(a) Flow cytometry histogram showing the expression of GD2 and B7-H3 in two human NB cell lines, CHLA-255 and LAN-1. Representative of three independent experiments. (b) Representative flow cytometry histograms showing the expression of CARs in human T cells transduced with retroviral vectors encoding CD19.28ζ, GD2.28ζ, GD2.BBζ, B7-H3.28ζ, and B7-H3.BBζ CARs. (c-e) Representative flow cytometry plots (c) and quantification of residual CHLA-255 (d) and LAN-1 (e) cells labelled with GFP and co-cultured with CAR-T cells at the T cell to tumor cell ratio of 1 to 5. On day 5, NB cells (GFP+) and CAR-T cells (CD3+) were enumerated by flow cytometry. Data are shown as individual values and the mean ± SD, n = 6 independent co-cultures using CAR-T cells generated from 6 different donors. (f,g) Summary of IFN-γ (f) and IL-2 (g) released by CAR-T cells in the culture supernatant after 24 hours of co-culture with NB cells as measured by ELISA. Data are shown as individual values and the mean ± SD, n = 6 independent co-cultures using CAR-T cells generated from 6 different donors. (h) Representative CFSE dilution of CSFE-labeled CAR-T cells co-cultured with NB cells for 5 days at the T cell to tumor cell ratio of 1 to 1 (red histogram). CFSE-labeled CAR-T cell alone (grey histogram) was used as negative control. Representative of three independent experiments.