cells and lower percentage of isotype switched memory (CD19 1 CD27. 1 IgM-) B cells in controls compared to 22qdel patients (p 5 0.099, p 5 0.055, p 5 0.13).
Abstracts S93
J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 2
Cd161 Defines Functional Memory Subset Of Cd4 And Cd8 In Asthma Y. Gernez, K. Atkuri, N. T. Guyen, L. Herzenberg, R. Tirouvanziam, K. Nadeau; Stanford, school of Medicine, Stanford, CA. RATIONALE: Machura et al. (2008) showed that the CD8 1 CD45RO1 T memory cells were increased in blood from children with allergic asthma. They did not find any significant difference in the CD41 memory compartment. We (Gernez et al. JACI 2007) have shown that expression of the C-type lectin receptor CD161, which is an activation marker, can be used to discriminate between atopic and allergic patients. We compared the functional characteristics of CD4 and CD8 subsets in blood from allergic asthmatics and heatlhy controls by combining conventional memory markers (CD45RO and CD62L) with CD161. METHODS: We analyzed peripheral blood from human subjects (9 allergic asthmatics -AA- and 7 heathly controls -HC-) obtained under Stanford IRB-approved protocols using direct single-cell analysis by multiparametric flow cytometry, without any exogenous stimulation. RESULTS: Within the CD81 compartment, the percentage of CD45RO1 cells was increased in allergic asthmatics, as shown previously. We found that this increase is caused by an increase in both central (CD45RO 1 CD62L1; AA:381/-28 vs HC:8 6 8%; P 5 0.005) and effector (CD45RO 1 CD62L-; AA:24 6 13 vs HC:10 6 13%; P 5 0.03) memory subsets within the CD8 1 CD161- compartment, not the CD8 1 CD1611 compartment. In addition, the CD4 1 CD1611 activated central memory subset was significantly increased in allergic asthmatics (AA:49 6 10 vs HC:44 6 3%; P 5 0.012). No change was observed among CD4 1 CD1611 and CD4 1 CD1612 with regards to expression of CD45RO and CD62L memory markers in asthma patients. CONCLUSIONS: CD161 can be used as a subset marker to further refine the definition of memory subsets of CD4 and CD8 T cells associated with the course of allergic asthma.
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Semaphorin 4C: a Unique B-cell Molecule Expressed Following Th2 stimulation O. Hajoui1, J. Guay1, S. Al-Mot1, Q. Hamid1, B. Mazer2; 1Meakins Christie Laboratories, Montreal, QC, Canada, 2McGill University/Montreal Children’s Hosp., Montreal, QC, Canada. RATIONALE: Semaphorins provide crucial attractive and repulsive signals in axon guidance. Semaphorins and their ligand (plexins) have been detected immune cells but their role is unclear. Using Affymatrix Human gene arrays, we observed for the first time induction of SEMA4C in activated human B-lymphocytes. We have studied the regulation of SEMA4C mRNA and protein in human Tonsillar B cells. METHODS: Purified B cells were isolated from human tonsils. SEMA4C mRNA was assessed by RT-PCR. Protein expression was analyzed by Western blot and Flow cytometry using monoclonal anti-SEMA4C antibodies. Paraffin fixed sections from nasal polyps and tonsils were stained with anti-SEMA4C antibodies and anti-plexin B2. RESULTS: No SEMA4C mRNA was detected in unstimulated B cells; the combination of anti-CD40 plus IL-4 induced significant SEMA4C mRNA at 24 hours. CD40-IL4 increased CD191/SEMA4C1 B-cells significantly (4.72% control versus 23.29% stimulated B-cells). The synthesis of SEMA4C protein in B-cells required both CD40 and IL-4 or IL-13, as there was no detectable SEMA4C when B cells were cultured with anti-CD40 or cytokines alone. Addition of IFNy diminished SEMA4C mRNA and protein. In contrast, SEMA4D expression in B cells was strongly induced by CD40 stimulation alone. The presence of SEMA4C and it’s ligand plexinB2 was confirmed in paraffin sections of nasal polyp and tonsil tissue by immunohistochemistry. CONCLUSIONS: Semaphorin 4C is uniquely expressed by B cells stimulated by Th2 cytokines, suggesting that it may play a role in directing IgE producing cells towards Plexin-B2, a matrix protein. This suggests further study into models of cell trafficking via the Semaphorin-Plexin network.
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Immunophenotyping of B Cell Subsets in Infants and Adults with 22q11.2 Deletion Syndrome R. M. Zemble1, Y. Z. Du2, K. E. Sullivan3, N. T. Luning Prak2; 1Hospital of the University of Pennsylvania, Philadelphia, PA, 2University of Pennsylvania, Philadelphia, PA, 3Children’s Hospital of Philadelphia, Philadelphia, PA. RATIONALE: To evaluate the B-lymphocyte compartment in subjects with 22q11.2 deletion syndrome (22qdel), we performed immunophenotyping analysis on peripheral blood B-cell subsets. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from a cohort of patients with 22qdel and controls with ages ranging from 5 months to 48 years. PBMCs were stained with antibodies against CD5, CD19, CD20, CD24, CD27, CD38, IgD, and IgM and analyzed by flow cytometry. Immunophenotyping data analysis was performed on B-cell subsets in adults (15 y) and infants (3.5 y). Statistical analysis was performed using the Mann-Whitney test. RESULTS: In adults and infants with 22qdel compared to controls, there were no significant differences in total B-cell count or percentages of memory (CD19 1 CD271) or unswitched memory (CD19 1 CD27 1 IgM1) B cells. In infants there was a trend towards a higher percentage of naı¨ve (CD19 1 IgM 1 CD27-) and transitional (CD19 1 CD27-CD38bright) B cells and lower percentage of isotype switched memory (CD19 1 CD27 1 IgM-) B cells in controls compared to 22qdel patients (p 5 0.099, p 5 0.055, p 5 0.13). In adults there were no significant differences between controls and 22qdel patients in the percentages of isotype switched memory, transitional or naı¨ve B cells. CONCLUSIONS: In a limited cohort, there were no significant differences between 22qdel patients and control subjects in the total number of peripheral blood B cells or the composition of B-cell subsets. Adults with 22qdel had a lower percentage of transitional B cells, a higher percentage of unswitched memory B cells and trends towards higher percentages of isotype switched memory and total memory B cells compared to infants with 22qdel.
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Npr1 Deficiency Upregulates Regulatory T Cells (Tregs) Through Impaired Dendritic Cell (DC) Development J. Wang1, W. Zhang2, K. Li1, D. Chen2, G. Liu1, R. F. Lockey1, S. S. Mohapatra2; 1James A Haley Veteran’s Hospital, Tampa, FL, 2University of South Florida, Tampa, FL. RATIONALE: Natriuretic peptide receptor A (Npr1) is a membranebound guanylate cyclase expressed in many types of immune cells, including Tregs and DCs, suggesting that Npr1 may play a role in immune regulation. Npr1 knockout mice exhibit reduced lung inflammation, however the mechanism remains unclear. The hypothesis that Npr1 deficiency impairs the development of DCs resulting in more immature DCs, while promoting the production of Tregs was tested in this study. METHODS: Balb/C mice with Npr1 knockout (KO) and Foxp3-RFP (red fluorescent protein) knock-in were generated. Foxp3-positive Tregs were detected by flow cytometry of RFP expression. Bone marrow-derived DC cells (BMDCs) were generated from WT and Npr1 KO mice and used for adoptive transfer. Tregs were induced from naı¨ve CD4 T cells in the presence of DCs from WT and Npr1 KO mice. RESULTS: Npr1 KO mice had fewer CD4 and CD8 T cells than WT and heterozygotes. Also, Npr1 KO mice had significantly more Foxp3-positive Tregs, and more immature BMDCs than WT mice. Adoptive transfer of BMDCs from Npr1 KO mice into WT increased the number of Tregs both in vivo and in vitro co-culture with naı¨ve T cells. The levels of phospho-Akt and phospho-Stat3 (pAkt and pStat3) were higher in BMDCs from Npr1 KO mice. CONCLUSIONS: Increased Tregs and fewer mature DC cells in Npr1 KO mice may account for the decreased inflammation in these mice. The increased levels of pAkt and pStat3 in Npr1 KO mice may promote survival of immature DCs and Tregs.
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