Fig 6. CD40 ligation enhances SEA-induced Breg cell development.

(A, B) Splenic B cells from naïve mice were cultured with 20 μg/ml SEA or medium as control, with or without addition of anti-CD40 co-stimulatory antibody. After 3 days of culture, supernatants were analyzed for IL-10 and IL-6 by ELISA (A) and B cell CD86 expression by flow cytometry (B). Summary of 6 experiments. (C) Splenic B cells were stimulated for 3 days with anti-CD40 mAb (2 μg/ml) and SEA (20 μg/ml) or anti-CD40 alone, and subsequently co-cultured with CD4⁺CD25^- sorted splenic T cells. After 4 days, the frequency of CD25⁺Foxp3⁺ Treg cells within the CD4 T cell population was determined by flow cytometry. Summary of 4 experiments. (D-F) Mice were i.p. injected with two doses of 100 μg SEA or PBS as control. CD40 ligand was blocked in vivo by i.p. injection of 200 μg hamster anti-mouse CD40 ligand or 200 μg hamster IgG as control (Ctrl) for 4 times, every 4 days starting at day -1 before SEA/PBS treatment. At day 14 after the first SEA/PBS injection, splenic B cells were in vitro restimulated with SEA for 2 days. Summary of 2 experiments (N = 8). (D) Fold increase of IL-10 in supernatants from B cells of SEA-injected versus PBS-injected mice. (E) Intracellular IL-10 expression of B cells after addition of Brefeldin A to the last 4 hours of the culture. (F) Mean fluorescence intensity of CD86 on B cells. Significant differences are indicated with * p < 0.05, ** p < 0.01, *** p < 0.001 as tested by Mann-Whitney test. ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 indicates significant difference by one-sample t-test of log-transformed data (D) or to respective medium control by Mann-Whitney test.