Type 1 diabetes is a T cell-mediated autoimmune disease. At present there is no effective therapy to prevent or cure this disease. Identification of prediabetic individuals by sensitive tests able to detect autoreactive T cells in blood is highly desirable. Also, there is an imperious need for the generation of new animal models to examine beta-cell recognition by human T cells, and testing immunotherapeutic strategies. MHC-Il/peptide molecules are the only physiological antigen-specific ligands for CD4 T cells. We demonstrated that soluble, dimeric MHC class II/peptide chimeras (DEF) prevent and reverse diabetes in mice through the immunomodulation of autoreactive CD4 T cells. Human DEF-like chimeras generated in the lab also immunomodulate human diabetes-related CD4 T cells. Our tetrameric DEF molecules can also be used to identify and characterize diabetes-related T cells in blood by FACS analysis. Our newly generated animal model for Type 1 diabetes (PBL-DR4), which develops insulitis by human T cells has a great potential. Our hypotheses are that "scoring diabetes-related CD4 T cells and determining their diabetogenicity in PBL-DR4 mouse are valuable strategies for the early diagnosis of Type 1 diabetes. Human DEF chimeras will down-regulate the diabetogenic function of human autoreactive T cells in vivo." The specific aims for the R21 phase are: 1. Identification and characterization human diabetogenic CD4 T cells in blood. 2. Immunocharacterization of PBL-DR4 mouse model. The specific aims for the R33 phase are: 1. Phenotypic and functional characterization of autoreactive T cells in blood of at-risk relatives 3. Evaluation of DEF antidiabetogenicity in PBL-DR4 mice. Human DEF molecules may provide valuable reagents for the early diagnosis of Type 1 diabetes, and at the same time a new antigen-specific approach for the prevention and treatment of this disease.