Shi-Kang Zhai

Intestinal Microflora and Diversification of the Rabbit Antibody Repertoire

The rabbit establishes its primary Ab repertoire by somatically diversifying an initial repertoire that is limited by restricted VH gene segment usage during VDJ gene rearrangement. Somatic diversification occurs in gut-associated lymphoid tissue (GALT), and by about 1–2 mo of age nearly all Ig VDJ genes are somatically diversified. In other species that are known to establish their primary Ab repertoire by somatic diversification, such as chicken, sheep, and cattle, diversification appears to be developmentally regulated: it begins before birth and occurs independent of exogenous factors. Because somatic diversification in rabbit occurs well after birth in GALT, the diversification process may not be developmentally regulated, but may require interaction with exogenous factors derived from the gut. To test this hypothesis, we examined Ab repertoire diversification in rabbits in which the appendix was ligated shortly after birth to prevent microbial colonization and all other organized GALT was surgically removed. We found that by 12 wk of age nearly 90% of the Ig VDJ genes in PBL were undiversified, indicating that intestinal microflora are required for somatically diversifying the Ab repertoire. We also examined repertoire diversification in sterilely derived remote colony rabbits that were hand raised away from contact with conventional rabbits and thereby acquired a different gut microflora. In these remote colony rabbits, GALT was underdeveloped, and 70% of the Ig VDJ genes in PBL were undiversified. We conclude that specific, currently unidentified intestinal microflora are required for Ab repertoire diversification.

Development of the antibody repertoire in rabbit: gut‐associated lymphoid tissue, microbes, and selection

Summary: Rabbits generate their antibody repertoire in three stages. First, a neonatal repertoire is generated by B lymphopoiesis in fetal liver and bone marrow and is limited by preferential VH gene segment usage. Between 4 and 8 weeks after birth a complex primary antibody repertoire is developed by somatically diversifying the neonatal repertoire through somatic hypermutation and a somatic gene conversion‐like mechanism in gut‐associated lymphoid tissue (GALT). In rabbits, unlike other species, the development of the primary antibody repertoire through somatic diversification of Ig genes appears to be dependent on intestinal microbial flora. The primary antibody repertoire is subsequently modified during antigen‐dependent immune responses in which VDJ genes further diversify both by somatic hypermutation and by a gene conversion‐like mechanism (the secondary repertoire). During the various stages of development, the antibody repertoire is modified and shaped by selective processes. In this review, we discuss the roles of GALT, microbes, and B‐cell selection in generating antibody diversity in rabbits.

B Lymphocyte Development in Rabbit: Progenitor B Cells and Waning of B Lymphopoiesis

In mammals that use gut-associated lymphoid tissues for expansion and somatic diversification of the B cell repertoire, B lymphopoiesis occurs early in ontogeny and does not appear to continue throughout life. In these species, including sheep, rabbit, and cattle, little is known about the pathway of B cell development and the time at which B lymphopoiesis wanes. We examined rabbit bone marrow by immunofluorescence with anti-CD79a and anti-μ and identified both proB and preB cells. The proB cells represent the vast majority of B-lineage cells in the bone marrow at birth and by incorporation of 5-bromo-2′-deoxyuridine, they appear to be a dynamic population. PreB cells reach maximum levels in the bone marrow at 3 wk of age, and B cells begin to accumulate at 7 wk of age. We cloned two VpreB and one λ5 gene and demonstrated that they are expressed within B-lineage cells in bone marrow. VpreB and λ5 coimmunoprecipitated with the μ-chain in lysates of 293T cells transfected with VpreB, λ5, and μ, indicating that VpreB, λ5, and μ-chains associate in a preB cell receptor-like complex. By 16 wk of age, essentially no proB or preB cells are found in bone marrow and by PCR amplification, B cell recombination excision circles were reduced 200-fold. By 18 mo of age, B cell recombination excision circles were reduced 500- to 1000-fold. We suggest that B cell development in the rabbit occurs primarily through the classical, or ordered, pathway and show that B lymphopoiesis is reduced over 99% by 16 wk of age.

Analysis of the 3′ Cμ region of the rabbit Ig heavy chain locus

The immunoglobulin D (IgD) antibody class was, for many years, identified only in primates, rodents and teleost fish. The limited distribution of IgD among vertebrates suggested that IgD is a functionally redundant antibody class that has been lost by many vertebrate species during evolution. The recent identification of IgD in artiodactyls, however, suggests that IgD might be more widely expressed among vertebrates than previously thought, possibly serving a unique role in immunity. IgD expression has been searched for but not detected in rabbits. In order to search directly for a rabbit Cdelta locus encoding the constant region of IgD, we determined the nucleotide sequence of 13.5 kb of genomic DNA downstream of the rabbit Cmu locus. We did not find a rabbit Cdelta locus in this region, but found instead that this region is densely populated by repetitive elements, including a long interspersed DNA element repeat, six C repeats, and two processed pseudogenes. We conclude that the rabbit probably does not express IgD because there is no Cdelta locus immediately downstream of the rabbit Cmu locus.

The evolution of the immunoglobulin heavy chain variable region (IgVH) in Leporids: an unusual case of transspecies polymorphism

In domestic rabbit (Oryctolagus cuniculus), three serological types have been distinguished at the variable domain of the antibody H chain, the so-called VHa allotypes a1, a2, and a3. They correspond to highly divergent allelic lineages of the VH1 gene, which is the gene rabbit utilizes in more than 80% of VDJ rearrangements. The sharing of serological VHa markers between rabbit and snowshoe hare (Lepus americanus) has suggested that the large genetic distances between rabbit VH1 alleles (9–14% nucleotide differences) can be explained by unusually long lineage persistence times (transspecies polymorphism). Because this interpretation of the serological data is uncertain, we have determined the nucleotide sequences of VH genes expressed in specimens of Lepus species. Two sequence groups were distinguished, one of which occurred only in hare specimen displaying serological motifs of the rabbit VHa-a2 allotype. Sequences of this group are part of a monophyletic cluster containing the VH1 sequences of the rabbit a2 allotype. The fact that this “transspecies a2 cluster” did not include genes of other rabbit VHa allotypes (a1, a3, and a4) is incompatible with the existence of a common VHa ancestor gene within the species, and suggests that the divergence of the VHa lineages preceded the Lepus vs Oryctolagus split. The sequence data are furthermore compatible with the hypothesis that the VHa polymorphism can be two times older than the divergence time between the Lepus and Oryctolagus lineages, which was estimated at 16–24 million years.

Allelic variation at the VHa locus in natural populations of rabbit (Oryctolagus cuniculus, L.)

The large interallelic distances between the three rabbit Ig VHa lineages, a1, a2 and a3, suggest that the persistence time of the VHa polymorphism could amount to 50 million years, which is much longer than that of MHC polymorphisms. Rabbit originated in the Iberian Peninsula where two subspecies coexist, one of which is confined to Southwestern Iberia (Oryctolagus cuniculus algirus). We studied the VH loci in the original species range to obtain a better understanding of the evolutionary history of this unusual polymorphism. Serological surveys revealed that sera from the subspecies algirus, when tested with VHa locus-specific alloantisera, showed either cross-reactivity (“a-positive” variants) or no reaction at all (“a-blank”). Using RT-PCR, we determined 120 sequences of rearranged VH genes expressed in seven algirus rabbits that were typed as either a-positive or a-blank. The data show that the VH genes transcribed in a-positive rabbits are closely related to the VH1 alleles of domestic rabbits. In contrast, a-blank rabbits were found to preferentially use VH genes that, although clearly related to the known VHa genes, define a new major allotypic lineage, designated a4. The a4 sequences have hallmark rabbit VHa residues together with a number of unprecedented amino acid changes in framework region 2 and 3. The net protein distances between the VHa4 and the VHa1, a2, and a3 lineages were 20, 29, and 21% respectively. We conclude that at least four distantly related lineages of the rabbit VHa locus exist, one of which seems to be endemic in the Iberian range.

A Single 3′α hs1,2 Enhancer in the Rabbit IgH Locus

Multiple cis-acting elements including the intronic enhancer and the 3′α enhancer (3′αE) regulate expression of the Ig heavy chain genes during B cell development. A 3′αE is composed of DNase I-hypersensitive sites, hs1,2, hs3a,b, and hs4, found 3′ of the murine Cα gene as well as 3′ of both human Cα genes, Cα1 and Cα2. Rabbits have 13 Cα genes, and we tested whether a 3′αE is associated with each of these genes. To identify 3′αE regions we developed a rabbit hs1,2 probe and used this to search for enhancer homologues of human hs1,2 in a genomic fosmid library. We identified a single hs1,2 fragment 8-kb downstream of Cα13, the presumed 3′-most Cα gene. We also identified and partially sequenced a new Cα gene, Cα14, located 6 kb upstream of Cα13. Genomic Southern blot analysis confirmed that the rabbit genome contains only one hs1,2 enhancer region. We tested the enhancer activity of the hs1,2 with the SV40, VH, and Iα promoters using the luciferase reporter gene in transient transfection assays and found that it significantly enhanced the activity of SV40 and VH promoters and slightly enhanced an Iα promoter. We conclude that the rabbit has a single hs1,2 enhancer that resides at the 3′ end of the IgH gene cluster and may constitute one of the cis-elements regulating the expression of IgH genes.

B lymphocyte deficiency in IgH‐transgenic rabbits

We developed IgH‐transgenic rabbits carrying a productive VDJ‐Cμ Tg and found the rabbits were B cell‐deficient, with a 50–100% reduction in serum IgM and IgG levels. The bone marrow of newborn Tg rabbits contained severely reduced levels of preB cells and almost no B cells. The few preB cells present in the bone marrow were large, cycling cells that expressed the VDJ‐Cμ Tg, indicating that the block in B cell development likely occurred at or before the transition from large (early) preB to small (late) preB cells. By immunoprecipitation, the Tg μ‐chain paired with VpreB and λ5, suggesting that the B cell deficiency is not due to an inability to form a preB cell receptor. Despite the block in B cell development, a few B cells, expressing predominantly endogenous μ‐chains, began the second stage of development in GALT. B cells were localized in and beneath the follicle‐associated epithelium of GALT prior to B cell follicle formation, suggesting to us that B cell follicle formation is initiated near the follicle‐associated epithelium, possibly through contact with intestinal microbiota. These IgH‐Tg rabbits should provide a useful model for studies of B cell development both in bone marrow and in GALT.

Chemokine-Mediated B Cell Trafficking during Early Rabbit GALT Development

Microbial and host cell interactions stimulate rabbit B cells to diversify the primary Ab repertoire in GALT. B cells at the base of appendix follicles begin proliferating and diversifying their V-(D)-J genes around 1 wk of age, ∼5 d after B cells first begin entering appendix follicles. To gain insight into the microbial and host cell interactions that stimulate B cells to diversify the primary Ab repertoire, we analyzed B cell trafficking within follicles during the first week of life. We visualized B cells, as well as chemokines that mediate B cell homing in lymphoid tissues, by in situ hybridization, and we examined B cell chemokine receptor expression by flow cytometry. We found that B cells were activated and began downregulating their BCRs well before a detectable B cell proliferative region appeared at the follicle base. The proliferative region was similar to germinal center dark zones, in that it exhibited elevated CXCL12 mRNA expression, and B cells that upregulated CXCR4 mRNA in response to signals acquired from selected intestinal commensals localized in this region. Our results suggest that after entering appendix follicles, B cells home sequentially to the follicle-associated epithelium, the follicular dendritic cell network, the B cell/T cell boundary, and, ultimately, the base of the follicle, where they enter a proliferative program and diversify the primary Ab repertoire.