T Cell Receptor (TCR) Structure of Autologous Melanoma-reactive Cytotoxic T Lymphocyte (CTL) Clones: Tumor-lnfiltrating Lymphocytes Overexpress In Vivo the TCR f3 Chain Sequence Used by an HLA-A2-restricted and Melanocyte-lineage-specific

HLA-A2 § melanomas express common melanoma-associated antigens (Ags) recognized in vitro by autologous cytotoxic T lymphocytes (CTL). However, it is not known whether tumor Ags can drive in vivo a selective accumulation/expansion of Ag-specific, tumor-infiltrating T lymphocytes (TIL). Therefore, to evaluate this possibility, 39 CTL clones isolated from several independent mixed lymphocyte tumor cultures (MLTC) of TIL and peripheral blood lymphocytes (PBL) of an HLA-A2 + melanoma patient and selected for T cell receptor (TCR)-dependent, HLArestricted tumor lysis, were used for analysis of TCR c~ and ~ chain structure by the cDNA polymerase chain reaction (PCR) technique with variable gene-specific primers followed by sequencing. Despite absence of oligoclonality in fresh TIL and PBL, as well as in T ceils of day 28 MLTC (day of cloning), sequence analysis of TCR c~ and ~ chains of TIL clones revealed a dominance of a major category of melanoma-specific, HLA-A2-restricted T cells expressing a Vc~8.2/JotAP511/Cot and Vf12.1/DB1/J31.1/C/31 TCR. The same TCR was also found in 2 out of 14 PBL clones. The other PBL clones employed a Vow2.1 gene segment associated with either Vfl13.2, 14, or w22. Clones A81 (Vce2.1/JotIGRJoL04/Ccr and V314/D31/Jfll.2/Cj31) and A21 (Vce8.2/JoeAP511/Col and V/32.1/D/31/J31.1/C31), representative of the two most frequent TCR of PBL and TIL, respectively, expressed different lyric patterns, but both were HLA-A2 restricted and lysed only HLA-A2 + melanomas and normal melanocytes, thus indicating recognition of two distinct HLA-A2-associated and tissue-related Ags. Finally, by the inverse PCR technique, the specific TCR 3 chain (V32.1/Dfll/J31.1/C3I) expressed by the dominant TIL clone was found to represent 19 and 18.4% of all V/32 sequences expressed in the fresh tumor sample and in the purified TIL, respectively, but <0.19% of Vfl2 + sequences expressed in PBL. These results are consistent with the hypothesis that a clonal expansion/accumulation of a melanocyte-lineage-specific and HLA-A2-restricted T cell clone occurred in vivo at the site of tumor growth.