Fig. 3

Generation of GD2/CAR-T cells in vitro. a Schematic representation of GD2-based CAR constructs containing the CD3 ζ cytosolic domain in combination with the CD137 costimulatory module (GD2.BBζ CAR). VL variable L chain, L linker, VH variable H chain, and TM transmembrane region. b The expression of CAR-GD2 was assessed by FACS analysis using the anti-idiotypic antibody 1A7 raised against anti-GD2 mAb 14G2a. The graph shows representative expression levels of CAR-GD2 in non-transduced T cells and GD2.BBζ CAR-T cells. c The overall transduction efficiency of CAR-T cell manufacture. d The expression of CAR-GD2 in CD4⁺ and CD8⁺ T lymphocytes after the gene transfer. Following the selection of GD2⁺ T cells, GD2.BBζ CAR-T cells consisted of 49.8% CD8⁺ T cells and 40.1% CD4⁺ T cells. Following the selection of GD2⁻ T cells, non-transduced T cells consisted of 54.1% CD8⁺ T cells and 42.7% CD4⁺ T cells. e Activation marker expression of GD2.BBζ CAR-T cells on 9 days after initial activation. f Exhaustion marker expression of GD2.BBζ CAR-T cells on 9 days after initial activation. g Tcm phenotypic features of GD2.BBζ CAR-T and non-transduced T cells were evaluated by FACS analysis on day 9 of culture initial activation. Mean positive rates ± SD from three different T cell lines are shown