Timing, genetic requirements and functional consequences of somatic hypermutation during B-cell development.

While somatic antibody mutants are rare in the preimmune repertoire and in primary immune responses, they dominate secondary and hyperimmune responses. We present evidence that somatic hypermutation is restricted to a particular pathway of B-cell differentiation in which distinct sets of B-cell clones are driven into the memory compartment. In accord with earlier results of McKean et al. (1984) and Rudikoff et al. (1984), somatic mutation occurs stepwise in the course of clonal expansion, before and after isotype switch, presumably at a rate close to 1 X 10(-3) per base pair per generation. At this rate, both selectable and unselectable mutations accumulate in the rearranged V region genes. The distribution of replacement mutations in the V regions shows that a fraction of the mutations in CDRs is positively selected whereas replacement mutations are counterselected in the FRs. By constructing an antibody mutant through site-specific mutagenesis we show that a point mutation in CDR1 of the heavy chain, found in most secondary anti-NP antibodies, is sufficient to increase NP binding affinity to the level typical for the secondary response. Somatic mutation may contribute to the immune repertoire in a more general sense than merely the diversification of a specific response. We have evidence that clones producing antibodies which no longer bind the immunizing antigen can be kept in the system and remain available for stimulation by a different antigen. Somatic mutations are 10 times less frequent in DJH loci than in either expressed or non-expressed rearranged VDJH or VJ loci. We therefore conclude that a V gene has to be brought into the proximity of the DJH segment in order to fully activate the hypermutational mechanism in these loci.