Cancer Immunotherapy: Cytotoxic Activity of γδ T-Cells Expanded Ex Vivo by the Third Generation Bisphosphonate Zoledronate.

Bisphosphonates (BPs), widely used to treat bone diseases, have recently been attracted much interest for their antitumor activity and have been reported to exert direct antitumor effects on several cancer cell lines via the inactivation of Ras proteins. BPs inhibit farnesyl pyrophosphate (FPP) synthetase in the mevalonate pathway and deplete cellular pools of PPs such as farnesyl PP and geranylgeranyl PP which are indispensable for the activation of Ras proteins. In addition to their direct antitumor activity, BPs expand γδ T-cells which are potent effector cells and also induce the accumulation of isopentenyl PP as a tumor antigen in target cancer cells. The purpose of this study was to clarify the cytotoxic activity of γδ T-cells expanded ex vivo by the potent third generation BP zoledronate (ZOL). Peripheral blood mononuclear cells of five healthy donors were incubated with different concentrations of ZOL and interleukin-2. After 14 days incubation, 1 μM ZOL increased the absolute number of γδ T-cells 500–800 fold. Expanded γδ T-cells were of the Vγ9Vδ2 subset and cytokine levels of IL-2, -4, -5, -10, TNF-α and IFN-γ were not elevated at resting i.e. before contact to target cancer cells. In vitro cytotoxic activities of γδ T-cells against the luciferase-labeled small cell lung cancer (SCLC) cell line SBC-5 were examined by a newly developed cytotoxic assay using an in vivo imaging system (Xenogen, Alameda, CA) and video microscopy, Leica AS MDW (Leica Microsystems Inc., Bannockburn, IL). γδ T-cells killed SBC-5 cells pre-treated with 5 μM ZOL for 12 h after 1.5–3.0 h contact with the target cells whereas untreated SBC-5 were rarely killed. SBC-5 cells pretreated with 5 μM ZOL showed a marked increase in their sensitivity to lysis by γδ T-cells, percentages of specific lysis were 42% and 55% at effector/target (E/T) ratios of 5:1 and 10:1, respectively, while those of untreated SBC-5 cells were 8% and 13% at E/T ratios of 5:1 and 10:1, respectively. In vivo efficacy of γδ T-cells was investigated in mice xenografted subcutaneously with SBC-5 cells. Pretreatment with 80 μg/kg ZOL enhanced significantly antitumor activity of γδ T-cells also in vivo. These findings showed that ZOL stimulated the proliferation of γδ T-cells significantly and that the cytotoxic activity of γδ T-cells required pre-treatment of target cells with ZOL, indicating the potential use of autologous ex vivo expanded γδ T-cells for cancer immunotherapy.