During the last 15 years several laboratories have attempted to generate rabbit monoclonal antibodies, mainly because rabbits recognize antigens and epitopes that are not immunogenic in mice or rats, two species from which monoclonal antibodies are usually generated. Monoclonal antibodies from rabbits could not be generated, however, because a plasmacytoma fusion partner was not available. To obtain a rabbit plasmacytoma cell line that could be used as a fusion partner we generated transgenic rabbits carrying two transgenes, c-myc and v-abl. These rabbits developed plasmacytomas, and we obtained several plasmacytoma cell lines from which we isolated hypoxanthine/aminopterin/thymidine-sensitive clones. One of these clones, when fused with spleen cells of immunized rabbits, produced stable hybridomas that secreted antibodies specific for the immunogen. The hybridomas can be cloned and propagated in nude mice, and they can be frozen without change in their ability to secrete specific monoclonal antibodies. These rabbit-rabbit hybridomas will be useful not only for production of monoclonal antibodies but also for studies of immunoglobulin gene rearrangements and isotype switching.Monoclonal antibodies (mAbs) from rabbits have not been available because no rabbit plasmacytomas, from which a hybridoma fusion partner could be generated, have been identified. The availability of rabbit mAbs is, however, highly desirable for several reasons. First, rabbits are known to produce antibodies to many antigens that are not especially immunogenic in mice (1-5). For example, Bystryn et al. (2) directly compared rabbit and mouse antibodies directed against human melanoma cells and showed that they recognize different epitopes. Second, rabbit antibodies are generally of high affinity. Third, because most mAbs are generated in mice and rats, relatively few mAbs are available that react with mouse or rat immunogens. Because of this desire for rabbit mAbs several laboratories developed mouse-rabbit heterohybridomas, but this technology has had limited success. The earliest mouse-rabbit heterohybridomas were unstable and/or secreted only light (L) chain (6-9). Raybould and Takahashi (5) reportedly overcame this problem by using normal rabbit serum (NRS) instead of fetal calf serum (FCS) as a supplement to the culture medium. However, Verbanac et al. (10) described major problems with this method. For example, they found that the heterohybridomas were highly unstable and had to be subcloned every 4-6 weeks to avoid loss of antibody secretion. In our laboratory, we obtained no more than two to five hybridomas per fusion when using the method described by Raybould and Takahashi (5). Further, these heterohybridomas were difficult to clone, and the clones were generally unstable and did not secrete antibody over a prolonged period of time. Thus it became clear that heterohybridomas were not a satisfying solution and that rabbit-rabbit hybridomas were needed to stably produce monoclonal rabbit antibodies. We have now developed a fus...

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.

Somatic DNA rearrangements in B lymphocytes, including V(D)J gene rearrangements and isotype switching, generally occur in cis, i. e., intrachromosomally. We showed previously, however, that 3 to 7% of IgA heavy chains have the V H and C␣ regions encoded in trans. To determine whether the trans-association of V H and C␣ occurred by trans-chromosomal recombination, by trans-splicing, or by trans-chromosomal gene conversion, we generated and analyzed eight IgA-secreting rabbit hybridomas with trans-associated V H and C␣ heavy chains. By ELISA and by nucleotide sequence analysis we found that the V H and C␣ regions were encoded by genes that were in trans in the germline. We cloned the rearranged VDJ-C␣ gene from a fosmid library of one hybridoma and found that the expressed V H and C␣ genes were juxtaposed. Moreover, the juxtaposed V H and C␣ genes originated from different IgH alleles. From the same hybridoma, we also identified a fosmid clone with the other expected product of a trans-chromosomal recombination. The recombination breakpoint occurred within the S ͞S␣ region, indicating that the trans-association of V H and C␣ genes occurred by trans-chromosomal recombination during isotype switching. We conclude that trans-chromosomal recombination occurs at an unexpectedly high frequency (7%) within the IgH locus of B lymphocytes in normal animals, which may explain the high incidence of B-cell tumors that arise from oncogene translocation into the IgH locus.Ig genes undergo somatic recombination both during early B-cell development and again during antigen-induced immune responses. The V, D, and J gene segments recombine in proB and preB cells leading to the expression of IgM and IgD on the surface of mature B cells (1). Later, during isotype switching, the VDJ genes of these B lymphocytes rearrange to downstream C H genes leading to the expression of the IgG, IgA, or IgE isotypes (2-4). Although the mechanism for class switching has not been elucidated, we know that most switch rearrangements occur in or around switch regions that are characterized by a series of tandem repeat structures found 5Ј of C , C␥, C␣, and C genes (2, 5).Although switch recombination occurs generally by intrachromosomal DNA recombination between V H and C H genes in cis, and Pernis et al. (7) identified transassociated rabbit IgG molecules in which V H and C H are derived from genes in trans. These investigators used antibodies specific for V H and C␥ allotypes and showed that, in IgH heterozygous rabbits, some of the IgG molecules had the V H allotype encoded by one IgH allele and the C␥ allotype encoded by the other IgH allele. Subsequently, Knight et al. (8) identified trans-associated secretory IgA molecules from colostrum and showed that they represented as many as 8% of total IgA molecules.The trans-association of V H and C␣ could result from transchromosomal recombination, from trans-splicing of RNA, or from trans-chromosomal gene conversion. Support for each of these mechanisms has been reported. Kipps and Herzenberg ...

The 13 nonallelic IgA H chain genes of rabbit are differentially expressed in vivo. They can be grouped into those expressed at high levels (Cα4, Cα5, Cα6, Cα9, Cα10, Cα12, and Cα13), those expressed at low levels (Cα1, Cα2, Cα7, and Cα11), and those that are not expressed (Cα3 and Cα8). We tested whether the differential in vivo expression is due to differential responses of the Iα promoters to TGF-β stimulation. We stimulated the rabbit B cell line 55D1 with TGF-β and, using single-cell RT-PCR, found that expression of germline (GL) transcripts of α3 and α8 could not be induced. By luciferase reporter gene assay and EMSA we found that the promoters of the unexpressed isotypes Cα3 and Cα8 are defective, thereby explaining the absence of IgA3 and IgA8 in vivo. When comparing the promoter activities of the other isotypes we found that the activities did not reflect the degree of in vivo expression. Instead, the promoters of the isotypes expressed at high or low levels promoted expression of the luciferase gene to a similar degree, except for the Iα4 promoter, which had much higher activity. Also the degree to which TGF-β induced GL expression of the various isotypes in 55D1 B cells did not reflect in vivo expression. However, most of the TGF-β-stimulated cells expressed GL mRNA of multiple isotypes; no isotype was expressed preferentially. These results suggest that the final switch to a single isotype is regulated in a step subsequent to GL transcription, rather than by induction of GL transcripts by the Iα promoter.