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 t he CD5(+) and CD5(-) subsets, individual CD19(+)/IgM(+)/CD5(+) or CD5( -) B cells were sorted and non-productive as well as productive V-H ge ne rearrangements were amplified from genomic DNA and sequenced, In bo th subsets, the V(H)3 family was overrepresented largely as a result o f preferential usage of a small number of specific individual family m embers, In the CD5(+) B cell subset, all other V-H families were found at a frequency expected from random usage, whereas in the CD5(-) popu lation, V(H)4 appeared to be overrepresented in the nonproductive repe rtoire, 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 rearr angements 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 f rom 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 betw een the B cell subsets in CDR3 length, the number of N-nucleotides or evidence of exonuclease activity, Somatically hypermutated V(H)DJ(H) r earrangements were significantly more frequent and extensive in CD5(-) compared to CD5(+) IgM(+) B cells, indicating that IgM(+) memory B ce lls were more frequent in the CD5(-) B cell population, Of note, the f requency of specific V-H genes in the mutated population differed from that in the nonmutated population, suggesting that antigen stimulatio n imposed additional biases on the repertoire of IgM(+) B cells, These results indicate that the expressed repertoire of IgM(+) B cell subse ts 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 tha t human peripheral blood CD5(+) and CD5(-) B cells represent different B cell lineages, with similarities to murine B-1a and B-2 subsets, re spectively.