Lentiviral Transduction of Activated T Cells

Human PBMCs (5 × 10⁶) isolated as described above were stimulated for 3 d with OKT-3 (100 ng ml⁻¹, BioLegend) in a culture medium containing IL-2 (100 IU ml⁻¹, Proleukin, Novartis). Activated T cells (1 × 10⁶) were resuspended in 2 × 10⁷ IU concentrated lentivirus particles, together with 10 mg ml⁻¹ protamine sulphate (Sigma-Aldrich) and IL-2 (100 IU), for a total volume of 20 μl, and incubated for 4 h. Then, 1 ml of fresh culture medium was added. The T cells were transduced again the following day in the same manner, and then cultured in 1 ml of culture medium containing IL-2 (100 IU ml⁻¹) for 7 d. Before further experiments, T cells were expanded for 2 weeks using a rapid expansion protocol as previously described^{38 (link)}.