A polymorphic CD40 Ligand (CD154) molecule mediates CD40-dependent signalling but interferes with the ability of soluble CD40 to functionally block CD154 : CD40 interactions

We report the characterization of a naturally occurring polymorphism in CD4 0 ligand (CD40L, CD154) expressed by activated T cells from a young female patient. This polymorphism encodes a nonconservative Gly --> Arg substituti on in amino acid 219 in the extracellular, CD40 binding domain of the molec ule. Studies carried out with 293 epithelial cells ectopically expressing t he polymorphic protein (CD154/G219R) revealed reduced levels of binding to different anti-CD154 monoclonal antibodies (mAb) and CD40-immunoglobulin (C D40-Ig). However, recognition of the polymorphic and wild-type CD154 molecu les by a polyclonal antiserum was comparable, suggesting that the polymorph ism affects the ability of the protein to interact with CD40 but does not s ignificantly alter its surface expression. To determine if reduced cross-li nking of CD40 mediated decreased functional effects, three CD40-dependent p roperties were measured. We found that pathways leading to the induction of surface CD23, CD80, and I gamma transcription were activated in response t o CD 154/G219R signalling. However, the decrease in affinity for CD40 by th e mutated CD154 affected the ability of CD40-Ig to efficiently interfere wi th the binding and effectively block induced CD80 expression. In contrast, we found that the 5c8 mAb, which recognized the polymorphic molecule to a s imilar extent as wild-type CD154, effectively blocked the interaction betwe en CD154/G219R and CD40 as measured by CD80 expression. These findings sugg est that naturally occurring polymorphisms in the CD 154 molecule may affec t the ability of CD40-mediated functions to be blocked by soluble CD40 or a nti-CD154 mAb in the therapeutic treatment of disease and graft rejection.