Blockade of miR-142-3p promotes anti-apoptotic and suppressive function by inducing KDM6A-mediated H3K27me3 demethylation in induced regulatory T cells

CD4+CD45RA+-naive T cells were sort purified using magnetic-activated cell sorting and induced as CD4+CD25+CD127？FOXP3+ iTregs, which were stimulated with anti-CD3/28 beads in the presence of fetal bovine serum (FBS) and interleukin-2 (IL-2). The iTregs were then harvested after treatment with a miR-142-3p inhibitor or mimic for 3 days on day 7. a Cell counts and b relative folds of expansion were recorded every 2 to 3 days. c, d Cell counts and relative folds of expansion of iTregs treated with miR-142-3p inhibitor or mimic were recorded every 2 to 3 days and compared to the control cells. e Flow detection and expression of forkhead box P3 (Foxp3) in iTregs treated with an inhibitor or mimic on day 10 (gated on CD4+CD127？ cells). Representative result. f Foxp3 expression in iTregs of each group was recorded at different time points (days 7, 10, and 13). g Cell apoptosis assay using Annexin V/PI (propidium iodide) staining and % Annexin V+ PI？ of iTregs from each group (gated on CD4+CD127？ cells). Representative result. h Flow detection and positive proportion of Ki67 staining of iTregs from each group (gated on CD4+CD127？ cells). Representative result. i Division index for CD8+ T cell mediated with anti-CD3 proliferation in vitro, at ratios from 1:2 to 1:16 (iTreg: peripheral blood mononuclear cells) as detected using carboxyfluorescein succinimidyl ester dye dilution. Mean？±？standard error of the mean values are presented. *P？<？0.05 and **P？<？0.0