tTregs treated with miR-146b-5p antagomir show increased FoxP3 and Ki67 expression and viability with enhanced suppressive function. Naive peripheral blood tTregs were sort-purified, expanded in vitro, and treated with or without scramble/antagomir for the final 2 days of culture. (A) miRNA was purified from each culture, and miR-146b expression was assessed by RT-PCR to determine knockdown efficiency (n = 3). (B) Representative example of Foxp3 vs CD127 staining in tTregs treated with antagomir compared with untreated and scramble groups (gated on CD4⁺ cells). Summary of overall percentage of Foxp3⁺CD127⁻ cells (C) and level of Foxp3 expression (D) in tTregs from each group (n = 5). (E) Percent suppression of in vitro, anti-CD3–mediated CD8⁺ T cell proliferation at ratios from 1:8 to 1:32 (tTregs:PBMCs) as determined by CFSE dye dilution (n = 5). (F, G) Representative flow images (F) and statistical analysis (G) of apoptosis tests were performed to analyze the survival ability in vitro. Viability was significantly enhanced after antagomir treatment (n = 3). (H) A higher relative Ki67 mean fluorescence intensity level was found after antagomir treatment (n = 5). Values indicate mean ± standard error of the mean (SEM) of these experiments. *P < .05; **P < .01.