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.

Diversification of the primary antibody repertoire begins during early follicle development in the rabbit appendix.

Rabbits generate a diversified primary antibody repertoire by somatically mutating, in gut-associated lymphoid tissue (GALT), an initial repertoire that is limited by preferential rearrangement of the 3'-most IGVH gene segment. To determine when repertoire diversification begins in GALT, we performed in situ hybridization on neonatal rabbit appendix sections with an activation-induced cytidine deaminase (AID) riboprobe, because AID is required for the mutational processes that diversify the primary antibody repertoire. We first detected AID mRNA expression around 1 week of age, in the basal region of developing follicles. By PCR-amplifying V-D-J genes from AID mRNA(+) B cells isolated by laser capture microdissection, we found evidence of somatic hypermutation, and one likely instance of somatic gene conversion. Our results suggest that V-(D)-J gene diversification begins during early postnatal appendix development, in B cells stimulated to enter a proliferative program by signals derived from select intestinal commensals.

B lymphocyte deficiency in IgH-transgenic rabbits.

We developed IgH-transgenic rabbits carrying a productive VDJ-Cmu 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-Cmu 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 mu-chain paired with VpreB and lambda5, 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 mu-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.

Suppression of B lymphopoiesis at a lymphoid progenitor stage in adult rabbits.

B lymphopoiesis in rabbits is robust early in ontogeny, but is arrested by 16 weeks of age at which time no proB or preB cells are found in bone marrow (BM). To determine if BM cells from adults retain B lymphopoietic potential, we transferred BM from adult green fluorescent protein (gfp) transgenic rabbits into young rabbits. We found gfp+ preB cells arising in the young recipients, indicating that BM cells from adults can differentiate into B cell precursors. We identified a population of MHCII-IL-7-binding BM cells in adults that collectively expresses Tdt, EBF and Pax5-genes known to be expressed in murine lymphoid progenitors. Upon co-culture with OP9 or OP9 delta-like 1+ stromal cells, we found that these cells both expanded in number and differentiated into B and T cell precursors, respectively, showing that early lymphoid progenitors, designated rLP for rabbit lymphoid progenitors, are present within the MHCII-IL-7-binding BM cell population. Further, IL-7 was required for rLPs to expand and differentiate into B cell precursors in vitro. The arrest of B lymphopoiesis in adults, however, is not likely due to the absence of IL-7, because the level of IL-7 transcripts was higher in BM from adults than in young rabbits. B lymphopoiesis was re-initiated in adults after sub-lethal irradiation as shown by the reappearance of B cell precursors and the presence of B cell receptor excision circles in BM. We conclude that B lymphopoiesis in adults is suppressed at a lymphoid progenitor stage (MHCII-IL-7 binding) of development.

Characterization of the T-cell receptor gamma locus and analysis of the variable gene segment expression in rabbit.

The genomic organization and expression of genes of the T-cell receptor gamma (TRG) locus are described for mice and humans, but not for species such as rabbits (Oryctolagus cuniculus), in which gammadelta T cells compose a sizeable proportion of T cells in the periphery. We cloned 200 kb of the rabbit TRG locus and determined the TRGV gene usage in adult and newborn rabbits by RT-PCR. We identified two TRGJ genes, one TRGC gene, and 22 TRGV genes, all of which encoded functional variable regions. One TRGV gene is the unique member of the TRGV2 subgroup, whereas the other genes belong to the TRGV1 subgroup. Evolutionary analyses of TRGV1 genes identified three distinct groups that can be explained by separate duplication events in the rabbit genome. Evidence of gene conversion between TRGV1.1 and TRGV1.6 was observed. Both TRGV1 and TRGV2 subgroup genes were expressed in the spleen, intestine, and appendix of adult rabbits, and the repertoire of TRGV genes expressed in these tissues was similar. In these tissues from newborns, and in skin from adults, only the genes from the TRGV1 subgroup were expressed. Greater TRGV-J junctional diversity was found in tissues from adult compared to newborn rabbits. Our analyses indicate rabbits have a larger germ line encoded TRG repertoire compared with that of mice and humans. In addition, we found TRGV gene usage is alike in most tissues of rabbits similar to that found in humans but in contrast to that found in mice.

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

The large interallelic distances between the three rabbit Ig V(H)a lineages, a1, a2 and a3, suggest that the persistence time of the V(H)a 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 V(H) 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 V(H)a 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 V(H) genes expressed in seven algirus rabbits that were typed as either a-positive or a-blank. The data show that the V(H) genes transcribed in a-positive rabbits are closely related to the V(H)1 alleles of domestic rabbits. In contrast, a-blank rabbits were found to preferentially use V(H) genes that, although clearly related to the known V(H)a genes, define a new major allotypic lineage, designated a4. The a4 sequences have hallmark rabbit V(H)a residues together with a number of unprecedented amino acid changes in framework region 2 and 3. The net protein distances between the V(H)a4 and the V(H)a1, a2, and a3 lineages were 20, 29, and 21% respectively. We conclude that at least four distantly related lineages of the rabbit V(H)a locus exist, one of which seems to be endemic in the Iberian range.

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-mu 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 lambda5 gene and demonstrated that they are expressed within B-lineage cells in bone marrow. VpreB and lambda5 coimmunoprecipitated with the mu-chain in lysates of 293T cells transfected with VpreB, lambda5, and mu, indicating that VpreB, lambda5, and mu-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' Cmu 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.