N-WASP deletion impairs GC formation and causes defective B-cell response in vitro and in vivo. Flow cytometry analysis showing severe reduction of the proportion of (A) bone marrow (BM) recirculating B220^hi lymphocytes and (B) splenic MZ B cells in all indicated knockout models as compared with wild-type (WT) mice. (C) Left panel: Reduced proportion of PNA⁺GL7⁺ GC B cells in naïve B/DcKO compared with naïve B/WcKO mice. Right panels: Immunofluorescence staining of optimal cutting temperature frozen spleen sections with anti-B220 (blue), anti-monocyte plus macrophage (anti-MOMA; green) monoclonal antibodies, and peanut agglutinin (PNA; red) demonstrating the presence of PNA⁺ GC B cells in naïve B/WcKO mice, but not in unimmunized WT and B/DcKO mice. Marked reduction of B220⁺ lymphocytes surrounding MOMA⁺ macrophages in B/WcKO and in B/DcKO mice is indicative of MZ defect. Shown are representative images from 3 different mice per group for WT and B/DcKO (original magnification ×20). (D) Impaired proliferation (as indicated by dilution of carboxyfluorescein succinimidyl ester (CFSE) and viability of sorted spleen Fo (upper panels) but not MZ (lower panels) B cells from B/DcKO mice upon stimulation with anti-IgM and CpG. (E) Immunofluorescence staining of spleen sections reveals a reduction of PNA⁺ GC formation in B/DcKO mice at day 7 after boosting immunization with TNP-KLH. Shown are representative images from 3 different mice per group for WT and B/DcKO (original magnification ×10). (F) Defective IgG1-specific anti-TNP responses in B/DcKO mice upon immunization with TNP-KLH. Data from individual mice are shown in representative dot plots. Statistical analysis was performed by one-way analysis of variance with Bonferroni post hoc analysis (FACS analysis of lymphocyte populations) and two-way analysis of variance with Bonferroni post hoc analysis (enzyme-linked immunosorbent assay detection of anti-TNP antibodies). *P < .05, **P < .01, ****P < .0001. OD, optical density.