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.
.