NBh cell depletion impairs the expansion of splenic IgG class-switched PBs and PCs after polysaccharide immunization. (A) IFA of spleens stained for MMP9 (green), TNP (red), and MOMA-1 or B220 (blue) from WT mice before (d0) and after (d3) i.v. immunization with TNP-Ficoll. Bars, 100 µm. (B) Flow cytometric analysis of the frequency of splenic Ly6G⁺Ly6C⁺ NBh cells and Ly6G^loLy6C⁺ monocytes/macrophages before and after treatment of WT mice with control (Ctrl) or 1A8 mAbs for 3 d. FSC, forward side scatter; SSC, side scatter. (C and D) Flow cytometric analysis of frequencies and absolute numbers of total B220⁻CD138⁺ PBs/PCs and class-switched B220⁻CD138⁺ PBs/PCs expressing IgG2b, IgG2c, or IgG3 from spleens of WT mice i.v. immunized with TNP-Ficoll (d7) after continuous i.p. treatment with nonneutrophil-depleting control mAb or neutrophil-depleting 1A8 mAb from day −3 before immunization to day 7 after immunization. (E) ELISA of serum TNP-specific IgG2b, IgG2c, IgG3, and IgM from WT mice treated and immunized as in A. Late 1A8 mAb indicates treatment with 1A8 mAb starting at day 3 instead of day −3. RU, relative units. Data show one representative experiment of at least three with similar results (A and B) or summarize at least two experiments with at least four mice in each experimental group (C–E). Error bars, SEM. *, P < 0.05; **, P < 0.01 (two-tailed Student’s t test).