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.