This proposal combines immunological and biochemical investigations of antigen presentation in the autoimmune diabetes of the NOD mouse. The main goal is to identify the spectrum of peptides from islet beta cells that are selected by the class II MHC molecule of the NOD mouse, I-A g7 (referred to as g7), the biochemical features of these selected peptides and the library of CD4 T cells reactive with them. We combine mass spectrometry identification, and analysis, of peptides obtained from new cell lines that express beta cell genes as well as the g7 molecule.(The lines are fusions of beta cells or insulinomas with presenting cells; or insulinomas transfected with the genes encoding the g7 molecule.) These cell lines grow well, express abundant g7 molecules and strongly stimulate several T cell clones an indication that they express beta cell genes. We plan to search for the g7-bound peptides that stimulate a number of T cell clones, and will also be doing the reverse, that is with the proteins and peptides at hand, search for T cells reactive to them in the diabetic mouse. We also plan to search for T cells to established beta cell antigens, The peptides will be studied for their biochemical features (length, anchoring residues, binding parameters), source and abundance on the presenting cell. These parameters will be correlated with the diabetogenic potential of the T cell clones, i.e. their capacity to induce diabetes in NOD.SCID under different experimental manipulations. We are keen in comparing T cells that cause diabetes on their own - and which may be the primary cells that drive the process - to others that may not be pathogenic. And to establish the reasons for these differences having the information on the chemistry of the g7-bound peptides. Our goal is not to catalogue peptides and T cells but rather to establish strong biochemical and quantitative parameters that supersede the many empiricisms of antigen presentation. Eventually the expectation is to determine how narrow or broad is the library of autoreactive T cells, the chemical parameters that determine the selection of strong or weak peptides and how these influence the pathogenic potential of a CD4 T cell.
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