Zoledronic acid

Zoledronic acid, LPS (lipopolysaccharide), puromycin, and G418 were obtained from Sigma‐Aldrich. Antibodies against human HLA‐G (sc‐21799) and β‐actin (sc‐47778) were purchased from Santa Cruz Biotechnology. PD‐L1 antibody (17952‐1‐AP) and PD‐L1 (22C3) were purchased from Proteintech and Agilent Technologies, respectively. Antibodies specific for CD3ε (NB600‐1441), PD‐L1 (FAB1561R, FAB1561G), CD4 (FAB3791R), and CD8 (NBP2‐34590AF488) were purchased from Novus Biologicals. HLA‐G (PE: #335906, Alexa Fluor 488: #335918), PD‐1 (#367412), CD3 (#317318), CD8 (#344704), CD56 (#304611), CD66b (#305104), and Vδ2 (#331418) were purchased from BioLegend. Anti‐Tyk2 (phospho‐Tyr1054/1055) (orb505746) antibody was obtained from Biorbyt. Antibodies specific for phospho‐ZAP70/Syk (Tyr319, Tyr352) (#MA5‐36963) and HLA‐G (MEM‐G/2: #MA1‐19394, 87G: MA1‐10356) were purchased from Thermo Fisher Scientific. Antibodies for CD14 (#12‐0149‐42), TCRγδ (#12‐9959‐42), TCRαβ (#11‐9955‐42), and NKG2D (#12‐5878‐42) were purchased from eBioscience. Vγ9 (#555732), hMito (ab92824), and VHH (iFluor 647: A02019, HRP: A2016) were purchased from BD Pharmingen, Abcam, and GenScript, respectively. Phospho‐Stat2 (Tyr690) (#77366) and HLA‐G (#79769) were obtained from Cell Signaling Technology. Recombinant human interleukin 2 (IL‐2) was obtained from Thermo Fisher Scientific.

Pitavastatin (Livalo, Adooq), zoledronic acid and risedronate (Selleck), and GGTI-2133, Tipifarnib, farnesol, geranylgeraniol and mevalonate (Sigma-Aldrich) were prepared as 20 mM solutions in DMSO except zoledronic acid which was dissolved in H₂O.

Glyoxal, pyrrolidine dithiocarbamate (PDTC), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue (MTT) and zoledronic acid (ZA) were purchased from Sigma-Aldrich. Cyclosporin A (CsA, Sandimmun^® 50 mg/mL) was from Novartis Pharma SAS (Rueil-Malmaison, France). The cytokines TNF and IFNγ were purchased from R&D Systems (Lille, France).

Two lines of human osteosarcoma cells were used in this study. U-2 OS cells were obtained from Dr. Sheau-Yann Shieh (Institute of Biomedical Sciences, Sinica Academia, Taipei, Taiwan) and MG-63 cells were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Both lines of cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 100 U/mL of penicillin, and 100 g/mL of streptomycin (Gibco). Zoledronic acid and cisplatin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Ursolic acid, N-acetyl-l-cysteine, acridine orange, and 3-methyladenine were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

3 X 10⁷ cryopreserved PBMC were thawed in a 37°C water bath and washed with RPMI 1640 medium. The PBMC were resuspended at a concentration of 1 X 10⁷cells/mL in 10 mL RPMI 1640 medium supplemented with 1 μM zoledronic acid (ZA) (Sigma-Aldrich) and 0.1 mg/mL recombinant human IL2 (Thermo Fisher Scientific). The cells were expanded in T25 or T75 tissue culture flasks (Greiner Bio, VWR, Radnor, PA, USA) for a minimum of two weeks prior to use for in vitro and in vivo experiments. During the first 10 days of expansion, the medium was supplemented with 0.1 mg/mL of recombinant human IL2 twice in the first week after which IL2 supplementation was continued at 0.01 mg/mL. After a minimum of two weeks in culture, the mixed cell population contained 20-90% Vδ2 T cells as determined by flow cytometry.

Zoledronic acid (synonymous with Zoledronate) (ZA) and Magnesium Chloride (MgCl₂) were both purchased from Sigma-Aldrich, dissolved in sterile water and added to cell cultures of both proliferating and differentiated U937 cells, at the concentrations that are indicated in detail in the Results section.