Recombinant lentivirus production and T cell transduction

Lentivirus was produced by transient transfection of HEK 293T cells with LV-MAX™ lentiviral packaging mix (Thermo Fisher) and corresponding transfer plasmid using Lipofectamine™ 3000 transfection reagent (Thermo Fisher). The media were replaced with Opti-MEM I Reduced Serum Media (Gibco) 6 h post-transfection. The lentiviral supernatant was harvested 48 h post-transfection and concentrated with Amicon ultra centrifugal filter units (Millipore). Jurkat T cells were transduced by resuspending 1 × 10⁵ cells in 200 μl medium at a multiplicity of infection (MOI) of 3. The culture medium was refreshed after 24 h incubation, and the cells were incubated for an additional 48 h before use. Primary T cell transduction was performed as described previously⁵¹. Briefly, CD4⁺ and CD8⁺ T cells were enriched from human leukopaks of healthy donors (Biological Specialty Corporation) and activated with human T-activator CD3/CD28 Dynabeads (Gibco) at a 1:1 bead to cell ratio and cultured in complete T cell growth medium (TexMACS medium (Miltenyi Biotec) supplemented with 5% human AB serum (Sigma), 12.5 ng/mL IL-7 (Miltenyi Biotec), and 12.5 ng/mL IL-15 (Miltenyi Biotec)). T cells were transduced twice with lentivirus at 24 and 48 h after activation at MOI of 6, followed by expansion in 50 ml bioreactor tubes (TubeSpin, TPP) at 1−3 × 10⁶ cells/ml on a tube roller (Thermo Fisher) with a setting of 5 rpm. T cell products were harvested when 100-folds T cell expansion was achieved, typically on day 9 or 10, and cryopreserved in a 1:2 mixture of T cell complete growth medium and CS10 (STEMCELL) for in vitro and in vivo experiments.