This approach is especially powerful to study rabbit germinal center B cells because rabbit B cells rearrange only a few V H genes and the majority do not rearrange the second allele. We adopted an experimental approach that combined the techniques of microdissection of single Ag-specific B cells with a PCR-based sequencing strategy that avoids PCR artifacts. The present study was initiated to investigate V H sequences in splenic germinal centers during T cell-dependent immune responses to a protein or a hapten in adult rabbits. The finding of only highly diversified VDJ sequences in the adult rabbit is in keeping with the notion that the rabbit, like the chicken, develops its B cell repertoire early in life and depends upon self-renewing cells in the periphery to maintain its B lymphocyte pool throughout life. This led them to conclude that B lymphopoiesis is limited in adults ( 20). ![]() ![]() In addition, reduced levels of recombination signal joints (VD and DJ excision circles) were found in bone marrow of adult rabbits compared with the levels found in bone marrow of newborn rabbits. Using an RNase protection assay, they reported that by adulthood, essentially all expressed VDJ genes in cells from appendix, peripheral blood, and bone marrow were diversified. By 6–8 wk of age, all but 1 of 35 sequences analyzed were diversified, and the average number of nucleotide changes per V H region increased to 12. Recently, this group reported that by 4 wk of age, expressed VDJ genes from peripheral blood leukocytes had, on average, three nucleotide changes per V H region, with approximately 75% of the genes showing some diversification. that just as lymphopoiesis does not occur in adult chickens, little or no B lymphopoiesis occurs in adult rabbits ( 20). Although considerable repertoire diversity is generated in young rabbits, mechanisms for continued generation of B cell receptor diversity are retained in adult life, where they may confer survival advantage. Although a major population of B lymphocytes may be generated early in life, diversified extensively, and maintained by self-renewal in the periphery, some sources of cells with sequences close to germline do exist in adult rabbits and appear in the developing germinal centers. The attractive idea that the rabbit, like the chicken, develops its B cell repertoire early in life and depends upon self-renewing cells in the periphery to maintain its B lymphocyte pool throughout life, is challenged by the current finding. We found that contrary to published reports, adult rabbits indeed have newly diversifying B cell receptors in splenic germinal centers. Clonally related cells underwent diversification by hypermutation and gene conversion. ![]() In seven different germinal centers, we found one to four different clones with two to seven members. About 58% (82/140) of the sequences had unique CDR3 regions and were unrelated. In the course of the study, the objective of which was to analyze diversification of rearranged V HDJ H sequences, we were surprised to find cells 7 and 10 days after immunization with rearranged V H1a2 as well as a-negative (圓3 and x32) sequences that were identical or close to germline (10 or fewer changes). We used PCR to amplify rearranged V HDJ Hgenes in single cells collected by micromanipulation from splenic germinal centers of immunized adult rabbits.
0 Comments
Leave a Reply. |