Abstract Type 1 diabetes (T1D) results from immune-mediated destruction of islet beta cells within the pancreas, which leads to the inability of the body to produce insulin, dysregulation of glucose metabolism, and disabling and potentially life-threatening damage to organs throughout the body. Development of new methods for early T1D diagnosis and new clinical approaches to arrest the autoimmune response are limited by incomplete information on T1D-associated T cells and the pathways they use to target pancreatic islets. Clinical and genome-wide association studies (GWAS) reveal that specific MHC class II (MHC-II) genes provide the strongest genetic predisposition to T1D, along with other key T1D-associated single nucleotide polymorphisms (SNPs) including genes that influence T cell development, signaling, and function. How expression of particular MHC-II alleles predisposes T cell repertoires towards autoimmunity and why their heterozygous expression does not confer a similar predisposition remains unresolved. We propose to identify tissue- and disease- specific processes that underlie T1D susceptibility by comparing autoimmune T cell development and function in host that solely express T1D-associated MHC-II allele with their disease-protected heterozygous counterparts. We will focus on studies of a novel Non-Obese Diabetic mouse model we have generated in which the MHC-II polymorphic residues most closely associated with T1D susceptibility have been substituted with the most common variants, I-Ag7 β56P/57D (I-Ag7-PD) using CRISPR /Cas9 techniques. Studies of individual with and without T1D will be used to reveal commonalities between the mouse model and human disease. Our preliminary data suggests homozygous expression of I-Ag7 in NOD mice allows high affinity β-islet specific CD4 T cells to develop, which following their activation in the periphery, differentiate into effector populations that are required for pancreatic targeting and to induce the activation of pathogenic CD8 T cell clones. Heterozygous expression the T1D protective allele, I-Ag7 β56P/57D (I-Ag7-PD), however, turns this autoimmune circuit is turned off at least in part during T cell development. In NOD mice that co-express I-Ag7-PD and I-Ag7 (I-Ag7-PD/WT), significant negative selection to the I-Ag7-restricted T cell repertoire occurs, including the targeting of β-islet specific CD4 T cells. However, contrary to prevailing models, negative selection occurs despite I-Ag7-PD having an inability to present β-islet to CD4 T cells in vivo. Proposed experiments will determine thymic selection events that regulate the overall affinity of self- and/or foreign-reactive T cell repertoires. Studies of the mature T cell compartment will identify key events that allow T cell to acquire pathogenic potential, leading to the targeting pancreatic β-islets. These studies will provide important insights into the fine-tuning of T cell repertoires by thymic selection events and differentiation p...