Enhanced IL-21 expression on CD4⁺ T cells in il2ra^−/− and DKO mice is responsible for death of MZ B cells. (A) Expression of IL-21 mRNA in the spleen as determined by real-time PCR. Values show averages ± SEM of 3 mice per group. (B-D) IL-21 production measured by flow cytometry. Splenocytes of individual mice (n = 3/group) were stimulated with PMA and ionomycin for 4-hour prior staining of surface CD4 and intracellular IFN-γ and IL-21. (B) Dot plot profiles of IL-21 and IFNγ expression gated on CD4⁺ cells of individual samples representative for a group. Numbers indicate average percentage ± SD of IFN-γ⁺CD4⁺ cells. (C) Histograms represent the overlay of IL-21 expression stained by a soluble IL-21R-Fc gated on all CD4⁺ (left panel), CD4⁺IFNγ⁺ (middle panel), or CD4⁺IFNγ⁻ (right panel) cells of WT (dashed line) and il2ra^−/− (continuous line) splenocytes. The solid gray line indicates staining with a control-Fc. (D) Bar graph shows averages ± SEM of the geometric MFI of staining with IL-21R-Fc gated on IFNγ⁺ and IFNγ⁻ cells. Data shown are representative of 2 independent experiments. The dashed line indicates the MFI obtained when staining with a control-Fc. (E) MZ and FO B cells were purified as described in “Ex vivo follicular and MZ B-cell stimulation” and cultured with indicated concentrations of cytokines for 6 hours before measurement of viability by staining with 7-AAD. The bar graph shows viability relative to a medium control in each condition. Baseline viability in the absence of cytokines was ∼80% for FO B and ∼55% for MZ B cells. Similar results were obtained in 2 independent experiments. (F) Reciprocal roles of IL-2 and IL-21 in regulating MZ and FO B cells during inflammation. IL-2 is required for Treg development and function and might directly support survival of MZ B cells. In the absence of IL-2, the lack of functional Tregs results in the deregulated activation of CD4 cells, leading to secretion of IFNγ and IL-21. The latter directly and preferentially induces death of MZ B over FO B cells.