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. 2020 Mar 4;217(5):e20190472. doi: 10.1084/jem.20190472

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© 2020 Yang et al.

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Figure 7. — IL-21 inhibits IgE and promotes IgG1 responses of cultured human B cells depending on the strength of CD40 and IL-4 signals. Total B cells (A–E) or naive B cells (F–H) were purified from human tonsils and cultured for 7 d with anti-CD40 (as indicated) and IL-4 (20 ng/ml, except in H), in the presence or absence of IL-21 (20 ng/ml). (A–C) Representative flow cytometry (A) and quantification of the frequency (B) and total number (C) of IgG⁺ and IgE⁺ cells among IgD⁻ cells from cultures of total B cells with the indicated concentrations of anti-CD40 and a fixed concentration of IL-4 (20 ng/ml) in the presence or absence of IL-21. (D) Representative flow cytometry showing proliferation of total B cells cultured from human tonsils with anti-CD40 (100 ng/ml) and IL-4, in the presence or absence of IL-21, as measured by dilution of CellTrace Violet. (E) Frequency of activated (IgD⁻) cells among total tonsil B cells after culture with anti-CD40 (as indicated) and IL-4, in the presence or absence of IL-21, quantified as a percentage of live cells. (F–H) Representative flow cytometry (F) and quantification (G and H) of the frequency of IgG4⁺, IgG1⁺, and IgE⁺ B cells, quantified as a percentage of IgD⁻ cells, after culturing naive B cells. (F) Naive B cells were cultured with anti-CD40 (20 ng/ml) and IL-4 in the presence or absence of IL-21. Cells were pregated as IgD^– (left panels) to identify IgG4⁺ cells; gates for IgG1⁺ and IgE⁺ cells were then drawn within the IgG4⁻ population (right panels). IgG1⁺ and IgG4⁺ populations were gated sequentially to account for cross-reactivity of the anti-IgG1 antibody with IgG4. (G) Naive B cells were cultured with a variable concentration of anti-CD40 as indicated with a fixed concentration of IL-4 (20 ng/ml), in the presence or absence of IL-21. (H) Naive B cells were cultured with a fixed concentration of anti-CD40 (100 ng/ml) and a variable concentration of IL-4, as indicated, in the presence or absence of IL-21. Dots represent data points from individual donors. Bars represent arithmetic (B, E, G, and H) or geometric (C) means. n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (t tests with the Holm-Sidak correction for multiple comparisons). Data were pooled from 10 experiments (B, C, and E), five experiments (G), or two experiments (H). In D, each donor is from a different experiment, and the results for each donor were replicated in another experiment.