PROJECT SUMMARY Type 1 diabetes (T1D) is caused by autoimmune destruction of insulin-producing pancreatic beta cells. While significant progress has been made in the past decades, our understanding of T1D pathogenesis remains far from complete. Nonobese diabetic (NOD) mice develop spontaneous T1D and have been used extensively to obtain mechanistic insight of this autoimmune disease. Pathogenic CD4 T cells promote T1D by secreting inflammatory cytokines, enhancing the immunogenic functions of antigen presenting cells (APCs), and help cytotoxic CD8 T cells that directly kill beta cells through the recognition of peptide-MHC I complexes. Autoreactive T cells and APCs are controlled by FOXP3+ regulatory CD4 T cells (Tregs), and defects associated with Tregs have been reported in T1D patients. Recently, a diabetogenic role of CD4 T cell-derived interleukin (IL)-21 has emerged. IL-21 promotes T1D development in part through its ability to support the survival and function of diabetogenic CD8 T cells. Interestingly, NOD islet-infiltrating IL-21+ CD4 T cells have a unique phenotype distinct from those in lymphoid tissues. Importantly, NOD mice genetically rendered IL-21- deficient are completely resistant to diabetes. Significantly higher frequencies of circulating IL-21+ CD4 T cells have been found in subjects with beta-cell autoimmunity than in healthy controls, consistent with a role of IL-21 in promoting T1D development in humans. Collective results from both mouse and human studies strongly indicate a central role of IL-21+ beta-cell autoreactive CD4 T cells in T1D pathogenesis. Both genetic susceptibility and environmental factors contribute to T1D development. The PTPN22 R620W variant is one of the top genetic risk factors that are associated with T1D development in humans. PTPN22 has been shown to regulate cells within the innate and adaptive immune systems, but additional effort is needed to identify its T1D-related function. NOD mice deficient in PTPN22 or carrying the 619W knock-in allele (orthologous to human 620W) developed accelerated autoimmune diabetes compared to the wildtype control, consistent with its role in T1D. Our preliminary results showed that Ptpn22-/- CD4 T cells were more diabetogenic than the wildtype counterpart. We further demonstrated by flow cytometry and paired single-cell RNA sequencing (scRNA-seq) and scTCR-seq that CD4 T cells were more prone to be IL-21+ effectors than Tregs in islets of NOD.Ptpn22-/- mice, supporting a role of PTPN22 in their fate decision at the site of autoimmune destruction. The overall hypothesis is that PTPN22 regulates T1D by controlling the balance of IL-21+ CD4 T cells and FOXP3+ Tregs. We will determine if the balance of IL-21+ CD4 T cells and FOXP3+ Tregs is altered by the Ptpn22 619W risk allele and mechanistically identify how PTPN22 controls the differentiation and accumulation of IL-21+ CD4 T cells and FOXP3+ Tregs. By completing the proposed experiments, we will be able to provide i...