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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. 7. Inverting the orientation of the B7-H3-specifc CAR and GD2-specific CAR does not alter the beneficial effects of dual targeting CAR-T cells with split costimulation and shared CD3ζ in vitro.

Extended Data Fig. 7.

(a) Schematic representation of retroviral vectors encoding B7-H3.28ζ, GD2.BB and B7-H3.28ζ/GD2.BB CARs. (b) Representative flow cytometry plots of 5 independent experiments showing the expression of CARs. (c) Summary of the transduction efficiency of the CARs. Data are shown as individual values and the mean ±; SD, n = 5 or 7 independent experiments using CAR-T cells generated from 5 or 7 different donors; ***p = 0.002, ****p <0.0001 by one-way ANOVA with Tukey’s multiple comparison test adjusted p value. (d-f) CAR-T cells were co-cultured with CHLA-255-GFP at T cell to tumor cell ratio of 1 to 5. IFN-γ (e) and IL-2 (f) released by CAR-T cells were measured by ELISA. On day 5, tumor cells (GFP+) and CAR-T cells (CD3+) number were measured by flow cytometry (d). Data are shown as individual values and the mean ± SD, n = 3 independent co-cultures using CAR-T cells generated from 3 different donors ; *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. (g) Schema of the repetitive multi-round co-culture experiments. Tumor cells were seeded in 24-well plates 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 day 4, 7, and 10, all T cells were collected and transferred into a new well in which 5 × 105 NB cells were seeded one day before. T cells and tumor cells, and cytokine were quantified at each cycle. (h-o) Multi-round co-culture with NB cell lines CHLA-255 (h-k) and LAN-1 (l-o) cells as described in (g). Summary of percentage of residual CHLA-255 (h) and LAN-1 (l) cells and number of T cells (i, m) at the end of each round of co-culture. Summary of IFN-γ (j, n) and IL-2 (k, o) released by CAR-T cells in the culture supernatant after 24 hours of co-culture with CHLA-255 (j, k) and LAN-1 (n, o) cells. Data are shown as individual values and the mean ± SD, n = 6 independent co-cultures using CAR-T cells generated from 6 different donors; *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.