Relevance of individual CD5 extracellular domains on antibody recognition, glycosylation and co-mitogenic signalling

CD5 is a type I glycoprotein which modulates T‐ and B‐cell receptor‐mediated signals and is expressed by thymocytes, mature T cells and a subset of mature B cells. The extracellular region of CD5 is composed of three scavenger receptor cysteine‐rich domains (D1, D2, D3) for which only limited functional and structural data are available. Using cell transfectants expressing ectodomain‐deficient CD5 molecules or CD5 immunoglobulin fusion proteins, we analysed individual CD5 domains with respect to monoclonal antibody binding specificity, glycosylation, and co‐mitogenic signalling. Our results show the presence of N‐linked oligosaccharides on D1 and D2, but not on D3. D1, the most amino‐terminal domain, is predicted to be the most appropriately placed domain for an interaction with a ligand. This domain is recognised by a large panel of well characterised CD5 mAbs, reflecting its higher immunogenicity. In an attempt to develop mAbs with specificity for the more conserved membrane‐proximal domains, we generated a unique mAb, named 83‐C4, whose binding mapped to D3. Co‐stimulatory studies revealed no significant differences between anti‐D1 and anti‐D3 mAbs. The high interspecies conservation of D3 implies a conserved role of this domain in CD5 function and the 83‐C4 mAb promises to be a valuable tool in exploring this.