Non-secretory apoptotic killing of tumor cells by human natural killer cells is mediated by multiple cell membrane bound ligands of the TNF family

Our recent studies have shown that freshly isolated non-activated human peripheral blood natural killer (NK) cells utilize a non-secretory/apoptotic mechanism to destroy tumor cells, in addition to the well known secretory/necrotic killing. We define herein the role of cell membrane bound molecules of the TNF family of ligands in the apoptotic killing mechanism of NK cells. Purified human NKcells showea by RTPCR and immunofluorescence analyses constitutive expression of the TNF, LT-α, LT-αβ and FasL mRNAs and/or cell membrane bound proteins. On the other hand, various solid tissue derived tumor cell lines exhibited the corresponding cell surface receptors, including TNFR1. TNFR2, LT-βR and Fas. Rapid induction of DNA fragmentation in solid tissue derived tumor cell lines by their co-incubation with NK cells, evidenced by 1h [³H]-thymidine release assay, was significantly inhibited by individual treatment with either anti-TNF, anti-LT-α, anu-TNFRl, anti-TNFR2 or anti-Fas blocking antibodies, or with either TNFR2.Fc or LT-R.Fc constructs. Furthermore, while co-incubation of tumor cells with either recombinant TNF, LT-α or FasL was without any apparent cytotoxic effect, combined treatment with either TNF plus FasL or LT-α plus FasL induced a rapid DNA fragmentation. Our findings show that the rapid apoptotic killing of tumor cells by NK cells is mediated by a simultaneous ligation of the cell membrane TNFR, LT-βR and Fas on tumor target cens by the corresponding ligands expressed on the effector cells.