Fig. 3.

mA3 promotes TYC antibody mutations in specific V_H genes. (A and B) The percentages of (A) AID hotspot and (B) mA3 hotspot mutations relative to the number of C or G mutations were evaluated in GC B-cell V_H genes from WT (n = 3) and mA3 KO (n = 4) mice. Data are shown for V_H1–19 and V_H1–26, which are the major V_H genes in the FV-specific mAb panel in Fig. 1G. The percentage of TCC mutations in V_H1–18 is shown in B. Differences were evaluated using a two-tailed unpaired Student t test with P < 0.05 considered as significant. (C) Prevalence of nonsynonymous TYC mutations in WT versus mA3 KO IgH sequences. TYC mutations from B6 WT (n = 16,680 noncollapsed sequence reads from three mice) and B6 mA3 KO (n = 51,096 noncollapsed sequence reads from four mice) sequences were combined, and those sequence reads that resulted in amino acid changes were evaluated in 2 × 2 contingency analyses. (Left) Five nonsynonymous V_H1–19 TYC substitutions found in >0.4% of sequence reads in WT mice were compared with sequence reads in KO mice by Fisher’s exact test. Percentages from the entire V_H1–19 sequence dataset are shown in parentheses. (Right) IMGT collier-de-perles plot (27) showing the relative positions of the predicted amino acid mutations. (D) Ontogeny of nonsynonymous V_H1–19 TYC mutations in individual B6 WT mice. Phylogenetic trees were constructed using the neighbor-joining method, and clades supported with >70% bootstrap are indicated. Each dot within each line corresponds to a unique sequence. Note that M39I was detected in all three mice and that lineages with two TYC mutations could emerge.