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 V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1 TCR. The same TCR was also found in 2 out of 14 PBL clones. The other PBL clones employed a V alpha 2.1 gene segment associated with either V beta 13.2, 14, or w22. Clones A81 (V alpha 2.1/J alpha IGRJ alpha 04/C alpha and V beta 14/D beta 1/J beta 1.2/C beta 1) and A21 (V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1), 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 (V beta 2.1/D beta 1/J beta 1.1/C beta 1) expressed by the dominant TIL clone was found to represent 19 and 18.4% of all V beta 2 sequences expressed in the fresh tumor sample and in the purified TIL, respectively, but < 0.19% of V beta 2+ 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.
Division of Experimental Oncology D, Istituto Nazionale Tumori, Milan, Italy.
M
M
Sensi
S
S
Salvi
C
C
Castelli
C
C
Maccalli
A
A
Mazzocchi
R
R
Mortarini
G
G
Nicolini
M
M
Herlyn
G
G
Parmiani
A
A
Anichini
D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm; D001483:Base Sequence; D002478:Cells, Cultured; D002999:Clone Cells; D004247:DNA; D015789:HLA-A2 Antigen / Q000276:immunology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology*; D008544:Melanocytes / Q000276:immunology*; D008545:Melanoma / Q000276:immunology* / Q000473:pathology; D058950:Melanoma-Specific Antigens; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000276:immunology; D051381:Rats; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D015789:HLA-A2 Antigen; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins; D016693:Receptors, Antigen, T-Cell, alpha-beta; D004247:DNA