D-2-hydroxyglutarate supports a tolerogenic phenotype with lowered major histocompatibility class II expression in non-malignant dendritic cells and acute myeloid leukemia cells

Abstract

D-2-hydroxyglutarate (D-2-HG) accumulates in patients with acute myeloid leukemia (AML) with mutated isocitrate dehydrogenase (IDH) and in other malignancies. D-2-HG suppresses antitumor T-cell immunity but little is known about potential effects on non-malignant myeloid cells. Here we show that D-2-HG impairs human but not murine dendritic cell differentiation, resulting in a tolerogenic phenotype with low major histocompatibility class II expression. In line with this, IDH-mutated AML blasts exhibited lower expression of HLA-DP and were less susceptible to lysis by HLA-DP-specific T cells. Interestingly, besides its expected impact on DNA demethylation, D-2-HG reprogrammed metabolism towards increased lactate production in dendritic cells and AML. Vitamin C accelerated DNA demethylation, but only the combination of vitamin C and glycolytic inhibition lowered lactate levels and supported major histocompatibility complex class II expression. Our results indicate an unexpected link between the immunosuppressive metabolites 2-HG and lactic acid and suggest a potentially novel therapeutic strategy with combinations of anti-glycolytic drugs and epigenetic modulators (hypomethylating agents) or other therapeutics for the treatment of AML.

Figure 1.

D-2-hydroxyglutarate inhibits human dendritic cell differentiation. (A) Intracellular levels of 2-hydroxyglutarate (2-HG) were analyzed by liquid chromatography tandem mass spectrometry in peripheral blood mononuclear cells (N=3), T cells (after 3 days of culture, N=3), fibroblasts (N=4), monocytes (N=4) and macrophages (N=3 after 7 days) from healthy donors incubated with or without 10 mM D-2-HG (20 h). (B) Viability of dendritic cells (DC) from humans (N=11 donors) and mice (N=4 mice) were analyzed after 7 (human DC) or 10 (murine DC) days of culture. (C) Transmission electron microscopy of DC cultured for 7 days in the presence or absence of 10 mM D-2-HG. One representative experiment out of four is shown at a magnification of 10,000x. Vacuoles of ten cells per donor and condition (N=40 for control and D-2-HG treatment) were counted. (D) Untreated human monocytes (N=3) and monocyte-derived DC cultured with or without 20 mM D-2-HG were analyzed on day 4 (N=4) and 7 (N=31). Murine DC (N=4) were cultured for 10 days with or without 20 mM D-2-HG. HLA-DP (human) or I-A/I-E (murine) cell surface expression was analyzed by flow cytometry and normalized to the expression of the control (monocyte-derived DC at day 7 of culture, without D-2-HG). (E) Representative histogram of HLA-DP (human) or I-A/I-E (murine) expression analyzed by flow cytometry. (F) HLA-DR surface expression on untreated monocytes (N=4) before the start of culture in comparison to the expression on DC on days 4 (N=3) and 7 (N=46) of culture (with and without 20 mM D-2-HG) as determined by flow cytometry. (G) Levels of cytokines (interleukin-12 [N=10]), interleukin-10 [N=6], tumor necrosis factor [N=8] and interleukin-6 [N=8]) were determined by enzyme-linked immunosorbent assay (ELISA) in supernatants of DC stimulated with lipopolysaccharide (100 ng/mL) for 24 h. (H) Human CD4 T cells (10⁵) were stimulated with DC (10⁴) from an allogeneic donor differentiated in the presence or absence of 20 mM D-2-HG. On day 7 of the mixed lymphocyte reaction, T-cell proliferation (N=6) was measured by cell counting. (I) Interferon-γ secretion of T cells (N=6) was analyzed in supernatants of day 5 cultures by ELISA and normalized to control. Symbols represent individual donors analyzed in independent experiments and horizontal bars mark median values. For two-group comparisons a Mann-Whitney test or Wilcoxon test was used. For multiple-group comparisons the Kruskal-Wallis and post-hoc Dunn test were performed. P<0.05 was considered statistically significant (*P<0.05, ***P<0.01, ***P<0.001). PBMC: peripheral blood mononuclear cells; w/o: without; MO: monocytes; MFI: mean fluorescence intensity; IL: interleukin; TNF: tumor necrosis factor; IFN: interferon.

Figure 2.

D-2-hydroxyglutarate induced changes in amino acid and glucose metabolism. (A) Concentrations of metabolites in supernatants of dendritic cells (DC, N=8) cultured with or without 20 mM D-2-hydroxyglutarate (D-2-HG) measured on day 7 of culture. Dashed lines indicate the concentrations in RPMI medium. (B) Western blot analysis of lactate dehydrogenase A (LDHA) and MYC in DC cultured with or without 20 mM D-2-HG for 7 days. (C, D) Scatter plots showing the summary and quantification of actin-normalized LDHA (C) and MYC (D) signals relative to the control. (E) pH values were monitored in the absence or presence of 20 mM D-2-HG or L-2-HG or 10 mM lactic acid every 5 minutes for 7 days by PreSens technology; the mean values of six independent experiments are shown. (F) Bisulfite-converted DNA of DC was analyzed by MassARRAY Epityper analysis. Four loci showing active DNA demethylation during monocyte-derived DC differentiation (CLEC10A, CCL13, CD207, C10ORF78) were analyzed. Data represent the mean of three different donors and are presented as heatmaps. The methylation ratio (including 5-methylcytosine and 5-hydroxymethylcytosine, which cannot be distinguished after bisulfite treatment) at single CpG dinucleotides (individual boxes) is indicated by shades of yellow to blue (yellow: no methylation, dark blue: 100% methylation). Gray boxes indicate CpG that were not detected by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. A bar plot presentation of representative CpG (marked with asterisks) indicating active demethylation is shown in Online Supplementary Figure S2H and methylation ratios of all single CpG units for individual donors are provided in Online Supplementary Table S1. (A-E) Symbols represent individual donors analyzed in independent experiments and horizontal bars mark median values. For two-group comparisons, the Mann-Whitney test was used; in (E) two-way analysis of variance and a post-hoc Tukey test were performed. P<0.05 was considered statistically significant (*P<0.05, ***P<0.01, ***P<0.001). w/o: without; Ala: alanine; Asn: asparagine; Asp: aspartic acid; OA: oxaloacetate; TCA: tricarboxylic acid cycle; α-KG: α-ketoglutarate; Glu: glutamic acid; Pro: proline; Arg: arginine; DC: dendritic cells; MO: monocytes; VitC: vitamin C; 5mC: 5-methylcytosine; 5hmC: 5-hydroxymethylcytosine.

Figure 3.

Vitamin C and lactate dehydrogenase inhibitor treatment counteract D-2-hydroxyglutarate-induced effects. (A) Scatter plot showing lactate level in supernatants of dendritic cells (DC) treated with 20 mM D-2-hydroxyglutarate (D-2-HG) or L-2-hydroxyglutarate (L-2-HG) or 10 mM lactic acid (started on day 0), 1 mM vitamin C (VitC, started on day 0) plus the lactate dehydrogenase inhibitor GNE140 (1 µM, started on day 2), and 1 mM VitC plus the lactate dehydrogenase inhibitor NCI-737 (0.1 µM, started on day 2) for 7 days. (B) HLA-DR and (C) HLA-DP expression on DC treated for 7 days with D-2-HG, L-2-HG or 10 mM lactic acid was analyzed by flow cytometry. (D-G) Representative histograms of HLA-DP expression in DC treated with D-2-HG and inhibitors (1 mM Vit C, 1 µM GNE140) as indicated in the Figure. Numbers indicate median fluorescence intensity values. (H) HLA-DP (N=5), (I) HLA-DR (N=5), (J) CD1a (N=4) and (K) DC-SIGN (N=4) surface expression on DC cultured with D-2-HG and inhibitors (1 mM VitC, 1 µM GNE140) as indicated in the Figure and analyzed by flow cytometry. Symbols represent individual donors analyzed in independent experiments and horizontal bars mark median values. For multiple-group comparisons, one way analysis of variance and a post-hoc Dunnett, Friedman or Dunn test were performed. P<0.05 was considered statistically significant (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). MFI: mean fluorescence intensity; GNE: GNE140.

Figure 4.

Primary acute myeloid leukemia blasts treated with exogenous D-2-hydroxyglutarate show altered metabolism and MHC class II expression. (A) Scatter plots showing concentrations of metabolites in supernatants of acute myeloid leukemia (AML) cells cultured with or without 20 mM D-2-hydroxyglutarate (D-2-HG) measured on day 7 of culture (N=6). Dashed lines indicate the respective concentrations in the culture medium without cells. (B) Cell yield (dashed line indicates starting cell counts) and (C) viability of AML blasts (N=9) after 7 days of culture with or without 20 mM D-2-HG. (D) Lactate levels (4 days, N=3; 7 days, N=9) of isocitrate dehydrogenase (IDH) wild-type AML cells cultured with or without 20 mM D-2-HG. (E) HLA-DP surface expression of IDH wild-type AML blasts cultured for either 4 or 7 days separated by lactate concentrations below and above 10 mM. (F) Correlations of lactate concentration and HLA-DP expression are shown. AML samples cultured without D-2-HG are displayed in black, samples treated with D-2-HG are shown in orange; samples after 4 days are shown as squares and those after 7 days as dots (<10 mM N=6, >10 mM N=14). For two-group comparisons a Wilcoxon test or Mann-Whitney U test was performed. P<0.05 was considered statistically significant (*P<0.05, **P<0.01). (F) Spearman rank correlation coefficient was calculated. w/o: without; Ala: alanine; Asn: asparagine; Asp: aspartic acid; OA: oxaloacetate; TCA: tricarboxylic acid cycle; α-KG: α-ketoglutarate; Glu: glutamic acid; Pro: proline; Arg: arginine; MFI: mean fluorescence intensity.

Figure 5.

Endogenous D-2-hydroxyglutarate produced by mutated IDH reduces MHC class II protein expression in primary acute myeloid leukemia blasts. (A) Intracellular levels of 2-hydroxyglutarate (2-HG) were analyzed by liquid chromatography tandem mass spectrometry in acute myeloid leukemia (AML) blasts with wild-type isocitrate dehydrogenase (IDH) (N=6), mutated IDH1 (N=6) or mutated IDH2 (N=6). (B) Transcription levels of MHC class II α and β chain genes (HLA-DP, -DQ and -DR) of AML blasts expressing wild-type or mutated IDH were analyzed in RNA-sequencing data from The Cancer Genome Atlas (TCGA), including wild-type (N=155) and IDH-mutated (N=35) AML blasts from different patients at primary AML diagnosis. (C) CIITA expression data of AML exported from TCGA (for numbers see above). (D) Surface expression of HLA class II molecules (HLA-DP [wild-type N=14, mutated N=8], -DQ [wild-type N=11, mutated N=8] and –DR [wild-type N=11, mutated N=8]) on primary AML blasts expressing mutated or wild-type IDH analyzed by flow cytometry. (E) Total HLA-DP expression of AML blasts expressing mutated (N=4) or wild-type (N=4) IDH was evaluated by western blot analysis. Specific IDH mutations are depicted. (F) Primary HLA-DPB1*04:01⁺ AML blasts expressing wild-type (black symbols) or mutated IDH (orange symbols) were analyzed for their recognition by CD8 T cells expressing an HLA-DPB1*04:01-specific T-cell receptor upon RNA electroporation in a standard 5 h [Cr] release assay at the indicated effector (T cell)-to-target (AML blast) ratios. Specific lysis of IDH wild-type (N=3) and IDH2 mutated (N=3) AML blasts is shown. (G) As a control, CD8 T cells were electroporated without RNA (mock). (A-D) Symbols represent individual donors analyzed in independent experiments and horizontal bars mark median values. For two-group comparisons a Mann-Whitney test was used and for multiple-group comparisons a Kruskal-Wallis and post-hoc Dunn test were performed. P<0.05 was considered statistically significant (*P<0.05, **P<0.01). wt: wild-type; mut: mutated; RKPM: reads per kilo base per million mapped reads; TCR: T-cell receptor; E:T: effector-to-target cell ratio.

Figure 6.

Vitamin C impairs survival of cultured primary acute myeloid leukemia cells and increases MHC class II expression in combination with GNE140. Primary cultured AML cells expressing wild-type (black) and mutant isocitrate dehydrogenase (IDH) (orange) were analyzed before (d0) and after 4 and 7 days of culture in the absence or presence of 2 mM vitamin C (VitC) and 1 µM GNE140, a lactate dehydrogenase inhibitor. (A) Representative FACS plots with live gating on primary cultured AML with or without treatment on day 7. (B) Summarized data on viability kinetics of untreated AML cells expressing wild-type (black) and mutant IDH (orange). (C) Percentage of living cells and (D) HLA-DP and (E) HLA-DR levels in the presence of 2 mM VitC and 1 µM GNE140 on day 7, normalized to the percentage of living, untreated cells. For multiple-group comparisons, a Kruskal-Wallis or Friedman test and post-hoc Dunn test were performed. P<0.05 was considered statistically significant (*P<0.05, **P<0.01). wt: wild-type; mut: mutated; w/o: without; GNE: GNE140; VitC: vitamin C.