The applicant seeks a VA Career Development Award-2 (CDA-2) to further our understanding of the pathogenesis of β-cell loss in type 2 diabetes (T2D) and support his career development at the Veteran’s Affairs Puget Sound Health Care System (VAPSHCS). His expertise in cell and molecular biology, islet inflammation, and islet amyloid deposition, along with his strong mentors (Steven Kahn and Jane Buckner), collaborators (Andrew Oberst and Matthias von Herrath), Advisory Committee (Rebecca Hull, Jerry Palmer, Alvin Powers), and location at the VAPSHCS, make him uniquely qualified to execute the proposed studies. With the support of this award, he will investigate novel and underappreciated aspects of β-cell death in T2D, interact with internationally-recognized diabetologists and immunologists, and undertake didactic and laboratory-based immunology training. These activities will allow the candidate to author additional high impact publications, gain research independence, and position himself to successfully compete for a VA Merit Review Award. The scientific goals of this proposal are to define the roles of necroptosis (a novel form of cell death) and T cells in islet amyloid-induced β-cell loss in order to develop new treatments for individuals afflicted by T2D. Islet amyloid is a pathologic hallmark of T2D, where it elicits islet inflammation and β-cell death. Investigation of the mechanisms of amyloid-induced β-cell death have focused largely on apoptosis signaling. Preliminary data in this application show for the first time that a recently described alternative form of cell death signaling called necroptosis is activated by islet amyloid formation. These data also show that components of this cell death pathway, including receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL), are present in β cells. A central characteristic of necroptosis is its ability to elicit immune responses to cellular antigens that are released by this form of lytic cell death. Consistent with this process occurring in response to islet amyloid, preliminary data show that amyloid-laden islets exhibit significantly higher T cell abundance and mRNA expression. Based on these findings and the literature, I hypothesize that islet amyloid-induced TNFα production by macrophages leads to RIPK3-mediated β-cell necroptosis, which results in T lymphocyte recruitment to amyloid-laden islets and exacerbation of amyloid-induced β-cell dysfunction and death. To test this hypothesis, the following specific aims are proposed: Specific Aim 1: Determine the role of RIPK3 signaling in amyloid-induced β-cell death, β-cell dysfunction, and islet immune responses. The contribution of RIPK3 signaling to islet amyloid-induced β-cell death will be determined in vitro using 1) a RIPK3 inhibitor, and 2) islets from Ripk3-/- mice crossed with amyloid-prone human IAPP (hIAPP) transgenic mice. These Ripk3-/- x hIAPP mice will also be used to deter...