T-CELL RECEPTOR (TCR) STRUCTURE OF AUTOLOGOUS MELANOMA REACTIVE CYTOTOXIC T-LYMPHOCYTE (CTL) CLONES - TUMOR-INFILTRATING LYMPHOCYTES OVEREXPRESS IN-VIVO THE TCR BETA-CHAIN SEQUENCE USED BY AN HLA-A2-RESTRICTED AND MELANOCYTE-LINEAGE SPECIFIC CTL CLONE

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, HLA-restricted tumor lysis, were used for analysis of TCR alpha and beta 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 cells of day 28 MLTC (day of cloning), sequence analysis of TCR alpha and beta chains of TIL clones revealed a dominance of a major category of melanoma-specific, HLA-A2-restricted T cells expressing a Valpha8.2/JalphaAP511/Calpha and Vbeta2.1/Dbeta1/Jbeta1.1/Cbeta1 TCR. The same TCR was also found in 2 out of 14 PBL clones. The other PBL clones employed a Valpha2.1 gene segment associated with either Vbeta13.2, 14, or w22. Clones A81 (Valpha2.1/JalphaIGRJalpha04/Calpha and Vbeta14/Dbeta1/Jbeta1.2/Cbeta1) and A21 (Valpha8.2/JalphaAP511/Calpha and Vbeta2.1/Dbeta1/Jbeta1.1/Cbeta1), representative of the two most frequent TCR of PBL and TIL, respectively, expressed different lytic 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 beta chain (Vbeta2.1/Dbeta1/Jbeta1.1/Cbeta1) expressed by the dominant TIL clone was found to represent 19 and 18.4% of all Vbeta2 sequences expressed in the fresh tumor sample and in the purified TIL, respectively, but <0.19% of Vbeta2+ 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.