We are interested in how the class II MHC I-Ag7 interacts with peptides during antigen processing. An understanding of the chemistry and biology of this set of molecules becomes important for explaining immunological diabetes. The I-Ag7 molecules are essential for the development for the development of diabetes in NOD mice: such molecules show some unique properties both chemical and biological. Our proposal is based on the premise that to understand the chemistry of peptide interactions with I-Ag7 we need to examine the peptides that it prefers to select from APC. We want to identify the sets of peptides by chemically extracting them from I-Ag7 molecules of APC. These peptides will be purified, sequenced and quantitated by mass spectrometry analysis. Once these peptides are identified, we will study their binding properties using synthetic peptides that have the same sequence as the naturally processed peptides-this will lead us to determine their affinities for I-Ag7 and binding kinetics; and to establish the residues that allows them to bind. Our plans includes examining cell lines that express I-Ag7, two of which will also express a known protein antigen [hen egg white lysozyme and glutamic acid decarboxylase]. Besides informing us of the peptides that are selected, the HEL line will be important in that we will be able to evaluate and manipulate the immune response to the selected peptides. Thus, we include here biological experiments that examine the T cell response to the peptides of HEL. We also plan an evaluation of how effective is T cell tolerance to this antigen by examining transgenic mice that express different amounts of the HEL protein. The importance of the GAD line relates to the relevance of this auto-antigen in diabetogenesis. We plan an evaluation of an insulinoma line that bears I-Ag7 and which presents peptides to several diabetogenic T cells. Thus our purpose is to carry out a definitive biochemical examination that will allow us to place the class II diabetogenic molecule in their proper biological context. From these experiments we should be able to determine the features of peptides that APC prefer to select during processing to proteins, including peptides relevant to diabetogenesis.
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