Antibodies to distinct epitopes on the CD40 molecule co-operate in stimulation and can be used for the detection of soluble CD40.

The B-cell surface protein, CD40, belongs to the tumour necrosis factor/nerve growth factor (TNF/NGF) receptor family and plays a crucial role in T cell-dependent B-cell activation. Ligation of this receptor with antibodies or its recently defined ligand, gp39, generates an intracellular signal that, when combined with triggering of surface immunoglobulin or the interleukin-4 (IL-4) receptor, induces a variety of stimulatory effects in B cells. In this study we provide further evidence for the importance of receptor cross-linking in generating this signal and we also report on the presence of a soluble form of CD40. A new CD40 monoclonal antibody (mAb), 17:40, was found to synergize with other CD40 antibodies (mAb89 and S2C6) in inducing proliferation as well as IgE synthesis in IL-4-treated tonsillar B cells. However, both this mAb and mAb89 failed to co-operate with a soluble construct of the CD40 ligand, whereas such co-operation was seen with the S2C6 antibody. Cross-inhibition experiments showed that the 17:40 mAb recognized an epitope that was clearly distinct from that seen by S2C6 and mAb89. Although directed to separate epitopes, both 17:40 and mAb89 completely blocked binding of gp39 to its receptor, while the S2C6 mAb only partially interfered with this binding. The findings suggest a close relationship between the degree of receptor clustering and the strength of the delivered signal. With the access to antibodies recognizing distinct structures on CD40 we also established a sandwich enzyme-linked immunosorbent assay for quantitative determinations of the antigen. With this assay we could demonstrate the presence of a soluble form of CD40 (sCD40) in culture supernatants. The fact that sCD40 also retained its ligand-binding capacity indicates that it may have an important regulatory role and modulate the T cell-dependent stimulation via CD40. Both the finding of soluble receptors and the need for receptor clustering are features that CD40 share with other members of the TNF/NGF receptor family.