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S-2-hydroxyglutarate regulates CD8+ T-lymphocyte fate

Abstract

R-2-hydroxyglutarate accumulates to millimolar levels in cancer cells with gain-of-function isocitrate dehydrogenase 1/2 mutations. These levels of R-2-hydroxyglutarate affect 2-oxoglutarate-dependent dioxygenases. Both metabolite enantiomers, R- and S-2-hydroxyglutarate, are detectible in healthy individuals, yet their physiological function remains elusive. Here we show that 2-hydroxyglutarate accumulates in mouse CD8+ T cells in response to T-cell receptor triggering, and accumulates to millimolar levels in physiological oxygen conditions through a hypoxia-inducible factor 1-alpha (HIF-1α)-dependent mechanism. S-2-hydroxyglutarate predominates over R-2-hydroxyglutarate in activated T cells, and we demonstrate alterations in markers of CD8+ T-cell differentiation in response to this metabolite. Modulation of histone and DNA demethylation, as well as HIF-1α stability, mediate these effects. S-2-hydroxyglutarate treatment greatly enhances the in vivo proliferation, persistence and anti-tumour capacity of adoptively transferred CD8+ T cells. Thus, S-2-hydroxyglutarate acts as an immunometabolite that links environmental context, through a metabolic–epigenetic axis, to immune fate and function.

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Figure 1: VHL-HIF signalling regulates 2-hydroxyglutarate levels.
Figure 2: Hypoxic induction of 2-hydroxyglutarate depends on Hif-1α in CD8+ T lymphocytes.
Figure 3: S -2HG alters phenotypic marker expression of CD8 + T lymphocytes.
Figure 4: S -2HG treatment promotes in vivo homeostatic renewal, persistence and anti-tumour capacity of transferred cells.
Figure 5: S -2HG alters global H3K27me3 in CD8 + T lymphocytes.
Figure 6: S -2HG alters global 5hmC and 5mC in DNA of CD8 + T lymphocytes.

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Acknowledgements

We thank the Cambridge Institute PK Bioanalytics Core facility for MS measurements; A. Cowburn, A. Wood, K. Lodge and E. Chilvers for human PBMCs; B. Jaggs for help with mouse work. P.A.T. was funded by CRUK, the MRC (1495954) and Wellcome Trust. J.R.G. was funded by CRUK. A.P. was funded by Marie-Curie IEF. A.T.P. was funded by UCSD NIH Grant (5T32GM007240-36). A.W.G. was funded by the NIH (A1096852, A1072117), Leukemia and Lymphoma Society, and Pew Scholars Fund. K.L.L., J.Y., G.S.C. and L.P. were funded by the Singapore National Research Foundation and Singapore Ministry of Education, the NMRC Clinician Scientist (NMRC/CIRG/1389/2014.) and the Swedish Research Council. R.S.J. and co-workers are funded by the Wellcome Trust (grant WT092738MA), the Swedish Cancer Foundation (Cancerfonden), and the Swedish Research Council (Vetenskapsrådet). This work is dedicated to the memory of Lorenz Poellinger.

Author information

Authors and Affiliations

Authors

Contributions

P.A.T. and A.P. designed experiments and carried out ex vivo and in vivo experiments, analysed the data, and wrote the manuscript. A.T.P., A.D., A.W.G. and R.S.J. carried out the initial metabolome survey. D.M, P.V., J.S., A.A. and C.E.E. aided in experimental execution and analysis. K.L.L., G.S.C., J. Y. and L.P. carried out ChIP and DIP experiments. J.R.G, K.L.L., L.P. and A.W.G. designed experiments and analysed data. R.S.J. designed experiments, analysed data, wrote the manuscript and administered the project.

Corresponding author

Correspondence to Randall S. Johnson.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 VHL–HIF-1α regulate central carbon metabolism and 2HG levels in CD8+ T lymphocytes.

a, Illustration of central carbon metabolism in CD8+ lymphocytes, including glycolysis and the tricarboxylic acid cycle, depicting relative levels of detected metabolites between Vhlfl/fl (n = 5), Vhlfl/fldLckcre (n = 5) and Hif-1afl/flVhlfl/fldLckcre (n = 3) CD8+ T-lymphocyte groups. b, Glucose consumption and lactate production in Vhlfl/fl and Vhlfl/fldLckcre CD8+ T lymphocytes 7 days after activation with anti-CD3 and anti-CD28 antibodies (n = 4 mice per genotype). c, Immunoblot analysis for Hif-1α and LaminB1, using nuclear extracts prepared from Vhlfl/fl and Vhl−/− CD8+ T lymphocytes cultured in 21% oxygen. d, Rank of metabolite loadings in PC1 from PCA. e, Immunoblot analysis for HIF-1α, HIF-2α and β-tubulin, on whole-cell extracts prepared from RCC4 and 786-O renal cancer cell lines, with and without expression of functional VHL. f, Deletion efficiency of Vhl in Vhlfl/fl MEFs following infection with adeno-Cre virus, n = 3 individual preparations. Accompanying immunoblot analysis for HIF-1α and β-tubulin, on whole-cell extracts. Two-tailed t-test (b), one-way ANOVA for multiple comparisons (a). Error bars denote s.d. and each dot represents an individual mouse in a and b. *P < 0.05, **P < 0.01, ***P < 0.001. For immunoblot source images, see Supplementary Fig. 1.

Extended Data Figure 2 Hif-1α-dependent metabolic alterations underlie S-2HG production in CD8+ T lymphocytes.

a, Example calculation of intracellular 2HG concentration. b, 1H-NMR analysis for 2HG from CD8+ T lymphocytes cultured as in Fig. 2b. c, Deletion efficiency of Hif1a or Hif2a in CD8+ T lymphocytes, isolated from Hif1afl/fldLckcre or Hif2afl/fldLckcre mice (n = 4 mice). d, e, Total 2HG levels, normalized to viable cell count or protein content, in Hif1afl/fl and Hif1afl/fldLckcre (d) and Hif2afl/fl and Hif2afl/fldLckcre (e) CD8+ T lymphocytes cultured as in Fig. 2b (n = 4 mice per genotype). f, Illustration outlining the workflow for metabolite extraction, deletion efficiency and viability experiments in Hif1afl/fl, Hif1afl/fldLckcre, Hif2afl/fl and Hif2afl/fldLckcre CD8+ T lymphocytes. Also shown are viability measurements at day 4 (n = 4 mice per genotype). g, Total amount of 2HG in Hif1afl/fl (n = 6) and Hif1afl/fldLckcre (n = 7) CD8+ T lymphocytes, at indicated times following activation (n 4 mice per time point). h, Heat map indicating qPCR measurement of expression of enzymes involved in central carbon metabolism in CD8+ T lymphocytes cultured as in Fig. 2b (n = 4 mice per condition). i, Liquid chromatography–tandem mass spectrometry (LC–MS/MS) quantification of total intracellular succinate, fumarate and malate levels in CD8+ T lymphocytes isolated from C57BL/6J mice and cultured as in Fig. 2b (n = 7 mice). j, Heat map indicating qPCR measurement, in Hif1afl/fl (n = 4) and Hif1afl/fldLckcre (n = 3) CD8+ T lymphocytes growing in 1% oxygen, of expression of enzymes implicated in the hypoxic production of S-2HG. k, qPCR validation of shRNA-knockdowns in CD8+ T lymphocytes isolated from C57BL/6J mice. ln, LC–MS/MS quantification of S- and R-2HG in CD8+ T lymphocytes isolated from C57BL/6J mice, with shRNA-mediated knockdown of Mdh1 (l), Mdh2 (m) and Ldha (n) (n = 4 pools of 4 mice per pool). o, Validation of Pdk1–Flag and Ldha–Flag expression in Hif1afl/fldLckcre CD8+ T lymphocytes by immunoblot analysis for Flag. p, LC–MS/MS quantification of total intracellular glutamate levels in CD8+ T lymphocytes cultured as in Fig. 2b; n = 7 mice. q, r, LC–MS/MS quantification of total intracellular glutamate levels in Hif1afl/fl, Hif1afl/fldLckcre, Hif2afl/fl and Hif2afl/fldLckcre CD8+ T lymphocytes cultured as in Fig. 2b; n = 4 mice per genotype. s, Immunoblot of cytosolic fractions for phospho-Pdh-E1α (S232) and total Pdh-E1α in CD8+ T lymphocytes cultured in 1% oxygen in the presence of the indicated concentration of DCA for 48h. tv, Total intracellular concentration of 2HG (t), 2HG normalized to viable cell count or protein content (u) and glutamate (v) in CD8+ T lymphocytes from C57BL/6J mice cultured as in Fig. 2b and treated with 5mM DCA for the latter 48 h of culture (n = 4 mice). Two-way ANOVA for grouped data (df, q, r, tv). Paired t-test for matched comparisons (i, p), one-way ANOVA for multiple matched comparisons (g, ln). Error bars denote s.d. and each dot represents an individual mouse in g, i and p. NS, non-significant, *P < 0.05, **P < 0.01, ***P < 0.0001. Experiments were performed with indicated numbers of mice from multiple occasions. For immunoblot source images, see Supplementary Fig. 1.

Extended Data Figure 3 Naive and expanding primary CD8+ T lymphocytes do not possess mutations in Idh1 or Idh2 that can explain the presence of high levels of 2HG

. a, Illustration outlining the workflow for mutational analysis of Idh1 and Idh2. b, Sanger sequencing chromatograms validating the presence of wild-type Idh1as compared to the C57BL/6J NCBI reference sequence. c, Alignment of mouse and human IDH1 protein indicating conservation of active site arginine residues. d, Sanger sequencing chromatograms validating the presence of wild-type Idh2 as compared to the C57BL/6J NCBI reference sequence. e, Alignment of mouse and human IDH2 protein indicating conservation of active site arginine residues.

Extended Data Figure 4 Kinetics of 2HG labelling in 21% and 1% oxygen, by U-13C-glucose and U-13C-glutamine.

a, Proposed mechanism by which Hif-1α controls S-2HG production in CD8+ T lymphocytes and 13C-labelling strategy using U-13C-glucose (m+6) and U-13C-glutamine (m+5) to label endogenous 2HG. Red and green represent pathways promoted and inhibited respectively by HIF-1α in hypoxia. b, Isotopologue distribution of 2HG (as a percentage of the total pool) in CD8+ T lymphocytes, after labelling with U-13C-glucose for 1, 5 and 24 h in both 21% and 1% oxygen conditions (n = 3 mice per time point). c, Isotopologue distribution of 2HG (as a percentage of the total pool) in CD8+ T lymphocytes, after labelling with U-13C-glutamine for 1, 5 and 24 h in both 21% and 1% oxygen conditions (n = 3 mice per time point). Error bars, s.d.

Extended Data Figure 5 S-2HG treatment promotes Hif-1α stability and alters the phenotypic and functional properties of CD8+ T lymphocytes in a Hif-1α-independent manner.

a, b, Immunoblot analysis of nuclear and cytosolic fractions, prepared from CD8+ T lymphocytes cultured in 21% (a) and 1% (b) oxygen, for Hif-1α, Hdac1, phospho-Pdh-E1α (S232) and total Pdh-E1α. Cells were activated for 48 h with anti-CD3 and anti-CD28 antibodies and then expanded for a further 4 days in the presence of IL-2, followed by treatment with the indicated concentration of S-2HG for 16 h. The arrow indicates Hif-1α protein. c, Glucose consumption and lactate production of C57BL/6J, Hif1afl/fl (n = 12) and Hif1afl/fldLckcre (n = 4) CD8+ T lymphocytes treated with or without 500 μM S-2HG-octyl ester. d, Vegfa production of wild-type C57BL/6J, Hif1afl/fl (n = 16) and Hif1afl/fldLckcre (n = 4) CD8+ T lymphocytes treated with or without 500 μM S-2HG-octyl ester. e, Representative flow cytometry plots of IFN-γ versus TNF-α in SIINFEKL re-stimulated OT-I CD8+ T lymphocytes, as a function of increasing doses of S-2HG-octyl ester for 7 days. Associated quantification and statistics are shown in the graphs below. f, Specific killing of EG7-OVA cells by OT-I CD8+ T lymphocytes (n = 3 mice per condition). g, CFSE dilution assay (n = 4 mice per condition) at day 3 of CD8+ T lymphocytes activated with anti-CD3 and anti-CD28 antibodies and cultured with or without 500 μM S-2HG-octyl ester from day 0. Associated quantification and statistics are shown in the graph on the right. h, Viability and annexin V assay of CD8+ T lymphocytes treated with increasing S-2HG doses for 4 days (n = 4 mice). i, Viability of CD8+ T lymphocytes cultured with 300 μM S-2HG-octyl ester for the indicated number of days (n = 4 mice). j, Amount of IFN-γ protein in the media of wild-type C57BL/6J, Hif1afl/fl (n = 8) and Hif1afl/fldLckcre (n = 4) CD8+ T lymphocytes treated for 24 h with or without 500 μM S-2HG-octyl ester. k, Viability of Hif1afl/fl (n = 8) and Hif1afl/fldLckcre (n = 4) OT-I CD8+ T lymphocytes activated with 1,000 nM SIINFEKL peptide and cultured for 7 days with or without 500 μM S-2HG-octyl ester in the absence of IL-2 supplementation from day 0. l, Expression of Ifng mRNA in CD8+ T lymphocytes treated for either 24 h or 7 days with or without 500 μM S-2HG-octyl ester. n = 4 mice per group. m, CD44 and CD62L surface expression on OT-I CD8+ T lymphocytes treated with increasing doses of S-2HG for 7 days. Cells were activated with 1,000 nM SIINFEKL peptide; n = 3 mice. Gated on live, CD8+ cells. n, Illustration outlining the workflow for the experiment. Percentage of CD62Lhi CD8+ T lymphocytes, treated for 7 days with 500 μM S-2HG-octyl ester (left) or vehicle (right), followed by washout or maintenance of the compound and follow up every three days, for 9 more days (n = 4 mice). Gated on live, CD8+ cells. o, p, CD44 and CD62L surface expression on Hif1afl/fl and Hif1afl/fldLckcre (o) or Hif2afl/fl and Hif2afl/fldLckcre (p) CD8+ T lymphocytes treated with or without 500 μM S-2HG-octyl ester for 1, 7 and 10 days following treatment. Data are representative of 3 (o) or 2 (p) mice per genotype. Gated on live, CD8+ cells. q, Flow cytometric characterization of indicated phenotypic markers on Hif1afl/fl and Hif1afl/fldLckcre (n = 4) CD8+ T lymphocytes treated for 7 days with 500 μM S-2HG-octyl ester. Gated on live, CD8+ cells. r, Validation of L2hgdh–Flag expression in CD8+ T lymphocytes from C57BL/6J mice by immunoblot analysis for Flag. The arrow indicates L2hgdh–Flag protein. s, qPCR validation of L2hgdh knockdown in CD8+ T lymphocytes isolated from C57BL/6J mice. t, CD127 surface expression in response to L2hgdh knockdown (n = 4). Representative flow cytometry histogram of CD127 surface levels on transduced (GFP+) CD8+ T lymphocytes in response to shScramble or shL2hgdh 3 in 21% or 1% oxygen is shown on the right. u, qPCR quantification of Prdm1, Sell, Eomes, Tcf7, Bcl6 and Ccr6 expression in CD8+ T lymphocytes treated for 1 or 7 days with or without 500 μM S-2HG-octyl ester. Paired t-test for matched comparisons (g) and two-way ANOVA for grouped data (c, d, j, k, l, q). One-way ANOVA for multiple comparisons (i, n, t). Error bars denote s.d. and each dot in c, d, g, j, k, l, q, t represents an individual mouse. NS, non-significant, *P < 0.05, **P < 0.01, ***P < 0.001. gMFI, geometric mean fluorescence intensity. Experiments were performed with indicated numbers of mice from multiple occasions. For immunoblot source images, see Supplementary Fig. 1.

Extended Data Figure 6 Ex vivo treatment of CD8+ T lymphocytes with S-2HG promotes in vivo homeostatic proliferation and recall of adoptively transferred cells.

a, Diagram outlining the homeostatic proliferation experiments in Fig. 4a–c. Representative flow cytometry plots are shown for each pool before and after adoptive transfer. Flow cytometry plots show viable CD8+ cells. b, Diagram outlining the recall experiments in Fig. 4f. c, Representative flow cytometry plots of recalling CD45.1+CD8+ T lymphocytes in indicated organs on day 7 after vaccination (day 37 after transfer).

Extended Data Figure 7 S-2HG does not inhibit mTOR signalling at the doses necessary for the formation of memory-like CD8+ T lymphocytes.

Immunoblot analysis on cytosolic extracts for mTOR signalling in CD8+ T lymphocytes treated with the indicated doses of S-2HG for 24 h. For immunoblot source images, see Supplementary Fig. 1.

Extended Data Figure 8 S-2HG does not induce Bcl-2 or Bcl-XL that can explain the in vivo persistence of adoptively transferred CD8+ T lymphocytes.

a, qPCR quantification of Bcl2 and Bcl2l1 (Bcl-XL) mRNA levels in response to 500 μM S-2HG-octyl ester treatment for either 1 or 7 days (n = 4 mice). b, Immunnoblot analysis for Bcl-2 and Bcl-XL protein in response to increasing doses of S-2HG-octyl ester for 9 days. c, qPCR quantification of Bcl2 and Bcl-XL mRNA levels in response to 300 μM S-2HG-octyl ester treatment for either 1, 7 or 9 days (n = 4 mice). d, Representative flow cytometry histograms of Bcl-2 and Bcl-XL abundance in CD8+ T lymphocytes treated with 300 μM S-2HG-octyl ester for 9 days. Quantification and associated statistics are shown in the graph on the right (n = 3 mice). e, Immunoblot analysis confirming the expression of Bcl-XL–Flag and Bcl-2–Flag in OT-I in CD8+ T lymphocytes. fh, CD62L (f), CD127 (g) and CD44 (h) surface expression in OT-I CD8+ T lymphocytes transduced with retrovirus expressing either Bcl-2–Flag or Bcl-XL–Flag and treated with the indicated concentration of S-2HG-octyl ester for 7 days (n = 2 mice). i, Representative flow cytometry histograms of CD62L, CD127 and CD44 surface expression in OT-I CD8+ T lymphocytes transduced with retrovirus expressing either Bcl-2–Flag or Bcl-XL–Flag and treated with the indicated concentration of S-2HG-octyl ester for 7 days. The associated statistics of these flow cytometry data are shown in f, g and h. **P < 0.01, NS, non-significant. Paired t-test for matched comparisons (d) and two-way ANOVA for grouped data (a). One-way ANOVA of matched samples for multiple comparisons (c, f, g, h). Error bars denote s.d. and each dot in a and c represents an individual mouse. Experiments were performed with indicated numbers of mice from at least two occasions. For immunoblot source images, see Supplementary Fig. 1.

Extended Data Figure 9 S-2HG induces global histone H3 methylation changes in CD8+ T lymphocytes.

a, Immunoblot analysis on nuclear extracts for histone H3 methylation marks in activated CD8+ T lymphocytes treated with the indicated doses of S-2HG for 7 days. b, Representative flow cytometry histograms of H3K27me3 staining as a function of increasing S-2HG-octyl ester concentration. c, H3K27me3 staining in CD8+ T lymphocytes treated with or without 500 μM S-2HG-octyl ester and stained with or without fluorophore-conjugated C36B11 antibody. d, qPCR measurement for expression of Utx in unstimulated and stimulated CD8+ T lymphocytes; n = 4 mice. Expression for Utx is displayed for each mouse individually. e, Representative flow cytometry plots of CD44 versus CD62L expression, with associated statistics, on activated CD8+ T lymphocytes after 4 days of treatment with 500 μM S-2HG-octyl ester or 1 μM GSKJ4. Gated on live, CD8+ cells. n = 3 mice. f, Representative flow cytometry plots of CD44 versus CD62L expression on CD8+ T lymphocytes with shRNA-mediated knockdown of Utx, 7 days after transduction. Gated on live CD8+GFP+ cells. Graph on right shows knockdown hairpin fidelity for Utx. g, IgG control ChIP–qPCR for H3K4me3, H3K27me and RNA Pol II at and around the TSS for CD62L, in freshly isolated naive or activated CD8+ T lymphocytes treated with or without 500 μM S-2HG-octyl ester for 7 days. Each profile shows the fold change over the non-binding control primer. Each dot represents an individual primer pair. A pool of n = 6 mice was used for each condition and error bars denote s.e.m. One-way ANOVA for multiple matched comparisons (e). Each dot in e represents an individual mouse. Error bars (e, f) denote s.d. **P < 0.01. Experiments were performed with indicated numbers of mice from at least two occasions. For immunoblot source images, see Supplementary Fig. 1.

Extended Data Figure 10 S-2HG induces global changes in the content of 5hmC and 5mC in genomic DNA of CD8+ T-lymphocyte genomic DNA.

a, Representative flow cytometry plots of CD44 versus CD62L expression on CD8+ T lymphocytes with shRNA-mediated knockdown of Tet2, 7 days after transduction. Gated on live CD8+GFP+ cells. Graph on right shows knockdown hairpin fidelity for Tet2 and error bars denote s.d. b, IgG control DIP-qPCR for 5mC and 5hmC at and around the TSS for CD62L, in freshly isolated naive or activated CD8+ T lymphocytes treated with or without 500 μM S-2HG for 7 days. Each profile shows the fold change over the non-binding control primer. Each dot represents an individual primer pair. A pool of n = 6 mice was used for each condition and error bars denote s.e.m.

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Tyrakis, P., Palazon, A., Macias, D. et al. S-2-hydroxyglutarate regulates CD8+ T-lymphocyte fate. Nature 540, 236–241 (2016). https://doi.org/10.1038/nature20165

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