Somatic mutation is inversely correlated with CD45RB expression among GC B cells. IgV_H4 IgM transcripts were sequenced from different tonsillar B-cell subsets that were sorted into separate RB^−/lo and RB⁺ fractions. (A) Mutation frequencies (average number of mutations per transcript in the region spanning FW1 through FW3) were calculated for GC B cells from 4 independent tonsils. Combined data (T81 + T95 + T100 + T101) are also presented (T-TOTAL). (B) Mutation distributions among RB^−/lo and RB⁺ GC B cells were calculated as the percentage of sequences with mutation counts that fall within the indicated range. Centrally encircled numbers indicate sample sizes (n values) for each RB fraction. (C) IgV_H4 mutation analyses were performed for pre-GC and Pblast B cells to verify that sorted populations used in subsequent AID expression experiments (Figure 3) were phenotypically consistent with conventionally accepted mutation patterns for these cell types (ie, mutation frequency pre-GC < GC {9i} Pblast). In each case, 2 independent tonsils were used as the source of pre-GC (T100 and T101) or Pblast (T85 and T86) B cells. Pre-GC and Pblast pools were independently compared against values for GC B cells that were isolated from the same respective tonsils (ie, [T100 + T101] pre-GC vs [T100 + T101] GC; [T85 + T86] Pblast vs [T85 + T86] GC). (D) Mutation distributions were calculated for pre-GC and Pblast B cells as in panel B. (E) Mutation frequencies were determined for RB-subdivided naive, GC, and memory B cells that were all isolated from the same tonsil (T95). Note that the correlative relationship between RB expression and SHM is opposite for the naive and memory B-cell pools which, respectively, represent early and late stages of peripheral B-cell development. (F) Mutation distributions were calculated for T95 (IgM⁺IgD⁺CD38⁻CD27⁻) naive and GC B cells as in panel B. Where appropriate, standard deviations are included as error bars. Statistically significant differences between compared populations (RB^−/lo vs RB⁺ in panels A and E; or GC vs pre-GC/Pblast in panel C) are indicated by 1, 2, or 3 asterisks (P ≤ .05, P ≤ .01, or P ≤ .001, respectively).