miR-19b is indispensible for IFNγ production from differentiated Th1 cells. (A-B) CD4⁺CD25⁻ T cells were sorted from LNs and spleens of WT, f/+, and KO littermates, activated by anti–CD3/CD28 Abs under Th1-skewing conditions for 4 days with 50 ng/mL of recombinant mouse IL-12 (Peprotech), 10 μg/mL of purified anti–IL-4 (11B11), and 50 U/mL of recombinant mouse IL-2 (Peprotech). (A) The percentage of viable cells producing IFN-γ and the mean florescence intensity (MFI) of IFN-γ were determined by intracellular staining after 4 hours of stimulation with 0.9nM PMA and 0.5 μg/mL of ionomycin (Sigma-Aldrich) in the presence of 5 μg/mL of brefeldin A (Sigma-Aldrich) and 2μM monensin (eBioscience). The Abs used were anti–IFNγ-APC (BioLegend) and anti–IL-4-PE (BD). The bar graph summarizes the means ± SEM from 3 independent experiments. (B) The mRNA levels of T-bet and IFN-γ from CD4⁺ T cells differentiated under the Th1-skewing conditions were quantified by qPCR. Data were normalized to a reference gene, SDHA, and are shown as relative to WT. The bar graph shows means ± SEM from 3 independent experiments. (C) As described in Figure 1E, CD4⁺CD25⁻ conventional T cells of KO mice were primed and transduced with retrovirus encoding individual miRNAs within the miR-17-92 cluster, and then cultured under the Th1-skewing condition for 4 days. The percentages of IFN-γ- or IL-4–producing cells and the MFI of IFN-γ signal were measured by intracellular cytokine staining. Left: representative FACS plot; right: means ± SEM from 3 independent experiments. Statistical analysis was done by comparison with mock. ***P < .001.