Identification of human T cell receptor γδ-recognized epitopes/proteins via CDR3δ peptide-based immunobiochemical strategy

Human T lymphocytes, bearing T cell receptor (TCR) gamma delta, play an important role in anti-tumor/microbe immune responses. However, few tumor antigens recognized by TCR gamma delta have been defined so far. To investigate antigenic epitopes/proteins recognized by gamma delta T cells, we have established a new immunobiochemical strategy that uses complementarity-determining region 3 of TCR delta chain (CDR3 delta) peptide-mediated epitope/protein-binding assays. CDR3 delta peptides synthesized using the CDR3 region in TCR V delta 2 chain were validated for their binding specificity to target cells or tissues. These CDR3 delta peptides were then employed as probes to pan putative epitopes in a 12-mer random peptide phage-displayed library and to identify putative protein ligands within tumor protein extracts by affinity chromatography and liquid chromatography/electrospray ionizationtandem mass spectrometry analysis. As a result, we have identified nine peptides and two proteins for TCR gamma delta, including human mutS homolog 2 ( hMSH2) and heat shock protein (HSP) 60. All nine tested epitope peptides not only bind to gamma delta T cells but also functionally activate gamma delta T cells in vitro. Identification of HSP60 confirms the validity of this method as HSP60 is an identified ligand for TCR gamma delta. We show that hMSH2 is expressed on the surface of SKOV3 tumor cells, and cytotoxicity of V delta 2 gamma delta T cells to SKOV3 cells was blocked by anti-hMSH2 antibody, suggesting that hMSH2 may be a new ligand for TCR gamma delta. Taken together, our findings provide a novel immunobiochemical strategy to identify epitopes/proteins recognized by gamma delta T cells.