miR-19b and miR-17 suppress iTreg differentiation. (A) 5C.C7 T cells were primed, transduced with retrovirus encoding GFP alone or miR-17-92/GFP, and cultured under iTreg-differentiation conditions for 6 days with 0.2-1.0 ng/mL of recombinant human TGFβ (Peprotech), 50 U/mL of recombinant mouse IL-2 (Peprotech), 10 μg/mL of anti–IL-4 (11B11), 10 μg/mL of anti–IFN-γ (XMG1.2), and 10 μg/mL of anti–IL-6 (BD Biosciences). The percentage of Treg cells within the CD4⁺GFP⁺ population was measured by Foxp3 staining (eBioscience). The bar graph summarizes means ± SEM from 4 independent experiments. (B) CD4⁺CD25⁻ T cells sorted from LNs and spleens of WT, f/+, and KO littermates were cultured under iTreg-differentiation conditions for 6 days with the indicated TGF-β doses, and the percentage of CD25⁺Foxp3⁺ Treg cells was assessed. Left: representative FACS plot; right: statistical analysis of 4 independent experiments at the indicated TGF-β dose. (C) 5C.C7 T cells were transduced with individual miRNAs from the miR-17 or miR-19 families, and cultured under iTreg-differentiation conditions for 6 days. The percentage of CD25⁺Foxp3⁺ cells was measured by flow cytometry. Bar graphs summarize the means ± SEM of 3 independent experiments. *P < .05; **P < .01; ***P < .00; and ns, no significance.