Interleukin 2 Activation of Cytotoxic T-Lymphocytes Infiltrating into Human Metastatic Melanomas

Tumor infiltrating lymphocytes (TIL) were isolated from 22 tumors obtained from 15 patients with metastatic melanoma. In 18 of the'22 tumors, a substantial number of lymphocytes was isolated with an average lymphoid cell:tumor cell ratio of 1.26. The TIL were predominantly cytotoxic/suppressor T-lymphocytes with an average of 87% Leu4+, 61% Leu2a+, and 18% Leu3a+ cells. There were less than 2% natural killer cells, B-cells or macrophages. An average of 3.8% (range, <0.1 to 8.6%) of freshly isolated TIL bound to autologous tumor cells. Prior to culture, none of the tumor-binding cells (TBC) was cytotoxic as judged by trypan blue exclusion. The frequency of TBC increased to 11.6% after 2 days of culture, and 10% of these TBC developed cytotoxic activity. When interleukin 2 was added to cultures, the frequency of TBC increased, and the frequency of cytotoxic TBC was 2-fold higher compared to control cultures. After 10 days of culture with interleukin 2, TIL increased in number with a concomitant disappearance of tumor cells, whereas there were severe decreases of lymphocytes and no decrease of tumor cells in control cultures. TIL were cultured for 8 to 10 days with recombinant interleukin 2 and tested for cytotoxicity against autologous and allogenic tumor cells and K562 targets in a 4-h 51Cr release assay. rIL2-cultured TIL from all nine patients tested exhibited the highest levels of lysis against autologous tumor cells. Of the nine TIL samples, five exhibited an apparent specificity for autologous melanoma, while four specimens killed both allogenic and autologous melanoma. The ability of TIL to kill K562 targets appeared to parallel the ability to kill allogenic targets. For comparison, recombinant interleukin 2-cultured peripheral blood mononuclear cells from the same patients were assayed for cytotoxic activity against autologous and allogenic melanomas. Unlike some TIL, none of the peripheral blood mononuclear cells exhibited specificity for autologous tumor cells. In summary, TIL isolated from metastatic melanoma patients were predominantly cytotoxic T-lymphocytes with the ability to recognize and kill autologous tumor cells after in vitro culture; interleukin 2 induced proliferation of TIL and augmented their cytotoxic activity such that they eliminated autologous tumor cells.