The emphasis in the current application is to identify TSHr structures to which TSH and autoantibodies with different biological effects bind. TSHr-derived peptides and polyclonal anti-peptide antibodies will be used to identify regions of the TSHr to which TSH and autoantibodies bind and exert their effects. A panel of monoclonal antibodies will be generated which contains stimulatory, growth- promoting and TSH-blocking antibodies. These monoclonal antibodies together with the full-length recombinant TSHr extracellular domain, peptides and mutant cDNA transfected cells, will be used to map epitopes on TSHr to which they bind. Functionally characterized monoclonal antibodies will be used in competitive inhibition studies to determine the binding specificities of Ig from patients with Graves' disease. Together these studies are expected to lead to a better understanding of the relationship of TSHr structure to its function. Systematic studies to characterize T cell responses will be carried out using in vitro cell proliferation assays with the extracellular domain of the TSHr protein as antigen. T cell subsets responding to TSHr will be determined by enriching for CD4+ and CD8+ T cells by selected depletion. Using TSHr-specific T cell lines and overlapping peptides that span the entire extracellular domain, T cell epitopes will be mapped. Next, to establish the relevance of these T- cell peptides to the disease, the sequence of naturally processed peptides in patients will be determined.
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