3.3. TR1419-28BBζ CAR-T cells exhibited dual antitumor effects
To further investigate whether TR1419-28BBζ CAR-T cells can mediate tumor apoptosis through binding to death receptor TRAIL-R1, we first generated TRAIL-R1 (full length) and TRAIL-R1 (truncation) lentivirus vectors. The TRAIL-R1 (full length) was composed of full TRAIL-R1, and the death domain of TRAIL-R1 was truncated in the TRAIL-R1 (truncation) [28] (Fig. 3A). We produced 293T (TRAIL-R1 full length) and 293T (TRAIL-R1 trunc) cells by lentiviral transfecting the associated plasmids (Fig. 3B).
Next, we generated TR1419-28BBζ CAR-T cells and TR1419∆ζ-T cells by lentiviral transduction, which transfection rates were detected by flow cytometry (Fig. 3C). Then, we performed cytotoxicity assays using TR1419-28BBζ CAR-T cells and TR1419∆ζ-T cells as effector cells, and different 293T cells as target cells. As for the TRAIL-R1 negative 293T cells, TR1419-28BBζ CAR-T cells and TR1419∆ζ-T cells had no significant killing effect and secretion of effector cytokines, compared with the mock T cells (Fig. 3D-F). The TR1419-28BBζ CAR-T cells and TR1419∆ζ-T cells effectively killed the 293T (TRAIL-R1 full length) cells, whereas TR1419-28BBζ CAR-T cells displayed significantly higher specific killing compared to TR1419∆ζ-T cells (Fig. 3D). Meanwhile, the data showed that much higher levels of IFN-γ and Granzyme B were produced by TR1419-28BBζ CAR-T cells compared to TR1419∆ζ-T cells when co-culturing with 293T (TRAIL-R1 full length) cells. However, no significant difference was found in cytokine secretion between TR1419∆ζ-T cells and mock T cells (Fig. 3E-F). As for the 293T (TRAIL-R1 trunc) cells, TR1419∆ζ-T cells had no significant killing effect and secretion of effector cytokines compared with the mock T cells, but TR1419-28BBζ CAR-T cells displayed significantly killing effect and IFN-γ and Granzyme B production (Fig. 3D-F). The results above suggested that TR1419-CAR-T cells can not only effectively induce T-cell mediated cytotoxicity toward tumor cells, but also mediate tumor cell apoptosis through activation of the death receptor-dependent apoptotic pathway, indicating dual anti-tumor activity.
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