Analysis of the heavy chain repertoire of human peripheral B cells using single-cell polymerase chain reaction.
The VH gene repertoire of human peripheral B cells was analyzed using PCR analysis of individual blood B cells. Because genomic DNA of single B cells was analyzed, data from both productive and nonproductive VDJ rearrangements were obtained. Nine out of 75 B cells contained both functional and nonfunctional rearrangement products, whereas 62/75 had a single productive VDJ rearrangement. The distribution of VH families was ordered in accordance with the germline complexity, although a bias toward VH3 and some of its members was found. This bias was noted in both the productively and nonproductively rearranged repertoires, indicating that it resulted from molecular and not selective processes. Evidence for negative selection of certain VH3 and VH4 family members was noted in that they were found less often as productive than nonproductive VDJ rearrangements. In addition, evidence for positive selection based on CDR3 was obtained, in that JH6 and DXP'1 were found at a higher frequency in the productively compared with the nonproductively rearranged repertoire. The CDR3 segments were markedly heterogenous, with a much greater degree of variability noted in the nonproductive VDJ rearrangements. Finally, a much greater frequency of mutations was noted in the nonproductively rearranged VH genes within individual B cells. These results have begun to delineate the human peripheral B cell repertoire and identify processes that shape it both positively and negatively.
Brezinschek
H P
HP
Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas 75235, USA.
Brezinschek
R I
RI
Lipsky
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PE
D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D005260:Female; D005434:Flow Cytometry; D005784:Gene Amplification / Q000235:genetics; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain / Q000235:genetics; D005803:Genes, Immunoglobulin; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000032:analysis* / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods
D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region