Figure 4. C3ar1/C5ar1 signaling is needed for B2 cell expression of CD40, AID and Bcl-6 and CD4⁺ cell production of IL-21.

A) WT B2 cells prelabeled with CFSE were incubated at 37°C for 5 d with anti-IgM F(ab’)₂/ anti-CD40 (3 μg/ml each) in the absence or presence of C3ar1/C5ar1 pharmaceutical antagonists (RA) (100 ng/ml) and proliferation assessed by CFSE dilution. Cells treated with RA overlapped the shaded area corresponding to unstimulated cells B) C3ar1^−/−C5ar1^−/− B2 cells prelabeled with CFSE were incubated as in A) and proliferation assessed by CFSE dilution. C3ar1^−/−C5ar1^−/− B2 cells overlapped the shaded area corresponding to unstimulated cells. C) WT and C3ar1^−/−C5ar1^−/− B2 cells were incubated at 37°C for 48 h with anti-IgM F(ab’)₂/CD40L (3 μg/ml each) ± RA (100 ng/ml) (left) or C5a (300 ng/ml) ± RA (100 ng/ml) (right), after which CD40 expression was quantified by flow cytometry. D) WT B2 cells were incubated at 37°C for 72 h with LPS (1 μg/mL) and IL-4 (10 ng/mL) ± RA (300 ng/mL) after which AID and Bcl-6 mRNA expression were quantified by qPCR. E) WT CD4⁺ and C3ar1^−/−C5ar1^−/− CD4⁺ cells were incubated at 37°C for 48 h with anti-CD3/anti-CD28 Dynabeads (1:1 bead-to-cell ratio) in the absence or presence of RA (300 ng/mL) after which IL-21 was assayed by ELISA. N=3, p< .001