Lentiviral Transduction of Activated T Cells

T cells were stimulated with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher Scientific, San Diego, CA) and were expanded in T cell media (47.5% Click’s media [Irving Scientific, Santa Ana, CA], 47.5% RPMI-1640, 5% human serum, 2 mM L-glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin) supplemented with 100 IU/mL IL-2. Forty-eight hours following stimulation, T cells were transduced with a lentivirus vector encoding ARI2h.³¹ (link) Subsequently, T cells were split and cytokines were refreshed every 1 to 2 days for a further 7 days of culture, unless indicated otherwise. For experiments in which T cells were exposed to G-CSF in culture, 10 ng/mL recombinant G-CSF (Miltenyi Biotech) was added to T cells that were resting in T cell media for 3 days prior to stimulation. ARP-1, U266, ARP-1-GFP-ffLuc, and U266-GFP-ffLuc cell lines were obtained, cultured, and modified to express GFP-ffLuc, where appropriate, as previously described.³¹ (link) All cultured cells were incubated at 37°C with 5% CO₂. Live cells were routinely counted using Trypan blue exclusion.