Abstract
Aerobic glycolysis regulates T cell function. However, whether and how primary cancer alters T cell glycolytic metabolism and affects tumor immunity in cancer patients remains a question. Here we found that ovarian cancers imposed glucose restriction on T cells and dampened their function via maintaining high expression of microRNAs miR-101 and miR-26a, which constrained expression of the methyltransferase EZH2. EZH2 activated the Notch pathway by suppressing Notch repressors Numb and Fbxw7 via trimethylation of histone H3 at Lys27 and, consequently, stimulated T cell polyfunctional cytokine expression and promoted their survival via Bcl-2 signaling. Moreover, small hairpin RNA–mediated knockdown of human EZH2 in T cells elicited poor antitumor immunity. EZH2+CD8+ T cells were associated with improved survival in patients. Together, these data unveil a metabolic target and mechanism of cancer immune evasion.
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References
Ladell, K. et al. A molecular basis for the control of preimmune escape variants by HIV–specific CD8+ T cells. Immunity 38, 425–436 (2013).
Almeida, J.R. et al. Superior control of HIV–1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover. J. Exp. Med. 204, 2473–2485 (2007).
Harari, A. et al. An HIV–1 clade C DNA prime, NYVAC boost vaccine regimen induces reliable, polyfunctional, and long-lasting T cell responses. J. Exp. Med. 205, 63–77 (2008).
Gaucher, D. et al. Yellow fever vaccine induces integrated multilineage and polyfunctional immune responses. J. Exp. Med. 205, 3119–3131 (2008).
Precopio, M.L. et al. Immunization with vaccinia virus induces polyfunctional and phenotypically distinctive CD8+ T cell responses. J. Exp. Med. 204, 1405–1416 (2007).
Wherry, E.J. T cell exhaustion. Nat. Immunol. 12, 492–499 (2011).
Zheng, Y., Zha, Y., Driessens, G., Locke, F. & Gajewski, T.F. Transcriptional regulator early growth response gene 2 (Egr2) is required for T cell anergy in vitro and in vivo. J. Exp. Med. 209, 2157–2163 (2012).
Crespo, J., Sun, H., Welling, T.H., Tian, Z. & Zou, W. T cell anergy, exhaustion, senescence, and stemness in the tumor microenvironment. Curr. Opin. Immunol. 25, 214–221 (2013).
Gubser, P.M. et al. Rapid effector function of memory CD8+ T cells requires an immediate-early glycolytic switch. Nat. Immunol. 14, 1064–1072 (2013).
Chang, C.H. et al. Posttranscriptional control of T cell effector function by aerobic glycolysis. Cell 153, 1239–1251 (2013).
Viré, E. et al. The Polycomb group protein EZH2 directly controls DNA methylation. Nature 439, 871–874 (2006).
Cao, R. et al. Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298, 1039–1043 (2002).
Tumes, D.J. et al. The polycomb protein Ezh2 regulates differentiation and plasticity of CD4+ T helper type 1 and type 2 cells. Immunity 39, 819–832 (2013).
Tong, Q. et al. Ezh2 regulates transcriptional and posttranslational expression of T-bet and promotes Th1 cell responses mediating aplastic anemia in mice. J. Immunol. 192, 5012–5022 (2014).
Pagès, F. et al. Effector memory T cells, early metastasis, and survival in colorectal cancer. N. Engl. J. Med. 353, 2654–2666 (2005).
Zhang, L. et al. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N. Engl. J. Med. 348, 203–213 (2003).
Kryczek, I. et al. Phenotype, distribution, generation, and functional and clinical relevance of Th17 cells in the human tumor environments. Blood 114, 1141–1149 (2009).
Marrack, P. & Kappler, J. Control of T cell viability. Annu. Rev. Immunol. 22, 765–787 (2004).
Kryczek, I. et al. Human TH17 cells are long–lived effector memory cells. Sci. Transl. Med. 3, 104ra100 (2011).
Miranda, T.B. et al. DZNep is a global histone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation. Mol. Cancer Ther. 8, 1579–1588 (2009).
McCabe, M.T. et al. EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature 492, 108–112 (2012).
Ciofani, M. & Zuniga–Pflucker, J.C. Notch promotes survival of pre–T cells at the beta–selection checkpoint by regulating cellular metabolism. Nat. Immunol. 6, 881–888 (2005).
Amsen, D. et al. Instruction of distinct CD4 T helper cell fates by different notch ligands on antigen-presenting cells. Cell 117, 515–526 (2004).
Wirtz–Peitz, F., Nishimura, T. & Knoblich, J.A. Linking cell cycle to asymmetric division: Aurora–A phosphorylates the Par complex to regulate Numb localization. Cell 135, 161–173 (2008).
Oberg, C. et al. The Notch intracellular domain is ubiquitinated and negatively regulated by the mammalian Sel-10 homolog. J. Biol. Chem. 276, 35847–35853 (2001).
Cui, T.X. et al. Myeloid-derived suppressor cells enhance stemness of cancer cells by inducing microRNA101 and suppressing the corepressor CtBP2. Immunity 39, 611–621 (2013).
Varambally, S. et al. Genomic loss of microRNA-101 leads to overexpression of histone methyltransferase EZH2 in cancer. Science 322, 1695–1699 (2008).
Sander, S. et al. MYC stimulates EZH2 expression by repression of its negative regulator miR-26a. Blood 112, 4202–4212 (2008).
Curiel, T.J. et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat. Med. 10, 942–949 (2004).
Galon, J. et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 313, 1960–1964 (2006).
Heagerty, P.J., Lumley, T. & Pepe, M.S. Time-dependent ROC curves for censored survival data and a diagnostic marker. Biometrics 56, 337–344 (2000).
Scholler, J. et al. Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells. Sci. Transl. Med. 4, 132ra153 (2012).
Ahmed, R., Bevan, M.J., Reiner, S.L. & Fearon, D.T. The precursors of memory: models and controversies. Nat. Rev. Immunol. 9, 662–668 (2009).
Rosenberg, S.A., Restifo, N.P., Yang, J.C., Morgan, R.A. & Dudley, M.E. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat. Rev. Cancer 8, 299–308 (2008).
Gattinoni, L. et al. A human memory T cell subset with stem cell–like properties. Nat. Med. 17, 1290–1297 (2011).
Zou, W. & Restifo, N.P. TH17 cells in tumour immunity and immunotherapy. Nat. Rev. Immunol. 10, 248–256 (2010).
Powell, D.J. Jr., Dudley, M.E., Robbins, P.F. & Rosenberg, S.A. Transition of late-stage effector T cells to CD27+CD28+ tumor-reactive effector memory T cells in humans after adoptive cell transfer therapy. Blood 105, 241–250 (2005).
Wei, S., Zhao, E., Kryczek, I. & Zou, W. Th17 cells have stem cell-like features and promote long-term tumor immunity. OncoImmunology 1, 516–519 (2012).
Yang, K. et al. T cell exit from quiescence and differentiation into Th2 cells depend on Raptor-mTORC1-mediated metabolic reprogramming. Immunity 39, 1043–1056 (2013).
Muranski, P. et al. Th17 cells are long lived and retain a stem cell–like molecular signature. Immunity 35, 972–985 (2011).
Varambally, S. et al. The Polycomb group protein EZH2 is involved in progression of prostate cancer. Nature 419, 624–629 (2002).
Tan, J. et al. Pharmacologic disruption of Polycomb-repressive complex 2–mediated gene repression selectively induces apoptosis in cancer cells. Genes Dev. 21, 1050–1063 (2007).
Fiskus, W. et al. Combined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3–deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells. Blood 114, 2733–2743 (2009).
Kryczek, I. et al. B7–H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma. J. Exp. Med. 203, 871–881 (2006).
Curiel, T.J. et al. Blockade of B7–H1 improves myeloid dendritic cell–mediated antitumor immunity. Nat. Med. 9, 562–567 (2003).
Zou, W. et al. Stromal–derived factor–1 in human tumors recruits and alters the function of plasmacytoid precursor dendritic cells. Nat. Med. 7, 1339–1346 (2001).
Kryczek, I. et al. Expression of aldehyde dehydrogenase and CD133 defines ovarian cancer stem cells. Int. J. Cancer 130, 29–39 (2012).
Yamamoto, N. et al. Role of Deltex-1 as a transcriptional regulator downstream of the Notch receptor. J. Biol. Chem. 276, 45031–45040 (2001).
Kryczek, I. et al. IL–22CD4 T cells promote colorectal cancer stemness via STAT3 transcription factor activation and induction of the methyltransferase DOT1L. Immunity 40, 772–784 (2014).
Lin, D.Y., Wei, L.J. & Ying, Z. Checking the Cox model with cumulative sums of martingale-based residuals. Biometrika 80, 557–572 (1993).
Acknowledgements
We thank D. Postiff, M. Vinco, R. Craig and J. Barikdar for tissue procurement core at the University of Michigan; G. Lv, W. Dong and L. Li for assistance; R. Zhang (Wistar Institute) for shEZH2 plasmids; P. King for discussions; and B. Leclair and D. Leclair for support. Supported by the US National Institutes of Health (the Intramural Research Program; and CA123088, CA099985, CA156685, CA171306 and 5P30CA46592), the Chinese Ministry of Science and Technology (973 program, 2015CB554000), the Wuhan Union Hospital Research Fund, the Ovarian Cancer Research Fund, and Marsha Rivkin Center for Ovarian Cancer Research.
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E.Z., T.M. and I.K. designed and performed most of the experiments, interpreted the data and drafted the paper; W.L., K.W., L.Z., S. Wei, J.C., S. Wan, L.V., W.S. and I.S. performed experiments; Y.W., Y.L., S.V., A.M.C., T.H.W., V.E.M., J.K., H.W., Y.Z., Z.W., R.L. and G.W. provided reagents or clinical specimens and clinopathological information and interpreted the data. W.Z., I.K. and G.W. supported, conceived of and supervised the research, designed experiments and wrote the paper.
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Supplementary Figure 1 Distribution and cytokine profile of EZH2+ T cells.
(a) EZH2+ T cells in different tissues. Snap-frozen tissues were stained for CD3 (red) and EZH2 (green). Magnification 40X, representative stainings for N = 15 (colitic colon) or N = 10 (tonsil and spleen) patients tested.
(b) Gating strategy to define polyfunctional T cells. Based on FSC, SSC and T cell staining, single CD3+CD4+ cells and CD3+CD8+ cells were gated for further analysis. To define cytokine profile for CD8+ T cells, CD8+ T cells were analyzed on the basis of TNF-α expression (gates R1 and R2). IFN-γ and granzyme B expression were analyzed within TNF-α-negative cells. This allows identification of the triple-negative population (R13), single positive TNF–IFN-γ+GranB– (R11), TNF–IFN-γ+GranB– cells (R14), and double positive and TNF–IFN-γ+GranB+ population (R12). Analysis of TNF-positive (R2) cells allows assessment to the percentage of triple-positive (polyfunctional) TNF+IFN-γ+GranB+ population (R22), double positive subsets TNF+IFN-γ+GranB– (R21), TNF+IFN-γ–GranB+ (R24), and single positive TNF+IFN-γ-GranB– (R23). Therefore, the percentages of cells from gates R11, R14 and R23 demonstrate the frequency of single positive cells, while the total percentages of cells from R12, R21, and R24 are double-positive cells. Polyfunctional CD4+ T cells were similarly analyzed for TNF-α, IFN-γ and IL-2 expression.
(c) Effect of cisplatin on CD8+ T cell apoptosis. CD8+ T cells were activated with anti-CD3 and anti-CD28. On day 4 cisplatin was added into the culture and apoptosis was measured on day 5 with Annexin V and 7-AAD staining. Representative data for one of 5 different donors is shown.
(d) Effect of cisplatin treatment on EZH2 expression in CD8+ T cells. Freshly isolated peripheral blood CD8+ T cells were stimulated with anti-CD3/anti-CD28 antibodies. EZH2 expression was measured in the cells on day 4 (pre-treated cells) and compared with the cells treated with cisplatin on day 4 and harvested 24 hours later. Data presented as mean ± SEM, N = 6 donors for pre-treated cells and N = 4 donors for cisplatin-treated cells. *P < 0.05.
Supplementary Figure 2 Effects of DZNep on pro-apoptotic and anti-apoptotic gene expression.
(a) Kinetic expression of EZH2 in T cells. T cells were stimulated with anti-CD3 and anti-CD28 antibodies for the indicated time. EZH2 protein was detected with Western blotting. One representative experiment of 6 is shown.
(b,c) Effects of DZNep on Bcl-2 and pro-apoptotic gene expression. T cells were activated in the presence of 5 µM DZNep for 2 days. Expression of Bcl-2 (b), Bak, Bax and BIM (c) was quantified by real-time PCR. Results are shown as the relative expression to control (mean ± SEM). N = 5, Wilcoxon rank-sum test, *P < 0.05.
Supplementary Figure 3 Effect of Notch signaling inhibitor on T cell viability and Bcl-2 promoter activity.
(a) Effect of Notch signaling inhibitors on cell viability. CD8+ T cells were activated with anti-CD3 and anti-CD28 in the presence of the γ-secretase inhibitors, DAPT or GSI-I. The cells were harvested after 3 days and counted on a hemocytometer using Trypan blue exclusion of dead cells. Data presented as mean ± SEM. N = 4, Mann-Whitney U testet, *P < 0.05.
(b) Effects of Notch signaling on Bcl-2 promoter activity. 293T cells were co-transfected with hBCL-2-EGFP vector, control vector, and vectors encoding Notch dominant negative (Notch-DN) or Notch intracellular domain (Notch-IC) for 2 days. The promoter activity was analyzed by FACS and expressed as the percentage of GFP expression (mean ± SD). N = 4, Mann-Whitney U test, *P < 0.05.
Supplementary Figure 4 Effects of tumor and glucose on T cell function and tumor inhibitory B7 expression.
(a,b) Effects of tumor and glucose on polyfunctionality of CD4+ T cells. CD4+ T cells were activated with anti-CD3 and anti-CD28 for 3 days in normal medium, tumor medium, or these media supplemented with glucose. Polyfunctional profile was assessed by FACS. Results are shown as the mean of double (a) and triple (b) positive cells ± SEM of 3 donors. Mann-Whitney test, *P < 0.05, compared to medium.
(c) Effect of different concentrations of glucose on the total numbers of double- and triple-positive cells. CD8+ T cells were activated with anti-CD3 and anti-CD28 in different concentrations of glucose. The cells were analyzed by flow cytometry after 3 days. Data are shown as the mean ± SD, N = 4 donors. *P < 0.05.
(d) Effect of glucose restriction on T cell apoptosis. CD8+ T cells were stimulated for 3 days with anti-CD3 and anti-CD28 antibodies. Apoptosis was analyzed by Annexin V stanining. Data are presented as the mean ± SD, N = 4 donors, Wilcoxon rak-sum test, *P < 0.05.
(e) Effect of glucose on the expression of tumor B7H1 and B7H4. Ovarian tumor cells were cultured for 24 hours in the presence or absence of glucose. B7H1 and B7H4 expression was determined by FACS. One of two independent experiments is shown.
(f) Effect of 2-DG on viability of CD8+ T cells. CD8+ T cells were activated with anti-CD3 and anti-CD28 for 3 days in the presence or absence of 2-DG. Data are shown as the mean ± SEM of 4 donors. Wilcoxon rak-sum test, *P < 0.05.
Supplementary Figure 5 Effects of tumor, microRNAs and glucose on T cell function.
(a) Representative staining of EZH2 in T cells cultured under normal or tumor conditions. CD8+ T cells were activated with anti-CD3 and anti-CD28 for 2 days in the presence of medium (red), tumor (blue), and tumor plus glucose (grey). EZH2 expression was determined by FACS. Results are shown as EZH2 expression in histogram. N = 8.
(b) Effect of glucose on T cell EZH2 expression. CD8+ T cells were activated with anti-CD3 and anti-CD28 for 4 days in media containing different levels of glucose. The level of EZH2 was measured by flow cytometry and demonstrated as the mean fluorescence intensity (MFI). Data are shown as the mean ± SEM, N = 3, Mann-Whitey test, *P < 0.05.
(c) Expression of miR-223, miR-106b, and miR-181a in T cells. CD8+ T cells were activated with anti-CD3 and anti-CD28 for 12 hours. MicroRNAs were measured with qPCR. Results are shown as the relative microRNA expression ± SD, N = 3, Mann-Whitney U test, *P < 0.05.
(d) Effect of glucose on miR-101 and miR-26a expression. CD8+ T cells were stimulated for 24 hours with anti-CD3 and anti-CD28 in media containing different levels of glucose. microRNAs were measured by qPCR. Data are shown as the mean ± SD, Mann-Whitney U test, N = 3 donors. *P < 0.05.
(e) Viability of CD8+ T cells after transfection with miR-101 and miR-26a mimics. CD8+ T cells were nucleofected with microRNA101 mimic, micorRNA26a mimic, or control oligonucleotide, and activated with anti-CD3 and anti-CD28 for 48 hours. The number of live cells was determined on the basis of trypan blue exclusion of dead cells during hemocytometer counting. Data presented as mean ± SEM, N = 4, Wilcoxon rank-sum test, *P < 0.05.
Supplementary Figure 6 Relationship between EZH2, microRNAs and T cell function.
(a) Effects of microRNA mimics on EZH2 expression. Fresh blood CD8+ T cells were transfected with empty plasmid (control band) or plasmid encoding EZH2 overexpression, then treated with microRNAs mimics. After 24 hours, T cells were harvested and EZH2 was analyzed by Western blotting. Representative data, one of 3 donors tested.
(b-d) Effects of microRNA mimics on Bcl2 (b) and Hey1 (c) expression and polyfunctional CD8+ T cells (d). Fresh blood CD8+ T cells were transfected with control plasmid or plasmid encoding EZH2 overexpression, and subsequently co-transfected with microRNA mimics. The cells were activated with anti-CD3 and anti-CD28 after 3 days. Bcl2 and Hey1 expression was determined by real-time PCR. Polyfunctional CD8+ T cells were analyzed by FACS. Data presented as mean ± SEM, N=5 donors, Wilcoxon rank-sum test, *P < 0.05.
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Zhao, E., Maj, T., Kryczek, I. et al. Cancer mediates effector T cell dysfunction by targeting microRNAs and EZH2 via glycolysis restriction. Nat Immunol 17, 95–103 (2016). https://doi.org/10.1038/ni.3313
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DOI: https://doi.org/10.1038/ni.3313
