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Eur. J. Immunol. 2006. 36: 207–215

Clinical immunology

Clinical immunology

Induction of experimental autoimmune encephalomyelitis in transgenic mice expressing ovalbumin in oligodendrocytes Yi Cao*1, Catherine Toben1, Shin-Young Na1, Kirsten Stark1, Lars Nitschke*1, Alan Peterson2, Ralf Gold3, Anneliese Schimpl1 and Thomas Hnig1 1

2 3

Institute for Virology and Immunobiology, University of Wrzburg, Wrzburg, Germany Laboratory of Developmental Biology, McGill University, Montreal, Canada Institute for MS Research, University of Gttingen and Gemeinntzige HertieStiftung, Gttingen, Germany

We have used the 50 flanking sequence of the myelin basic protein gene known to include the core promoter and a strong oligodendrocyte (ODC)-specific enhancer to target expression of the well-studied model antigen ovalbumin (OVA) to ODC in transgenic mice. OVA protein was detected in a tissue- and cell-specific manner in these "ODC-OVA" mice. Without immunization, CD4 T cells and B cells remained ignorant of the neo-self antigen expressed in the central nervous system (CNS), as indicated by unimpaired development and lack of activation of OVA/IAb-specific TCR transgenic T cells in these mice, and the ability to mount normal OVA-specific recall and antibody responses. Upon immunization with OVA in complete Freund's adjuvant, about half of the transgenic mice developed neurological symptoms characteristic of experimental autoimmune encephalomyelitis (EAE). Mononuclear infiltrates in the brain and spinal cord contained both macrophages and T cells, similar to classical models of EAE induced by immunization with CNS antigens in adjuvant. The wealth of immunological reagents available to study and manipulate the OVA-specific response should make this new model useful for the investigation of components and mechanisms involved in CNSspecific autoimmunity.

Introduction Transgenic mice expressing well-characterized model antigens in a tissue-specific manner have proven extremely useful for the investigation of mechanisms involved in autoimmune disease. In particular, studies * The first two authors contributed equally to this work. Correspondence: Dr. Thomas Hnig, Institute for Virology and Immunobiology, University of Wrzburg, Versbacher Str. 7, D-97078 Wrzburg, Germany Fax: +49-931-201-49243 e-mail: [email protected] Abbreviations: CNS: central nervous system  HA: hemagglutinin  MBP: myelin basic protein  MS: multiple sclerosis  ODC: oligodendrocyte f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Received 30/6/05 Revised 20/9/05 Accepted 10/11/05 [DOI 10.1002/eji.200535211]

Key words: Autoimmunity  EAE  Ovalbumin  Transgene

on autoimmune diabetes in mice expressing the OVA antigen in pancreatic islets have generated a wealth of information on the development of type 1 diabetes, due to the availability of mouse lines expressing MHC class I and class II restricted transgenic TCR that recognize defined OVA-derived peptides and their variants [1]. With regard to multiple sclerosis (MS), an autoimmune disease of the central nervous system (CNS), no such transgenic mouse model is available so far. An MS model using a well-defined antigen in which all components of specific antigen recognition are available would be of particular interest because all lymphocyte subsets and a

* Present address: Department of Genetics, University of Erlangen, Erlangen, Germany www.eji.de

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variety of effector mechanisms have been implicated both in the human disease itself and in the various rodent models of EAE [2, 3]. So far, one mouse strain has been described that harbors a transgene directing the expression of a model antigen under the control of a CNS-specific promoter: The "GFAP-HA" mouse, which expresses viral hemagglutinin (HA) under the control of the glial fibrillary acidic protein promotor in astrocytes of the CNS and glial cells of the enteric nervous system [4]. Although HA-specific TCR transgenic CD4 cells developing in these mice remain ignorant of, and reactive to the HAantigen, immunization with the relevant peptide in CFA fails to induce autoimmunity [5]. On the other hand, introduction of a class I-restricted transgenic TCR in this model led to the early death of the double transgenic pups due to an acute bowel inflammation caused by autoimmune destruction of HA-expressing enteric glial cells [6]. Oligodendrocytes (ODC) express most of the major self antigens relevant for the classical EAE models and probably also for MS [2]. We therefore sought to target OVA-specific CD4 and CD8 T cells to ODC. Transgenic mice were generated expressing OVA under the control of the myelin basic protein (MBP) promoter lacking the upstream enhancer element required for its activity in Schwann cells, and previously shown to confer selective expression in ODC [7, 8]. Here, we show that ODCspecific expression of OVA is indeed observed in these animals, and that CD4 T cells and B cells of the ODC-OVA mice appear ignorant of this artificial self antigen as they mount normal recall and antibody responses against OVA. Importantly, active immunization induces mild EAE in about 50% of ODC-OVA mice, indicating that the OVA expressed in ODC indeed provides a target for autoimmune attack.

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both in brain and in spinal cord (Fig. 1B, C). Importantly, OVA expression was maintained in adult mice, indicating that the most 50 module contained in the promoter/ enhancer construct, which is required for MBP expression in the post-weaning period, is functional. No protein was detected by Western blotting or immunohistochemistry in sciatic nerve (Fig. 1C), spleen, thymus, kidney, heart, lung, stomach, colon, liver, testicle, uterus and ovary (not shown). Immunohistochemistry performed on brain sections revealed distinct OVA-positive cells in young adult transgenic but not in WT mice (Fig. 2A, B). Double labeling with anti-OVA and anti-20 , 30 cyclic nucleotide 30 phosphodiesterase identified the OVA-expressing cells as ODC (Fig. 2C–E). For further experiments, line 2 was selected. In reference to the ODC-specific expression of OVA, these mice will be referred to as ODC-OVA mice.

Results ODC-specific expression of OVA in ODC-OVA mice In an attempt to express the well-studied model antigen OVA as a tissue-specific autoantigen in the brain, transgenic mice were generated with an OVA cDNA under the control of an oligodendrocyte-specific MBP promotor construct, containing the 6.5-kb proximal part of the MBP promoter [7, 8] (Fig. 1A). For future studies on the requirement for antigen persistence in chronic inflammation of the CNS, the transgene was flanked with loxP sites, thereby allowing its deletion upon activation of an inducible Cre transgene. Three transgenic founder lines were obtained and confirmed by PCR. Of these, two (lines 2 and 7) expressed the OVA protein at levels readily detectable by Western blotting f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 1. Expression of OVA protein in the CNS of mice with an OVA transgene under the control of the proximal 6.5-kb MBP promoter. (A) Construct and location of PCR primers used for typing. (B) Western blots of protein extracts from brain (lane 1: WT, lane 2: ODC-OVA 4 wk old, lane 3: 12 wk old). (C) Western blots of protein extracts from spinal cord (lane 1: WT, lane 2: ODC-OVA 4 wk old, lane 3: ODC-OVA 12 wk. old) and sciatic nerve (lane 4: WT, lane 5: ODC-OVA 4 wk old). The weak band observed in the extract from WT brain (panel B, lane 1) is an unrelated band of slightly higher molecular weight consistently observed in non-transgenic brain extracts. www.eji.de

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TCR transgenic CD4 cells are ignorant of OVA in ODC-OVA mice Clonal thymic deletion and peripheral T cell activation are very sensitive means of detecting even minute amounts of 'self' antigens in vivo. Accordingly, OT-II mice expressing a transgenic TCR on CD4 T cells reactive with OVA peptide 323–339 presented by IAb [9] were crossed with ODC-OVA mice, and cellularity as well as expression of the OT-II TCR and of activation markers was compared between these double transgenic and OT-II single transgenic mice. As shown in Fig. 3A, the features distinguishing OT-II from C57BL/6 WT mice, i.e. a marked reduction in peripheral CD8 T cells as well as thymic CD8 single-positive cells, and a mild reduction in CD4, CD8 double-positive thymocytes were maintained in the double transgenic mice. Moreover, levels of the transgenic TCR (Fig. 3B) and of the CD4 coreceptor (not shown) on cells expressing the OT-II receptor were indistinguishable in OT-II single and OT-II/ODC-OVA double transgenic mice, indicating that neither immature thymocytes nor mature CD4 T cells were recogniz-

Clinical immunology

ing their cognate antigen. This notion was further supported by identical levels of CD44, CD62L, CD69 and CD25 on CD4 T cells from OT-II single and OT-II/ODCOVA double transgenic mice (data not shown). To test for a possible induction of functional anergy, LN cells from WT, single transgenic and double transgenic mice were stimulated with OVA 323–339 in vitro (Fig. 3C). Anti-CD3 stimulation served as a control. Cells from OT-II and OT-II/ODC-OVA double transgenic mice responded equally well to the antigenic peptide, whereas as expected, neither C57BL/6 WT nor single transgenic ODC-OVA mice mounted a detectable response. Taken together, these experiments show that OVAderived peptides presented by IAb are not recognized by developing or mature T cells in these mice. Accordingly, it can be assumed that OVA expressed by ODC in the CNS does not reach the thymus or the periphery of ODC-OVA mice in amounts sufficient to generate stimulatory IAb/ peptide complexes on APC via processing and presentation. Induction of EAE by immunization with OVA WT and ODC-OVA mice were immunized subcutaneously with OVA in CFA, and monitored for symptoms of EAE and for weight loss (Table 1 and Fig. 4). About half (14/26 in line 2 and 2/5 in line 7) of the transgenic, but none of the control mice developed neurological symptoms characteristic of classical EAE between 8 and 20 days post-infection, which, however, only rarely progressed beyond grade 1 (limp tail). Of 16 mice with EAE, 2 developed complete paraplegia before they were sacrificed for histological analysis. Development of EAE was accompanied by typical weight loss, which was absent in immunized WT animals and animals not displaying disease (Fig. 4). Histopathology of active OVA-induced EAE in ODC-OVA mice

Figure 2. Detection and cellular localization of OVA in the brain of transgenic mice of line 2. Binding of polyclonal OVA-specific antibodies detected by alkaline peroxidase conjugated secondary reagent identifies OVA in the brain of the transgenic (B), but not of the wild-type mouse (A). (C–E) Double fluorescent labeling for OVA (C) and 2,3–cyclic nucleotide 3-phosphohydrolase (CNPase, (D) in the brain of the transgenic mouse localizes OVA to oligodendrocytes as shown by overlay (E). f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

As illustrated in Fig. 5 and listed in Table 1, cellular infiltrates were observed in the white matter of the brain and the spinal cord of ODC-OVA mice undergoing EAE, but were absent in WT mice immunized with OVA. In the CNS, the majority of the infiltrating cells were present outside of the perivascular cuff. In one case of severe EAE, infiltrates were also observed in the meninges of the brain (Table 1). Immunohistochemistry revealed both T cells and macrophages at the sites of inflammation. Furthermore, in both of the severely affected animals studied, focal lesions were observed in the white matter that included demyelination, myelin degeneration, and the presence of degenerative and necrotic cells (not shown). www.eji.de

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Figure 3. OVA expressed in ODC-OVA mice is ignored by thymocytes and CD4 T cells expressing an OVA+IAb specific transgenic TCR. (A) CD4/CD8 profiles of thymocytes and LN from ODC-OVA and OT-II single and double transgenic mice. Numbers give percentages of events in quadrants. (B) Level of transgenic TCR expression in OT-II single and OT-II/ODC-OVA double transgenic mice shown as staining intensity with Vbeta5-specific antibody on gated CD4+Valpha2+ LN cells. (C) Proliferative in vitro response of whole LN cells from OT-II single and OT-II/ODC-OVA double transgenic mice.

Antibody production and recall responses in OVAimmunized ODC-OVA mice In other model systems of EAE, immunization with CNS antigens in CFA primarily activates autoreactive CD4 cells and antibodies, both of which can contribute to CNS inflammation, depending on the model studied [10]. To investigate if an anti-OVA response can be induced in both of these components of the adaptive immune system in ODC-OVA mice, their proliferative recall response to OVA protein as well as their serum antibody titers were compared to those induced by the same immunization protocol in WT C57BL/6 mice. Whereas unimmunized WT or transgenic mice did not exhibit detectable proliferative responses (Fig. 6A) or f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

serum antibody titers (Fig. 6B) to OVA, both groups of mice responded equally when immunized with OVA in CFA. Thus, as already suggested by the phenotype of OTII/ODC-OVA double transgenic mice, the CD4 T cell compartment is fully reactive to OVA in mice expressing OVA in ODC. Furthermore, there is no tolerization of the B cell compartment towards OVA in these animals, making this model useful for the study of T and B cell contributions to CNS-specific autoimmunity.

Discussion We present the initial characterization of a new transgenic mouse model for human MS, in which EAE www.eji.de

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Clinical immunology

Table 1. Summary of clinical and histological observations in ODC-OVA mice with active EAE Onset (days

Histological analysis

Animal no.

Strain

Sex

Clinical grade

post-immunization)

Duration

Inflammation/ Days after onset

Distribution

1

2

Female

1

14

5

+/25

Med, SPb)

2

2

Female

1

14

6

+/26

Med, SP

3

2

Female

1

14

5

+/25

Med, SP

+++/2

Med, SP, Mng

4

2

Female

5

8

>2

5

2

Female

1

12

5

6 7 8 9

2 2 2 2

Female Male Male Female

1 1 1 4

16 14 14 8

a)

+/11

Med, SP

>1

a)

+/1

Med, SP

>1

a)

+/1

Med, SP

>1

a)

+/1

Med, SP

>3

a)

+++/3

Med, SP

a)

+/1

Med, SP

10

2

Male

1

14

>1

11

2

Male

1

20

6

+/7

Med, SP

12

2

Female

1

14

8

+/16

Med, SP

13

2

Female

1

13

5

+/18

Med, SP

14

2

Female

1

12

10

++/12

Med, SP

+/3

Med, SP

+/21

Med, SP

15

7

Female

1

14

>3

16

7

Female

1

16

6

a) b)

a)

Sacrificed on day after onset given. Med: Medulla; SP: Spinal cord; Mng: Meninge.

can be induced by immunization with the model antigen OVA. Key features of this model are (1) selective expression of the transgene in ODC, (2) absence of tolerance with regard to CD4 T cells and B cells, and (3) mononuclear infiltration and development of neurological disease in response to OVA immunization in CFA. In two mouse lines derived from two independent transgenic founders that expressed OVA in the CNS, this expression was restricted to ODC, in agreement with the reported cell-type specificity inferred on the MBP promoter by deletion of the upstream enhancer [7]. The absence of detectable OVA protein in the peripheral nervous system (Fig. 1C) and of infiltrates or lesions in the sciatic nerve (not shown) indicates that the promoter/enhancer construct used successfully targeted the transgene to the CNS alone, making ODC-OVA mice a useful model for CNS-specific autoimmunity (MS) without simultaneous features of autoimmunity in the peripheral nervous system (Guillain-Barr syndrome). Importantly, the ectopically expressed model antigen OVA appears to be sufficiently sequestered in ODC to obtain a state of immunological ignorance in CD4 T cells and B cells of unimmunized mice. Interestingly, this even holds true for double transgenic ODC-OVA/OT-II mice, in which the majority of all T cells are OVA-specific CD4 T cells. A similar observation has been made in f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

double transgenic mice expressing HA in astrocytes together with a class II-restricted HA-specific TCR [5]. In that case, however, even active immunization did not lead to an inflammation of the CNS or to neurological symptoms, whereas both could be induced in our ODCOVA mice. It is noteworthy that in both transgenic systems, CD8 T cells apparently have an easier access to the neo-self antigen. Thus, in the HA-GFAP system, double transgenic mice expressing a class I-restricted TCR die at an early age from acute enteritis due to destruction of the enteric nervous system [6]. Similarly, ODC-OVA/OT-I double transgenic mice develop an acute CD8-T cell mediated EAE, a phenomenon currently under investigation. These findings are in line with the constitutive presentation of these model antigens in the class I pathway, whereas cross presentation by class II-positive cells is required for recognition by CD4 T cells. Classical EAE models rely on immunization with brain antigens in adjuvant, a protocol preferentially inducing Th1 cells and antibodies. Indeed, either Th1 cells by themselves or both Th1 cells and antibodies have been shown to contribute to EAE development, dependent on the rodent strain and the antigen employed [2, 11]. As a possible alternative to CFA, which allows induction of mild EAE in about half of www.eji.de

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Figure 4. Weight changes in mice immunized with OVA in CFA. (A) ODC-OVA mice without EAE symptoms (n = 7), (B) ODC-OVA mice with EAE symptoms (n = 7) and (C) WT mice (n = 9).

Figure 5. Histopathology of EAE induced by OVA-immunization in ODC-OVA mice. ODC-OVA and WT mice were immunized with OVA/CFA. Brain (A) and spinal cord (B) of a transgenic mouse with grade 4 EAE display infiltration by inflammatory cells (arrows, A) and nervous tissue damage (arrow, B); Immunohistochemical staining shows CD3+ T cells (C) and macrophages (arrows, D) in the CNS of the transgenic mouse. f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Figure 6. Proliferative recall response and antibody production in CFA/OVA immunized transgenic and WT mice. (A) Lymph node cells from OVA transgenic mice and WT mice immunized with OVA in CFA 15 days earlier were stimulated for 3 days with OVA before pulsing with [3H]thymidine. (B) Serum levels of OVA-specific antibody in OVA-immunized OVA transgenic mice and WT mice were measured by ELISA at day 10 p.i.

ODC-OVA mice but only rarely supports its progression to a fulminant stage, we also tried to induce disease with OVA conjugated to CpG oligonucleotides, previously shown to induce massive Th1 and CTL responses [12, 13]. This conjugate was, however, unable to induce EAE in ODC-OVA mice (data not shown). Furthermore, we have attempted to increase the incidence and severity of EAE by depletion of CD25+ regulatory T cells in either single ODC-OVA or double transgenic OT-II/ODC-OVA mice. However, in spite of effective depletion, we did not observe an increased susceptibility. This is in contrast to EAE in MBP-specific class II-restricted TCR transgenic mice where CD4+CD25+ regulatory T cells effectively prevented disease [14]. It will be of interest to investigate whether other types of regulatory T cells suppress more fulminant disease development in our system. The reproducible induction of EAE in about half of ODC-OVA animals provides a solid basis for such mechanistic studies, as well as for experimental therapies. The well-characterized reagents available in www.eji.de

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the OVA system, which beyond the TCR transgenic OT-I and OT-II lines include mAb leading to demyelination, peptides suitable for stimulation and tetramer staining, and peptide variants allowing immunomodulation will facilitate these experiments and will make this model useful for further dissection of the mechanisms by which immune responses to CNS antigens lead to inflammation and permanent damage. One feature of particular interest for future studies is the potential to delete the "neo-autoantigen" by Cre-mediated deletion of the floxed transgene. This will allow distinguishing between chronic antigen-specific responses and "antigen-spread". Finally, both bacterial and viral vectors expressing OVA have been extensively characterized, making the ODC-OVA model suitable for the study of the importance of anti-microbial immune responses, in particular to antigens cross-reactive with brain antigens, in the induction of EAE and MS.

Materials and methods Generation of ODC-OVA transgenic mice To generate the pMBPOVA construct, the standard cloning vector pSP72 2.5 kb (Promega, GeneBank accession number X65332) was modified by inserting an additional NotI restriction site into the Bglll site with a 22 nucleotide long linker. Two directly repeated loxP sites were included to allow deletion of the transgene by Cre recombinase. Before inserting the second loxP site, the 1.8-kb OVA cDNA was released from pAc-NEO-OVA [15] and cloned into the EcoRI site of the vector. Prior to insertion of the 6.5-kb MBP promoter, an oligonucleotide linker containing KpnI and SalI restriction sites was cloned into the respective sites in pSP72LX/HOVA. Before removing the 6.5-kb MBP promoter from the pMBP clone #8 plasmid [7], a ClaI restriction site was destroyed by blunting (Klenow; MBI Fermentas, Heidelberg, Germany) before re-ligating with T4 ligase (Gibco-Invitrogen, Karlsruhe, Germany). The modified 6.5-kb MBP promoter was subsequently released with KpnI and SalI and ligated 50 upstream of the OVA cDNA into the respective sites in pSPOVAHKS, generating pMBPOVAHKS. The second loxP site contained within an oligonucleotide and supporting an AseI overhang was cloned together with the 0.68-kb polyA tail (ClaI/AseI) into ClaI/NotI sites in pMBPOVAHKS, resulting in pMBPOVA. The MBPOVA construct was released with NotI/XhoI from prokaryote sequences and eluted from a 0.8% TAE agarose gel using the Qiagen Quiaquick kit and a TE-based (pH7.5) injection buffer. B. Kanzler (Freiburg, Germany) kindly carried out microinjections of the linearized pMBPOVA construct into C57BL/6 oocytes. Genotyping of ODC-OVA transgenic mice Ova was detected by PCR in DNA from tails of ODC-OVA transgenic mice using the primers (50 TGAGGAFATGCCAGACAGAT) and (50 TTCCAGGATTCGGAGACAGT). PCR reagents f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Clinical immunology

were purchased from PAN Biotech GmbH (Aidenbach, Germany). After 35 cycles (30 s 94 C, 30 s 57 C, 90 s 72 C), the amplified fragment of 733 bp was detected on a 2% agarose gel containing ethidiumbromide. As positive controls, primers specific for mouse A1 were used. SDS-PAGE and Western blot for OVA expression About 500 mg brain, spinal cord or sciatic nerve tissues were removed and put in 1 mL of 1% Triton X-100 (10 mM sodium phosphate pH 7.5, 150 mM NaCl) containing a mixture of protease inhibitors (PMSF, phenanthroline and N-ethylmaleimide, 2 mM each) (Sigma), minced finely and homogenized with oscillation at 4 C for 24 h. After centrifugation for 10 min at 15 000  g in an Eppendorff centrifuge, the supernatants were taken and stored frozen (–20 C) until use. Total protein extracts were boiled in SDS-PAGE reducing buffer for 5 min and subsequently separated by SDS-PAGE and transferred to a polyvinylidene difluoride transfer membrane. The membrane was incubated in 0.1% Tween 20 in PBS containing 5% nonfat dry milk overnight at 4 C, incubated with rabbit polyclonal anti-OVA (Sigma) for 1 h at room temperature, washed with 0.1% Tween 20 in PBS, and incubated with peroxidase-conjugated anti-rabbit immunoglobulin (Sigma) for 1 hour at room temperature. After washing, membranes were incubated with the Western Blot Chemiluminescence Reagent (NEN Life Science Products, Boston, MA) for 1 min at room temperature. After draining excess detection reagent, the blots were exposed to X-ray film from 5 s to 5 min and then developed. Induction and evaluation of EAE Mice were injected s.c. at flanks and base of backs with 200 lg OVA whole protein (Sigma, Deisenhofen, Germany) in PBS emulsified in an equal volume of CFA containing Mycobacterium tuberculosis (Sigma) at a final concentration of 1 mg/mL. Two i.p. injections of 400 ng pertussis toxin (Sigma) were given 24 and 72 h later. Mice were weighed and scored for disease on a daily basis. Disease severity was assessed using a scale ranging from 0 to 5 [16, 17]: 1, limp tail; 2, partial hind leg paralysis; 3, total hind leg paralysis; 4, hind and front limb paralysis; 5, moribund or dead. All experiments were performed according to the Bavarian state regulations for animal experimentation and approved by the responsible authorities. Animals were kept at standard conditions with free access to food and water. Histology and immunohistochemistry Mice were anesthetized with pentobarbital and transcardially perfused with saline followed by 4% of paraformaldehyde. Spinal cord, brain, optic nerve, sciatic nerve, and other organs were removed and processed for routine paraffin embedding and histological staining. Immunohistochemistry was performed with 5-lm paraffin sections as described [18]. If necessary, antigen unmasking was achieved by heat pretreatment of sections for 30 min in 10 mM citric acid buffer (Mac-3) or 1 mM EDTA (CD3) in a microwave oven (850 W). After blocking of unspecific binding www.eji.de

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sites with 10% BSA, sections were incubated overnight at 4 C with the appropriate primary antibody in 1% BSA. Secondary antibodies were used as indicated below. After blocking of endogenous peroxidase with H2O2, the peroxidase-based ABC detection system (DAKO, Hamburg, Germany) was employed with DAB as the chromogenic substrate. Specificity of staining was confirmed by omitting the primary antibody as a negative control. T cells were labeled by rat anti-CD3 (Serotec, Wiesbaden, Germany; 1:300) and macrophages by rat antimouse Mac-3 (PharMingen; 1:200), each with a rabbit anti-rat secondary antibody (1:100, Vector via Linavis, Wertheim, Germany). Staining for OVA was done using rabbit anti-OVA antiserum (Sigma). Pancreas sections of transgenic RIP-mOVA mice expressing OVA in islet b cells [1] were used as positive control for OVA staining, and normal mouse spleen sections for CD3 and Mac-3. To study the localization of OVA in the CNS, cryostat sections of brains and spinal cords were examined for double labeling with rabbit anti-OVA (Sigma) and mouse anti-20 , 30 cyclic nucleotide 30 phosphodiesterase (CNPase, Sternberger Monclonals via Szabo Scandic, Vienna, Austria). Brain and spinal cord were removed and frozen in Tissue-Tek OCT. The cryostat sections were fixed with –20 C acetone for 20 min. The sections were treated with normal goat serum for 30 min and treated with Vector M.O. M. Immunodetection Kit (Vector Laboratories, Burlingame, USA). The thoroughly washed slides were sequentially treated with rabbit anti-OVA, goat-antirabbit-Cy2, mouse anti-CNPase, biotinylated anti-mouse IgG, and streptavidin-Cy3 for 1 h at room temperature. ELISA The 96-well ELISA plates were coated with 10 lg/mL OVA in carbonate coating buffer pH 9.6 for 12 h at 4 C. Plates were blocked for 2 h at 37 C with 0.2% gelatin and washed twice in PBST (PBS with 0.1% Tween 20). After washing, 100 lL of diluted sera (1:200, 1:400, 1:800, 1:1600) in PBS were added and incubated for 2 h at room temperature. Sera from naive WT mice were used as negative controls while mouse anti-OVA mAb was used as a positive control. After washing in PBST, the plates were incubated for 1 h at room temperature with alkaline phosphatase-conjugated goat anti-mouse IgG (cchain) and goat anti-mouse IgM (l-chain). The reaction product was visualized using p-nitrophenylphosphat in diethanolamin buffer and the absorbance read at 405 nm. Flow cytometry Single-cell suspensions of freshly isolated thymocytes and pooled LN cells were resuspended in PBS (0.1% BSA, 0.02% NaN3) at a concentration of 1  106cells/mL followed by incubation at 4 C for 20 min with 50 lL of properly diluted mAb. Four-color stainings were carried out with mAb either directly labeled with FITC, R-PE, allophycocyanin, or were biotinylated. For the latter, avidin-Cy-chrome (PE-Cy5) was used as a secondary reagent for detection. After staining, cells were washed twice with PBS/BSA/NaN3. Relative fluorescence intensities were then measured with FACScalibur (Becton Dickinson, Heidelberg, Germany) using CellQuestTM software (Becton Dickinson). f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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T cell proliferation A total of 2  105 LN cells/0.2 mL supplemented RPMI 1640/ 5% FCS were incubated at 37 C in triplicate in round-bottom 96-well tissue culture plates with various concentrations of indicated OVA (grade V, Sigma) or synthetic OVA peptides, 323–339 (ISQAVHAAHAEINEAGR) or 257–264 (SIINFEKL) for 72 h. In parallel, soluble anti-CD3e mAb (5 lg/mL) was added as a polyclonal stimulator to the cell culture. During the last 16 h of the culture [3H] thymidine (1 lCi/well) was added after which its incorporation into DNA of proliferating lymphocytes was measured using a Betaplate scintillation counter.

Acknowledgements: We thank Drs. B. Kanzler and T. Boehm for microinjection of our constructs into C57BL/ 6 oocytes, and Dr. C. Kurts for providing tissue samples and reagents for OVA-specific immune histology. This project was supported by DFG through SFB 581 and by the IZKF Wrzburg.

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