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. Author manuscript; available in PMC: 2022 Nov 17.
Published in final edited form as: Sci Transl Med. 2021 Nov 17;13(620):eabh0272. doi: 10.1126/scitranslmed.abh0272

Fig. 4. IL-10 promotes DNMT3A KO CAR T cell survival.

Fig. 4.

(A) Activated human T cells were electroporated with Ctrl (mCherry), DNMT3A, IL-10, or DNMT3A and IL-10 (DKO) RNPs. At day 3, gene-edited T cells were transduced with retroviral vector encoding HER2.ζ-CAR. (B to E) CAR T cells were incubated with U373 tumor cells at a 2:1 E:T ratio in the presence of exogenous IL-15 and restimulated with fresh tumor cells on a weekly basis until the T cells stopped killing tumor cells. (B) Cell culture supernatants were harvested 24 hours after each stimulation, and an IL-10 ELISA was used to determine IL-10 cytokine production. Data are presented as means ± SEM (n = 5). (C) Gene-edited CAR T cell expansion was measured after weekly stimulations with U373 cells for HER2.ζ-CAR. Each graph represents one healthy donor. (D) Left: A summary graph of CAR T cell expansion (relative to Ctrl) for all donors up to five stimulations is shown (n = 4 donors). Right: A summary graph of CAR T cell expansion (relative to Ctrl) for all donors in the presence of exogenous IL-10 is shown (n = 3 donors). A two-way ANOVA with Sidak’s multiple comparisons test was used to compare DNMT3A KO and DNMT3A IL-10 DKO at each stimulation; ns, not significant; **P < 0.01. Data are presented as means ± SEM. (E) MPIs of Ctrl, IL-10 KO, DNMT3A KO, and DKO CAR T cells were measured before stimulation and after 4 weeks of chronic stimulation (n = 3). Data were analyzed by an unpaired t test; *P < 0.05. Each dot represents data from a single donor. (F) The total number of DMRs between week 4 DNMT3A KO (D3A KO), IL-10 KO, and DKO CAR T cells is shown. (G) Representative methylation profiles are shown for the CD28, LEF1, and TCF7 loci among week 4 Ctrl, DNMT3A KO, IL-10 KO, and DKO CAR T cells. DMRs are represented by a purple box. (H) GO analysis of DMRs between week 4 DNMT3A KO, IL-10 KO, and DKO CAR T cells is shown. VEGFA-VEGFR2, vascular endothelial growth factor A–vascular endothelial growth factor receptor 2; GPCRs, G protein–coupled receptors; miRNA, microRNA; ECM, extracellular matrix. TYROBP, protein tyrosine kinase-binding protein; NRF2-ARE, nuclear erythroid 2-related factor 2-antioxidant response element; ATM, ATM Serine/Threonine Kinase.