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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

Figure 6. Dual targeting with split co-stimulation and shared D3ζ promote TCR tonic signaling.

Figure 6.

(a) Schema of CAR-T cell stimulation and sample preparation for RNAseq. Both GD2.28ζ and GD2.28ζ/B7-H3.BB CAR-T cells were stimulated with 1 μg/mL 1A7 Ab and 1 μg/mL B7-H3-Fc protein coated plate for 24 hours, and then transferred to a new plate without any pre-coating and cultured for 4 more days. CAR-T cells were collected for RNAseq at days 0, 1 and 5. (b) RNAseq analysis of non-stimulated GD2.28ζ and GD2.28ζ/B7-H3.BB CAR-T cells. (c-f)) Gene set enrichment analysis (GSEA) of glycolytic (c), IFN-γ signaling pathways (d), TCR upregulated (e) and downregulated genes (f) in non-stimulated CAR-T cells expressing GD2.28ζ or GD2.28ζ/B7-H3.BB. (g) qPCR validation of TCR-related genes upregulated and down regulated in GD2.28ζ vs. GD2.28ζ/B7-H3.BB CAR-T cells in the absence of antigen stimulation; n = 4 independent donors, and data are shown as individual values and the mean + SD in C, *p <0.05, **p<0.01, two-tailed p value determined by unpaired t test. (h) Basal phosphorylation of CAR-CD3ζ, Erk1/2, and Akt in GD2.28ζ and GD2.28ζ/B7-H3.BB CAR-T cells in the absence of antigen stimulation. Data are from one experiment, representative of three independent experiments. (i) Time course of CAR-CD3ζ, Erk1/2, and Akt phosphorylation in GD2.28ζ and GD2.28ζ/B7-H3.BB CAR-T cells after CAR cross-linking (1A7 Ab for GD2.CAR and B7-H3-Fc protein for B7-H3.CAR). Data are from one experiment, representative of three independent experiments.