Katherine L. Knight

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.

Generation of antibody diversity in rabbits

Most rabbit B lymphocytes use the same VH gene in V(D)J gene rearrangements and undergo somatic diversification by gene conversion and hypermutation. Recent experiments have shown that V(D)J genes in essentially all rabbit B cells diversify shortly after birth and that this diversification occurs in the gut-associated lymphoid tissue. Still to be determined is whether this diversification is developmentally programmed or is driven by exogenous microbial antigens.

Group 1 CD1 Genes in Rabbit

CD1 is an Ag-presenting molecule that can present lipids and glycolipids to T cells. The CD1 genes were first identified in the human, and since then, homologs have been identified in every mammalian species examined to date. Over a decade ago, CD1B and CD1D homologs were identified in the rabbit. We have extended this earlier study by identifying additional CD1 genes with the goal of developing the rabbit as an animal model to study the function of CD1 proteins. We constructed a thymocyte cDNA library and screened the library with CD1-specific probes. Based on nucleotide sequence analyses of the CD1+ cDNA clones obtained from the library, we have identified two CD1A genes and one CD1E gene as well as determined the complete sequence of the previously identified CD1B gene. The CD1E+ cDNA clones lacked the transmembrane and cytoplasmic domains and, if translated, would encode for a soluble or secreted CD1E protein. In addition, expression studies demonstrated that the CD1 genes were expressed in peripheral lymphoid tissues as well as in skin, gut, and lung. Of interest is the finding that CD1A2, CD1B, and CD1E genes were found to be expressed by rabbit B cell populations. The rabbit, with a complex CD1 locus composed of at least two CD1A genes, one CD1B gene, one CD1D gene, and one CD1E gene, is an excellent candidate as an animal model to study CD1 proteins.

Somatic diversification of IgH genes in rabbit

Summary: Rabbits have helped elucidate one of the major immunologic puzzles, namely the genetic control of antibody diversity. The primary IgH antibody repertoire in rabbits is dominated by B cells that use the same germline VH‐gene segment in VDJ gene rearrangements. The VDJ genes of essentially all B lymphocytes undergo somatic diversification within the first few weeks of the rabbits life. Such diversification occurs both by a somatic gene conversion‐like mechanism as well as by somatic hyper‐pointmutation. The diversification that occurs early in ontogeny takes place in gut‐associated lymphoid tissues and potentially depends on external factors such as microbial antigens. Few, if any. new B lymphocytes develop in adult rabbits and we discuss how the antibody repertoire is maintained throughout life. Finally, we discuss the molecular mechanism of somatic gene conversion of Ig genes, including die possibility that this involves the use of RAD5 1, an enzyme required for gene conversion‐mediated mating type switch in yeast.

RABBIT MAJOR HISTOCOMPATIBILITY COMPLEX IV. EXPRESSION OF MAJOR HISTOCOMPATIBILITY COMPLEX CLASS II GENES

The rabbit MHC class II DP, DQ, and DR alpha and beta chain genes were transfected into murine B lymphoma cells. The transfected cells expressed R‐DQ and R‐DR molecules on the cell surface but they did not express the R‐DP genes either on the cell surface or at the level of mRNA. Northern blot analyses showed that the R‐DP genes were expressed, albeit at low levels, in rabbit spleen. Similar analyses showed that the R‐DQ and R‐DR genes were expressed at high levels in rabbit spleen. A new monoclonal anti‐rabbit class II antibody, RDR34, has been developed and shown to react with the R‐DR transfected cells and not with the R‐DQ transfected cells. The previously described monoclonal anti‐rabbit class II antibody, 2C4, reacted with the R‐DQ transfected cells and not with the R‐DR transfected cells. Thus, 2C4 and RDR34 MAbs are specific for the R‐DQ and R‐DR molecules, respectively. Each of the antibodies reacted with approximately 50% of rabbit spleen cells as shown by immunofluorescent antibody studies.

Leukemia, Lymphoma, Embryonic Carcinoma and Hepatoma Induced in Transgenic Rabbits by the c-myc Oncogene Fused with Immunoglobulin Enhancers

Transgenic rabbits were developed with the rabbit c-myc proto oncogene fused to the immunoglobulin heavy or light chain enhancers, E µ or E κ .Rabbits transgenic for E µ -mycdeveloped leukemia very early in life, 17–21 days of age, and were terminally ill. The disease was diagnosed as acute lymphoblastic leukemia resembling childhood leukemia. The white blood cell counts were increased as much as 100-fold, the spleens were enlarged and various organs were infiltrated by the neoplastic lymphocytes. Analysis of the DNA showed that immunoglobulin heavy and light chain genes were rearranged, showing the leukemia to be of the B cell type. Since several different rearranged VDJ genes could be cloned from each of three rabbits we concluded that the leukemias were polyclonal. The blood lymphocytes, as well as the cells cloned from various tissues, appear to be lymphoblastic, that is, they are large, have a large nucleus and have very little cytoplasm. These cells do not express surface immunoglobulin, but they have a low concentration of cytoplasmic IgM and they secrete small amounts of light chain.