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. Author manuscript; available in PMC: 2016 May 18.
Published in final edited form as: Nat Immunol. 2015 Nov 2;17(1):95–103. doi: 10.1038/ni.3313

Fig. 5. Tumor controls T cell EZH2 by microRNA101 and microRNA26 via glucose restriction.

Fig. 5

(a,b) Effect of tumor associated glucose restriction on T cell EZH2 expression. CD4+ (a) and CD8+ (b) T cells were activated with anti-CD3 and anti-CD28 for 3 days in medium or tumor conditioned medium with or without glucose supplementation. EZH2 protein expression was determined by flow cytometry. Results are shown as the mean fluorescence intensity (MFI) of EZH2 ± SEM in 3 donors, Mann-Whitney U test, *P < 0.05

(c) Effect of 2-DG on T cell EZH2 expression. T cells were activated with anti-CD3 and anti-CD28 for 3 days in the presence or absence of 2-DG. EZH2 protein expression was determined in viable T cells by flow cytometry. Results are shown as the relative EZH2 expression to the control group ± SEM in 3 donors, Mann-Whitney U test, *P<0.05.

(d,e) Dynamic changes of microRNA101 and microRNA26a in activated CD8+ T cells. CD8+ T cells were activated with anti-CD3 and anti-CD28 in the presence or absence of 2-DG. MicroRNA101 (d) and microRNA26a (e) were measured by qPCR in different time points. Results are shown as the relative microRNA expression. One of 2 donors is shown.

(f,g) Effect of tumor associated glucose restriction on microRNA expression in CD8+ T cells. Memory CD8+ T cells were pre-incubated overnight in medium or tumor-conditioned supernatants with or without glucose supplementation, then washed and stimulated for 12 hours with anti-CD3 and anti-CD28 antibodies. The abundance of microRNA101 (f) and microRNA26a (g) was measured by qPCR. Data are presented as the mean ± SEM, N = 3, Mann-Whitney U test, *P < 0.05.

(h–j) Effects of microRNA mimics on T cell EZH2 expression, polyfunctional profile, and apoptosis. Memory CD8+ T cells were nucleofected with microRNA101 mimic, micorRNA26a mimic, or control oligonucleotide, and activated with anti-CD3 and anti-CD28 for 48 hours. T cell EZH2 expression (h), polyfunctionality (i), and apoptosis (j) were determined by flow cytometry. Results are shown as the relative mean of EZH2 expression (h), the mean percentage of triple positive T cells (i), and the mean of Annexin V+ cells (j) ± SEM, N = 4, Mann-Whitney U test, *P < 0.05.