Project Summary Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by T cell destruction of insulin- producing pancreatic b cells. This results in lifelong dependence on exogenous insulin therapy and increases the risk of complications in many organ systems. Advances in understanding the mechanisms by which T cells mediate their autoimmune functions may provide new therapeutic targets for T1D. It is established that CD4 T cells and CD8 T cells are both required for the development of T1D. Studies found that the cytokine IL-21 functions as a critical signal produced by CD4 T cells to help CD8 T cells destroy insulin-producing b cells. However, the direct effects of IL-21 on β cell-reactive CD8 T cell effector function remain incompletely understood. Therefore, the long-term goal of this project is to elucidate molecular mechanisms regulating autoreactive CD8 T cell function. Preliminary scRNA-seq data presented in this proposal identified Pim1 as a possible modulator of activated diabetogenic CD8 T cell function. PIM1 belongs to a serine/threonine protein kinase family that functions downstream of the JAK-STAT pathway and is involved in regulating many processes including cell survival and metabolism. It was also demonstrated that IL-21 induced the expression of PIM1 and its downstream targets in CD8 T cells, while inhibition of PIM kinase in the presence of IL-21 resulted in reduced expression of PIM1 downstream targets. These data led to the hypothesis that IL-21-induced PIM1 kinase is necessary for supporting the sustained cytolytic function and metabolic demands of autoreactive CD8 T cells that are required for T1D development. Aim 1 will determine the role of PIM1 kinase in β cell-autoreactive CD8 T cell cytolytic function in T1D development. These experiments will determine if PIM1 is necessary and sufficient for T1D development. Additionally, the immunological phenotype of autoreactive CD8 T cells following inhibition and overexpression of PIM1 will be assessed. Aim 2 will elucidate the role of PIM1 kinase in b cell- autoreactive CD8 T cell signal transduction and metabolism in T1D. This project will be conducted at the Medical College of Wisconsin and Versiti Blood Research Institute with multidisciplinary support from experts in immunology and diabetes. Rigorous research and bioinformatic training in addition to presentations at national meetings and publication of manuscripts in immunology and T1D will support the development of an independent, academic physician-scientist. Overall, this proposal will advance knowledge of how b cell-autoreactive CD8 T cells function during the development of T1D. This is in line with the mission of NIDDK, since the results of this project may identify PIM1 as a novel therapeutic target for T1D.