Transduction of T cells was carried out in RetroNectin (Takara Bio, Tokyo, Japan)–coated 24-well plates, which were preloaded with viral supernatant. T cells (0.5 × 106) suspended in 0.5 ml of T cell medium + IL-2 (400 IU) were combined with 1.5 ml of viral supernatant and centrifuged for 40 min, 1000g at room temperature. Typically, 12 × 106 T cells per donor were plated for transduction.

Timing of transduction differed between γδT cells and αβT cells due to expansion dynamics. γδT cell expansion was stimulated with 5 μM zoledronic acid (Actavis, New Jersey, USA) and IL-2 (100 IU/ml) (Aldesleukin), and transduction was performed at day 5. At day 8 of culture (day 3 after transduction), cells were pooled, washed, and plated at 2 × 106 cells/ml in T cell medium + IL-2 (100 IU/ml) (24-well plates, Nucleon Delta Surface, Thermo Fisher Scientific, Massachusetts, USA). Transduction efficiency was determined by flow cytometry at day 10 (day 5 after transduction).

In the case of αβ T cells, transduction was performed 72 hours after stimulation with anti-CD3/CD28 antibodies in the presence of IL-2 (100 IU/ml) (Aldesleukin), with replating 3 days after transduction. Signaling analysis and cytotoxicity assays were performed 6 days after transduction in both cases.

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