Improving the efficiency of the TriVST transduction. (A) PBMCs were stimulated using the Ad/LCL protocol in the presence of IL-4 and IL-7 and transduced on day 3 with the GD2.CAR vector. Co-expression of the GD2.CAR on pentamer positive CMV (A2-NLV), EBV (A2-CLG) and AdV (A24-TYF)-specific T-cells was analyzed by flow cytometry after the 3rd stimulation to confirm the transduction of triVSTs. The dot plots of one representative donor are shown. (B) GD2.CAR-transduced and non-transduced triVSTs from three donors were analyzed in IFN-γ ELISPOT with serial dilutions of overlapping pepmix library spanning adenovirus hexon. Responses were analyzed by two-way ANOVA and no statistical significant difference (p > 0.5) between transduced and non-transduced cells were found (C) The transduction efficiency with or without centrifugation of the retrovirus vector at 2000 × G onto the retronectin-coated plates is shown. (D) The transduction efficiency of VSTs after the indicated centrifugation time for the retroviral supernatant. Data represent the mean +/− sd of 2 donors. (E) Surface expression levels of GD2.CAR were upregulated in early transduced (ET) CD4+ and CD8+ T cells (left panel) and in late transduced (LT, right panel) triVSTs upon stimulation with LCLs and LCLs transduced with Ad5f35-pp65 (LCL Adpp65) for 48 hours without cytokines. Results are shown as mean ± SD, n = 3.