CSR, SHM, and antigen-driven selection in Igh^CGG/+ B cells. (a) Igh ^CGG/+ B cells (CD45.2/2) were adoptively transferred into WT (CD45.1/1) recipients, which were immunized 1 d later with CGG in alum. CSR to IgG₁ was determined in GC B cells (CD19⁺TCRβ^–Fas⁺CD38^lo) from donor (CD45.2/2) and recipient (CD45.1/1) mice by flow cytometry. Graph shows percentage of IgG₁⁺ GC B cells in five mice from two independent experiments. Proportions of switched cells in the same mouse are connected by a line. P = 0.23 (unpaired t test). Numbers within flow plots indicate percentage of cells in the designated gate. (b) Left: Serum titer of IgG₁⁺/human Cκ⁺ Ig in three mice adoptively transferred with Igh^CGG/+ B cells and immunized with CGG. Graph shows data for three mice from two independent experiments. Right: Western blot for human Igκ in serum of mice adoptively transferred with Igh^CGG/+ B cells and immunized with CGG 14 d after immunization. NR, nonreducing conditions; R, reducing conditions (DTT). Blot is representative of two experiments. (c–f) GC B cells from adoptive transfer experiments as in panel a were single sorted, and the entire Igh^CGG/+ allele was PCR-amplified and sequenced. (c) Nucleotide mutation frequencies along the Igh^CGG/+ locus, calculated as the fraction of times a particular nucleotide was mutated from the original sequence. (d) Total mutation frequency per region (normalized to sequence length). Each symbol represents one cell. (e) Number of nucleotide mutations in VDJ_H and VJκ in Igh^CGG/+ GC B cells compared with WT GC B cells carrying the same rearrangement selected by CGG immunization, as reported previously (Tas et al., 2016; only data from LN#2 GC2 in Fig. 4 from Tas et al., 2016 are analyzed). Data from two transfers of Igh^CGG/+ B cells are pooled and compared with the WT. **, P ≤ 0.001; ****, P ≤ 0.0001 (unpaired t test). (f) Comparison of nucleotide mutation patterns between Igh^CGG/+ B cells and WT GC B cells carrying the same rearrangement. Mutation patterns are shown for FR1 to FR4 regions of IgH and CDR1 to FR4 regions of Igκ. Shared nucleotide mutation positions are shown in red. Asterisk indicates a C119>G mutation, which confers an approximately eightfold gain in affinity. Data from two transfers of Igh^CGG/+ B cells are pooled and compared with the WT. (g) Reconstructed phylogeny of Igh^CGG/+ GC B cell VJ Igκ sequences from multiple GCs in one LN. Clades of expanded clones are indicated in red, green, and blue. Cells containing the high-affinity C119>G mutation are indicated in green.