Expansion and Cryopreservation of γδ T Cells

γδ T cells were maintained in RPMI 1640 medium (Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 1% L-glutamine (Lonza, Walkersville, MD, USA), and 1% penicillin/streptomycin (Lonza, Walkersville, MD, USA). On day 0, PBMCs were stimulated with 3 μM of zoledronic acid (Daewoong Pharmaceutical Co, Ltd., Seoul, Korea) in the presence of 1000 U/mL of IL-2 (Proleukin; Novartis, East Hanover, NJ, USA) and seeded at 5 × 10⁵ cells/mL in a 24-well plate. From day 7, γδ T cells were treated with IL-2 (1000 U/mL) every 3–4 days, stimulated weekly with irradiated (100 Gy) aAPCs at a 2:1 T:aAPC ratio. On day 21, cells were frozen using a controlled-rate freezer (Thermo Fisher Scientific, Waltham, MA, USA) and cryopreserved in liquid nitrogen until further use. For cytotoxicity assays, frozen γδ T cells were thawed and cultured overnight in the presence of IL-2 (1000 U/mL).