Project Summary The goal of the proposed research is to understand the immunologic mechanisms by which specific major histocompatibility complex (MHC) class II molecules provide resistance to develop autoimmune type 1 diabetes (T1D) by evaluating insulin specific T cell responses, particularly focused on insulin B chain amino acids 9-23 (B:9-23). For many autoimmune disorders, including T1D, specific human leukocyte antigen (HLA) alleles confer disease risk as HLA-DQ8 and/or DQ2 are present in approximately 90% of all T1D patients. Strikingly, the polymorphic HLA-DQ6 (DQB*06:02) allele provides dominant protection from T1D. The objective of the proposed studies is to bridge the gap of knowledge regarding MHC class II disease resistance and self- antigen T cell response in T1D. Our long-term goal is to understand how insulin specific T cell responses can be manipulated for preventative and therapeutic purposes, ideally using mutated insulin B:9-23 as antigen specific therapy. The central hypothesis is that the MHC class II genotype shapes autoimmunity to insulin B:9- 23, determining susceptibility or protection from T1D. We plan to test our hypothesis using mutated insulin B:9- 23 peptides to determine T cell phenotypes in the context of MHC class II genotypes in murine and human diabetes. In specific aim 1, we will utilize a spontaneous mouse model of autoimmune diabetes including a model with a single amino acid mutation in the MHC class II beta chain (β57Ser→Asp) implicated in diabetes resistance. Insulin B:9-23 specific T cells restricted to risk and protective MHC class II molecules will be tracked using fluorescent tetramers. Experiments will be done to test the hypothesis that a protective MHC class II molecule leads to the generation of B:9-23 regulatory T cells (Tregs) that suppress islet autoimmunity; either through converting a pathogenic T cell to become regulatory and/or by differentiating B:9-23 specific Tregs restricted to the protective MHC class II molecule. In the second aim, we will study insulin and insulin B:9-23 specific T cell responses from well-characterized human subjects at risk for developing T1D and subjects without islet autoantibodies in a prospective longitudinal study. We now have the ability to measure both inflammatory and regulatory T cell responses to insulin with mutated B:9-23 peptides from the peripheral blood, which will allow for an increased understanding of disease pathogenesis in preclinical T1D. Further, healthy controls having the protective DQB*06:02 allele will be studied to isolate, characterize and T cell receptor (TCR) sequence B:9-23-specific Tregs, thereby creating reagents to assess the structural interactions of a diabetes resistant trimolecular complex, TCR-B:9-23-DQ6. A B:9-23/DQ6 tetramer will be produced to stain and phenotype ex vivo T cells from DQ6 individuals. The findings from this research are directly applicable to the design of insulin antigen specific therapies, monitoring and ti...