Traf5−/− B cells are refractory to CD40L stimulation in vitro. CD40L-mediated cell proliferation (A), gene expression (B), and IgM and IgG1 production (C) in B cells from unimmunized WT and KO mice were assessed using MTT assay, real-time RT-PCR, and ELISA, respectively. (A) Proliferative responses of splenic naïve B cells cultured with the indicated concentrations of Fc-CD40L for 3 days. (B) Expression levels of CD40L-target genes, Tnfaip3, Tnfsf4, and Cd80, in B cells treated with 30 µg/mL Fc-CD40L for 7 h. Data are normalized to Stx5a expression and presented as mean ± standard deviation (n = 3) from a representative experiment of two independent experiments showing similar results. (C) IgM and IgG1 production of splenic naïve B cells cultured with combinations of Fc-CD40L (10 µg/mL), anti-CD40 Ab (4 µg/mL), and IL-4 (10 ng/mL) for 4 days, evaluated by ELISA. * p < 0.05 ((A,C): Student’s t-test; (B): one-way ANOVA with Tukey’s post hoc test).