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Reductive carboxylation epigenetically instructs T cell differentiation

Nature volume 621, pages 849–856 (2023)Cite this article

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Abstract

Protective immunity against pathogens or cancer is mediated by the activation and clonal expansion of antigen-specific naive T cells into effector T cells. To sustain their rapid proliferation and effector functions, naive T cells switch their quiescent metabolism to an anabolic metabolism through increased levels of aerobic glycolysis, but also through mitochondrial metabolism and oxidative phosphorylation, generating energy and signalling molecules1,2,3. However, how that metabolic rewiring drives and defines the differentiation of T cells remains unclear. Here we show that proliferating effector CD8+ T cells reductively carboxylate glutamine through the mitochondrial enzyme isocitrate dehydrogenase 2 (IDH2). Notably, deletion of the gene encoding IDH2 does not impair the proliferation of T cells nor their effector function, but promotes the differentiation of memory CD8+ T cells. Accordingly, inhibiting IDH2 during ex vivo manufacturing of chimeric antigen receptor (CAR) T cells induces features of memory T cells and enhances antitumour activity in melanoma, leukaemia and multiple myeloma. Mechanistically, inhibition of IDH2 activates compensating metabolic pathways that cause a disequilibrium in metabolites regulating histone-modifying enzymes, and this maintains chromatin accessibility at genes that are required for the differentiation of memory T cells. These findings show that reductive carboxylation in CD8+ T cells is dispensable for their effector response and proliferation, but that it mainly produces a pattern of metabolites that epigenetically locks CD8+ T cells into a terminal effector differentiation program. Blocking this metabolic route allows the increased formation of memory T cells, which could be exploited to optimize the therapeutic efficacy of CAR T cells.

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Fig. 1: Deletion of IDH2 inhibits RC in TE cells and promotes the differentiation of TM cells.
Fig. 2: Inhibition of IDH2 improves the function of CAR T cells.
Fig. 3: IDH2 inhibition leaves an epigenetic imprint.
Fig. 4: IDH2 inhibition alters the balance of metabolites, which dictates epigenetic memory.

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Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information. RNA-seq data are available in the NCBI’s GEO database under the accession code GSE192395. ATAC-seq data are available in the NCBI’s GEO database under the accession code GSE192394. Source data are provided with this paper.

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Acknowledgements

We thank C. Vuillefroy de Silly for technical assistance; G. Gyülvészi for scientific discussion; and M. Mittelbrunn and S.-S. Im for providing TFAM and IDH2 KO splenocytes. Metabolomic analysis was performed at the VIB Metabolomics Core at the University of Leuven; RNA library preparation and RNA-seq were performed at the Lausanne Genomic Technologies Facility, University of Lausanne; and ATAC-seq was performed at the Gene Expression Core Facility at the Ecole Polytechnique Fédérale de Lausanne. D.M. is supported by the ISREC Foundation, PHRT Foundation, Ligue Genevoise Contre le Cancer, Fondation Dr Henri Dubois-Ferrière Dinu Lipatti and the Swiss Innovation Agency (Innosuisse). M.W., P.-C.H. and P.R. were supported in part by a grant from Roche. P.R. was supported in part by the SNSF grant 310030_182735 and Oncosuisse KFS-4404-02-2018. P.-C.H. is funded in part by a European Research Council Starting Grant (802773-MitoGuide), a European Molecular Biology Organization (EMBO) Young Investigator award, SNSF project grants (31003A_182470), the Cancer Research Institute (Lloyd J. Old STAR award) and a Melanoma Research Alliance Established Investigator award. N.M.-P. was funded by the EMBO and the European Federation of Immunological Societies (EFIS). C.A. receives funding from Swiss Cancer Research KFS-4542-08-2018-R, Stiftung für Krebsbekämpfung, Fondation Leenaards, Helmut Horten Stiftung and Fondation Muschamp. J.A.R. was a recipient of a Swiss Government Excellence Scholarship.

Author information

Author notes

  1. These authors jointly supervised this work: Pedro Romero, Ping-Chih Ho, Mathias Wenes

Authors and Affiliations

  1. Department of Oncology, University of Lausanne, Lausanne, Switzerland

    Alison Jaccard, Tania Wyss, Alessio Bevilacqua, Jhan-Jie Peng, Fabien Franco, Kung-Chi Kao, Pedro Romero, Ping-Chih Ho & Mathias Wenes

  2. Ludwig Institute for Cancer Research Lausanne, University of Lausanne, Épalinges, Switzerland

    Alison Jaccard, Jan A. Rath, Alessio Bevilacqua, Jhan-Jie Peng, Christine Von Gunten, Fabien Franco, Kung-Chi Kao, Nicolas Camviel, Caroline Arber & Ping-Chih Ho

  3. Translational Data Science (TDS) Group, AGORA Cancer Research Center, Swiss Institute of Bioinformatics (SIB), Lausanne, Switzerland

    Tania Wyss

  4. Department of Genomic Medicine, Pfizer-University of Granada-Junta de Andalucía, Centre for Genomics and Oncological Research (GENYO), Granada, Spain

    Noelia Maldonado-Pérez & Francisco Martín

  5. Department of Oncology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland

    Jan A. Rath, Christine Von Gunten, Nicolas Camviel & Caroline Arber

  6. AGORA Cancer Research Center, Lausanne, Switzerland

    Jan A. Rath, Anouk Lepez, Christine Von Gunten, Nicolas Camviel, Denis Migliorini, Caroline Arber & Mathias Wenes

  7. Swiss Cancer Center Léman, Lausanne, Switzerland

    Jan A. Rath, Anouk Lepez, Christine Von Gunten, Denis Migliorini, Caroline Arber & Mathias Wenes

  8. Center for Molecular and Clinical Immunology, Chang Gung University, Taoyuan, Taiwan

    Jhan-Jie Peng

  9. Brain Tumor and Immune Cell Engineering Group, Faculty of Medicine, University of Geneva, Geneva, Switzerland

    Anouk Lepez, Denis Migliorini & Mathias Wenes

  10. Center for Translational Research in Onco-Hematology, University of Geneva, Geneva, Switzerland

    Anouk Lepez, Denis Migliorini & Mathias Wenes

  11. Metabolomics Expertise Center, Center for Cancer Biology, VIB, KU Leuven, Leuven, Belgium

    Bart Ghesquière

  12. Laboratory of Applied Mass Spectrometry, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium

    Bart Ghesquière

  13. Department of Oncology, Geneva University Hospitals (HUG), Geneva, Switzerland

    Denis Migliorini

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  1. Alison Jaccard
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  2. Tania Wyss
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Contributions

A.J. conceived, designed and performed most experiments, analysed data and wrote the manuscript. T.W. performed bioinformatics analyses. N.M.-P., A.L., N.C. and C.V.G. performed experiments on human T cells. J.A.R. contributed to BCMA CAR T cell experiments and analysis. A.B. provided assistance with Seahorse experiments. J.-J.P., F.F. and K.-C.K. helped with cloning. B.G. supervised metabolomics experiments. C.A., D.M. and F.M. designed and supervised human CAR T cell experiments and provided scientific input. C.A. edited the manuscript. P.R. and P.-C.H. provided scientific input, supervised the project and edited the manuscript. M.W. conceived the study, designed experiments, analysed data, supervised the project and wrote the manuscript.

Corresponding authors

Correspondence to Pedro Romero, Ping-Chih Ho or Mathias Wenes.

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Competing interests

P.R. and M.W. are inventors on a patent application filed by the University of Lausanne related to memory induction by IHD2 inhibition and its application in cellular therapies. The remaining authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 RC is a unique metabolic feature of rapidly proliferating TE cells.

a, Experimental set-up of expanding effector (TE, blue) and resting memory (TM, red) T cell differentiation. OVA-specific CD8+ T cells from OT1 mice were activated with SIINFEKL (OVA) peptide and cultured with IL-2 for 3 days and with IL-15 for 4 additional days. TE cells were collected at day 3 and TM cells were collected at day 7 post-activation. Before collection, cells were cultured with [U-13C]-glutamine for 2 h and labelling patterns were analysed by mass spectrometry. b, Bar graphs representing the percentages of citrate isotopologues m + 0, m + 1, m + 2, m + 3, m + 4, m + 5 and m + 6 detected by [U-13C]-glutamine labelling in TE or TM cells. (n = 4 biological replicates per group). c–e, Percentages of aspartate (c), malate (d) and fumarate (e) isotopologues m + 0, m + 1, m + 2, m + 3 and m + 4 detected by [U-13C]-glutamine labelling in TE or TM cells. f–h, Ratios of m + 3 over m + 4 isotopologues of aspartate (f), malate (g) and fumarate (h) detected by [U-13C]-glutamine labelling in TE and TM cells. (n = 4 biological replicates per group). i–k, Percentage of isotopologues of palmitate (i), myristate (j) and stearate (k) labelling from [U-13C]-glutamine in TE or TM cells. (n = 3 biological replicates per group (TM cells) and n = 2 biological replicates per group (TE cells)). l, Glutamine consumption in mouse CD8+ TE and TM cells over 24 h at day 3 and day 7 post-activation respectively. (n = 3 biological replicates per group). m, Percentages of KI67+ out of CD8+ T cells in TE and TM cells. (n = 6 biological replicates per group, pooled data from 2 independent experiments). n, Experimental set-up of yellow fever vaccine administration to healthy volunteers, from which yellow fever tetramer+ CD8+ T cells were isolated from the blood at indicated time points. o, Gene expression in CD8+ T cells from healthy human volunteers vaccinated with yellow fever vaccine. Shown here are IDH3A, B and G reads per kilobase of transcript per million reads mapped in indicated cell subsets isolated from peripheral blood. (n = 3 (TE), n = 5 (TM) and n = 6 (TN) biological replicates). p, mRNA expression of Idh3a in indicated T cell subsets isolated from mouse spleens at day 7 or day 28 post-LM-OVA infection. Data are represented as fold change as compared to SLECs, normalized to β2-microglobulin expression. (n = 3 biological replicates). q, mRNA expression of Idh2 in indicated T cell subsets isolated from spleens of mice at day 7 or day 28 post-LM-OVA infection. Data are represented as fold change as compared to SLECs, normalized to β2-microglobulin expression. (n = 4 biological replicates). r, Representative immunoblot for IDH2 in control Scr or IDH2 deleted cells. s–v, Percentage of m + 1 cis-aconitate (s), fumarate (t), malate (u) and succinate (v) labelling from [1-13C]-glutamine in Scr or IDH2 deleted CD8+ T cells. (n = 3 biological replicates per group). Data represent mean ± s.e.m. and were analysed by unpaired, two-tailed Student’s t-test (c–h,l,m,s–v), multiple unpaired, two-tailed t-test (b,i–k) or one-way ANOVA using Tukey’s multiple comparison test (o–q). Only relevant statistical comparisons are shown. For gel source data, see Supplementary Fig. 1.

Source data

Extended Data Fig. 2 HIF1 drives IDH2 expression and RC in CD8+ T cells.

a, mRNA expression of Idh2 in mouse CD8+ TE cells upon treatment with DMSO and HIF-1α inhibitor (HIF-1αi). Data are represented as fold change as compared to DMSO-treated, normalized to β2-microglobulin expression. (n = 3 biological replicates per group). b,c, Representative immunoblot (b) and quantification (c) of IDH2 in mouse CD8+ T cells upon treatment with DMSO and HIF-1αi. A dashed line indicates where the immunoblot membrane was cropped (b) (n = 3 biological replicates per group). d,e, mRNA expression of Glut1 (d) and Pdk1 (e) in mouse TE cells treated with HIF-1αi. Data are represented as fold change as compared to DMSO-treated, normalized to β2-microglobulin expression. (n = 3 biological replicates per group). f–h, Representative immunoblot (f) and quantification of HIF-1α (g) and IDH2 (h) in TE or TM cells upon treatment with control or DMOG. (n = 3 biological replicates per group). i, Representation of human and mouse Idh2 transcripts, promoters and extended promoters with HIF-1α- recognized sequences highlighted in red. j, Ratio of α-KG over citrate in TE or TM cells at day 3 and day 7 post-activation respectively. (n = 4 biological replicates). k, Ratio of α-KG over citrate in mouse CD8+ TE cells treated with either 5 mM sodium dichloroacetate (DCA) or an equimolar sodium chloride control solution (-). (n = 3 biological replicates per group). l,m, Percentages of citrate m + 5 (l) and m + 4 (m) detected by 4 h [U-13C]-glutamine labelling in mouse CD8+ TE cells treated with either 5 mM sodium dichloroacetate (DCA) or an equimolar sodium chloride control solution (-). (n = 3 biological replicates per group). n, Ratio of m + 5 over m + 4 isotopologues of citrate detected by [U-13C]-glutamine labelling in mouse CD8+ TE cells treated with either 5 mM sodium dichloroacetate (DCA) or an equimolar sodium chloride control solution (-). (n = 3 biological replicates per group). o, Percentage of IL-2+ cells out of transferred Thy1.1+ CD8+ T cells upon ex vivo restimulation at day 28 post-primary infection. p,q, Percentage of TCF1+ cells out of transferred Thy1.1+ CD8+ T cells in the blood at day 8 (p) and day 28 (q) post-primary infection. r, Percentage of TCF1+ cells out of transferred Thy1.1+ CD8+ T cells in the spleen at day 7 post-secondary challenge. s, Percentage of CD44+CD62L+ cells out of transferred Thy1.1+ CD8+ T cells in the spleen at day 7 post-secondary challenge. (o–q, n = 11 (Scr gRNA) and n = 13 (Idh2 gRNA) biological replicates per group, pooled data from 3 independent experiments. r,s, n = 9 (Scr gRNA) and n = 8 (Idh2 gRNA) biological replicates per group, pooled data from 2 independent experiments.). Data represent mean ± s.e.m. and were analysed by unpaired, two-tailed Student’s t-test (a,c–e,j–s) or two-way ANOVA using the original FDR test of Benjamini and Hochberg (h,i). Only relevant statistical comparisons are shown. For gel source data, see Supplementary Fig. 1.

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Extended Data Fig. 3 Genetic deletion of IDH2 promotes the differentiation of memory CD8+ T cells.

a, Experimental set-up of OT1CD4CreCas9 CD8+ T cell retroviral transduction with Idh2 gRNA or control scramble (Scr) gRNA, followed by ACT into mice that are subsequently infected with LM-OVA. 34 days post-primary infection, Thy1.1-positive cells were isolated and transferred in new hosts followed by LM-OVA infection ( = secondary infection). b, Percentage of Thy1.1+ CD8+ out of live cells over the course of the primary infection. c–e, Percentage of TCF1+ (c), IFNγ+ TNF+ IL-2+ (d) and CD44+CD62L+ (e) cells out of transferred Thy1.1+ CD8+ T cells in the blood at day 28 post-primary infection. (b–e, n = 4 biological replicates per group). f, Representative flow cytometry graphs showing the percentage of TCF1+ cells out of transferred Thy1.1+ CD8+ T cells in the spleen at day 28 post-primary infection. g, Graphs representing the percentage of CD44+CD62L+ cells out of transferred Thy1.1+ CD8+ T cells in the spleen at day 34 post-primary infection. h,i, Percentage of CD44+ CD62L+ (h) and TCF1+ (i) cells out of transferred Thy1.1+ CD8+ T cells in the spleen, lymph nodes (LN) and bone marrow (BM) at day 34 post-primary infection. (h,i, n = 5 biological replicates per group). j–l, Percentage of Thy1.1+ cells out of CD8+ T cells in the spleen (j), lymph nodes (k) and bone marrow (l) at day 34 post-secondary infection with LM-OVA. m, Percentage of IFNγ+TNF+ cells out of transferred Thy1.1+ CD8+ T cells in the spleen at day 34 post-secondary infection with LM-OVA. (j–m, n = 4 (Idh2 gRNA) and n = 6 (Scr gRNA) biological replicates per group). Data represent mean ± s.e.m. and were analysed by unpaired, two-tailed Student’s t-test.

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Extended Data Fig. 4 Pharmacological inhibition of IDH2 boosts the formation of memory-like CD8+ T cells.

a, TCA-cycle schematic with indication of two different IDH2 inhibitors. b,c, Bar graphs representing the percentage of m + 1 citrate labelling from [1-13C]-glutamine in DMSO, AGI-6780-conditioned CD8+ T cells (b) or AG-221-conditioned cells (c). (n = 3 biological replicates per group). d, Experimental set-up of in vitro T cell culture. OVA-specific CD8+ T cells from the spleen of OT1 mice were activated with OVA and IL-2 and cultured in the presence of DMSO or IDH2i for 7 days, with IL-7 supplementation from day 3. e,f, Representative flow cytometry histograms indicating the relative expression of CD62L (e) and of the indicated cell-surface proteins (f) on the surface of CD8+ T cells at day 7 post-activation upon inhibition of IDH2 using AG-221 (IDH2i) or control (DMSO) treatment. g, Cell count at 24 h, 48 h, 72 h and 7 days post-activation of DMSO- or IDH2i-conditioned CD8+ T cells. (n = 4 biological replicates, data pooled from 2 independent experiments). h, Percentages of TCF1+ CD8+ T cells at day 7 post-activation upon DMSO or IDH2i conditioning. (n = 2 biological replicates). i,j, Representative histogram indicating the relative expression of CD62L (i) and bar graph showing the percentage of CD62L+ CD8+ T cells (j) treated with AGI-6780 at day 7 post-activation. (n = 5 biological replicates, data pooled from 4 independent experiments). k,l, Representative histogram indicating the relative expression of CD62L (k) and bar graph showing the percentage of CD62L+ CD8+ T cells (l) treated with an IDH1 inhibitor (AG-120) at day 7 post-activation. (n = 2 biological replicates, data pooled from 2 independent experiments). m, Percentages of CD62L+ CD8+ T cells 7 days post-activation of splenocytes from IDH2 wild-type or knockout mice cultured with or without IDH2i (n = 2 biological replicates, data pooled from 2 independent experiments). n, Experimental set-up of DMSO- or IDH2i-conditioned T cells transferred into mice that are subsequently infected with LM-OVA. o,p, Graphs representing the percentage of SLECs (KLRG1+CD127−) (o) and MPECs (KLRG1−CD127+) (p) out of transferred CD8+ T cells in the spleen at day 14 post-infection. q, Percentage of TCM (CD44+CD62L+) cells out of transferred CD8+ T cells in the spleen at day 28 post-infection. r, Percentage of IFNγ+TNF+ cells out of transferred CD8+ T cells upon ex vivo restimulation at day 28 post-infection. s, Percentage of IFNγ+TNF+ cells out of transferred CD8+ T cells upon ex vivo restimulation over one year post-infection. (n–q, n = 3(IDH2i) and n = 5 (DMSO) biological replicates per group). Data represents mean ± s.e.m. and were analysed by unpaired, two-tailed Student’s t-test (b,c,g,h,j,l,o–s) or two-way ANOVA using the original FDR test of Benjamini and Hochberg (m). Only relevant statistical comparisons are shown.

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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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Extended Data Fig. 6 Pharmacological inhibition of IDH2 boosts the antitumour function of adoptively transferred cells in a Yumm1.7 melanoma model.

a, Experimental set-up of in vivo Yumm1.7-OVA tumour model. b–d, Tumour growth curve (b), weight (c) and representative photo (d) of B16-OVA tumour-bearing mice either left untreated (unt.) or transferred with T cells conditioned with DMSO or IDH2i (AG-221). e, Number of transferred DMSO- or IDH2i-conditioned CD8+ T cells per tumour-draining lymph node, analysed by flow cytometry 25 days post-Yumm1.7-OVA tumour engraftment. f, Percentages of TCF1+ cells out of transferred CD8+ T cells in tumour-draining lymph node 25 days post-Yumm1.7-OVA tumour engraftment. g, Percentages of CD44+CD62L+ cells out of transferred CD8+ T cells in the spleen 25 days post-tumour engraftment. h, Number of transferred DMSO- or IDH2i-conditioned CD8+ TILs per milligram of tumour, analysed by flow cytometry 25 days post-Yumm1.7-OVA tumour engraftment. i,j, Number of transferred PD-1+ TCF1+ cells (i) and PD-1+TCF1− cells (j) DMSO- or IDH2i-conditioned CD8+ TILs, analysed by flow cytometry 25 days post-tumour engraftment. k,l, Percentages of PD-1+TCF1+ cells (k) and PD-1+TCF1− cells (l) out of transferred CD8+ TILs. m, Bar plots representing the percentage of IFNγ and TNF production by transferred TILs upon ex vivo restimulation with OVA peptide. (b–m, n = 8 (unt.), n = 11 (DMSO) and n = 10 (IDH2i) biological replicates per group, pooled data from 2 independent experiments). n, Survival curve of mice treated with OVA-specific CD8+ T cells conditioned with DMSO or IDH2i, with α-PD-1 or IgG control combination treatment. (n = 8 biological replicates per group). o, Number of transferred DMSO- or IDH2i-conditioned TILs with α-PD-1 or IgG control combination treatment per milligram of tumour, analysed by flow cytometry 25 days post-Yumm1.7-OVA tumour engraftment. p, Percentage of IFNγ+TNF+IL-2+ CD8+ TILs upon ex vivo restimulation with OVA peptide. (o–q, n = 5 (DMSO+IgG and IDH2i+IgG), n = 6 (IDH2i + α-PD-1) and n = 8 (DMSO + α-PD-1) biological replicates per group). Data represent mean ± s.e.m. and were analysed by unpaired, two-tailed Student’s t-test (b,e–m), one-way ANOVA using Tukey’s multiple comparison test (c), two-way ANOVA corrected for multiple testing by the original FDR of Benjamini and Hochberg (o,p) or log-rank test (n). Only relevant statistical comparisons are shown.

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Extended Data Fig. 7 Inhibition of IDH2 induces memory features in human T cells.

a,b, MFI of CD62L (a) and representative histogram (b) at day 9 post-activation on the surface of human CD8+ T cells isolated from healthy volunteer PBMCs, measured by flow cytometry. PBMCs from healthy donors were activated with anti-CD3/CD28 beads for 5 days and cultured 4 additional days prior phenotypic analyses. The cells were cultured in the presence of DMSO or IDH2i during the entire experiment (a–c). (n = 7 donors, pooled data from 2 independent experiments). c, Percentages of human CD8+ TSCM-like cells, characterized as CD45RA+, CD62L+, CD45RO−, CCR7+, CD95+, CD28+, CD27+. (n = 7 donors, pooled data from 2 independent experiments). d, Experimental set-up of NAML6 tumour experiment. Human T cells were transduced with anti-CD19 CAR T cells, expanded during 12 days in the presence of DMSO or IDH2i and subsequently transferred into tumour-bearing NSG mice. e–g, Human MILs from patients with multiple myeloma were activated with anti-CD3/CD28 beads for 5 days and cultured 4 additional days prior phenotypic analyses. The cells were cultured in the presence of DMSO or IDH2i during the entire experiment. Shown here is MFI of CD62L (e) and representative histogram (f), and percentage of TSCM (g) at day 9 post-activation in human CD8+ MILs. (n = 6 donors, pooled data from 2 independent experiments). h, Experimental set-up of NCI-H929 tumour experiment. Human T cells were transduced with anti-BCMA CAR T cells, expanded during 10 days in the presence of DMSO or IDH2i and subsequently transferred into tumour-bearing NSG mice. i, BLI quantification of total flux (p/s) (representative of 2 independent experiments). j–l, Fold-change expansion at day 9 post-activation of human PBMCs (j), human MILs (k) and human CAR T cells (l) cultured with DMSO or IDH2i. (n = 3 (j), n = 6 (k) and n = 2 donors (l), pooled data from 2 independent experiments (k). m, Representation (row z-scores) of differentially accessible regions in mouse CD8+ T cells after DMSO or IDH2i conditioning (ATAC-seq). n, Bar plots representing log2 fold change in mRNA expression of Sell, Tcf7 and Ccr7 upon IDH2i-compared to DMSO-conditioned CD8+ T cells (RNA-seq). Data represent mean ± s.e.m. and were analysed by paired two-tailed Student’s t-test (a,c,e,g,j–m).

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Extended Data Fig. 8 Inhibition of IDH2 induces metabolic compensation.

a, MitoSOX MFI in DMSO- or IDH2i-treated cells upon addition of H2O2 at indicated concentrations, measured by flow cytometry. (n = 5 biological replicates, pooled data from 2 independent experiments). b, Number of live TFAM WT and TFAM KO T cells per millilitre upon DMSO or IDH2i treatment. (n = 3 biological replicates, pooled data from 2 independent experiments). c, Percentage of CD62L+ out of CD8+ TFAM WT and KO T cells upon DMSO or IDH2i treatment. (n = 3 biological replicates, pooled data from 2 independent experiments). d, Percentage of CD62L+ out of Thy1.1+ CD8+ T cells at day 7 post-activation upon deletion of indicated genes. (n = 6 (Scr, Ogdh and Sdhb) and n = 5 (Idh2) biological replicates per group, pooled data from 3 independent experiments). e, Immunoblots for OGDH and SDHB in control Scr and OGDH- or SDHB-deleted cells. (Representative of 2 independent experiments). f, Percentages of citrate m + 5 detected by [U-13C]-glutamine labelling in DMSO or IDH2i-treated T cells. (n = 3 biological replicates per group). g, Percentages of m + 3 succinate, fumarate and malate detected by 2 h [U-13C]-glutamine labelling in DMSO or IDH2i-treated T cells. (n = 3 biological replicates per group). h, Percentages of m + 4 succinate, fumarate and malate detected by 2 h [U-13C]-glutamine labelling in DMSO or IDH2i-treated T cells. (n = 3 biological replicates per group). i, Percentages of m + 5 α-KG detected by 2 h [U-13C]-glutamine labelling in DMSO or IDH2i-treated T cells. (n = 3 biological replicates per group). j, Percentages of m + 2 acetyl-CoA, detected by [U-13C]-glucose labelling for indicated time in DMSO or IDH2i-treated T cells. (n = 3 biological replicates per group). k, Percentages of m + 2 citrate, succinate, fumarate and malate detected by 2 h [U-13C]-glucose labelling in DMSO or IDH2i-treated T cells. (n = 3 biological replicates per group). l, Percentages of citrate m + 2 detected by [U-13C16]-palmitate labelling in mouse CD8+ T cells treated with DMSO or the IDH2 inhibitor (IDH2i). (n = 3 biological replicates per group). m–o, Quantification of basal OCR (m), ATP-linked OCR (n) and spare respiratory capacity (SRC; o) from data presented in Fig. 4d. (n = 4 biological replicates, pooled data from 2 independent experiments). p, Percentage of CD62L+ T cells in control Scr or ΟGDH-deleted CD8+ T cells, treated with IDH2i. (n = 4 biological replicates, pooled data from 2 independent experiments). q, Percentage of CD62L+ T cells in DMSO or IDH2i-conditioned CD8+ T cells, treated with etomoxir (FAOi). (n = 5 biological replicates, pooled data from 2 independent experiments). r, Glucose consumption in DMSO- or IDH2i-treated mouse CD8+ T cells over 24 h, at day 3 post-activation. (n = 3 biological replicates per group). s, Intracellular abundances of indicated amino acids (arbitrary units, a.u) measured by mass spectrometry upon DMSO or IDH2i treatment. (n = 9 biological replicates, pooled data from 3 independent experiments). t,u, CD98 MFI in DMSO- or AG-221- (t) or AGI-6780- (u) treated CD8+ T cells, measured by flow cytometry at day 3 post-activation. (n = 9 (AG-221) and n = 3 (AGI-6780) biological replicates, pooled data from 4 (AG-221) and 2 (AGI-6780) independent experiments). Data represent mean ± s.e.m. and were analysed by unpaired two-tailed Student’s t-test (f,i,l–o,r,t,u), multiple unpaired, two-tailed t-tests using Benjamini and Hochberg method (g,h,j,k,s), one-way ANOVA using Tukey’s multiple comparison test (d) or two-way ANOVA using the original FDR test of Benjamini and Hochberg (a–c,p,q). Only relevant statistical comparisons are shown. For gel source data, see Supplementary Fig. 1.

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Extended Data Fig. 9 Inhibition of IDH2 leaves an epigenetic imprint.

a, Bar graphs representing permissive and repressive histone marks in DMSO or IDH2i-conditioned CD8+ T cells. b, Immunoblot and quantification of indicated histone marks in DMSO or IDH2i-conditioned CD8+ T cells. (n = 2 (H3K36me2), pooled from 2 independent experiments, n = 3 (H3K79me3, H3K14ac) and n = 5 (H3K27ac) biological replicates, pooled data from 3 independent experiments). c, Immunoblot and quantification of total acetylated lysines in DMSO or IDH2i-conditioned CD8+ T cells. (n = 3 biological replicates, data representing 2 independent experiments). d, Barcode plots representing gene set enrichment analysis of a memory signature on H3K4me3-enriched regions in OT1 cells. NES, normalized enrichment score; Running ES, running enrichment score. e, H3K4me3 ChIP–seq tracks at Sell, Ccr7 and Tcf7 loci from mouse naive (TN, green), effector (TE, blue) and memory (TM, pink) OT1 cells and from human naive (TN, green), effector memory (TEM, blue) and central memory (TCM, pink) CD8+ T cells from the peripheral blood of healthy adults. f, Immunoblot and quantification of H3K4me3 histone mark in DMSO- or IDH2i-conditioned MILs. (n = 5 biological replicates, pooled data from 2 independent experiments). g, Intracellular abundance of α-KG in DMSO or IDH2i-conditioned CD8+ T cells, supplemented with indicated doses of cell-permeable α-KG (n = 2 biological replicates). h, Immunoblot quantification of H3K4me3 in DMSO or IDH2i-conditioned CD8+ T cells, supplemented with α-KG. (n = 4 biological replicates, pooled data from 4 independent experiments). i, Immunoblot quantification of H3K4me3 in T cells treated with DMSO, fumarate or 2-HG. j, Percentage of CD62L+ T cells upon DMSO, fumarate or S-2-HG treatment and α-KG supplementation. (i,j, 3 biological replicates, pooled data from 3 independent experiments). k, Representative histogram indicating the relative expression of CD62L on the surface of CD8+ T cells treated with 0.5, 1 or 2 mM succinate (dimethyl succinate, Sigma-Aldrich) at day 7 post-activation. Data represent mean ± s.e.m. and were analysed by unpaired (c,i,j) or paired (d) two-tailed Student’s t-test or two-way ANOVA using the original FDR test of Benjamini and Hochberg (e–h,k). Only relevant statistical comparisons are shown. For gel source data, see Supplementary Fig. 1.

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Extended Data Fig. 10 Compensatory metabolism upon IDH2 inhibition influences histone modification and memory T cell differentiation.

a, KDM5 activating potential represented by the ratio of fold increase in Km value for α-KG versus the sum of fold increased IC50 concentrations of inhibitory metabolites (2-HG, succinate, fumarate and malate), calculated as: (concentration α-KG/Km α-KG)/((concentration 2-HG/IC50 2-HG) + (concentration succinate/IC50 succinate) + (concentration fumarate/IC50 fumarate) + (concentration malate/IC50 malate)). Ratio based on cellular metabolite concentrations calculated from absolute metabolite quantifications. (n = 6 biological replicates, pooled data from 2 independent experiments). b, Immunoblot quantification of H3K4me3 in DMSO- and IDH2i-treated CD8+ T cells, supplemented with α-KG and/or KDM5 inhibitor. (n = 2 biological replicates, pooled data from 2 independent experiments). c,d, Immunoblot (c) and quantifications (d) of H3K27ac and H3K14ac in DMSO or IDH2i-conditioned CD8+ T cells, treated with etomoxir (FAOi) and supplemented with acetate. (n = 3 biological replicates). e, Immunoblot quantification of H3K27ac and H3K14ac in DMSO or IDH2i-conditioned CD8+ T cells, treated with ACLYi. (n = 3 biological replicates). f, Percentage of CD62L+ T cells in DMSO or IDH2i-conditioned CD8+ T cells, treated with ACLYi. (n = 5 biological replicates, pooled data from 2 independent experiments). g,h, Immunoblot (g) and quantification (h) of H3K27ac in DMSO, IDH2i-, DMSO+HATi-and IDH2i+HATi-conditioned CD8+ T cells. (n = 3 biological replicates, pooled data from 3 independent experiments). i, CD62L expression in DMSO, IDH2i-, DMSO+HATi- and IDH2i+HATi-conditioned cells. (HATi = histone acetyltranferase p300/CBP inhibitor, 8 μM C646). (Representative blot and histogram from 3 independent experiments with 3 biological replicates). j, Tumour growth curve of B16-OVA tumours from mice treated with either DMSO, IDH2i-, DMSO+HATi- or IDH2i+HATi-conditioned OVA-specific CD8+ T cells. (n = 13 (IDH2i), n = 14 (DMSO+HATi, IDH2i+HATi) and n = 15 (DMSO) biological replicates per group, pooled data from 2 independent experiments). k, Pluripotent naive CD8+ T cells are characterized by low metabolic activity and permissive H3K4me3 deposition at pro-memory genes, including Sell, Ccr7 and Tcf7. After activation, TE cells increase glutamine metabolism along both the oxidative and reductive pathways (represented by two red arrows on the left panel of the figure). RC generates citrate and acetyl-CoA, which is a key metabolite supporting cell growth and function. This specific TE cell metabolism creates a unique metabolite composition (represented by balanced levels of α-KG on one side and succinate, fumarate, malate and 2-HG on the other side) enabling the activity of the α-KG-dependent histone demethylase KDM5. KDM5-mediated demethylation of H3K4me3 in activated T cells induces a repressive chromatin state at pro-memory genes, which prevents the maintenance of pluripotency and facilitates terminal effector differentiation. Blocking RC through inhibition of IDH2 does not hinder T cell proliferation and function, as compensatory fatty acid oxidation can refuel acetyl-CoA pools, with the support of glutamine anaplerosis, which is now redirected entirely in the oxidative branch of the TCA cycle (represented by the larger red arrow on the right panel of the figure), altogether enhancing cellular OCR. This leads to a perturbation of the metabolite balance by increasing the levels of the TCA intermediates succinate, fumarate, malate and 2-HG (represented by a shift in the balance upon IDH2 inhibition). The accumulation of these inhibitory metabolites impairs KDM5 demethylase activity, and elevated H3K4me3 is likely to foster a permissive chromatin state at pro-memory genes, allowing for the maintenance of pluripotency and memory differentiation. Data represent mean ± s.e.m. and were analysed by unpaired two-tailed Students t-test (a,j) or two-way ANOVA using the original FDR test of Benjamini and Hochberg (b,d–f,h). Only relevant statistical comparisons are shown. For gel source data, see Supplementary Fig. 1.

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Supplementary Figure 1

This file contains uncropped immunoblot images with size marker indications.

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Supplementary Figure 2

This file contains figures exemplifying the flow cytometry gating strategies for mouse and human cells.

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Jaccard, A., Wyss, T., Maldonado-Pérez, N. et al. Reductive carboxylation epigenetically instructs T cell differentiation. Nature 621, 849–856 (2023). https://doi.org/10.1038/s41586-023-06546-y

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