CD3-LVs activate cytokine-cultured T cells and induce cytokine production and proliferation. PBMCs isolated from adult blood were cultured overnight in the presence of IL-2 only and then transduced with the indicated CD3-LVs or with VSV-LV. Controls were left untransduced (ut) or activated with αCD3/αCD28 antibodies (1 µg/mL αCD3, 3 µg/ml αCD28) until first medium exchange. (A-B) Expression of the activation markers CD69 (A) and CD25 (B) on all viable cells was followed for 6 days by flow cytometry. The number of activation marker positive cells are shown. N = 3 donors, mean ± standard error of the mean. *P < .05, **P < .01, ***P < .001, ****P < .0001 by 2-way ANOVA with Dunnett's correction. (C-E) One day postincubation with vector particles or recombinant antibodies, cytokines secreted into the cell culture supernatant were quantified. Scatter bar diagrams show the concentration of interferon-γ (C), tumor necrosis factor-α (D), and IL-2 (E) for each condition. Mean ± SD from 1 experiment with n = 3 triplicates each are shown. (F) PBMCs were stained with CellTrace Violet (CTV) before transduction ± costimulation to follow cell proliferation over time. Histograms show the fluorescence of CTV at day 5 posttransduction. Data are representative of 3 different donors. (G) Bar diagrams display T-cell expansion after 6 days compared with day 0 ± costimulation. N = 3 donors, mean ± SD. ns, nonsignificant; *P < .05 by 1-way ANOVA with Dunnett's correction. (H) Bar diagrams show percentages of GFP⁺ cells gated from all viable single cells ± costimulation at day 6 posttransduction. N = 3 donors. Mean ± SD. ns, nonsignificant by 2-way ANOVA with Sidak's correction.