We used techniques of immunogenetics and molecular biology to study rabbit immunoglobulins, and other genes including RAG-1 and RAG-2, which are necessary for gene rearrangements to occur during lymphocyte development. We investigated the development of anatomical sites such as appendix follicles and germinal centers in gut-associated lymphoid tissues and the regulated expression and sequence diversification of Ig genes during lymphoid cell development. Whereas B cells with rearranged VH1 predominate in normal rabbits, in homozygous Alicia mutant rabbits (ali/ali) the VH1 gene is deleted and B cells with upstream VH genes rearrange. We found differences between appendix development in normal and ali/ali rabbits based on immunohistochemistry, analyses of cell proliferation and apoptotic death. The development of the appendix in mutants appears to be retarded compared to normals. As populations of B cells bearing VHa2-like epitopes develop in mutant animals, appendix development appears more like normals. A higher proportion of B cells expressing the a2 allotype may receive strong signals to survive rather than undergo apoptosis. VHa2-positive B cells express high levels of Bcl-2 protein compared to a2-negative B cells. This suggests that B cells with FR allotypic motifs may become resistant to programmed cell death via the Bcl-2 pathway. The a2 allotype probably plays functional role(s) in selection and effective expansion of B cells in the appendix. We have now identified CD5 as a ligand for B-cell surface immunoglobulin. Immobilized F(ab')2 fragments isolate CD5 molecules from appendix cell lysates. We purified and biotinylated F(ab')2 fragments from sera of normal (a2+) and VH mutant animals (a2-) and used them as probes. By flow cytometry, VHa2+ F(ab')2 bind IgM+ B cells more strongly than VHa2- F(ab')2. This interaction as well as F(ab')2 binding to appendix germinal centers can be blocked by anti-CD5 antibodies. Cell attachment assays also suggest that the B cell-surface glycoprotein CD5 is a ligand for B cell surface immunoglobulin framework region sequences. Interactions of VH framework region structures with endogenous ligands such as CD5 may affect maintenance and selective expansion of particular B cells.
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