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.