengtsson & N. Ahlborg R&D Department, Mabtech AB, Stockholm, Sweden Purpose/Objective: IL-21 is primarily secreted by activated T cells, in particular CD4+ follicular and Th17 T cells, and regulates the development of T cells, B cells, NK cells and dendritic cells. Dysregulation of IL-21 has been reported to contribute to diseases such as SLE and RA. The fundamental relevance of IL-21 as well as its involvement in inflammatory and autoimmune diseases highlights the need for development and evaluation of suitable immunoassays facilitating analysis of IL-21. Materials and methods: Monoclonal antibodies to human IL-21 were generated and used to develop sensitive capture ELISA and ELISpot assays. The assays were used to analyze IL-21 secretion by peripheral blood mononuclear cells (PBMC) after polyclonal (PHA) or specific stimulation (Tetanus toxoid; Candida albicans extract). Results: Polyclonal activation of PBMC induced IL-21 production detectable by ELISpot and ELISA. Increasing PBMC concentrations and pro-longed incubation times increased the number of IL-21secreting cells found by ELISPOT. IL-21 levels in supernatants, analyzed by ELISA, showed a reversed relationship with increasing cell concentrations and incubation times associated with a decrease in levels. In PBMC activated with specific stimuli, IL-21-producing cells could be detected by ELISpot whereas IL-21 levels in supernatants were not measurable by ELISA. Similar results were obtained when analyzing IL-4 by ELISpot and ELISA, in parallel. IL-17 production after specific stimuli, on the other hand, was detectable both by ELISpot and ELISA. Conclusions: Our data show that ELISpot can detect IL-21-producing PBMC under conditions where IL-21 levels in PBMC supernatants are not measurable by ELISA. Similar results were obtained with IL-4, a cytokine known to be difficult to detect by ELISA in supernatants from specifically activated PBMC due to consumption by cellular receptors. Difficulties to measure IL-21 by ELISA could thus depend on a low IL21 secretion level per cell and/or receptor consumption. Further investigations of this aspect are ongoing. The results emphasize the importance of using suitable methods for analysis of IL-21. Key words: human IL21; human IL17; human IL4; ELISpot; ELISA


Abstracts 671 P1580 Factors associated with parietal cell autoantibodies in the general population A. Cabrera,* D. Almeida, A. Arencibia,à A. Gonza´lez,§ M. Carretero, M. C. Rodrı´guez,§ V. Gil– & B. Brito§ *Research Unit, Nuestra Senõra de la Candelaria University Hospital, University of La Laguna, Santa Cruz de Tenerife, Spain, Immunology Unit, Nuestra Senõra de la Candelaria University Hospital, Santa Cruz de Tenerife, Spain, àDigestive Diseases Department, Nuestra Senõra de la Candelaria University Hospital, Santa Cruz de Tenerife, Spain, §Research Unit, Nuestra Senõra de la Candelaria University Hospital, Santa Cruz de Tenerife, Spain, –Research Unit, Miguel Hernańdez University, Alicante, Spain Purpose/Objective: The presence in serum of parietal cell autoantibodies (PCA) is a characteristic of autoimmune gastritis. We determined the prevalence of PCA in the general population and investigate their association with type 2 diabetes, insulin resistance and lifestyle factors related with autoimmune gastritis. Materials and methods: A cross-sectional study was performed, involving 429 individuals enrolled in a cohort study of the general population of the Canary Islands. All participants underwent physical examination, provided a blood sample and responded to a questionnaire regarding health and lifestyle factors. Serum concentrations of PCA, soluble CD40 ligand (sCD40L), C-peptide and glucose (to determine insulin resistance) were measured. The association of PCA with the other factors was determined with bivariate analysis, and losgistic regression models were used to adjust the associations for age and sex. Results: The prevalence of PCA was 7.8% (95% CI = 10.3 5.3). The factors associated with PCA were female sex (P = 0.032), insulin resistance (P = 0.016), menopause (P = 0.029) and sCD40L (P = 0.019). Alcohol consumption (P = 0.006) and smoking (P = 0.005) were associated with low prevalences of PCA. After adjustment for age and sex, the association with PCA was confirmed for smoking [OR = 0.1 (0.0 0.9)], alcohol consumption [OR = 0.3 (0.1 0.9)], insulin resistance [OR = 2.4 (1.1 4.9)], female sex [OR = 2.4 (1.1 5.3)], sCD40L [OR = 3.7 (1.2 11.4)] and menopause [OR = 5.3 (1.2 23.3)]. Conclusions: Smoking and alcohol consumption acted as protective factors against the appearance of PCA in the general population, whereas female sex, menopause, insulin resistance and elevated serum sCD40L were risk markers for PCA. In patients who smoke or drink alcohol, clinicians should be cautious when using PCA to rule out autoimmune gastritis.

P1581 Hematogonal cell populations in routine multiparametric flow cytometry analysis E. Caranfil,* I. Funingana,* C. Barzu,* G. Grigore & M. Zlei *Medicine, University of Medicine and Pharmacy ‘‘Grigore T. Popa’’ Iasi, Iasi, Romania, Laboratory of Molecular Biology, Regional Institute of Oncology Iasi, Iasi, Romania Purpose/Objective: Context: Multiparametric flow cytometry is a critical method in diagnosis and monitoring the evolution of neoplastic haematological processes, allowing quantitative classification, based on key molecular expression, of the cellular population structure in patient samples. Marker list selection and staining combinations are both crucial in defining normal and malignant proliferation, developmental and lineage cell identity, and may be useful to evaluate the minimal residual disease (MRD) and the postchemotherapy regenerating bone marrow. Objective: To determine the feasability of using retrospective (repetitive) flow cytometry multiparametric data in documenting the

MRD evolution and describing the regenerating haematological compartments. Materials and methods: Repetitive bone marrow aspiration cell samples (six samples), covering a 6 year interval (2006 2011) of a B-acute lymphoblastic leukemia studied case (first diagnosed at age 15) were analysed using up to six color flowcytometry (FACS CaliburCellQuest; FACS Canto II-FACS Diva). Staining protocol targeted with monoclonal antibodies the following molecules: CD45, CD19, CD22, CD20, CD10, CD34, CD38, TdT. Results: Retrospective analysis of the six iterative bone marrow sample cell populations documents the complete therapeutical response of the tumor cell line (which was a phenotypically-homogenous B-cell precursor expressing CD19, CD10, CD34, CD22, intracellular CD79a and TdT), and, lately, the expansion of a B-cell precursor compartment that was heterogenous and thus not evocative for relapse occurence (below 10%, CD45+ low CD19+ CD10+ CD34-/+, 0.6% CD22+ low CD20 s-/+, 3% CD38+ high). Conclusions: Multiparametric flow cytometry can discriminate between regenerative postchemotherapeutical nature of cell expansions versus MRD or relapse.

P1583 Immune system genes expression in athletes with allergies M. Boldyreva,* S. Tsarev,* M. Chulkina, A. Savilova, O. Burmenskaya, D. Trofimov, N. Shartanovaà & N. Ilyinaà *Immunogenetics, SSC Institute of Immunology FMBA of Russia, Moscow, Russia, R&D, JSC ‘‘DNA Technology’’, Moscow, Russia, à Clinical, SSC Institute of Immunology FMBA of Russia, Moscow, Russia Purpose/Objective: The aim of this study is to identify biomarkers for monitoring the functional state of the immune system in elite athletes. Materials and methods: The expression of genes (mRNA) IL-1b, TNF-a, IL-8, IL18; TLR2, TLR4, TLR9, IFN-a, IFNc, IL28, IL29, CD45, CD68, CD56, GATA3, TBX21, RORS2, Foxp3, TGF-b1 has been investigated. Quantitative mRNA was investigated by real-time PCR with reverse transcription and with normalization with respect to gene GUSB, HPRT1 B2M. We examined 22 athletes from the youth team of Russia on weightlifting. All the athletes were divided into three groups. Group 1 (n = 10) consisted of healthy athletes. Group 2 (n = 7) consisted of athletes who had chronic infection in anamnesis (frequent respiratory viral infection, chronic tonsillitis, herpes virus infection). Group 3 (n = 5) included athletes who had any manifestations of allergy in anamnesis and/or having a positive skin test and/or have elevated levels of total IgE in serum, but no allergy symptoms at the time of clinical examination. Epithelial cells scraping of the nasopharynx and peripheral blood mononuclear cells were used as a samples for the study of gene expression. Results: The performed investigation demonstrated increased expression of genes involved in the inflammatory IL1, IL8 and innate immune receptors TLR2, TLR4, and reducing the expression of the transcription factor RORC2, providing feedback regulation of inflammation only in the scraping of the epithelial cells and only in athletes with allergies without exacerbation. Conclusions: These results indicate that changes in immune function only in athletes with allergies (without exacerbation) in place of the primary immune cells contact with inhaled air, which may contain pathogens of respiratory infections and/or allergens.


672 Poster Session: Myeloid Cell Development P1584 Immunological features of IgA nephropathy development M. Yurkevich,* V. Pilotovich, K. Komisarov, H. Ivanchik* & M. Zafranskaya* *Department of Immunology, Belarusian Medical Academy of PostGraduate Education, Minsk, Belarus, Department of Nephrology, Minsk City Hospital N 1, Minsk, Belarus Purpose/Objective: IgA nephropathy (IgAN) is a major cause of endstage kidney disease but at this moment there are no reliable prognostic criteria for the assessment of IgAN severity and none of the factors initiating this disease has been identified. Defects in systemic and mucosal immunity and immune complex clearance by mesangial cells (MCs) may be of importance in IgAN initiation. The aim of this study was to estimate the contribution of immune cells and IgA clearance via IgA-binding receptors to IgAN development. Materials and methods: MCs were isolated from nephrobiopsies of patients with IgAN (n = 6) and non-IgA-mesangioproliferative glomerulonephritis (n = 4) by incubation with 1 mg/ml collagenase I type and were characterized by vimentin+ CD90+44+31-14-45- phenotype. Peripheral blood lymphocytes from patients with IgAN (34 ± 5 years, n = 28) and other glomerulopathies (31 ± 4 years, n = 15) were analyzed using CD3/CD5-PC7, CD4/CD8-PC5, CD19-PE, HLA DR/ CD25-PE, Vc2 TCR-Fitc, CD45RO-ECD monoclonal antibodies by means of flow cytometry FC500 (Beckman Coulter, USA). The level of serum immunoglobulins was determined by ELISA (VecktorBest, Russia). Results: We have observed increased level of serum IgA only in 62% patients with IgAN, while the presence of IgA in the kidney mesangium was the basic diagnostic criterion of this disease. High level of IgA receptor CD71 (81.2% (74.6% ‚ 85.4%) expression was detected in all MCs cultures. Other IgA-binding receptors (CD89, ASCRR) were not determined in MCs. At the same time IgAN patients are characterized by significantly decreased percentage of cd T cells in peripheral blood compared with non-IgAN patients (1.8% (1.1% ‚ 2.7%) and 3.7% (1.8% ‚ 6.4%), respectively). Compared to patients with normal frequencies of CD8+ T cells (30.3% (27.0% ‚ 33.9%); IgAN patients with CD8+ T cells number exceeding 38% (40.2% (38.3% ‚ 41.4%) had decreased whole blood protein concentration, increased serum creatinine andsevere proteinuria (up to 2 g/day) (P < 0.05).

P1586 In vivo quantitative fluorescence imaging of matrix metalloproteinases activity in the CAIA mouse model of rheumatoid arthritis Z. Stencel, S. Allden, M. Catley & S. Shaw UCB, Pharmacology, Slough, United Kingdom Purpose/ObjectiveMatrix metalloproteinases including MMP-9 are known to be involved in diseases including inflammatory arthritis and contribute to pathological processes such as joint destruction. In RA patients MMP-9 is localized to sites of inflammation in synovium and is elevated in synovial fluid, serum and plasma (Ahrens et al., 1996, Gruber et al., 1996). Similarly in the collagen antibody induced arthritis (CAIA) mouse model MMP-9 mRNA levels are also elevated in the footpad (Chia et al., 2008). The aim of this study was to determine in vivo MMPs activity at the site of inflammation at the acute and chronic stages of mouse CAIA using a fluorescence imaging techniques. In addition MMP-9 levels were measured in the serum. Materials and methods: CAIA was induced in male Balb/c mice by an i.p. injection of 4 mg of monoclonal antibody cocktail to collagen II followed by 25 lg LPS 24 h later. 1 h prior to antibody cocktail administration mice were treated with 30 mg/kg Enbrel s.c. q.a.d. The MMPSense 680, a fluorescent imaging agent, was used to detect in vivo activity of MMP-2, -3, -9 and -13 in hind paws at the peak of disease (d5) and in chronic stage (d12). Directly post imaging, serum samples were collected and MMP-9 concentration was measured using a mouse total MMP-9 immunoassay. Results: Control mice showed first clinical signs of swelling 48 h post LPS challenge that peaked 72 h post challenge. The anti-TNF treatment showed significant inhibition on disease throughout the experiment. The quantitative in vivo imaging showed significantly higher levels of MMP activity in inflamed paws compared to the negative control at acute and chronic stage. Positive control serum MMP-9 concentration was elevated compared to naı¨ve serum in both, acute and chronic stages. The Enbrel treated mice showed a similar level of MMP activity as naı¨ve mice at acute stage, however at the chronic stage MMP activity was significantly higher than naı¨ve mice. This correlated with serum MMP-9 levels that were comparable to naı¨ve levels at d5 and were significantly elevated in the chronic stage of disease. In addition the measured MMPs fluorescence correlated with clinical score in the chronic stage. Conclusions: Activity of MMPs was significantly increased in vivo at site of inflammation during acute and chronic stage in the CAIA model. The localised MMP activity correlated with measured MMP-9 concentration in serum from all treatment groups. Moreover the MMP activity could be inhibited by anti-TNF treatment in this model. This indicates that MMP-9 is a valuable biomarker for disease progression in CAIA model and could be used as an addition to clinical score.

Figure 1. Morphology (A) and phenotype (B) of MCs culture. Conclusions: The high level of CD71 expression on MCs of patients with various glomerulopathies is an evidence of the prevalence of immune system’s abnormalities over kidney clearance in IgAN. We suggest that CD8+ and cd T cells involve in IgAN development and progression and may be used as possible criteria for the assessment of this disease severity.


Abstracts 673 P1588 LC/DAD analysis of serum biogenic amines in patients with diabetes mellitus, chronic urticaria and Hashimoto’s thyroiditis

P1589 Levels of inflammatory mediators in gingival crevicular fluid from patients with periodontal disease before and after treatment

J. Trifunovic,* M. Jadranin, A. Damjanovic,à S. Raskovic,§ M. N. Djurovic,– D. Draskovic,– G. Pudar,** V. Tesevic, I. Juranicàà & Z. Juranicà *Organic Chemistry, Faculty of Chemistry, Innovative Center, University of Belgrade, Belgrade, Serbia, Center for Chemistry, Institute for Chemistry Technology and Metallurgy, University of Belgrade, Belgrade, Serbia, àExperimental Oncology, Institute for Oncology and Radiology of Serbia, Belgrade, Serbia, §Immunology and Allergology, Institute of Immunology and Allergology Clinical Center of Serbia, Belgrade, Serbia, – Endocrinology, Institute of Endocrinology Clinical Center of Serbia, Belgrade, Serbia, **Endocrinology Diabetes and Metabolism Diseases, University Clinical Center ‘‘Zvezdara’’, Belgrade, Serbia, Organic Chemistry, Faculty of Chemistry University of Belgrade, Belgrade, Serbia, àà Organic Chemistry, Institute for Chemistry Technology and Metallurgy, University of Belgrade, Belgrade, Serbia

P. M. Alberga,* A. Romero, M. Correnti & L. Escalona *Postgrado en Ciencias Fisiolo´gicas, Instituto de Medicina Experimental, Caracas, Venezuela, Facultad de Odontologıá, Instituto de Investigacion Odontologica, Caracas, Venezuela

Purpose/Objective: The biogenic amines putrescine (Put), histamine (His), spermidine (Spd), N-acetyl putrescine (NAP), N-acetyl spermidine (NAS), dopamine (Dop), epinephrine (Epi), norepinephrine (NE) and spermine (Spm) are a group of naturally occurring compounds exerting a large number of biological effects. This study was commenced to elucidate the role of biogenic amines as possible diagnostic markers for three autoimmune diseases: Diabetes mellitus, Chronic urticaria, and Hashimoto’s thyroiditis. Materials and methods: This study involved 20 patients with Diabetes mellitus, 20 patients with Chronic urticaria, eight patients with Hashimoto’s thyroiditis, and 20 healthy volunteers. We precipitated serum proteins using 0.4 M HClO4. At pH 8.0 we performed derivatization with dansyl-chloride. 50 ll of prepared serum samples were injected into LC/DAD, in conditions of gradient elution, on C18 column. Commercially available Put, His, Spd, NAP, NAS, Dop, Epi, NE, and Spm were dissolved in different concentrations in ultra pure water; treated in the same way as serum samples and injected into LC. Calibration curves were made by plotting peak area values against the respective concentrations of standards. The qualitative analysis was done using the method of retention time, and quantitative analysis using external calibration. The recovery study was carried out using real serum sample from healthy control, by spiking techniques. Results: Retention times were 6.6 min for NAS, 8.8 min for NAP, 9.1 min for Put, 10.1 min for His, 13.2 min for Spd, 13.9 min for NE, 14.7 min for Epi, 14.9 min for Dop, and 15.4 min for Spm, respectively. Obtained data showed excellent linearity of calibration curves for Put, His, Spd, NAP, NAS, Dop, Epi, NE, and Spm. Compared to controls, His levels in Diabetes mellitus patients were statistically higher; in Chronic urticaria patients levels of Put and His were lower; Spd, NE and Epi levels were enhanced; Put was statistically lower and Spd higher in Hashimoto’s thyroiditis patients. Chronic urticaria patients were the only in whose serum NE was found. NAP, NAS, Dop, and Spm were under limits of detection. Conclusions: Preliminary results from this study showed diverse distribution of investigated biogenic amines indicating different activation of metabolic pathways controlling biogenic amine biosynthesis and degradations in analyzed autoimmune diseases.

Purpose/Objective: The purpose of the present research was to determine the levels of IL-1a, IL-1b, TNF-a, MMP-3 and MMP-8 in gingival crevicular fluid (GCF) of subjects with chronic periodontitis before and after non surgical treatment. Materials and methods: Clinical measurements were carried out in 11 patients diagnosed with chronic periodontitis and 11 periodontally healthy controls. The clinical indexes evaluated were: gingival index (GI), plaque index (PI), bleeding on probing (BOP), probing depth (PD) and attachment loss (AL). The measurements were taken at six sites per tooth in all teeth in each subject. GCF samples were taken from one tooth per quadrant, and the levels of these mediators were measured using an ELISA test. Results: A statistically significant difference (P < 0.05) was observed in all the clinical parameters between patients and control group before and after periodontal treatment. Correlations between levels of mediators with the clinical parameters were not observed. Statistically significant differences were found between patients and control group in relation with levels of inflammatory mediators (P < 0.05) before and after treatment. The levels of IL-1a, IL1-b, TNF-a, MMP-3 and MMP-8 decreased in 44.2%, 48.35%, 53.28%, 62.16% y 34.03% respectively. Conclusions: Periodontal therapy reduced the levels of the inflammatory mediators evaluated in this study, which were significantly associated with the severity of periodontal disease. This research was supported by CDCH PG: 10-00-7070-2007.

P1590 Measuring autoantibodies against IL-17F and IL-22 in autoimmune polyedocrine sydnrome type I B. E. V. Oftedal,* A. Meager, E. S. Husebyeà & A. S. B. Wolff* *Endocrinology, Institute of Medicine, Bergen, Norway, Biotherapeutics, The National Institute for Biological Standards and Control, South Mimms, UK, àEndocrinology, Institute of Medicine, Haukeland University Hospital, Bergen, Norway Purpose/Objective: Patients with autoimmune polyendocrine syndrome type I (APS I) have at least two of the three disease components adrenal insufficiency, hypoparathyroidism and chronic mucocutaneous candidiasis. Various other organ-specific autoimmune manifestations are common, as well as a number of ectodermal symptoms. The underlying cause of APS I is mutations in the gene encoding the autoimmune regulator protein AIRE. Deficiency of this protein leads to loss of immunologic tolerance and release of autoreactive T-cells from thymus into the periphery. Patients frequently develop high titers of autoantibodies against molecular targets in their affected organs. The pathological role of these antibodies is unknown, but they have become important markers for APS I and other autoimmune conditions. Autoantibodies against interleukin (IL) -17A, IL-17F and IL-22 have recently been described in patients with APS I, and their presence is reported to be highly correlated to chronic mucocutaneous candidiasis (CMC). The aim of this study was to develop a robust high-throughput radioligand binding assays (RLBA) measuring IL-17F and IL-22 antibodies, and to compare them with current enzyme-linked immunosorbent assays (ELISA) of IL-17F and IL-22; moreover to correlate the presence of these antibodies to the presence of CMC. Materials and methods: A total of five RLBAs were developed based on IL-17F and IL-22 monomers and homo- or hetero dimers. As these


674 Poster Session: Myeloid Cell Development interleukins are small molecules, hence difficult to express in vitro, they were fused as dimers, which proved to increase the efficiency of expression. Sera from 25 APS-I patients were analysed by the different RLBAs and ELISA. Results: Analysing the presence of these autoantibodies in 25 Norwegian APS I-patients revealed that the different RLBAs detected anti-IL17F and anti-IL-22 with high specificity, using both homo- and heterodimers. The RLBAs based on dimer proteins are highly reproducible with low inter- and intra-variation. Conclusions: The RLBAs have the advantages of high throughput and easily standardisation compared to ELISA, thus proving excellent choices for the screening of IL-17F and IL-22 autoantibodies.

P1594 Preanalytical and analytical factors affecting assessment of extracellular vesicles as biomarkers E. Buza´s, B. Gyo¨rgy, T. G. Szabo´, K. Szabo´-taylor, K. Pa´loćzi, A. Kittel, G. Y. Nagy & A. Falus Department of Genetics Cell- and Immunobiology, Semmelweis University, Budapest, Hungary Purpose/Objective: Current research in the field of extracellular vesicles (including exosomes, microvesicles and apoptotic bodies) is undergoing a revolutionary development. Extracellular vesicles are considered both as disease-specific biomarkers and therapeutic vehicles. Our goal was to identify preanalytical and analytical factors that may have an impact on the detection of cell-derived extracellular vesicles. Materials and methods: We tested the effect of different anticoagulant tubes, shaking, storage at different temperatures, centrifugation forces and times and the effect of size filtration on the number and structure of blood plasma derived extracellular vesicles assessed by flow cytometry, transmission electron microscopy, atomic force microscopy, dynamic light scattering and qNANO techniques. Results: Our data indicate that the number of platelet derived microvesicles is strongly influenced by the type of anticoagulation. Shaking and storage of blood plasma samples at 37C proved to be strong inducers of platelet derived microvesicles. Forced filtration caused fragmentation of extracellular vesicles. Furthermore, we found that protein aggregates (such as immune complexes, biotin-avidin complexes) shared biophysical parameters with microvesicles and resulted in microvesicle-mimicking signals during flow cytometry. We developed a differential detergent lysis method to differentiate microvesicles from protein aggregates. Here we show that by using our improved method of microvesicle assessment, important new insights are gained into the pathomechanism of diseases such as rheumatoid arthritis. Conclusions: Correct assessment of extracellular vesicles in biological fluids requires standardization of both preanalytical and analytical parameters.

P1595 Preoperative serum levels of CA 242 in colorectal carcinoma M. Al-Kaabe,* S. Al-Fatlawi, J. Al-Musawi,à Q. Al-terrahi§ & M. Alsaidi– *Pathology and Foriensic Medicine, Medical College, Al Kut, Iraq, Al Sadar Teaching Hospital, Kidney Transplantation Center, Al Najaf, Iraq, à Medical Microbiology, Medical College, Al Najaf, Iraq, §Pathology and Foriensic Medicine, Medical College, Al Najaf, Iraq, –Microbiology & Immunology, Medical College, Al Kut, Iraq Purpose/Objective: Biochemical markers for colorectal carcinoma (CRC) are potentially useful in screening for early disease, aid in

diagnosis, determining prognosis, surveillance of patients undergoing curative resection and monitoring the treatment of advanced disease. Materials and methods: Preoperative serum levels of CA242was determined using ELISA technique for 35 patients with colorectal carcinoma, 25 patients with benign colorectal diseases (ulcerative colitis) and 10 volunteers as control group. Results: CA242 showed to be high distributed in patients with colorectal carcinoma which represented 37.1% as compared to patients with ulcerative colitis and control group which represented (16% and 0%) respectively (P = 0.026). The overall sensitivity of the CA242 test was 37.1% and the corresponding specificity was88.6%, using cut-off level 20 U/ml for CA 242. In regard to effect of age on the level of CA242 in study groups, it was showed to be high distributed in young age from (36 to 65) years which represented 17.1% (P = 0.088). Regarding to gender, CA242 showed to be high distributed in females which represented 61.5% as compared to males which represented 38.5% (P = 0.149). In regard to effect of grade (degree of differentiation) on the level of CA242, it showed to be high distributed in poorly differentiated colorectal adenocarcinoma which represented 38.5%, while in well and moderately differentiated colorectal adenocarcinoma which represented (30.7% and 15.4%) respectively (P = 0.588). When the stage of disease takes in a consideration CA 242 was 7.7%, 7.7%, 15.4% and 38.5% in stage (I, IIA, IIB, IIIB and IV) respectively (P = 0.218). Conclusions: In conclusion these findings suggest that CA242 elevated level and high sensitive in patients with colorectal carcinoma and may be used as an indicator of disease activity and may be considered as prognostic factor for patients with colorectal carcinoma. CA242 high distribution in young age may be attributed to incidence of colorectal carcinoma in Iraq occur in young age, but the cause of why was CA242 in females higher than males remain unclear. In patients with ulcerative colitis, CA242 may be used as predicting factor for malignant transformation.

P1596 Serum ghrelin and adiponectin levels are increased in underweight COPD patients, but leptin and proinflammatory cytokines are not changed A. K. Uzum,* M. M. Aydin, Y. Tutuncu,* E. Kiyan, B. Omer,à N. C. Ozbey* & F. Alagol* *Internal Medicine Endocrinology & Metabolism, Faculty of Medicine, Istanbul University, Istanbul, Turkey, Pulmonary Diseases, Faculty of Medicine, Istanbul University, Istanbul, Turkey, àClinical Biochemistry, Faculty of Medicine, Istanbul University, Istanbul, Turkey Purpose/Objective: Weight loss, muscle wasting, and tissue depletion are common features reported in COPD patients and they are all related with systemic inflammation. We investigated the relations between pulmonary functions, circulating levels of inflammatory mediators and metabolic parameters in underweight COPD patients. Materials and methods: Biochemical, hormonal, and anthropometric parameters, serum levels of adiponectin (ApN), ghrelin, leptin, hsCRP, IL-6, IL-1b, IL-8, TNF-a and pulmonary functions were evaluated. COPD patients were grouped according to Global Initiative for Chronic Obstructive Lung Disease criterion. Patients and control subjects were all male. Group 1: Mild-moderate COPD patients (n = 18; with a mean age of 66.4 ± 9.2 years; range 45 79 years and body mass index (BMI): 19.7 ± 1.5 kg/m2), group 2: Severe-very severe COPD patients (n = 32; with a mean age of 65.9 ± 10.0 years; range 45 80 years; BMI: 19.3 ± 1.6 kg/m2), group 3: Control group was composed of healthy subjects with normal BMI and pulmonary function tests (n = 17; with a mean age of 50.2 ± 8.4 years; BMI: 21.85 ± 1.5 kg/m2). The mean duration of COPD was 7.1 ± 6.4 years for group 1 and 7.3 ± 5.1 years for group 2. All patients were clinically


Abstracts 675 stable for at least 3 months and were not smoking for at least 6 months. Results: ApN concentration was higher in group 1 (43.3 ± 28.6 ng/ ml; P < 0.05) and group 2 (59.9 ± 31.8 ng/ml; P < 0.001) when compared with control group (23.5 ± 13.6 ng/ml). Ghrelin concentrations were higher in both COPD groups (1281.0 ± 1173.7 and 1840.0 ± 403.6 pg/ml; P < 0.05) than the controls (554.0 ± 281.9 pg/ ml). No significant increase for leptin, IL-1b, TNF-a, IL-8 was found between groups. IL-6, hsCRP levels were higher in group 1 than control group. ApN was negatively correlated with BMI and FEV1. Skinfold thickness were decreased in COPD patients when compared with control group. In the whole group, FEV1 was positively correlated with BMI, skinfold thicknesses of biceps, triceps and abdomen, insulin, tryglyceride whereas was negatively correlated with age, pack/years, HDLChol and ApN. We detected increased SHBG level with decreased insulin and HOMA-IR which mayindicate increased insulin sensitivity. Conclusions: We can conclude that antiinflammatory effect of ApN and ghrelin had been more evidentin severe-very severe COPD patients. This work was supported by Scientific Research Projects Coordination Unit of Istanbul University. Project number: 2245.

P1597 Significance of T helper 17 immunity in hepatitis C virus recurrence in orthotopic liver transplantation F. B. G. F. B. Giner,* G. S. G. Salgado,* M. B. M. Brunet, O. M. O. Millań, M. L. H. M. Lo´pez-Hoyos,à D. V. H. D. ValeroHervas,§ A. M. A. Minguela,* M. M. M. Miras,– R. A. R. A´lvarez* & M. M. M. Muro* *Immunology Service, Hospital Universitario Virgen de la Arrixaca, Murcia, Spain, Pharmacy Service, Hospital Clinic, Barcelona, Spain, à Immunology Service, Hospital Marque´s de Valdecilla, Santander, Spain, § Immunology Service, Hospital 12 de Octubre, Madrid, Spain, –Digestive Medicine Service, Hospital Universitario Virgen de la Arrixaca, Murcia, Spain Purpose/Objective: Hepatitis C virus (HCV) recurrence is universal following orthotopic liver transplantation (OLT) with an accelerated rate of fibrosis in the allograft compared to that in the native liver in chronic HCV. CD4+ T cells play an important role in HCV immunity and T-helper type 17 (Th17) cells have also been implicated in inflammatory proccesses associated with liver fibrosis. Our aim was to determinate the role of CD4+ Th17 cells in HCV recurrence in liver allograft transplantation. Materials and methods: Thirty OLTr and eighty four healthy donors (HD) from Murcia Region, Spain was included in this study. To quantify the frequency of CD4+ Th17 cells, the OLTr and HD bloods was cultured during 4 h in humidified 5% CO2 at 37C and used for an intracytoplasmatic flow cytometry assay. To measure IL-17 cytokine levels, we used a specific ELISA test (Quantikine Human IL-17 Immunoassay R&D System) after cultured the OLTr and HD bloods with Concanavaline A (ConA-Sigma). Statistical analysis was performed by using SPSS vs15.0 for Windows (SPSS, Chicago, IL). MannWhitney test was used to determinate significant differences in the frecuency of CD4+ Th17 cells or IL-17 cytokine levels between three groups (AR = Acute Rejection, NAR = Non-Acute Rejection and Control). Two-sided level of significance was set at P < 0.05. Results: We analyzed the frequency of CD4+ Th17 cells in non-HCV recurrence group, HCV recurrence and controls. The frequency of CD4+ Th17 cells in patients with HCV-recurrence increase since the beginning of orthotopic liver transplantation in comparison to NHCV group. Moreover IL-17 cytokine levels were higher in patiens with HCV-recurrence than in NHCV in basal situation.

Conclusions: This data suggest that the CD4+ Th17 cells plays an important role in HCV immunity.

P1598 Skin immunity in complex regional pain syndrome: epidermal langerhans cell density is significantly different between CRPS and non-CRPS affected limbs S. Osborne,* S. W. Edwards,* A. Goebel & R. Mootsà *Integrative Biology, University of Liverpool, Liverpool, UK, Pain Research Institute, University of Liverpool, Liverpool, UK, àInstitute of Aging and Chronic Disease, University of Liverpool, Liverpool, UK Purpose/Objective: We are investigating the role of the immune system within Complex Regional Pain Syndrome (CRPS) affected tissues in order to elucidate any possible role for immune cells in the generation or maintenance of chronic pain. Currently we are focused on cutaneous immunity and in particular on epidermal dendritic cells known as Langerhans cells (LCs). We hope that by characterizing any differences in immune cell infiltrate, phenotype, and function within CRPS limbs we can further understand the pathophysiology of the disease and discover new diagnostic indicators or avenues for therapeutic intervention. Materials and methods: Two 6mm skin punch biopsies were taken from CRPS patients (n = 6), one from a CRPS affected limb and another from a non-CRPS affected contra-lateral control limb. Biopsies were then incubated in 2 mM EDTA for a minimum of 2 h before separating the dermis and epidermis. Epidermal sheets were then stained for LCs in a two step procedure using the cell specific antibody CD1a. Co-staining with HLA-DR and appropriate controls were also performed. Epidermal sheets were imaged using 3 dimensional confocal microscopy and LC densities calculated as cells/mm2 of epidermis. Results: LC density in CRPS affected limbs was 484 ± 44 cells/m2 and in CRPS non-affected limbs was 587 ± 57 cells/mm2. These values were found to be significantly different using a paired T-test analysis (P = 5). This effect was dependent on direct MSC * B cell contact as shown using trans-well systems in co-culture assays. Preliminary data on candidate contact signals will be presented. Conclusions: These results suggest that allogeneic human bone marrow derived MSC enhanced the activation and proliferation of CD19+ B cell populations in vitro through a contact dependent mechanism, and may limit the utility of MSC therapy for autoimmune or idiopathic conditions with a major B cell contribution to pathology such as SLE.

P1623 Induction of allo-specific IL-10-producing T cells by lentiviral vector-mediated IL-10 gene transfer G. Locafaro, G. Andolfi, M. G. Roncarolo & S. Gregori Department of Regenerative Medicine Stem Cells and Gene Therapy, San Raffaele Telethon Institute for Gene Therapy, Milano, Italy Purpose/Objective: Type 1 regulatory T (Tr1) cells are a subset of CD4+ regulatory T cells (Tregs) induced in the periphery characterized by high levels of IL-10 production and regulatory activity. During the last decade, Tregs cell-based therapy has become an attractive nonpharmacological therapeutic option for immunomodulation in different pathological settings. Although, several protocols to generate human Tr1 cells have been developed in vitro, the resulting population includes a significant fraction of contaminating non-Tr1 cells, representing a major drawback for clinical application of Tr1 cell therapy. To overcome this limitation we developed a lentiviral vector (LV) encoding for human IL-10 (LV-IL-10) and we demonstrated that enforced LV-IL-10-mediated expression confers Tr1 phenotype and function to human polyclonal CD4+ T cells. In the present study we applied the LV-IL-10 platform for generating antigen-specific human Tr1 cells suitable for cell therapy. Materials and methods: Human CD4+ T cells were stimulated with allogeneic in vitro differentiated DC and activated T cells have been isolated based on the co-expression of early T cell activation markers. FACS-sorted T cells were expanded in vitro with IL-2 and transduced with LV co-encoding IL-10 and the marker gene, DNGFR. Resulting IL-10 engineered T cells were functionally characterized. Results: The co-expression of early T cell activation markers allowed the selection of allo-specific T cells upon in vitro re-activation. FACSsorted T cells can be easily transduced with LV-IL-10 and selected DNGFR+ IL-10-transduced CD4+ T cells expressed, upon allo-specific stimulation, high levels of IL-10, and are anergic. We are currently investigating whether IL-10 engineered T cells acquired suppressive activity. Conclusions: We develop a suitable method to generate allo-specific IL-10 engineered T cells. These results represent the first step for the


682 Poster Session: Myeloid Cell Development development of antigen-specific IL-10-producing T cells and will contribute to increase the success of Tr1-based immunotherapy, inducing tolerance to selected antigens, while minimizing general immune suppression.

P1624 Lack of long pentraxin 3 (PTX3) in bone marrow-derived mesenchymal stem cells impairs wound healing in mouse C. Cappuzzello,* A. Doni, E. Dander,* F. Pasqualini, M. Nebuloni,à B. Bottazzi, A. Mantovani, A. Biondi,§ C. Garlanda & G. D’Amico* *Research Center ‘‘M. Tettamanti’’, Universita` Milano-Bicocca, Monza, MB, Italy, Istituto Clinico Humanitas, Istituto di Ricovero e Cura a Carattere Scientifico, Rozzano, MI, Italy, àPathology Unit L. Sacco, Department of Clinical Sciences, L. Sacco Hospital Universita` degli Studi di Milano, Milan, MI, Italy, §Research Center ‘‘M. Tettamanti’’, Clinica Pediatrica Universita` Milano-Bicocca Ospedale San Gerardo, Monza, MB, Italy Purpose/Objective: Although several studies have shown the capacity of mesenchymal stem cells (MSCs) to repair and regenerate different tissues, the mechanisms underlying these processes are not understood. In the present study we analyzed the role of the long pentraxin 3 (PTX3) in enhancing the wound closure. PTX3 is a multifunctional protein produced by MSCs after activation with inflammatory cytokines, which is involved in innate immunity, inflammation and extracellular matrix deposition. Materials and methods: PTX3-deficient MSCs (PTX3-/-MSCs) were collected from bone marrow of PTX3 knockout mice. After 3 5 culture passages, cells were tested for the expression of specific surface markers, their ability to differentiate into mesengenic lineages and their capacity to abrogate T cell proliferation. Finally, equal number of both PTX3-/- and WT MSCs were implanted into excisional wounds created by a biopsy punch on the back of allogenic WT and PTX3-/- mice. Wound area was measured up to 14 day and calculated using an image analysis program. The wound specimens were collected at 2, 7 and 14 days and processed for histology. Results: By analyzing MSCs obtained from bone marrow of PTX3 knockout mice we demonstrated that, similarly to WT MSCs, PTX3-/-MSCs displayed typical fibroblastoid morphology, were consistently devoid of contaminating hematopoietic cells and expressed common MSC markers. In addition, these cells were able to differentiate into adipocytes and osteoblasts, and drastically decreased the mitogen-induced proliferation of PBMCs, in a dose dependent manner. Importantly, in a mouse model of wound healing, PTX3-/-MSCs showed an highly significant defect in wound closure compared to WT MSCs at each time point. Histologic evaluation of skin samples from mice treated with PTX3-/-MSCs showed a reduction in the granulation tissue at day 7, a significant increase of polymorphonuclear cells in the wound bed and an enhancement of the fibrin deposition at the 2nd day after injury. Accordingly, PTX3-/-MSCs also failed the ulcers closure in PTX3 knockout mice. Conclusions: We demonstrated that PTX3 deficiency does not alter the phenotype or the capacity of MSCs to differentiate into mesengenic lineages; of relevance, the production of PTX3 represents an essential requirement for MSC ability of enhancing tissue repair.

P1625 Lentivirus-induced dendritic cells co-expressing GM-CSF, IFN-a and pp65 for boosting immunity against cytomegalovirus A. Daenthanasanmak,* G. Salguero,* A. Schneider,* S. Borchers,* C. Figueiredo, B. Eiz-Vesper, A. Ganser* & R. Stripecke* *Department of Hematology Hemostasis Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany, Transfusion Medicine, Hannover Medical School, Hannover, Germany Purpose/Objective: Off the shelf vaccines such as proteins, peptides, and DNA for reducing the risks of cytomegalovirus (CMV) reactivation after hematopoietic stem cell transplantation have so far not shown major efficacy in clinical trials. One possible explanation is the underlying immune suppressed or immune dysfunctional status of the host, which might be partly alleviated with Donor Lymphocte Infusions (DLI). We are seeking to harness the effects of DLI, by providing the lymphocytes with improved homeostatic and antigenic stimulation provided by engineered induced dendritic cells (iDCs). Materials and methods: We evaluated integrase-defective lentiviral vector (IDLV) co-expressing the differentiated cytokines GM-CSF/ IFN-a in combination with CMV antigen, pp65. Overnight transduction of human monocytes with IDLV generated highly viable (>21 days) and immunophenotypically stable DCs, called Self-differentiated Myeloid derived Lentivirus-induced DC (SmyleDC). Results: In vitro cultured SmyleDCs demonstrated stable secretion of GM-CSF/IFN-a and several endogenously produced inflammatory cytokines (IFN-c, IL-2, -5, -6, -8). Expansion of multi-antigenic CMVreactive effector and memory CTLs were obtained after a single in vitro re-stimulation with SmyleDCs co-expressing pp65 as evaluated by tetramer analyses and IFN-c ELISPOT. In order to evaluate the effects of anti-CMV CTLs expansion in vivo, immune deficient NOD. Rag1-/-.IL2rc-/- (NRG) mice were immunized with SmyleDC/pp65 and, 7 days later, mice were infused with autologous CD8+ T cells from a CMV seropositive donor. SmyleDCs enhanced T cell engraftment and rapid expansion of multi-antigenic pp65 reactive CTLs in peripheral blood and spleen. Conclusions: Due to their capacity to precondition the host with a suitable homeostatic and antigenic environment prior to lymphocytes infusion, SmyleDC/pp65 is a promising immune regenerative cell product for the post-transplantation setting. Development of tricistronic lentiviral vectors and evaluation of potency and safety in humanized mouse models transplanted with human hematopoietic stem cells are ongoing for further clinical development.

P1627 Mesenchymal stromal cells isolated from bone marrow from multiple sclerosis patients reveals immunosuppressive properties G. L. V. de Oliveira,* K. C. R. Malmegrim,* A. M. Colombini,* P. B. Palma,* D. T. Covas & E. A. Donadià *Department of Immunology, National Institute of Science and Technology in Stem Cells and Cell Therapy, University of Saõ Paulo, Ribeiraõ Preto, Brazil, Department of Clinical Medicine, National Institute of Science and Technology in Stem Cells and Cell Therapy, University of Saõ Paulo, Ribeiraõ Preto, Brazil, àDepartment of Clinical Medicine, School of Medicine of Ribeiraõ Preto, University of Saõ Paulo, Ribeiraõ Preto, Brazil Purpose/Objective: Introduction/objective: Mesenchymal stromal cells (MSCs) have shown immunosuppressive and immunoregulatory functions. These properties of MSCs suggest their potential role in tolerance induction in autoimmune diseases. We evaluated the antiproliferative capacity of MSCs derived from multiple sclerosis (MS) patients, their ability to induce regulatory T cells and secrete IL-10 and TGF-b in vitro.


Abstracts 683 Materials and methods: MSCs were derived from bone marrow aspirates from MS patients (N = 10) and healthy controls (N = 10) and isolated by plastic adherence. For the inhibition assays, peripheral blood mononuclear cells (PBMCs) were labeled with CFSE and cocultivated (1:2, 1:5 and 1:10 ratios) with patients’ and controls’ MSCs MSCs in the presence of PHA at 37C in a 5% CO2 for 5 days. For the immune-regulation assays, non-labeled PBMCs were cocultivated with patients’ and controls’ MSCs. After 5 days of coculture, Tcell proliferation was assessed by CFSE method and the percentage of CD4+CD25hiFoxp3+ was assessed by flow cytometry. IL-10 and TGFb was measured in coculture supernatants by CBA flex kit. The results were analyzed by Mann-Whitney t test. This study was approved by the local ethics committee. Results: We observed significant differences (P = 0.002) in proliferation mean percentage when comparing PBMCs plus PHA (71.3 ± 12.9%) with patients’ MSCs plus PBMCs at 1:2 (33.3 ± 13.4%), 1:5 (39.1 ± 21.9%) and 1:10 ratios (46.6 ± 20.1%). There were no differences (P > 0.05) in percentages of CD4+CD25hiFoxp3+ regulatory T cells recovered after 5 days of ˜ MSCs compared to controls. IL-10 and coculture with patientsO TGF-b secretion were significantly decreased (P < 0.05) in supernatants derived from cocultures with patients’ MSC (IL-10: 53.8 ± 49.4 pg/ml; TGF-b: 59.4 ± 24.6 pg/ml) compared to controls (IL-10: 109.9 ± 64.6 pg/ml; TGF-b: 226.6 ± 153.2 pg/ml). Conclusions: Although the MSCs isolated from MS patients were able to suppress T-cell proliferation, they showed lower secretion of IL-10 and TGF-b cytokines. These data suggest that MSCs from MS patients have immunosuppressive capacity in vitro, however more studies should be conducted before their use in autologous clinical applications. Financial support: FAPESP.

P1628 Modulation of helper T cell responses using recombinant FoxP3 transfection A. Virksaite, S. Zigmantas & L. Zaliauskiene Fermentas UAB, R&D, Vilnius, Lithuania Purpose/Objective: Cationic polymer based transfection reagents are widely used as non-viral gene delivery vectors. The goal was to investigate if cationic polymers could be adapted for protein transport as well. Recombinant FoxP3 was chosen as a model protein for transfection into CD4+CD25- T cells and potential induction of regulatory T cell phenotype and function. Materials and methods: Full length FoxP3 gene was amplified by RTPCR from cDNA of Balb/C mouse CD4+CD25+ cells. Protein was expressed in E. coli ER2566 strain and purified by affinity chromatography under denaturing conditions; activity was confirmed by EMSA. Mouse CD4+CD25- T cells were transfected with recombinant FoxP3 using cationic polymer as transfection reagent. Protein localization after transfection was assessed by Western blot. T cell suppression was evaluated based on CFSE stained CD4+CD25- T cell proliferation 72 h

after activation with anti-CD3, ELISA assay was performed to monitor IL-2 levels in the culture medium. Results: Recombinant FoxP3 was found in the nucleus 3 h after transfection. Transfected CD4+CD25- T cells were able to suppress CFSE labeled CD4+CD25- T cell proliferation to a similar extent as natural regulatory T cells (Fig). IL-2 secretion was blocked as well; however, this effect was highly dependent on FoxP3 transfection efficiency. Conclusions: Cationic polymer can deliver functional FoxP3 protein and exert regulatory T cell phenotype to CD4+CD25- T cells.

P1629 Optimization for contact time dependent effect of human mesenchymal stem cells on the CD4 effector memory T cells M. Kim,* H. Jeong, W. Wee* & W. Leeà *Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea, Laboratory of Corneal Regenerative Medicine and Ocular Immunology, Seoul Artificial Eye Center, Seoul National University Hospital Clinical Research Institute, Seoul, Korea, àDepartment of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, Korea Purpose/Objective: Mesenchymal stem cells (MSCs) have been known to have anti-inflammatory properties in various inflammatory diseases. However, the reported effects of MSCs on inflammation highly vary, which is partly attributed to the lack of standardized analysis. This study was aimed at analyzing contact time-dependent effects of human mesenchymal stem cells (hMSCs) on IL-17 and IFN-c secretion by CD4+ effector memory T cells for the optimization. Materials and methods: Bone marrow-derived hMSCs were cocultured with human CD4+ T cells at a MSC:T cell ratio of 1:10. The suppressive effects of MSCs were evaluated by assessing their effects on the proliferation of CD4+ T cells by carboxyfluorescein diacetate succinimidyl ester (CFSE) stainingand the secretion of IFN-c and IL-17A by CD 4+ memory T cells at different time points of contact and culture duration. The levels of IL-17 and IFN-g were determined in supernatants from the co-culture using commercial kits for enzyme-linked immunosorbent assay (ELISA). Results: An earlier contact point between hMSCs and CD4+ T cells resulted in a greater suppressive effect on proliferation andboth IFN-c and IL-17A secretionin an indoleamine 2,3-dioxygenase (IDO)-independent manner. While, a later contact point time of hMSCs to activated T cells after 3 days or longer and duration more than 4 days dropped most suppressive effect of hMSCs down on effector memory CD4 T cells. Conclusions: Our study demonstrated that an earlier initial contact time, prior to the fully activated state of T cells, enhances the suppressive effects of hMSCs on effector memory CD4+ T cells. It also suggests that identifying an appropriate therapeutic window time would be critical in clinical trials with hMSC therapy for inflammatory disease.

P1630 Phenotypical/functional characterization of in vitro-expanded multipotent mesenchymal stromal cells from patients with type 1 diabetes J. N. U. Yaochite,* K. C. R. Malmegrim, K. A. Lima,* K. L. Prata, R. M. F. Bolzoni, P. V. B. Palma, D. T. Covas & J. C. Voltarelli *Basic and Applied Immunology, School of Medicine of Ribeiraõ Preto, University of Saõ Paulo, Ribeiraõ Preto, Brazil, Regional Hemotherapy Center, School of Medicine of Ribeiraõ Preto, University of Saõ Paulo, Ribeiraõ Preto, Brazil Purpose/Objective: Mesenchymal stromal cells (MSCs) are progenitor cells with capacity to modulate the immune response, migrate to injury


684 Poster Session: Myeloid Cell Development site and promote tissue repair, thus representing a potential treatment for autoimmune/inflammatory diseases. However, it is not established whether MSCs have functional impairments in type 1 diabetes (T1D) setting. The objective of this study was to evaluate and compare phenotypical and functional characteristics from T1D-MSCs with control-MSCs for future clinical applications. Materials and methods: MSCs cultures were derived from bone marrow aspirates of healthy controls (control-MSCs) and newly diagnosed T1D-patients (T1D-MSCs). T1D-MSCs were characterized using the following characteristics: morphology, circularity average (ViCell-XR analyzer), expression of cell surface antigens (flow cytometry), adipogenic differentiation capacity (in vitro assay), gene expression profile by microarray analyses (Agilent Technologies) and migration capacity (in vitro assay). Results were compared with those of control-MSCs. Results: MSC derived from T1D patients showed typical spindleshaped morphology. The presence of surface markers (CD45, CD90, CD105, CD73, HLA-II) and cell circularity average (16.41 ± 0.69 lm T1D-MSCs · 15.91 ± 0.59 lm control-MSCs) did not differ from control-MSCs. T1D-MSCs exhibited the same differentiation potential towards the adipogenic lineage when compared to control-MSCs. Gene expression analyses reveled about 2978 differentially expressed genes (P < 0.05, fold change: 2). We observed an up-regulation of chemokine receptors genes (CCR3, CXCR5) and down-regulation of genes related with focal adhesion in T1D-MSCs. According to these results, MSCs from T1D-patients exhibit higher capacity to migrate through an 8lm membrane pore using 50% FBS as chemoattraent (786.1 ± 106.8 cells T1D-MSCs · 385.8 ± 61.02 cells control-MSCs, P = 0.006). Conclusions: MSCs from T1D-patients exhibit the same morphological, immunophenotype properties and adipogenic differentiation potential as their healthy counterparts. On the other hand, T1D-MSCs exhibited differences in the gene expression profile and also a higher ability to migrate than control-MSCs. Further studies are needed to investigate the immunomodulatory/regenerative features of T1DMSCs prior to their potential use in clinical trials.

P1631 Role of interferon-gamma in the activation of mesenchymal stem cells: analysis in a model of acute kidney injury P. B. Costa,* M. B. Silva,* P. Semedo,* M. A. Reis, A. P. Silvaà & N. O. S. Camara§ *Medicine, Federal University of Sao Paulo, Saõ Paulo, Brazil, Pathology, Federal University of Triangulo Mineiro, Uberada, Brazil, à Education and Research Institute, Albert Einstein Hospital, Saõ Paulo, Brazil, §Immunology, University of Saõ Paulo, Saõ Paulo, Brazil Purpose/Objective: The stem cell therapy is flourishingas an alternative to acute kidney injury (AKI). It is known that in the lesion environment, pro-inflammatory cytokines, such as TNFa and IFNc, activate the therapeutic role of MSCs. However little is known about this mechanism. This study assesses the role of IFNc in the activation of MSC regenerative properties in acute renal models. Materials and methods: MSC from IFNc receptor knockout animals (IFNcR KO) and from wild type animals (WT C57/Bl6) were isolated from adipose tissue. After isolation and its culture, both cells were administrated in renal ischemia-reperfusion (IR) model. C57/Bl6 mice were subjected to bilateral IR by clamping both renal pedicles for 45 min. Four hour after reperfusion, 2.105 MSCs from IFNcR KO or from WT were intraperitoneally administered to each animal. After 24 h of IR, animals were sacrificed. Results: Both treatments with MSC showed significant reduction of serum urea and creatinine, but the treatment with IFNcR KO MSC was less effective. Acute tubular necrosis (ATN) analyses corroborate with functional assays. Both cells treatments havereduced IL-6 mRNA

expression when compared to untreated animals. However, IL-6 mRNA expression was greater in animals treated with IFNcR KO MSC when compared to treatment with WT MSCs. The IL-10 mRNA expression was higher in animals treated with WT MSC when compared to untreated and KO MSC group. PCNA analysis by immunohistochemistry was increased in treated animals with WT MSCs than animals treated with IFNcR KO MSC. At Tunel assays, it was observed that the group treated with IFNcR KO MSC had more apoptosis than others. In addition, In vitro studies have shown that IFNcR KO MSC have a lower proliferative rate than WT MSC. However, when WT MSC were incubated with recombinant IFNc (20 ng/ml) the proliferative rate is increased. Conclusions: The presence of IFNc receptor at MSC is not essential for tissue repair, since functional parameters and ATN are not different in both cells treated animals. On the other hand, immunomodulation response differs. MSC response to IFNc may play role on it, since proliferative rate and apoptosis are altered in the animals treated IFNcR KO MSC. More research is needed to better understand the role of IFNc in activation and response of MSCs and their implication in cell therapy. Support: FAPESP, CNPq.

P1632 Salvage T cell therapy for resistant viral diseases after stem cell transplantation M. Uhlin, J. Gertow, S. Berglund, M. Okas, M. Uzunel, E. Watz, P. Ljungman, M. Maeurer & J. Mattsson Deaprtment of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden Purpose/Objective: Epstein-Barr virus (EBV), cytomegalovirus (CMV) and adenoviral reactivations are frequent complications after allogeneic SCT because of a lack of T cell control due to extensive immunosuppression. Cytotoxic T lymphocytes (CTLs) that recognize viral antigens are the most important immune effector mechanism controlling the persistent viral infections. First-line treatment for viral reactivation is dose reduction of the immunosuppressive drugs and/or anti-viral therapy. For PTLD this is followed by rituximab (anti-CD20 monoclonal antibody). Despite these multiple treatment strategies, the l mortality from drug-resistant viremia after SCT is still considerable. Another treatment approach is adoptive transfer of virus specific CTLs from the donor. The standard method of adoptive T cell immunotherapy is laborious and timeconsuming and is often too late to be administrated to the patient. Materials and methods: We have developed a clinical separation protocol for virus specific CTLs based on labeling with multimeric complexes containing recombinant HLA molecules together with virus derived peptides. By combining this labeling technique with a secondary magnetic sorting we have managed to get a high purity of specific CTLs. This high purity diminish the risk of creating GVHD in the recipient even if the adoptive transfer of cells is done in an allogeneic or haplo-identical setting. Results: We first used this protocol in an 18 year old patient with lifethreatening PTLD. The patient developed an EBV associated lymphoma involving lungs, liver and both kidneys and also showed extremely high EBV titers in blood. It was decided to give her EBV specific CTLs from her mother. 2 months after the given EBV specific CTL infusion the EBV associated lymphoma was in complete regression. After this we have further successfully treated seven patients with life threatening viral disease (Adeno, CMV and EBV) with good efficacy. We could see a clinical and immunological response in six out of eigth patients. In five out of six of these responding patients we have been able to identify infused T cells using chimerism analysis and SNP markers specific for the CTL donor. Conclusions: This method opens up the possibility to rapidly treat patients which are in acute need of T cell therapy and cannot wait for


Abstracts 685 prolonged expansion techniques or cannot tolerate standard treatment regiment.

P1634 T cells engineered to express high but not lower affinity T cell receptors recognize human tumor antigen cross-presented by the stroma and eradicate large tumors M. Leisegang,* T. Kammerto¨ns, T. Blankenstein & W. Uckert* *Molecular Cell Biology and Gene Therapy, Max-Delbru¨ck-Center for Molecular Medicine, Berlin, Germany, Institute of Immunology, Charite´, Berlin, Germany Purpose/Objective: Engineering of T cells with tumor-reactive T cell receptors (TCR) TCR gene therapy provides a powerful approach for adoptive therapy of cancer. The anti-tumor effect of TCR-engineered T cells is largely determined by the transgenic TCR that recognizes tumor cell-derived peptides presented on the patient’s tumor tissue. However, current therapeutic approaches are impeded because although function of selected TCR can be analyzed in vitro, parameters that predict in vivo efficacy are not known. We developed a mouse model to evaluate the therapeutic effect of TCR gene therapy on established cancer. Materials and methods: In order to establish a versatile, syngeneic model system for TCR gene therapy, we generated human MHCtransgenic mice that accept cancer cells modified to express human

tumor antigens of choice. T cells were engineered with human tyrosinase-specific TCR of high or lower affinity. The anti-tumor effect of the T cells was analyzed in vitro and by adoptive transfer into mice bearing established, tyrosinase-positive cancer. Therapeutic efficacy was assessed by monitoring tumor growth. Cross-presentation of antigen by tumor stroma was analyzed in parallel by functional assays and by in situ confocal microscopy. Results: Adoptive transfer of T cells engineered with high affinity tyrosinase-specific TCR resulted in complete rejection of cancer, whereas lower TCR affinity selected for escape variants and relapse. The tumor-derived human tyrosinase was cross-presented by tumor stroma cells. Although T cells engineered with both the high or lower affinity TCR showed identical tumor cell killing in vitro, only T cells expressing the high affinity TCR recognized cross-presented antigen on the tumor stroma. Conclusions: This in vivo model provides a versatile, pre-clinical test system of adoptive T cell therapy of cancer to predict whether TCRengineered T cells eradicate large tumors or select escape variants. Our results indicate that only TCR gene therapy with high affinity TCR prevents relapse, as opposed to use of lower affinity TCR which are typically isolated from the tolerant human repertoire against tumorassociated antigens. We suggest that the high affinity of the TCR enables the T cells to recognize and perhaps eliminate tumor stroma cells cross-presenting the melanoma antigen tyrosinase, resulting in bystander elimination of escape variants.



Poster Session: Gene Therapy P1636 A good manufacturing practice procedure to generate therapeutic numbers of highly pure anti-leukemic virus-specific T-cells M. M. van Loenen,* R. de Boer,* P. A. G. van Liempt,* R. S. Hagedoorn,* P. Meij, J. H. F. Falkenburg* & M. H. M. Heemskerk* *Hematology, Leiden University Medical Center, Leiden, Netherlands, KFT, Leiden University Medical Center, Leiden, Netherlands Purpose/Objective: Second half of 2012, we aim to start a clinical trial to treat patients with hihg risk acute leukemia with a donor-derived HA-1TCR transduced (td) virus-specific T-cell product as early as 8 weeks after allogeneic stem cell transplantation. This T-cell product is selected based on well-defined specificities predicted to result in selective Graft versus Leukemia effect without Graft versus Host Disease. Materials and methods: To obtain therapeutic cell numbers, one of the inclusion criteria is presence in donor peripheral blood of ‡1 virusspecific T-cell population with a frequency of ‡0.05% of T-cells. MHCStreptamers will be used to isolate minimally one and maximally two virus-specific T-cell populations from donor leukocytes. MHCStreptamer incubation will allow magnetic bead based cell selction of virus-specific T-cells of interest. Subsequently, these T-cells will be transduced with the HA-1-TCR. The process of isolation of pure populations of virus-specific T-cells and transduction with good manufacturing practice (GMP)-grade retroviral supernatant encoding the HA-1-TCR has been validated with three test procedures at large scale in the cleanroom. Results: To pass the in process (IP) testing, T-cells needed to be ‡50% pure for the respective virus-specific tetramer after MHC-Streptamer isolation. In addition, release criteria for the cell product after transduction and subsequent culturing were positive staining for CD3/ TCRab ‡95% of the cells and ‡60% antigen-specific, as measured with virus- and HA-1-tetramers. Moreover, transduction efficiency of ‡5% as measured with HA-1-tetramers is a prerequisite. Conclusions: All HA-1-TCR td virus-specific T-cell products met the criteria for IP testing and quality control testing, and were highly reactive against HA-1-positive leukemic cells. Here, we present a GMPgrade procedure, to generate therapeutically relevant numbers of defined antigen-specific T-cells.

P1638 Downregulation of CTLA4 by vector-mediated RNAi in human leukaemia T cells R. Vassileva,* J. Curtin* & J. Kelly *Dublin Institute of Technology, School of Biology, Dublin, Ireland, National Children’s Research Centre, Our Lady’s Children’s Hospital, Dublin, Ireland Purpose/Objective: In this study a gene therapy delivery system to overexpress small interfering RNA (siRNA) was designed and used to silence the expression of human CTLA4. We generated a stable T cell line expressing the CTLA4-specific siRNA that can be used as a tool for analysis of the role of CTLA4 in T cell down-regulatory pathways. Materials and methods: A small hairpin RNA (shRNA) duplex designed to target the human CTLA4 mRNA transcript (siCTLA4) was cloned under the control of a U6 promoter in a previously described pBSU6neo vector. As a negative control a scrambled version of that sequence (scrCTLA4) was used. The efficacy of each plasmid was estimated by transient co-transfections in HeLa cells with a plasmid encoding CTLA4 expression, which was detected using Western blot and real-time PCR. Three stable T cell lines that have separately integrated pBSU6neo siCTLA4, pBSU6neo scrCTLA4 and pBSU6neo

into the cellular genome have been developed and silencing efficacy was estimated by RT-PCR analysis. A stem loop RT-PCR method followed by real-time quantification was optimized to detect the expression of the mature siRNA in the new cell lines. Results: We generated successfully a novel shRNA expressing vector pBSU6neo siCTLA4 encoding a sequence designed to silence CTLA4 expression. The ability of this vector to downregulate over 80% of induced CTLA4 expression compared to negative controls was confirmed in co-transfection experiments. Further stable T cell lines that incorporate the new plasmids were developed as a model to investigate the role of CTLA4 in functional assays. It was confirmed that the endogenous CTLA4 expression induced upon stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin is silenced in the T cell line which constitutively expresses the CTLA4-specific siRNA. Conclusions: In this study we demonstrated the successful downregulation of human CTLA4 expression using RNA interference. We suggest that the new developed T cell line with silenced CTLA4 expression could be used as a new model for the investigation of the role of CTLA4 as a suppressor of T cell activation.

P1639 Enhancing the potency of plasmid DNA vaccine via the incorporation of CpG motifs C. H. Huang,* E. X. L. Loo,* G. H. Soh,* L. H. Chua,* H. M. Wen,* X. Fan, Z. Li, K. Y. Chua,* I. C. Kuo* *Paediatrics, National University of Singapore, Singapore, Singapore, Shanghai Public Health Clinical Centre, Fundan Universisty, Shanghai, China Purpose/Objective: Plasmid DNA immunization is a potential approach against allergy, infectious diseases or tumors as it conferred many advantages over other vaccine approaches. In contrast to rodent models, most DNA vaccines exhibited low immunogenicity in nonhuman primates and humans. This study aimed at enhancing the immunogenicity of plasmid DNA vaccines by incorporating additional CpG motifs into the pVAX1 backbone. Materials and methods: The D and K type CpG motifs were inserted into the pVAXBlot5 construct. The resulted plasmid, pVAXBlot5DTKT, encoded a major allergen from the dust mite Blomia tropicalis for monitoring the antigen specific immune responses. The stimulatory properties of pVAXBlot5, pVAXBlot5-DTKT or DTKT oligonucleotides were evaluated by measuring the mediators released from the coculture with human peripheral blood mononuclear cells (PBMCs). The prophylactic effects of the modified construct on Th2 immune responses were evaluated in a Blo t 5 induced murine allergy model. The immunogenicity of modified and unmodified constructs was also assessed in rhesus macaques by measuring Blo t 5 specific IgE and IgG as well as Blot 5 specific IFN-c and IL-4 producing cells by ELISPOT assays. Results: Co-culture of PBMCs with pVAXBlot5-DTKT elicited higher levels of pro-inflammatory cytokines/chemokines such as IL-6, TNF-a, MIP-1a and MIP-1b as compared to pVAXBlot5 or DTKT oligonucleotides stimulation at 5 and 24 h. In vivo evaluation in mice revealed that both pVAXBlot5-DTKT and pVAXBlot5 attenuated Blot 5 specific IgE. The modified pVAXBlot5-DTKT induced higher levels of Blot 5 specific IgG2c and IFN-c when compared to pVAXBlot5. Monkeys immunized with both pVAXBlot5 and pVAXBlot5-DTKT and challenged with the Blot 5 protein showed lower Blot 5 specific IgE as compared to monkeys immunized with the pVAX1. The pVAXBlot5DTKT immunized group also produced higher levels and fast kinetics of Blot 5 specific IgG when compared to those immunized with pVAXBlot5. Furthermore, the numbers of Blot 5 and Blot 5 peptides specific IFN-c producing cells were higher from pVAXBlot5-DTKT immunized monkeys.


Abstracts 687 Conclusions: The optimal use of CpG motifs as the molecular adjuvant to enhance the priming of immune responses induced by DNA vaccine is still a feasible approach.

P1640 Entire requirement of endogenous type I interferon for the efficacious cancer treatment with viral vector-encoded interleukin-12 S. Hervas-Stubbs,* J. I. Quetglas,* J. R. Rodriguez-Madoz,* ˜ o,* E. Casales,* J. I. Riezu-Boj,* M. C. Ochoa,* U. Manchen M. Sanmamed,* N. Thieblemont, C. Smerdou* & I. Melero* *CIMA, Gene Therapy and Hepatology, Pamplona, Spain, Universite´ Paris Descartes, Hoˆpital Necker, Paris, France Purpose/Objective: To date little is known about how the innate IFNa/b response affects the efficacy of cancer treatments based in viral vector delivery of therapeutic genes. Using Semliki Forest virus encoding IL-12 (SFV/IL-12), a promising RNA viral vector for tumor treatment, we have explored this. Materials and methods: SFV/IL-12 was administered intratumorally to MC38 tumor-bearing mice and tumor growth, IFNa/b production and/or anti-tumoral CTL response were monitored. The IFNa/b production elicited by SFV/IL-12 was compared in WT, IPS1)/) and TRIF)/) mice. The role of macrophages, pDCs and conventional DCs (cDCs) in the IFNa/b response was analyzed by depleting these populations with clodronate or anti-mPDCA-1 mAb administration to WT mice or by treated CD11c/DTRGFP mice with Diphtheria toxin, respectively. Finally, the requirement of IFNa/b for the anti-tumoral efficacy of SFV/IL-12 was tested in mice deficient for the IFNa/b receptor (IFNAR) and in mice treated with a neutralizing IFNAR mAb. Results: Intratumoral injection of SFV/IL-12 induces IFNa/b production which is impaired in IPS1)/) and, to some extent, in TRIF)/) mice. Using bone-marrow (BM) chimeric mice we show that both hematopoietic and stromal cells are involved in the IFNa/b response. Macrophages, pDCs and cDCs are all implicated in the IFNa/b production. The therapeutic activity of SFV/IL-12 against MC38 tumor is absolutely lost in IFNAR)/) mice and when IFNAR signaling is blocked in vivo with anti-IFNAR mAb. This is also true with other tumor model efficiently cured by SFV/IL-12, such as TC1. The lack of efficacy is not related to an impaired expression of the transgene because IFNAR)/) mice even express higher levels of IL-12 and other reporter SFV-encoding genes, such as Luciferase. Using BM chimeric mice were only hematopoietic or stromal cells were IFNAR)/), we show that the IFNAR absolute requirement is operational at hematopoietic-derived cells. In accordance with the described mechanism of action of SFV/IL-12, tumor-specific CTLs are outstandingly expanded upon SFV/IL-12 intratumoral injection. However, this does not happen when tumor-bearing mice are IFNAR)/), or have been treated with anti-IFNAR mAb, or if they are deficient for IPS1. Conclusions: All in all, our data show that the efficacy of tumor immunotherapy with viral vector-encoded IL-12 is totally dependent on type I IFNs, unrevealing an unexpected mechanism of action of a therapy that is being translated to patients.

P1641 Exploiting CD8 co-receptor to improve the function of genetically modified CD4 T cells for cancer immunotherapy I. Chua,* M. Ahmadi,* S. Xue,* A. Holler,* R. Zamoyska, H. J. Stauss* & E. C. Morris* *UCL, Immunology and molecular pathology, London, United Kingdom, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Purpose/Objective: TCR gene transfer can generate tumour antigenspecific T cells for adoptive immunotherapy. Many TCR genes isolated

for tumour immunotherapy are MHC class-I restricted TCR and when transduced into CD8 T cells endow potent anti-tumour protection effects in-vivo. Attempts to engage the CD4 T cell using the MHC-I restricted TCR often resulted in suboptimal antigen specific responses. In this study, we show that the anti-tumour function of genetically modified CD4 T cells can be enhanced by introducing the CD8 coreceptor. Materials and methods: The MHC-I restricted F5 TCR recognized influenza-A nucleoprotein (NP) presented by Db. Retroviral constructs containing the F5-TCR or the CD8 co-receptor were used to transduce murine CD4 T cells for in-vitro functional studies or in-vivo tumour protection studies. Irradiated mice were injected subcutaneously with tumour cells expressing NP and luciferase. These mice were subsequently injected with genetically modified CD4 T cells and the tumour growth was monitored. Surviving mice were re-challenged with irradiated tumour cells. Results: The in-vitro function of CD4 T cells transduced with F5-TCR shows that IL-2 and IFN-c production is enhanced with CD8 coreceptor. In addition, introducing a L58R mutation in the CD8 coreceptor can further increase this effect. In-vivo studies shows that introducing the TCR alone did not endow CD4 T cells with significant tumour protection. However adding CD8 co-receptor to the CD4 T cells enhances tumour protection. The genetically modified CD4 T cells persist for >3 months in surviving mice and when re-challenged with antigen the CD4 T cells with both F5-TCR and CD8 co-receptor have greater proliferative capacity and have more central memory phenotype cells. Conclusions: The CD8 co-receptor enhances the in-vitro and in-vivo function of genetically modified CD4 T cells transduced with MHC-I restricted TCR. These genetically modified CD4 T cells can provide tumour protection, persist indefinitely and proliferate on re-challenge. The effects of the CD8 co-receptor can be further enhanced by introducing the L58R mutation.

P1642 Generation of human islet antigen-specific regulatory T cells for treatment of type 1 diabetes C. M. Hull, T. Tree, M. Peakman, J. Maher & M. Estorninho Immunology Infection and Inflammatory Disease, King’s College London, London, UK Purpose/Objective: Type 1 diabetes (T1D) is an autoimmune disease characterised by the destruction of insulin producing beta cells in the pancreas. Research has shown that the ability of natural regulatory T cells (nTregs CD4+, CD25hi, FoxP3+) to suppress effector T cells in T1D is defective leading to the proposal of adoptive cell therapy, in which nTregs isolated from patients are enhanced for their regulatory capacity and re-infused, as a possible therapy. Support for this comes from the NOD mouse model of T1D where it has been shown that infusion of polyclonal nTregs can suppress disease with more effective suppression seen if the infused nTregs are specific for an antigen involved in disease. Therefore this research focuses on the development of methods for lentiviral transduction of T cell receptors (TCRs) into human nTregs, allowing for redirection of a patient’s polyclonal pool towards antigens involved in disease. Materials and methods: T cells with a known specificity were clonotyped in order to identify the allele usage of the V, (D) and J regions of the TCR chains and the sequence of the V region. To sequence TCR chains cDNA was produced from RNA via 5’ RACE which was then used to produce TCR alpha and beta chains using constant region primers. Each cDNA product was then sequenced and re-amplified to add restriction sites for sub-cloning in to an expression vector for future production of lentivirus . This will involve the transfection of 293T cells with the viral backbone (product of sub-cloning), viral envelope and encapsidation plasmid using polyethylenimine.


688 Poster Session: Myeloid Cell Development Results: Clonotyping data has identified the sequences and allele usage of three TCRs specific for peptides of: the tyrosine phosphatase IA-2, insulin and haemagglutinin (HA). From these results V region primers were designed allowing for the alpha and beta chains of each of the TCRs to be re-amplified in order to add restriction sites to the cDNA products for subcloning in to the expression vector. Conclusions: In conclusion, previous work has shown that addition of antigen specific nTregs can suppress the development of diabetes. Therefore the redirection of a patient’s nTregs towards an antigen involved in disease may represent a powerful therapeutic tool. This ectopic expression of a TCR can be induced using lentiviral transduction and therefore the alpha and beta chains of three TCRs specific for antigens involved in T1D have been cloned to produce lentiviral vectors.

P1643 Immune responses to transgene and retroviral vector in patients treated with ex vivo engineered T-cells C. Lamers,* P. Elzakker van,* S. Steenbergen van,* J. Oosterwijk, S. Sleijfer,* R. Debets* & J. Gratama* *Medical Oncology, Erasmus MC University Medical Centre, Rotterdam, Netherlands, Experimental Urology, Radbout University Medical Centre, Nijmegen, Netherlands Purpose/Objective;Adoptive transfer of autologous T-cells that are gene-engineered to express antigen-specific receptors represents a promising experimental therapy to provide tumor-specific immunity to cancer patients. We studied safety and proof of concept of therapy with T-cells gene-engineered with a Chimeric Antigen Receptor (CAR) in patients with metastatic Renal Cell Carcinoma (RCC). Materials and methods: In 12 patients with metastatic renal cell carcinoma (RCC), we performed a study with autologous T-cells genetically retargeted with a chimeric antibody receptor (CAR) directed towards carbonic anhydrase IX (CAIX), an antigen highly expressed in RCC. As we observed a limited (3 months in transfused patients. In contrast, Treg levels remained the same between BS and AS


Abstracts 693 in non-transfused patients. Functional assays showed that Tregs were functional post-ABT and could suppress Teff proliferation efficiently. In culture, isolated Tregs secreted TGF-b1. All cytokines and TNFRI and II plasma levels increased on d0 AS (IL-6, TNF-RI and II significantly), in transfused patients immediately AS, and decreased by d7 AS. In contrast, TGF-b1 levels decreased on d0 AS and increased by d7. No differences in cytokine or receptor levels were observed in nontransfused patients AS.

Conclusions: Experiments indicate the influence of TsF exosomes on MF dependent on the dose and mechanisms of exosomal stimulation. Low dose of TsF in vivo stimulation seems to activate natural cytotoxicity of MF which in turn may lead to elimination of CS T effector lymphocytes. The effect of in vitro higher dose stimulation with TsF suppresses MF activity. Present study was the first step to define the exact regulatory mechanisms induced by the interaction between TsFs and macrophages.

P1662 Generation of human antigen-specific regulatory T cells by MHCclass I restricted T cell receptor gene transfer L. E. Nickolay,* A. Skowera,* J. S. Bridgeman, A. K. Sewell, M. Peakman* & T. I. M. Tree* *Department of Immunology Infection and Inflammatory Disease, Kings College London, London, UK, Institute of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK

Conclusions: ABT induces the immediate production of plasma cytokines, especially pro-inflammatory IL-6 and TNF-RI and II that decreases by d7. In parallel, ABT induces the proliferation of Tregs. These Tregs are functional, and secrete TGF-b1 that reaches BS plasma levels by d7. The Tregs seem to be Th3 inducible Tregs. Homeostasis is reached by 3 months post-ABT.

P1661 Exosomal T cell suppressor factor inhibits the generation of reactive oxygen intermediates in murine peritoneal macrophages K. Nazimek, B. Nowak, W. Ptak & K. Bryniarski Immunology, Jagiellonian University College of Medicine, Krako´w, Poland Purpose/Objective: Contact sensitivity (CS) reaction in mice is regulated by antigen specific T CD8+ suppressor lymphocytes that produce biologically active exosomes carrying T cell suppressor factor (TsF). Macrophages (MF) as effector cells of classical CS reaction mediated by Th1 lymphocytes and as antigen presenting cells may be the target cells for TsF. The aim of our experiments was to define if exosomal TsF with different hapten specificity affect macrophages in the production of reactive oxygen intermediates (ROIs). Materials and methods: Production of TsF (tolerogenesis) is induced by intravenous application of a high dose of hapten-labelled syngeneic erythrocytes 7 days before epicutaneous immunization with the same hapten (trinitrophenol or oxazolone). Peritoneal MF are induced by mineral oil i.p. injection in naı¨ve or tolerized donors. The ability of MF to generate ROIs was measured as luminol-dependent chemiluminescence after in vitro activation of macrophages by zymosan (in case of naı¨ve MF in the presence of hapten-specific T cell suppressor factors). Results: MF from in vivo tolerized donors expressed the increased ROIs production in comparison to appropriate control. On the other hand the significant inhibition of generation of ROIs by MF was observed in the in vitro presence of either trinitrophenol- or oxazolone- specific TsF in comparison to untreated MF.

Purpose/Objective: The adoptive transfer of regulatory T cells (Tregs) as a therapy in autoimmune diseases has been well demonstrated in mouse models of disease. In the case of Type 1 diabetes, this therapy could potentially prevent or dampen down the auto-reactive immune response to the islets of the pancreas, a process in which CD8+ T cells play a pivotal role. However, this therapy is limited by the number of antigen-specific Tregs that can be isolated and expanded from patients. To overcome this hurdle, lentiviral gene transfer technology can be utilised to engineer populations of Tregs to express any T cell receptor (TCR) of choice, re-directing their antigen specificity. Furthermore, by generating Tregs whose TCR is MHC class I restricted and islet antigen specific, these cells can be targeted directly to the site of the inflammation and similarly against the CD8+ T cells that elicit a large proportion of islet cell damage. Materials and methods: An HLA-A2 restricted CD8+ T cell clone specific for a peptide in the preproinsulin signal peptide region has previously been isolated from a type 1 diabetic patient and designated the 1E6 clone. In order to produce lentivirus, the packaging cell line HEK 293Ts are transfected with plasmids containing the 1E6 TCR, lentiviral packaging and integrating genes. Supernatant from these cells containing lentivirus is then used to transduce the TCRb deficient Jurkat cell line JRT3 T-3.5. Results: We have created a stably transduced Jurkat cell line expressing the 1E6 TCR, which led to the reassembly of the full CD3 complex on the cell surface. However MHC tetramers+ the 1E6 cognate peptide fail to stain these cells, suggesting that in the absence of the CD8 coreceptor, MHC and peptide cannot bind to cell surface of the 1E6+ cells. Furthermore, peptide specific activation of these cells shows that they cannot be activated by APCs expressing cognate peptide by both CD69 up-regulation and IL-2 secretion. Conclusions: In conclusion we have demonstrated that we can generate stably transduced cells expressing a HLA-A2 restricted preproinsulin-specific TCR. However, our data has revealed a dependency on the presence of CD8 for this TCR to function. Thus, in order to transduce Tregs with an MHC class I restricted islet antigen specific TCR, a CD8 independent TCR will need to be identified to be used in this system.

P1664 Human dendritic cell based in vitro screening models mimicking Th1, Th2, Th17 and Treg responses J. E. Wern, M. Gad & S. S. Jensen Immune Targeting, Bioneer A/S, Hoersholm, Denmark Purpose/Objective: Dendritic cells (DCs) play a central role in regulating the immune system both under normal conditions, but also


694 Poster Session: Myeloid Cell Development under pathogenic conditions like autoimmunity, allergy and infection. This function of DCs makes them attractive target cells for therapeutic intervention in inflammatory diseases. We have designed human monocyte derived DC-based in vitro screening models for identification of anti-inflammatory compounds for use in Th1 or Th17 mediated autoimmunity or Th2 mediated allergic reactions. Further, we have setup a Treg platform, with tolerogenic DCs differentiating CD4+ T cells into IL-10 secreting Tr1 cells, in order to evaluate Treg promoting compounds. Materials and methods: Monocyte derived DCs were incubated with pro- or anti-inflammatory DC cocktails with or without prior addition of immune suppressive reagents. The DC cocktails were evaluated by FACS phenotypic analysis, ELISA cytokine measurement and T cell proliferation [mixed lymphocyte reaction (MLR)]. Generation of Treg cells was further evaluated by classical T cell suppression assay (PKH26 proliferation). Results: Nine different Th1 cocktails consisting of TLR agonists, cytokines and lipids were designed based on their ability to induce secretion of IL12p70 and TNFa from DCs. All cocktails induced DC expression of HLA-DR, CD40, 80, 83 and 86, indicating development of the immature DCs into mature DCs. Furthermore, DCs were able to induce a Th1 response defined by high IFN-g secretion in a MLR. The model was validated with anti-inflammatory drugs at both DC and T cell level using dexamethasone (Dex) and rapamycin. Two different Th17 cocktails were used to induce an inflammatory Th17 response. DCs stimulated with the Th17 cocktails secreted IL23 and differentiate CD4+ T cells towards an IL17A secreting Th17 phenotype in a MLR. Dex or the small molecule inhibitor STA5326 was used to validate the model. A single Th2 cocktail was developed modulating DCs to promote a IL5 and IL13 secreting Th2 phenotype. The model was validated with Dex which was able to suppress the Th2 response. Finally, a Treg platform was setup with tolerogenic DCs differentiating CD4+ T cells into IL-10 secreting Tr1 cells. Conclusions: We have designed human in vitro DC-based screening models for identification of immune-modulatory drugs for treatment of inflammatory or allergic diseases.

P1666 IL-2 and rapamycin alone or combined, friend or foe for type 1 diabetes? A. Baeyens, L. Perol, H. Sadeck, G. Foucarde & E. Piaggio Therapy and Autoimmunity, UMR7211, Paris, France Purpose/Objective: Regulatory T cells (Treg) play a major role in controlling the pathogenic autoimmune process in type 1 diabetes (T1D). We have recently shown that a short course of low-dose IL-2 administration cures new onset T1D in non obese diabetic (NOD) mice, by specifically activating pancreas infiltrating Treg and reducing interferon-gammaproduction by pancreas infiltrating T cells. However, only 60% of mice afford remission and only half of them remain normoglycemic for lifelong. Here we attempted to improve the rate of T1D remission in NOD mice. Materials and methods: For that we tested different high doses of IL-2 and the combination of low dose IL-2 and rapamycin (RAPA) in T1D. Results: First, we tested the capacity of different IL-2 doses to prevent T1D. Surprisingly, only with a ten times increasing ofthe curative IL-2 dose, T1D development was accelerated and higher IL-2 dosis were toxic to the mice. Of note, NOD mice of different sex and ages showed a significantly different susceptibility to high-dose IL-2 diabetes acceleration or toxicity. Second, we tested the combination of IL-2 with (RAPA), which is an immunossupressor used in human transplantation and which can selectively eliminate effector T cells (Teff) while sustaining an increase of Treg in vitro. It has already been described that long-term RAPA treatment can prevent diabetes, so we

directly tested the RAPA+ IL-2 combination in a curative schedulle in new onset diabetic NOD mice. Of note, RAPA+ IL-2 could not induce T1D remission, associated with an absence of control of the production of IFN-gamma by pancreas infiltrating T cells. Moreover, in IL-2- cured mice, RAPA annulated the T1D remission in a reversible way. We are now studying the underlying mechanism. Conclusions: Our results have an impact on IL-2 and RAPA treatment on human T1D, as new clinical trials are ongoing or are been implemented using these drugs in patients.

P1667 Immunomodulatory effects of different hASC clonogenic populations on human PBMNc P. Martıńez-Peinado,* M. I. A.-G. de Leoń, E. Roche-Collado, M. Garcıá-Irles,* F. M. M. De la Calle* & J. M. Sempere-Ortells* *Biotechnology, University of Alicante, San Vicente del Raspeig, Spain, Bioengineering, University Miguel Hernańdez, Elche, Spain Purpose/Objective: Previous studies have shown that mesenchymal stem cells (MSC) are able to modulate the immune response of different leukocyte populations, i.e. Tregs, through mechanisms yet to be elucidated. Most of these studies have used heterogenic cell populations. However in the present study we analyze the immunomodulatory effect of five different MSC clones obtained from human lipoaspirates (hASCs). Objectives: (1) To identify the membrane phenotype and Th1/Th2 cytokine production profile of each clone. (2) To assess the modulating effect of clones on the proliferation of PBMNc in cocultures at different ratios (MSC:PBMC; 1:10 and 1:5) (3) To establish differences between the behavior of the different clones, especially according to their specific immunomodulatory ability. Materials and methods: Flow cytometry (FC: EPICS XL, Coulter, FACSCalibur, BD) anddirect immunofluorescence for membrane phenotype. Cytokines by FC, CBA techniques (BD) and Flow-cytomix (eBioscience). Proliferation through FC and CFSE technics (Sigma). Results: All of the clones showed different intensities of autofluorescence (FITC emission spectrum) and different expression of CD90, CD105, CD73 and CD44 being negative for CD34 and CD45. All the clones secreted high to very high levels of IL-6 and IL-8 and mild to moderate levels of IL-1§ and TNF-a, both spontaneously and after LPS-stimulation. Two out of them also showed a slight secretion of IL12 and INF-c, and two more clones a slight secretion of IL-10 and/or IL-4. Cocultures carried out under different stimuli (LPS, PHA and anti-CD3/anti-CD28) showed different degrees of decrease in lymphocyte proliferation for all of the clones, reaching this immunosuppressant effect maximum values for one of these clones (two times more). Conclusions: The Th1/Th2 profile as well as the membrane phenotype of each clone can influence in their ability to modulate various leukocyte populations. This different immunomodulating effects obtained by using clonogenic ASCs populations instead of heterogenic whole populations could be of interest for being applied with clinical purposes in the field of cell therapy.

P1668 Immunosuppression of ICR mouse strain with 5-FU administration T. Lutsenko Department of Cell Regulatory Mechanisms, Institute of Molecular Biology and Genetics National Academy of Sciences of Ukra, Kiev, Ukraine Purpose/Objective: It has been already reported the experience with a variety of dosage schedules therefore an intravenous administration of


Abstracts 695 5-Fluorouracil (5-FU) is recommended, undiluted given by rapid injection of 15 mg/kg/day · 5 followed by 7.5 mg/kg on alternate days to the point of slight toxicity and so on. Our aim was to develop a halflethal dose of 5-FU for ICR mouse strain, to determine the effect on the myeloid and lymphoid cells, to detect white blood cell abnormality. Materials and methods: We’ve chosen virgin ICR female mouse strains, 2 2.5 months age. Each mouse had a 25 g weight. Intravenous administrations of 5-FU were in the following doses: 16, 32, 40, 48-mg/ kg/day during four times in volume 100 ll for each time. Intravenous administrations of saline drip were used as a control. 5-FU was administered daily for 4 days, and every second day for 8 days. Blood sampling was performed before the introduction of 5-FU, and after the first administration on 5th, 7th, 12th and 30th days. Gorjaev’s count chamber was used for measuring the number of white blood cells. For performing differential white blood cell counts May-Grunwald and Romanowsky stain and microscopy were applied. Results: ICR mouse strain contains 10 15 million leukocyte cells per ml in a blood. After course of administration of 5-FU 48 mg/kg/day for during four times * the lethal dose was established. With the introduction of 40 mg/kg/day during 4 days, on the 5th day after the first injection of 5-FU, the total leukocyte count was from 3 to 4 millions cells/ml, which is three times less than normal. Also neutropenia was detected. The total number of leukocytes was restored to half rate, increased number of neutrophils detected. Doses of 32 and 16 mg/kg/day during four times and during every second day administration for 8 days didn’t showed such significant results. Conclusions: To determine the half lethal dose of 5- FU we used the following concentrations: 16, 32, 40, 48-mg/kg/day during four times in volume of 100 ll for each time. 40-mg/kg/day during four times reduced the total number of leukocytes to the minimum 3 4 million/ ml. All animals were survived after this dose and restored their hematopoiesis in 30 days after first administration of 5- FU. Such kind of suppression may be used for further studies in the allogeneic transplantation and be helpful for engraftment of donor cells in the body of the recipient.

P1669 Investigating the role of splice variant CTLA-4 in cellular immunity A. Gazali,* F. Kaiser,* F. Ward & S. Thompson* *Academic Rheumatology, Kings College London, London, UK, School of Medicine, University of Aberdeen, London, UK Purpose/Objective: Cytotoxic T Lymphocyte Antigen 4 (CTLA-4, CD152) is a costimulatory molecule expressed on T helper cells which can bind to CD80 or CD86 molecules on antigen presenting cells and transmits an inhibitory signal to T cells. Various forms of CTLA-4 have been described in the literature. Our focus is mainly on a novel splice variant of CTLA-4 (svCTLA-4) produced by alternative mRNA splicing and importantly not by proteolytic digestion or shedding of membrane-bound CTLA-4. This transcript was first described by Magistrelli et al. in 1999 and it was demonstrated that unactivated human peripheral blood T lymphocytes could produce svCTLA-4. In contrast to membrane-bound CTLA-4, this protein lacks transmembrane and intracellular regions. Little is known about the immune regulatory functions of this protein. Materials and methods: Aliquots of 2.5 · 10(5) PBMC were diluted in 1000 ll RPMI1640 supplemented with 2 mM L-glutamine, sodium pyruvate, HEPES, Penicillin & Streptomycin and 10% FCS. PBMC were incubated in medium alone or with PMA & ionomycin at a concentration of 10 ng/ml and 1 lg/ml at 37C in a humidified atmosphere with 5% CO2. Cell supernatants were harvested every 24 h in vitro culture for further study of svCTLA-4 levels. Ninety-six wells plates (Nunc Maxisorp, Thermo Fisher Scientific, Roskilde, Denmark) were used for svCTLA-4 ELISA. The coating antibody (Anti-human pan CTLA-4 antibody, BNI3 clone, BD

Pharmingen) was added into each well & incubated at 37C for 90 min. Plates were blocked with 3% BSA in PBS for 1 h at 37C. Four replicates of samples were aliquoted into each well & incubated at 37C overnight. A pool of high titre plasma was used as the standard & was diluted in 0.2% BSA-PBS. After that, the detection antibody (Anti-soluble CTLA-4 biotinylated antibody, JMW-3B3 clone (specific for the splice variant form), obtained from Dr Frank Ward from University of Aberdeen) was added & incubated at RT in the dark for 2 h. Next, enzyme [Extravidin-alkaline phosphatase (Sigma-Aldrich)] was added and incubated for 90 min at RT in dark. Then, phosphatase substrate (Sigma Aldrich) was added into each well. Plates were read at 410 nm absorbance after 1 h of development. Results: Our data demonstrates that this protein can be secreted by PBMC in in vitro culture. Upon stimulation of PBMC by PMA & ionomycin initially levels of svCTLA-4 were downregulated (until 48 h) in culture. SvCTLA-4 secretion was increased from 72 h in culture suggesting a possible role in controlling immune activation. Conclusions: CTLA-4 is known to play an important role in mediating the function of regulatory T cells. Therefore, we would like to further investigate the effect of svCTLA-4 on T regulatory cell function.

P1670 Investigating the role of the aryl hydrocarbon receptor (AHR) in thymic-derived regulatory T cells (Tregs) in vivo D. M. Richards,* F. Cvetkovski,* M. Platten & M. Feuerer* *Immune Tolerance, German Cancer Research Center, Heidelberg, Germany, Department of Neurooncology, University Hospital of Heidelberg, Heidelberg, Germany Purpose/Objective: Recently, CD4+ Foxp3+ Tregs have emerged as key elements within the immune system, controlling the establishment and maintenance of immunologic tolerance and immune homeostasis. Many cells in the immune system express AHR and its activation has been shown to suppress a number of T cell responses in vivo. Interestingly, evidence suggests that activation of AHR induces Tregs that are suppressive in multiple in vivo models of autoimmune disease. Kynurenine (Kyn), a tryptophan catabolite constitutively produced by human tumor cells, was recently identified as an endogenous ligand of AHR. In this context, Kyn was shown to suppress the anti-tumor immune response and promote tumor survival. It was demonstrated that addition of exogenous Kyn to in vitro cultures led to an AHRdependent increase in Treg induction. The purpose of this study was to determine if expression of AHR and interactions with endogenous ligands is important for generation, maintenance and function of thymic-derived Tregs in vivo. Materials and methods: To test the importance of AHR expression on Tregs in vivo, bone marrow chimeras were produced using an equal mixture of AHR-deficient and Foxp3-DTR bone marrow to reconstitute lethally irradiated Foxp3-DTR recipients. Foxp3-DTR mice express the human diphtheria toxin receptor (DTR) under the control of the Foxp3 promoter. Injection of DT leads to 98% depletion of Foxp3-expressing cells. Upon depletion of AHR-sufficient Treg cells, all remaining Tregs would lack AHR while all other hematopoietic cell populations would include at least 50% wildtype cells. Results: Congenic markers (CD90 and CD45) were used to identify the origin of each cell population and we found that both donors were represented in equal proportions in the CD4+ and CD8+ T cell populations. Both donors were equally present in the Foxp3+ CD4+ population of PBS treated mice suggesting that AHR-deficient Tregs had no competitive disadvantage in the steady state compared to wildtype Tregs. In addition, within 2 weeks following Treg depletion, the Foxp3+ CD4+ population returned to control levels and was nearly entirely comprised of cells originating from AHR-deficient donor. This


696 Poster Session: Myeloid Cell Development suggests that AHR-deficient Tregs were able to successfully respond to homeostatic pressure and fill the space left following Treg depletion. Conclusions: Our results suggest that expression of AHR on Tregs is not required for the development and expansion of the Treg compartment in vivo. However, we have some evidence suggesting that expression of AHR might play a role in the function of these Tregs.

P1672 L-plastin phosphorylation: a novel target for the immunosuppressive drug dexamethasone in primary human T cells B. Schulte, G. H. Wabnitz, H. Kirchgessner, B. Jahraus, C. Stober, D. J. H. van den Boomen, F. Michalke & Y. Samstag Immunology, Universita¨tsklinikum Heidelberg, Heidelberg, Germany Purpose/Objective: Activation of naı¨ve T cells requires costimulation via TCR/CD3 plus accessory receptors, which enables the dynamic rearrangement of the actin cytoskeleton and immune synapse maturation. Signaling events induced following costimulation may thus be valuable targets for therapeutic immunosuppression. Phosphorylation of the actin-bundling protein L-plastin represents such a costimulatory signal in primary human T cells. Phosphorylated L-plastin has a higher affinity toward F-actin. However, the importance of the L-plastin phosphorylation for actin cytoskeleton regulation upon antigen recognition remained unclear. Here, we aim to analyze whether phosphorylation of L-plastin is important for immune synapse formation and/or maturation and whether the immunosuppressive glucocorticoid dexamethasone plays a role in L-plastin phosphorylation and synapse formation. Materials and methods: Human peripheral T cell isolation. Primary T cell transfection. Immune synapse via multispectral imaging flow cytometry. Gel electrophoresis and Western blotting. Cell cycle analysis and flow cytometry. Cell viability assay. Results: We demonstrate that phosphorylation of L-plastin is important for immune synapse maturation. Thus, expression of nonphosphorylatable L-plastin in untransformed human peripheral blood T cells leads to reduced accumulation of LFA-1 in the immune synapse and to a diminished F-actin increase upon T-cell activation. Interestingly, Lplastin phosphorylation is inhibited by the glucocorticoid dexamethasone. In line with this finding, dexamethasone treatment leads to a reduced F-actin content in stimulated T cells and prevents maturation of the immune synapse. This inhibitory effect of dexamethasone could be reverted by expression of a phospho-mimicking L-plastin mutant. Conclusions: Our data introduce costimulation-induced L-plastin phosphorylation as an important event for immune synapse formation and its inhibition by dexamethasone as a novel mode of function of this immunosuppressive glucocorticoid.

P1673 Lipopolysaccharide-induced M2 to M1 macrophage transformation for IL-12p70 production is blocked by Candida albicans mediated up-regulation of EBI3 expression X. Q. Wei, X. F. Zheng, Y. X. Hong, H. Rogers, B. Song, M. A. O. Lewis & D. Willliams Tissue Engineering and Reparative Dentistry, Dental School, Cardiff, UK Purpose/Objective: The response of M1 and M2 macrophages to C. albicans in the presence of LPS stimulation has not previously been reported. In this study, we examined plasticity of macrophages transforming from M2 to M1 in the presence of LPS and the impact C. albicans has on this transformation. Materials and methods: Bone marrow cells extracted from C57/black mice femur bones were cultured with either 10 ng/ml rmGM-CSF or

10 ng/ml of mlM-CSF/IL-4 for 7 days before seeding the cells into 24 well plates for LPS stimulation, with or without Heat-Kill Candida (HKC). Released cytokines were subsequently measured by ELISA and gene expression determined by real-time RT-PCR. In some experiments, cytokine proteins were examined by Western blot. To confirm intracellular EBi3/p40/35 protein interaction, IL-12p70 biscistronic expression vector and EBI3 expression vector were constructed in pcDNA3.1A. CHO cells were transfected with expression vectors before detection of the protein by ELISA, Western blot (WB) and/or Immuno-precipitation WB. Results: M1 cells stimulated by Candida albicans produced much higher TNFa compared with M2 cells. LPS converted M2 macrophages to the M1 phenotype with higher IL-12p70 production. Candida albicans stimulated a higher Epstein-Barr Virus induced protein 3 (EBI3) which can couple with IL-12p35 to form IL-35, a heterodimer soluble protein (EBI3/p35) with an immune suppressing activity. M2 cells stimulated with C. albicans suppressed IL-12p70 production (induced by LPS) in a dose dependent manner. This suppression resulted from competition between EBI3 and IL-12p40 for IL-12p35 binding. EBI3 expression was higher in M2 compared with M1 cells. Candida albicans also induced higher levels of EBI3 expression than LPS in M2. However, LPS and C. albicans stimulated similar levels of IL-12p35 in M2 cells. This suggests EBI3 induced by C. albicans competes with IL-12p40 for p35, leading to suppression of LPS induced IL-12p70. To confirm this hypothesis, an IL-12p70 expression CHO cell line was cloned by transfection with a biscistronic IL-12p40 and p35. The over expressed EBI3 largely suppressed IL-12p70 secretion in all cell clones. This reduction of IL-12p70 production was associated with EBI3/p35 heterodimer detection in the cell culture medium. Conclusions: This result demonstrated that Candida de-sensitises tissue M2 macrophages to transform to M1 phenotype in the presence of LPS, by suppressing IL-12p70 production. This may lead to the avoidance of an unnecessary Th1 response during the resolving phase of infection.

P1674 Low dose of cyclophosphamide alleviates the suppression of contact sensitivity response mediated by macrophages K. Nazimek, E. Sikora, B. Nowak, M. Ptak, W. Ptak & K. Bryniarski Immunology, Jagiellonian University Medical College, Krakow, Poland Purpose/Objective: Intravenous transfer of hapten-conjugated macrophages (Mf) to naı¨ve recipients results in the suppression of contact sensitivity reaction (CS) as an effect of IL-10 secretion by donors’ Mf and recipients regulatory T cells. A single administration of low dose of cyclophosphamide (CY) inhibits the suppression of CS response. The aim of our study was to test in the in vivo experiment how CY treatment influences both populations * donors macrophages and regulatory T cells in recipients and leads to the alleviation of suppression of CS reaction. Materials and methods: To estimate CS oil-induced peritoneal Mf harvested from naı¨ve or CY low dose (50 mg/kg) treated donors were labeled with TNP and transferred i.v. to naı¨ve or CY treated recipients or recipients injected i.v. with anti-IL-10 and/or antiTGF-b mAbs. Some grop7 days later ear thickness was measured just before the PCL challenge and 24 h later ear swelling responses were measured. Results: Treatment with CY of Mf donors and/or recipients as well as recipients with anti-IL-10 mAb significantly inhibits the suppression while anti-TGF-b mAb therapy is inactive. Conclusions: A single administration of low dose of CY into either donors or recipients restores the ability of Mf to induce significant CS reaction as a result of elimination of suppressive properties of Mf and/ or depletion of population of regulatory T cells in recipients or


Abstracts 697 elimination of their suppressive activities. Observed inhibition was probably a consequence of decreased secretion of IL-10 by both populations of cells as a result of CY low dose treatment.

P1675 Macrophages present a regulatory phenotype after chronic cold stress in mice: correlation with increased 11ß-hydroxysteroid dehydrogenase expression R. Sesti-Costa, M. D. C. Ignacchiti, S. Chedraoui-Silva, L. F. Marchi & B. Mantovani Biochemistry and Immunology, Faculdade de Medicina de Ribeiraõ Preto, University of Saõ Paulo, Ribeiraõ Preto, Brazil Purpose/Objective: Susceptibility to infections, autoimmune disorders and tumor progression are strongly influenced by activity of endocrine and nervous system in response to a stressful stimulus. When this adaptive system is switched on and off efficiently, the body is able to recover from the stress imposed. However, when this system is activated repeatedly or sustained, as during chronic or excessive stress, an allostatic load is generated, which can lead to disease over long periods of time. Chronic stress has immunosuppressive effects as illustrated by increase in severity and duration of infectious diseases and by the impairment of immune response to vaccines. However, the cells and the mechanisms involved in this immunossupression are not completely known. We investigated the effects of chronic stress on main functions of macrophages. Materials and methods: Balb/c mice were chronically stressed by 4C/ 4 h during 7 days and macrophages were evaluated for their ability to phagocytosis, superoxide and cytokines production and stimulate T cells. Results: We found that chronic cold stress in mice regulates macrophages functions to a regulatory pattern. This regulation is shown by diminished phagocytosis, lower TNF-a and IL-6 and higher IL-10 production. Besides, resting macrophages are able to stimulate T cells with a regulatory cytokines profile after chronic stress. Furthermore, macrophages of stressed mice have increased expression of 11bhydroxysteroid dehydrogenase, an enzyme that converts inactive glucocorticoid into its active form, and have lower capacity to control Trypanosoma cruzi infection in vivo. Conclusions: As stress is a common aspect of modern life and takes part in the etiology of many diseases, the results of this study are important to improve the knowledge about the neuro-immuneendocrine interactions occurring during stress and show the role of macrophages in immunosuppression induced by chronic stress. Support: CNPq, CAPES, FAPESP and FAEPA.

P1676 Mast cells are a target of tick saliva H. Langhansova,* M. Klein, E. Schmitt & J. Kopeckyà *Laboratory of Parasite Immunology, Institute of Parasitology Biology centre of the AS CR, Ceske Budejovice, Czech Republic, Institute for Immunology, University Medical Centre of the Johannes GutenbergUniversity Mainz, Mainz, Germany, àDepartment of Medical Biology, Faculty of Science, University of South Bohemia, Ceske Budejovice, Czech Republic Purpose/Objective: Mast cells concentrated beneath the epithelial surface of the skin play an important role during feeding of bloodsucking arthropods including ticks. Since tick feeding period can exceed 10 days, active modulation of the host immune response by tick saliva is required for the tick to complete its blood meal. However, whether the tick saliva affects also mast cell physiology and functions was not known.

Materials and methods: We tested the effect of Ixodes ricinus tick saliva on the degranulation and cytokine production by ionomycinand/or IgE receptor-activated bone marrow-derived murine mast cells. Results: The most affected mast cell cytokine was interleukin 9; its production after 48 h upon saliva treatment was decreased by approx. 80% compared with the IL-9 production in control untreated cells. Interleukin-4 and IL-13 were also significantly inhibited by the saliva, whereas IL-6 levels were only moderately reduced. In contrast, production of TNF-a after 4-h co-culture with tick saliva was not affected. Saliva had also no direct impact on the level of mast cell degranulation. Conclusions: We demonstrated the direct effect of tick saliva on the production of cytokines in mast cells; these effects can change the cytokine balance in the tick feeding site and contribute to the modulation of host immune responses. Grant support: GACR P302/11/J029

P1677 Notch family signalling between MSC and immune cells E. Cahill & L. M. Tobin Biology, National University of Ireland Maynooth, Kildare, Ireland Purpose/Objective: Mesenchymal Stem or Stromal Cells (MSC) are immunomodulatory cells derived from bone marrow. MSC mediated immune modulation involves the induction of regulatory T cell populations and inhibition of dendritic cell maturation. This study sought to (1) determine the contact dependent signalling pathways between MSC and dendritic cells that block DC maturation; (2) identify the parallel signal between MSC and naı¨ve CD4+ T cells leading to Treg induction; (3) determine the induction of tolerogenic DC by MSC. Materials and methods: MSC and either DC or CD4+ T cells were used. An eGFP*FOXP3 reporter system was used to measure Treg induction. Notch signalling was blocked using Gamma Secretase Inhibitor (GSI) on either T cells or dendritic cells and expression of GFP-FoxP3 and maturation markers were examined by FACS. Immature DC co-cultured with MSC were pulled back after 48 h and co-cultured with CD4+ T cells from DO11.10 mice in the presence or absence of cognate antigen. After 72 h, proliferation was examined and Treg determined. Results: Use of GSI prevented the MSC mediatedinduction/expansion of CD4+ FoxP3+ T cellsto controllevels. When GSI was used with DC and MSC, the immature DC characteristics sustained by MSC were lost. These DC induced regulatory T cells and inhibited antigen driven T cell proliferation in a Notch dependent manner. Conclusions: These data demonstrate that the Notch-Notch ligand pathway is involved in MSC mediated induction of Treg. Inhibition of the Notch pathway abrogates MSC modulation of DC function but the exact receptors/ligand interaction here have yet to be determined. Using lentiviral transduction, a stable lines of silenced MSC were created. Using these cells the specific ligand involved in MSC immunomodulation can be determined.

P1678 Rapamycin inhibits innate and adaptive immune functions of human plasmacytoid dendritic cells P. Boor,* S. Mancham,* L. J. W. van der Laan, H. J. Metselaar* & J. Kwekkeboom* *Gastroenterology and Hepatology, Erasmus MC University Medical Centre, Rotterdam, Netherlands, Surgery, Erasmus MC University Medical Centre, Rotterdam, Netherlands Purpose/Objective: Rapamycin is an immunosuppressive drug used to prevent organ transplant rejection and cancer. Plasmacytoid


698 Poster Session: Myeloid Cell Development dendritric cells (PDC) are important in innate immunity as they are the principal producers of IFN-a, and in adaptive immunity by presentation of antigens to T cells. PDC are critically involved in immunity to viral infections and in the pathogenesis of auto-immune disorders like Systemic Lupus Erythematosus (SLE). Here we report that innate and adaptive immune functions of human PDC are differentially regulated by Toll-Like Receptors (TLR) and CD40, and that rapamycin inhibits both innate and adaptive immune functions of PDC. Materials and methods: Human PDC were isolated from blood of healthy volunteers and stimulated with loxoribin, CpG or CD40L in the presence or absence of rapamycin and tested for their cytokine production and T-cell stimulatory capacity. Results: Human PDC activated by TLR-7 or TLR-9 ligands produced high amounts of IFN-a, but exhibited a weak T cell stimulatory capacity. Conversely, PDC activated by CD40-ligation failed to produce IFN-a, but induced robust allogeneic T cell proliferation and effector functions, among which production of pro-inflammatory cytokines IFN-gamma, TNF-alpha and IL-17. A clinically relevant concentration of rapamycin (20 ng/ml) inhibited the phosphorylation of S6 and the production of IFN-a by PDC, and suppressed the capacity of PDC to stimulate allogeneic T cell effector functions. Reduction of T-cell stimulatory capacity was most pronounced when rapamycin was added during activation of PDC via CD40, and was associated with inhibition of CD40 expression on PDC. Conclusions: Activation of human PDC via TLR stimulates their innate immune functions, while activation via CD40 stimulates their adaptive immune functions. Rapamycin inhibits both innate and adaptive immune functions of human PDC, and may therefore constitute a novel treatment option for SLE, but increase susceptibility to viral infections.

P1680 Soluble MHC class II antigens as potent regulators of the immune response K. Bakela & I. Athanassakis Biology, University of Crete, Heraklion, Greece Purpose/Objective: Soluble MHC-II (sMHCII) molecules are present in body fluids of healthy individuals and are considered to be involved in the maintenance of self tolerance, while they have also been related to various diseases. Their concentration has been shown to increase during in vivo antigen-specific tolerogenic stimulation. At the cellular level, sMHCII proteins compete with membrane MHCII for TCR binding and induce Src kinases activation. The aim of the present study was to define the physical and chemical properties of sMHCII and examine their ability to affect responsiveness and cytokine secretion profile of total spleen cells, total T-cells or CD4+ cells. Materials and methods: Soluble MHC-II proteins were isolated from serum of mice tolerized with human serum albumin (HSA). Purity was verified through ELISA, SDS-PAGE, Western blot analysis and specific biosensor assays. Protein concentration was calculated using the Lowry method. Total spleen cells or T-cells were isolated from control or immunized BALB/c male mice, while CD4+ T-cells were purified using Flow Cytometry Sorter. The effect of sMHCII on the different cell populations tested as to cell proliferation and marker expression was evaluated using incorporation assays and Flow Cytometry analysis respectively. Cytokine production was tested by ELISA. Results: The obtained results showed that the sMHCII proteins were capable to suppress an antigen-specific immune response by reducing the number of active cells. Thus, sMHCII induced control CD25+ and CTLA+ T-cell populations, while reducing CD28+ T-cells. Furthermore, it was shown that sMHCII induced secretion of the cytokine IL10, while stimulating the proliferation of suppressive CTLA-4+ cells, through the negative regulation of CD4+ and CD28+ cells.

Conclusions: The results presented here showed that sMHCII can negatively regulate an antigen-specific immune response in vivo as well as in vitro by inducing immunosuppressive cell populations and mediators. It is proposed that sMHCII molecules could play a very important role in immune homeostasis, as they could mediate antigenspecific tolerance.

P1681 Structure and function of oxazolone specific T suppressor factor in contact sensitivity response E. Sikora,* B. Nowak,* K. Nazimek,* M. Ptak,* P. W. Askenase, W. Ptakà & K. Bryniarskià *Immunology, Jagiellonian University Medical College, Krakow, Poland, Internal Medicine, Yale University School of Medicine, New Haven, CT, USA, àImmunology, Jagiellonian University College of Medicine, Krako´w, Poland Purpose/Objective: Contact hypersensitivity (CHS) in mice is mediated and regulated by different subpopulations of T-cells. High antigen dose induced-suppressor T (Ts) lymphocytes inhibit CHS reaction through release of soluble suppressor factor (TsF). The aim of our study was to establish the nature of OX-TsF, factor specific for CHS elicited by oxazolone (OX). Materials and methods: OX-TsF production is induced in mice by i.v. immunization with OX, followed by skin sensitization with the same hapten. OX-TsF is obtained as a culture supernatant of Ts and may be purified by phenol-chloroform extraction into a mixture of nucleic acids (PCE) and then DNA (QDNA) and RNA (QRNA) fractions may be separated on chromatography columns. Alternatively, suppressive exosomes may be isolated from culture supernatant of Ts by ultracentrifugation. Biological activity of OX-TsF and its preparations was determined in vivo in CHS model. Results: OX-TsF displays the ability to inhibit both the elicitation of CHS in naı¨ve recipients of T effector cells (Tef) and ongoing allergy in actively sensitized mice. The function of OX-TsF was observed in both PCE and QRNA, but not QDNA, fractions of the tested factor and was characterized by a dose-response effect. RNA nature of OX-TsF was confirmed by its sensitivity to RNase A treatment. Additionally, electrophoresis on agarose gel proved that the RNA fraction of TsF comprises particles of the size of 10 90 nucleotides. TsF performs its function in antigen-specific manner, which is observed in both supernatants and exosomes obtained from cultures of Ts induced by different haptens. The mechanism of action of OX-TsF may tentatively involve apoptosis of the Tef. Conclusions: It seems likely that the observed suppressive effect, mediated in vivo by short RNAs transported antigen-specifically in exosomes in the future may be used as a therapeutic approach to the diseases which result from the impaired function of effector T cells.

P1682 Study of the effect of CAMPATH on cord blood and peripheral blood cells F. Y. Lee, A. Madrigal & B. Shaw Cancer Research, Anthony Nolan Trust- UCL, London, UK Purpose/Objective: CAMPATH is an anti-CD52 monoclonal antibody used as an immunosuppressive drug that effectively depletes T cells in the pre-transplant conditioning regimen. Materials and methods: CD52 expression of the resting and activated immune cells were checked. Both resting and activated samples were treated with CAMPATH and compared the the non-treated samples. Results: CD52 expression on resting and activated peripheral blood (PB) and cord blood (CB) cells was studied. For resting CB cells, the


Abstracts 699 level of CD52 expression on B cells and T cells was very comparable (569.2 ± 44.19), natural killer (NK) cells expressed the lowest level of CD52 (278.4 ± 31.64), and CD52 was not expressed on resting CB stem cells. For resting PB, B cells expressed the highest CD52 expression (540.8 ± 68.5), followed by T cells (433.1 ± 67.15), NKT cells (349 ± 74.67), and NK cells (301.4 ± 59.5). CD52 density was shown to be significantly higher in CB T cell subsets than PB T cell subsets except for Treg cells where CD52 expression was comparable in both CB and PB (451.2 ± 53). In contrast, CD52 expression in activated cells was slightly higher in PB than CB, with T cell subsets expressing the highest level (657.3 ± 27.70 and 496.8 ± 4.872 respectively) followed by NK cells (415.0 ± 16.15). However, both PB and CB activated Treg expressed similar level of CD52 density (396.4 ± 9.948). A viability study was designed to study the potency of CAMPATH in inducing PB and CB immune cell death. Although different concentrations were used, CAMPATH induced apoptosis and necrosis in a comparable manner, having the same effects on both CB/PB T and B cells. No difference was shown for both CB/PB NK cells. A maximum response towards CAMPATH treatment was observed approximately 24-h post administration for all cell types. Conclusions: A better understanding of the impact of CAMPATH on immune cells would be useful to contribute to the future clinical application of the drug in order to achieve optimal efficacy.

P1683 T cell-selective mutated interleukin-2 AIC284 efficiently protects Lewis rats from experimental autoimmune encephalomyelitis N. Beyersdorf,* A. Weishaupt, S. Werner,* N. Wolf,* G. Ko¨llner, D. Paulsen,à T. Hu¨nig* & T. Kerkau* *Chair for Immunology, Institute for Virology and Immunobiology, Wu¨rzburg, Germany, Department for Neurology, University Hospital Wu¨rzburg, Wu¨rzburg, Germany, àAiCuris GmbH & Co. KG, Wuppertal, Germany Purpose/Objective: The cytokine interleukin-2 (IL-2) critically influences the survival and function of Foxp3+ regulatory T cells (Treg cells) in vivo. Therefore, targeting Treg cells via the high affinity IL-2 receptor, CD25, constitutes a novel therapeutic approach for autoimmunity. As anti-cancer therapy with IL-2 has revealed substantial toxicities due to activation of conventional memory T cells and NK cells expressing intermediate affinity IL-2 receptors, a mutated human IL-2 molecule, termed AIC284, has been developed to reduce these side effects. Materials and methods: AIC284 shows similar binding to the human high affinity IL-2 receptor as wild-type (wt) IL-2 but about 104-fold less binding capacity to the intermediate affinity IL-2 receptor. In search for an appropriate rodent animal model to analyze the therapeutic potential of AIC284 in autoimmunity we observed that AIC284 only bound less well (100-fold) than wt IL-2 to intermediate affinity IL-2 receptors of the rat but not to those of the mouse. Therefore, we treated rats immunized with guinea pig myelin basic protein (gpMBP) to develop experimental autoimmune encephalomyelitis (EAE) for the first 4 days after the immunization with daily doses of 100 mg AIC284 or wt human IL-2 or 250 ml phosphatebuffered saline s.c. Results: Treatment with AIC284 or wt IL-2 protected the rats similarly well from EAE, both, clinically and histologically. Further analyses revealed that application of AIC284 and wt IL-2 inhibited the generation of gpMBP-specific pathogenic, i.e. IFNg-producing, CD4+ T cells and strongly reduced the infiltration of leukocytes into the spinal cord (SC). As expected, protection from EAE was associated with increased frequencies and absolute numbers of Foxp3+ Treg cells among CD4+ T cells. Conclusions: As far as the precise therapeutic mechanism is concerned, wt IL-2 and AIC284 appear to differ as wt IL-2 strongly

expanded Treg cells expressing the activation marker CD8 within secondary lymphoid organs (SLO) while AIC284 induced only a slight increase in CD8+ Treg cell numbers in SLO but instead favored their accumulation in the SC. Experiments directly addressing the contribution of CD8-expressing Treg cells in AIC284-mediated protection from EAE are ongoing. This study was supported by a grant from AiCuris GmbH & Co. KG, Germany.

P1684 The compartment of human natural CD4+CD25++CD127- regulatory T cells is filled with cells reactive to various antigens N. Litjens, K. Boer, C. C. Baan & M. G. H. Betjes Internal Medicine Nephrology and Transplantation, Erasmus Medical Center, Rotterdam, Netherlands Purpose/Objective: The population of regulatory CD4+CD25++CD127)FOXP3+ T cells (Tregs) is composed of thymusderived (natural, nTregs) or Tregs induced in the periphery (iTregs). nTregs have a highly demethylated TSDR FOXP3 and are believed to be important for control of autoreactive T cells. Tregs are important to control alloreactive T cells and could be used for cell therapy to induce tolerance to an allograft. Recognition of natural alloantigen-specific Tregs could facilitate a targeted approach for such a therapy. Expression of CD40L (CD154) can be used to detect antigen-specific CD4+ T cells. In this study, we used specific expression of CD154 to study the presence of antigen-specific Tregs. Materials and methods: Highly enriched fractions of Tregs and CD4+ effector T cells (CD4+Teff) were sorted and stimulated with various antigens in the presence of aCD40 to maintain CD154 on the cell surface. Subsequently, these CD154-expressing Tregs were sorted and tested either immediately for their suppressive capacity or following polyclonal (using CD3/CD28 beads) or antigen-specific expansion, respectively. The demethylation of TSDR FOXP3 was determined for the different Treg-fractions. In addition, in depth phenotypic analyses were performed on the different Treg-fractions. Results: Using different viral, vaccination as well as alloantigens, we detected similar frequencies of antigen-specific CD154+T cells in both fractions with great specificity and sensitivity. Ag-specific CD154+Tregs had a memory phenotype, no cytokine production after polyclonal stimulation, stable FOXP3 expression and a highly demethylated TSDR FOXP3. Sorted CD154+ Tregs were superior in suppressing Ag-specific responses when compared to CD154)Tregs. CD154+ Tregs could be efficiently expanded in an Ag-specific manner, which enhanced their suppressor activity. After booster vaccination, the ratio of Ag-specific CD4+ Teff:Treg temporarily increased substantially due to an increase in CD154+CD4+ Teff but not Tregs. Conclusions: These data show for the first time that the compartment of circulating human nTregs is filled with Ag-specific T cells for a variety of antigens. Isolation and expansion of Ag-specific nTregs may be of potential benefit for Treg-therapy to induce tolerance in the setting of kidney transplantation.

P1685 The effect of all-trans retinoic acid on histocompatibility receptor distribution at the foeto-maternal interface P. Jain, A. Blanch, A. Jabeen, S. Hakam, P. Laissue & N. Fernandez Department of Biological Sciences, University of Essex, Colchester, UK Purpose/Objective: The field of reproductive immunology was created for understanding the tolerance strategies by which the semiallogenic foetus evades the maternal immune system. Multiple mechanisms underlie the tolerance shown by the maternal immune system. A chief role is played by the Human Leukocyte Antigen -G (HLA-G), a member of the non-classical Major Histocompatibility


700 Poster Session: Myeloid Cell Development Complex (MHC) class I molecule. HLA G has a unique pattern of expression; low polymorphism and tissue distribution that enable it to aid the process of fetal implantation by preventing it from being recognized as ‘foreign’. In early work1 it was shown that the MHC receptors (HLA-G and HLA-E) and that their distribution is linked to the cytoskeleton. Certain retro-retinoids are known to induce growth arrest and death by apoptosis in cell lines. It was demonstrated2 that Factin is a functional target for Retro-Retinoid Action. Hence, F- actin reorganization is an early event in RA induced apoptosis. Lack of MHC class I a and the expression of these non classical antigens has long been hypothesized to be a major factor in immune tolerance. This study is mainly focused on investigating the expression of HLA class I b, and assessing whether Retinoic Acid plays a role in the regulation of the functional expression of HLA-G. Materials and methods: The techniques used in order to establish this study are flow cytometry, protein analysis and microscopic analysis. Results: Our results revealed that Retinoic Acid up regulates surface HLA-G expression significantly in a dose and time dependant manner. Proliferation and Cytotoxicity assays further confirmed this effect and results were supported by the fact that the effects of ATRA could be inhibited by various concentrations of 9-cis RA, the isomer and antagonist of ATRA. Image analysis with Nikon Viewer software identified changes in morphology in HLA-G expression after ATRA induction, includingminor changes in the cytoskeleton. Conclusions: All trans Retinoic Acid acts as a stimulator at certain concentration for HLA-G, and can induce apoptosis beyond 50 lM. The regulation of the functional expression of HLA-G by Retinoic Acid could induce a physiological role such as apoptosis. This study is also, a novel approach for studying ATRA mediated (using cytoskeleton as a functional target) apoptosis in embryonic cell lines. As the results were consistent in both cell lines, we can also propose ACH-3P as a model cell line for trophoblast investigation. References 1. Fernàndez, E. M., O’Toole, P. J., Morrison, I. E., Cherry, R. J., Fernàndez, N. (2003) Interaction of HLA-DR with actin microfilaments. Human Immunology, 64(3), 327 * 37. 2. Korichneva, I. and H*mmerling, U. (1999) F-actin as a functional target for retro-retinoids: a potential role in anhydroretinol-triggered cell death. Journal of Cell Sciences, 112 (15):2521 8.

P1686 The influence of opioids treatment on the immune response in mice: the role of macrophages I. Filipczak-Bryniarska,* B. Nowak, E. Sikora, K. Nazimek, M. Ptak, J. Woron,* J. Wordliczek,* W. Ptak & K. Bryniarski *Pain Treatment and Paliative Care, Jagiellonian University Medical College, Krakow, Poland, Immunology, Jagiellonian University Medical College, Krakow, Poland Purpose/Objective: Immune responses mediated by lymphocytes and macrophages could be modulated by opioids (OPs) via their cell surface receptors. The aim of our study was to establish the role of opioid (morphine, fentanyl or methadone) treated murine macrophages (MF) in the regulation of humoral and cell-mediated responses in CBA/J mice. Materials and methods: For this purpose macrophages obtained from opioid treated mice were either fed with sheep erythrocytes (SRBC) or labeled with TNP hapten and transferred i.v. into naı¨ve recipients. Humoral response was estimated by plaque forming assay (PFA) and direct haemagglutination assay (HA) determining sera titres of antiSRBC IgM and IgG antibodies. Contact hypersensitivity reaction (CHS) was tested by ear swelling measurement after challenge withTNP/PCL hapten. Macrophage properties in vitro were assessed in FACS analysis of surface markers expression, ELISA for cytokine

secretion and chemiluminescence assay for oxygen intermediates (ROIs) production. Results: The induction of humoral response by opioid treated Mf was strongly inhibited. Numbers of PFC were lower by 30% (morphine or fentanyl treatment) to 57% (methadone) as compared with control animals. This same holds true for Ab titers. CHS reaction was differentially affected by OPs. While all tested OPs increased the early phase of reaction, the late phase is increased only in morphine therapy and affected by fentanyl and methadone. The impairment of MF antigen-presentation ability was also observed as decreased expression of CD80, CD86 and MHC class II in FACS analysis. The secretion of proinflammatory cytokines by MF obtained from donors treated with OPs was inhibited andmorphine or methadone but not fentanyl therapy caused an enhancement of IL-10 as well as ROIs production by MF. Conclusions: MF are the target cells for opioid activity in modulation of immunity. The inhibitory action of OPs on humoral and cellular responses seems to be an effect of impaired ability of MF to present antigen.

P1687 The role of Dickkopf-3 in immune responses J. Ludwig, M. Papatriantafyllou, M. Meister, G. Moldenhauer, H. J. Gro¨ne, C. Niehrs, G. Ha¨mmerling, T. Oelert & B. Arnold Molecular Immunology, German Cancer Research Center, Heidelberg, Germany Purpose/Objective: It is so far unknown to which extend tissue cells influence the type and strength of a local immune response. Recently, we demonstrated that Dickkopf-3 (Dkk3) is essential to maintain peripheral CD8 T cell tolerance in a TCR-transgenic mouse model (Papatriantafyllou, M; Proc Natl Acad Sci U S A, 2012, 109:1631 1636). Since Dkk3 is highly expressed by tissue cells of immune-privileged sites, as e.g. the brain and the eye, it is of interest whether the local production of Dkk3 contributes to immuno-suppression. Materials and methods: In vitro T and B cells were stimulated and proliferation and secreted cytokines were analyzed. For these experiments either Dkk3)/) versus wt cells or addition of exogenous Dkk3 versus control were compared. In vivo EAE was induced in Dkk3)/) and wt mice. T cells were purified from the brain at the peak of disease or during the recovery phase and analyzed. To investigate B cell responses Dkk3)/) and wt mice were immunized and antibody titers were determined at different time points. Results: In order to investigate whether Dkk3 has an impact on immune responses in the brain, experimental autoimmune encephalitis (EAE) was compared in Dkk3)/) and wt mice. Animals lacking Dkk3 exhibited a prolonged disease compared to Dkk3 sufficient animals. This was associated with changes in T cell subset composition and the local cytokine milieu. Comparing immune responses in organs lacking Dkk3 expression as the spleen by in vivo cytotoxicity assay no general state of hyperresponsiveness in Dkk3 deficient T cells was observed. In vitro we found a decrease in proliferation, expression of CD25 and secretion of IL-2 upon stimulation of T cells in the presence of exogenous Dkk3. B cell responses were also affected by Dkk3. Mice deficient for Dkk3 showed altered antibody responses after immunization with TNP-BSA or TNP-Ficoll in comparison to wt mice. Furthermore, B cells from Dkk3)/) mice showed changes in cytokine expression in vitro. Conclusions: Based on these data we conclude that Dkk3 is modulating T cell responses and antibody production.


Abstracts 701 P1688 Tolerization of diabetogenic CD4+ T cells by intranasal treatment with CTA1R7K-DD containing specific peptide through the induction of FoxP3+ Treg cells S. Cardell, L. Fahleń-Yrlid, L. Mark, L. Lo¨fbom & N. Lycke Department of Microbiology and Immunology, University of Gothenburg, Gothenburg, Sweden Purpose/Objective: Type I diabetes (T1D) results from immune destruction of insulin producing b-cells in the pancreas islets. Diabetogenic CD4+ T cells are key cells in the autoimmune process. To achieve tolerization of diabetogenic CD4+ T cells would therefore be an important advancement in the development of treatment of T1D. We previously described that a mutated (R7K), enzyme killed, form of the cholera toxin A1 subunit based adjuvant CTA1R7K-DD induces specific tolerance rather than enhancement of immunity. Intranasal (i n) treatment with CTA1R7K-DD containing a type II collagen peptide reduced in vitro recall responses to the peptide, and moreover, ameliorated collagen induced arthritis in mice. Here, we use CTA1R7K-DD to investigate tolerization of diabetogenic CD4+ T cells of the nonobese diabetic (NOD) mouse, exploring diabetogenic TCR transgenic BDC2.5 CD4+ T cells. Materials and methods: NOD or TCR transgenic BDC2.5 NOD mice were treated i n with CTA1R7K-peptide-DD fusion proteins, where the inserted peptide was Ins9-23 or PS3 (a strong agonist peptide for the BDC2.5 TCR), respectively. Results: I n treatment of BDC2.5 NOD mice with CTA1R7K-PS3-DD reduced proliferation and IFN-c production to in vitro PS3 peptide stimulation. Transfer of CD4+ BDC2.5 T cells to NOD.scid mice results in T1D development in 80 100% of recipient mice. In contrast, 95% of recipients of cells from BDC2.5 NOD mice treated with the CTA1R7K-DD peptide construct remained healthy. The i n treatment resulted in systemic increase in the frequency of CD4+ BDC2.5 transgenic T cells expressing FoxP3, most enhanced in draining mediastinal lymph nodes. Conclusions: We demonstrate that i n treatment with CTA1R7Kpept-DD induced tolerance in peptide specific diabetogenic CD4+ T cells, and resulted in an increased frequency of Treg cells. The data suggest that CTA1R7K-peptide-DD may prevent T1D development by the induction of peptide specific CD4+ Treg cells.

P1689 Treg differentiation induces by the immunosuppressive enzyme IL4I1 C. Cousin, F. Castellano & V. Frenkel INSERM U955, Cre´teil, France Purpose/Objective: IL4I1 is an L-phenylalanine oxidase which inhibits the TCRf chain expression and the proliferation of T lymphocytes in vitro [Boulland ML et al. (2007) Blood 110(1):220 227]. IL4I1 is highly expressed by macrophages infiltrating tumors and tumor cells of some cancers, particularly different B-cell lymphomas

[Carbonnelle-Puscian A et al. (2009) Leukemia 23(5):952 60]. We recently showed in a mouse model that IL4I1 inhibits the cytotoxic antitumor T-cell response [Lasoudris F et al. (2011) European Journal of Immunology 41:1629 38]. Materials and methods: To evaluate the hypothesis that IL4I1 could contribute to the enrichment of regulatory T cells (Treg) in tumors, we used a recombinant monocytic cell line expressing IL4I1 [Marquet J et al. (2010) European Journal of Immunology 40(9):2557 68] to stimulate naı¨ve CD4+ T cells and studied the effect on T helper cell differentiation in the presence or absence of polarizing stimuli. Results: The phenotypic analysis of the population obtained showed an enrichment in CD25high FoxP3+ Treg endowed with in vitro suppressive activity. The mechanisms involved and the phenotypic characteristics of these cells are currently under investigation. Conclusions: Thus, IL4I1 may participate to tumor escape both via inhibition of effector T cell proliferation and functions and Treg enrichment in the tumor microenvironment.

P1690 Use of the quantitative DNA methylation assay to measure Treg frequencies in the ovarian cancer patients I. Vancurova,* L. Sojka, A. Fialova´* & R. Spisek *Department of Immunology, 2nd Faculty of Medicine Charles University and University Hospital Motol, Prague, Czech Republic, Research Department, Sotio a.s., Prague, Czech Republic Purpose/Objective: The type of immune cells that are present within the tumor microenvironment can play a crucial role in the prognosis of patients. However, little is known about the dynamics of the tumorinfiltrating immune cells during disease progression. We studied the immune cells infiltrating the tumor tissues of ovarian cancer patients at different stages of disease. Materials and methods: We used the flow cytometry for detection Th17 cells and a dominant population of FoxP3+ Helios+ activated regulatory T cells (Tregs) in disseminated tumors (stage III-IV). So far, the most specific marker for human naturally occurring Treg cells is the demethylation in the TSDR region of the transcription factor FoxP3. To measure nTreg frequencies we used methyl sensitive real time PCR assay (MS-qPCR) that is strictly specific for nTreg cells. Results: The early stages of development of ovarian carcinomas were characterized by a strong Th17 immune response. A population of naturally occuring Treg cells was measured. We found that the frequency of nTregs significantly increases during the progression of the ovarian cancer. Conclusions: Our findings demonstrate for the first time an alteration in the tumor-infiltrating immune cell pattern during ovarian cancer development and cleary show a dynamic shift from an active antitumor immune response represented by the presence of effector T cells, which prevail over Tregs in the early stages of EOC, to a significant immune suppression in the advanced stages of disease.



Poster Session: Immuno-Reset P1691 A synthetic peptide that restores the inhibitory CD31 signaling as first-in class multifunctional anti-inflammatory drug G. Caligiuri, M. Clement, G. Fornasa, K. Guedj, M. Morvan, J. Khallou-Laschet, A. T. Gaston & A. T. Nicoletti Immunopathology and Immunomodulation of Cardiovascular Diseases, Inserm U698, Paris, France Purpose/Objective: In addition to adaptive immune cells, platelets and endothelial activation contributes to dissemination of chronic inflammatory diseases, such as multiple sclerosis. CD31, a homophilic ITIM receptor exerts a global regulatory function on leukocytes, endothelial cells and platelets, but it is rapidly lost by cleavage upon cell activation. The aim of this study was to evaluate the inhibitory potential of a 8aa retro-inverso CD31-agonist synthetic peptide (P8RI) on cell activation in vitro and on the clinical course of Experimental Autoimmune Encephalomyelitis (EAE) in vivo. Materials and methods: The in vitro effect of the peptide (5 100 lM) was evaluated on the activation of Human T cells by cytometric analysis of calcium mobilization in fluo 3-AM labelled Jurkat T cells stimulated with anti-CD3 antibodies and F(ab’)2 anti-Ig fragments. The effect of P8RI on expression of VCAM-1 in response to IFNc/ TNFca stimulation by endothelial cells (HMEC/D3 cells) by immu-

nofluorescence microscopy and cytometry. Finally, the anti-inflammatory effect of P8RI on platelets was tested on the release of soluble P-selectin (sCD62P) and IL-1b in response to tissue factor. Curative potential of P8RI was assessed on C57Bl6 mice (female, 8 week-old) subjected to MOG-induced EAE and clinically examined daily. When the clinical score reached a value ‡ 1, mice were randomized to receive either P8RI (2.5 mg/kg/day, sc) or prednisone (5 mg/kg/day, sc), or the vehicle alone (PBS, vol/injection = 50 ll). Results: P8RI significantly reduced calcium mobilization in TCRstimulated Jurkat T cells. In addition, it reduced the expression of VCAM-1 on stimulated endothelial cells as well as the release of sCD62P and IL-1b by tissue factor-stimulated platelets. In the curative EAE model, body weight and blood cell count were not affected by either the P8RI (n = 14) or prednisone (n = 14) as compared to control mice (n = 13). The time course of the clinical score was significantly bent by P8RI (P < 0.01 versus PBS, MANOVA). Of note, the extent of protection was even greater that that observed with prednisone (P < 0.05) up to 12 days of follow-up. Conclusions: P8RI could be the first-in-class immune-regulatory drug that acts by rescuing a physiologic regulatory mechanism that is common to key cells involved in chronic inflammatory diseases. Due to the fact that it does suppress/deplete resting blood cells, nor blocks a given immune pathway, its use may carry significantly fewer side effects as compared to other biologicals such as lymphocyte and cytokine neutralizing antibodies or soluble receptors.


Abstracts 703

Poster Session: Immunotherapy for Cancer P1692 A high throughput phenotypic screen for Cbl-b antagonists on primary human T-cells I. Sternberger, G. Schmiedel, I. Winzenborg, B. Magsig & M. Slack Evotec AG, Cellular Assays, Hamburg, Germany Purpose/Objective: Cbl-b is a well validated target for anti-tumor immune therapy. It functions in controlling T cell anergy and thereby contributes to tumor growth and metastasis. Cbl-b is a member of the E3 ubiquitin ligase family and mediates ubiquitin attachment to key signaling molecules in immune cells. The Cbl-b protein consists of several conserved domains that mediate the interaction with a high number of proteins. This, together with the large size of Cbl-b protein, poses a challenge for a robust biochemical screen. Cbl-b causes numerous unique effects in T and NK cells and thus allows a number of specific cellular functional assays for inhibition of Cbl-b. Therefore, Cbl-b is a prototypic target for a cellular, phenotypic screen. In order to identify Cbl-b antagonists, a set of approximately 80 000 compounds was screened. To this end, human PBMCs have been employed in a phenotypic assay measuring IL2 secretion. Materials and methods: Human PBMCs were isolated from precharacterized donors, stimulated with CD3- and CD28-antibodies and used to screen 80 000 lead like compounds of Evotec’s small molecule library at a concentration of 10 lM, phorbol ester stimulation was employed as a positive control. After overnight incubation, tissue culture supernatants were harvested and used to determine IL2 content by HTRF. Results: Primary human PBMCs were obtained from pre-characterized volunteer donors. In order to identify Cbl-b antagonists, a phenotypic read-out was employed: Cells were stimulated with CD3/CD28 antibodies and Interleukin 2 content was measured after overnight incubation. Compounds that increased IL2 secretion in stimulated cells were selected. Counter screening was performed in the absence of cells, in order to rule out HTRF-related artifacts, and with naı¨ve, non stimulated cells, to rule out pathway-unrelated stimulation of IL2 secretion. Screening of 80 000 substances revealed 39 confirmed, specific hits. The hit substances were clustered and revealed three different compound series in addition to numerous singleton hits. Hit expansion was performed by 2D based nearest neighbour searches and ca. One-sixty compounds together with the original hits were selected to be tested in 11 point concentration titration experiments on purified CD4+ T-cells. Conclusions: Disease relevant, primary human T cells have successfully been employed in HTS using phenotypic, non-engineered readouts and relevant chemical matter has been identified.

P1693 A mass spectrometry approach to identify HPV16 T cell epitopes for therapeutic vaccine design S. Hoppe,* R. Blatnik,* A. K. Grabowska,* M. Wu¨hl,* T. Ruppert, U. Warnken,à M. Schno¨lzerà & A. B. Riemer* *Immunotherapy and -Prevention, German Cancer Research Center, Heidelberg, Germany, ZMBH, Core Facility for Mass Spectrometry and Proteomics, Heidelberg, Germany, àFunctional Proteome Analysis, German Cancer Research Center, Heidelberg, Germany Purpose/Objective: To rationally design therapeutic HPV vaccines it is important to know which T cell epitopes are present on HPVtransformed malignant cells. HPV affects the cellular antigen processing machinery, thus not every epitope derived from viral proteins is necessarily presented by MHC molecules on the cancer cell surface. Human papillomavirus (HPV) 16 has been identified as the causative agent in 50% of all cervical cancer cases and in virtually all

extra-cervical mucosal HPV-induced tumors. The transforming potential of high-risk HPVs is mediated by two consistently expressed viral oncoproteins, E6 and E7. As the induction and maintenance of the malignant phenotype depends on these two proteins, they are ideal targets for immunotherapy. A therapeutic vaccine that is applicable to everyone without prior HLA typing needs to contain epitopes for all HLA types. Definition of epitopes for the five major HLA supertypes allows >95% population coverage. To date, HPV T cell epitopes have mostly been determined by indirect methods and for the most prevalent HLA type, HLA-A2. In this study, we aim to directly identify HPV16 E6 and E7 T cell epitopes for the HLA-A3/HLA-A11 and HLA-A24 supertypes by a mass spectrometry (MS) approach. Materials and methods: Several web-based prediction algorithms were used to predict prospective epitopes from the HPV16 E6 and E7 proteins for the above mentioned HLA supertypes. These candidate epitopes were tested for actual HLA binding in competition-based cellular binding assays. The presence of binding peptides on HPV16transformed cells was analyzed by nano-UPLC-ESI-MS2 and -MS3 of immunoprecipitated HLA-A2-peptide complexes. Results: In silico prediction resulted in 48 candidate epitopes for HLAA3, 86 for HLA-A11 and 66 for HLA-A24. The majority of predicted peptides were found to bind to their respective HLA allele. Several novel binding peptides derived from HPV16 E6 and E7 were identified. MS analysis was successfully established and validated for HLA-A2. Conclusions: We show that ascertaining the actual cellular presentation of HPV T cell epitopes is feasible. Having shown proof-of-concept for this approach for HLA-A2, it will now be extended to other HLA supertypes. Verified epitopes are the basis of rational therapeutic vaccine design and also important for immunomonitoring purposes.

P1696 A toxoplasma gondii vaccine strain transforms immunosuppressive ovarian cancer dendritic cells into immunostimulatory cells that trigger therapeutic antitumor immunity D. Bzik, K. L. Sanders, & B. A. Fox Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon, NH, USA Purpose/Objective: Targeting tumor-associated immunosuppression may be necessary to stimulate effective therapeutic immunity against lethal epithelial tumors such as ovarian cancer, one of the most aggressive and most frequently occurring epithelial cancers. We examined efficacy and mechanisms of Toxoplasma gondii vaccine immunotherapy targeting CD11c+ dendritic cells that exhibit proangiogenic and immunosuppressive activity and are the most abundant leukocyte present in the ovarian carcinoma microenvironment. Materials and methods: Attenuated nonreplicating T. gondii; transplantable ID8-Defb29/Vegf-A and ID8-Vegf-A tumors; knockout mice; FACS analysis; cytokine analysis; tumor microenviroment. Results: We show that an avirulent nonreplicating vaccine strain (cps) of Toxoplasma gondii preferentially targets and invades immunosuppressive CD11c+ dendritic cells in the ovarian carcinoma microenvironment. Tumor CD11c+ dendritic cells actively invaded by cps were activated into immunostimulatory phenotypes that expressed markedly increased levels of co-stimulatory molecules CD80 and CD86. In response to cps treatment of the immunosuppressive ovarian tumor environment, IL-12 was rapidly stimulated and antigen-presenting cells regained the ability to efficiently process and present antigen. Treatment of an extremely aggressive transplantable ovarian carcinoma (ID8-Defb29/Vegf-A) with cps provided a significant therapeutic survival benefit, and remarkably, cps treatment induced the rejection of a lethal aggressive model (ID8-Vegf-A) of established ovarian carcinoma. The therapeutic benefit of cps treatment was dependent on expression of IL-12 and interferon gamma. Surprisingly, the thera-


704 Poster Session: Myeloid Cell Development peutic effect was independent of MyD88 signaling as well as immune status to Toxoplasma gondii. Conclusions: Our results establish that cps preferentially targets and activates tumor-associated dendritic cells to trigger the stimulation of potent antitumor responses. Immunotherapy based on cps reawakens natural immunity in immunosuppressive tumor environments and provides a new potent vaccine platform for engineering effective cancer vaccines based on the stimulation of tumor cell-specific CD8+ T cells.

P1697 A tumor directed antibody_IL-15_IL-15Radomain fusionprotein for cancer immunotherapy V. Kermer, V. Baum, R. E. Kontermann & D. Mu¨ller Biomedical Engineering, Institute of Cellbiology and Immunology, Stuttgart, Germany Purpose/Objective: Cytokines driving the immune response are powerful tools for cancer immunotherapy, but their application is generally limited by severe systemic toxicity. Targeted approaches by means of antibody-cytokine fusion proteins might enable to focus the cytokine activity to the tumor site, thereby reducing unwanted side effects. Here we investigated the possibility to improve the efficiency of IL-15 presentation in a targeted approach by the incorporation of an IL-15Ra chain fragment, mimicking physiological trans-presentation. Therefore, an antibody cytokine fusion protein (scFv_RD_IL-15) composed of an antibody moiety (scFv) targeting the tumor stromal fibroblast activation protein (FAP), an extended IL-15Rasushi domain (RD) and IL-15 and according control proteins devoid of one of the components were generated. Materials and methods: FAP-transfected B16 melanoma cells were used as target cells for in vitro and in vivo experiments. Binding capacity of the scFv to FAP was analysed by flow cytometry. Cytokine activity of soluble and targeted fusion proteins was analyzed in terms of proliferation of cytokine growth-dependent cell lines (determined by MTT-assay) and human PBMC subsets (CFSE-staining/flow cytometry) and cytotoxicity of T cells (CD107a/flow cytometry). Analysis of pharmacokinetic properties and the therapeutic effect in a lung metastasis model were performed in C57/BL6 mice. Results: All components of the fusion proteins showed full functionality: the scFv mediated specific binding to target-expressing cells and IL-15 stimulated the proliferation of cytokine-dependent cell lines and PBMCs. Soluble and target-bound scFv_RD_IL-15 achieved stronger proliferation and cytotoxicity of unstimulated and activated T cells, respectively compared to scFv_IL-15. Furthermore the scFv_RD_IL-15 revealed best anti tumor effects in a lung metastasis mouse model compared to an untargeted or RD missing version of the protein. Conclusions: Thus, an antibody_RD_IL-15 fusion protein appears as a promising approach to further improve the antitumor effect of IL-15.

P1698 Activation of human dendritic cells for immunotherapy in ovarian carcinoma E. Brencicova,* A. Jagger, H. Evans, A. Laios,à S. Montalto,§ A. A. Ahmed,à S. Raju-Kankipati,§ L. S. Taams & S. Diebold* *Peter Gorer Department of Immunobiology, King’s College London, London, UK, Centre for Molecular and Cellular Biology of Inflammation (CMCBI), King’s College London, London, UK, àNuffield Department of Obstetrics and Gynaecology, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK, §Department of Gynaecological Oncology, St Thomas’ Hospital, London, UK Purpose/Objective: In this study, we investigate the impact of ovarian carcinoma (OC)-associated ascites fluid (AF) on dendritic cell (DC) activation by Toll-like-receptor (TLR) agonists in vitro. DC have the

potential to instigate a tumour-specific adaptive immune response, but their ability to induce differentiation of naı¨ve lymphocytes into effector cells in lymphoid tissues is dependent on their activation status. We are interested in studying whether DC activation by TLR agonists * an approach often attempted in tumour immunotherapeutic concepts * is impeded by OC environment and if so, how these effects can be eleviated. Materials and methods: AF was obtained from patients with epithelial OC or benign ovarian tumours with informed consent. Monocytederived DC from healthy volunteers were cultured overnight in the absence or presence of R848, LPS or poly I:C and AF. To investigate the role of different AF components, cytokine-neutralizing antibodies were added to selected cultures. DC activation was assessed by surface marker expression and cytokine production. Mixed leukocyte reactions with allogeneic naı¨ve T-cells were performed to evaluate DC functionality. Results: Our results show that AF reduces the up-regulation of the costimulatory molecule CD86 and partially inhibits the production of the pro-inflammatory cytokines IL-6, IL-12 and TNF-a in TLR-activated monocyte-derived DC. We further observe an impaired T-cell stimulatory capacity of monocyte-derived DC upon activation with TLR agonists in the presence of AF, which indicates that suppressed activation status of the monocyte-derived DC directly affects their function as antigen-presenting cells. We have identified IL-10 as the pivotal component in AF jeopardizing DC activation, and although further factors may play a role, our data show that selective blocking of IL-10 by neutralizing antibodies is sufficient to largely restore DC activation and function in vitro. Conclusions: IL-10 is the key agent impeding TLR-mediated DC activation within OC environment in vitro. By selective neutraliziation, the immunosuppressive effects of IL-10 can be offset and DC functionality reinstated. Our findings can provide substantial insight into the feasibility and execution of DC-based vaccines and other immunotherapeutic intervention strategies in OC.

P1699 Actively personalized multi-peptide vaccination for primary liver cancers a novel therapeutic approach for overcoming residual disease? N. Trautwein,* M. Lo¨ffler,* M. Walzer,* D. Zieker, P. Horvath, C. Schroeder,à P. Bauer,à A. Ko¨nigsrainer, H. G. Rammensee* & S. Stevanovic* *Immunology, Interfaculty Institute for Cell Biology, Tu¨bingen, Germany, Surgery, University Hospital Tu¨bingen, Tu¨bingen, Germany, àHuman Genetics, Medical Genetics, Tu¨bingen, Germany Purpose/Objective: Primary liver cancers, mainly hepatocellular carcinomas (HCC), are among the five most common cancers globally and a leading cause of cancer related death. More than half a million patients are diagnosed with HCC every year. Also in Europe and the USA incidence numbers have been rising over the last decades. With chemotherapies and other adjuvant strategies showing only limited benefit, available therapeutic options are scarce. Therefore primary malignancies of the liver are a promising entity for targeted immune interventions after surgery. Materials and methods: In order to evaluate this novel targeted adjuvant therapeutic approach, we focused on naturally processed and presented MHC-ligands eluted directly from tumor tissue. Our interest is particularly centered on mutated peptides representing neo-epitopes derived from specific mutations of the tumor exclusively present on malignant tissue. Those tumor specific peptides lack central tolerance and should thus be highly immunogenic. To identify those peptides we employ a dual approach. On the one hand MHC-ligands are isolated by means of immunoaffinity chromatography of MHC molecules followed by acidic


Abstracts 705 elution of peptides. These peptides are subsequently identified using tandem mass spectrometry and database dependent search algorithms. On the other hand genomic mutations forming the basis for tumor specific MHC-ligands are analyzed by next generation exome sequencing. Based on sequencing results differences between tumor and germline genome are determined and specific databases are generated. Results: Tumor specific peptides are predicted using SYFPEITHI database to facilitate a targeted search for mutated peptides in the MS2data. In addition to tumor tissue adjacent benign tissue of patients was analyzed in the same fashion. So far sample pairs from several patients have been investigated in this manner. Conclusions: Based on this approach, we aim at providing an actively personalized vaccine cocktail comprising individual patient- and tumor-specific peptides for adjuvant tumor therapy. This approach is completely new and may offer the chance of overcoming residual disease, which is a major cause of tumor related death and relapse so far.

P1701 Adoptive immunotherapy utilizing genetically modified T cells in an orthotopic breast cancer model L. B. John,* C. Devaud,* C. P. M. Duong,* C. Yong,* L. X. J. Wang,* W. Z. Wei, R. L. Anderson,à Y. Cao,à M. H. Kershaw* & P. K. Darcy* *Cancer Immunology Research, Peter MacCallum Cancer Centre, East Melbourne, Australia, Department of Pathology, Karmanos Cancer Institute, Detroit, MI, USA, àCancer Cell Biology Research, Peter MacCallum Cancer Centre, East Melbourne, Australia Purpose/Objective: Adoptive immunotherapy involving genetic modification of T cells with chimeric antigen receptors (CARs) is a promising approach for the treatment of cancer. We have recently shown that the anti-tumour effects observed in Her-2 transgenic mice bearing established Her-2+ lung metastases, treated with anti-Her-2 T cells was not associated with any autoimmune pathology to normal tissue (breast, brain) expressing the target antigen. However, the efficacy and safety of this approach against tumor located directly within normal tissue expressing the Her-2 antigen has not been evaluated. Materials and methods: We generated a spontaneously metastatic breast carcinoma cell line, E0771-LMC expressing the human Her-2 antigen which displayed comparable growth kinetics of primary tumor and formation of spontaneous lung metastases as the parental cells following orthotopic injection into the mammary fat pad (MFP). We then examined antigen-specific function by CAR T cells by investigating cytokine secretion, killing and proliferation in vitro. In adoptive transfer studies, primary tumor growth and survival of mice was evaluated in the Her-2 transgenic mouse model. Results: We first demonstrated antigen-specific function by CAR T cells in vitro. In adoptive transfer studies, we demonstrated significant regression of established primary Her-2+ tumours injected into the MFP and enhanced survival of mice treated with anti-Her-2 T cells compared to control T cells. Increasing the dose of anti-Her-2 T cells led to complete tumour eradication in a proportion of mice. The reduction in tumour growth correlated with localisation of transferred T cells at the tumour site. Interestingly, we observed no overt toxicity in mice following therapy. Future studies will examine the efficacy of anti-Her-2 T cells against spontaneous metastases in this model and examine more closely breast and brain tissue by immunohistochemistry for potential signs of pathology. Conclusions: In conclusion, our study highlights for the first time that CAR T cells can distinguish and specifically eradicate Her-2+ breast cancer cells without on target toxicity to surrounding normal tissue expressing the target antigen.

P1702 Anti-CD20 treatment induces long-term adaptive immune response by blocking CD4+ Foxp3+ Treg expansion and provoking an increase in CD4+ IFNgamma+ Th1 cell subset C. Deligne, A. Metidji & J. L. Teillaud Cordeliers Research Center UMRS872, Team 14 ‘‘Antibody BioEngineering’’, Paris, France Purpose/Objective: We have recently demonstrated that anti-CD20 mAb exerts a long-lasting anti-tumor protection through the induction of an adaptive immune response (Abes et al., Blood, 2010). This requires the presence of CD4+ cells at the initiation of treatment but not CD8+ cells, whereas both cell types are needed after tumor challenge for further protection. It is unclear how anti-CD20 mAb treatment impacts CD4+ T cell subsets, enabling the development of this adaptive response despite the fact that tumor cells induce regulatory T cells (Treg). Thus, we have investigated if anti-CD20 mAb treatment modifies the balance between T cell subsets and has an impact on the expansion of Treg induced by tumor cells. Materials and methods: C57Bl/6 mice were injected with EL4huCD20 cells and treated with the anti-CD20 mAb CAT-13 (Abes et al., 2010). CD3+CD4+ T cell subsets were analyzed by intracellular staining with anti-IFNc, -IL10, -IL17, and -Foxp3 antibodies. The logrank test was used to compare survival. In some experiments, Treg were depleted by anti-CD25 injection. In other experiments, a neutralizing anti-TGFb antibody was injected repeatedly. Results: Adoptive transfer of CD4+ T cells from CAT-13-treated surviving mice into naı¨ve recipients, followed by EL4-huCD20 injection, showed that CD4+ T cells are responsible for the induction of the long-term protection. Analysis of CD4+ T cells, at different time points after tumor cell injection, initially revealed the expansion of Treg in both untreated and CAT-13-treated mice. However, after completion of anti-CD20 treatment, CAT-13-treated mice exhibited a strong Th1 subset increase while Treg diminished markedly. When Treg were depleted by anti-CD25 treatment before tumor injection, the survival of CAT-13-treated mice was not increased, although most of the mice not treated with CAT-13 were protected. This suggests that combination of Treg depletion before tumor injection with anti-CD20 treatment does not potentiate anti-tumor protection. Finally, injections of neutralizing anti-TGFb did not increase survival of CAT-13treated animals, although it delayed death of untreated animals. Conclusions: These data demonstrate that the balance between Treg and Th1 compartments, essential for controlling tumor progression, is fine-tuned by therapeutic mAb to switch the immune response from a pro- to an anti-tumor response.

P1703 Anti-IL-20 monoclonal antibody alleviates inflammation within oral cancer and suppresses tumor growth H. Yu-Hsiang & C. Ming-Shi Department of Biochemistry and Molecular Biology, National Cheng Kung University, Tainan, Taiwan Purpose/Objective: IL-20 is a proinflammatory cytokine involved in rheumatoid arthritis, atherosclerosis, and osteoporosis. However, little is known about the role of IL-20 in oral cancer. Materials and methods: We explored the function of IL-20 in tumor progression of oral cancer. IL-20 expression levels in tumorous and non-tumorous oral tissue specimens from 40 patients with four different stages oral cancer were analyzed with IHC staining and RTQPCR. Clinical oral tumor tissue expressed higher IL-20 and its receptor subunits than did non-tumorous oral tissue. The roles of IL-20 were examined in oral cancer OEC-M1 and OC-3 cells. Results: In vitro, IL-20 promoted TNF-a, IL-1b, MCP-1, CCR4, and CXCR4, and enhanced proliferation, migration, ROS production, and


706 Poster Session: Myeloid Cell Development colony formation of oral cancer cells with activated STAT3 and AKT/ JNK/ERK signals. To evaluate the therapeutic potential of anti-IL-20 monoclonal antibody 7E to treat oral cancer, an ex vivo tumor growth model was used. In vivo, 7E reduced tumor growth and inflammation within oral cancer cells. Conclusions: IL-20 promoted oral tumor growth, migration, and tumor-associated inflammation. Therefore, IL-20 may be a novel target for treating oral cancer, and anti-IL-20 monoclonal antibody 7E may be a feasible therapeutic.

Results: We could, in one patient, detect enhanced direct ex-vivo Tcell cytotoxicity against tumour-cells during treatment with AZA. Interestingly, tumour-cells were better recognized at later time-points during treatment, while T-cell functionality seemed to decline. In a larger group of patients, specific T-cell recognition of Cancer-Testis Antigens (CTA) was detected using a panel of 43 known CTAs. Conclusions: Our data supports the hypotheses that AZA-treatment has an additional immunological effect that could be boosted in combination with immunotherapy.

P1704 Anti-tumoral effects of anti-CD115 monoclonal antibody through inhibition of tumor associated macrophages and osteoclasts

P1709 Cancer immunotherapy by adoptive transfer of tumor-specific Th2 cells

L. Fend, J. Kintz, C. Reymann, N. Accart, R. Rooke, J. Y. Bonnefoy, X. Preville, & H. Haegel Immunopharmacology, TRANSGENE S.A, Illkirch Graffenstaden Cedex, France

A. Corthay,* C. Hammarstro¨m,* M. Fauskanger, O. A. W. Haabeth, M. Zangani, H. Haraldsen,* B. Bogen & K. B. Lorvik *Department of Pathology, University of Oslo, Oslo, Norway, Department of Immunology, University of Oslo, Oslo, Norway

Purpose/Objective: Some aspects of tumor progression are controlled by Tumor-Associated Macrophages (TAMs) and metastasis-induced bone destruction by osteoclasts. Both cell types depend on the CD115CSF-1 pathway for their differentiation and function. We studied the effects of targeting CD115 with a function-blocking monoclonal antibody (mAb) in 3 different mouse cancer models. Materials and methods: Anti-CD115 mAb, isotype control or PBS was administered to (1) mice bearing sub-cutaneous EL4 tumors, (2) MMTV-PyMT mice developing spontaneous mammary tumors, and (3) mice injected with human breast cancer cells metastatic to bone. Results: In mice bearing solid EL4 tumors, the anti-CD115 mAb depleted F4/80 macrophages and reduced tumor growth, resulting in prolonged mouse survival. In MMTV-PyMT mice, primary tumor growth was not detectable in mice treated with the anti CD115 mAb but became palpable only after termination of the immunotherapy. In the breast cancer model of bone metastasis, the anti CD115 mAb showed potent therapeutic effect by inhibiting the differentiation of osteoclasts and their bone destruction activity. Conclusions: CD115 represents a promising target for cancer immunotherapy, since a blocking antibody may not only inhibit the growth of a primary tumor through TAM depletion, but also metastasisinduced bone destruction through osteoclast inhibition.

P1706 Azacytidine treatment sensitize tumour cells to T-cell mediated cytotoxicity in patients with myeloid malignancies

à

M. K. Brimnes,* A. O. Gang, I. H. Dufva, R. Lyngaa, M. B. Treppendahl,§ M. H. Andersen, K. Grønbæk,§ P. T. Straten & S. R. Hadrup *Department of Rheumatology, Institut for Inflammtions Research, Copenhagen Univeristy Hospital Rigshospitalet, Copenhagen, Denmark, Department of Haematology, Center for Cancer Immune Therapy, University Hospital Herlev, Herlev, Denmark, àDepartment of Haematology, University Hospital Herlev, Herlev, Denmark, §Department of Haematology, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark Purpose/Objective: We investigated a potential immunological effect of azacytidine (AZA) treatment in patients with myeloid malignancies, following the hypotheses that AZA-treatment may induce T-cell recognition of tumour-cells. Materials and methods: Cytotoxicity of T cells was analysed by CD107a expression in co-cultures of purified tumour cells and CD8 T cells. Evaluation of CTA specific T cells was done using combinatorial encoded MHC-multimers after 7 days peptide restimulation.

Purpose/Objective: Adoptive transfer of tumor-specific T lymphocytes is a promising strategy to cure cancer. Materials and methods: We tested adoptive transfer of tumor-specific Th2 cells for the treatment of mice with major histocompatiblity complex (MHC) class II-negative MOPC315 myeloma. Results: Transfer of tumor-specific Th2 cells efficiently eradicated myeloma and conferred long-lasting immunity. Tumor-specific Th2 cells produced interleukin-4 (IL-4), IL-5, and IL-13, and kept a stable Th2 phenotype in vivo upon transfer. Successful Th2-based immunotherapy did not require B cells, natural killer (NK) T cells, CD8+ T cells, or interferon-c. Upon transfer, tumor-specific Th2 cells were shown to induce an inflammatory reaction at the tumor site. Tumorspecific Th2 cells eradicated myeloma by rendering tumor-infiltrating M2-type macrophages tumoricidal. Conclusions: These results suggest that adoptive transfer of tumorspecific Th2 cells could be a very efficient treatment against multiple myeloma and other human malignancies.

P1710 CD28 aptamers as immune response modulators F. Pastor, M. M. Soldevilla, H. Villanueva, S. Inoges, A. L. de Cerio & M. Bendandi CIMA, Oncology, Pamplona, Spain Purpose/Objective: CD28 is one of the main costimulatory receptors responsible for the proper activation of T lymphocytes. Agonistic antibodies and recombinant-protein blockers for the CD28 receptor have already been identified and shown to alter the immune response. Soluble protein therapeutic molecules derived from cell-based procedures need to pass through a lot of high-cost regulatory processes to get into the clinic. Aptamers are single-strand oligonucleotides that exhibit similar affinity and specificity to that of an antibody and its ligand. Aptamers can easily be chemically synthesized, therefore reducing the cost of the clinical regulatory approval process. Materials and methods: In this study, two aptamers to the CD28 receptor were selected by SELEX (Systematic Evolution of Ligands by Exponential Enrichment). In vitro immunoassays were performed to characterize the immunomodulatory aptamers. The agonistic CD28 aptamer was tested as an adjuvant in a mouse idiotypic vaccine protocol for follicular lymphoma. Results: We have isolated and characterized two aptamers that bind to the CD28 receptor. One of them interferes with the binding of CD28 to its ligand (B7), precluding the costimulatory signal. Vice versa, dimerization of any of the two anti-CD28 aptamers is enough to


Abstracts 707 provide an artificial costimulatory signal. Different dimer structures were tested, and they have shown different costimulatory properties. In a follicular lymphoma model, the agonistic CD28 aptamer enhances the antitumor immune response of the idiotypic vaccine. Conclusions: The CD28 aptamers described could be used to modulate the immune response whether by blocking the interaction with B7 for certain autoimmune diseases or by enhancing the vaccineinduced immune response in cancer immunotherapy.

P1711 CD4 cells expressing an MHC class I restricted TCR can rescue CD8 cells tolerized to tumour-associated antigen S. Ghorashian,* A. Holler,* B. Carpenter, M. Ahmadi,* E. Nicholson,* M. Zech,* S. A. Xue,* C. Bennett, H. J. Stauss* & R. Chakraverty *UCL, Department of Immunology, London, United Kingdom, UCL, Department of Haematology, London, United Kingdom Purpose/Objective: The efficacy of T cell therapies for cancer may be limited because tumour-associated antigens (TAA) are also self-antigens (Ag). Exposure to TAA on normal cells may lead to tolerance via deletion of Ag-specific T cells or their anergy/exhaustion. We designed a model of tolerance to TAA in which T cell receptor (TCR)-transduced CD8 T cells recognise pMDM2, a TAA that is also a ubiquitous self-Ag. We aimed to determine the mechanism of tolerance and whether this is reversed by CD4 help. Materials and methods: CD8 T cells transduced with pMDM2specific TCR (MDM-CD8) were transferred to sub-lethally irradiated B6 mice expressing cognate MDM2 antigen (MDM100:Kb). MDMCD8 cells were traceable using a congenic marker. To assess their function we transferred CFSE-labelled target cells pulsed with MDM2 peptide and compared killing at 16 h to that of a control population. Results: Four weeks after transfer, MDM-CD8 cells were detectable, but showed defective Ag-specific killing of target cells. To determine whether tolerance was dependent upon recognition of cognate Ag within the host, we transferred MDM-CD8 cells into BALBc mice (where MDM100:Kb can not be expressed). In a host environment free of cognate Ag, they retained a full capacity to kill target cells presenting Ag. To determine the effect of CD4 T cell help, we co-transferred MDM-CD8 cells and transgenic OT-II CD4+ cells. OT-II cells were primed with dendritic cells (DCs) loaded with cognate pOVA323-339 or irrelevant peptide. After activation through their TCR, OT-II cells increased both the frequency of MDM-CD8 cells and their cytotoxic functions, indicating that CD4 help can overcome CD8 tolerance to TAA. A major limitation to providing CD4 help in T cell cancer therapies is a lack of known MHC class II-restricted TAA. We therefore tested whether CD4 cells transduced with the MHC Class I-restricted MDM2 TCR (MDM-CD4) could provide help upon transfer to Ag-expressing hosts. Co-transfer of MDM-CD4 along with MDM-CD8 cells improved Ag-specific killing of target cells when compared to either population alone. Conclusions: CD4 cells rendered capable of responding to an MHC class I-restricted TAA by TCR transfer can rescue tolerance in a CD8 population with the same specificity. This is potentially a novel way to circumvent defective immune responses arising in adoptive populations due to prolonged exposure to TAA.

P1713 Chemoimmunotherapy of MC38 mouse colon carcinoma: effect of cyclophosphamide treatment followed by dendritic cell-based vaccines on induction of anti-tumor immunity J. Kicielin´ska,* J. Wojas-Turek, J. Rossowskaà & E. Pajtasz-Piasecka* *Laboratory of Experimental Anticancer Therapy, Institute of Immunology and Experimental Therapy, Wroclaw, Poland, Laboratory of Virology, Institute of Immunology and Experimental Therapy, Wroclaw, Poland, àLaboratory of Glycobiology and Cell Interactions, Institute of Immunology and Experimental Therapy, Wroclaw, Poland Purpose/Objective: The aim of this study was to analyze the effect of cyclophosphamide (CY) administration followed by bone marrowderived dendritic cells (BM-DC) activated ex vivo with tumor antigens and applied with genetically modified dendritic cells of line JAWS II on trigger the antitumor response. Materials and methods: Vaccines consisted of BM-DC activated with tumor lysate (BMDC/TAg) and IL-2-gene transduced JAWS II (JAWS II/IL-2) cells orneo-gene modified JAWS II/Neoused as a control. C57BL/6 mice were inoculated s. c. with MC38/0 colon carcinoma cells and on the 14thday, when tumors were palpable, mice were injected, i. p., with CY (150 mg/kg body weight). Starting on the 17thday, the cell vaccines were injected p.t., in 3 weekly intervals. Tumor growth delay was estimated. Spleens were obtained 1 week after the last injection. Splenocytes obtained from mice treated with CY +/- vaccines containing genetically modified cells or BM-DC/TAg cells were examined for their capacity of responding against growing tumor. Thus, the phenotype of freshly isolated cells as well as their ex vivo reactivity after TAg restimulation were analyzed. Results: The administration of the CY combination with BM-DC/TAg and/or JAWS II/IL-2 strongly enhanced the effect of the cytostatic used alone. By contrast, the use of JAWS II/Neo cells caused no therapeutic effect. The combined therapy exploiting genetically modified DCs resulted in increase in percentage of CD8+, CD4+ and CD49b+ cells. The 5-day cocultivation of splenocytes with MC38/0 cells induced cytotoxic activity to some extent associated with increase in percentage of CD107a+ cells, augmented IFN-c production and was accompanied by changes in percentage of CD8+, CD4+, CD49b+ activated lymphocytes. The effect depended on the type of applied vaccines. Conclusions: The obtained results suggest the strong augmentation of cytostatic effect by application of DC-based vaccines and may help to understand the mechanisms of the anti-tumor immune reactions initiated by combined chemoimmunotherapy of experimental murine tumors. The financial support of the study is granted by the National Science Center of Poland (decision number DEC-2011/01/N/NZ4/01725).

P1714 Circulating specific antibodies enhance systemic cross-priming by delivery of complexed antigen to dendritic cells in vivo W. van Maren, N. van Montfoort, S. M. Mangsbo & F. Ossendorp Tumor Immunology, Leiden University Medical Center, Leiden, Netherlands Purpose/Objective: DCs loaded with antigen-antibody complexes (ICs) have shown to confer tumor protection in mice. In the case of therapeutic antibodies directed to tumor-associated antigens (TAA), T cells may contribute to the induced anti-tumor response. To demonstrate the role of circulating antibodies in the priming of a naı¨ve T cells, we applied a system in which mice, naı¨ve for OVA, with circulating hapten (TNP)-specific antibodies were used. Materials and methods: Mice were either vaccinated for a specific hapten (TNP) to induce hapten-specific antibodies, or passively transfered with the hapten-specific antibodies. These mice were then


708 Poster Session: Myeloid Cell Development injected with the TNP-OVA constructs to induce an immune response. The induction of an immune response was measure by in vivo antigen presentation assays, in vivo cytotoxicity assays and in vivo uptake experiments. Results: Mice with circulating hapten-specific antibodies showed significantly enhanced cross-presentation of the injected antigen compared with mice that lacked these antibodies. Increased systemic CD8 and CD4 T cell proliferation was seen after TNP-OVA administration, demonstrating effective MHC class I and II (cross-) presentation. The enhanced cross-presentation in mice with circulating antigen-specific antibodies was associated with improved antigen capture by APCs. Importantly, CD11c+ APCs were responsible for the enhanced and sustained cross-presentation, although CD11c- APCs had initially captured a significant amount of the injected antigen. These results indicate that circulating antibodies aid in vivo antigen delivery to DCs, which improves vaccination by increased antigen uptake and FcR-mediated DC maturation. Conclusions: In vivo formation of antigen*antibody immune complexes improves MHC class I cross-presentation, and CD8+ T-cell activation. This demonstrates that humoral immunity can aid the initiation of systemic cellular immunity. These findings have important implications for the understanding of the action of therapeutic antibodies against tumor-associated antigens intensively used in the clinic nowadays.

P1715 Combining a bispecific antibody with costimulatory antibody ligand fusion proteins in a human and murine model system for targeted cancer immunotherapy N. Hornig, K. Reinhardt, R. E. Kontermann & D. Mu¨ller Antibody Engineering, Institute of Cell Biology and Immunology, Stuttgart, Germany Purpose/Objective: Bispecific antibodies have shown to be able to retarget cytotoxic T cells to tumor cells in a MHC-independent manner, triggering effector cell activation and consecutive tumor cell killing. Considering that costimulation is an essential requirement not only to initiate T cell activation, but also for cell expansion and the development of T cell effector functions, we propose a combinatorial approach by a recombinant bispecific antibody and targeted costimulatory ligands of the B7 and TNF family in order to achieve a highly efficient antitumoral immune response. Therefore, appropriate model systems were generated that take into account targeting of different tumor antigens, combinations of costimulatory ligands and mouse or human effector cells. Materials and methods: Immunostimulatory effects of the recombinant proteins (bispecific antibody + antibody-ligand fusion proteins) were assayed by incubation on tumor cells in presence of unstimulated human PBMC/isolated naı¨ve CD8+ T cells or murine splenocytes. Cytokine release was monitored via ELISA, while proliferation (CFSE labeling), activation and differentiation marker expression and cytotoxic potential (CD107a staining) of T cells was detected via flow cytometry. Results: Model systems involving tumor cells expressing 2 target antigens for the combined application of a bispecific antibody and costimulatory antibody-ligand fusion proteins with human and mouse specificity were established. In both experimental settings, bispecific antibody-induced T-cell stimulation could be enhanced by targeted costimulation with an antibody-4-1BBL fusion protein in a concentration-dependent and ligand-specific manner. Furthermore, in the human model system, modulation of T cell response by the application of the bispecific antibody together with two costimulatory fusion proteins (B7.2, 4-1BBL) resulted in enhanced proliferation and activation marker expression, differentiation into an activation-experienced memory phenotype and in an increase of cytotoxic potential.

Conclusions: In our model systems we could show that the combination of a bispecific antibody and costimulatory antibody-ligand fusion proteins appears as a promising strategy for cancer immunotherapy.

P1716 Controlled local delivery of CTLA-4 blocking antibody induces CD8+ T cell dependent tumor eradication without systemic toxicity M. Fransen, F. Ossendorp, R. Arens & C. J. M. Melief Leiden University Medical Center, IHB, Leiden, Netherlands Purpose/Objective: Blockade of CTLA-4 by monoclonal antibodies improves anti-tumor T cell responses in both pre-clinical models and clinical trials and is now approved for treatment of melanoma patients. However, treatment with CTLA-4 blocking antibodies is associated with auto-immune and inflammatory side-effects. In order to reduce side-effects we propose using a local administration for CTLA-4 blocking antibodies, previously shown to be very effective in CD40 agonistic antibody treatment of tumor. Material and methods: We use an in vivo tumormodel to explore administration strategies of CTLA-4 blocking antibodies. By injecting the antibodies in a subcutaneous slow-release delivery formulation in the tumor area, we show that an eightfold lower dose of antibody is as effective in activating a tumor-eradicating T cell response as systemic delivery. Results: Lower dose and slow release together result in 1000-fold decreased levels of antibody in the serum, reducing adverse events and the risk of auto-immunity. The main target and effector cells in this tumor-model are endogenous CD8+ T cells, whereas CD4+ T cells do not play a prominent role in the antibody-mediated tumor eradication. Conclusions: These results call for exploration of a similar delivery principle in the clinic to reduce toxic side effects of CTLA-4 blocking antibody.

P1717 Culture expansion alters synthesis and content of actin binding proteins in T lymphocytes and mesenchymal stromal cells (MSCs) I. Weber, K. Kollar, M. Giesen, E. Seifried & R. Henschler DRK-Institute of Transfusion Medicine and Immune Hematology, University Hospital Frankfurt, Frankfurt am Main, Germany Purpose/Objective: The expansion of T lymphocytes and MSCs in culture is pursued for adoptive therapy, however deficits in the migration potential and function of expanded cells have been reported. We asked here whether major elements of the cytoskeleton, including actin binding proteins (APBs) such as the actin-polymerizing factors cofilin and profilin, small intercalating molecules filamin A and alpha actinin, as well as components engaging into focal adhesion complexes paxillin, talin and vinculin are subject to alterations during culture in these two cell types. Materials and methods: Magnetically separated CD3+ T lymphocytes from peripheral blood were expanded using immobilized anti-CD3 and anti-CD28 microbeads. MSCs were expanded from human bone marrow according to standard protocols. Cell size was monitored microscopically and by flow cytometry, using size-calibrated micro beads. Quantitation of ABP RNA was performed using qRT-PCR and of ABP proteins by flow cytometry in permeabilized cells. Results: T lymphocytes expanded 15 ± 1 fold within 7 days, but over the culture period, the mean diameter of T lymphocytes increased from 7 ± 0.3 to 15 ± 4.4 lm reflecting a ninefold increase in cell volume from a mean 208 to 1828 lm3. However, on a per cell base, protein levels of ABPs remained constant except for vinculin, which showed a statistically significant 1.6-fold increase. In contrast, RNA levels of all investigated ABPs decreased between five and 50-fold


Abstracts 709 during culture expansion, indicating major alterations in ABP protein turnover in the expanded cells. MSCs revealed two cell populations with mean diameters of 38 ± 5 and 71 ± 9.6 lm, respectively. Compared to expanded T lymphocytes, MSCs showed a four to13fold lower content of all investigated ABPs on both, the protein and mRNA levels. Conclusions: In conclusion, culture expansion resulted in maintenance of absolute levels of ABPs in T lymphocytes, but drastic decreases in the relation between ABPs and cytoplasmic volume. The strong decrease in transcription of ABP mRNAs indicates major alterations in the turnover of ABPs protein in both T cells and MSCs. Our data point to role of alterations in ABP turnover in the functional deficits observed in cultured versus freshly isolated cells which are used for immunotherapies in patients.

P1719 Cytosolically targeted isRNA induces melanoma destruction via type I interferon signaling on myeloid cells in the tumor microenvironment M. Renn, T. Bald, J. Landsberg, A. Sporleder, S. Mikus & T. Tu¨ting Laboratory for experimental Dermatology, Clinic for Dermatolgoy and Allergology, Bonn, Germany Purpose/Objective: Activation of the ubiquitously expressed cytosolic RNA recognition receptors with immunostimulatory RNA (isRNA) emerges as a new treatment option for cancer. This mimics a viral infection and induces a strong type I interferon response. Materials and methods: Here we investigated the cell type specific role of type I IFNs using Ifnar1 competent and deficient melanoma cell lines established from Hgf-Cdk4R24C mice in combination with global and tissue-specific Ifnar1 knockout mice. Results: pI:C complexed with polyethylenimine but not naked pI:C induces regression of primary cutaneous melanomas in Hgf-Cdk4R24C. This was accompanied by a strong infiltration of immune cells mostly of myeloid origin. In vitro, we observed similar chemokine release and apoptosis of pI:C-PEI treated Ifnar1 competent as well as deficient melanoma cells. In contrast, BM-derived Ifnar1-/-macrophages and DCs showed an impaired chemokine release compared to wildtype cells after pI:C-PEI stimulation. In vivo, pI:C-PEI had significant antitumor activity against transplanted Ifnar1-competent or -deficient melanoma cells in syngeneic wildtype but not in Ifnar1 knockout mice, demonstrating the importance of type I interferon signaling in the tumor microenvironment, but not in tumor cells. Because myeloid cells were the predominant immune cell population infiltrating the tumor after pI:C-PEI therapy, we treated Ifnar1 competent melanoma cells in LysMCre · Ifnar1fl/fl mice, that lack the type I IFN receptor on myeloid cells. Again, treatment efficacy was largely abrogated, indicating that myeloid cells are the primary target of type I interferons. To further delineate if cytotoxic cells were required for tumor destruction, we depleted NK and CD8 T-cells with monoclonal antibodies. Whereas the elimination of NK cells strongly reduced treatment efficacy, the elimination of CD8 T-cells had no affect. Conclusions: Taken together, our data suggest that melanoma therapy with cytosolically targeted immuno-stimulatory RNA largely depends on a functional type I IFN system in myeloid cells of the tumor microenvironment and the presence of NK cells.

P1720 Effective activity of cytokine-induced killer (CIK) cells against autologous putative cancer stem cells in solid tumors L. Gammaitoni,* G. Mesiano,* L. Giraudo,* V. Leuci,* M. Todorovic,* F. Picciotto, F. Carnevale-Schianca,à G. Grignani,à M. Agliettaà & D. Sangiolo* *Oncological Sciences, Institute for Cancer Research and Treatment, Candiolo, Italy, Dermatologic Surgery, Institute for Cancer Research and Treatment, Candiolo, Italy, àMedical Oncology, Institute for Cancer Research and Treatment, Candiolo, Italy Purpose/Objective: Targeting the putative subset of cancer stem cells (CSC) within solid tumors is a crucial and still unmet issue as CSC are considered responsible for chemo-resistance and disease relapses. Cytokine-Induced Killer (CIK) Cells are ex-vivo expanded T lymphocytes, endowed with a promising MHC-independent tumor killing activity against several solid tumors. We investigated the ability of CIK cells to kill autologous metastatic cells including the subset of putative CSC. Materials and methods: CIK cells were expanded from PBMC of 10 patients with metastatic melanoma (n = 5) or bone and soft tissue sarcomas (n = 5); their tumor killing ability was assessed with standard cytotoxicity essays against autologous targets obtained from biopsies of metastatic samples. To identify putative CSC, we transduced bulk autologous metastatic tumor cells with a lentiviral vector encoding for the enhanced Green Fluorescent Protein (eGFP) regulated by the human OCT4 promoter. The underlying idea is that CSCs can be visualized based on their exclusive ability, proper of both normal and cancer stem cells, to activate the OCT4 promoter and consequently express eGFP. Results: We efficiently generated metastatic autologous cells lines. The average presence of GFP+ putative melanoma CSC (mCSC) and sarcoma CSC (sCSC), within the bulk metastatic tumor cell population, was 12% and 15% respectively; these data were consistent with levels of protein expression. Both mCSC and sCSC displayed on average a 3 times reduced proliferative potential compared with their GFP- counterpart after 14 days of culture (n = 6), showing a slowgrowing phenotype typical of CSC. CIK cells efficiently killed both autologous mCSC and sCSC; the average specific killing was 71%, 57%, 44% and 41% against mCSC (n = 4), 82%, 72%, 71% and 60% against sCSC (n = 4) at 40:1, 20:1, 10:1 and 5:1 effector/target ratios respectively. The specific killing against CSC overlaid that observed against differentiated GFPmetastatic cells (Fig. 1).

Conclusions: We reported for the first time the preclinical activity of an immunotherapy approach with CIK cells against autologous CSC enriched fractions of metastatic melanomas and sarcomas. These data set biologic basis for further clinical investigations and picture CIK cells as promising candidates for effective immunotherapy in currently incurable clinical settings.


710 Poster Session: Myeloid Cell Development P1721 Elimination of MDSC and Treg cells after combined immunotherapy with cyclophosphamide and dendritic cell vaccines in MC38 tumor bearing mice J. Rossowska,* E. Pajtasz-Piasecka, A. Stepasiuk,* J. Wojas-Turek,à E. Piaseckià & D. Dus* *Laboratory of Glicobiology and Intracellular Interactions, Institute of Immunology and Experimental Therapy, Wroclaw, Poland, Laboratory of Experimental Therapy, Institute of Immunology and Experimental Therapy, Wroclaw, Poland, àLaboratory of Virology, Institute of Immunology and Experimental Therapy, Wroclaw, Poland Purpose/Objective: The microenvironment of solid tumors is characterized by excessive amounts of immunosuppressive factors e.g. VEGF, TGF-b, IL-10 which cause dysfunctions of immune competent cells as well as activation of suppressive cells. The tumor-infiltrating suppressive cells e.g. T regulatory cells (Treg), tumor associated macrophages and myeloid-derived suppressor cells (MDSCs) interact with one another and have an important impact on cancer disease progression. Recent work highlighted the immunostymulatory and antiangiogenic effects of low dose of cyclophosphamide (CY). Thus, it could be successfully combined with new-generation anti-cancer vaccines. The aim of the study was to estimate the anti-tumor effect of low dose of CY combined with dendritic cells stimulated with tumor antigens (BM-DC/TAg) and/or genetically modified dendritic cells producing IL-12. Materials and methods: Mice with s.c. growing, advanced MC38 tumor were treated with CY followed by three consecutive injections of BM-DC-based vaccines. Tumors were measured every 2 days and the tumor growth inhibition was calculated. Seven days after the last injection of BM-DC based vaccines spleens and tumors were collected and the activation of systemic and local anti-tumor response were evaluated. Results: It was observed that CY combined with BM-DC/TAg and/or BM-DC/IL-12 caused the highest tumor growth inhibition. Moreover, in these groups the immunohistological analysis of tumor tissues confirmed the most intensive influx of CD4+ and CD8+ lymphocytes into tumor. Restimulated in vitro spleen cells also showed increased anti-tumor activity. Spleen cells obtained from mice treated with CY and BM-DC/TAg and/or BM-DC/IL-12 showed the highest cytotoxic activity toward MC38 cells and among them the highest percentages of CD8+CD107a+ and CD49b+CD107a+ cells were detected. It was also observed that numbers of MDSC and Treg cells in spleens were different in all examined groups and decreased in mice treated with CY and BM-DC/TAg and/or BM-DC/IL-12. Conclusions: Obtained results suggest that anti-tumor effect of chemoimmunotherapy with CY and DC-based vaccines is dependent on successful elimination of suppressor cells. This work was supported by the Ministry of Science and Higher Education (grants: N N401 316239).

in vitro and in vivo efficacy of novel antagonistic antibodies against c-Met. Materials and methods: A diverse panel of human antibodies against c-Met was generates and characterized. The lead clones were subcloned in various monovalent and bivalent formats to determine the optimal format for c-Met targeting. The different antibody formats were tested in various in vitro assays including ligand inhibition, target downmodulation, receptor phosphorylation, proliferation and migration assays as well as in vivo xenograft models. Results: We have generated a diverse antibody panel against c-Met including clones that could effectively block the ligand for c-Met and inhibit viability and growth of cells. Residual agonistic activity that was observed in some assays and conditions by bivalent IgG and could be modulated by either generating monovalent or engineering the backbone of the antibody to make the antibody more rigid. Efficacy in multiple cell lines both in vitro and in vivo models was shown with these engineered formats. Conclusions: Here we present an overview of the required characteristics of therapeutic antibodies against c-Met with regard to epitope and antibody format. We were able to generate full c-Met antagonists by expressing antibodies in a monovalent format, without compromising the characteristics of the original IgG1 molecules. These novel antibodies have potential for successful targeting of c-Met expressing cancers.

P1723 Expression of ganglioside GM1 in osteosarcoma and Alzheimer’s disease and its significance in B-cell functions G. kodandaraman,* M. M. Carneiro,* M. Goncalves,* J. Casanova, V. Alves,* I. Santanaà & P. Rodrigues-Santos* *Immuno- CNC, Cento for Neuroscience and Cell Biology, Coimbra, Portugal, Faculcidade de Medicina, Universidade de Coimbra, Coimbra, Portugal, àImmuno- CNC, Centro Hospitalar Universitario de Coimbra, Coimbra, Portugal Purpose/Objective: Introduction: In the immune system GM1 is found on the surface of natural killer cells (NK), T cells (both CD4+ and CD8+), B cells, plasmocytes and monocytes. The very low incidence of cancer among the AD patients, has led us to study the differences in GM1-expression in various B lymphocyte subpopulations of osteosarcoma (OS) and AD patients, and whether they show any alterations in the expression of the cytokines and cell-surface markers. Materials and methods: Methodology: Peripheral blood mononuclear cells were isolated from blood samples were collected from OS patients (before starting chemotherapy, n = 6), early stage AD patients

P1722 Exploring novel therapeutic antibodies against c-Met J. Neijssen, B. De Goeij, L. Wiegman, A. Labrijn, J. Schuurman & P. Parren Antibody Science, Genmab BV, Utrecht, Netherlands Purpose/Objective: c-Met, a receptor tyrosine kinase overexpressed in many types of cancer, represents an attractive target for antibody therapy. Use of traditional bivalent IgG is often problematic due to agonistic activity of the antibody. Binding of bivalent IgG can induce dimerization of c-Met and induce downstream signaling, ultimately resulting in cell proliferation, invasion and survival. Here we present various strategies to circumvent the problem of agonism and we show


Abstracts 711 (n = 10) and matched healthy controls (no infection or chronic inflammatory disease, n = 10). The GM1-expressing B lymphocytes were identified using FITC-labeled CTxB. The changes in cell surface markers expression and intracellular cytokine production were determined by flow cytometry. Data were analyzed using a non-parametric one way ANOVA, and differences were considered different for P < 0.05.

Cell populations

Mean ± SEM

P value

Il-10

2.59 ± 0.47

0.0011***

AD

CT

1.66 ± 0.28

OS Tgf-B CT

CT

6.77 ± 1.50

3.915

5.715

2.03 ± 0.55

OS

3.60 ± 2.15

Plasmocytes-AD

1.16 ± 0.24

CT

0.83 ± 0.40

0.233

OS

0.54 ± 0.30

0.130

AD

88.33 ± 1.70

169.6

5.780 0.0355**

CT

Baff-r

21.77

1.601

12.89 ± 5.01 AD

1.670 3.720

0.0181*

2.89 ± 0.93

OS Cd-69

5.21 ± 1.45

17730

0.652

4.57 ± 1.72 AD

2.025 96.970

0.0057**

1.02 ± 0.27

OS il-6r

1.71 ± 0.27

MS

1.430

95.81 ± 2.36 AD

Median

57.43

1.535 0.150 0.5164 (NS)

0.0455*

1.016

88.550

CT

80.80 ± 2.69

90.350

OS

78.83 ± 6.74

80.610

0.6752

177.4

AD, Alzeimer’s Disease; Os, osteosarcoma; CT, controls; SE, standard error; MS, mean square; NS

activity, cytokine production and ability to stimulate antigen specific T cells. Results: Our results demonstrate that although D5 DCs were larger in size compared to D3 DCs, they exhibited similar phagocytic capacity. Viability of D5 and D3 DCs was comparable (80%). Poly (I:C) activated D5 DCs expressed higher levels of costimulatory and surface molecules CD80, CD86 and HLA-DR compared to Poly (I:C) activated D3 DCs. Nevertheless LPS activated D5 and D3 were phenotypically similar. The cytokine production was generally stronger when LPS was used as maturation stimuli. Surprisingly LPS activated D3 DCs were able to produce higher levels of IL-12p70 compared to D5 DCs. Furthermore Poly (I:C) activated D5 DCs secreted higher amounts of IL-6, IFN-a and TNF-a compared to D3 DCs. Importantly, Day-3 and Day-5 DCs were able to induce comparable numbers of antigenspecific T cells. Conclusions: In this study, we identified monocyte-derived DCs generated in CellGro for 3 days and activated using Poly (I:C) as similarly potent clinical-grade DCs when compared to DCs produced by the standard 5 day protocol. These results provide the rationale for the testing of the faster protocols for DCs generation in the clinical trials. Generation of fast DCs markedly reduces the time, work load and cost associated with in vitro culture of DCs precursors and thus may facilitate the use of DCs in clinical trials of cellular immunotherapy.

not significant. P value < 0.05 [***, **. *].

Conclusions: Discussion: Our data showsignificantly higher levels of expression of GM1 on the surface of peripheral B cells of OS and AD patients versus Controls (OS > AD > Cnt). Moreover, our present data show, for the first time, that the increased levels of expression of IL-6R, IL-10, TGF *b on GM1+ B cells might link GM1-expression to B cell function, thus opening new avenues for therapeutic modulation of these cells in the context of cancer and neurodegeneration.

P1724 Fast 3 days poly (I:C)-activated dendritic cells generated in CellGro for use in cancer immunotherapy trials are fully comparable to standard 5 days DCs I. Truxova,* K. Pokorna,* S. Partlova, R. Spisek* & J. Fucikova *Research Department, SOTIO a.s., Prague, Czech Republic, Department of Immunology, Second Faculty of Medicine, University Hospital Motol, Charles University, Prague, Czech Republic Purpose/Objective: Dendritic cells (DCs) are professional antigenpresenting cells capable of inducing immune responses. DC-based vaccines are normally generated using a standard 5 7 days protocol by culturing blood monocytes in the presence of cytokines IL-4 and GMCSF. In order to shorten the vaccine production for the use in the cancer immunotherapy protocol and to generate DCs in GMP quality in shorter time we have developed fast DC protocol by comparing standard DCs (5 days) and fast DCs (3 days). Materials and methods: In this study, we tested D5 versus D3 DCs generation using GMP culture media CellGro and subsequent activation by two activation stimuli * Poly (I:C) (TLR-3) and LPS (TLR-4). We evaluated DCs morphology, viability, phagocytic

P1725 Functionalized dendrimer for the in vivo specific genetic regulation of tumor immunity P. Serafini,* J. Vella,* P. Daftarian & A. Delafuente* *Microbiology and Immunology, University of Miami, Miami, FL, USA, Biochemestry, University of Miami, Miami, FL, USA Purpose/Objective: While mature dendritic cells (DC) can support a strong tumor immunity leading to tumor immune surveillance or eradication, tumor conditioned myeloid cells (TCMC, aka Myeloid Derived Suppressor Cells, MDSC, and Tumor Associated Macrophage, TAM) promote tumor progression, invasion and metastasis. A specific modulation of these populations can be the key for the effective immune therapeutic treatment of malignacies. To boost tumor immunity while reversing TCMC tolerogenic program we used functionalized dendrimer to direct DNA encoding tumor antigen/s into immunogenic APC and STAT3 specific shRNA into suppressive TCMC. To this aim we generate two classes of nanoparticles in which a targeting peptide is covalently linked to G5 PAMAM dendrimers that serve as a loading surface for the desired nucleic acid. The first nanoparticle (HAPD) specifically target dendritic cells, the second one (4PD) instead deliver its cargo particularly to TCMC. Materials and methods: MHC class II or IL4 derived targetting peptides were coupled to the dendrimer by maleidoamide chemistry and the complex purified by HPLC. Nucleic acids were loaded on the funcionalized dendrimer by electrostatic interaction at RT in 80%) or PBMCs from healthy donors using immunoprecipitation. In order to identify HLA presented peptides, liquid chromatography coupled mass spectrometry (LC-MS/MS) based peptide sequencing was employed. The identified ligands were mined for leukemia associated peptides via comparing the HLA ligandomes of AML-patients with those of healthy donors, by analyzing gene expression databases and literature research. In order to identify veritable tumor specific peptides, acquired data has been mined for peptides derived from established leukemia associated mutations comprised in the COSMIC database (http://www.sanger.ac.uk/genetics/CGP/cosmic/). Results: Having identified more than 6000 HLA ligands out of 6 AML patients, several new AML associated peptides (e.g. ligands derived from tumor protein p53, c-myc oncogene, KIS) were obtained by cross checking for occurrence on healthy tissues, especially on PBMCs. The sequences of identified LEUKAPs were verified by LC-MS/MS based peptide sequencing of their synthetic counterparts. Identified candidate peptides are currently checked for their immunogenicity after in vitro T cell priming. Conclusions: This study provides several new leukemia associated antigens, for the first time directly obtained from AML patients‘ HLA ligandomes, offering promising options for a more efficient treatment of AML in the future.

P1728 HPMA copolymer-modified IL-2 possesses superior biological activity to free IL-2 in vivo P. Votavova´,* J. Tomala,* V. Subr, K. Ulbrich, B. Rihova* & M. Kovar* *Department of Immunology and Gnotobiology, Institute of Microbiology Academy of Sciences of the Czech Republic, Prague, Czech Republic, Department of Biomedical Polymers, Institute of Macromolecular Chemistry of Sciences of the Czech Republic, Prague, Czech Republic Purpose/Objective: Interleukin-2 possesses strong stimulatory activity for activated T and NK cells and thus it is an attractive molecule for immunotherapy. However, its unfavourable pharmacological properties, extremely short half-life and severe toxicities associated with highdose IL-2 are the most serious and limiting drawbacks. In order to increase IL-2 half-life and stability in vivo, we covalently conjugated IL2 to synthetic semitelechelic polymeric carrier based on N-(2-Hydroxypropyl) methacrylamide (HPMA). Thus, we synthesized IL-2pHPMA conjugate containing an average 2 3 polymer chains per IL-2 molecule. Materials and methods: Expansion of CD8 ± T lymphocytes, NK and Treg cells in vivo. Purified OT-1 CD8+ T cells (Ly5.2) were labeled with CFSE and injected i.v. into B6.SJL recipients (Ly5.1) on day 1. On day 2 the mice were injected i.p. with PBS, SIINFEKL peptide plus IL-2 (50 lg), and SIINFEKL peptide plus IL-2-pHPMA (1 lg of IL-2). Splenocytes were isolated day 7 and relative expansion of Ly5.2+ CD8+ T cells, NK cells, memory CD8+ T cells and T regulatory cells was analyzed. Kinetics of IL-2-pHPMA in blood. B6.SJL mice were injected i.v. with PBS, free IL-2 (2 lg) and IL-2-pHPMA (2 lg of IL-2). Blood sera were isolated at various time points after injection. Serum IL-2 concentrations were determined by sandwich ELISA with anti-mouse JES6.1A12 and anti-mouse JES6.5H4-biotin mAbs. Determination of MTD. B6.SJL mice were injected i.p. with PBS or IL-2-pHPMA (25, 50 and 75 lg of IL-2) on day 0 . Weight of each mouse was recorded prior to injection (day -1) and for 6 days after. Average % weight change for each group was plotted. Results: IL-2-pHPMA conjugate was found to be significantly more potent than free IL-2 in terms of expansion of NK, NKT, cdT, memory


Abstracts 713 CD8+ T and also T regulatory cells in vivo. IL-2-pHPMA conjugate also potently expanded adoptively transferred OT-1 CD8+ T cells activated with SIINFEKL peptide while free IL-2 at the same dosage (1 lg IL-2 i.p. daily for 4 days) showed minimal activity. Moreover, activated naı¨ve OT-1 CD8+ T cells expanded by IL-2-pHPMA conjugate established a robust population of long-lived cells with memory phenotype, which were able to express effector functions upon reactivation. We also determined IL-2-pHPMA half-life in circulation upon intravenous administration. We found that short half-life of IL-2 is greatly prolonged by modification of IL-2 with HPMA polymeric carrier. Finally, we determined maximal tolerated dose of IL-2pHPMA which is about 80 lg in case of single i.v. administration. Conclusions: IL-2-pHPMA conjugate showed much longer biological half-time (~4 h versus