Figure 9. Somatic mutation in IgM primary repertoires.

The ratio of replacement mutations in CDR-H1 and CDR-H2 (R _CDR) to the total number of mutations (M _v) is plotted on the y-axis versus M _v on the x-axis. The 95% confidence limit for the probability of random mutations is shaded in gray. Data points falling outside this limit represent IgM sequences with a significant occurrence of replacement mutations in the CDRs and are considered indicative of antigen selection. Data points are numbered according to their observed frequency. The overall paucity of somatic mutation in the V_H gene among humanized B cells is visually apparent. Replacement mutations in CDR-H1 or CDR-H2 are rare. Whereas this result is expected for humanized samples HuMs-1 and HuMs-2 (naïve CD19⁺IgD⁺CD27^– splenic B cells), and expected for HuMs-ImmB (newly formed, immature bone marrow B cells), HuMs-3 was prepared from bulk spleen and could conceivably have captured a subpopulation of somatically mutated human IgM⁺ memory cells. Whereas greater than 99% of humanized HuMs B cells show no evidence for antigen selection (nonrandom mutation), HuPBC cells show an approximately ten-fold (4.83%) to twenty-fold (7.65%) greater proportion of antigen-selected sequences.