The aim of this study is to analyze, at the molecular level, the T cell receptors (TCRs) associated with collagen induced arthritis (CIA) in the DBA/1 mouse (I-Aq). Towards this aim, the TCR genes will be isolated from T cell hybridomas or clones which recognize peptides derived from type II collagen associated with the MHC class II protein, I-Aq. The genes will be tailored and cloned for expression in vectors designed to secrete TCRs as single Valpha, Vbeta domains, Fv-like Valpha:Vbeta heterodimers, Fab-like Valpha-Calpha:Vbeta-Cbeta heterodimers or Fab-like TCR-immunoglobulin Valpha-CH1:Vbeta-Ckappa chimeras, using Escherichia coli as a host. The expressed proteins will be purified and analyzed to assess their multimeric state. This will be followed by the development of competition and direct binding assays to determine the ability of the recombinant TCRs to bind to cognate peptide-MHC complexes. Structure-function studies of the expressed TCRs will be carried out, and this will involve the use of site-directed mutagenesis to map the TCR residues which are involved in mediating peptide binding and specificity. PCR-directed, random mutagenesis will be used in an attempt to increase the binding affinity of the recombinant TCRs for cognate peptide-MHC complexes.
The aim of this work is to develop novel strategies for the immunotherapy of murine CIA. For example, the information gained from the structural studies will be used to design suitable peptides for use in vaccination to both block and reverse CIA. The soluble TCR fragments will also be used in vaccination. Production of recombinant TCRs will provide a valuable source of protein for use in immunization to generate anti-clonotypic antibodies which could have uses in passive immunization and diagnosis. Higher affinity variants of these TCRs, generated by random mutagenesis, will also be tested as blocking reagents in therapy. Ultimately, the study will be extended to the analysis of TCRs associated with RA in humans, with the long term goal of applying the therapeutic approaches used in the murine model to this disease.
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