Project Summary Type 1 diabetes (T1D) is a tissue-specific autoimmune disorder. Strong genetic association with the HLA class II gene locus suggests the importance of CD4 T cells in initiating and driving the disease process. We and others have isolated T cells from the pancreas of T1D organ donors and identified T cell epitopes specific to insulin, and its precursor preproinsulin, as well as fusion peptides. An important next step is to elucidate the phenotypes of such islet-derived T cells in health and disease. Concurrently, the previous studies uncovered the fact that antigens for the majority of T cells in the islets are still unknown, and we do not even know whether those T cells are islet antigen-specific. Identifying antigen specificity of islet-CD4 T cells is critical to dissect disease pathogenicity and heterogeneity to develop antigen-specific immunotherapy to halt disease progression using appropriate antigens in a given individual. Therefore, the goal of this grant is to identify antigen specificity of CD4 T cells in the islets and link antigen-specific responses to T cell phenotypes. Multiple lines of evidence suggest that there may be `common' or preferred CD4 T cell epitopes within subsets of T1D patients. First, the vast majority of T1D patients have risk HLA class II alleles, and specific class II alleles are associated with development of an initial islet autoantibody (e.g. insulin autoantibodies develop in those with HLA-DR4, and glutamic acid decarboxylase-65 antibodies with HLA-DR3). Second, CD4 T cells having the same antigen specificity (e.g. proinsulin peptides and hybrid insulin peptides) are detected in the pancreata from multiple T1D organ donors, and the same reactivity is found in the blood of those with early stages of T1D. Finally, CD4 T cells specific to these epitopes have an inflammatory phenotype in the blood of T1D patients compared to non-diabetic controls. This leads us to hypothesize that islet autoimmunity is promoted and regulated by CD4 T cells reacting to `common' antigens that are shared by T1D patients having specific HLA class II molecules. We will determine the proportion of tissue-specific CD4 T cells in the islets (Aim 1) and seek to identify epitopes targeted by CD4 T cells in the islets of T1D organ donors (Aim 2). Furthermore, we will determine the molecular phenotype of islet antigen-specific T cells in the pancreatic lymph nodes and spleen of organ donors with and without T1D (Aim 3). The successful completion of this proposal will: (1) identify antigens and epitopes targeted by CD4 T cells from pancreatic islets across the stages of T1D development and (2) elucidate the molecular phenotype and features that render these T cells pathogenic. Thus, these studies will enhance our understanding of human T1D pathogenesis, dissect disease heterogeneity, and aid in improving the design of clinical trials evaluating antigen-specific immunotherapy for diabetes prevention.