Islet autoantigen specific T cells can be found in both T1D patients and healthy individuals.  However, these T cells with the same antigen specificity are expected to differ in their phenotypic characteristics. We will investigate CD4 and CD8 T cells in nPOD samples from T1D patients, prediabetic and healthy subjects allowing us to compare T cell specificities and phenotypes at different stages of the disease. We have generated tetramers representing the most common HLA-DR types associated with T1D risk (DR401, 404 and 405, 301, DQ8) or protection (DR1501, DRB5 0101 and DQ 0602) containing immunodominant peptides from the major diabetes autoantigens GAD65, Proinsulin/insulin, IA2 and Zn-T8.  The approach is based on the utilization of tetramers to identify and isolate autoreactive T cells from pancreatic lymph node tissue, peripheral blood (and possibly bone marrow) offers an advantage that phenotypic analyses are performed on T cells responding to a single, known antigen mediated with a defined MHC restriction.  Tetramer-based technology also allows determination of the functional phenotype of autoantigen specific T cells, for example memory, avidity/affinity, cytokine profiles, homing properties, and these cells can be sorted for gene expression studies and TcR analysis. In the cases when both peripheral blood lymphocyte and pancreatic lymph node samples are available the proposed study would provide valuable information on the predictive value of blood samples as biomarker in the evaluation of the disease process.  Collaboration with other nPOD investigators allows our project to generate new knowledge on the key features and the population dynamics of CD4+ T cells directly associated with islet autoimmunity and beta-cell biology in human diabetes.