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Volume 272, Number 8, Issue of February 21, 1997 pp. 5275-5282
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Affinity and Kinetics of the Interaction between Soluble Trimeric OX40 Ligand, a Member of the Tumor Necrosis Factor Superfamily, and Its Receptor OX40 on Activated T Cells

(Received for publication, August 13, 1996, and in revised form, November 15, 1996)

Aymen Al-Shamkhani ^§ , Susan Mallett , Marion H. Brown , William James ^§ and A. Neil Barclay

From the  Medical Research Council Cellular Immunology Unit and the ^§ Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom

OX40 ligand (OX40L) and OX40 are members of the tumor necrosis factor and tumor necrosis factor receptor superfamilies, respectively. OX40L is expressed on activated B and T cells and endothelial cell lines, whereas OX40 is expressed on activated T cells. A construct for mouse OX40L was expressed as a soluble protein with domains 3 and 4 of rat CD4 as a tag (sCD4-OX40L). It formed a homotrimer as assessed by chemical cross-linking and gel filtration chromatography. Radiolabeled sCD4-OX40L bound to activated mouse T cells with a high affinity (K_D = 0.2-0.4 nM) and dissociated slowly (k_off = 4 × 10⁵ s¹). The affinity and kinetics of the OX40L/OX40 interactions were studied using the BIAcore^TM biosensor, which measures macromolecular interactions in real time. The extracellular part of the OX40 antigen was expressed as a soluble monomeric protein and immobilized on the BIAcore sensor chip. sCD4-OX40L bound the OX40 with a high affinity (K_D = 3.8 nM), although this was lower than that determined on the surface of activated T cells (K_D = 0.2-0.4 nM), where there is likely to be less restriction in mobility of the receptor. In the reverse orientation, sOX40 bound to immobilized sCD4-OX40L with a stoichiometry of 3.1 receptors to one ligand, with low affinity (K_D = 190 nM) and had a relatively fast dissociation rate constant (k_off = 2 × 10² s¹). Thus if the OX40 receptor is cleaved by proteolysis, it will release any bound ligand and is unlikely to block re-binding of ligand to cell surface OX40 because of the low monomeric affinity.

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