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).