The majority of human T cell antigens relevant to T1D have been defined using algorithms which predict high affinity peptide binding to MHC. However, direct evidence showing that these antigens are presented to T cells by human islets and APCs in T1D patients has not been established for the vast majority of these peptides. A major goal of our project is to identify T cell peptide antigens relevant to T1D which meet the following criteria: 1) the peptides are naturally processed and presented by human islets/APCs and 2) there are T cells specific for these antigens in the PLN and/or islets of T1D patients. To achieve this goal, we are eluting peptides from class I MHC on HLA-A2+ human islets and identifying them by mass spectrometry to define naturally presented antigens. We are also verifying natural antigen presentation by using antigen-specific T cell lines as “biosensors” of antigen presentation on islets and APCs. Islet-specific peptides that are shown to be naturally presented will be used to make MHC-peptide tetramers and analyzed by CyTOF technology to measure the frequency, phenotype, and function of T cells specific for these antigens in T1D pancreatic draining lymph nodes obtained from the nPOD tissue bank. By verifying the natural antigen presentation of islet-derived peptides and the presence of activated T cells specific for these peptides in T1D patients, we will be able to differentiate between peptide antigens with bonafide relevance to T1D from islet peptides that are irrelevant to T1D. CyTOF analysis of the frequency, phenotype, and function of pathogenic T cells specific for disease-relevant islet antigens in T1D patients should be informative with respect this disease, potentially enabling sophisticated and early diagnoses and the ability to assess the efficacy of therapeutic intervention in human patients.