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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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
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