Oncometabolite d-2HG alters T cell metabolism to impair CD8+ T cell function

Abstract

Gain-of-function mutations in isocitrate dehydrogenase (IDH) in human cancers result in the production of d-2-hydroxyglutarate (d-2HG), an oncometabolite that promotes tumorigenesis through epigenetic alterations. The cancer cell-intrinsic effects of d-2HG are well understood, but its tumor cell-nonautonomous roles remain poorly explored. We compared the oncometabolite d-2HG with its enantiomer, l-2HG, and found that tumor-derived d-2HG was taken up by CD8⁺ T cells and altered their metabolism and antitumor functions in an acute and reversible fashion. We identified the glycolytic enzyme lactate dehydrogenase (LDH) as a molecular target of d-2HG. d-2HG and inhibition of LDH drive a metabolic program and immune CD8⁺ T cell signature marked by decreased cytotoxicity and impaired interferon-γ signaling that was recapitulated in clinical samples from human patients with IDH1 mutant gliomas.

Fig. 1.. d-2HG impairs CD8⁺ T cell proliferation, cytotoxicity, and IFN-γ signaling in an acute and reversible fashion.

(A) Total 2HG levels in activated CD8⁺ T cells after 24-hour treatment with increasing concentrations of d-2HG or l-2HG. Total ion count was normalized to cell number (n = 3). (B) Intracellular 2HG concentration determined by assuming a T cell volume of 500 fl (n = 3). (C) Left: CellTrace Violet dilution assay at day 3 of CD8⁺ T cells activated by CD3/CD28 mAbs in the presence of 20 mM d-2HG, 20 mM l-2HG, or left untreated. Stained, naive cells (shaded gray) that did not proliferate are shown. Right: Division index corresponds to the average number of cell divisions that a cell in the total cell population has undergone (n = 3). (D) CellTrace Violet dilution assay according to experimental design described in fig. S1E to assess reversibility of proliferation phenotype. (E) Schematic of a cytotoxic CD8⁺ T cell releasing granzyme B and IFN-γ. (F) Intracellular granzyme B levels of CD8⁺ T cells activated by CD3/CD28 mAbs in the presence of 20 mM d-2HG, 20 mM l-2HG, or left untreated for 3 days. (G) Percentage degranulation as assessed by CD107a/b staining of CD8⁺ T cells activated by CD3/CD28 mAbs and restimulated by CD3 mAb in the presence of 20 mM d-2HG, 20 mM l-2HG, or left untreated. In the washout condition, CD8⁺ T cells were activated in the presence of 20 mM d-2HG, and the oncometabolite was subsequently washed out before restimulation. In the acute condition, d-2HG was added solely at the time of restimulation. In all other conditions, the metabolites were kept for the entirety of the assay (n = 3). (H) Percentage of IFN-γ⁺ CD8⁺ T cells after intracellular cytokine staining of CD8⁺ T cells activated with phorbol myristate acetate (PMA) and ionomycin for 4 hours in the presence of 20 mM d-2HG, 20 mM l-2HG, or left untreated (n = 3). (I) Mean fluorescent intensity for IFN-γ after intracellular cytokine staining of CD8⁺ T cells activated with PMA and ionomycin for 4 hours in the presence of 20 mM d-2HG, 20 mM l-2HG, or left untreated (n = 3). (J) IFN-γ levels in the medium of CD8⁺ T cells activated with 20 mM d-2HG, 20 mM l-2HG, or left untreated for 24 hours (n = 3). (K) Volcano plot showing distribution of the top down-regulated and up-regulated genes in CD8⁺ T cells activated in the presence of 20 mM d-2HG or left untreated. Statistically significant ISGs with a fold change >1.5 are marked in pink (n = 3). (L) Antigen-specific killing of B16 ovalbumin-positive tumor cells by OT1 CD8⁺ T cells that were activated in the presence of 20 mM d-2HG, 20 mM l-2HG, or left untreated. In the washout condition, d-2HG was removed before co-culture seeding of tumor cells and T cells. In the acute condition, d-2HG was added at the time of tumor cell and T cell co-culture. In all other conditions, the metabolites were kept for the entirety of the assay (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way and two-way ANOVA). Data are representative of at least two independent experiments.

Fig. 2.. d-2HG alters glycolysis in CD8⁺ T cells.

(A) Schematic of experimental design to assess the acute (24 hours) effects of 20 mM d-2HG and 20 mM l-2HG on steady-state metabolite levels in activated CD8⁺ T cells. (B) Log₂ fold changes of glycolytic intermediates in 20 mM d-2HG–treated versus 20 mM l-2HG–treated CD8⁺ T cells relative to control. F6P, fructose 6-phosphate; G6P, glucose 6-phosphate; DHAP, dihydroxyacetone phosphate; GA3P, glyceraldehyde 3-phosphate; PEP, phosphoenolpyruvic acid (n = 3). (C) Log₂ fold changes of key glycolytic metabolites that are secreted into or consumed from the medium of d-2HG–treated, l-2HG–treated, or untreated CD8⁺ T cells (n = 3). (D) Schematic of expected incorporation of heavy carbons into glycolytic intermediates after ¹³C₆ glucose is provided for 24 hours. (E) Ion intensities of glycolytic intermediates and their respective ¹³C-isotopologs after 24-hour cotreatment of ¹³C₆ glucose with 20 mM d-2HG, 20 mM l-2HG, or control (n = 3). (F) Levels of secreted pyruvate isotopologs in the medium of 20 mM d-2HG–treated, 20 mM l-2HG–treated, and untreated CD8⁺ T cells after providing ¹³C₆ glucose for 24 hours (n = 3). (G) Levels of secreted lactate isotopologs in the medium of 20 mM d-2HG–treated, 20 mM l-2HG–treated, and untreated CD8⁺ T cells after providing ³C₆ glucose for 24 hours (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way ANOVA). Data are representative of at least two independent experiments.

Fig. 3.. d-2HG inhibits LDH-A activity in vitro and in CD8⁺ T cells.

(A) Schematic of LDH reaction. (B) Intracellular lactate (M+3)/pyruvate (M+3) ratio of CD8⁺ T cells after 24-hour cotreatment of ¹³C₆ glucose with 20 mM d-2HG or control (n = 3). (C) Schematic of the metabolic consequences of antimycin A (AA) treatment on glucose catabolism. (D) Intracellular lactate (M+3)/pyruvate (M+3) ratio after brief 20 mM d-2HG pretreatment, followed by AA and ¹³C₆ glucose cotreatment (n = 3). Solid bars, −AA; striped bars, +AA. (E) In vitro enzymatic assessment of 3 mM l-2HG, 3 mM d-2HG, 80 μM oxamate, and 10 nM GSK2837808A on LDH-A activity. Oxamate and GSK2837808A are known LDH inhibitors and were used as a control (n = 3). (F) Lineweaver-Burk plot for d-2HG. (G) NAD⁺/NADH ratio of CD8⁺ T cells treated for 24 hours with control, 1 mM nicotinamide mononucleotide (NMN), 20 mM d-2HG, or 20 mM l-2HG. NMN raises intracellular NAD⁺ levels and was therefore used as a positive control (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test, one-way and two-way ANOVA). Data are representative of at least two independent experiments.

Fig. 4.. Cytosolic NAD(H) imbalance drives mitochondrial membrane hyperpolarization in d-2HG–treated CD8⁺ T cells.

(A) Relative contribution of mitochondrial oxidative phosphorylation and glycolysis to ATP production in response to d-2HG after 24 hours of treatment (n = 10). (B) Quantification of basal respiration in response to 20 mM d-2HG after 24 hours of treatment (n = 10). (C) Mitochondrial membrane potential as assessed by tetramethylrhodamine ethyl ester (TMRE) fluorescence in CD8⁺ T cells treated with 20 mM d-2HG, 20 mM l-2HG, or control for 3 days. (D) TMRE assay according to experimental design described in fig. S4F. (E) Mitochondrial membrane potential as assessed by TMRE fluorescence in CD8⁺ T cells treated with 20 mM d-2HG, 20 mM l-2HG, or control for 2 hours. (F) Schematic of targets of rotenone, AA, oligomycin, and FCCP. (G) Effects of inhibition of ETC complexes on mitochondrial membrane potential in control and d-2HG–treated CD8⁺ T cells (n = 3). (H) Quantification of spare respiratory capacity in response to 20 mM d-2HG after 24 hours of treatment (n = 10). (I) Schematic of targets of AOA. (J) Mitochondrial membrane potential as assessed by TMRE fluorescence in CD8⁺ T cells treated with 20 mM d-2HG or control for 24 hours in the presence or absence of 1 mM AOA (n = 3). (K) Schematic of LbNOX mechanism of action. (L) Western blot of CD8⁺ T cells overexpressing empty vector (EV) or FLAG-tagged cytosolic LbNOX. (M) NAD⁺/NADH ratio of CD8⁺ T cells overexpressing EV or cytosolic LbNOX after a 24-hour-long treatment with 20 mM d-2HG or control (n = 3). (N) TMRE staining of EV or LbNOX-overexpressing CD8⁺ T cells treated with 20 mM d-2HG or left untreated for 24 hours (n = 3). (O) Schematic of the target of rotenone. (P) Quantification of division index of CD8⁺ T cells activated in the presence or absence of 20 mM d-2HG for 3 days and cotreated with 1 nM rotenone (n = 3). (Q) Quantification of intracellular ROS levels as assessed by CellROX staining in CD8⁺ T cells treated with 20 mM d-2HG or left untreated for 1 day. Hydrogen peroxide (H₂O₂, 100 mM) and 10 mM NAC were used as positive and negative controls, respectively (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test, one-way and two-way ANOVA). Data are representative of at least two independent experiments.

Fig. 5.. LDH inhibition recapitulates the effects of d-2HG on CD8⁺ T cell metabolism, proliferation, cytotoxicity, and IFN-γ signaling.

(A) Lactate (M+3)/pyruvate (M+3) ratio in CD8⁺ T cells treated with 20 mM d-2HG, 20 mM oxamate, 10 μM GSK2837808A, or left untreated for 24 hours (n = 3). (B) NAD⁺/NADH ratio of CD8⁺ T cells after a 24-hour-long treatment with 20 mM d-2HG, 20 mM oxamate, 10 μM GSK2837808A, or control (n = 3). (C) Quantification of basal respiration in response to 20 mM d-2HG, 20 mM oxamate, 10 μM GSK2837808A, or control after 24 hours of treatment (n = 10). (D) Left: Mitochondrial membrane potential as assessed by TMRE fluorescence in CD8⁺ T cells treated with 20 mM d-2HG, 20 mM oxamate, 10 μM GSK2837808A, or control for 24 hours. Right: quantification of mean fluorescence intensity for TMRE (n = 3). (E) Left: CellTrace Violet dilution assay at day 3 of CD8⁺ T cells activated in the presence of 20 mM d-2HG, 20 mM oxamate, 10 μM GSK2837808A, or left untreated. Right: quantification of division index (n = 3). (F) Quantification of intracellular granzyme B expression in CD8⁺ T cells activated in the presence of 20 mM d-2HG, 20 mM oxamate, 10 μM GSK2837808A, or control for 3 days (n = 3). (G) Percentage degranulation assessed by CD107a/b staining in CD8⁺ T cells activated in the presence of 20 mM d-2HG, 20 mM oxamate, 10 μM GSK2837808A, or left untreated (n = 3). (H) Percentage of IFN-γ⁺ CD8⁺ T cells after intracellular cytokine staining of CD8⁺ T cells activated with PMA and ionomycin for 4 hours in the presence of 20 mM d-2HG, 20 mM oxamate, 10 μM GSK2837808A, or left untreated (n = 3). (I) Mean fluorescent intensity for IFN-γ after intracellular cytokine staining of CD8⁺ T cells activated with PMA and ionomycin for 4 hours in the presence of 20 mM d-2HG, 20 mM oxamate, 10 μM GSK2837808A, or left untreated (n = 3). (J) Cell number–normalized levels of IFN-γ in the cell culture medium of CD8⁺ T cells activated in the presence of 20 mM d-2HG, 20 mM oxamate, 10 μM GSK2837808A, or control for 2 days (n = 3). (K) Relative mRNA expression of ISGs in CD8⁺ T cells activated in the presence of 20 mM d-2HG, 20 mM oxamate, 10 μM GSK2837808A, or control for 24 hours (n = 3). (L) Specific killing of B16 ovalbumin-positive tumor cells by OT1 CD8⁺ T cells activated in the presence of 20 mM d-2HG, 20 mM oxamate, 10 μM GSK2837808A, or control (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way ANOVA). Data are representative of at least two independent experiments.

Fig. 6.. Altered metabolic and cytotoxicity signatures in IDH1-mutant relative to IDH1^WT cancers.

(A) Schematic depicting experimental setup. (B) Quantification of 2HG levels by liquid chromatography–mass spectrometry in the tumor interstitial fluid (TIF) from IDH1^WT and IDH1^R132H MC38 and B16 tumors (n = 10 to 12). (C) Quantification of the percentage of CD8⁺ T cells (of CD3⁺CD45⁺ cells) in spleen and tumor of IDH1^WT and IDH1^R132H MC38 and B16 syngeneic tumor mouse models (n = 6 to 10). (D) Quantification of the percentage of Ki67⁺ CD8⁺ T cells in spleen and tumor of IDH1^WT and IDH1^R132H MC38 and B16 syngeneic tumor mouse models (n = 6 to 10). (E) Quantification of the percentage of IFN-γ⁺, IL-2⁺, and TNF-α⁺CD8⁺ T cells isolated from IDH1^WT or IDH1^R132H MC38 or B16 tumors on day 16 after inoculation and after ex vivo restimulation with PMA and ionomycin for 4 hours in the presence of 0 or 2.5 mM d-2HG to recapitulate the 2HG levels measured in the TIF (n = 8). (F) MSI-based quantification of 2HG levels in tissue sections of human IDH1^R132H gliomas subdivided by grade. The upper limit of 2HG quantification based on the calibration curve was 30 mM; therefore, any value beyond that was collapsed to 30 mM. A, astrocytoma; OD, oligodendroglioma; I-II, low-grade; III-IV, high-grade. (G) Per-section average lactate ion counts across IDH1^WT GBM (n = 5) and IDH1^R132H (n = 4) astrocytoma, grade IV patients. (H) Per-pixel lactate and 2HG ion counts across all sections from case #3, an IDH1^WT GBM patient, and case #19, an IDH1^R132H glioma patient. Sections were annotated based on high cellularity (HC), low cellularity (LC), nontumor (NT), and transition (T). (I) Three-dimensional (3D) UMAP embedding of single-cell CyCIF data derived from human brain tumor sections colored by HDBSCAN cluster identifying a population of CD8⁺ T cells (cluster 2, black arrow). (J) 3D UMAP embedding color mapped according to CD8 signal intensity showing that cluster 2 cells (black arrow) are characterized by high levels of CD8 expression. (K) Overlay of matched MSI and CyCIF images from case #18, sample 3 of an IDH1^R132H astrocytoma. Spatial positions of cluster 2 cells are shown in green; Hoechst nuclear dye (DNA) is shown in gray. Grid superimposed on the image shows equal-sized microregions of tissue with x and y intervals corresponding to the pixel size of the original MSI image used to compute statistics in (L) and (M). (L) Welch’s t tests comparing mean tissue density–corrected d-2HG levels in microregions of tissue with and without CD8⁺ T cells in case #18, sample 3. (M) Welch’s t tests comparing mean tissue density–corrected d-2HG levels in microregions of tissue with and without CD8⁺ T cells in case #19, sample 1. (N) Spearman’s rank-order correlation showing anticorrelation between mean tissue density-corrected d-2HG levels and CD8⁺ T cell counts in microregions of tissue in case #18, sample 3. (O) Spearman’s rank-order correlation showing anticorrelation between mean tissue density–corrected d-2HG levels and CD8⁺ T cell counts in microregions of tissue in case #19, sample 1. (P) Heatmap showing expression of top DE up-regulated genes in CD8⁺ and CD4⁺ T cells in IDH^WT GBM relative to IDH-mutant gliomas. Gene expression is zero-centered and given in units of ln(TP100K+1). (Q) Grouping of the top up-regulated genes into hallmark gene sets. (R) Density plot of expression of top DE up-regulated genes in the IFN subcluster for CD8⁺ T cells in IDH^WT and IDH-mutant samples. (S) Density plot of expression of the top DE up-regulated CD8⁺ IDH^WT genes in the chemokine–IFN-γ subcluster for T cells in and IDH-mutant samples. (T) Density plot of expression of the top DE up-regulated genes in cytotoxicity-NK subcluster for CD8⁺ T cells in IDH^WT and IDH-mutant samples. (U) Overlay of the top DE ISGs from mouse RNA sequencing onto the top 400 DE up-regulated genes in the CD4⁺ and CD8⁺ T cell populations of IDH-mutant and IDH^WT tumors showing the relative expression of genes in common between the two datasets. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test and two-way ANOVA). Data are representative of at least two independent experiments.