PTX3 from NBh cells delivers NF-κB–inducing signals and elicits IgG CSR and production in MZ B cells. (A) qRT-PCR of mRNAs for AID (Aicda) and Iγ2b-Cγ2b and ELISA of IgG2b in MZ B cells from WT mice co-cultured with or without NBh cells from WT or Ptx3^−/− mice in the presence or absence of LPS for 2 d (mRNAs) or 5 d (IgG). Results are normalized to mRNA for the B cell–specific molecule CD79a and presented as relative expression (RE) as compared with MZ B cells incubated with medium alone. (B) qRT-PCR array of NF-κB–dependent gene products in MZ B cells isolated from 2-d co-cultures with NBh cells from WT or Ptx3^−/− mice. In these co-cultures, NBh cells from Ptx3^−/− mice were supplemented or not supplemented with exogenous PTX3. Results are normalized to mRNA for β-actin and presented relatively to mean expression. The color code indicates high (red), mean (black), and low (green) expression. (C–E) ELISA of serum SP-specific IgG2b, IgG2c, and IgG3 (C) and flow cytometric analysis of frequencies of MZ, transitional (T)/B-1 and FO B cells (D and E) after i.v. injection of live SP in WT mice, Ptx3^−/− mice adoptively transferred with PTX3-sufficient (from WT mice) NBh cells, or Ptx3^−/− mice adoptively transferred with PTX3-deficient (from Ptx3^−/− mice) NBh cells. RU, relative units. Data summarize at least two experiments with at least three mice in each experimental group (A and C–E) or show one representative experiment of two with similar results (B). Error bars, SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed Student’s t test).