Fig 1: Established CARs have specific reactions to target antigens. (A) Characteristic FC (left) and histograms (right) of CD69 expression levels of different CAR-T cells following 12 h of provoking by target antigens (recombinant human GPC3 and PD-1 proteins). (B and C) Concentrations of IL-2 and IFN-? released by the different CAR-T cells secreted upon encountering target antigens for 24 h, as examined by ELISA. Data are presented as the mean ± SD. *P<0.05, **P<0.01 and ***P<0.001. ns, not significant; CAR, chimeric antigen receptor; FC, flow cytometry; GPC3, glypican-3; PD-1, programmed death 1.
Fig 2: Double-target CAR-T cells show cytolytic potency to target HCC cells. (A) Cytotoxicity of GPC3/PD-1-CAR-T cells to the four indicated HCC cell lines at various E:T ratios evaluated by an LDH cytotoxicity assay. (B) Comparison of the toxicity of GPC3/PD-1-CAR-T cells to HuH7 cells with different GPC3 levels at E:T ratios of 4:1, 8:1, 16:1 and 32:1. Un-transduced T cells served as control. (C) Comparison of the toxicity of various CAR-T cells against HuH7 at different E:T ratios. Un-transduced T cells served as control. (D) Levels of GrB and perforin measured using ELISA after co-culturing different CAR-T cells with HuH7 at an E:T ratio of 1:1 for 24 h. (E) Comparison between the cytotoxicity of GPC3/PD-1-CAR-T cells and that of HuH7, PD-1+ T and T cells at various E:T ratios, as assessed by an LDH assay. (F) The death rate of HuH7 cells under a series of concentrations of GrB and perforin for 12 h. (G) The death rates of HuH7, PD-1+ T and T cells after co-culturing in the indicated culture medium for 12 h. Data are presented as the mean ± SD. *P<0.05, **P<0.01 and ***P<0.001. ns, not significant; CAR, chimeric antigen receptor; HCC, hepatocellular carcinoma; GPC3, glypican-3; PD-1, programmed death 1; E:T, effector-to-target; LDH, lactate dehydrogenase; GrB, granzyme B.
Fig 3: Double-target CAR-PD-1+ T cells exhibit high toxicity to tumour cells that highly express PD-L1. (A) Characteristic FC diagrams of the percentages of residual PD-L1+-HuH7 cells after a 3-day coculture with CAR-PD-1+-T or CAR-PD-1-T cells at an E:T ratio of 1:1 (left). GPC3 is a marker for HuH7 cells and CD3 for CAR-T cells. Un-transduced PD-1--T or PD-1+-T cells were used as controls. Measurement of ratios of residual tumour cells in each paired group (right). (B) Comparison of the cytotoxicity of GPC3/PD-1-CAR-PD-1+-T and GPC3-CAR-PD-1+-T cells combined with anti-PD-1 mAb to PD-L1+-HuH7 at different E:T ratios. (C) Concentrations of IL-2 and IFN-? after co-culturing GPC3/PD-1-CAR-PD-1+-T cells and GPC3-CAR-PD-1+-T cells + anti-PD-1 mAb with PD-L1+-HuH7 at an E:T ratio of 4:1 for 24 h, as measured by ELISA. Un-transduced PD-1+-T cells were used as controls (one-way ANOVA). Data are presented as the mean ± SD. *P<0.05 and ***P<0.001. ns, not significant; CAR, chimeric antigen receptor; PD-1, programmed death 1; GPC3, glypican-3; IFN, interferon; E:T, effector-to-target; FC, flow cytometry.
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