Fig. 6

IL-21 signaling promotes B cell expansion, antibody production, and T–B cell interaction in Act1^−/− mice. a Flow cytometry analysis of marginal zone B cells (B220⁺CD21^hiCD23^lo), follicular B cells (B220⁺CD21^intCD23^hi), T1 transitional B cells (B220⁺IgM^hiCD21^low), T2 transitional B cells (B220⁺IgM^hiCD21⁺), Germinal center B cell (B220⁺GL7⁺Fas⁺), and plasma cell (B220^-CD138⁺) from spleen of C57B/L6J background 8-month-old wild-type, Act1^−/−, Il21r^−/−, and Act1^−/− Il21r^−/− mice. The graphs indicate the absolute cell numbers in spleen. b Naive T cells isolated from 6-week-old MOG_35–55 immunized wild-type and Act1^−/− mice with indicated phenotypes were cultured with sorted dendritic cells from spleen of same mice in the presence of MOG_35–55 (2 μg/ml) under Th0 or Th17-poralizing conditions, followed by co-culturing with CFSE-labeled B220⁺ B cells isolated from MOG_35–55 immunized WT or Act1^−/− mice in the presence of MOG_35–55 (2 μg/ml) with or without addition of anti-IL-21 neutralizing antibody. B cell (CD45⁺B220⁺) proliferation was assessed by CFSE dilution on day 3. c Naive T cells isolated from the indicated 6-week-old MOG_35–55 immunized wild-type and Act1^−/− mice were cultured with sorted dendritic cells from spleen of same mice in the presence of MOG_35–55 (2 μg/ml) under Th0 or Th17-poralizing conditions, followed by co-culturing with B cells from immunized mice with indicated genotypes in the presence of MOG_35–55 (2 μg/ml) for 10 days followed by ELISA to determine the production of immunoglobulin in the supernatant. d B220⁺ B cells from WT, Act1^−/−, Il17ra^−/−, Il17rc^−/−, and Il17rb^−/− mice were stimulated with IL-21 for the indicated times, followed by western blot analysis with the indicated antibodies. STAT3 phosphorylation was quantified as a ratio of phosphorylated-to-total STAT3. Data presented as fold of induction of the cells from knockouts over the wild-type cells. e B220⁺ B cells were stimulated with IL-21 for 1 h and immunoprecipitated with anti-IL21R followed by western analysis with anti-STAT3, anti-Act1, and anti-IL21R. STAT3 levels of the immunoprecipitates were quantified as percentage of total STAT3 in the cell lysates. Data presented as fold of induction of the cells from knockouts over the wild-type cells. N = 3–5/group. Mean ± SEM. *p < 0.05; **p < 0.01. Two-tailed Student’s T test. All the data presented were from two independent experiments