The adaptive arm of the human immune system provides exquisite protection from pathogens, but the highly variable receptors can turn on self-tissues when immunoregulatory checkpoints are broken due to genetic risk and inflammatory events. A breakdown in tolerance impacting T cells is thought to be a critical immune checkpoint during the natural history of type 1 diabetes (T1D). Of the approximately 150 independent loci identified by genome-wide association studies as contributing to polygenic T1D risk, the major histocompatibility complex (MHC) remains the largest factor conferring risk due to its influence over thymic selection of the T cell receptor (TCR) repertoire and impact on peripheral T cell activation. Despite this critical link, little is known about how high-risk alleles like HLA-DR4 impact TCR selection, activation thresholds, and distribution across tissues. Our compelling preliminary data suggest that the risk-associated DR4/DQ8 haplotype, which has been linked with insulin autoreactivity, is hyper-expressed on the surface of antigen presenting cells (APCs) and associated with a unique TCR signature. Additional gene variants impacting T cell co-stimulation and differentiation are also enriched in subjects with T1D, yet their mechanistic contributions toward T1D autoimmunity remain poorly characterized. Project 3 proposes to address knowledge gaps governing these key aspects influencing the TCR repertoire and activation requirements in health and disease. We hypothesize that MHC risk, and additional non-MHC protein-coding risk variants, lead to aberrant T cell activation and differentiation thresholds and result in the loss of T cell tolerance in T1D. Specifically, T1D-associated risk variants alter the functional avidity of the MHC:peptide:TCR complex, and risk variants in the molecules SIRPG and CD226 alter T cell signaling, resulting in activation of autoreactive effector T cells as well as defective immunoregulation by regulatory T cells (Treg). These studies aim to investigate the mechanisms by which MHC class II (Aim 1) and non-MHC risk variants (Aim 2) control autoreactive T cells through the analysis of pancreatic draining lymph nodes (pLN), spleen, and peripheral blood from subjects with T1D and those at-risk for disease development (collected through Core A). We will leverage polygenic risk scoring and broad immunophenotyping data generated by Core B to direct case selection and analysis with state-of-the-art single-cell profiling and focused functional assays utilizing both genotype-selected and gene-edited samples. To investigate individual clones and risk alleles, we will modify primary human T cell specificity and function through lentiviral gene expression systems and CRISPR/Cas9 genome engineering for use in ex vivo pancreas slice culture and in vitro isogenic cellular modeling systems, in collaboration with Projects 1 & 2, respectively. Data from this Project are expected to inform on the T cell activation checkpoint...