BADERC- David Hafler

David Hafler, M.D.

Determinants of T Cell Autoreactivity in Type 1 Diabetes

We will determine what factors prevent pre-diabetic individuals from developing diabetes. This project will investigate the regulation of IL-4 in a subset of T cells which are implicated in the progression of human type 1 diabetes (IDDM). In human disease, incomplete concordance of disease progression between identical twins both with autoreactive T cells and autoantibodies suggests that other factors contribute to the incidence and progression of IDDM. We have recently described a subset of regulatory V24JaQ T cells that are reduced in frequency and are Th1-like (lacking IL-4 secretion) in diabetic twins as compared to non-diabetic twins. In addition, the non- or slow-progressors to disease had high levels of the cytokines IL-4 and gFN in their circulation; this may reflect a more Th0- or Th2-like phenotype for these individuals. We have set up collaborations with laboratories investigating T cell signaling events to address the question, what signaling events lead to the loss of IL-4 secretion in the invariant Vo RTK polypeptides, or in the case of the preassembled IR ((zH), rather than on the crude, unidentified PKC (a)S657(>E kinase, which may or may not be (>PKCa24JaQ T cells in individuals with diabetes. Expression of the nuclear factors, GATA-3, c-maf, NIP45, and NF-AT species, that have been identified as IL-4 transcription factors, will be quantitated by northern blot under conditions of activation and conditions of exogenous factors to bias cells toward a Th1 or Th2 phenotype. We will also transfect c-maf and GATA-3 into non IL-4 secreting Va24JaQ T cells to determine if there is a dominant negative signal preventing IL-4 secretion. Secondly, we will determine what proximal signal transduction elements are activated in the Va24JaQ T cells. We will examine phosphorylation of STAT 6 on tyr-641, which correlates with its DNA binding activity in addition to examining STAT6 DNA binding activity on EMSAs. In addition, as there are defects in Ca²⁺ flux in Va24JaQ T cells from diabetics that do not secrete IL-4, we will survey early TCR associated signaling events. Besides examining targeted pathways, we will use other, more broader approaches, to isolate target genes which may regulate IL-4 expression in V24JaQ T cells from subjects with diabetes. We will use subtraction methods coupled with PCR (RDA), and DNA microchip array hybridization. Lastly, we will examine whether the cytokine secretion phenotype of Va24JaQ T cells from the diabetics can be altered to a Th0/Th2 phenotype using the data generated from the first three aims. The eventual goal of the project is to develop a therapy that can be administered to pre-diabetics that prevent their progression into diabetics.

References:

1. Wilson, S.B., Kent, S.C., Patton, K.T., Orban, T., Jackson, R.A., Exley, M., Porcelli, S., Atkinson, M.A., Balk, S.P., Strominger, J.L., Hafler, D.A. Extreme Th1 bias of regulatory Va24JaQ T cells in type 1 diabetes. 1998; Nature 391:177-181.

2. Milner. J. D., Kent, S. C., Ashley, T. A., Wilson, S. B., Strominger, J. L. and D. A. Hafler. 1999. Differential responses of invariant Va24JaQ T cells and MHC class II restricted CD4+ T cells to dexamethasone. Journal of Immunology. 163: 2522-2529.

3. Kent, S. C., Hafler, D. A., Strominger, J. L. and S. B. Wilson. 1999. Noncanonical Va24JaQ T cells with conservative a chain CDR3 region amino acid substitutions are restricted by CD1d. Human Immunology. 60: 1080-1089.

4. Wilson, S. B., Kent, S. C., Horton, H., Bollyky, P. L., Hafler, D. A., Strominger, J. L. and M. Byrne. 2000. Transcriptional profiling of Va24JaQ T cells uncovers multiple differences between IL-4 secreting and IL-4-null clones. Proceedings of the National Academy of Sciences USA 97:7411-7416.

5. Viglietta, V., Kent S. C., Orban, T., and D. A. Hafler. 2002. GAD65-reactive T cells are activated in patients with autoimmune type 1a diabetes. Journal of Clinical Investigation 109: 895-903.

6. Kent, S.C., Clemmings, S., Merritt, D., Viglietta, V., Kenyon, N., Ricordi, C., Teyton, L., Hering, B., and D. A. Hafler. NKT cells from human Type 1a diabetic pancreatic draining lymph nodes (PLN) have a loss of IL-4 cytokine secretion. Submitted, 2003.

7. Lim DG, Slavik JM, Bourcier K, Smith KJ, Hafler DA. Allelic variation of MHC structure alters peptide ligands to induce atypical partial agonistic CD8+ T cell function. J Exp Med. 2003;198:99-109.

8. Viglietta V, Baecher-Allan C, Weiner HL, Hafler DA. Loss of Functional Suppression by CD4+CD25+ Regulatory T Cells in Patients with Multiple Sclerosis. J Exp Med. 2004;199:971-9.






			David Hafler, M.D. Determinants of T Cell Autoreactivity in Type 1 Diabetes We will determine what factors prevent pre-diabetic individuals from developing diabetes. This project will investigate the regulation of IL-4 in a subset of T cells which are implicated in the progression of human type 1 diabetes (IDDM). In human disease, incomplete concordance of disease progression between identical twins both with autoreactive T cells and autoantibodies suggests that other factors contribute to the incidence and progression of IDDM. We have recently described a subset of regulatory V24JaQ T cells that are reduced in frequency and are Th1-like (lacking IL-4 secretion) in diabetic twins as compared to non-diabetic twins. In addition, the non- or slow-progressors to disease had high levels of the cytokines IL-4 and gFN in their circulation; this may reflect a more Th0- or Th2-like phenotype for these individuals. We have set up collaborations with laboratories investigating T cell signaling events to address the question, what signaling events lead to the loss of IL-4 secretion in the invariant Vo RTK polypeptides, or in the case of the preassembled IR ((zH), rather than on the crude, unidentified PKC (a)S657(>E kinase, which may or may not be (>PKCa24JaQ T cells in individuals with diabetes. Expression of the nuclear factors, GATA-3, c-maf, NIP45, and NF-AT species, that have been identified as IL-4 transcription factors, will be quantitated by northern blot under conditions of activation and conditions of exogenous factors to bias cells toward a Th1 or Th2 phenotype. We will also transfect c-maf and GATA-3 into non IL-4 secreting Va24JaQ T cells to determine if there is a dominant negative signal preventing IL-4 secretion. Secondly, we will determine what proximal signal transduction elements are activated in the Va24JaQ T cells. We will examine phosphorylation of STAT 6 on tyr-641, which correlates with its DNA binding activity in addition to examining STAT6 DNA binding activity on EMSAs. In addition, as there are defects in Ca²⁺ flux in Va24JaQ T cells from diabetics that do not secrete IL-4, we will survey early TCR associated signaling events. Besides examining targeted pathways, we will use other, more broader approaches, to isolate target genes which may regulate IL-4 expression in V24JaQ T cells from subjects with diabetes. We will use subtraction methods coupled with PCR (RDA), and DNA microchip array hybridization. Lastly, we will examine whether the cytokine secretion phenotype of Va24JaQ T cells from the diabetics can be altered to a Th0/Th2 phenotype using the data generated from the first three aims. The eventual goal of the project is to develop a therapy that can be administered to pre-diabetics that prevent their progression into diabetics. References: 1. Wilson, S.B., Kent, S.C., Patton, K.T., Orban, T., Jackson, R.A., Exley, M., Porcelli, S., Atkinson, M.A., Balk, S.P., Strominger, J.L., Hafler, D.A. Extreme Th1 bias of regulatory Va24JaQ T cells in type 1 diabetes. 1998; Nature 391:177-181. 2. Milner. J. D., Kent, S. C., Ashley, T. A., Wilson, S. B., Strominger, J. L. and D. A. Hafler. 1999. Differential responses of invariant Va24JaQ T cells and MHC class II restricted CD4+ T cells to dexamethasone. Journal of Immunology. 163: 2522-2529. 3. Kent, S. C., Hafler, D. A., Strominger, J. L. and S. B. Wilson. 1999. Noncanonical Va24JaQ T cells with conservative a chain CDR3 region amino acid substitutions are restricted by CD1d. Human Immunology. 60: 1080-1089. 4. Wilson, S. B., Kent, S. C., Horton, H., Bollyky, P. L., Hafler, D. A., Strominger, J. L. and M. Byrne. 2000. Transcriptional profiling of Va24JaQ T cells uncovers multiple differences between IL-4 secreting and IL-4-null clones. Proceedings of the National Academy of Sciences USA 97:7411-7416. 5. Viglietta, V., Kent S. C., Orban, T., and D. A. Hafler. 2002. GAD65-reactive T cells are activated in patients with autoimmune type 1a diabetes. Journal of Clinical Investigation 109: 895-903. 6. Kent, S.C., Clemmings, S., Merritt, D., Viglietta, V., Kenyon, N., Ricordi, C., Teyton, L., Hering, B., and D. A. Hafler. NKT cells from human Type 1a diabetic pancreatic draining lymph nodes (PLN) have a loss of IL-4 cytokine secretion. Submitted, 2003. 7. Lim DG, Slavik JM, Bourcier K, Smith KJ, Hafler DA. Allelic variation of MHC structure alters peptide ligands to induce atypical partial agonistic CD8+ T cell function. J Exp Med. 2003;198:99-109. 8. Viglietta V, Baecher-Allan C, Weiner HL, Hafler DA. Loss of Functional Suppression by CD4+CD25+ Regulatory T Cells in Patients with Multiple Sclerosis. J Exp Med. 2004;199:971-9.