Fig.3.TR1419-28BBζ CAR-T cells mediated dual tumour cell killing. (A) Schematic representation of TRAIL-R1 (full length) and TRAIL-R1 (truncation). TRAIL-R1 (full length) comprised the extracellular region, the transmembrane domain, and the intracellular region with death domain. TRAIL-R1 (truncation) comprised the extracellular region, the transmembrane domain, and the intracellular region without death domain, SP: signal peptide, TM: transmembrane domain, DD: death domain. (B) 293T cells were transfected with the lentiviral expression TRAIL-R1 trunc and TRAIL-R1 full length plasmids, respectively. Then, the expression of TRAIL-R1 in 293T cells was determined by flow cytometry. (C) Mock (untransduced), TR1419-28BBζ CAR-T cells or TR1419∆ζ-T cells were evaluated by flow cytometry for scFv expression. (D) Cytotoxic activity of TR1419-28BBζ CAR- and TR1419∆ζ-T cells against 293T, 293T (TRAIL-R1 trunc) and 293T (TRAIL-R1 full length) was determined by RTCA assay. (E, F) IFN-γ and Granzyme B production by TR1419-28BBζ CAR-T cells or TR1419∆ζ-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 triplicate wells. *p < 0.05, **p< 0.01 and ***p < 0.001.

3.4. The costimulatory domains did not influence the phenotypes of TR1419-CAR-T cells

It has been reported that the phenotypic and functional differences of the second and third generation CAR-T cells are related to the selection of target antigen and the design of scFv structure [29,30], which suggested that we should select the most appropriate CARs based on our own scFv structure. To explore which CARs structure is the most appropriate for scFv from TR1419, we first constructed CD28-containing (TR1419-28ζ) and 4-1BB-containing (TR1419-BBζ) TR1419-CARs (Fig. 4A). For the following studies, we generated TR1419-28ζ, TR1419-BBζ and TR1419-28BBζ CAR-T cells by lentiviral transduction. The surface expression of CAR on the T cells was measured by flow cytometry. Three TR1419-CARs showed comparable transfection rates and mean fluorescence intensity (Fig. 4B). Meanwhile, TR1419-28ζ, TR1419-BBζ and TR1419-28BBζ CAR-T cells exhibited similar cell surface T cell phenotypes, of which approximately 60% were Tn (Fig. 4C). CD8+TR1419-28BBζ CAR-T cells showed higher levels of PD-1 and LAG-3 expression than CD8+TR1419-28ζ CAR-T cells. However, CD4+ TR1419-28ζ CAR-T cells showed higher levels of TIM-3 expression than CD4+TR1419-28BBζ CAR-T cells (Figure 4D). (Fig. 4D).