Fig. 5 IL-5–mediated signaling is critical for the development of CD1dintCD5+ Breg precursor cells and IL-10+ Breg cells. IL-5–mediated signaling is critical.

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Fig. 5 IL-5–mediated signaling is critical for the development of CD1dintCD5+ Breg precursor cells and IL-10+ Breg cells. IL-5–mediated signaling is critical for the development of CD1dintCD5+ Breg precursor cells and IL-10+ Breg cells. (A) The histograms show the amount of IL-5 in LNs from KitW-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. Hyuk Soon Kim et al. Sci Adv 2019;5:eaav8152 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).