Molecular mechanisms and selective influences that shape the kappa gene repertoire of IgM(+) B cells

To analyze the human kappa chain repertoire and the influences that shape it, a single cell PCR technique was used that amplified V kappa J kappa rearrangements from genomic DNA of individual human B cells. More than 350 productive and 250 nonproductive V kappa J kappa rearrangements were sequenced, Nearly every functional V kappa gene segment was used in rearrangements, although six V kappa gene segments, A27, L2, L6, L12a, A17, and O12/O2 were used preferentially, Of these, A27, L2, L6, and L12a showed evidence of positive selection based on the variable region and not CDR3, whereas A17 was overrepresented because of a rearrangement bias based on molecular mechanisms, Utilization of J kappa segments was also nonrandom, with J kappa 1 and J kappa 2 being overrepresented and J kappa 3 and J kappa 5 underrepresented in the nonproductive repertoire, implying a molecular basis for the bias. In B cells with two V kappa J kappa rearrangements, marked differences were noted in the V kappa segments used for the initial and subsequent rearrangements, whereas J kappa segments were used comparably, Junctional diversity was generated by n-nucleotide addition in 60% and by exonuclease trimming in 75% of the V kappa J kappa rearrangements analyzed. Despite this large degree of diversity, a strict CDR3 length was maintained in both productive and nonproductive rearrangements, More than 23% of the productive rearrangements, but only 7% of the nonproductive rearrangements contained somatic hypermutations. Mutations were significantly more frequent in V kappa sequences derived from CD5(-) as compared with CD5(+) B cells, These results document that the gene segment utilization within the V kappa repertoire is biased by both intrinsic molecular processes as well as selection after light chain expression. Moreover, IgM(+) memory cells with highly mutated kappa genes reside within the CD5(-) but not the CD5(+) B cell compartment.