The specific aims of this project are to identify peptides derived from a known autoantigen in insulin-dependent diabetes mellitus, glutamic acid decarboxylase (GAD65), that are bound to the diabetes-related MHC proteins HLA-DR3, HLA-DR4, HLA-DQ2, and HLA-DQ8 and presented to T cells. The experimental model will be class II-null (AbetaO) mice into which one or two HLA transgenes, the non-obese diabetic (NOD) mouse background genes, and/or the CD1 knockout have been introduced. Initially, the double transgenic HLA-DR3, HLA-DQ8 mouse AbetaO/DR3, DQ8) and the same mouse into which the background genes of the NOD mouse have been introduced (AbetaO/DR3, DQ8/NOD) will be used. The AbetaO/DR3, DQ8 mouse spontaneously develops insulitis accompanied by autoreactivity to GAD65. In order to identify the peptides these mice will be immunized with GAD65, following which HLA-DR3 and HLA-DQ8 will be isolated from regional lymph nodes. A newly developed very rapid microscale procedure for isolating HLA proteins will be employed. The peptides extracted from the HLA proteins will be analyzed in the Harvard Microchemistry Facility using several techniques. T cell hybridomas from the immunized transgenic animals will be prepared and used for specific detection of the GAD65 peptides. Any peptides identified will be tested for their immunogenicity to the transgenic mice and can also be used to examine at-risk diabetic subjects prospectively for the development of T cell reactivity to these peptides. These studies may help both in understanding the dual roles of HLA-DR and HLA-DQ proteins in the genesis of this disease, and in defining peptides that might be useful in induction of tolerance in at-risk subjects.
