Human insulin-dependent diabetes mellitus (IDDM) is thought to result from the autoimmune destruction of the insulin-production islet cells of the pancreas. The autoimmune hypothesis is supported by the association of the disease with autoantibodies specific for islet cell antigen (ICA) and human leukocyte antigen (HLA) alleles that convey susceptibility and resistance, and also by rodent models of IDDM that clearly show T cell-mediated autoimmune etiology. In the rodent models, CD4 and CD8 cells both seem to contribute to the destruction of islet cells. Presently, it is unclear whether the lessons learned from the rodent models also hold for the human disease. In particular, it has been difficult to obtain consistent data in support of the hypothesis that activated, ICA-specific T cells are present in patients, as opposed to healthy controls, whether these cells were primed in vivo to a Th1-type or Th2-type memory state, and whether they belong to the CD4 or CD8 category. The lack of this central information can be attributed to technical limitations in the measurement of T cell reactivity in freshly isolated cellular material, which are the primary goal of this application to overcome. The first limitation is the difficulty in detecting the low frequency antigen-reactive cells. The Investigator has overcome this limitation by developing a cytokine ELISPOT system that has single cell resolution such that it is able to detect the cytokine response of a single antigen-specific T cells within a million bystander cells. The second limitation is the failure of the present assays to detect CD8 cell responses. This application has two specific aims. 1) To modify the cytokine ELISPOT system in a manner that it can also detect CD8 responses to ICA, also at single cell resolution. The another limitation is the inability of standard assays to detect T cell responses to cryptic determinants on autoantigens, due to which the bulk of the autoreactive repertoire may be missed. 2) To use a modification of the ELISPOT assay that should detect responses to cryptic determinants on ICA as well. By further expanding the limits of T cell diagnostics, this proposal provides the tools and the initial measurements required to understand the very cells that mediate the disease and thereby foster new therapeutic controls.