Our project aims to define islet-specific T cell receptor (TCR) sequence repertoires directly from pancreas to pursue two goals; 1) to identify antigens targeted by T cells contributing to the development of type 1 diabetes (T1D); and 2) to pursue the potential of TCR sequences to be used as biomarker.   We hypothesize that common antigens targeted by T cells negatively (Treg) and positively (Tpath) contributing to the development of T1D in multiple patients are present.  Identification of such antigens and TCR repertoires will lead us to develop antigen-specific immunotherapy and immunodiagnostic tools.  We directly sequence TCR alpha and beta chains of T cells in the pancreas as well as in different T cell subsets in the immune organs and peripheral blood of patients having T1D utilizing the high-throughput Illumina and 454 sequencing technologies.  TCRs detected in the pancreas are expressed on TCR-null T cell hybridoma cells using the retroviral expression system and are tested for the response to known antigens as well as the islet cells.  Once TCR pairs responding to the islet cells are determined, we will further assess pathogenicity in inducing diabetes by generating retrogenic animals.  In addition, presence and frequency of pancreas-derived TCR sequences in the peripheral organs of the same patients as well as those having the same HLA alleles will be evaluated to “trace” T cells detected in the pancreas.  Given evidence that a large number of public TCR sequences detected from multiple individuals are present, we believe that TCRs associated with the development of T1D exist and are able to be used for diagnosis in combination with other T cell assays (e.g. tetramer analysis).  Common antigens associated with the pathogenesis and regulation of T1D development will gain our insights to develop antigen-specific immunotherapy for T1D.