Defective up-regulation of FcγRIIb expression on GC B cells in FcγRIIB^wild/H1 KI mice. (A) Cloning strategy for the generation of the FcγRIIB^wild/H1 KI mice. A PCR product corresponding to the promoter and three first exons from Fcgr2b from haplotype I (1,271 bp) was first fused with the 5′ homology arm (1,996 bp from haplotype III). The fusion product was then cloned into the targeting vector (pCR_LPacINeo; Ozgene). The 3′ homology arm (3,905 bp from haplotype III) was then amplified and cloned in the targeting vector. (B–G) Surface expression of FcγRIIb was assessed by flow cytometry 14 d after NP-KLH immunization on naive (PNA⁻B220⁺) and GC B cells (PNA⁺B220⁺; B), memory B cells (IgG1⁺/PNA⁻CD38⁺; C), splenic PCs (CD138⁺B220^lo; D), and BM PCs (CD138⁺B220^lo; G). Surface FcγRIIb expression was also assessed by flow cytometry on unimmunized mice for splenic follicular B cells (CD21⁺CD23⁺), marginal zone B cells (CD21⁺CD23^lo), and transitional B cells (CD21^loCD23^lo; E) and for BM pre-B cells (IgM⁻B220⁺), immature B cells (IgM⁺B220⁺), and recirculating mature B cells (IgM^hiB220⁺; F). (left) Representative dot plot of the gating strategy used to determine FcγRIIb expression on each subset. (middle) Representative overlay of FcγRIIb expression by FcγRIIb^wild/H1 KI, WT, and FcγRIIb KO mice is shown for PNA⁺B220⁺ GC B cells. (right) Geometric mean fluorescence intensity (MFI) of FcγRIIb expression on the different cell subsets. (H) Splenocytes were stimulated with 20 µg/ml goat anti–mouse IgM F(ab′)2 or intact goat anti–mouse IgM for 72 h at 37°C. The expression of FcγRIIb on activated B cells was assessed by flow cytometry. The mean fluorescence intensity of FcγRIIb on activated B cells was plotted for WT and FcγRIIb^wild/H1 KI mice. For all experiments, n ≥ 4 mice per group, and data are representative of at least three independent experiments. Error bars represent SEM, and p-values were determined using the Mann–Whitney two-tailed test with a risk of 5% (*, P < 0.05; **, P < 0.01).