A CD40-CD95L fusion protein interferes with CD40L-induced prosurvival signaling and allows membrane CD40L-restricted activation of CD95.

We analyzed a novel bifunctional fusion protein, CD40ed-CD95Led, consisting amino-terminally of the extracellular domain of CD40 and carboxy-terminally of the extracellular domain of CD95L. On cells lacking CD40L, this fusion protein is poorly active with respect to CD95 activation [median effective dose (ED50)>1 microg/ml], but it stimulates CD95 signaling with high efficiency upon binding to membrane-expressed CD40L (ED50<1 ng/ml). Thus, cell surface immobilization mediated by the CD40 part of the molecule unmasks the high-latent, CD95-stimulating capacity of the otherwise poorly active CD95L fusion protein. Moreover, interaction of the CD40 part of CD40ed-CD95Led with CD40L prevents the activation of cellular CD40. The CD40ed-CD95Led fusion protein therefore simultaneously blocks antiapoptotic CD40 activation and induces CD95-mediated apoptosis. Indeed, T47D cells displaying an antiapoptotic autocrine CD40-CD40L signaling loop were significantly more sensitive toward CD40ed-CD95Led than toward soluble CD95L artificially activated by crosslinking. Fusion proteins of RANK and CD95L (RANKed-CD95Led) and CD40 and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) (CD40ed-TRAILed), with domain architectures similar to CD40ed-Cd95Led, displayed RANKL-dependent CD95 and CD40L-dependent TRAILR2 activation, respectively, indicating the principle feasibility of this fusion protein design.

Tumor necrosis factor-receptor 2 is up-regulated on lamina propria T cells in Crohn's disease and promotes experimental colitis in vivo.

Tumor necrosis factor (TNF) plays a pivotal role in the pathogenesis of Crohn's disease (CD). However, little is known about the role of TNF receptors (TNF-R) in this disease. Here, we found that TNF-R2 (in contrast to TNF-R1) was significantly up-regulated on lamina propria and peripheral blood T cells in CD compared to control patients. To directly test the functional role of TNF-R2 in Th1-mediated experimental colitis in vivo, we took advantage of transgenic animals overexpressing TNF-R2 in T cells. Reconstitution of SCID mice with CD4+ CD62L+ T cells from TNF-R2 transgenic mice led to an earlier wasting syndrome, a more severe colitis and augmented Th1 cytokine production than reconstitution with cells from wild-type littermates. In addition, TUNEL staining revealed a significantly decreased apoptosis rate of lamina propria mononuclear cells in mice reconstituted with TNF-R2 transgenic T cells compared to mice reconstituted with wild-type T cells. In summary, our data suggest a critical regulatory role of TNF-R2 signaling for disease exacerbation in Th1-mediated chronic colitis. Taken together with the increased expression of TNF-R2 in CD, selective targeting of TNF-R2 signaling thus emerges as a potentially novel approach to the treatment of CD.