Lactate Limits T Cell Proliferation via the NAD(H) Redox State

Abstract

Immune cell function is influenced by metabolic conditions. Low-glucose, high-lactate environments, such as the placenta, gastrointestinal tract, and the tumor microenvironment, are immunosuppressive, especially for glycolysis-dependent effector T cells. We report that nicotinamide adenine dinucleotide (NAD⁺), which is reduced to NADH by lactate dehydrogenase in lactate-rich conditions, is a key point of metabolic control in T cells. Reduced NADH is not available for NAD⁺-dependent enzymatic reactions involving glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate dehydrogenase (PGDH). We show that increased lactate leads to a block at GAPDH and PGDH, leading to the depletion of post-GAPDH glycolytic intermediates, as well as the 3-phosphoglycerate derivative serine that is known to be important for T cell proliferation. Supplementing serine rescues the ability of T cells to proliferate in the presence of lactate-induced reductive stress. Directly targeting the redox state may be a useful approach for developing novel immunotherapies in cancer and therapeutic immunosuppression.

Keywords: 3-phosphoglycerate; T cell metabolism; glycolysis; immunometabolism; lactate metabolism; nicotinamide adenine dinucleotide; redox metabolism; serine.

Figure 1.. L-Lactate Lowers the NAD⁺:NADH Ratio and Induces a GAPDH Block

(A and B) Murine CD4⁺CD25⁻ T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE), co-stimulated for 3 days with anti-CD3ε/CD28-coated beads, and exposed to NaCl, Na L-lactate, HCl, and L-lactic acid at the indicated doses in low-glucose media (30 mg × dL⁻¹). The pH of the HCl condition was adjusted to match the L-lactic-acid-containing media. Teff proliferation was analyzed by CFSE dilution through flow cytometry. (A) shows representative and (B) pooled data from 11 independent experiments (two-way ANOVA with multiple comparison of treatment condition and dose). (C and D) Phenformin (70 mg kg⁻¹ day⁻¹) was administered to C57BL/6J mice. (C) 2 h after phenformin injection, CG4⁺ venous blood gas showed increased L-lactate (6/grp). (D) C57BL/6J mice received an major histocompatibility complex (MHC)-mismatched BALB/c heart transplant and were treated with phenformin (70 mg kg⁻¹ day⁻¹) for 14 days or DMSO vehicle control (3/grp, log rank [Mantel-Cox] test). (E) L-lactate derivative analysis from modified media with 20 mM [¹³C₃] L-lactate and 1.11 mM [¹²C₆] D-glucose shows that 16 h anti-CD3ε/CD28 stimulated Tconvs oxidize L-lactate into pyruvate and metabolize it in the Krebs cycle. MID denotes mass isotopomer distribution, and M0–M6 denote the number of [¹³C] per indicated molecule. Data are pooled from three independent experiments. (F) Hypothesis: L-lactate-mediated reduction of NAD⁺ to NADH blocks the GAPDH forward reaction. (G) Tconvs were isolated from C57BL/6J mice and co-stimulated for 16 h with CD3ε/CD28 mAb-coated beads and 20 mM NaCl, Na lactate, or Na pyruvate. NAD:NADH was measured by cycling. Pyruvate increased although lactate decreased the proportion of oxidized NAD⁺. Data are pooled from seven independent experiments (paired one-way ANOVA) (H) Tconvs were stimulated with CD3ε/CD28 mAb-coated beads and cultured in low-glucose medium (30 mg × dL⁻¹) for 48 h ± 20 mM NaCl, Na L-lactate, or Na pyruvate and 1 μM heptelidic acid (GAPDHi) or vehicle control (H₂O). Metabolites were extracted and analyzed via LC-MS. The heatmap shows increased (red) and decreased (blue) glycolytic intermediates ion count normalized to the NaCl and vehicle control (dotted line indicates GAPDH reaction). Addition of Na pyruvate led to a depletion of pre-GAPDH triose phosphate, although Na L-lactate (NADH reduction) led to a decline in post-GAPDH metabolites, similar to direct GAPDH inhibition. (I) 10⁶ CD8⁺ T cells were co-stimulated and cultured with ±20 mM NaCl, Na L-lactate, GAPDHi, or Na pyruvate, and glucose was measured in the supernatant. Data are pooled from seven independent experiments (one-way ANOVA). *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars indicate SEM.

Figure 2.. Reduced NAD⁺:NADH Redox State Limits Glycolysis in T Cells

(A) Schematic of the electron transport chain (ETC) complexes I–V at the inner mitochondrial membrane with complex V denoting ATP synthase and how oligomycin (oligo), FCCP (cyanide-4-[trifluoromethoxy]phenylhydrazone), rotenone (Rot), and antimycin (anti) can act as NAD:NADH polarizing agents. (B and C) Tconvs were stimulated with CD3ε/CD28 mAb-coated beads for 48 h and then exposed to 1.25 μM oligomycin, 1 μM FCCP, or 1 μM rotenone for 15 min. NAD and NADH were measured by (B) NAD cycling or (C) LC-MS (paired one-way ANOVA; four independent experiments). Oligomycin and rotenone increased NADH reduction, although mitochondrial uncoupling (FCCP) increased NAD oxidation. (D) Tconvs were stimulated with CD3ε/CD28 soluble mAb and exposed to oligomycin, FCCP, or rotenone as in (B) and (C). Fluorescence lifetime imaging microscopy showed prolonged NADH autofluorescence with FCCP uncoupling, consistent with mitochondrial NAD oxidation. Scale bar, 10 μm. Data are representative of four independent experiments. (E–G) Tconv cells were stimulated with anti-CD3ε/CD28 beads and 25 U interleukin-2 (IL-2) × mL⁻¹ for 16 h (E and F) or 2 h (G). Oligomycin increases ECAR as expected. In stimulated Tconv, mitochondrial uncoupling (oxidized NAD⁺) through FCCP further increases ECAR, which can be brought back to the oligomycin level via rotenone and antimycin (reduced NADH). (E) shows representative and (F) and (G) pooled data from five (F) and four (G) independent experiments, respectively (paired one-way ANOVA). (H) LC-MS total ion counts of glycolysis metabolites from Tconv treated as in (C). *p < 0.05 (Student’s t test versus untreated; data pooled from four independent experiments). Dotted line indicates GAPDH reaction. (I) NAD and NADH measurements in murine Tconv-stimulated CD3ε/CD28 mAb-coated beads for 3 days and exposed to the nicotinamide phosphoribosyl-transferase (Nampt) inhibitor FK866 at 100 nM with or without 100 μM of the NAD precursor nicotinamide riboside (NR). Data are pooled from three independent experiments (one-way ANOVA). (J) Murine Tconvs were CFSE labeled and stimulated with CD3ε/CD28 mAb for 3 days in low-glucose media (30 mg × dL⁻¹). Adding 100 nM FK866 to inhibit NAD regeneration was toxic to dividing T cells. Enriching NAD⁺ through administration of NR rescued the dividing T cells from FK866 toxicity. Adding NR along augmented T cell proliferation is shown. (K) Quantitative data to added NR in low-glucose media; data pooled from four independent experiments (paired Student’s t test). *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars indicate SEM.

Figure 3.. L-Lactate Depletes Post-GAPDH Glycolytic Intermediates

(A and B) Tconvs were co-stimulated with CD3ε/CD28 mAb-coated beads and cultured in low-glucose medium (30 mg × dL⁻¹) ± 20 mM NaCl or Na L-lactate, with either 0.5 μM ionomycin, 1 μM of the GAPDH inhibitor (GAPDHi) heptelidic acid, or vehicle controls. After 3 days, proliferation was analyzed via CFSE dilution. (A) shows representative and (B) quantitative data pooled from three independent experiments (paired one-way ANOVA). (C) Lactate inhibits GAPDH and PGDH, although oxalate blocks pyruvate kinase. (D and E) Tconvs were co-stimulated with CD3ε/CD28 mAb-coated beads and cultured in low-glucose medium (30 mg × dL⁻¹) in serine/glycine-free media ± 20 mM NaCl or Na L-lactate, with either 0.4 mM serine or 1 mM Na oxalate or vehicle controls. After 3 days, proliferation was analyzed via CFSE dilution. (D) shows representative and (E) quantitative data pooled from four independent experiments (paired one-way ANOVA). *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars indicate SEM.

Figure 4.. L-Lactate Impairs Glucose-Derived Serine Production

Tconvs were co-stimulated with CD3ε/CD28 mAb-coated beads for 20 h and were then labeled with [¹³C₆] D-glucose in serine and glycine free media for 3 h with either 20 mM of NaCl or Na L-lactate (L-lac) and 1 mM NaCl or Na oxalate (Ox). Metabolites were extracted and analyzed for derivative analysis, with M+1−10 indicating the number of [¹³C] labeling per molecule. (A–E) Prior to GAPDH, L-lactate and oxalate lead to an accumulation of M+6 glucose and M+3 triose-P. Oxalate and lactate also lead to some reverse glycolysis (M+3 tracing in hexose 6P and fructose 1,6-BP). (F–J) Post-GAPDH glycolytic intermediates: oxalate inhibits pyruvate kinase reaction. Both L-lactate and oxalate diminish the amount of pyruvate made from glucose. (K–M) L-lactate decreases and oxalate increases glucose-derived serine. (N) The pentose phosphate pathway is not diminished by L-lactate, if anything augmented by L-lactate/oxalate. (O) Heatmap of total ion counts of ATP shows equal M+5 but diminished M+7 to M+9 with L-lactate treatment. Data are derived from three independent samples per condition (one-way ANOVA or Student’s t test of the highest enriched isotopologue).