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S.71. HLA-DRB1-Presented Peptides in Inflamed Synovium from Patients with Rheumatoid Arthritis or Antibiotic-Refractory Lyme Arthritis
S152 expression of CD25 and GITR was comparable between groups, expression of CD62L and CD69 was dramatically lower in SSc patients. Further, Tregs from SSc patients were unable to effectively inhibit the proliferation of CD4+effector T cells, a characteristic that correlated strongly with decreased CD69 and TGFβ expression. Co-incubation of Tregs from healthy donors with plasma from SSc patients fully abrogated suppressive activity. Activation of Tregs from healthy donors or SSc patients with PHA significantly upregulated CD69 expression that could be inhibited by SSc plasma. Filtration of SSc plasma samples revealed that the responsible factor for these phenomena is b10kD. Conclusion. These results indicate that soluble factors in SSc plasma inhibit Treg function specifically associated with altered Treg CD69 and TGFβ expression. These data suggest that aberrant function of Tregs may lead to immune dysfunction and autoimmunity in patients with SSc. doi:10.1016/j.clim.2009.03.447
S.71. HLA-DRB1-Presented Peptides in Inflamed Synovium from Patients with Rheumatoid Arthritis or Antibiotic-Refractory Lyme Arthritis Elise Drouin1, Robert Seward1, Catherine Costello2, Allen Steere1. 1Massachusetts General Hospital and Harvard Medical School, Charlestown, MA; 2Boston University, Boston, MA
Abstracts
S.73. Defective Retinoic Acid Expansion of Foxp3+Regulatory T Cells in Lupus Eric Sobel, Edward Butfiloski, Carla Cuda, Laurence Morel. University of Florida, Gainesville, FL Retinoic acid (RA) is the key metabolite from Vitamin A with pleiotropic effects on multiple tissues in developmental and maintenance processes. More recently, murine models have shown that RA plays a key role in maintaining the peripheral homeostasis between regulatory Foxp3+T cells (Treg) and inflammatory IL-17 producing T cells (Th17) in a TGFβ-dependent manner. A number of autoimmune diseases, including systemic lupus erythematosus, have been associated with impaired Treg number and/or function. Our work in the NZM2410 mouse model of lupus has shown an impaired amplification of the TGFβ-dependent induction of Treg. Here we first show that RA also amplified Tregs induced by TGFβ from human peripheral CD4+T cells stimulated with anti-CD3, anti-CD28 and IL-2. This suggests that the role of RA in T cell homeostasis uncovered in the mouse extends to humans. Furthermore, when Tregs were induced from peripheral CD4+T cells obtained from lupus patients, the addition of RA reduced rather than amplified the Treg pool. This result first validates the result that we have obtained in the NZM2410 model, and indicates that defects in the response of T cells to RA may contribute to the reduced Treg pool that has been reported in lupus patients. doi:10.1016/j.clim.2009.03.449
Autoimmunity is thought to play a pathogenic role in rheumatoid arthritis (RA) and antibiotic-refractory Lyme arthritis (LA). Susceptibility for both diseases is associated with specific HLA-DRB1 molecules that contain the “RA-shared epitope”; a sequence of amino acids predicted to confer peptide binding specificity. Although it has been proposed that these MHC class II molecules may trigger autoimmunity by presenting autoantigenic peptides to CD4+Tcells, the inflamed synovial HLA-DRB1-presented peptides of RA and LA patients have never been characterized. Using mass spectrometry, we analyzed synovial tissue samples from two RA and two LA patients; 1,427 peptides (220 to 464/patient) derived from 167 proteins were identified, the largest number of HLA-DRpresented peptides identified to date in individual samples from patients with a particular disease. Five epitopes identified were unique to the RA or the LA patients, suggesting they might be disease-specific. However, the largest number of shared epitopes (38) were found in the only two patients who shared the same DR allele, DRB1⁎0101. Most of these peptides were predicted to have a common binding register that could be utilized by both DRB1⁎0101 and 0401 (RA-shared epitope alleles). Ten peptides post-translationally modified by S-cysteinylation were identified in one RA patient. As a next step, patients' peptides will be synthesized and tested for reactivity with that patient's peripheral blood and synovial fluid T cells. The elucidation of HLA-DRB1-presented peptides in affected tissues, which could include potential autoantigens, provides an important tool for Tcell epitope discovery in autoimmune diseases. doi:10.1016/j.clim.2009.03.448
S.74. Gamma Delta Cells Recognizing the B:9-23 Peptide of NOD Mice Li Zhang1, Niyun Jin2, Jm Wands2, Maki Nakayama1, Willi Born2, George Eisenbarth1. 1University of Colorado Denver, Aurora, CO; 2National Jewish Health, Denver, CO Type 1 diabetes is an autoimmune disease mediated by multiple antigen specific T cells. Proinsulin/insulin is regarded as an important and perhaps primary auto-antigen. Many diabetogenic γδ T cells recognize an insulin-epitope represented by insulin2 B:9-23. The insulin recognizing γδ T cells in type 1 diabetes have not been studied. We generated insulin2 B:9-23 peptide recognizing γδ T cells from NOD mice. The original insulin2 B:9-23 recognizing γδ Tcell hybridoma, named SP9D11, was generated from splenocytes of BDC12-4.1 TCRα+Cα-/-NOD mice and utilized γ4 chain pairing with a new delta chain encoded with TRAV4-2/DV gene. A TCR αβ chains loss variant hybridoma cell line that was transduced with the SP9D11 TCR preserved responsiveness to insulin peptides. The SP9D11 γδ T cells in vitro also recognized multiple truncated and single amino acid mutated insulin 2B:9-23 peptides and islets of NOD mice without addition of antigen-presenting cells. Furthermore, multiple γδ T cells including γ1, γ2 and γ4 cells paired with different δ chains were generated from BDC12-4.4 TCR-α+Cα-/-NOD mice, and responded to insulin 2B:9-23 peptide. As important, up to 4.6% CD3 positive NOD islet infiltrating cells were γδ Tcells and multiple insulin2 B:923 recognizing γδ T cells were generated from pancreatic lymph node cells of NOD mice. In conclusion, multi-clonal γδ T
S.71. HLA-DRB1-Presented Peptides in Inflamed Synovium from Patients with Rheumatoid Arthritis or Antibiotic-Refractory Lyme Arthritis Elise Drouin1, Robert Seward1, Catherine Costello2, Allen Steere1. 1Massachusetts General Hospital and Harvard Medical School, Charlestown, MA; 2Boston University, Boston, MA
Abstracts
S.73. Defective Retinoic Acid Expansion of Foxp3+Regulatory T Cells in Lupus Eric Sobel, Edward Butfiloski, Carla Cuda, Laurence Morel. University of Florida, Gainesville, FL Retinoic acid (RA) is the key metabolite from Vitamin A with pleiotropic effects on multiple tissues in developmental and maintenance processes. More recently, murine models have shown that RA plays a key role in maintaining the peripheral homeostasis between regulatory Foxp3+T cells (Treg) and inflammatory IL-17 producing T cells (Th17) in a TGFβ-dependent manner. A number of autoimmune diseases, including systemic lupus erythematosus, have been associated with impaired Treg number and/or function. Our work in the NZM2410 mouse model of lupus has shown an impaired amplification of the TGFβ-dependent induction of Treg. Here we first show that RA also amplified Tregs induced by TGFβ from human peripheral CD4+T cells stimulated with anti-CD3, anti-CD28 and IL-2. This suggests that the role of RA in T cell homeostasis uncovered in the mouse extends to humans. Furthermore, when Tregs were induced from peripheral CD4+T cells obtained from lupus patients, the addition of RA reduced rather than amplified the Treg pool. This result first validates the result that we have obtained in the NZM2410 model, and indicates that defects in the response of T cells to RA may contribute to the reduced Treg pool that has been reported in lupus patients. doi:10.1016/j.clim.2009.03.449
Autoimmunity is thought to play a pathogenic role in rheumatoid arthritis (RA) and antibiotic-refractory Lyme arthritis (LA). Susceptibility for both diseases is associated with specific HLA-DRB1 molecules that contain the “RA-shared epitope”; a sequence of amino acids predicted to confer peptide binding specificity. Although it has been proposed that these MHC class II molecules may trigger autoimmunity by presenting autoantigenic peptides to CD4+Tcells, the inflamed synovial HLA-DRB1-presented peptides of RA and LA patients have never been characterized. Using mass spectrometry, we analyzed synovial tissue samples from two RA and two LA patients; 1,427 peptides (220 to 464/patient) derived from 167 proteins were identified, the largest number of HLA-DRpresented peptides identified to date in individual samples from patients with a particular disease. Five epitopes identified were unique to the RA or the LA patients, suggesting they might be disease-specific. However, the largest number of shared epitopes (38) were found in the only two patients who shared the same DR allele, DRB1⁎0101. Most of these peptides were predicted to have a common binding register that could be utilized by both DRB1⁎0101 and 0401 (RA-shared epitope alleles). Ten peptides post-translationally modified by S-cysteinylation were identified in one RA patient. As a next step, patients' peptides will be synthesized and tested for reactivity with that patient's peripheral blood and synovial fluid T cells. The elucidation of HLA-DRB1-presented peptides in affected tissues, which could include potential autoantigens, provides an important tool for Tcell epitope discovery in autoimmune diseases. doi:10.1016/j.clim.2009.03.448
S.74. Gamma Delta Cells Recognizing the B:9-23 Peptide of NOD Mice Li Zhang1, Niyun Jin2, Jm Wands2, Maki Nakayama1, Willi Born2, George Eisenbarth1. 1University of Colorado Denver, Aurora, CO; 2National Jewish Health, Denver, CO Type 1 diabetes is an autoimmune disease mediated by multiple antigen specific T cells. Proinsulin/insulin is regarded as an important and perhaps primary auto-antigen. Many diabetogenic γδ T cells recognize an insulin-epitope represented by insulin2 B:9-23. The insulin recognizing γδ T cells in type 1 diabetes have not been studied. We generated insulin2 B:9-23 peptide recognizing γδ T cells from NOD mice. The original insulin2 B:9-23 recognizing γδ Tcell hybridoma, named SP9D11, was generated from splenocytes of BDC12-4.1 TCRα+Cα-/-NOD mice and utilized γ4 chain pairing with a new delta chain encoded with TRAV4-2/DV gene. A TCR αβ chains loss variant hybridoma cell line that was transduced with the SP9D11 TCR preserved responsiveness to insulin peptides. The SP9D11 γδ T cells in vitro also recognized multiple truncated and single amino acid mutated insulin 2B:9-23 peptides and islets of NOD mice without addition of antigen-presenting cells. Furthermore, multiple γδ T cells including γ1, γ2 and γ4 cells paired with different δ chains were generated from BDC12-4.4 TCR-α+Cα-/-NOD mice, and responded to insulin 2B:9-23 peptide. As important, up to 4.6% CD3 positive NOD islet infiltrating cells were γδ Tcells and multiple insulin2 B:923 recognizing γδ T cells were generated from pancreatic lymph node cells of NOD mice. In conclusion, multi-clonal γδ T