MiR-31 facilitates Th17/Treg imbalance and increases extrafollicular CD4 T-cell differentiation while inhibiting Treg stability. (A-C) Splenocytes (CD45.2⁺) from WT or miR-31 KO B6 mice were injected together with WT T cell–depleted (TCD) BM (CD45.1⁺) into lethally irradiated BALB/c mice. Frequencies of IL-17⁺ or Foxp3⁺ cells in gated live donor H2K^b+CD45.2⁺ CD4 T cells are shown in recipient skin-draining LNs (A) and spleens (B) on day 50 to 60 after BMT. Frequencies of CD62L^loPSGL-1^lo cells in gated live H2K^b+CD45.2⁺CD44⁺CD4 T cells are shown in recipient spleens on day 60 after BMT (top WT, bottom KO). Histograms for neuropilin 1 (Nrp1), PD-L1, and Bcl2 expression on gated H2K^b+CD45.2⁺Foxp3⁺ cells are shown (C). Data shown are from 1 representative of 3 individual experiments (n = 4-5 mice per group in each experiment). (D) CD4⁺CD25⁻ T cells isolated from WT or miR-31 KO mice were stimulated with CD11c⁺ dendritic cells from BALB/c together with IL-2 (5 ng/mL), transforming growth factor β (5 μg/mL), and retinoic acid (40 nM). Frequency of Foxp3⁺ cells in gated live H2K^b+CD4⁺ cells is shown on day 5. (E-F) CD25^hi-enriched induced Tregs (iTregs) were cultured in IL-2 (5 ng/mL), IL-12 (5 ng/mL), or IL-6 (10 ng/mL) in the presence of BALB/c APCs. Frequencies of Nrp1⁺ cells in gated live H2K^b+CD4⁺Foxp3⁺ cells (E) and Foxp3⁺ cells in gated live H2K^b+CD4⁺ cells (F) are shown. (G) Enriched iTregs (CD45.2⁺) were cocultured with B6 T cells (CD45.1⁺) at different ratios as indicated on the x-axis in the presence of BALB/c APCs. Frequencies of IFN-γ⁺ cells and live/dead dye⁺ cells in gated CD45.2⁺CD4⁺ cells and CFSE^low cells in gated live CD45.1⁺ CD8 T cells are shown on day 4. (H) Enriched iTregs were injected together with BM cells (Rag1^−/−) into lethally irradiated BALB/c mice followed by T_eff cell (CD45.1⁺) transfer 3 days later. Expression of IFN-γ and CD25 on gated H2K^b+CD45.2⁺Foxp3⁺ cells is shown 4 days after T_eff transfer. Data shown are from 1 representative of 2 individual experiments. *P < .05, **P < .01, ***P < .001, ****P < .0001. MFI, mean fluorescence intensity.