ABSTRACT. Type 1 diabetes (T1D) results from complex interactions between over 150 independent loci imparting disease susceptibility and environmental factors that break immune tolerance, leading to the immune- mediated destruction of insulin-producing pancreatic -cells. Among these exists a small number of coding variants, which may represent rational therapeutic targets to restore immune tolerance, yet the cellular and molecular mechanisms by which risk variants alter immune function remain poorly characterized. The CD226 candidate gene contains a protein coding variant (rs763361) linked to multiple autoimmune disorders, including T1D. CD226 functions as a costimulatory molecule that competes with the negative regulators, TIGIT and CD96, for binding to CD155 or CD112 expressed on antigen presenting cells (APCs). Our published and preliminary data, supported by the initial phase of this R01, suggest that CD226 signaling destabilizes the regulatory T cell (Treg) phenotype. Specifically, genetic deletion of Cd226 attenuated disease development in the non-obese diabetic (NOD) mouse model of T1D, both in genomic knockout (gKO) and Treg-specific conditional KO (cKO) lines, with reduced ex-Treg frequency in the pancreatic lymph nodes (pLN) of gKO animals. Moreover, CD226– human Tregs display increased purity, stability, and suppressive function. Single-cell transcriptional profiling and flow cytometric analysis of tissue-resident T cells isolated from T1D organ donor pancreas and pLN identified an imbalance of CD226 and TIGIT expression on CD8+ T cells. We hypothesize that the T1D-associated risk variant in CD226 results in immune checkpoint dysregulation that leads to defects in peripheral immune tolerance, specifically resulting in Treg instability prior to T1D onset. To test this, we propose three Specific Aims. 1) We will perform single cell multi-omic profiling and adoptive transfer studies to identify the cellular and molecular basis by which CD226 contributes to defective immune tolerance in the NOD mouse, using our Cd226 gKO and Treg cKO strains. 2) We will assess the expression profile and functional impact of the CD226 risk variant using banked organ donor tissues and human peripheral blood samples derived from individuals with and at risk for T1D. These efforts will involve in situ profiling of genotype-selected pancreas and pLN samples via spatial transcriptomics and high-content imaging, along with CRISPR/cas9 gene-editing of CD226 in primary cells with T cell receptor (TCR) gene transfer to generate isogenic, autoreactive Treg, CD4+, and CD8+ T cells for in vitro functional studies. 3) We will test candidate therapeutics targeting the CD226 costimulatory axis in vivo using NOD mice and in vitro using human cells. Hence, the proposed studies will employ novel animal strains along with gene editing and TCR gene transfer techniques in human lymphocytes, which we pioneered over the prior grant term, to inform on the contributions of the CD2...