We have recently demonstrated that enzymatically modified peptides derived from beta cell antigens are preferentially recognized by autoreactive CD4+ T cell clones isolated from the peripheral blood of patients with T1D. Furthermore, T cells that recognize these modified peptides are present in the peripheral blood of patients with T1D and have a high affinity, enabling their detection by flow cytometry. Importantly, the expression and activity of enzymes responsible for these modifications are upregulated in response to cellular stress and inflammation. Therefore, modified peptides are likely to form in stressed islets and to elicit pathogenic T cell responses. Consistent with this notion, T cells that recognize two or three of these modified peptides were shown to be present in islets and pancreatic lymph nodes (PLN) of two organ donors with T1D. The goal of this project is to corroborate and extend our study of T cell responses to modified beta cell antigens are relevant to the pathogenesis of T1D by identifying and studying T cells that recognize these peptides within PLN from nPOD donors. We will identify and isolate these T cells using HLA class II tetramers. We will then characterize these cells by examining their cytokine profiles and sequencing their T cell receptors. Finally, we will compare these attributes with those of T cells with the same peptide specificity that are found in peripheral blood to define unique attributes of T cells that are most relevant to disease.