Insulin-dependent diabetes mellitus (IDDM) is one of the most common autoimmune diseases in humans. Environmental and genetic factors combine to bring about the onset the disease. The mechanism of initiation of T cell activation is still unknown, but a number of islet beta cell-expressed proteins have been shown to be targeted. Susceptibility to IDDM is most strongly determined by DQ/I-A beta MHC class II molecules which contain serine, alanine, or valine at position 57. We propose to address and characterize in depth, molecular aspects of the autoimmune process leading to beta cell destruction in the NOD mouse, the best characterized model of diabetes. Our approach is to produce IA-peptide complexes and their T cell receptors and carry out functional, structural, and in vivo experiments. We will express and purify the unique MHC class II molecule of the NOD mouse, I-Ag7 using a Drosophila melanogaster expression system with which we have had success with many other immunological molecules. The purified molecules will be used to characterize both peptide and invariant chain binding, and also for crystallization trials and x-ray structure determination. Two T cell receptors, BDC2.5 and R28, which are restricted on this molecules will also be expressed in a soluble form in the fly system. Both of these TCRs are pathogenic in transgenic mice and BDC2.5 causes insulitis and diabetes. The autoantigens for these 2 TCRs are still unknown. One of our aims is to determine which peptide(s) these 2 TCRs recognize in the context of I-Ag7. In vitro selection by phage display will be used to determine the natural ligands and to construct mimics, or mimotopes, of these ligands. Natural ligands and mimotopes in association with I-Ag7 will be used to measure the affinity of BDC2.5 and R28. We will attempt to crystallize and collaboratively determine the crystal structures these TCRs and TCR/p-MHC complexes. Soluble I- Ag7 peptide molecules will also be used to attempt to induce apoptosis and deletion of specific T cells in a TCR transgenic mouse and in a double transgenic diabetic mouse. Depending on the success of these experiments, treatment will be attempted in the NOD mouse for the prevention of islet graft rejection. This proposal should provide a great deal of functional and structural information about I-Ag7 molecules and their interactions with autoimmune TCRs.
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