Fig. 4

PD-L1.BB CSR engagement enhances 4-1BB expression and inhibits PD-1/PD-L1signals. a, b Statistical analysis of 4-1BB expression on HER2.28ζ and HER2.28ζ/PD-L1.BB CAR-T cells after stimulation. CAR-T cells were stimulated by either a irradiated SKOV3^PD-L1 or b SKOV3 cells (E: T = 2: 1) in culture medium, and 4-1BB expression was assessed by flow cytometry at the indicated time points. c Representative flow cytometry plots showed PD-1 expression on CD4⁺ and CD8⁺ CAR-T cells on day 5 after stimulation (E: T = 2: 1). d Statistical analysis of PD-1 expression on CD4⁺ and CD8⁺ CAR-T cells. e PD-L1 expression on CAR-T cells. CAR-T cells were incubated with irradiated SKOV3^PD-L1 cells (E: T = 2: 1) for 5 days, either in culture medium or MPE-supernatant (Pt1, as Supplementary Table. S1). f The cytotoxicity and cytokine release of HER2.28ζ and HER2.28ζ/PD-L1BB CAR-T cells before and after PD-1 blockade. CAR-T cells were stimulated with irradiated SKOV3^PD-L1 cells, and treated with PD-1 blocking antibody (Tislelizumab, 10 μg/ml) for 3 days. Then the post-stimulated CAR-T cells were co-incubated with SKOV3^PD-L1 (E: T = 2: 1) for 24 h. The cytotoxic activity of CAR-T cells was analyzed by CCK-8, and effector cytokine (IFN-γ, IL-2 and TNF-α) in culture supernatant were measured by ELISA. P-values were determined by one-way ANOVA with Tukey’s multiple comparison test adjusted P value (a, b, f) or unpaired two-tailed t-test d. *P <0.05; **P <0.01, ns, not significant. Data show the mean ± SD from three independent experiments