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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. 9. MSLN and CSPG4 dual targeting CAR-T cells with split co-stimulation and shared CD3ζ show sustained T cell activation and proliferation in vitro.

Extended Data Fig. 9.

(a) Representative flow cytometry plots showing the expression of CARs. (b) Summary of the transduction efficiency of the CARs (n = 7 or 9 independent experiments using CAR-T cells generated from 7 or 9 different donors). Data are shown as individual values and the mean + SD. (c-e) CAR-T cells co-cultured with GFP labeled H2052 cell at T cell to tumor cell ratio of 1 to 5. IFN-γ (d) and IL-2 (e) released by CAR-T cells. On day 5, tumor cells (GFP+) and CAR-T cells (CD3+) were measured by flow cytometry (c). 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 the CSPG4.BBζ group, n = 5 independent co-cultures using CAR-T cells generated from 5 different donors for the other groups; *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001 by one-way ANOVA with Tukey’s multiple comparison test adjusted p value, the full list of p values can be found in the source data. (f) Schema of the multi-round co-culture experiments of CAR-T cells and H2052. Tumor cells were seeded one day prior to the addition of T cells. At day 0, CAR-T cells were added at T cell to tumor cell ratio of 1 to 5. At the end of each round of co-culture, which are at days 5, 9, 13 and 17, one third of T cells were collected and transferred into a new well with 2.5 × 105 H2052 cells that were seeded one day before. T cells and tumor cells and cytokine released by CAR-T cells were quantified at each round of co-culture. (g-h) Summary of IFN-γ (g) and IL-2 (h) released by CAR-T cells in the multi-round co-culture with H2052 as described in (f). 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 the CSPG4.BBζ group, n = 4 independent co-cultures using CAR-T cells generated from 4 different donors for the other groups; *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001 by one-way ANOVA with Tukey’s multiple comparison test adjusted p value, the full list of p values can be found in the source data.