Tetramer-guided analysis of TCR β-chain usage reveals a large repertoire of melan-A-specific CD8+ T cells in melanoma patients

The assessment of the TCR repertoire expressed by tumor-specific CD8(+) T lymphocytes has been hampered to date by the difficulty of targeting the analysis to lymphocytes directed against a single epitope, In the present study we have used fluorescent A2/Melan-A tetramers in conjunction with anti-CD8 and anti-TCR beta-chain variable (BV) mAbs to analyze by flow cytometry the BV segment usage by Melan-A-specific CD8(+) T cells in tumor-infiltrated lymph nodes (TILN) and tumor-infiltrating lymphocytes (TIL) from A2 melanoma patients. Analysis of TILN populations revealed small proportions of A2/Melan-A tetramer(+) cells expressing many different BV together with over-representation of A2/Melan-A tetramer(+) cells expressing certain BVs, The BV usage by A2/Melan-A tetramer(+) lymphocytes in TIL was more restricted than that in TILN, Moreover, the predominant BV segments were quite distinct in populations derived from different patients. A2/Melan-A tetramer(+) cells expressing the dominant BVs found in TILN could also be found in the corresponding peptide-stimulated autologous PBMC, although A2/Melan-A tetramer(+) lymphocytes expressing additional BVs were also identified. Together, these results suggest that a large and diverse repertoire of Melan-A-specific T cells using different BV TCR segments is available in A2 melanoma patients.