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. V/H framework region sequences (a2 allotype) probably play functional role(s) in selection and effective expansion of B cells in the appendix. We identified the B-cell surface glycoprotein CD5 as a ligand for B-cell surface immunoglobulin. Immobilized F(ab')2 fragments isolated CD5 molecules from appendix cell lysates. We purified and biotinylated F(ab')2 fragments from sera of normal (a2+) and V/H mutant animals (a2-) and used them as probes. By flow cytometry, V/Ha2+ F(ab')2 bind IgM+ B cells more strongly than V/Ha2- 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 CD5 is a ligand for B-cell surface immunoglobulin framework region sequences. Interactions of V/H framework region structures with endogenous ligands such as CD5 may affect maintenance and selective expansion of particular B cells. An extension of these studies to human B cells in collaboration with Drs. G. Marti and G. Silverman showed that human immunoglobulin binds to CD5-expressing cell lines. Staining was inhibited when these cells were first preincubated with anti-CD5 antibody. We isolated CD5 molecules from EBV-CLL cell lysates using human immunoglobulin coupled to tosyl-activated dynabeads. If we preincubated cell lysates with anti-CD5 coupled beads, CD5 molecules were not isolated. Rabbits were immunized to make classical anti-DNP hapten antibody responses in order to study clonal V/H and V/L region diversification in germinal centers during antibody responses. Individual cells from germinal centers were collected by micromanipulation. The rearranged genes for antibody heavy and light chains in single cells were PCR-amplified and sequenced. Preliminary results show clonally related sequences that have probably diversified by both hypermutation and gene-conversion-like mechanisms. Antibodies that appear to recognize rabbit mb-1 (CD79a) were generated and characterized. These antibodies along with commercially available anti-CD79b are being used to further characterize populations of developing B lymphocytes in the young rabbit appendix.
Showing the most recent 10 out of 15 publications
