Fig. 6: GD2-B7H3 are metabolically fit and resilient to in vivo and in vitro exhaustion.

a Summary of the data representing expression of exhaustion markers PD1 and LAG3 enumerated by flow cytometry in UT, B7H3 CAR-T cells, GD2-B7H3 T cells co-cultured with CHLA255 NBL cells at indicated times. b Summary of the data representing expression of activation markers CD25 and CD27 enumerated by flow cytometry in UT, B7H3 CAR-T cells, and GD2-B7H3 gated CAR-T cells co-cultured with CHLA255 NBL cells at indicated times. c, left: Oxygen consumption rate (OCR) as measured by Seahorse assay in UT, B7H3 CAR-T cells, and GD2-B7H3 gated CAR-T cells co-cultured with CHLA255 NBL cells for 48 h. Right: Summary of the data representing oxygen consumption reserve in GD2-B7H3 T cells and B7H3 CAR-T cells. d GSEA enrichment plot of significantly ranked pathways (MsigDB C5 gene ontology) from RNA sequencing data of GD2-B7H3 T cells and B7H3 CAR-T cells co-cultured with CHLA255 NBL cells for 3 days (tumor cells are eliminated in either group after co-culture). e Heatmap of gene expression of exhaustion-related transcription factors (TBX21, EOMES, PRDM1, IKZF2), inhibitory receptors (LAG3, HAVCR2, CTLA4, BTLA, CD244), and transcription factors reported being preferentially expressed in memory T cells (KLF6, JUN, JUNB) from RNA sequencing data of GD2-B7H3 T cells and B7H3 CAR-T cells isolated post co-culture with CHLA255 NBL cells for 3 days (tumor cells are eliminated in either group after co-culture). f Histogram of PD1, LAG3, TIM3 protein expression in T cells co-cultured repeatedly with CHLA255 cells for 12 days. g Bioluminescence images of CHLA255 tumor-bearing mice treated 1 day after tumor injection with i.v. injection of 1 × 10⁷ GD2-B7H3 or B7H3 CAR-T cells previously co-cultured repeatedly with CHLA255 cells for 12 days. Surviving mice were followed for a minimum of 100 days post-tumor inoculation. h Summary data representing CAR-T-cell count in peripheral blood of animals in Fig. 5g at week 1 and week 5 post CAR-T-cell infusion. Data shown are representative of three independent experiments (a–e), mean ± SD (a–c). Two-tailed t test (a–c, h). n = 4 mice (GD2-B7H3, B7H3). Data shown are representative of three individual mice, remaining images are included in the Supplementary Information (e). Individual BLI and source data are provided as a Source Data file.