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.

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 Vhl^fl/fl (n=5), Vhl^fl/fldlck^cre (n=5) and Hif1α^fl/fl Vhl^fl/fldlck^cre (n=3) CD8⁺ T-lymphocyte groups. b/ Glucose consumption and lactate production in Vhl^fl/fl and Vhl^fl/fldlck^cre CD8⁺ T-lymphocytes 7 days after activation with αCD3 and αCD28 antibodies. n=4 mice per genotype from two independent experiments. c/ Immunoblot analysis for HIF-1α and LaminB1, using nuclear extracts prepared from Vhl^fl/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 Vhl^fl/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 Hif1α or Hif2α in CD8⁺ T-lymphocytes, isolated from Hif1α^fl/fldlck^cre or Hif2α^fl/fldlck^cre mice. n=4 mice. d/ Total 2HG levels, normalized to viable cell count or protein content, in Hif1α^fl/fl and Hif1α^fl/fldlck^cre CD8⁺ T-lymphocytes cultured as in Fig. 2b. n=4 mice per genotype. e/ Total 2HG levels, normalized to viable cells or protein content, in Hif2α^fl/fl and Hif2α^fl/fldlck^cre 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 Hif1α^fl/fl, Hif1α^fl/fldlck^cre, Hif2α^fl/fl and Hif2α^fl/fldlck^cre CD8⁺ T-lymphocytes. Also shown are viability measurements at day 4. n=4 mice per genotype. g/ Total amount of 2HG in Hif1α^fl/fl (n=6) and Hif1α^fl/fldlck^cre (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/ 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 Hif1α^fl/fl (n=4) and Hif1α^fl/fldlck^cre (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. l/ LC-MS/MS quantification of S- and R-2HG in CD8⁺ T-lymphocytes isolated from C57BL/6J mice, with shRNA-mediated knockdown of Mdh1; n=4 pools of 4 mice per pool. m/ LC-MS/MS quantification of S- and R-2HG in CD8⁺ T-lymphocytes isolated from C57BL/6J mice, with shRNA-mediated knockdown of Mdh2; n=4 pools of 4 mice per pool. n/ LC-MS/MS quantification of R-2HG in CD8⁺ T-lymphocytes isolated from C57BL/6J mice, with shRNA-mediated knockdown of Ldha; n=4 pools of 4 mice per pool. o/ Validation of Pdk1-FLAG and Ldha-FLAG expression in Hif1α^fl/fldlck^cre 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 Hif1α^fl/fl, Hif1α^fl/fldlck^cre, Hif2α^fl/fl and Hif2α^fl/fldlck^cre 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. t/ Total intracellular concentration of 2HG in CD8⁺ T-lymphocytes from C57BL/6J mice cultured as in Fig. 2b and treated with 5mM DCA for the latter 48h of culture; n=4 mice. u/ Total intracellular amount of 2HG, nomralized to viable cell count or protein content in CD8⁺ T-lymphocytes from C57BL/6J mice cultured as in Fig. 2b and treated with 5mM DCA for the latter 48h of culture; n=4 mice. v/ Total intracellular amount of glutamate in CD8⁺ T-lymphocytes from C57BL/6J mice cultured as in Fig. 2b and treated with 5mM DCA for the latter 48h of culture; n=4 mice. Two-way ANOVA for grouped data (d, e, f, q, r, t, u, v). Paired t-test for matched comparisons (i, p), one-way ANOVA for multiple matched comparisons (g, l, m, n). 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. Naïve 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 Idh1 as 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. Error bars denote s.d. 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 denote s.d.

Extended Data Figure 5. S-2HG treatement 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 (a) 21% and (b) 1% oxygen, for HIF-1α, HDAC1, phospho-PDH E1α (S232) and total PDH-E1α. Cells were activated for 48 h with αCD3+αCD28 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 hours. The arrow indicates HIF-1α protein. c/ Glucose consumption and lactate production of C57BL/6J, Hif1α^fl/fl (n=12) and Hif1α^fl/fldlck^cre (n=4) CD8⁺ T-lymphocytes treated with or without 500 μM S-2HG-octyl ester for 16 hours as in Extended Data Fig. 5a. d/ VEGF-A production of wild type C57BL/6J, Hif1α^fl/fl (n=16) and Hif1α^fl/fldlck^cre (n=4) CD8⁺ T-lymphocytes treated with or without 500 μM S-2HG-octyl ester for 16 hours as in Extended Data Fig. 5a. e/ Representative flow cytometry plots of IFN-γ vs 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. Total splenocytes were activated for 48 h with 1000 nM SIINFEKL and then expanded for a further 4 days in the presence of IL-2 followed by treatment with 500 μM S-2HG-octyl ester for 24 h. OT-I CD8⁺ T-lymphocytes were incubated with target and control cells for 4 hours; n=3 mice per condition. g/ CFSE dilution assay, with associated statistics (n=4 mice per condition) at day 3 of CD8⁺ T-lymphocytes activated with αCD3+αCD28 antibodies and cultured with or without 500 μM S-2HG-octyl ester from day 0. Data are representative of 4 mice. 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, Hif1α^fl/fl (n=8) and Hif1α^fl/fl dlck^cre (n=4) CD8⁺ T-lymphocytes treated for 24 h with or without 500 μM S-2HG-octyl ester. k/Viability of Hif1α^fl/fl (n=8) and Hif1α^fl/fl dlck^cre (n=4) OT-I CD8⁺ T-lymphocytes activated with 1000 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 1000 nM SIINFEKL peptide; n=3 mice. Gated on live, CD8⁺ cells. n/ Illustration outlining the workflow for the experiment. Left panel: %CD62L^High CD8⁺ T-lymphocytes, treated for 7 days with 500 μM S-2HG-octyl ester, followed by washout or maintenance of the compound and follow up every 3^rd day, for 9 more days; n=4 mice. Right panel: %CD62L^High CD8⁺ T-lymphocytes, treated for 7 days with vehicle, followed by addition of 500 μM S-2HG-octyl ester or vehicle and follow up every 3^rd day, for 9 more days; n=4 mice. Gated on live, CD8⁺ cells. o/ CD44 and CD62L surface expression on Hif1α^fl/fl and Hif1α^fl/fl dlck^cre 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 mice per genotype. Gated on live, CD8⁺ cells. p/ CD44 and CD62L surface expression on Hif2α^fl/fl and Hif2α^fl/fl dlck^cre 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 2 mice per genotype. Gated on live, CD8⁺ cells. q/ Flow cytometric characterisation of indicated phenotypic markers on Hif1α^fl/fl (n=4) and Hif1α^fl/fl dlck^cre (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 knockdowns 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 Figure 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 Figure 4f. c/ Representative flow cytometry plots of recalling CD45.1⁺ CD8⁺ T-lymphocytes in indicated organs on day 7 post vaccination (day 37 post 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 Bclxl 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 Bclxl 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. f-h/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, b). 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. ns=non-significant, **p<0.01, gMFI=geometric mean fluorescence intensity. 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 histione 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/ Represetative flow cytomery plots of CD44 vs 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 vs CD62L expression on CD8⁺ T-lymphocytes with shRNA-mediated knockdown of Utx, 7 days after trasnduction. 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 naïve 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. Error bars denote s.e.m. One-way ANOVA for multiple matched comparisons (e). Each dot in c represents an individual mouse. Error bars 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 vs CD6L expression on CD8⁺ T-lymphocytes with shRNA-mediated knockdown of Tet2, 7 days after trasnduction. Gated on live, CD8⁺GFP⁺ cells. Graph on right shows knockdown hairpin fidelity for Tet2. b/ IgG control DIP-qPCR for 5mC and 5hmC at and around the TSS for CD62L, in freshly isolated naïve 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. Error bars denote s.e.m.

Figure 1. VHL-HIF signalling regulates 2-hydroxyglutarate levels

a/ Unsupervised hierarchical clustering and heat map of all detected metabolites. b/ PCA of metabolomes. Percentage variance of each PC is in parenthesis. c/ Metabolites ranked in order of decreasing p-value . d/ 2HG levels in Vhl^fl/fl (n=7), Vhl^fl/fldlck^cre (n=4) and Hif1α^fl/fl Vhl^fl/fldlck^cre (n=4) CD8⁺ T-lymphocytes activated with αCD3 and αCD28 antibodies and then cultured in IL-2 for a further 5 days. Each dot represents a mouse. e/ 2HG levels in RCC4 and 786-O cells with reconstitution of VHL; n=3. f/ 2HG levels in MEFs with deletion of VHL; n=3. Unpaired t-test (e-f) and one-way ANOVA (d). Error bars denote s.d.; ns = non-significant, *p<0.05, **p<0.01, ***p<0.001.

Figure 2. Hypoxic induction of 2-hydroxyglutarate depends on HIF-1α in CD8⁺ T-lymphocytes

a/ 2HG in naïve and activated cells, n≥4 mice per time point. b/ 2HG in cells cultured at 21% or 1% oxygen 48 h; n=12 mice. c/ 2HG in cells cultured at 21% (n=15), 10% (n=6), 5% (n=10) or 1% (n=6) oxygen for 48 h. d/ Enantioselective MS for S- and R-2HG. e/ 2HG in human cells( n=4 healthy donors). f/ Enantioselective MS for S- and R-2HG from cells in e. g-h/ 2HG in Hif1α^fl/fl and Hif1α^fl/fl dlck^cre (g) or Hif2α^fl/fl and Hif2α^fl/fl dlck^cre (h) cells, cultured as in b; n=4 mice per genotype. i/ 2HG in splenic extracts from Hif1α^fl/fl (n=20) and Hif1α^fl/fl dlck^cre (n=15) mice. j/ 2HG in urine from Hif1α^fl/fl (n=49) and Hif1α^fl/fl dlck^cre (n=25) mice. k/. S-2HG in cells with shLdha. n=4 individual transductions. l/ Ldha-FLAG (n=6) or Pdk1-FLAG (n=4) over-expression in Hif1α^fl/fl dlck^cre cells. The dotted line represents S-2HG levels in Hif1α^fl/fl cells n=4/6 individual tranductions. m/ ¹³C-isotopologue profile of 2HG in cells cultured as in b ; n=7 mice per condition. Unpaired t-test ( i, j). Paired t-test (b, l, m). One-way ANOVA (c, k) and two-way ANOVA (g, h). Error bars denote s.d.; each dot in a, b, c, i and j represents a. ns = non-significant, *p<0.05, **p<0.01, ***p<0.001.

Figure 3. S-2HG alters phenotypic marker expression of CD8⁺ T-lymphocytes

a/ Media IL-2 levels from S-2HG-treated cells. b/ Expression of Il2 mRNA in S-2HG-treated cells. c/ CD62L and CD127 expression on from S-2HG-treated OT-I cellsn=4 mice. d/ % CD62L^HighCD44^Low and % CD62L^LowCD44^High cells with S-2HG treatment. n=4 mice. e/CD62L expression on cells treated with S-2HG and transduced with empty or L2hgdh-FLAG over-epression vectors. f/ Representative flow cytommetry plots of KLRG1 vs CD127 (n=4) on cells from e. Associated statistics are shown. g/ S-2HG level in response to shRNA against L2hgdh (n=4). h/ CD62L expression in response to L2hgdh knockdown (n=4). Representative flow cytometry histogram of CD62L surface levels is shown on the right. Two-way ANOVA (a, b, e, f). One-way ANOVA (c, d, g, h). Error bars denote s.d. .Each dot in a, b, e, f, h, represents a mouse. ns=non-significant, *p<0.05, **p<0.01, ***p<0.001. gMFI=geometric mean fluorescence intensity.

Figure 4. S-2HG treatment promotes in vivo homeostatic renewal, persistence and anti-tumour capacity of transferred cells

a/ Recovery of co-transferred CD45.1⁺ OT-I cells, from spleens (n=6). b/In vivo CFSE levels in cells from a. c/ % of cells in a that divided 0-9 times in vivo. d/ Representative flow cytometry plots and associated statistics of recovered cells from spleens (n=6). e/ Representative analysis of cells, in d, relative to naïve cells (n=6). f/ Recovery of co-transferred cells, from spleens, lymphnodes and livers of vaccinated mice (n=6). g/ Lymphodepleted mice bearing EG7-OVA tumours treated with no T-cells (n=7) or OT-I cells cultured with (n=6) or without (n=6) S-2HG. Error bars denote s.e.m. h/ Lymphoreplete mice bearing EG7-OVA tumours treated with no T-cells (n=6) or OT-I cells cultured with (n=6) or without (n=6) S-2HG. Error bars denote s.e.m. Paired t-test (a, b, f), unpaired t-test (d), one-way ANOVA (g, h). Error bars denote s.d. (not in g and h). Each dot in a, b, d and f represents a mouse. ns=non-significant, *p<0.05, **p<0.01, ***p<0.001. gMFI=geometric mean fluorescence intensity.

Figure 5. S-2HG alters global H3K27me3 in CD8⁺ T-lymphocytes

a/Immunoblot analysis for H3K27me2 and H3K27me3 in cells treated with S-2HG. b/ H3K27me3 in OT-I cells treated with S-2HG. n=6 mice. c/ H3K27me3 abundance in Hif1α^fl/fl (n=4) and Hif1α^fl/fl dlck^cre (n=4) cells treated with S-2HG. d/ CD62L expression by cells with shUtx #3. e/In vivo H3K27me3 levels in CD8⁺ populations. n=6 mice. f/ ChIP-qPCR for H3K4me3, H3K27me and RNA pol II around the TSS for CD62L. A pool of n=6 mice was used. Error bars denote s.e.m. One-way ANOVA (b, c). Error bars denote s.d. and each dot in c and e represents a mouse. ns=non-significant, *p<0.05, **p<0.01, ***p<0.001. gMFI=geometric mean fluorescence intensity. For immunoblot source images, see Supplementary Fig. 1.

Figure 6. S-2HG alters global 5hmC and 5mC in DNA of CD8⁺ T-lymphocytes

a/ 5hmC and 5mC in gDNA. n=4 mice. b/ 5hmC in gDNA from cells treated with S-2HG. n=4 mice. c/ 5mC in gDNA from cells treated with S-2HG-. n=4 mice. d/ CD62L expression on CD8⁺ T-lymphocytes 7 days after transduction with shTet2 #3. e/ DIP-qPCR for 5mC and 5hmC around the TSS for CD62L. A pool of n=6 mice was used. Error bars denote s.e.m. One-way ANOVA (b and c). Error bars denote s.d. and each dot in b and c represents a mouse. ns= non-significant, *p<0.05, ***p<0.001. gMFI=geometric mean fluorescence intensity.