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.