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Stable human regulatory T cells switch to glycolysis following TNF receptor 2 costimulation

Nature Metabolism volume 2, pages 1046–1061 (2020)Cite this article

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Abstract

Following activation, conventional T (Tconv) cells undergo an mTOR-driven glycolytic switch. Regulatory T (Treg) cells reportedly repress the mTOR pathway and avoid glycolysis. However, here we demonstrate that human thymus-derived Treg (tTreg) cells can become glycolytic in response to tumour necrosis factor receptor 2 (TNFR2) costimulation. This costimulus increases proliferation and induces a glycolytic switch in CD3-activated tTreg cells, but not in Tconv cells. Glycolysis in CD3–TNFR2-activated tTreg cells is driven by PI3-kinase–mTOR signalling and supports tTreg cell identity and suppressive function. In contrast to glycolytic Tconv cells, glycolytic tTreg cells do not show net lactate secretion and shuttle glucose-derived carbon into the tricarboxylic acid cycle. Ex vivo characterization of blood-derived TNFR2hiCD4+CD25hiCD127lo effector T cells, which were FOXP3+IKZF2+, revealed an increase in glucose consumption and intracellular lactate levels, thus identifying them as glycolytic tTreg cells. Our study links TNFR2 costimulation in human tTreg cells to metabolic remodelling, providing an additional avenue for drug targeting.

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Fig. 1: Analysis of glycolysis in Tconv and tTreg cells after CD3–CD28-mediated activation.
Fig. 2: TNFR2 costimulation drives tTreg cell proliferation while identity and suppressive function are maintained.
Fig. 3: tTreg cells express glycolytic enzymes on TNFR2 costimulation.
Fig. 4: TNFR2 costimulation induces glycolysis in CD3-activated tTreg cells without increasing net lactate secretion.
Fig. 5: Tracing of [13C6]glucose shows increased flux through the glycolysis pathway and TCA cycle in tTreg cells on TNFR2 costimulation.
Fig. 6: TNFR2-induced glycolysis maintains tTreg cell identity and suppressive function.
Fig. 7: TNFR2 costimulation drives glycolysis in tTreg cells via PI3K and mTOR.
Fig. 8: In vivo effector Treg cells that express high levels of TNFR2 are primarily tTreg cells and have increased glycolytic activity.

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Data availability

All RNA-seq data are available in the GEO database under accession code GSE138603 and GSE138604. GSEAs were performed with GSEA software using the hallmark gene sets listed in the Molecular Signatures Database (MSigDB) available through https://www.gsea-msigdb.org/gsea/index.jsp. The proteomics data set used in this study is published by Cuadrado et al.14. All other data that support the findings of this study are available from the corresponding author on reasonable request.

Code availability

For RNA-sequencing, the script Itreecount was used to count the number of reads per gene. Itreecount is publicly available through https://github.com/NKI-GCF/itreecount.

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Acknowledgements

We thank the employees of the Flow Cytometry Facility and Genomics Facility of the Netherlands Cancer Institute and the Flow Cytometry Core Facility at Leiden University Medical Center for their technical assistance, M. Giera and S. Kostidis of the Center for Proteomics and Metabolomics Facility at Leiden University Medical Center for valuable discussions regarding the metabolomics experiments on freshly sorted cells and E. Schrama for assistance in flow-cytometric assays. M. A. Aslam was supported by the Institute of Molecular Biology and Biotechnology, Bahauddin Zakariya University, Multan, Pakistan. This work was supported by Oncode, ZonMW-TOP grant 40-00812-98-13071 and grants ICI-00014 and ICI-00025 from the Institute for Chemical Immunology, funded by ZonMW Gravitation.

Author information

Author notes

  1. These authors contributed equally: Sander de Kivit, Mark Mensink, Anna T. Hoekstra.

  2. These authors jointly supervised this work: Celia R. Berkers, Jannie Borst.

Authors and Affiliations

  1. Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands

    Sander de Kivit, Mark Mensink & Jannie Borst

  2. Oncode Institute, Leiden University Medical Center, Leiden, the Netherlands

    Sander de Kivit, Mark Mensink, Ilana Berlin & Jannie Borst

  3. Division of Tumor Biology & Immunology, The Netherlands Cancer Institute, Amsterdam, the Netherlands

    Sander de Kivit, Mark Mensink, Demi Both, Muhammad A. Aslam & Jannie Borst

  4. Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, the Netherlands

    Anna T. Hoekstra & Celia R. Berkers

  5. Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands

    Ilana Berlin

  6. Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, the Netherlands

    Rico J. E. Derks

  7. Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory, Amsterdam, the Netherlands

    Derk Amsen

  8. Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands

    Celia R. Berkers

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Contributions

S.d.K., M.M., J.B. and C.R.B. designed the study; S.d.K., M.M. and A.T.H. designed and performed the experiments and analysed the data; I.B. performed confocal microscopy experiments, analysed the data and contributed to the manuscript; R.J.E.D. performed targeted metabolomics analysis on freshly sorted cells and contributed to the manuscript; D.B. performed the short-term metabolic assays; M.A.A. provided assistance in analyses of the transcriptome data; S.d.K., M.M., J.B. and C.R.B. wrote the manuscript; D.A. provided conceptual advice in the study design and contributed to writing the manuscript.

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Correspondence to Celia R. Berkers or Jannie Borst.

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Extended data

Extended Data Fig. 1 Cell sorting strategy and characterization of naïve CD4+ Tconv and tTreg cells from human peripheral blood.

a, Total human CD4+ T cells were isolated from human PBMC using MACS and subjected to fluorescence-activated cell sorting. Within the live (PI-) and single cell gates (FSC-A/FSC-H), CD4+ T cells were selected for further discrimination between Tconv and tTreg cells using CD127 and CD25 expression levels (G1 and G2). Subsequently, naïve Tconv and tTreg cells were gated based on CD45RA expression and the novel marker GPA33 to obtain tTreg cells with high purity, resulting in CD25lowCD127highCD45RA+GPA33int Tconv cells and CD25highCD127lowCD45RA+GPA33high tTreg cells. b, Flow cytometric analysis of FOXP3, IKZF2 and total CTLA4 protein expression in the naïve Tconv and tTreg cells (representative of n=6). Mean fluorescence intensity (MFI) is shown in the plots. Sample size (n) represents cells from individual donors, analyzed in independent experiments.

Extended Data Fig. 2 Phenotypical and functional characterization of in vitro expanded Tconv and tTreg cells.

a, Schematic overview of Tconv and tTreg cell expansion cultures. The nature of “restimulation” on day 18 is indicated in the legends of the specific experiments. b, Flow cytometric analysis of FOXP3, IKZF2, cell surface CD25 and total CTLA4 protein levels in expanded Tconv and tTreg cells at 24 h after restimulation with anti-CD3/CD28 mAbs or control (-) (representative of n=6). c, Quantification of flow cytometric results obtained as indicated in b, showing the MFI for each protein (n=6), ****p=5.02x10-5, 3.34x10-6 and 5.58x10-5 for FOXP3, IKZF2 and CTLA4, respectively. d, Assessment of the suppressive capacity of tTreg cells on day 18 of the expansion cultures. Cells in the suppression assay were stimulated with agonistic mAb to CD3 and the assay was read out after 4 days. The percentage of dividing CD4+ and CD8+ Tconv cells is displayed (n=5), ****p=5.1x10-13, 4.99x10-13, 5.57x10-13, 8.67x10-13 and 3.32x10-11 for 1:1, 1:2, 1:4, 1:8 and 1:16 ratio versus 0:1 ratio, respectively, within CD3+CD4+ responder cells and ****p=5.81x10-13, 7.83x10-13, 2.67x10-12, 3.4x10-11 and 1.78x10-7 for 1:1, 1:2, 1:4, 1:8 and 1:16 ratio versus 0:1 ratio, respectively, within CD3+CD8+ responder cells. (c, d) Two-way ANOVA with Tukey’s post hoc test was used for statistical analysis. Data are presented as mean ± SEM. Sample size (n) represents cells from individual donors, analyzed in independent experiments (a–d).

Extended Data Fig. 3 PFK mRNA and protein expression by Tconv and tTreg cells upon CD3/CD28-mediated activation.

a, Venn diagrams depicting the number of overlapping and unique genes (left) or metabolites (right) that showed significant changes in abundance upon CD3/CD28-mediated activation for 24 h in Tconv and tTreg cells, as determined by transcriptomics and metabolomics (n=4). Statistical evaluation was performed using an unpaired two-sided Student’s t-test with the Benjamini-Hochberg method with FDR<0.05 (transcriptomics) or by unpaired two-sided Student’s t-test with p<0.05 (metabolomics). b, mRNA (top) and protein levels (bottom) of PFK isoforms in Tconv and tTreg cells at 24 h after stimulation as indicated. mRNA levels are expressed in TPM, as detected in our transcriptome data set (n=4), *p=0.0408. (c) Protein levels are expressed in LFQ intensity, as detected in an earlier proteomics data set14 (n=3). (b, c) Two-way ANOVA with Bonferroni’s post hoc test was used for statistical analysis. Data are presented as mean ± SEM. Sample size (n) represents independent expansion cultures of distinct donors (a–c).

Extended Data Fig. 4 Significant changes in glycolysis/TCA cycle intermediates in Tconv and tTreg cells activated via CD3/CD28.

Unpaired two-sided Student’s t-test was used for statistical analysis (*). Data is derived from 4 independent expansion cultures of distinct donors.

Extended Data Fig. 5 TNFR2 acts as a costimulatory receptor on tTreg cells.

a, Flow cytometric assessment of tTreg cell division by CTV dilution upon restimulation for 96 h as indicated. The percentage of dividing cells is depicted. FOXP3, IKZF2 and total CTLA4 protein expression is shown for each cell division (representative of n=3). b, Left panel: Flow cytometric assessment of 6-NBDG uptake activity in Tconv and tTreg cells that were restimulated for 24 h as indicated. Dashed lines indicate the modal 6-NBDG uptake for unstimulated cells. Right panel: quantification of 6-NBDG uptake data, based on the geometric MFI, normalized to unstimulated Tconv cells (n=3), ****p=5.88x10-5. Two-way ANOVA with Tukey’s post hoc test was used for statistical analysis. Data are presented as mean ± SEM. Sample size (n) represents cells from individual donors, analyzed in independent experiments.

Extended Data Fig. 6 Transcriptomic analysis of Tconv and tTreg cells upon TNFR2 costimulation.

a, PCA plot of the transcriptomes of Tconv and tTreg cells that were activated via either CD3 or CD3/TNFR2 for 24 h (n=3 independent expansion cultures from different donors). b, Heat map showing hierarchical clustering of the 2664 genes that were differentially expressed between the comparative conditions. Z-scores showing relative gene expression values are color-coded. Grey-scale boxes (right) indicate clusters. Statistical evaluation was performed according to ANOVA with the Benjamini-Hochberg method with FDR<0.005.

Extended Data Fig. 7 Significant changes in glycolysis/TCA cycle intermediates in Tconv and tTreg cells activated via CD3/TNFR2.

Unpaired two-sided Student’s t-test was used for statistical analysis (*). Data is derived from 4 independent expansion cultures of distinct donors.

Extended Data Fig. 8 Complete mass isotopologue analysis of glycolysis and TCA cycle intermediates upon [13C6]-glucose tracing in Tconv and tTreg cells.

Levels of all detected mass isotopologues of the indicated metabolites in Tconv and tTreg cells that were unstimulated or activated via CD3 alone or CD3/TNFR2 for 24 h in the presence of [13C6]-glucose, as measured by LC-MS (n=4 independent expansion cultures, as in Fig. 5), ***p=0.0002 for HexP, *p=0.0381 for F-1,6-BP, **p=0.0029 for DHAP, *p=0.0397 for pyruvate, *p=0.0137 for α-ketoglutarate and **p=0.0029 for malate. Two-way ANOVA with Bonferroni’s post hoc test was used for statistical analysis. Data are presented as mean ± SEM.

Extended Data Fig. 9 Phosphorylation of mTOR and S6 in Tconv cells.

a, Left panel: flow cytometric analysis of phosphorylated mTOR (Ser2448) levels in Tconv cells following stimulation for 24 h as indicated. Right panel: quantification based on MFI (n=4), *p=0.0131 and 0.0197 for αCD3/28 and αCD3/TNFR2 versus unstimulated, respectively. b, Left panel: flow cytometric analysis of phosphorylated S6 (Ser235/236) levels in Tconv cells following stimulation for 24 h as indicated. Right panel: quantification based on the MFI (n=4), **p=0.0026 and ***p=0.0003 for αCD3/28 and αCD3/TNFR2 versus unstimulated, respectively. (a, b) One-way repeated measures ANOVA with Bonferroni’s post hoc test was used for statistical analysis. c, Quantification of phosphorylated mTOR (Ser2448) levels based on MFI in ex vivo naïve Tconv and tTreg cells stimulated for 24 h as indicated (n=3), **p=0.0039. d, Quantification of phosphorylated S6 (Ser235/236) levels based on MFI in ex vivo naïve Tconv and tTreg cells stimulated for 24 h as indicated (n=4), *p=0.0175. e, Quantification of 6-NBDG uptake activity in ex vivo naïve Tconv and tTreg cells stimulated for 24 h as indicated (n=3), **p=0.0015. f, g, Quantification of flow cytometric analysis of phosphorylated mTOR (Ser2448) (f) or S6 (Ser235/Ser236) (g) levels in Tconv cells following stimulation for 24 h as indicated in presence or absence of selective inhibitors of PI3K (LY294002) or NIK (NIK-SMI1) in the culture medium (n=5), ****p=4.54x10-6, 2.13x10-6 and 5.2x10-10 for phosphorylated mTOR comparing medium versus LY294002 for αCD3, αCD3/28 and αCD3/TNFR2, respectively, and ****p=1.91x10-5, 8.55x10-7 and 4.64x10-9 for phosphorylated S6 comparing medium versus LY294002 for αCD3, αCD3/28 and αCD3/TNFR2, respectively. (c–g) Two-way ANOVA with Tukey’s post hoc test was used for statistical analysis. Data are presented as mean ± SEM. Sample size (n) represents cells from individual donors, analyzed in independent experiments.

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de Kivit, S., Mensink, M., Hoekstra, A.T. et al. Stable human regulatory T cells switch to glycolysis following TNF receptor 2 costimulation. Nat Metab 2, 1046–1061 (2020). https://doi.org/10.1038/s42255-020-00271-w

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