Fig. 1

PD-1 diminishes the advantages in central memory accumulation and long-term anti-tumor effects of CD8⁺ CAR-T cells with 4-1BB co-stimulation. a–c Construction and characterization of CD8⁺ CAR-T cells. Structures of 28ζ and BBζ CAR (a). CD8⁺ untransduced (UTD), 28ζ and BBζ Τ cells were incubated with target cells at the indicated E:T ratios for 24 h, after which target cell viability was determined according to bioluminescence (BLI) intensity (b). Tumor cells were incubated with listed T cells at E:T = 1:1 for 24 h, after which the supernatants were collected, and ELISA was performed to determine IFN-γ and IL-2 secretion (c). d–g Activation-induced memory differentiation and PD-1 expression of CD8⁺ CAR-T cells. HeLa cells expressing luciferase were adhered to the wells overnight, then purified CD8⁺ CAR-T cells were added at E:T = 1:10 without exogenous cytokines. The differentiation statuses of CAR-T cells were determined and the ratios of naive (T_Naive-like; CD45RA⁺CD62L⁺), central memory (T_CM; CD45RA⁻CD62L⁺), effector memory (T_EM; CD45RA⁻CD62L⁻), and most differentiated T (T_EMRA; CD45RA⁺CD62L⁻) cells were compared on day 0, 3, and 7 post-co-incubation (d). CAR-T cell expansions were determined on days 0, 3, and 7. At the indicated time point, suspended cells were collected, and dead cells were excluded by Trypan Blue staining. Live cells were then counted and compared with those of 28ζ cells on day 0 (e). The long-term tumoricidal activity of CAR-T cells was monitored via BLI assays (f). On days 0, 3, and 7, CAR-T cells were collected and PD-1 expression was determined using FACS (g). h–j PD-1 blockade increased T_cm subset and the persistence of CD8⁺ CAR-T cells. Luciferase-expressing HeLa cells were adhered to the well surface overnight, after which purified CD8⁺ CAR-T cells were added at E:T = 1:10 without exogenous cytokines. IgG and anti-PD-1 antibody were repeatedly added on days 0, 3, and 6 at a concentration of 20 μg/mL. On day 7 post-incubation, CAR-T cells were collected and their differentiation statuses were determined (h). CAR-T cell proliferation was determined on days 0, 3, and 7 and compared (i). Tumor cell proliferation was monitored via BLI assay and statistically analyzed (j). k, l PD-1 blockade enhances the central memory differentiation of CD8⁺ CAR-T cells in vivo. HeLa cells were subcutaneously inoculated into SCID-Beige mice. Ten days later, 5 × 10⁶ CD8⁺ CAR-T cells were injected once through the tail vein and the indicated antibodies were injected intraperitoneally four times every 3 days (n = 3 per group). On days 5 and 14 post-CAR-T cell infusion, tumor tissues were isolated, and single-cell suspensions were prepared. After excluding dead cells using Fixable Viability Dye eFluor™ 660, the differentiation status (k) and intratumoral accumulation (l) of CAR-T cells were determined between mice that received non-specific IgG or anti-PD-1 neutralizing antibody. m, n PD-1 blockade improved the anti-tumor effects of CD8⁺ CAR-T cells in vivo. Mice (n = 6 in each group) with established xenograft tumors were injected with 5 × 10⁶ CD8⁺ UTD or CAR-T cells once on day 0 and six times with IgG or anti-PD-1 antibody every 3 days after T cell infusion. Tumor volumes were calculated every 3 days after T cell injection (m). And the survival curves for mice with different treatments were demonstrated (n). Data presented as the means ± SD are representative of three independent experiments on CAR-T cells collected from at least three healthy donors. Statistical analysis was performed using t-test or ANOVA. Log-rank test was used to compare the difference of survival among mice. P-values < 0.05 are considered statistically significant. *P < 0.05; **P < 0.01; ***P < 0.005