Project Summary Type 1 diabetes (T1D) is a chronic autoimmune disease that lead to the destruction of insulin producing β cells over a period of years before clinical presentation and afterwards. However, not all β cells are killed since follow- up studies of individuals with long standing T1D have identified residual insulin production even years after the onset of disease. The premise of this work, based on these and other observations, is that there are adaptive responses of the β cells to the immunologic attack that may prevent their destruction. We previously found that some β cells undergo “dedifferentiation”, express lower levels of β cell transcription factors, have reduced immunogenicity, and are protected from killing. The overall goal of this proposal is to identify adaptive changes in β cells and use this information to enhance their survival in the setting of immune attack. We found that there was increased expression of modifiers of the epigenome such as DNMT3a and Tet2 in human and murine β cells in vitro, during exposure to inflammatory cytokines, or in vivo during autoimmunity. We found increased expression of TET2 in β cells in islets from humans with autoimmune pancreatitis and T1D in nPOD samples. However, from our analysis of β cells during diabetes progression in NOD mice, those that resist autoimmune killing have decreased expression of Tet2. Tet2 induces hydroxymethylation of methylated CpG site which is the first step in converting a repressive to permissive epigenetic mark. We created Tet2-/- NOD mice and in adoptive transfer studies, bone marrow transplants and direct cultures with inflammatory cytokines and diabetogenic immune cells showed that deletion of Tet2 prevents autoimmune killing of β cells. Our data indicates that the Tet2-deficient β cells are not only resistant to immune killing but also modify the autoimmune responses. We hypothesize that Tet2 can modify β cells and affect their susceptibility to autoimmune killing and plan to test this hypothesis in murine model systems and human cells. We will analyze on a single cell basis the transcriptome and epigenome (by ATACseq) of β cells from WT and Tet2-/- mice. We will identify the DNA binding sequences of Tet2 in β cells. To specifically identify the role of Tet2 in β cells and determine the relationship between timing of Tet2 expression and susceptibility to killing we will create mice with tissue specific deletion of Tet2 and induce the deletion at times throughout the development of T1D. We will analyze the differences in immune cells in Tet2 sufficient and -/- mice. In the 2nd aim we will analyze TET2 expression and associated gene expression and epigenetic signatures in human samples from nPOD, patients with autoimmune pancreatitis, and control subjects. Finally, we will assess the role of TET2 expression in human embryonic stem cell-derived- β cells with and without TET2 expression in vitro and after transplantation into mice. These studies will determin...