Fig. 3

Enhanced specific cytotoxic effects of CD133 CAR-T and PD-1 s cells against CD133 + HCC cell lines. A CAR expression in Mock, CD133 CAR-T and CD133 CAR-T and PD-1 s cells was assessed by flow cytometry after enrichment with anti-c-Myc-tag-biotin and anti-biotin microbeads. B RT-QPCR and western blot analysis of the CAR gene number and CD3ζ expression in Mock T, CD133 CAR-T, and CD133 CAR-T and PD-1 s cells. C The CD8-positive subset of CD133 CAR-T and PD-1 s cells and their CCR7 and CD45R expression by flow cytometric analysis. D Flow cytometry analysis of CD133 expression in HCC cell lines (SK-Hep-1 and Hep3B). E The upper images show Mock T, CD133 CAR-T and CD133 CAR-T and PD-1 s cells targeting Hep3B cells. The red arrow represents the CAR-T cells, and the yellow arrow represents the tumour. The lower image shows the proportion of Mock T, CD133 CAR-T cells or CD133 CAR-T and PD-1 s cells incubated with CD133 + HCC cell lines (Hep3B) after 24 h. F Specific lysis (ratio of 7AAD-positive cells to CD133 + tumour total cells) after 3 days of incubation of CAR-T cells with tumour cells. G Representative flow cytometric image of CD107a expression in Mock T cells, CD133 CAR-T cells, and CD133 CAR-T and PD-1 s cells incubated with tumour cells for 4 h. H The frequency of CD107a expression on Mock T, CD133 CAR-T, CD133 CAR-T and PD-1 s cells alone or incubated with Hep3B cells by flow cytometric analysis. I Cytokine production (IFNγ and TNFα) by Mock T cells, CD133 CAR-T cells, and CD133 CAR-T and PD-1 s cells with or without tumour cell incubation for 4 h was measured by ELISA. Mean ± SD, n = 4, two-tailed unpaired Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant