Zoledronic acid activates in vitro Vγ9Vδ2 cells and induces subset distribution in cancer patients in vivo. (A) PBMCs were incubated in vitro with IPP (▵) or zoledronic acid (□) at the indicated final concentrations and 10 U/mL IL-2. Then, 7 days later, cells were collected and the percentage of Vγ9Vδ2⁺ cells within the CD3⁺ population was assessed. (B) Vγ9Vδ2 T-cell subsets were analyzed by 3-color fluorescence-activated cell-sorter (FACS) analysis before or after in vivo treatment with zoledronic acid. The following antibodies were used in different combinations: anti-Vδ2 fluorescein isothiocyanate (FITC, IMMU389; Coulter, Miami, FL), anti-CD27 phycoerythrin (PE) (M-T271; BD Pharmingen, San Diego, CA), and anti-CD45RA PE–cytochrome 5 (Cy5, 2H4; Coulter). Data were acquired on a FACSCalibur instrument (BD Biosciences, San Diego, CA) and analyzed using CellQuest software (BD Immunocytometry Systems, San Jose, CA). Error bars indicate standard deviation.