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. 2014 May;50(100):107–122. doi: 10.1016/j.jaut.2014.01.032

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Crown Copyright © 2014 Published by Elsevier Ltd.

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).

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Fig. 4 — Phenotypic and functional characterization of the IL-33-induced Breg-like cells (Breg^IL-33) in the blood. A–C. Surface phenotype of Breg^IL-33: The IL-33-induced IL-10 producing B cells expressed high surface IgM (A), CD1d and CD25 (B), but down-regulated CD23 (FceRII) (B, C). Data shown in the table under (B) were results showing the Mean MFI (±SD) values calculated from results of 5 repeated experiments, as ratio of the IL-10⁻B, and IL-10⁺B, cell subset over the total B cells gated. Statistical analysis: p values (Student t test). D–F. In vitro Breg suppression assays: Immunosuppressive effects of the IL-33-induced Breg-like cells on B effector (Beff) cell proliferation (D), division (E), and its IL-10 dependency (F). D. CD23⁺ B cells (Beff, 10⁵) were cultured in the presence of anti-CD40 (2.5 μg/ml), with titrating (as indicated) doses of CD23⁻ B cells (Breg^IL-33) purified from the IL-33-injected mice. Cell proliferation was determined by thymidine incorporation at 48 h s. E. Breg^IL-33 (CD23^-) purified from hIL-33-injected WT mice (Wk-2) were primed with anti-CD40 (2.5 μg/ml) for 5 h before being added to CFSE-labelled CD23⁺ Beff cells. Cell division (CFSE dilution) was determined by flow cytometry at day 5 of culture in the presence or absence of LPS (0.5 μg/ml) or anti-IgM (5 μg/ml). F. IL-10 levels in the culture supernatants were quantified by ELISA (BD Biosciences).

Fig. 4 — Phenotypic and functional characterization of the IL-33-induced Breg-like cells (Breg^IL-33) in the blood. A–C. Surface phenotype of Breg^IL-33: The IL-33-induced IL-10 producing B cells expressed high surface IgM (A), CD1d and CD25 (B), but down-regulated CD23 (FceRII) (B, C). Data shown in the table under (B) were results showing the Mean MFI (±SD) values calculated from results of 5 repeated experiments, as ratio of the IL-10⁻B, and IL-10⁺B, cell subset over the total B cells gated. Statistical analysis: p values (Student t test). D–F. In vitro Breg suppression assays: Immunosuppressive effects of the IL-33-induced Breg-like cells on B effector (Beff) cell proliferation (D), division (E), and its IL-10 dependency (F). D. CD23⁺ B cells (Beff, 10⁵) were cultured in the presence of anti-CD40 (2.5 μg/ml), with titrating (as indicated) doses of CD23⁻ B cells (Breg^IL-33) purified from the IL-33-injected mice. Cell proliferation was determined by thymidine incorporation at 48 h s. E. Breg^IL-33 (CD23^-) purified from hIL-33-injected WT mice (Wk-2) were primed with anti-CD40 (2.5 μg/ml) for 5 h before being added to CFSE-labelled CD23⁺ Beff cells. Cell division (CFSE dilution) was determined by flow cytometry at day 5 of culture in the presence or absence of LPS (0.5 μg/ml) or anti-IgM (5 μg/ml). F. IL-10 levels in the culture supernatants were quantified by ELISA (BD Biosciences).