3.5. TR1419-28BBζ CAR-T cells proliferated more
rapidly than TR1419-28ζ CAR-T cells and
TR1419-BBζ CAR-T cells upon antigen stimulation
To explore the functional differences among
TR1419-28ζ, TR1419-BBζ and
TR1419-28BBζ CAR-T cells, we first examined the
expression of CD137 in TR1419CAR-T cells after 24
hours of stimulation with different concentrations of plate-bound
recombinant human TRAIL-R1 protein. While TR1419-28ζ,
TR1419-BBζ and TR1419-28BBζ CAR-T
cells exhibited similar CD137 expression against 0.5ug/ml, 0.01ug/ml and
0.005ug/ml antigen protein, TR1419-28BBζ CAR-T cells
induced higher CD137 expression against 0.1ug/ml and 0.05ug/ml antigen
protein (Fig. 5A). Then, we performed in vitro
tumor killing assays. TR1419-28ζ,
TR1419-BBζ and TR1419-28BBζ CAR-T
cells were co-cultured with various tumor targets, and Calcein AM
release-based cytotoxic cell assay was used to quantify tumor cell
killing. The results showed that TR1419-28ζ,
TR1419-BBζ and TR1419-28BBζ CAR-T
cells killed TRAIL-R1-expressing tumor cells with similar efficiency
(Fig. 5B).
The proliferation of TR1419-CAR-T cells upon antigen
stimulation was assessed using CFSE staining, and the CFSE fluorescence
declined following each cell division. The expression CFSE of
TR1419-CAR-T cells was detected after co-cultured with
TRAIL-R1 positive SW480 cells for 7 days. TR1419-28BBζ
CAR-T cells showed superior expansion compared with
TR1419-28ζ and TR1419-BBζ CAR-T
cells against TRAIL-R1 positive tumors, and no significant difference
was found in cell proliferation between TR1419-28ζ and
TR1419-BBζCAR-T cells (Fig.
5C). Meanwhile, we also detected the expression of PD-1 in
TR1419CAR-T cells after co-incubation with SW480. The
results indicated that PD-1 was more frequently expressed by
TR1419-28BBζCAR-T cells than
TR1419-28ζ and TR1419-BBζCAR-T
cells. However, the frequencies of PD-1+ CAR-T cells
between TR1419-28ζ and TR1419-BBζ
groups were similar (Fig. 5D). There has shown
that the highest level of PD-1 expression reflects T-cell exhaustion
status [31]. Thus, based on positive
PD-1 expression, PD-1+TR1419CAR-T
cells were divided into two subgroups: high PD-1 expressing cells
(PD-1high) and low PD-1 positive cells
(PD-1low). There was no significant difference in the
expression of PD-1low among the
TR1419-28ζ, TR1419-BBζ and
TR1419-28BBζ CAR-T cells. However,
PD-1high was more frequently expressed by
TR1419-28BBζCAR-T cells than
TR1419-BBζCAR-T cells (Fig.
5E). In summary, TR1419-28BBζ CAR-T cells
proliferated more rapidly than TR1419-28ζ CAR-T cells
and TR1419-BBζ CAR-T cells upon antigen stimulation,
but expressed higher levels of PD-1.