The main goal is to identify the peptides bound to the Kd class I MHC molecule and which trigger diabetogenic CD8 T cells. The project is based on the development of CD8 T cell clones found in the islets of NOD mice. From one of the clones, O. Kanagawa isolated the genes encoding the T cell receptor, and produced a transgenic mouse line that develops diabetes. Also available to us are other diabetogenic CD8 clones, including those from a second transgenic mouse produced elsewhere. We present evidence that the insulinoma line NIT-1 can be recognized by the T cell clones and that this line increases it content of Kd by treatment with interferon-gamma. The project makes use of high-technology chemistry and biology, where the peptides bound to Kd of the NIT-1 line are extracted and purified by a sequence of steps. The initial step is the purification of the KD protein, which is then followed by peptide isolation and a first step fractionation on reverse phase HPLC. The isolated peptides are purified by tandem mass spectrometry in our Department of Chemistry under Gross. We plan to use capillary HPLC on line to an ion trap Finnigan LCQ mass spectrometer. peptides will be sequenced by tandem mass spectrometry with the use of an automated computer program (Sequest). We believe we can identify peptides represented by a few picomoles in the initial extracts of NIT-1 cells. We also discuss the various alternatives for both the biological, as well as the biochemical, assays. Both Gross and Unanue's laboratories have proven experienced in their areas of expertise and a proven track-record of interactions. Identification of one or more peptides responsible for triggering diabetogenic CD8 T cells should lead to an understanding of autoimmune diabetes and to an evaluation of the possibilities of peptide immunotherapy.
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