Loss of miR-221/222 leads to defects in B cell CSR to IgG1/IgE in vitro and IgE production in vivo. (A) Δ or flox B cells were stimulated identically to Fig. 1 A and transfected with miR-221-3p (miR-221) or miR-222-3p (miR-222) or CM at day 0, and surface IgG1 was analyzed at day 2. Four mice per genotype were used, where each mouse was transfected with each mimic. Statistical analysis was performed by repeated measures one-way ANOVA with Dunnett’s post-test within each genotype. A representative experiment of three independent experiments is shown. (B) Expression of each miRNA relative to U6 small nucleolar RNA (snRNA) in WT C57BL/6 mouse B cells stimulated for 96 h. Data are the average of four biological replicates. Statistical analysis was performed by one-way ANOVA with Dunnett’s post-test. (C) Representative flow cytometry plots. CSR phenotype of Δ or flox B cells stimulated in anti-CD40 + IL-4 + IL-21 media for 96 h gated on live singlets to determine IgD^lo, IgG1⁺ cells. IgE⁺ cells are IgM⁻ and gated from an IgD^lo, IgG1⁻ population. (D) Quantification of the percentage of surface IgE⁺, IgG1⁺, or either IgG1⁺/IgE⁺ from C. A combined three independent experiments with at least three mice per genotype are shown. (E) Quantification of division number from cocultured Δ and flox B cells stimulated in anti-CD40 + IL-4 + IL-21 media for 96 h. A paired Student’s t test was used for each cocultured well. Combined from three independent experiments with at least two independent cocultures per experiment. CTV, CellTrace Violet. (F) CSR phenotype of Δ or flox B cells stimulated for 96 h in CSR conditions to induce IgG3, LPS alone; IgG2b, LPS + TGFβ1; or IgA, anti-CD40 + IL-5 + TGFβ1 + retinoic acid (with or without a proliferation-inducing ligand). *, P < 0.05; **, P < 0.01; ***, P < 0.001.