ANTIBODIES TO DISTINCT EPITOPES ON THE CD40 MOLECULE COOPERATE IN STIMULATION AND CAN BE USED FOR THE DETECTION OF SOLUBLE CD40

The B-cell surface protein, CD40, belongs to the tumour necrosis facto r/nerve growth factor (TNF/NGF) receptor family and plays a crucial ro le in T cell-dependent B-cell activation. Ligation of this receptor wi th antibodies or its recently defined ligand, gp39, generates an intra cellular signal that, when combined with triggering of surface immunog lobulin or the interleukin-4 (IL-4) receptor, induces a variety of sti mulatory effects in B cells. In this study we provide further evidence for the importance of receptor cross-linking in generating this signa l 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 oth er 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 CD 40 ligand, whereas such co-operation was seen with the S2C6 antibody. Cross-inhibition experiments showed that the 17:40 mAb recognized an e pitope that was clearly distinct from that seen by S2C6 and mAb89. Alt hough directed to separate epitopes, both 17:40 and mAb89 completely b locked binding of gp39 to its receptor, while the S2C6 mAb only partia lly interfered with this binding. The findings suggest a close relatio nship between the degree of receptor clustering and the strength of th e delivered signal. With the access to antibodies recognizing distinct structures on CD40 we also established a sandwich enzyme-linked immun osorbent assay for quantitative determinations of the antigen. With th is 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 regula tory 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 recept or family.