Correlation between SHM and CD45RO expression levels. A total of 547 IgV_H4 family IgM and IgG transcripts were amplified from RO-subdivided GC B cells. (A) The average number of mutations per V_H4 sequence was calculated and compared for each RO fraction. Standard deviations are represented by error bars. P values are included where intersample differences were statistically significant (P ≤ .05). N values for RO⁻ (□), RO^+/− (▩), and RO⁺ (■) fractions: T76 IgM (31, 24, 41), T76 IgG (76, 30, 58), T78 IgM (28, 38, 83), and T78 IgG (26, 55, 57), respectively. Total IgM and IgG mutation averages were, respectively, 7.9 and 11.2 for T76 and 9.1 and 18.9 for T78. (B) The number of unmutated (germ-line) IgM (□) and IgG (■) sequences among each RO fraction was quantified for T76 and T78. (C-D) The cumulative distribution of mutations among IgM and IgG sequences was determined for RO⁻ (thin line; ●), RO^+/− (dashed line; ×), and RO⁺ (thick line; □) fractions. Percentages represent the number of samples that have mutations within the indicated range divided by the sample size n value (range increases in 3-bp increments). Arrows highlight the percentage difference between RO⁻ and RO⁺ fractions at the RO⁺ average. For example, only 5% of T76 RO⁻IgM sequences have more than 10 mutations compared with 35% of RO⁻IgM sequences. Results are presented for (C) T76 and (D) T78. Note that RO^+/− values are typically medial to RO⁻ and RO⁺ values, suggesting that mutations progressively accumulate with respect to RO level.