Dennis Lanning

Role of Commensal Bacteria in Development of Gut-Associated Lymphoid Tissues and Preimmune Antibody Repertoire

Intestinal bacteria are required for development of gut-associated lymphoid tissues (GALT), which mediate a variety of host immune functions, such as mucosal immunity and oral tolerance. In rabbits, the intestinal microflora are also required for developing the preimmune Ab repertoire by promoting somatic diversification of Ig genes in B cells that have migrated to GALT. We studied the mechanism of bacteria-induced GALT development. Bacteria were introduced into rabbits in which the appendix had been rendered germfree by microsurgery (we refer to these rabbits as germfree-appendix rabbits). We then identified specific members of the intestinal flora that promote GALT development. The combination of Bacteroides fragilis and Bacillus subtilis consistently promoted GALT development and led to development of the preimmune Ab repertoire, as shown by an increase in somatic diversification of VDJ-Cμ genes in appendix B cells. Neither species alone consistently induced GALT development, nor did Clostridium subterminale, Escherichia coli, or Staphylococcus epidermidis. B. fragilis, which by itself is immunogenic, did not promote GALT development; hence, GALT development in rabbits does not appear to be the result of an Ag-specific immune response. To identify bacterial pathways required for GALT development, we introduced B. fragilis along with stress-response mutants of B. subtilis into germfree-appendix rabbits. We identified two Spo0A-controlled stress responses, sporulation and secretion of the protein YqxM, which are required for GALT development. We conclude that specific members of the commensal, intestinal flora drive GALT development through a specific subset of stress responses.

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.

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.

Microbial induction of B and T cell areas in rabbit appendix.

Gut-associated lymphoid tissue (GALT) development requires interaction with the intestinal microbiota. Because murine secondary lymphoid tissue development is driven by positive feedback interactions between B cells and stromal cells, we used in situ hybridization to determine whether intestinal commensals influence such interactions during rabbit appendix development. The features of positive feedback interactions we examined (CXCL13 mRNA expression, B cell accumulation and FDC differentiation) increased during early follicle development, but stalled in the absence of intestinal commensals. These features were reinitiated by commensals that stimulated follicle development and intrafollicular B cell proliferation. Our results suggest that rabbit appendix follicles develop in two phases: an initial phase of B cell recruitment to nascent follicles, possibly through positive feedback interactions, and a subsequent phase of intrafollicular B cell proliferation stimulated by intestinal commensals. In addition, we found that intestinal commensals stimulate appendix CCL21 mRNA expression and T cell area formation.

CHAPTER 28 – Immunoglobulin Genes and Generation of Antibody Repertoires in Higher Vertebrates: A Key Role for GALT

This chapter focuses on the immunoglobulin (Ig) genes and development of the antibody repertoire in species that generate their primary antibody repertoires through the extensive somatic diversification of V(D)J heavy and light chain genes. In contrast, the V hand V L of mouse and human, use combinatorial joining of multiple V, (D), and J gene segments to generate the primary repertoire, comprise multiple V hand V L groups. It is possible that species in which the germline genes belong to are only a single group, and therefore have a less diverse repertoire of V gene segments have evolved to utilize somatic hypermutation, gene conversion, or both for developing a highly diversified primary antibody repertoire. Further, the species with less diverse V-gene segments utilize gut-associated lymphoid tissue (GALT) as the site for B-cell expansion and generation of the primary antibody repertoire. It is likely that the somatic diversification in GALT that leads to the primary antibody repertoire is not antigen driven, but instead occurs as a polyclonal response either to an exogenous agent, as occurred in rabbits, or possibly to an endogenous agent, such as a retroviral superantigen in species such as chicken, in which exogenous molecules are not required. Further studies are needed to elucidate the mechanism by which both GALT development and the somatic diversification of the Ig genes are initiated.

Study of Sylvilagus rabbit TRIM5α species-specific domain: how ancient endoviruses could have shaped the antiviral repertoire in Lagomorpha

BackgroundSince the first report of the antiretroviral restriction factor TRIM5α in primates, several orthologs in other mammals have been described. Recent studies suggest that leporid retroviruses like RELIK, the first reported endogenous lentivirus ever, may have imposed positive selection in TRIM5α orthologs of the European rabbit and European brown hare. Considering that RELIK must already have been present in a common ancestor of the leporid genera Lepus, Sylvilagus and Oryctolagus, we extended the study of evolutionary patterns of TRIM5α to other members of the Leporidae family, particularly to the genus Sylvilagus. Therefore, we obtained the TRIM5α nucleotide sequences of additional subspecies and species of the three leporid genera. We also compared lagomorph TRIM5α deduced protein sequences and established TRIM5α gene and TRIM5α protein phylogenies.ResultsThe deduced protein sequence of Iberian hare TRIM5α was 89% identical to European rabbit TRIM5α, although high divergence was observed at the PRYSPRY v1 region between rabbit and the identified alleles from this hare species (allele 1: 50% divergence; allele 2: 53% divergence). A high identity was expected between the Sylvilagus and Oryctolagus TRIM5α proteins and, in fact, the Sylvilagus TRIM5α was 91% identical to the Oryctolagus protein. Nevertheless, the PRYSPRY v1 region was only 50% similar between these genera. Selection analysis of Lagomorpha TRIM5α proteins identified 25 positively-selected codons, 11 of which are located in the PRYSPRY v1 region, responsible for species specific differences in viral capsid recognition.ConclusionsBy extending Lagomorpha TRIM5α studies to an additional genus known to bear RELIK, we verified that the divergent species-specific pattern observed between the Oryctolagus and Lepus PRYSPRY-domains is also present in Sylvilagus TRIM5α. This work is one of the first known studies that compare the evolution of the antiretroviral restriction factor TRIM5α in different mammalian groups, Lagomorpha and Primates.

Genetic characterization of CCL3, CCL4 and CCL5 in leporid genera Oryctolagus, Sylvilagus and Lepus

The genetic diversity of C‐C motif chemokine receptor 5 (CCR5) ligands CCL3, CCL4 and CCL5 in the leporid genera Oryctolagus, Sylvilagus and Lepus was studied. Our results demonstrate that the three CCR5 chemokine ligands are under strong purifying selection as a result of possible functional binding constraints.