Article Figures & Data
Figures
- Figure S1. Time courses of human CD4+ T-cell activation and proliferation.
Human CD4+ T cells were cultured in 5 mM glucose and stimulated using anti-CD3/CD28 human T-cell activation beads. (A) Mean fluorescence intensity and standard error of the mean for the expression of the cell surface marker CD69+ at the indicated times up to 96 h (n = 5). (B) Cumulative number of proliferated cells as the percent of CD4+ T cells over time. Proliferation was tracked by flow cytometry using the cell proliferation dye eFluor450. Results are shown for four independent experiments performed in duplicate.
- Figure 1. Mitochondrial function and cellular glucose uptake in human CD4+ T cells.
Human eFluor450-labeled CD4+ T cells were stimulated using anti-CD3/CD28 beads and cultured in the presence of 5 mM glucose. (A, B) Proliferation was measured by dye dilution at 72 h (A, B). (B) Oligomycin was either added to the cell culture together with the stimulation beads (time point 0 h; A), and then washed out after 16 h for the remainder of cell culture or (B) added after 16 h and kept in the medium until the end of culture. (C) Glucose uptake during CD4+ activation was monitored in anti-CD3/CD28 bead-stimulated T cells using 2-NBDG after 0, 16, and 72 h. Data are represented as mean fluorescence intensity and mean + SD; Each point represents one independent experiment. (D) Mitochondrial reactive oxygen species production was determined using MitoSOX Red in unstimulated T cells, and after 10 min, 16 h, and 72 h of stimulation. Comparisons were made using two-tailed paired t tests in (A) and (B), repeated-measures one-way ANOVA in (C) and (D). *P ≤ 0.05 and **P ≤ 0.01.
- Figure 2. Respiratory signatures of human CD4+ T cells in the presence of metabolic inhibitors.
Human CD4+ T cells were stimulated with anti-CD3/CD28 beads for 48 h and the T-cell respiration signature was analyzed using an extracellular flux analyzer. (A, B, C) Mitochondrial ATP-linked respiration and (D, E, F) Glycolytic activity at the indicated concentrations of oligomycin, rotenone, and resveratrol added in real time during the measurement period in the presence of 5 mM glucose. (G, H) Effects of low and high glucose concentrations on mitochondrial ATP-linked respiration (panel G) and glycolytic activity (panel H). Comparisons were made using repeated-measured one-way ANOVA with Fisher’s least significant difference test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.
- Figure S2. Methodology of mitochondrial inhibition.
(A) Schematic of the metabolic perturbations used in the study. (B, C, D, E, F, G) Representative measurements of the oxygen consumption rate (panels B, C and D) and the extracellular acidification rate (ECAR; panels E, F and G) in the presence of different concentrations of different inhibitors. CD4+ T cells were cultured in 5 mM glucose and stimulated with anti-CD3/anti-CD28 beads for 48 h before measurement in the xf extracellular flux analyzer. Injection time points are indicated by red boxes on the x-axis. After baseline measurement at 22 min, different concentrations of Rotenone (panels B and E), Oligomycin (panels C and F) and Resveratrol (panels D and G) were injected. Subsequently, after 56 min, Oligomycin (2 μM) was injected followed by FCCP after 76 min and rotenone/oligomycin together with 2-DG after 97 min.
- Figure 3. Glucose uptake and reactive oxygen species production in the presence of metabolic inhibitors.
(A) CD4+ T cells were stimulated with anti-CD3/CD28 beads for 16 h in the presence of 5 mM glucose with or without 1 μM oligomycin, 1 μM rotenone, or 50 μM resveratrol. The cells were starved for the last 2 h and labeled glucose (2-NBDG) was added to the culture for 10 min, after which glucose uptake was measured by flow cytometry. Data are shown as the mean ± SEM (n = 3) mean fluorescence intensity. (B, C) T cells were left unstimulated (panel B) or were stimulated with anti-CD3/CD28 beads for 16 h (panel C) in the absence (control) or presence of the indicated inhibitor. Mitochondrial reactive oxygen species production was monitored using MitoSOX Red by flow cytometry and is shown as the mean ± SEM MitoSOX Red mean fluorescence intensity (n = 5). Comparisons to the control conditions without inhibitor or 5 mM glucose were made using repeated-measures one-way ANOVA, with Fisher’s least significant difference test *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.
- Figure S3. CD4+ glucose uptake and reactive oxygen species production at different glucose concentrations.
Human CD4+ T cells were cultured in 1, 5, or 25 mM glucose for 16 h, with and without anti-CD3/CD28 bead stimulation. (A) The mean fluorescence intensity in glucose uptake measured by flow cytometry using 2-NBDG. Each dot represents one donor. (B, C) Reactive oxygen species production in stimulated (B) or unstimulated (C) cells was measured by flow cytometry using MitoSOX Red. The mean fluorescence intensity was assessed at 16 h in cells exposed to 1, 5, or 25 mM glucose. (2-NBDG: n = 3; MitoSOX Red: n = 5.) Experiments for each condition were performed in duplicate.
- Figure 4. Effects of metabolic perturbations on T-cell function.
(A, B) T-cell activation markers CD69 (panel A) and CD25 (panel B) after 16 h of anti-CD3/CD28 stimulation in the absence (control) or at increasing doses of oligomycin (n = 3), rotenone (n = 3), and resveratrol (n = 4), each in the presence of 5 mM glucose, and in different glucose concentrations without additional inhibitor (n = 3). The surface marker expression of CD69 and CD25 is shown as the median with interquartile range of the frequency of positive cells T. (C) T-cell proliferation was measured by dye dilution at 72 h after stimulation of eFluor450-labeled CD4+ T cells in the absence or presence of oligomycin (n = 3), rotenone (n = 3), and resveratrol (n = 3) each in the presence of 5 mM glucose, and in different glucose concentrations (n = 8) without additional inhibitor. Data are shown as the median with interquartile range of the frequency of proliferated cells. (D, E) Intracellular cytokines were measured 6 h after stimulation with anti-CD3/CD28 beads in the absence or presence of oligomycin (1 μM) (n = 3), rotenone (1 μM) (n = 3), or resveratrol (50 μM) (n = 3), and in different glucose concentrations (n = 4) (panel D) as well as in different concentrations of resveratrol (panel E). Secretion inhibitor brefeldin A was added for the last 4 h. Data are shown as the relative fold value of the frequency of positive cells for each of the cytokines as compared to control conditions as median with interquartile range. Comparisons to the control conditions without inhibitor or 5 mM glucose were made using repeated-measures one-way ANOVA with individual variances computed for each comparison in (A, B, C), and paired t tests in (D, E). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.
- Figure S4. Representative flow analysis of activation, proliferation, and cytokine production of CD4+ T cells in the presence of metabolic inhibitors.
(A) Expression of the T-cell activation marker CD69 after 16 h of anti-CD3/CD28 bead stimulation without or in the presence of 0.1 μM oligomycin, 1 μM rotenone, or 50 μM resveratrol. (B) T-cell proliferation measured using the dye dilution method at 72 h after anti-CD3/CD28 bead stimulation of eFluor450-labeled CD4+ T cells without or with oligomycin, rotenone, or resveratrol. Intracellular cytokine production 6 h after anti-CD3/CD28 bead stimulation without or with oligomycin, rotenone, or resveratrol. The secretion inhibitor brefeldin A was added for the last 4 h of the stimulation time.
- Figure S5. FCCP rescue experiment of T cells from the effects of oligomycin treatment.
(A) Illustration of the expected mitochondrial effects of oligomycin in the absence or presence of FCCP on in the mitochondria and upon addition of FCCP. Blocking the ATP synthase by oligomycin disables the flux of electrons and protons through the mitochondrial membrane, and creates a redox imbalanced by the accumulation of NADH and inhibits the regeneration of NAD+ (left panel). This redox imbalance can be restored by uncoupling the electron and proton flux from ATP production by using the uncoupling agent Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (right panel). (B) The dependence of an intact redox balance, independent of ATP production during T-cell activation, represented by CD4+ CD69+ T cells is shown in (B). T cells were stimulated for 16 h using with anti-CD3/CD28 beads in the presence (red bars) or absence (grey bars) of 0.1 μM oligomycin. and for the rescue experiment, cells were also exposed to 0.1 μM oligomycin plus 1.5 μM FCCP (blue bars). The number of CD69-positive cells positive for CD69 was measured. Each point represents one independent experiment, performed in technical duplicates (n = 6). Comparisons were made using repeated-measures one-way ANOVA, with a single pooled variance was performed (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; and ****P ≤ 0.0001).
- Figure S6. Representative flow analysis of cytokine production of CD4+ T cells in the presence of metabolic inhibitors.
Intracellular cytokine production without stimulation (top row) and 6 h after anti-CD3/CD28 bead stimulation without (row 2) or with oligomycin (row 3), rotenone (row 4), or resveratrol (row 5). The secretion inhibitor brefeldin A was added for the last 4 h of the stimulation time.
- Figure S7. Effects of metabolic inhibitors on T-cell cytokine production.
(A, B, C) Intracellular T-cell cytokine production after 6 h of anti-CD3/CD28 stimulation in the absence or presence of the indicated concentrations of (A) oligomycin (n = 3), (B) rotenone (n = 3) and (C) glucose (n = 4). The secretion inhibitor brefeldin A was added for the last 4 h of the stimulation time. Data are shown as the fold-value in the frequency of positive cells for each cytokine relative to the control condition (no inhibitor and 5 mM glucose). Comparisons were made using repeated-measures one-way ANOVA, with individual variances computed for each comparison. *P ≤ 0.05 and **P ≤ 0.01.
- Figure 5. Effects of glucose deprivation during mitochondrial stress on T-cell functions.
(A, B, C) CD4+ T cells were cultured and stimulated for (A) 16 h to measure T-cell activation as the frequency of CD69+ cells, (B) for 72 h to measure T-cell proliferation, and (C) for 6 h to measure IFNγ production with the indicated glucose concentration without (black symbols) or with a high concentration (1 μM) for T-cell activation and cytokine production and low concentration (0.1 μM) for T-cell proliferation of the mitochondrial inhibitors oligomycin (blue symbols) or rotenone (red symbols). Each point represents one donor (n = 3). Data are represented as mean value. Comparisons to the control conditions of 5 mM glucose were made using repeated-measures one-way ANOVA with a single pooled variance and uncorrected Fishers’ least significant difference. *P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001.
- Figure S8. Volcano plots of the genes that were differentially expressed between the control and inhibitor treated cells for CD4+ CD45RO+ (left) and CD45RA+ (right) T cells.
(A, B, C) Cells were treated with (A) 0.1 µM oligomycin, (B) 1 µM rotenone, or (C) 50 µM resveratrol. The red dots denote differentially expressed genes that met the P-value threshold (adjusted P-value < 0.05) and the fold-change threshold (absolute log2 fold change ≥ 0.5). The blue dots denote genes that only met the P-value threshold, the green dots denote genes that only met the fold-change threshold, and the grey dots denote the genes that did not meet either threshold.
- Figure 6. RNAseq analysis of memory CD4+ T cells in the presence of metabolic inhibitors.
(A) Dot plot showing the results of the pathway enrichment analysis of differentially expressed genes in the memory CD4+ T cells determined using gprofiler2. The size of the dot is proportional to the ratio of the differentially expressed genes in a particular Reactome pathway and the total number of genes assigned to that pathway (pathway coverage). The color of the dot shows the pathway enrichment significance (adjusted P-value). The pathways with adjusted P-values of >0.05 are indicated with a red box. (B) Heat map showing the log2 fold changes of the differentially expressed genes associated with the OAS antiviral response and IFN pathways as well as heat shock proteins in memory and naive T cells after exposure to rotenone and oligomycin. (C) Heat map showing the log2 fold change values of cytokine secretion genes in memory T cells with resveratrol, rotenone, and oligomycin, and oligomycin in the presence of 1 mM glucose.
- Figure 7. Metabolic consequences of resveratrol on human CD4+ T cells.
(A) Heat map showing the log2 fold-change values of naive, memory and regulatory T cells when comparing the resveratrol treated samples to control conditions. HIC1 was transcriptionally down-regulated by resveratrol in all three CD4+ T cell subsets. A concomitant increase in SIRT1 and downstream genes such as TP53 and PPARA in some of these T cells subsets was also observed. (B) Representative flow analysis histograms of MitoTracker Deep Red FM in CD4+ T cells after 16 h in anti-CD3/CD28 unstimulated and bead-stimulated conditions in the presence (red) or absence (grey) of 50 μM resveratrol and 5 mM glucose. (C) Quantification of the mitochondrial mass changes shown in (B). The fold-values of the MitoTracker Deep Red FM Median fluorescence intensity for increasing resveratrol concentrations relative to no resveratrol for unstimulated (left) and stimulated (right) are shown as mean ± SD. (D) The spare respiratory capacity was measured using an extracellular flux analyzer 48 h after stimulation with anti-CD3/CD28 beads together with increasing resveratrol concentrations in the last 16 h of incubation. (E) Mitochondrial reactive oxygen species production was measured using MitoSOX Red at different resveratrol concentrations. (F) Cellular glucose uptake was measured in the presence of 50 μM resveratrol after 16 and 72 h using 2-NBDG. Comparisons to the control conditions without inhibitor were made using repeated-measures one-way ANOVA, with individual variances computed for each comparison. *P ≤ 0.05 and **P ≤ 0.01.
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