Preferential expansion and survival of B lymphocytes based on VH framework 1 and framework 3 expression: "positive" selection in appendix of normal and VH-mutant rabbits.

Abstract

B cells with a rearranged heavy-chain variable region VHa allotype-encoding VHI gene segment predominate throughout the life of normal rabbits and appear to be the source of the majority of serum immunoglobulins, which thus bear VHa allotypes. The functional role(s) of these VH framework region (FR) allotypic structures has not been defined. We show here that B cells expressing surface immunoglobulin with VHa2 allotypic specificities are preferentially expanded and positively selected in the appendix of young rabbits. By flow cytometry, a higher proportion of a2+ B cells were progressing through the cell cycle (S/G2/M) compared to a2-B cells, most of which were in the G1/GO phase of the cell cycle. The majority of appendix B cells in dark zones of germinal centers of normal 6-week-old rabbits were proliferating and very little apoptosis were observed. In contrast, in 6-week-old VH-mutant ali/ali rabbits, little cell proliferation and extensive apoptosis were observed. Nonetheless even in the absence of VH1, B cells with a2-like surface immunoglobulin had developed and expanded in the appendix of 11-weekold mutants. The numbers and tissue localization of B cells undergoing apoptosis then appeared similar to those found in 6-week-old normal appendix. Thus, B cells with immunoglobulin receptors lacking the VHa2 allotypic structures were less likely to undergo clonal expansion and maturation. These data suggest that "positive" selection of B lymphocytes through FR1 and FR3 VHa allotypic structures occurs during their development in the appendix.

Microbial induction of B and T cell areas in rabbit appendix

“…Early in postnatal life, newly developed B cells emigrate from the bone marrow and migrate to GALT, of which the appendix is a major site [29,30]. Within GALT, B cells proliferate extensively and diversify their V(D) J genes through somatic gene conversion and somatic hyper-point mutation [2][3][4][5]. These developmental processes are driven by select members of the intestinal microbiota [9,10].…”

“…In further contrast, rabbit B cells utilize a restricted number of different V, D and J gene segments during VDJ gene rearrangement, thereby generating a limited neonatal antibody repertoire. After leaving the bone marrow, newly developed B cells seed gut-associated lymphoid tissues (GALT), particularly the appendix and sacculus rotundus, where they proliferate and concomitantly diversify their V(D)J genes through gene conversion [2,3] and somatic hyper-point mutation [4,5]. These developmental events greatly expand the B cell population and extensively diversify the primary antibody repertoire to provide immune protection against foreign antigens.…”

Distinct Clonal Ig Diversification Patterns in Young Appendix Compared to Antigen-Specific Splenic Clones

“…1 and Refs. [12][13][14][15]. These cells are the hypothesized source of the primary repertoire, i.e., B cell precursors generated to exit to the periphery and respond to specific Ags (14).…”

“…Just as bursectomy leads to deficiencies in numbers of chicken B cells and their repertoire of specificities, neonatal appendectomy and removal of other GALT early in life leads to B cell deficiency (10) and diminished diversity of V H D H J H gene sequences in B cells of young rabbits (11). Remarkable development and expansion of B cells takes place in rabbit appendix between birth and 6 wk of age (10,(12)(13)(14). It is accompanied by diversification of rearranged V H D H J H sequences (14,15) and dependent upon the presence of normal gut flora (16 -18).…”

“…Growth and expansion of B cells is also accompanied by apoptotic death, suggesting that both negative and positive selective processes affecting the B cell repertoire are at work (12,13,19,20). Previously, existing technology did not permit determination of rearranged V H D H J H gene sequences within single cells (15).…”