Fig. 1

The disruption of SHP-1 enhanced the cytolysis of CAR T cells in vitro. (A)Optimize the protocol of gene knockout CAR-T cells. (B) U251 cells were transfected with CD133 cDNA, and CD133 expression on cell surface was evaluated by flow cytometry. (C) The CD133 CAR T and SHP-1 KO CD133 CAR T cells were co-cultured with U251-CD133 target cells at the effect-to-target ratios of 1:1, 4:1 and 16:1 respectively, and the survival of target cells were observed after 16 h. (D) The indicated CAR-T cells and target tumor cells were cocultured for 16 h at the effector-to-target ratios of 1:1, 4:1, 8:1 and 16:1 respectively, and the lysis of target cells were measured. (E) The expression of CD107a on the cell membrane of CD133 CAR-T and SHP-1 KO CD133 CAR T cells under the stimulation of CD3 antibody were evaluated by flow cytometry. (F)-(H) The supernatant was collected after CD133 CAR-T cells or SHP-1 KO CD133 CAR T cells were co-cultured with CD133 negative U251 cells (U251) or CD133 positive U251 cells (U251-CD133) for 16 h, and the levels of IL-2 (E), TNFα (F) and INF-γ (G) cytokines were determined by ELISA assay. *P < 0.05 or **P < 0.01 indicates a significant difference between the indicated groups (n = 3, two-way ANOVA in D and one-way ANOVA in F, G and H, and Tukey posttest). ns, not significant