The goal of this project is to further our understanding of the CD4 T cells and CD8 T cells that infiltrate the islets in autoantibody+ donors at risk for development of type 1 diabetes (T1D) and from donors with recent onset T1D. We will hand pick islets from digests of live pancreas slices from the two cohorts of donors, supplied by nPOD and examine them in two ways. First, we will culture single islets in conditions that favor T cell outgrowth from islets: this will add to the unique bank of islet-derived T cell lines we have generated. These T cell lines can be propagated in culture and used to test reactivities against specific epitopes, both conventional and neoepitopes, for both CD4 and CD8 T cells; these tests will also reveal the islet-infiltrating T cells HLA restriction and effector phenotype. Second, hand-picked islets will be analyzed for their transcriptome by sc-RNA-Seq with TCR sequencing; this method captures more in depth phenotypic and functional information about islet-infiltrating T cell. The TCR sequencing will allow for the construction of TCR transductants which will be used to examine reactivity to conventional and neoepitope and for HLA restriction. In addition to furthering our knowledge concerning the T cells that infiltrate the site of pathology, the islets, early in the disease process, information generated here could be used in constructing antigen-specific Tregs or used in tolerance induction protocols.
