Cloned human cytotoxic T lymphocyte (CTL) lines reactive with autologous melanoma cells. I. In vitro generation, isolation, and analysis to phenotype and specificity.

Abstract

Activation of peripheral blood lymphocytes (PBL) from a melanoma patient either in secondary MLC in which EBV-transformed B cells from the cell line JY were used as stimulator cells, or by co-cultivation with the autologous melanoma cells in a mixed leukocyte tumor cell culture (MLTC) resulted in the generation of cytotoxic activity against the autologous melanoma (O-mel) cells. From these activated bulk cultures four cloned cytotoxic T lymphocyte (CTL) lines were isolated.The CTL clone O-1 (T3+, T4+, T8-, OKM-1-, HNK-, and HLA-DR+), and O-36 (T3+, T4-, T8+, OKM-, HNK-, and HLA-DR+) were obtained from MLC, whereas the CTLC clones O-C7 (T3+, T4+, T8-, OKM-1-, HNK-, and HLA-DR+) and O-D5 (T3+, T4-, T8+, OKM-1-, HNK, and HLA-DR+) were isolated from autologous MLTC. All four CTL clones were strongly cytotoxic for O-mel cells but failed to lyse autologous fibroblasts and autologous T lymphoblasts. Moreover, the CTL clones lacked NK activity as measured against K562 and Daudi cells. Panel studies indicated that the CTL clones also killed approximately 50% of the allogeneic melanoma cells preferentially, whereas the corresponding T lymphoblasts were not lysed. Monoclonal antibodies against class I (W6/32) and class II (279) MHC antigens failed to block the reactivity of the CTL clones against O-mel and allogeneic melanoma cells, indicating that a proportion of human melanoma cells share determinants that are different from HLA antigens and that are recognized by CTL clones.In contrast to the CTL clones isolated from MLTC, the clones obtained from MLC also lysed JY cells, which initially were used as stimulator cells. The reactivity of O-36 against JY could be inhibited with W6/32, demonstrating that this reactivity was directed against class I MHC antigens. These results suggest that the lysis of O-mel and JY cells by O-36 has to be attributed to two independent specificities of this CTL clone. The specificity of the other cross-reactive CTL clone (O-1) could not be determined.The notion that individual CTL clones can have two specificities was supported by the following observations. The cytotoxic reactivity of both O-1 (T4+) and O-36 (T8+) against JY was blocked by monoclonal antibodies directed against T3 and human LFA-1, and against T3, T8, and human LFA-1, respectively. In contrast, none of these monoclonal antibodies was able to block the reactivity of 0-1 and 0-36a gainst 0-me1 cells. Thcey totoxic reactivity of 0-36 against J Y was abolished after trypsin treatment of this CTL clone, whereas its reactivity against 0-me1 cells was not affected. The anti-T3, anti-T8, and anti-LFA-1 monoclonala ntibodies also failed to inhibit the reactivity of 0-C7 and 0-D5 against 0-me1 cells. Thesed ata indicate that T3 and human LFA-1, or T3, T8, and human LFA-1 are required for the expression of cytotoxicity of 0-1 and 0-36 against JY, but that these antigens are not associated with the cytotoxic reactivity of the four CTL clones against 0-me1 cells.In addition to their "specific" cytotoxicityt,h e CTL clones were nonspecifically cytotoxic for Daudi and 0-me1 cells pretreated with PHA. This LDCC against 0-me1 (whichw as superimposed on the direct cytotoxicity against these target cells) and against Daudi was completely inhibitbeyd monoclonal antibodies directed against human LFA-1, whereas the direct cytotoxicity against 0-me1 remained.Taken together, these data indicate that it is feasible to obtain CTL clones that kill autologous and allogeneic melanoma cells preferentially. An initial analysis to the specificity of these CTL clones showed that the recognition and interaction mechanisms required for the reactivity against the antigens expressed on human melanoma cells are different from those involved in the reactivity against HLA antigens.