Extended Data Fig. 5: Pharmacological inhibition of IDH2 boosts the antitumour function of adoptively transferred cells in B16 melanoma tumor models.

a, Representative dot plots of anti-HER2-CAR expression on BFP- or anti-HER2-CAR- transduced CD8⁺ T cells and representative histogram of CD62L protein expression on the cell surface of HER2-CAR CD8⁺ T cells treated with DMSO or IDH2i, analysed by flow cytometry at day 7 post-activation. b, Representative histogram of HER2 protein expression and isotype control on the cell surface of B16-HER2 tumour cells compared to B16 tumour cells, analysed by flow cytometry. c, Experimental set-up of B16-HER2 tumour experiment. Polyclonal CD8⁺ T cells were transduced with blue fluorescent protein (BFP)-expressing vector or anti-HER2-CAR, expanded during 7 days in the presence of DMSO or IDH2i and subsequently transferred into B16-HER2 tumour-bearing mice. d,e, Weight (d) and photo (e) of B16-HER2 tumours from mice transferred with either DMSO or IDH2i-conditioned anti-HER2-CAR T cells. f,g, Number of transferred PD-1⁺TCF1⁺ cells (f) and PD-1⁺TCF1⁻ cells (g) DMSO- or IDH2i-conditioned CD8⁺ HER2-CAR TILs, analysed by flow cytometry 21 days post-tumour engraftment. h–k, Percentages of TCF1⁺ (h), PD-1⁺ (i), TIM3⁺ (j) and LAG3⁺ (k) cells out of transferred HER2-CAR TILs. l, Percentages of PD-1⁺TIM3⁺LAG3⁺ cells out of transferred HER2-CAR TILs. m, Percentage of granzyme B⁺ HER2-CAR⁺ CD8⁺ TILs upon ex vivo restimulation with PMA-ionomycin. (d–m, n = 13 (DMSO HER2-CAR), n = 12 (IDH2i HER2-CAR) and n = 8 (control, DMSO BFP and IDH2i BFP) biological replicates per group, pooled data from 2 independent experiments). n,o, Tumour growth curve (n) and weight (o) of B16-OVA tumour-bearing mice transferred with T cells conditioned with DMSO or IDH2i (AG-221). p, Percentages of CD44⁺CD62L⁺ cells out of transferred CD8⁺ T cells in the spleen 23 days post-tumour engraftment. q, Percentages of TCF1⁺ cells out of transferred CD8⁺ T cells in the spleen 23 days post-B16-OVA tumour engraftment. r, Number of transferred DMSO- or IDH2i-conditioned CD8⁺ TILs per milligram of tumour, analysed by flow cytometry 23 days post-B16-OVA tumour engraftment. s,t, Percentages of PD-1⁺TCF1⁺ cells (s) and PD-1⁺TCF1⁻ cells (t) out of transferred CD8⁺ TILs. u–y, Percentage of IFNγ and TNF (u), granzyme B (v), and CD107a (y) production and representative FACS plots of IFNγ and TNF (w) and granzyme B (x) production by transferred TILs upon ex vivo restimulation with OVA peptide. (n–y, n = 7 biological replicates per group). z, Tumour growth curve of B16-OVA tumour-bearing mice transferred with OT1 cells conditioned with DMSO, AG-221 or AGI-6780. (n = 7 biological replicates per group). Data represent mean ± s.e.m. and were analysed by unpaired, two-tailed Student’s t-test (f–v,y,z) and one-way ANOVA using Tukey’s multiple comparison test (d). Only relevant statistical comparisons are shown.

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