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

Abstract

To analyze the human kappa chain repertoire and the influences that shape it, a single cell PCR technique was used that amplified VκJκ rearrangements from genomic DNA of individual human B cells. More than 350 productive and 250 nonproductive VκJκ rearrangements were sequenced. Nearly every functional Vκ gene segment was used in rearrangements, although six Vκ 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 Jκ segments was also nonrandom, with Jκ1 and Jκ2 being overrepresented and Jκ3 and Jκ5 underrepresented in the nonproductive repertoire, implying a molecular basis for the bias. In B cells with two VκJκ rearrangements, marked differences were noted in the Vκ segments used for the initial and subsequent rearrangements, whereas Jκ segments were used comparably. Junctional diversity was generated by n- nucleotide addition in 60% and by exonuclease trimming in 75% of the VκJκ 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 Vκ sequences derived from CD5- as compared with CD5+ B cells. These results document that the gene segment utilization within the Vκ 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.