B cell–specific increased affinity maturation in FcγRIIb^wild/H1 KI mice. (A and B) Total NP₁₂-specific IgG1 antibody titer (A) and high-affinity NP₂-specific IgG1 antibody titer (B) were determined by ELISA 7, 14, 21, and 28 d after immunization with NP-KLH and 7 d after secondary immunization (day 35). Concentrations are expressed in relative units (RU). (C) The ratio of high-affinity anti-NP₂ and total anti-NP₁₂ IgG1 titer at each time point was determined. n = 8 mice per group, and data are representative of at least two independent experiments. (D) V_H mutation analysis of single cell sorted NP-specific GC B cells 11 d after immunization with NP-KLH (three pooled mice for each group). n = 54 sequences for the WT, and n = 62 sequences for the KI mice. Data are representative of three independent experiments. (E) The frequency of the high-affinity mutation W33L in V_H186.2 CDR1 was assessed after nested PCR and sequencing. (F) WT and FcγRIIb^wild/H1 KI µMT chimera mice were immunized with NP-KLH, and NP₂/NP₁₂ IgG1 ratio was determined by ELISA after 11 d. n = 12 mice for the WT, and n = 14 mice for the KI from three experiments pooled. (G) µMT chimera were immunized with NP-KLH, and GC B cells were sorted 11 d later from three pooled WT and three pooled KI chimera mice. The frequency of the high-affinity mutation W33L in V_H186.2 CDR1 was assessed after nested PCR and sequencing of each clone. n = 56 sequences for the WT, and n = 60 sequences for the KI chimera mice. Data are representative of two 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%.