PROJECT SUMMARY Type I Diabetes (T1D) is a T cell mediated autoimmune which is dramatically increasing in incidence. The pathogenesis of T1D is complex and involves the infiltration of β cell-specific CD8 T cells into the pancreas and progressive destruction of insulin-producing b cells, resulting in loss of glucose homeostasis. Utilizing the clinically relevant Non-Obese Diabetic (NOD) mouse model of T1D, the Schietinger lab recently discovered a β cell-specific stem-like CD8 T cell population in the pancreatic lymph node (pLN) which self-renews and gives rise to differentiated progeny that migrate to the pancreas and destroy β cells; pancreatic T cells are short-lived and stem-like T cells in the pLN must continuously seed the pancreas to sustain β cell destruction. In this application I aim to understand the underlying molecular and cellular mechanisms that determine autoimmune T cell differentiation. In aim 1, I will determine how and where stem-T cells self-renew and differentiate, employing imaging cytometry and spatial transcriptomics technologies. In aim 2, I will define the functional role of transcription factors associated with T cell stemness through gain and loss of function approaches. Finally, in aim 3, I will ask whether perturbation of pLN intranodal positioning or inhibition of stem-T cell associated signaling molecules disrupt autoimmune T cell differentiation and prevent T1D onset. These proposed studies are designed to obtain a deep mechanistic understanding of the spatiotemporal and molecular factors that determine autoimmune T cell differentiation which could lead to novel therapeutic targets for T1D and other T cell-mediated autoimmune diseases.