Analysis of the human V-H gene repertoire - Differential effects of selection and somatic hypermutation on human peripheral CD5(+)/IgM(+) and CD5(-)/IgM(+) B cells

To analyze the immunoglobulin repertoire of human IgM(+) B cells and the CD5(+) and CD5(-) subsets, individual CD19(+)/IgM(+)/CD5(+) or CD5(-) B cells were sorted and non-productive as well as productive V-H gene rearrangements were amplified from genomic DNA and sequenced, In both subsets, the V(H)3 family was overrepresented largely as a result of preferential usage of a small number of specific individual family members, In the CD5(+) B cell subset, all other V-H families were found at a frequency expected from random usage, whereas in the CD5(-) population, V(H)4 appeared to be overrepresented in the nonproductive repertoire, and also negatively selected since it was found significantly less often in the productive compared to the nonproductive repertoire; the V(H)1 family was significantly diminished in the productive rearrangements of CD5(-) B cells. 3-23/DP-47 was the most frequently used V-H gene segment and was found significantly more often than expected from random usage in productive rearrangements of both CD5(+) and CD5(-) B cells, Evidence for selection based on the D segment and the J(H) gene usage was noted in CD5(+) B cells. No differences were found between the B cell subsets in CDR3 length, the number of N-nucleotides or evidence of exonuclease activity, Somatically hypermutated V(H)DJ(H) rearrangements were significantly more frequent and extensive in CD5(-) compared to CD5(+) IgM(+) B cells, indicating that IgM(+) memory B cells were more frequent in the CD5(-) B cell population, Of note, the frequency of specific V-H genes in the mutated population differed from that in the nonmutated population, suggesting that antigen stimulation imposed additional biases on the repertoire of IgM(+) B cells, These results indicate that the expressed repertoire of IgM(+) B cell subsets is shaped by recombinational bias, as well as selection before and after antigen exposure. Moreover, the influences on the repertoires of CD5(+) and CD5(-) B cells are significantly different, suggesting that human peripheral blood CD5(+) and CD5(-) B cells represent different B cell lineages, with similarities to murine B-1a and B-2 subsets, respectively.