NEW ASSAY FOR TCR DIVERSITY IN TMJ RHEUMATOID ARTHRITIS

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The activated T-cells appear to be a central element in the pathogenesis of temporomandibular joint (TMJ) rheumatoid arthritis (RA). The mechanisms responsible for the activation of the T cells are not known. If the T cells are activated according to conventional mechanisms, then specific immunogen(s) should be recognized through the T-cell antigen receptor (TCR). Neither the immunogen(s), nor the TCR's involved have been defined at the molecular level. We hypothesize that a predominant form of TCR variable sequence can be found among the in vivo activated TMJ T lymphocytes. The primary objective of this proposal is to analyze the diversity TCR alpha\Beta V-region nucleotide sequences expressed by the in vivo activated T cells, and characterize the nucleotide sequences of any predominant receptors. To this end, we will select six suitable TMJ RA patients from which we can obtain in vivo activated synovial T-cells. We will develop a novel assay which combines "linker-anchored" PCR (LA-PCR) and Denaturing Gradient Gel Electrophoresis (DGGE) to quantitatively estimate the diversity of TCR V-region sequences. These cells will be analyzed by the LA-PCR plus DGGE. Predominant receptor species identified on the gel will be cloned and sequenced. Development of this assay would allow resolution of the clonality, and might in the future be needed to identify patient-specific pathogenic T cell clones. As a future effort, unique regions of these predominant receptors will be expressed in a recombinant system, and monoclonal antibodies specific for them will be generated. Identification of predominant T cell clones, and development of monoclonal antibody could lead directly to important medical and scientific applications. Monoclonal antibodies specific for predominant TCR might prove useful as new assays for diagnosis and for monitoring disease activity, and potentially in specific immunotherapy.