Fig.4. The costimulatory domains did not impact TR1419CAR-T phenotypes. (A) Diagram of the lentiviral expression cassette with the second generation TR1419-CARs containing the humanized scFv (TR1419) targeting TRAIL-R1, with a CD8α hinge domain, a CD28 transmembrane domain, a cytoplasmic CD28 or 4-1BB costimulatory domain, and a cytolytic CD3ζ domain. (B) Mock (untransduced), TR1419-28ζ, TR1419-BBζ or TR1419-28BBζ CAR T cells were evaluated by flow cytometry for scFv expression to detect lentiviral transduction of CARs. (C) Representative CAR-T cell phenotyping plot based on CD45RA and CCR7. CD45RA+CCR7+: naïve T cells, CD45RA-CCR7+: central memory T cells, CD45RA-CCR7-: effector memory T cells, CD45RA+CCR7-: effector T cells. (D) Quantification of inhibitory molecules (PD-1, LAG-3 and TIM-3) expression on CD4+CAR and CD8+CAR T cells on day 7 following lentiviral transfection. The analyses were performed using Students’ t tests. Data reflected the mean ± SEM of three independent experiments, *p< 0.05, **p < 0.01.