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. Author manuscript; available in PMC: 2021 Nov 5.
Published in final edited form as: Nat Cancer. 2021 Sep 23;2(9):904–918. doi: 10.1038/s43018-021-00244-2

Extended Data Fig. 4. Cytotoxic activity of the double CAR-T cells with shared CD3ζ is antigen dependent.

Extended Data Fig. 4.

(a) Flow cytometry plots showing the expression of B7-H3 and GD2 in Raji cells wild type and B7-H3 expression in Raji cells transduced with a retroviral vector encoding B7-H3 (Raji-B7-H3). Representative of three independent experiments. (b-d) CAR-T cells (B7-H3.BB, B7-H3.BBζ, GD2.28ζ, GD2.28ζ/B7-H3.BB, dNGFR.28ζ/B7-H3.BB and 28ζ/B7-H3.BB) were co-cultured with Raji-B7-H3 cell at 1 to 1 ratio, and 5 days later tumor cells (CD19+) and T cells (CD3+) were collected and enumerated by flow cytometry (b). Supernatants of the co-cultures were collected 24 hours later, and IFN-γ (c) and IL-2 (d) released by CAR-T cells were measured by ELISA. Data are shown as individual values and the mean ± SD, n = 3 independent co-cultures using CAR-T cells generated from 3 different donors for dNGFR.28ζ/B7-H3.BB group, and n = 5 independent co-cultures using CAR-T cells generated from 5 different donors for all the other groups; *p <0.05 (0.0228 in c, 0.0141 in d ), **p <0.01 (0.0025 in c, 0.0015 in d), ***p = 0.0005, ****p <0.0001 by one-way ANOVA with Tukey’s multiple comparison test adjusted p value. (e-g) CAR-T cells (CD19.28ζ, GD2.28ζ/B7-H3.BB, dNGFR.28ζ/B7-H3.BB and 28ζ/B7-H3.BB) were co-cultured with Raji cell wild type at 1 to 1 ratio, and 5 days later tumor cells (CD19+) and T cells (CD3+) were collected and enumerated by flow cytometry (e). Supernatants of the co-cultures were collected 24 hours later, and IFN-γ (f) and IL-2 (g) released by CAR-T cells were measured by ELISA. Data are shown as individual values and the mean ± SD, n = 4 independent co-cultures using CAR-T cells generated from 4 different donors for each group; ****p <0.0001 by one-way ANOVA with Tukey’s multiple comparison test adjusted p value.