Figure 6.
Metabolic profiling of NFATc1-dysfunctional T cells shows impaired glycolysis upon stimulation. (A) Summary dot plots showing the ratio of change of expression level of glucose transporter 1 (GLUT1) and glucose uptake using fluorescent glucose analog 2-NBDG after 24-hour stimulation of feeder-expanded T cells with soluble CD3/CD28 antibodies measured by gMFI. (B) Extracellular flux analysis. (Top) Graph showing oxidative consumption rate of HC (n = 3) and patient CD8+ T lymphoblasts after 30 minutes stimulation with CD3/CD28 beads and subjected to mitochondrial stress test. (Bottom) Graph showing extracellular acidification rate of HC (n = 3) and patient CD8+ T lymphoblasts, which were stimulated for 30 minutes with CD3/CD28 beads and subjected to glycolytic stress test. Experimental data were normalized to flow cytometric cell counts. (C, left) Immunoblot showing the protein expression levels of HK2, glucose transporter-3 (GLUT3), carnitine palmitoyl transferase 1a (CPT1a), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and lactate dehydrogenase (LDH) on patient and HC T lymphoblasts with (ST) and without (US) 24-hour stimulation using soluble anti-CD3/CD28 antibodies. (C, right) Graphs showing the quantification of the western blots averaged from 3 independent experiments displaying the expressions of HK2 and CPT1a. (D) Scheme summarizing the metabolic alteration observed in patient T lymphoblasts after stimulation. (E) Heatmap showing fold changes of metabolite concentrations (stimulated/unstimulated) in T lymphoblasts in patients and HCs measured by metabolomics after 24 hours of stimulation with soluble anti-CD3/CD28 antibodies. All data are mean ± SEM. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc test (A-B) or 2-way ANOVA with Bonferroni post hoc test (C) to correct for multiple comparison. AA, antimycin A; DHAP, dihydroxyacetonephosphate; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; GA3P, glyceraldehyde 3-phosphate; NAD, nicotinamide adenine dinucleotide; NAD(H), nicotinamide adenine dinucleotide+hydrogen; oligo, oligomycin; 3-PG, 3-phosphoglycerate; Rot, rotenone.

Metabolic profiling of NFATc1-dysfunctional T cells shows impaired glycolysis upon stimulation. (A) Summary dot plots showing the ratio of change of expression level of glucose transporter 1 (GLUT1) and glucose uptake using fluorescent glucose analog 2-NBDG after 24-hour stimulation of feeder-expanded T cells with soluble CD3/CD28 antibodies measured by gMFI. (B) Extracellular flux analysis. (Top) Graph showing oxidative consumption rate of HC (n = 3) and patient CD8+ T lymphoblasts after 30 minutes stimulation with CD3/CD28 beads and subjected to mitochondrial stress test. (Bottom) Graph showing extracellular acidification rate of HC (n = 3) and patient CD8+ T lymphoblasts, which were stimulated for 30 minutes with CD3/CD28 beads and subjected to glycolytic stress test. Experimental data were normalized to flow cytometric cell counts. (C, left) Immunoblot showing the protein expression levels of HK2, glucose transporter-3 (GLUT3), carnitine palmitoyl transferase 1a (CPT1a), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and lactate dehydrogenase (LDH) on patient and HC T lymphoblasts with (ST) and without (US) 24-hour stimulation using soluble anti-CD3/CD28 antibodies. (C, right) Graphs showing the quantification of the western blots averaged from 3 independent experiments displaying the expressions of HK2 and CPT1a. (D) Scheme summarizing the metabolic alteration observed in patient T lymphoblasts after stimulation. (E) Heatmap showing fold changes of metabolite concentrations (stimulated/unstimulated) in T lymphoblasts in patients and HCs measured by metabolomics after 24 hours of stimulation with soluble anti-CD3/CD28 antibodies. All data are mean ± SEM. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc test (A-B) or 2-way ANOVA with Bonferroni post hoc test (C) to correct for multiple comparison. AA, antimycin A; DHAP, dihydroxyacetonephosphate; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; GA3P, glyceraldehyde 3-phosphate; NAD, nicotinamide adenine dinucleotide; NAD(H), nicotinamide adenine dinucleotide+hydrogen; oligo, oligomycin; 3-PG, 3-phosphoglycerate; Rot, rotenone.

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