SummaryHLA-A2-restricted, CD3 +, CD8 + , ol/B + cytotoxic T cell (CTL) clones were isolated from peripheral blood (PBL) or tumor infiltrating lymphocytes (TIL) of two HLA-A2 + melanoma patients (9742 and 5810), to evaluate the possible recognition of autologous melanoma and of allogeneic HLA-A2-matched normal melanocytes. These CTL clones lysed not only fresh and cultured autologous melanoma cells, but also allogeneic HLA-A2 +, but not HLA-A2-, normal melanocytes. The lysis of autologous neoplastic cells and of melanocytes could be inhibited by an anti-HLA-A2 monoclonal antibody (mAb). Lysis of the normal melanocytes was not dependent on the presence of human or fetal calf serum in the culture medium. HLA-A2-restricted CTL clones recognized not only proliferating melanocytes cultured in complete melanocyte medium, but also melanocytes made quiescent by culture for up to 6 d in a basal medium devoid of exogenous factors such as phorbol ester (O-tetradecanoyl phorbol 13-acetate [TPA]), epidermal growth factor, insulin, and pituitary extracts. Analysis of specificity of four CTL clones (A75, A83, A94, and 119) from patient 9742, performed on a panel of 39 targets, indicated that the three HLA-A2-restricted CTL (A75, A83, and A94) lysed all but one of nine allogeneic melanomas expressing the HLA-A2 molecule with no reactivity on nine HLA-A2-allogeneic melanomas. Only a few instances of borderline reactivity were seen by the same effectors on 21 targets of nonmelanocyte lineage, including 12 carcinomas of different histology, four Epstein-Barr virus-transformed B cells (lymphoblastoid cell lines [LCL]), including the autologous LCL, four lines of normal fibroblasts, and normal kidney cells. Lack of reactivity on allogeneic targets of nonmelanocyte lineage occurred in spite of expression of HLA-A2 on 14 of these targets as determined by conventional tissue typing and cytofluorimetric analysis with four different anti-HLA-A2 mAb. These data indicate that tissue-related antigens can be expressed on normal and neoplastic cells of the melanocyte lineage and can be recognized in association with HLA-A2 by CTL clones from melanoma patients.
SummaryHLA-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.
To determine whether T-cell-receptor (TCR) usage by T cells recognizing a defined human tumor antigen in the context of the same HLA molecule is conserved, we analyzed the TCR diversity of autologous HLA-A2-restricted cytotoxic T-lymphocyte (CTL) clones derived from five patients with metastatic melanoma and specific for the common melanoma antigen Melan-A/MART-1. These clones were first identified among HLA-A2-restricted anti-melanoma CTL clones by their ability to specifically release tumor necrosis factor in response to HLA-A2.1+ COS-7 cells expressing this tumor antigen. A PCR with variable (V)-region gene subfamily-specific primers was performed on cDNA from each clone followed by DNA sequencing. TCRAV2S1 was the predominant ai-chain V region, being transcribed in 6 out of 9 Melan-A/MART-1-specific CTL clones obtained from the five patients. 13-chain V-region usage was also restricted, with either TCRBV14 or TCRBV7 expressed by all but one clone. In addition, a conserved TCRAV2S1/TCRBV14 combination was expressed in four CTL clones from three patients. None of these V-region genes was found in a group of four HLA-A2-restricted CTL clones recognizing different antigens (e.g., tyrosinase) on the autologous tumor. TCR joining regions were heterogeneous, although conserved structural features were observed in the complementarity-determining region 3 sequences. These results indicate that a selective repertoire of TCR genes is used in anti-melanoma responses when the response is narrowed to major histocompatibility complexrestricted antigen-specific interactions.
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