BADERC- David Hafler

David Hafler, M.D.

Function of T cells, B cells, monocytes and genetic polymorphisms in human Type 1 diabetes

My research group in human Type 1 diabetes has focused on autoantigenic recognition by T and B cell and their cellular functions, the function of pro-inflammatory monocytes, and the description and function of genetic polymorphisms expressed by immune cells. The goal of these investigations is to understand and manipulate the factors influencing the autoimmune responses in human Type 1 diabetes.

A central question in T cell mediated autoimmune diseases is the identification of the cognate self-target(s) recognized by T lymphocytes of pathogenic function. With access to PLN and spleen from Type 1 diabetic subjects (www.jdrfnpod.org and other collaborators), we found a high degree of T cell clonal expansion was observed in long-term diabetic PLNs and not in control subjects. The oligoclonally expanded T cells from the two DR4 expressing diabetic subjects recognized insulin A1-15 epitope restricted by the diabetes susceptibility allele, DRB1*0401. These are the first results to identify insulin reactive, clonally expanded T cells
from the site of auto-inflammatory drainage in human T1D. We are continuing this line of research by examining a panel of these difficult-to-obtain tissue samples from controls, autoantibody-positive, but without clinical disease subjects, and T1D subjects. The purpose of these studies is to identify, in the course of development of human T1D, the autoantigens that are recognized and with what HLA restrictions. We also are interested in the development of autoantigen-specific memory T cells in the periphery of those at-risk for Type 1 diabetes development.

The role of autoreactive B cells in antigen recognition, antigen-driven B cell expansion, costimulation and cytokine and chemokine secretion in the autoimmune response is not known in the development of T1D in humans. We propose to study, on a single cell basis, human PLN B cells and B cells from the periphery of T1D subjects bearing functional markers for reactivity with cognate self-target(s) by the newly developed micro-engraving technology that allows for the multiplexed detection of secreted products from cells in correlation with cell surface lineage markers and BCR sequence. Application of the method to a clinical sample from a recent onset Type 1 diabetic subject with a positive titer of anti-insulin antibodies showed that ~0.58% of circulating CD19+ B cells secreted proinsulin-reactive antibodies of the IgG isotype and 2-3% of circulating cells secreted IL-6. These data demonstrate the utility of microengraving for interrogating multiple phenotypes of single human cells concurrently and for detecting rare populations of cells by their secreted products.

The derivation of inflammatory T cells in Type 1 diabetes has not been determined. We have investigated the activation state of monocytes in patients with T1D, hypothesizing that in vivo activation of pro-inflammatory, circulating monocytes were driving the differentiation/expansion of CD4 cells into Th17/Th1 cells. We found a striking activation of a subset of CD16- monocytes isolated ex vivo from patients with T1D that both spontaneously secreted and expressed mRNA transcripts for IL-1β/IL-6. These in vivo activated monocytes from T1D subjects induced IL-17/IFN-γ secreting cells from central memory T cells as compared to monocytes from healthy control subjects and this in vitro induction was inhibited by a combination of an IL-6 blocking antibody and IL-1 receptor antagonist. These data suggest a mechanism by which an activated, pro-inflammatory innate immune system drives theexpansion of Th17 cells in patients with T1D through spontaneous secretion of IL-6 and IL-1β. We will investigate the pathways activated in these monocytes that lead to their proinflammatory phenotype.

References:

Wilson, S. B., Kent, S. C., Patton, K. T., Orban, T., Jackson, R. A., Exley. M.,Porcelli, S., Schatz, D. A., Atkinson, M. A., Balk, S. P., Strominger, J. L. and D. A. Hafler.1998. Extreme Th1 bias of invariant Vα24JαQ T cells in Type 1 Diabetes. Nature. 391:177-181.
Semana, G., Gausling, R., Jackson, R. A., and D. A. Hafler. 1999. T cell autoreactivity to proinsulin epitopes in diabetic patients and healthy subjects. Journal of Autoimmunity. 12:259-67.
Milner. J. D., Kent, S. C., Ashley, T. A., Wilson, S. B., Strominger, J. L. and D. A. Hafler. 1999. Differential responses of invariant Vα24JαQ T cells and MHC class II restricted CD4+ T cells to dexamethasone. Journal of Immunology. 163: 2522-2529.
Kent, S. C., Hafler, D. A., Strominger, J. L. and S. B. Wilson. 1999. Noncanonical Vα24JαQ T cells with conservative α chain CDR3 region amino acid substitutions are restricted by CD1d.Human Immunology. 60: 1080-1089.
Wilson, S. B., Kent, S. C., Horton, H., Bollyky, P. L., Hafler, D. A., Strominger, J. L. and M.Byrne. 2000. Transcriptional profiling of Vα24JαQ T cells uncovers multiple differences between IL-4 secreting and IL-4-null clones. 2000. Proceedings of the National Academy of Sciences USA 97:7411-7416.
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.
Kent, S. C., Chen, Y., Bregoli, L., Clemmings, S. M., Kenyon, N. S., Ricordi, C., Hering, B. J.,and D. A. Hafler. 2005. Expanded T cells from pancreatic lymph nodes of type 1 diabetic recognize an insulin epitope. Nature 435: 224-228.
Kent, S.C., Chen, Y., Clemmings, S. M., Viglietta, V., Kenyon, N., Ricordi, C., Hering, B. J., and D. A. Hafler. 2005. Loss of IL-4 secretion from human Type 1a diabetic pancreatic draining lymph node NKT cells. Journal of Immunology 175: 4458-4464.
Smith, R. N., Kent, S. C., Nagle, J., Selig, M., Iafrate, A. J., Najafian, N., Hafler, D. A.,Auchincloss, H., Orban, T., and E. Cagliero. 2008. Pathology of an islet transplant 2 years after transplantation: evidence for a non-immunological loss. Transplantation 86: 54-62.
Bradshaw. E. M., Kent, S.C., Tripuraneni, V., Orban, T., Ploegh, H. L., Hafler, D. A., and J. C. Love. 2008. Concurrent detection of secreted products from human lymphocytes by microengraving: cytokines and antigen-reactive antibodies. Clinical Immunology 129:10-18.
Maier, L. M., Lowe, C. E., Cooper, J., Downes, K., Anderson, D. E., Severson, C., Clark, P. M.,Healy, B., Walker, N., Aubin, C., Oksenberg, J. R., Hauser, S. L., Compston, A., Sawcer, S.,International Multiple Sclerosis Genetics Consortium, De Jager, P. L., Wicker, L. S., Todd, J.A., and D. A. Hafler. 2009. IL2RA genetic heterogeneity in multiple sclerosis and type 1 diabetes susceptibility and soluble interleukin-2 receptor production. PLoS Genet.5(1):e1000322. Epub 2009 Jan 2.
International Multiple Sclerosis Genetics Consortium (IMSGC). 2009. The expanding genetic overlap between multiple sclerosis and type I diabetes. Genes and Immunity. 10:11-4.






			David Hafler, M.D. Function of T cells, B cells, monocytes and genetic polymorphisms in human Type 1 diabetes My research group in human Type 1 diabetes has focused on autoantigenic recognition by T and B cell and their cellular functions, the function of pro-inflammatory monocytes, and the description and function of genetic polymorphisms expressed by immune cells. The goal of these investigations is to understand and manipulate the factors influencing the autoimmune responses in human Type 1 diabetes. A central question in T cell mediated autoimmune diseases is the identification of the cognate self-target(s) recognized by T lymphocytes of pathogenic function. With access to PLN and spleen from Type 1 diabetic subjects (www.jdrfnpod.org and other collaborators), we found a high degree of T cell clonal expansion was observed in long-term diabetic PLNs and not in control subjects. The oligoclonally expanded T cells from the two DR4 expressing diabetic subjects recognized insulin A1-15 epitope restricted by the diabetes susceptibility allele, DRB10401. These are the first results to identify insulin reactive, clonally expanded T cells from the site of auto-inflammatory drainage in human T1D. We are continuing this line of research by examining a panel of these difficult-to-obtain tissue samples from controls, autoantibody-positive, but without clinical disease subjects, and T1D subjects. The purpose of these studies is to identify, in the course of development of human T1D, the autoantigens that are recognized and with what HLA restrictions. We also are interested in the development of autoantigen-specific memory T cells in the periphery of those at-risk for Type 1 diabetes development. The role of autoreactive B cells in antigen recognition, antigen-driven B cell expansion, costimulation and cytokine and chemokine secretion in the autoimmune response is not known in the development of T1D in humans. We propose to study, on a single cell basis, human PLN B cells and B cells from the periphery of T1D subjects bearing functional markers for reactivity with cognate self-target(s) by the newly developed micro-engraving technology that allows for the multiplexed detection of secreted products from cells in correlation with cell surface lineage markers and BCR sequence. Application of the method to a clinical sample from a recent onset Type 1 diabetic subject with a positive titer of anti-insulin antibodies showed that ~0.58% of circulating CD19+ B cells secreted proinsulin-reactive antibodies of the IgG isotype and 2-3% of circulating cells secreted IL-6. These data demonstrate the utility of microengraving for interrogating multiple phenotypes of single human cells concurrently and for detecting rare populations of cells by their secreted products. The derivation of inflammatory T cells in Type 1 diabetes has not been determined. We have investigated the activation state of monocytes in patients with T1D, hypothesizing that in vivo activation of pro-inflammatory, circulating monocytes were driving the differentiation/expansion of CD4 cells into Th17/Th1 cells. We found a striking activation of a subset of CD16- monocytes isolated ex vivo from patients with T1D that both spontaneously secreted and expressed mRNA transcripts for IL-1β/IL-6. These in vivo activated monocytes from T1D subjects induced IL-17/IFN-γ secreting cells from central memory T cells as compared to monocytes from healthy control subjects and this in vitro induction was inhibited by a combination of an IL-6 blocking antibody and IL-1 receptor antagonist. These data suggest a mechanism by which an activated, pro-inflammatory innate immune system drives theexpansion of Th17 cells in patients with T1D through spontaneous secretion of IL-6 and IL-1β. We will investigate the pathways activated in these monocytes that lead to their proinflammatory phenotype. References:* Wilson, S. B., Kent, S. C., Patton, K. T., Orban, T., Jackson, R. A., Exley. M.,Porcelli, S., Schatz, D. A., Atkinson, M. A., Balk, S. P., Strominger, J. L. and D. A. Hafler.1998. Extreme Th1 bias of invariant Vα24JαQ T cells in Type 1 Diabetes. Nature. 391:177-181. Semana, G., Gausling, R., Jackson, R. A., and D. A. Hafler. 1999. T cell autoreactivity to proinsulin epitopes in diabetic patients and healthy subjects. Journal of Autoimmunity. 12:259-67. Milner. J. D., Kent, S. C., Ashley, T. A., Wilson, S. B., Strominger, J. L. and D. A. Hafler. 1999. Differential responses of invariant Vα24JαQ T cells and MHC class II restricted CD4+ T cells to dexamethasone. Journal of Immunology. 163: 2522-2529. Kent, S. C., Hafler, D. A., Strominger, J. L. and S. B. Wilson. 1999. Noncanonical Vα24JαQ T cells with conservative α chain CDR3 region amino acid substitutions are restricted by CD1d.Human Immunology. 60: 1080-1089. Wilson, S. B., Kent, S. C., Horton, H., Bollyky, P. L., Hafler, D. A., Strominger, J. L. and M.Byrne. 2000. Transcriptional profiling of Vα24JαQ T cells uncovers multiple differences between IL-4 secreting and IL-4-null clones. 2000. Proceedings of the National Academy of Sciences USA 97:7411-7416. 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. Kent, S. C., Chen, Y., Bregoli, L., Clemmings, S. M., Kenyon, N. S., Ricordi, C., Hering, B. J.,and D. A. Hafler. 2005. Expanded T cells from pancreatic lymph nodes of type 1 diabetic recognize an insulin epitope. Nature 435: 224-228. Kent, S.C., Chen, Y., Clemmings, S. M., Viglietta, V., Kenyon, N., Ricordi, C., Hering, B. J., and D. A. Hafler. 2005. Loss of IL-4 secretion from human Type 1a diabetic pancreatic draining lymph node NKT cells. Journal of Immunology 175: 4458-4464. Smith, R. N., Kent, S. C., Nagle, J., Selig, M., Iafrate, A. J., Najafian, N., Hafler, D. A.,Auchincloss, H., Orban, T., and E. Cagliero. 2008. Pathology of an islet transplant 2 years after transplantation: evidence for a non-immunological loss. Transplantation 86: 54-62. Bradshaw. E. M., Kent, S.C., Tripuraneni, V., Orban, T., Ploegh, H. L., Hafler, D. A., and J. C. Love. 2008. Concurrent detection of secreted products from human lymphocytes by microengraving: cytokines and antigen-reactive antibodies. Clinical Immunology 129:10-18. Maier, L. M., Lowe, C. E., Cooper, J., Downes, K., Anderson, D. E., Severson, C., Clark, P. M.,Healy, B., Walker, N., Aubin, C., Oksenberg, J. R., Hauser, S. L., Compston, A., Sawcer, S.,International Multiple Sclerosis Genetics Consortium, De Jager, P. L., Wicker, L. S., Todd, J.A., and D. A. Hafler. 2009. IL2RA genetic heterogeneity in multiple sclerosis and type 1 diabetes susceptibility and soluble interleukin-2 receptor production. PLoS Genet.5(1):e1000322. Epub 2009 Jan 2. International Multiple Sclerosis Genetics Consortium (IMSGC). 2009. The expanding genetic overlap between multiple sclerosis and type I diabetes. Genes and Immunity. 10:11-4.