Fig 1: Factors affecting transduction of primary T cells with lentivectors. (a–c) T‐cell activation marker (CD25, CD69) expression in CD3 T cells (n = 3 donors) was assessed by flow cytometry at 24‐ or 48‐h after stimulation by chemicals (Phorbol 12‐myristate 13‐acetate [30 nM] and ionomycin [1 μM], PMA/Io) or by different cell‐to‐bead (Dynabeads loaded with antihuman CD3 and antihuman CD28) ratios. Strategy for evaluating CD3 T cell activation is presented in Figure S1. Different stages of T‐cell activation are shown in (a) early activation (CD69+CD25−), (b) peak activation (CD69+CD25+), and (c) late activation (CD69−CD25+). (d) FRα‐CAR expression (% FRα‐CAR+ T cells on left y‐axis) and T‐cell viability (% Viability on right y‐axis) was assessed by flow cytometry after transducing stimulated and unstimulated primary T cells. (e–g) FRα‐CAR expression (% FRα‐CAR+ T cells on left y‐axis) and T‐cell viability (% Viability on right y‐axis) was assessed by flow cytometry after varying factors affecting transduction (e) size of the genetic payload (chimeric antigen receptor [CAR] only, 5.6 kb vs. T‐cell‐based delivery system comprising of CAR and nuclear factor of activated T cells response element [NFAT‐RE] inducible transgene, 7.2 kb), see Figure S2 for schematics, (f) lentivector pseudotype (RD114 vs. VSV‐g), and (g) lentivector transduction methods (Method a: spinoculation at 800g in 0.5 ml for 1.5 and 14.5 h in cell culture incubator; Method b: 1.0 ml reaction volume for 16 h in cell culture incubator; Method c: 0.1 ml reaction volume for 16 h in cell culture incubator). Transduction efficiency was determined after 5 days. All results are represented as mean ± SD. Statistical analysis and p values for (d) and (g) were determined by one‐way ANOVA and Tukey's multiple comparison test. Statistical analysis and p values for (e) were determined by Student's t test, two‐tailed. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.