TCR BETA-CHAIN VARIABLE REGION-DRIVEN SELECTION AND MASSIVE EXPANSIONOF HLA-CLASS I-RESTRICTED ANTITUMOR CTL LINES FROM HLA-A-ASTERISK-0201(+) MELANOMA PATIENTS

Recognition of a given melanoma Ag involves a limited array of T cell clones bearing a structurally defined TCR, The aim of this study was t o verify whether this information can be used to isolate and expand su ch anti-tumor effectors from fresh lymphocyte populations. We found th at one to three different TCR beta-chain variable (TCRBV) regions were significantly expanded in 4-wk mixed lymphocyte-tumor cultures (MLTC) from six HLA-A0201(+) melanoma patients, and that the T cells expres sing the expanded TCRBV regions were involved in HLA class-I-restricte d lysis of the tumor. T cell activation by mAbs to MLTC-selected TCRBV region and CD28 resulted in large scale expansion (1-10 x 10(9) cells ) of T cell lines, highly enriched for the expression of a single TCRB V region and for CD8(+) T cells, The TCRBV-driven selection was Equall y effective when applied to patients' or healthy donors' lymphocytes, and the T cell lines isolated from melanoma patients exerted HLA class I-restricted lysis of the autologous tumor. MLTC and TCRBV-selected l ines recognized allogeneic melanomas sharing HLA-B and -B alleles with the autologous tumor, but only two of the HLA-A2-restricted lines wer e directed to a known peptide from melanoma-associated Ags. Single-str and conformation polymorphism analysis indicated a polyclonal composit ion of both MLTC and TCRBV-selected lines, but expansion of clonotypes with identical CDR3 length was observed only in the MLTC lines, Thus, TCRBV-driven selection can be exploited to obtain large scale expansi on of antitumor CTL lines from melanoma patients.