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.

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Studies using the European rabbit Oryctolagus cuniculus contributed to elucidating numerous fundamental aspects of antibody structure and diversification mechanisms and continue to be valuable for the development and testing of therapeutic humanized polyclonal and monoclonal antibodies. Additionally, during the last two decades, the use of the European rabbit as an animal model has been increasingly extended to many human diseases. This review documents the continuing wide utility of the rabbit as a reliable disease model for development of therapeutics and vaccines and studies of the cellular and molecular mechanisms underlying many human diseases. Examples include syphilis, tuberculosis, HIV-AIDS, acute hepatic failure and diseases caused by noroviruses, ocular herpes, and papillomaviruses. The use of rabbits for vaccine development studies, which began with Louis Pasteur’s rabies vaccine in 1881, continues today with targets that include the potentially blinding HSV-1 virus infection and HIV-AIDS. Additionally, two highly fatal viral diseases, rabbit hemorrhagic disease and myxomatosis, affect the European rabbit and provide unique models to understand co-evolution between a vertebrate host and viral pathogens.

The rabbit establishes its primary Ab repertoire by somatically diversifying an initial repertoire that is limited by restricted V H 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. The Journal of Immunology, 2000, 165: 2012-2019.T he rabbit is one of a few vertebrate species known to make limited use of combinatorial joining of multiple V H, D H , and J H gene segments during Ig heavy chain gene rearrangement. Although there are more than 100 V H gene segments available within the rabbit Ig heavy chain locus, many of which appear to be potentially functional (1), the 3Ј-most V H gene segment, V H 1, is utilized in 80 -90% of VDJ gene rearrangements (2). Most of the remaining 10 -20% of VDJ gene rearrangements utilize only two other V H gene segments, V H x and V H y (3). Rabbit B cells diversify their VDJ genes in gut-associated lymphoid tissue (GALT) 4 at 1-2 mo of age through two targeted mutational processes: a somatic gene conversion-like mechanism that transfers tracts of nucleotide sequence from upstream V H gene segment donors into the rearranged V H gene segment (4), and somatic hypermutation that distributes point mutations throughout the entire VDJ gene (5-7). At that time, the B cell population is also expanded through proliferation in GALT. B cell proliferation and Ig VDJ gene diversification in GALT results in a large population of B cells that express a wide range of Ab specificities.Several studies have established the importance of GALT in B cell proliferation and VDJ gene diversification. Weinstein et al. (7), for example, determined VDJ gene nucleotide...

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