3.2. ScFv from TR1419 CAR was proved to mediate
tumor apoptosis in Jurkat cells
To explore whether scFv from TR1419 CAR could mediate
tumor apoptosis through activation of the death receptor-dependent
apoptotic pathway, we first constructed TR1419∆ζ
lentivirus vector, which was composed of scFv from
TR1419 fused to a CD8α hinge-transmembrane region, and
truncated CD3ζ region (Fig. 2A). We generated
TR1419-28BBζ or TR1419∆ζ-jurkat
cells by lentiviral transduction, and the surface expression of scFv on
the jurkat cells was measured by flow cytometry
(Fig. 2B). Then, the xCELLigence RTCA DP was
adopted to determine whether TR1419-28BBζ or
TR1419∆ζ-jurkat cells could mediate tumor apoptosis.
We have previously demonstrated
with CD19-CAR that CAR-Jurkat
cells have no ability to mediate the death of target antigen-positive
tumor cells (Fig. S2). However, our results showed that
TRAIL-R1-positive tumor cells were lysed by
TR1419-28BBζ-jurkat cells and
TR1419∆ζ-jurkat cells, and the specific cytotoxic
effect can be blocked by the soluble target antigen TRAIL-R1
(Fig. 2C).
Next, TR1419-28BBζ-jurkat cells and
TR1419∆ζ-jurkat cells were co-incubated separately
with SW480 cell lines for 2 h at a 1:1 ratio, then the expression of
AnnexinⅤ/7-AAD and caspase3 in SW480 cells was detected by flow
cytometry. The percentages of 7-AAD−Annexin
V+ early (25.3%) and 7-AAD+Annexin
V+ late (24.9%) apoptotic SW480 cells mediated by
TR1419-28BBζ-jurkat cells and
TR1419∆ζ-jurkat cells were significantly higher than
the control (Fig. 2D). The percentages of
Caspase3+ in SW480 cells mediated by
TR1419-28BBζ-jurkat cells and
TR1419∆ζ-jurkat cells were also significantly higher
than the control. Meanwhile, the soluble target antigen blocked 100% of
the above killing activity. Collectively, these results suggested scFv
from TR1419 CAR can mediate TRAIL-R1-positive tumor
apoptosis through activation of the death receptor-dependent apoptotic
pathway.