RAG1 and RAG2 in developing rabbit appendix subpopulations.

The appendix of young rabbits is a site of primary heavy chain variable region-gene diversification and B cell selection. Appendix cells from 6- to 9-wk-old rabbits were stained and sorted for surface CD43 and IgM. We found that the CD43+IgM- and double-negative CD43-IgM- cells contained RAG1 transcripts and RAG2 protein. The presence of RAG gene products in appendix raised the possibility that pro-/pre-B cells were present in young rabbit appendix. Although an early suggestion that RAG2 plays a role in variable region-gene diversification by gene conversion in chicken bursa was not supported by studies of RAG2 protein in this tissue, we produced anti-rabbit RAG2 Abs to determine whether RAG2 protein was present in rabbit appendix, where cells that recently underwent gene conversion are found. We detected RAG2 protein in the four subpopulations of rabbit appendix lymphocytes, distinguished by surface CD43 and IgM markers. The appearance of RAG gene products during different stages of B cell maturation may reflect the function of the young rabbit appendix as a site of both B cell development and diversification.

CD5 is a potential selecting ligand for B cell surface immunoglobulin framework region sequences.

In rabbits nearly all B lymphocytes express the glycoprotein CD5, in contrast to mice and humans, where only a small proportion of B cells express this molecule (Raman, C., and K.L. Knight. 1992. J. Immunol. 149:3858-3864). CD5+ B cells appear to develop early in ontogeny and be maintained throughout life by self-renewal. The function of CD5 on B cells is still unknown. We showed earlier that "positive" selection occurs during B lymphocyte development in the rabbit appendix. This selection favors B cell expressing surface immunoglobulins with VHa2 structures in the first and third framework regions (Pospisil, R., G.O. Young-Cooper, and R.G. Mage. 1995. Proc. Natl. Acad. Sci. USA. 92:6961-6965). Here we report that F(ab')2 fragments, especially those bearing VHa2 framework region determinants, specifically interact with the B cell-surface glycoprotein CD5. This interaction can be inhibited by anti-CD5 antibodies. Furthermore, immobilized F(ab')2 fragments selectively bind CD5 molecules in appendix cell lysates. Interactions of VH framework region structures with CD5 may affect maintenance and selective expansion of particular B cells and thus contribute to autostimulatory growth of autoimmune or transformed cells.

The rabbit B cell antigen receptor is non-covalently associated with unique heteromeric protein complexes: possible insights into the membrane IgM/IgD coexpression paradox.

We describe several proteins that are components of the rabbit B cell receptor complex. Two proteins (37 kDa and 42 kDa) were found in non-covalent association with IgM expressed on B cells from peripheral blood. These proteins were also immunoprecipitated by anti-B29 (Ig-beta) and anti-mb1 (Ig-alpha) monoclonal antibodies. As in the mouse and human, the IgM associated molecules were found as heteromeric structures with non-reduced apparent molecular weights of approximately 70-75 kDa. On rabbit B cells we also found these proteins in a 100-135 kDa complex which may represent trimeric or tetrameric structures. By Western blot, the 37 kDa protein was identified as rabbit Ig-beta (B29), suggesting that the 42 kDa protein is rabbit Ig-alpha. These data suggest that rabbit IgM is associated with both Ig-alpha/beta and Ig-(alpha beta)2 or alpha beta gamma complexes. When similar immunoprecipitation studies were performed on lysates made from B cells isolated from appendix follicles, we found two additional IgM associated protein complexes containing 34 kDa and 36 kDa proteins.

Secondary rearrangements and post-rearrangement selection contribute to restricted immunoglobulin DJH expression in young rabbit bone marrow.

Extrachromosomal circular DNA purified from bone marrow cells of 2-weeks-old rabbits was assayed by polymerase chain reaction to determine the relative rearrangement frequencies of immunoglobulin DH to JH genes in vivo. DH genes rearranged to individual JH genes with different frequencies. This bias did not correlate with potential sequence overlaps in the DH or JH coding sequences. The JH2 and JH4 genes were the preferred targets of recombination in primary rearrangements. Although primary rearrangements to JH6 were relatively infrequent, secondary rearrangements were detected. This assay also revealed previously undescribed JH pseudogenes with functional recombination signal sequences. Analyses of genomic VDJH indicated that B cells expressing VDJH4 heavy chains survived and dominated in the bone marrow environment due to secondary rearrangements and/or post-rearrangement selection.

Identification of rabbit genomic Ig-VH pseudogenes that could serve as donor sequences for latent allotype expression.

Synthetic DNA oligomers specific for the VHa allotypes of rabbit Ig genes have been used to identify latent allotypic sequences in homozygous a1 and a2 rabbits. Two Ig VH pseudogenes containing latent a3 regions have been cloned from the genome of a homozygous a2 rabbit. Analysis of the regions associated with allotype expression indicates that these two pseudogenes contain VHa- sequences in framework region 1 (FR1) and VHa3 sequences in FR3. One gene has undergone an unusual rearrangement with a third VH gene, deleting their intervening sequences and recombining in FR3 with sequences 5' to the leader exon. Our results demonstrate the presence of latent VH sequences in the genomic DNA of normal rabbits and suggest that a mechanism such as gene conversion is responsible for expression of genetically-unexpected Ig VH genes.