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Abstract: Previously described polyclonal or monoclonal antibodies (mAb) to rabbit CD5, raised against expressed recombinant protein or peptides, recognize CD5 on most rabbit B cells. The mAb KEN-5 was originally reported to recognize rabbit CD5. However, KEN-5 binds almost exclusively to T cells and only to a minor population of B cells. We show here that by Enzyme-linked Immunosorbent Assay (ELISA), KEN-5 binds to recombinant rabbit CD5. This interaction is partially inhibited by polyclonal goat anti-CD5 antibody. In addition, immunoprecipitations from lysates of surface biotinylated rabbit lymphocytes with KEN-5 or our anti-CD5 mAb isolate molecules that migrate identically on gels with the same approximate relative molecular mass of 67,000 M r. By flow cytometric analyses of individual cells from spleen, thymus and appendix, KEN-5 recognizes CD5-like molecules mainly on T cells and on 3–6% of IgM+ B cells. Immunohistochemical staining of splenic and appendix tissues and confocal immunofluorescent imaging confirm and extend results from flow cytometric analyses. Quantitation of fluorescent colocalization indicates that staining by KEN-5 colocalizes with staining by anti-CD5 on small percentage lymphocytes in splenic tissue sections. As CD5 has both N- and O-linked glycosylation, we hypothesised that differential binding of KEN-5 to T cells and B-cells may be explained by different glycan structures on the CD5 present on T compared to B cells. This hypothesis is supported by ELISA data that show that deglycosylation diminishes the binding of KEN-5 to recombinant rabbit CD5. Screening KEN-5 on an array with 406 glycans was inconclusive. Although we did not identify a strongly binding glycan structure, the data are suggestive that the epitope recognized by KEN-5 may be influenced by glycan structures. The epitope this mAb recognizes may either be the glycan itself, or more likely, is influenced by neighboring glycan structure. Our findings suggest that development, selection and function of different B- and T-cell subsets or their preferential survival may be directly or indirectly dependent on different glycan structures associated with CD5 or CD5-like molecules expressed on T cells compared to B cells. [Copyright &y& Elsevier]

Abstract: Although only a small proportion of mouse and human B cells are CD5+, most adult rabbit B cells express CD5. However, CD5 was not detectable on the majority of B cells in neonatal appendix 1 and 3days after birth. Cell trafficking studies demonstrated that CD5+ and CD5− CD62L+ B cells from bone marrow migrated into appendix. There, CD5+ B cells were preferentially expanded and predominated by ∼2weeks of age. In mutant ali/ali rabbits, VHa2+ B cells develop through gene conversion-like alteration of rearranged VH genes upstream of deleted VH1a2. Correlated appearance of individual CD5+ germinal centers and VHa2+ B-cells in mutant appendix suggests that CD5 binding positively selects cells with a2+ framework regions that bind CD5. Following negative and positive selection, cells with diversified rearranged heavy- and light-chain sequences exit appendix, migrate to peripheral tissues and constitute the preimmune repertoire of CD5+ B cells that encounter foreign antigens. [Copyright &y& Elsevier]

Abstract: Previous studies in our laboratory suggested that there was positive selection of B cells during early development in the appendix of normal and VH mutant (ali/ali) rabbits. Preferential expansion and survival of B lymphocytes was affected by the Ig VH frameworks 1 and 3 sequences expressed on the cell surface. We demonstrated a specific interaction between rabbit CD5 and the V region of rabbit heavy chains and suggested that CD5 is a potential selecting ligand for B-cell surface immunoglobulin framework region sequences. To further investigate the role of CD5 in rabbit B-cell selection and survival we prepared recombinant constructs and obtained stable expression of the three scavenger receptor cysteine-rich (SRCR) extracellular domains of rabbit CD5. Here we describe the production and purification of this expressed recombinant CD5 protein, polyclonal antibody obtained by immunization of a goat and initial production and characterization of specific mAbs against peptides selected from each sequenced SRCR domain. [Copyright &y& Elsevier]