Abstract
Increasing attention has been paid to human γδ T cells expressing Vγ2Vδ2 T cell receptor (also termed Vγ9Vδ2) in the field of cancer immunotherapy. We have previously demonstrated that a novel bisphosphonate prodrug, tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate (PTA), efficiently expands peripheral blood Vγ2Vδ2 T cells to purities up to 95–99% in 10–11 days. In the present study, we first examined the effect of PTA on farnesyl diphosphate synthase (FDPS) using liquid chromatography mass spectrometry (LC-MS) to analyze the mechanism underlying the PTA-mediated expansion of Vγ2Vδ2 T cells. We find that the prodrug induced the accumulation of both isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), direct upstream metabolites of FDPS. This indicates that not only IPP but also DMAPP plays an important role in PTA-mediated stimulation of Vγ2Vδ2 T cells. We next analyzed TCR-independent cytotoxicity of Vγ2Vδ2 T cells. When human lung cancer cell lines were challenged by Vγ2Vδ2 T cells, no detectable cytotoxicity was observed in 40 min. The lung cancer cell lines were, however, significantly killed by Vγ2Vδ2 T cells after 4–16 h in an effector-to-target ratio-dependent manner, demonstrating that Vγ2Vδ2 T cell-based cell therapy required a large number of cells and longer time when tumor cells were not sensitized. By contrast, pulsing tumor cell lines with 10–30 nM of PTA induced significant lysis of tumor cells by Vγ2Vδ2 T cells even in 40 min. Similar levels of cytotoxicity were elicited by ZOL at concentrations of 100–300 μM, which were much higher than blood levels of ZOL after infusion (1–2 μM), suggesting that standard 4 mg infusion of ZOL was not enough to sensitize lung cancer cells in clinical settings. In addition, Vγ2Vδ2 T cells secreted interferon-γ (IFN-γ) when challenged by lung cancer cell lines pulsed with PTA in a dose-dependent manner. Taken together, PTA could be utilized for both expansion of Vγ2Vδ2 T cells ex vivo and sensitization of tumor cells in vivo in Vγ2Vδ2 T cell-based cancer immunotherapy. For use in patients, further studies on drug delivery are essential because of the hydrophobic nature of the prodrug.
Highlights
Cancer is the leading cause of deaths in developed countries
After Raji Burkitt’s lymphoma cells were treated with tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino) ethylidene-1,1-bisphosphonate (PTA), an nitrogen-containing bisphosphonates (N-BPs) prodrug, the cell lysates were examined for isopentenyl diphosphate (IPP) and DMAPP
Since the success of cytotoxic T lymphocyteassociated protein-4 (CTLA-4) and programmed death-1 (PD-1) immune checkpoint inhibitors, increasing attention has been paid to cancer immunotherapy [1, 5]
Summary
Cancer is the leading cause of deaths in developed countries. Despite recent successes in cancer immunotherapy harnessing programmed death-1 (PD-1) [1,2,3,4,5] and cytotoxic T lymphocyteassociated protein-4 (CTLA-4) immune checkpoint inhibitors [6], significant limitations exist for the antibody-based immunotherapies. TCR is a membrane-anchored heterodimeric protein consisting of either α and β or γ and δ chains expressed as part of a complex with cluster of differentiation 3 (CD3). Most αβ T cells expressing α and β TCR chains recognize antigenic peptides in the context of the major histocompatibility complex (MHC) class I or class II molecules with the help of CD4 or CD8 co-receptors, whereas other αβ T cell subsets respond to lipid antigens and vitamin B precursors bound to monomorphic MHC class I-related proteins, such as CD1 family members and MR1 [9]. The precise mode of recognition by Vγ2Vδ2 T cells of the BTN complex has not been fully elucidated [26, 27]
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