The overarching goals of this project are to elucidate the roles of TCR??? pairing and thymic selection in creating the T cell repertoire specific for foreign antigens, and to identify how failures in this process lead to Type-1 Diabetes. We have identified TCRs with a variety of peptide-MHC (pMHC) reactivity patterns, some of which are self-tolerant and pMHC specific while others are overtly self-reactive and pMHC cross-reactive. Our preliminary studies show that this spectrum of TCR reactivity patterns occurs because particular TCR V gene residues can bind pMHC using a variety of different binding modes. The first set of experiments of this proposal will uncover how TCRs are created with different pMHC binding modes, and determine if a subset of pMHC binding modes are intrinsically self-reactive. The second set of experiments in this proposal will test the model that central tolerance functions to limit mature T cells from expressing TCRs with an enhanced pMHC cross-reactive phenotype, and that self-reactive T cells that also have enhanced pMHC cross-reactivity are the subset of lymphocytes which trigger the autoimmune cascade leading to Type-1 Diabetes. We will determine whether defects in negative selection in autoimmune susceptible NOD mice allow a subset of self-reactive T cells to develop that express TCRs with enhanced pMHC cross-reactivity. We predict cross-reactive T cells are the most difficult T cell subset to control throuh peripheral tolerance mechanism, and it is these T cells which become activated and trigger the autoimmune cascade causing T1D. These experiments proposal will identify molecular mechanisms that control the specificity of TCRs, and identify how defects in immune regulation allow self-reactive T cells to trigger Type 1 Diabetes.