A novel subset of T cells express the T cell receptor (TCR) gamma/delta. These T cells show preferential reactivity to and expand dramatically in response to mycobacterial antigens, including the heat shock protein 65 (HSP65). Here, HSP65-reactive gamma/delta T cell clones will be derived from rheumatoid arthritis (RA) synovial tissue by stimulation with recombinant mycobacterial HSP65 protein or its human homologue HSP58, or with transfectants expressing these proteins. The antigenic epitopes recognized by HSP-specific gamma/delta T cells will be identified using HSP peptides, or transfectants expressing HSP65 deletion mutants. The TCR gamma and delta repertoire of HSP65-reactive T cell clones will be examined by staining with V-gene specific mAb, PCR analysis, and nucleotide sequencing. The in vivo TCR gamma/delta repertoire in RA will be examined in synovial tissue and compared to that of peripheral blood of the same individuals. This will be performed by immunohistochemical staining and two-color FACS analyses using TCR gamma/delta framework and V-gene specific mAb, quantitative PCR, and nucleotide sequencing. In this way, we can determine if the TCR gamma/delta repertoire in joints is distinct from that of peripheral blood, and if the in vivo activated gamma/delta T cells show expansion of peripheral blood, and if the in vivo activated gamma/delta T cells show expansion of particular V-gene segments or V-J junctional regions. Such results would provide evidence of local antigen-driven expansion or of oligoclonality of these T cells. Studies of human RA suggest a role of HSP65-reactive T cells in tissue damage. For example, it has been hypothesized that one component of the mechanisms producing chronicity in RA is T cell reactivity to self heat shock proteins of stressed cells in the inflamed synovium. The analyses proposed here on HSP65-specific gamma/delta T cell clones, together with the in vivo repertoire studies will determine the nature of gamma/delta T cells, and may provide insights into their role in rheumatoid arthritis. In this Developmental and Feasibility study, the P.I. wishes to apply his expertise in the basic molecular analyses of human gamma/delta T cell receptors to examine the role of cells bearing this receptor in RA.
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