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Resveratrol stimulates the metabolic reprogramming of human CD4+ T cells to enhance effector function

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Science Signaling  17 Oct 2017:
Vol. 10, Issue 501, eaal3024
DOI: 10.1126/scisignal.aal3024
  • Fig. 1 Resveratrol modulates the TCR-stimulated activity of the NAD+-dependent deacetylase Sirt1 and blast formation in human CD4+ T cells.

    (A) Left: The presence of Sirt1 in freshly isolated quiescent human CD4+ T cells was assessed by staining with a Sirt1 polyclonal antibody (green) and the nuclear stain 4′,6-diamidino-2-phenylindole (DAPI) (blue). The presence and localization of Sirt1 were also assessed after 1 (D1) or 3 (D3) days under nonstimulating conditions (NS) or after TCR stimulation (TCR) in the absence or presence of 20 or 100 μM resveratrol (RVT). Images are representative of 50 cells in three independent experiments. Right: Sirt1 was also monitored by flow cytometric analysis of quiescent human CD4+ T cells and cells that were stimulated for 3 days through the TCR in the absence or presence of 20 or 100 μM resveratrol. Data are representative of four independent experiments. (B) Left: Sirt1 abundance in CD4+ T cells treated with 20 μM resveratrol was monitored by flow cytometry as a function of both FSC and SSC. The four numbered populations of cells were distinguished on the basis of their FSC-SSC characteristics (dot plot), and Sirt1 abundance in the indicated populations was further analyzed. Right: Histograms are representative of 10 independent experiments. Bottom: Sirt1 abundance and localization in cells from the indicated populations were also analyzed by immunofluorescence staining. Images are representative of three experiments. (C) Sirt1 deacetylase activity was monitored as a function of the generation of OAADPr generation, a reaction product of Sirt1-catalyzed NAD+-dependent protein deacetylation. CD4+ T cells were untreated or were stimulated through the TCR in the absence or presence of 20 or 100 μM resveratrol for 3 days before the amount of OAADPr in each sample was determined. Data are means ± SEM of six independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.0001 by one-way analysis of variance (ANOVA) and Tukey’s post hoc test.

  • Fig. 2 Low- and high-dose resveratrol block TCR-mediated cell cycle progression at distinct stages of the cell cycle.

    (A) Cell cycle entry after TCR stimulation at day 1 (D1; top) and day 3 (D3; bottom) in the presence or absence of resveratrol was monitored by simultaneous staining of DNA and RNA with 7-aminoactinomycin D and pyronin Y, respectively. Representative dot plots from five experiments of nonstimulated and TCR-activated CD4 T cells, in the absence or presence of resveratrol, are shown. The percentages of cells in G0-G1A phase (lower left quadrant), G1B phase (lower right quadrant), and S, G2, and M phases (upper right quadrant) are indicated. (B) T cell proliferation under the indicated conditions was monitored by carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling, and dilution of the fluorescent dye was assessed at 72 hours. The number of division peaks is indicated in each histogram. Data are representative of six experiments. (C) Schematic representation of cell cycle regulators that are altered upon TCR-mediated cell cycle entry. Cell cycle progression requires the expression of cyclins and Cdks, the F-box protein Skp2-dependent and ubiquitin-mediated degradation of the p27Kip1 Cdk inhibitor, and Cdk-mediated hyperphosphorylation of the pRb tumor suppressor and the related p130 pocket protein. Cdk1 activity and mitotic entry are regulated by the kinase Wee1 and the phosphatase of Cdc25. (D) The abundances of the cyclins-Cdks that regulate cell cycle entry, including cyclins D2, E1, A2, and B1, and Cdk4, Cdk6, Cdk2, and Cdk1 were monitored by Western blotting analysis on days 1 and 3 of activation. Data are representative of three independent experiments. The arrow indicates the hyperphosphorylated Cdk1 isoform. (E) The abundances of the Ki-67 proliferation marker, cell division inhibitors (pRb, p130, and p27), and the p27 regulator Skp2 under the indicated conditions were monitored by Western blotting analysis. Data are representative of three independent experiments. Arrows indicated hyperphosphorylated p130 and phosphorylated Skp2. Quantification of all panels is shown in fig. S1.

  • Fig. 3 TCR signaling is attenuated by high-dose, but not low-dose, resveratrol.

    (A) Top: Phosphorylation of ZAP-70 and ERK after TCR stimulation of human CD4+ T cells in the presence of 20 or 100 μM resveratrol was monitored by flow cytometry. Representative histograms at 1 min after stimulation are presented. Bottom: Quantification of the fold increase in MFIs of the indicated proteins in stimulated relative to nonstimulated CD4+ T cells. Data are means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.005 by one-way ANOVA and Tukey’s post hoc test. (B) The relative amounts of total and phosphorylated Akt in T cells 5 and 30 min after activation under the indicated conditions were determined by Western blotting analysis. Data are representative of three experiments. (C) The extent of phosphorylation of TSC2, mTOR, and S6 in CD4+ T cells 24 hours after stimulation under the indicated conditions was monitored by Western blotting analysis. Data are representative of three experiments. (D) Left: The cell surface expression of the CD69, IL-2Rα (CD25), and transferrin receptor (CD71) activation markers on CD4+ T cells stimulated under the indicated conditions were assessed by flow cytometry. Histograms are representative of three independent experiments. Right: Quantification of the percentages of positive cells under each condition. **P < 0.01 and ****P < 0.0001 by two-way ANOVA with Bonferroni’s post hoc test. Quantification data are shown in fig. S1.

  • Fig. 4 Low-dose resveratrol stimulates H2AX phosphorylation in TCR-stimulated CD4+ T lymphocytes.

    (A) Top: Freshly isolated quiescent CD4+ T cells were either left nonstimulated or were TCR-stimulated in the presence of resveratrol (20 and 100 μM) or aphidicolin. The amount of H2AX phosphorylation (γH2AX) was assessed at 24 hours by flow cytometry. Data are representative of four independent experiments. Bottom left: The percentages of γH2AX-positive cells were quantified after 2, 6, 12, and 24 hours of stimulation. Data are representative of four independent experiments. Bottom right: Means ± SEM of γH2AX-positive cells from three independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. ***P < 0.005. (B) Left: CD4+ T cells were cultured with the homeostatic cytokine IL-7 (10 ng/ml) in the absence or presence of resveratrol (20 and 100 μM). H2AX phosphorylation (γH2AX) was assessed at 24 hours by flow cytometry. Dot plots are representative of three experiments. Right: Means ± SEM of three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. (C) CD4+ T cells were TCR-stimulated in the presence or absence of resveratrol (20 and 100 μM) and in the presence or absence of IL-2 (50 U/ml). H2AX phosphorylation was assessed by flow cytometry as described earlier, and the increase in phosphorylation relative to that in cells stimulated by TCR engagement alone is shown. Data are means ± SEM of three experiments with statistical significance determined by one-way ANOVA with Tukey’s post hoc test. **P < 0.01 and ***P < 0.005.

  • Fig. 5 Resveratrol stimulates p53 phosphorylation and the ATR-mediated arrest of the cell cycle.

    (A) Phosphorylation of p53 at Ser15 and total p53 were monitored by Western blotting analysis of CD4+ T cells activated in the absence or presence of resveratrol (20 and 100 μM) or aphidicolin (1 μM). Representative blots at days 1 and 3 of activation and loading controls (LC) are shown. Data are representative of three experiments; quantification data are shown in fig. S1. (B) Phosphorylation of AMPK and p53 was assessed by Western blotting analysis of CD4+ T cells at day 1 after treatment as indicated with resveratrol and the AMPK inhibitor (compound C; 1 μM). Representative blots showing phosphorylated and total proteins under the indicated conditions are shown. Data are representative of three experiments; quantification data are shown in fig. S1. (C) Schematic model of the ATR and ATM signaling cascades culminating in cell cycle arrest. The former results in Chk1 activation, whereas the latter proceeds through Chk2 activation and the p53-mediated expression of p21. p21 directly inhibits Cdks, whereas Chk1 blocks cell cycle progression by activating Wee1 and preventing the Cdc25-mediated dephosphorylation of Cdk1. (D) The abundance and phosphorylation of Chk1 and Chk2 and the cell cycle regulators Wee1, Cdk1, Cdk2, p21, and Mcm2 were monitored by Western blotting analysis of cells treated under the indicated conditions. Blots of samples at days 1 and 3 are representative of three experiments. The arrow indicates hyperphosphorylated Cdk1. Quantification data are shown in fig. S1. (E) Phosphorylation of Mcm2 and p53 in cells was assessed by Western blotting analysis with the appropriate phosphospecific antibodies at day 1 after treatment with resveratrol and the ATR inhibitor (VE-821; 1 and 5 μM). The amounts of total p53 and γ-tubulin were assessed. Data are representative of three independent experiments. Quantification data are shown in fig. S1. (F) Top: H2AX phosphorylation (γH2AX) in response to resveratrol treatment was assessed at 24 hours after stimulation in the absence or presence of VE-821 (5 μM). Plots show the percentages of γH2AX-positive cells and are representative of three independent experiments. Bottom: Quantification of the MFI of γH2AX staining is shown as means ± SEM of three experiments. *P < 0.05 and **P < 0.01 by two-way ANOVA with Bonferroni’s post hoc test.

  • Fig. 6 TCR-stimulated CD4+ T cells exhibit increased transcription of p53-dependent metabolic target genes and an altered T cell metabolism after exposure to resveratrol.

    (A) The expression of 84 genes involved in DNA damage signaling pathways was evaluated in CD4+ T cells activated by TCR engagement alone as compared to TCR engagement in the presence of resveratrol (20 μM) using a polymerase chain reaction (PCR) array profile (Qiagen; fig. S3). Representative data in one of three samples at day 1 of stimulation are shown with only Bbc3 (PUMA) significantly induced in the latter conditions (4.2- to 6.4-fold induction, n = 3). (B) Transcripts of p53 metabolic target genes including TIGAR, PGM, GLS2, and SCO2 were monitored by quantitative reverse transcription PCR analysis of cells under the indicated conditions (day 3) and were normalized against the abundance of 18S ribosomal RNA. Data are means ± SEM of four independent experiments. *P < 0.05 and **P < 0.01 by paired t test. (C) Glut1 surface expression was monitored by flow cytometry, and representative histograms from three experiments under the indicated conditions (day 3) are shown, with quantifications shown in fig. S1. (D) 2-Deoxy-d[1-3H]glucose (2DG) uptake (n = 3), lactate production (n = 5), and extracellular acidification (n = 5) were monitored in cells under the indicated conditions at day 3. Data are means ± SEM of the indicated number of experiments. *P < 0.05, **P < 0.01, and ***P < 0.005 by one-way ANOVA with Tukey’s post hoc test. (E) Left: ASCT2 surface expression was monitored by flow cytometry, and representative histograms are shown. Middle: The ratio ± SEM of ASCT2 to Glut1 abundance in cells stimulated through the TCR in the presence or absence of resveratrol are presented as a function of the MFI of both transporters (n = 3; paired t test). Right: Uptake of l-2,3,4-[3H]glutamine was performed for 10 min, and mean counts per minute ± SEM for triplicate samples from three independent experiments at day 3 are presented. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. **P < 0.01. (F) Left: Cellular respiration was monitored on a Seahorse XF-24 analyzer, and OCRs of triplicate samples under basal conditions and in response to the indicated mitochondrial inhibitors are presented for cells on day 3 of activation. Mean basal consumption rates (OCR; picomoles/min per 106 cells) ± SEM of triplicate samples from three independent experiments are shown (upper right). Right: ATP was measured in cells under the same conditions by luminescent detection, and mean intracellular amounts ± SEM from data obtained in 10 independent experiments are presented. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. Mitochondrial superoxide anion was monitored using MitoSOX Red reagent, and the MFI ± SEM of triplicate samples are presented. Data were analyzed by paired t test. FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; Ant.A, antimycin A; Rot., rotenone. *P < 0.05, **P < 0.01, and ***P < 0.005.

  • Fig. 7 IFN-γ secretion by TCR-stimulated naïve and memory CD4+ T cells is markedly enhanced by low-dose resveratrol.

    (A) Left: Naïve and memory CD4+ T cells were isolated, as described in fig. S4A, and were stimulated through the TCR in the presence or absence of resveratrol (20 and 100 μM). Secretion of IL-2 and IFN-γ was monitored by intracellular staining at day 6, and representative dot plots of three representative experiments are shown. Right: Quantification of the mean percentages ± SEM of IFN-γ–secreting and double IL-2/IFN-γ–secreting cells are shown. Data were analyzed by two-way ANOVA with Bonferroni’s post hoc test. (B) Proposed model showing the effects of resveratrol on TCR-stimulated CD4+ T cells. Left: Sirt1 and p53 are interrelated, regulating mTOR signals and metabolic networks (115), and both are activated in response to TCR stimulation of human CD4+ T cells. The integration of TCR signals stimulates intracellular glycolysis and glutaminolysis, resulting in proliferation and effector function. Middle: In response to low-dose resveratrol, TCR-engaged CD4+ T cells undergo a genomic stress response, which results in an ATR- and Chk1-mediated S-G2 cell cycle arrest. Moreover, ATR-mediated p53 signaling decreases glycolysis and increases glutaminolysis. Under these conditions, wherein cell cycle progression is blocked and OXPHOS is augmented, there is a substantial increase in IFN-γ secretion. Right: In response to high-dose resveratrol, TCR-mediated mTOR and Sirt1 signaling pathways are markedly attenuated, leading to p27-mediated G1 cell cycle arrest.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/501/eaal3024/DC1

    Fig. S1. Quantification and statistical analyses of main data panels.

    Fig. S2. Effects of resveratrol on the formation of CD4+ T cell blasts and cell counts in response to TCR engagement.

    Fig. S3. Effect of resveratrol treatment on the expression of DNA damage signaling pathway genes.

    Fig. S4. Sorting of naïve and memory CD4+ T cells for the assessment of phenotype and cytokine secretion profiles.

    Fig. S5. TCR-mediated induction of HIF1A and c-Myc in human CD4+ cells is not altered by low-dose resveratrol.

    Table S1. Antibody list.

    Table S2. Primer sequences.

  • Supplementary Materials for:

    Resveratrol stimulates the metabolic reprogramming of human CD4+ T cells to enhance effector function

    Marco Craveiro, Gaspard Cretenet, Cédric Mongellaz, Maria I. Matias, Olivier Caron, Maria C. Pedroso de Lima, Valérie S. Zimmermann, Eric Solary, Valérie Dardalhon, Vjekoslav Dulić,* Naomi Taylor*

    *Corresponding author. Email: vjekoslav.dulic{at}igmm.cnrs.fr (V.D.); taylor{at}igmm.cnrs.fr (N.T.)

    This PDF file includes:

    • Fig. S1. Quantification and statistical analyses of main data panels.
    • Fig. S2. Effects of resveratrol on the formation of CD4+ T cell blasts and cell counts in response to TCR engagement.
    • Fig. S3. Effect of resveratrol treatment on the expression of DNA damage signaling pathway genes.
    • Fig. S4. Sorting of naïve and memory CD4+ T cells for the assessment of phenotype and cytokine secretion profiles.
    • Fig. S5. TCR-mediated induction of HIF1A and c-Myc in human CD4+ cells is not altered by low-dose resveratrol.
    • Table S1. Antibody list.
    • Table S2. Primer sequences.

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    Citation: M. Craveiro, G. Cretenet, C. Mongellaz, M. I. Matias, O. Caron, M. C. P. de Lima, V. S. Zimmermann, E. Solary, V. Dardalhon, V. Dulić, N. Taylor, Resveratrol stimulates the metabolic reprogramming of human CD4+ T cells to enhance effector function. Sci. Signal. 10, eaal3024 (2017).

    © 2017 American Association for the Advancement of Science

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