Fig.1.TR1419-28BBζ CAR-T cells had specific killing to tumor cells. (A) CAR construct with humanized anti-TRAIL-R1 scFv, CD8a hinge, CD28 transmembrane domain and intracellular co-stimulatory domain, CD137 intracellular co-stimulatory domain, and intracellular CD3ζ activation domain. (B) Expression rate of the CAR on T cells was confirmed by flow cytometry on day 7 following lentiviral transfection. Mock T cells were served as a negative control. (C-D) Expression of inhibitory receptors and subtypes on CAR-T cells was examined by flow cytometry, CD45RA+CCR7+: naïve T cells, CD45RA-CCR7+: central memory T cells, CD45RA-CCR7-: effector memory T cells, CD45RA+CCR7-: effector T cells. (E) Cytotoxic activity of TR1419-28BBζ CAR- or mock transduced T cells against TRAIL-R1 positive tumor cell lines. The effector cells were co-cultured for 6 h with target cells at E:T ratio of 10:1, 5:1 and 2.5:1, respectively. Cytotoxicity was determined by Calcein AM release-based cytotoxic cell assay. (F-G) IFN-γ and Granzyme B production by TR1419-28BBζ CAR-T cells or mock-transduced T cells was detected by ELISA when co-cultured with the indicated cells for 24 h. The analyses were performed using Students’ t tests. Data reflected the mean ± SEM of three separate experiments.*p < 0.05, **p < 0.01 and ***p< 0.001.