Studies of the mechanism of cytolysis by tumour-infiltrating lymphocytes

In order to determine the mechanism of tumour destruction by tumour-infiltr ating lymphocytes (TIL), we examined the ability of both CD4(+) and CD8(+) effector TIL, and TIL clones, to manifest granzyme-mediated and Fas-mediate d destruction of tumour targets. In many in vitro studies TIL have been sho wn to manifest anti-tumour reactivity, yet many tumours escape immunologica l destruction. To investigate the role of Fas expression and the concomitan t sensitivity to the inducibility of apoptotic death, we derived TIL from f our melanomas and one glioma. The glioma, and all but one of the melanomas, expressed Fas, but Fas-mediated apoptosis could only be detected if the ta rgets were treated with cyclohexamide. The melanomas and the glioma all exp ressed detectable cytoplasmic Bcl-2 protein, known to exert anti-apoptotic activity. Lysis of tumours by CD8-enriched cultures and CD8(+) clones was C a2+-dependent and could not be modified by an anti-Fas MoAb. In CD4-enriche d cultures or CD4(+) clones with cytotoxic potential against tumour cells, cytotoxicity was also Ca2+-dependent. As Ca2+-dependent cytotoxicity is usu ally the result of secretion of perforin/granzyme- B, we investigated the p resence of perforin in cytotoxic CD4(+) clones and demonstrated the presenc e of granular deposits of this enzyme in some of the CD4(+) clones. Althoug h an anti-Fas MoAb did not block the lysis of melanoma targets by CD4(+) cl ones, the examination of Fas-dependent targets demonstrated that these clon es also had the potential to kill by the Fas/Fas ligand system. These data suggest that the predominant mechanism in tumour killing by TIL appears to be perforin-granzyme-dependent, and that the solid tumour cell lines we stu died are less susceptible to Fas-mediated apoptosis. As non-apoptotic pathw ays may enhance tumour immunogenicity, exploitation of the perforin-granzym e-dependent cytotoxic T lymphocyte (CTL) pathways may be important for achi eving successful anti-tumour responses.