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* Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Wuerzburg, Wuerzburg, Germany; and
Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany
For many ligands of the TNF family, trimer stability and oligomerization status are crucial determinants of receptor activation. However, for the immunostimulatory ligands CD27L, CD40L, 41BBL, and glucocorticoid-induced TNF receptor ligand (GITRL) detailed information regarding these requirements is lacking. Here, we comprehensively evaluated the effect of trimer stability and oligomerization on receptor activation by these ligands. Treatment with soluble Flag-tagged CD27L, 41BBL, and GITRL minimally activated receptor signaling, while Flag-CD40L was highly active. Oligomerization with anti-Flag Abs further enhanced the specific activity of Flag-CD40L 10-fold and of Flag-41BBL more than 200-fold, but it failed to activate Flag-CD27L and Flag-GITRL. We next investigated the relevance of trimer stability by introducing the tenascin-C (TNC) trimerization domain, yielding stabilized Flag-TNC-ligand trimers. Oligomerization with anti-Flag Ab potently activated signaling by Flag-TNC-CD27L and Flag-TNC-GITRL and, albeit to a lesser extent, Flag-TNC-CD40L and Flag-TNC-41BBL. Forced hexamerization, by introducing an Ig Fc domain, revealed that hexameric derivatives of Flag-TNC-41BBL, Flag-CD40L, and Flag-TNC-GITRL all activate receptor signaling with high efficiency, whereas hexameric Flag-CD27L variant left inactive. Finally, we attempted to selectively activate receptor signaling on targeted cells, by using Ab fragment (single-chain fragment variable region, scFv)-ligand fusion proteins, an approach previously applied to other TNF ligands. Target cell surface Ag-selective activation was achieved for scFv-41BBL, scFv-CD40L, and scFv-GITRL, although the latter two displayed already significant activity toward Ag-negative cells. In conclusion, our data establish that trimeric CD40L is active, 41BBL requires hexamerization, GITRL requires trimer stabilization, and CD27L requires trimer stabilization and oligomerization. Furthermore, surface immobilization might be exploited to gain locally enhanced ligand activity.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Deutsche Krebshilfe (projects 106222, 107034-226), Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 487), and Interdisziplinäres Zentrum für Klinische Forschung der Universität Würzburg (project A49 and B7).
2 A.W., N.M., and A.F. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Harald Wajant, Department of Molecular Medicine, Medical Clinic and Polyclinic II, University of Wuerzburg, Roentgenring 11, 97070 Wuerzburg; Germany. E-mail address: harald.wajant{at}mail.uni-wuerzburg.de
4 Abbreviations used in this paper: GITRL, glucocorticoid-induced TNF receptor ligand; CFDA-SE, carboxyfluorescein diacetate succinimidyl ester; DC, dendritic cell; FAP, fibroblast activation protein; HMW, high molecular mass (weight); THD, TNF homology domain; TNC, tenascin-C; scFv, single-chain fragment variable region.
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