The applicants' overall objective is to elucidate the role of T cells in the pathogenesis of RA and to identify new molecular approaches for the treatment of the disease. Significant evidence has been accumulated suggesting an important role for T lymphocytes in the pathogenesis of RA. The hypothesis to be tested in this research grant application is that T cells infiltrating the synovial membrane or extravasating into the synovial fluid are the result of clonal expansion of T lymphocytes undergoing activation and proliferation in response to a yet unknown set of (auto) antigens.
The specific aims of this proposal are: (1) To determine whether fresh (not expanded in culture) mononuclear cells infiltrating the synovial membrane or extravasating into the synovial fluid of patients with RA, contain substantial proportions of monoclonal T cells. To identify the V, D, and J gene segments employed by the alpha/beta or gamma/ delta T-cell antigen receptors (TCR) of these T cells, by amplifying the alpha-, beta-, gamma-, and delta-chain TCR cDNAs by the polymerase chain reaction (PCR), followed by cloning and sequencing; (2) To develop alpha/beta TCR+ or gamma/delta TCR+ T-cell clones from T cells infiltrating the synovial membrane or extravasating into the synovial fluid of patients with RA that are specific either for Mycobacterium tuberculosis antigens or type II collagen. To characterize these T cell clones and determine their reactivity to recombinant 65 kD M. tuberculosis antigen; (3) To identify the gene segments employed by the alpha/beta or gamma/delta TCR of these antigen-specific T cell clones, using amplification by PCR of the TCR cDNAs, followed by cloning and sequencing. To compare the sequences of the alpha/beta or gamma/delta TCR of these antigen-specific T cell clones to those of fresh T cells infiltrating the synovial membrane or extravasating into synovial fluid, in order to determine whether clonal populations of these infiltrating or extravasating cells have the same antigenic specificities with those of antigen-specific T cell clones; (4) in the event that the molecular analysis reveals that particular alpha/beta or gamma/delta TCR gene segments are primarily used by T cells infiltrating the synovial membrane or extravasating into the synovial fluid the investigators will develop anti-TCR monoclonal antibodies (mabs) specific for these segments, and the investigators will characterize the T cells recognized by these monoclonals. It is anticipated that these antibodies will be useful for treatment in vivo of patients with RA. These studies they hope will significantly improve our understanding of RA and will permit the development of new molecular approaches for the treatment of the disease.
