Fig. 5. IL-5–mediated signaling is critical for the development of CD1d^intCD5⁺ B_reg precursor cells and IL-10⁺ B_reg cells.

(A) The histograms show the amount of IL-5 in LNs from Kit^W-sh/W-sh mice with or without the reconstitution of BMMCs. The data are expressed as the mean ± SEM from three independent experiments (n ≥ 3 per group for each experiment). *P < 0.05 versus phosphate-buffered saline (PBS) intravenous group by Student’s t test. (B) Representative flow cytometry images (middle) and histograms for the population of IL-10⁺ B cells and the amount of IL-10 in the culture medium (right) are shown. Data are expressed as the mean ± SEM from four independent experiments (triplicate for each experiment). *P < 0.05, **P < 0.01 versus medium group by Student’s t test. (C) Splenic CD19⁺ B cells (2 × 10⁶ cells per well) from WT mice were cultured with or without cytokines [TNF-α, 10 ng/ml; IFN-γ (interferon-γ), 10 ng/ml; IL-6, 10 ng/ml; IL-4, 5 ng/ml; IL-5, 10 ng/ml; and IL-13, 100 ng/ml] for 48 hours. Representative flow cytometry images (left) and histograms (right) show the mean fluorescence intensity (MFI) of CD125 (IL-5Rα) on B cells. The results are expressed as representative images (left) and the mean ± SEM (right) from three independent experiments (duplicate for each experiment). **P < 0.01 versus medium group by Student’s t test. (D) CD19⁺ B cells were stimulated with IL-4 (5 ng/ml), IL-5 (10 ng/ml), or IL-4 (5 ng/ml) + IL-5 (10 ng/ml) for 43 hours and subsequently stimulated with lipopolysaccharide (LPS) + phorbol 12-myristate 13-acetate (PMA), ionomycin, and monensin (PIM) for an additional 5 hours. The histograms of IL-10⁺ B cells by flow cytometry (left) and the amount of IL-10 in the culture medium (right) are shown. (E) CD19⁺ B cells (2 × 10⁶ cells per well) were cultured with or without IL-4 and IL-5 for 43 hours and subsequently stimulated with LPIM for 5 hours. Representative flow cytometry images (left) and histograms (right) show the MFIs of CD5 on B cells. (D and E) The results are expressed as the mean ± SEM from three independent experiments (triplicate for each experiment). *P < 0.05; **P < 0.01; n.s., not significant by one-way ANOVA with post hoc Tukey’s test. (F to H) Representative flow cytometry images are shown for various B cell subsets (F), and histograms show the frequencies of the B cell subsets (G) and IL-10⁺ B cells (H) in peripheral tissues from WT or Il5ra^−/− mice. Data are expressed as the mean ± SEM from three independent experiments (n ≥ 3 per group for each experiment). *P < 0.05; **P < 0.01; n.s., not significant versus WT by Student’s t test.