Increased GC reaction in FcγRIIb^wild/H1 KI mice. (A) 8 d after immunization with NP-KLH in alum, splenocytes were stimulated ex vivo, and the mean fluorescence intensity (MFI) of phospho-Syk and phospho-BLNK in GC B cells gated as B220⁺/GL7^hiFAS^hi and naive B cells gated as B220⁺/GL7⁻FAS⁻ was determined by flow cytometry. The ratio of naive and GC B cell mean fluorescence intensity is plotted. Data are representative of two or three independent experiments depending on the time points. n = 4 mice per group. (B) Splenic GC B cell numbers were determined by flow cytometry at day 8. Representative dot plots of WT and KI are shown. (C) GC formation in the spleen 8 d after immunization was assessed by immunohistology. Staining: anti-B220, B cell follicle; anti-CD8, T cell zone; and anti-KI67, GCs. Representative images of at least four mice per group are shown. Bars, 50 µm. (D and E) GC B cells (D) and NP-specific GC B cells (E) were enumerated at days 7, 8, 10, and 11 after immunization. n ≥ 4 mice, and data are representative of at least two experiments per time point. (F) 8 d after immunization, apoptosis and proliferation of GC B cells were assessed by staining apoptotic cells with CaspGLOW (left) and cells in cycle with anti-KI67 (right). n = 4 mice for the KI and 5 mice for the WT. Data are representative of at least two independent experiments. (G) The number of T_FH cells (CD4⁺/CXCR5^hiPD1^hi) per spleen was determined 11 d after immunization with NP-KLH in alum. n = 6 for the WT and 5 for the KI. Data are representative of three independent experiments. In all panels, error bars represent SEM, and p-values were determined using the Mann–Whitney two-tailed test with a risk of 5%.