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

Abstract

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 ؋ 10 ؊5 s ؊1 ). The affinity and kinetics of the OX40L/OX40 interactions were studied using the BIAcore™ 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 ؋ 10 ؊2 s ؊1 ). 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.

Real Time Kinetics of Insulin-like Growth Factor II (IGF-II) Interaction with the IGF-II/Mannose 6-Phosphate Receptor

“…cDNA was cloned in frame with the rat CD4 leader sequence, CD4 domains 3 and 4, and a biotin-accepting peptide and expressed in 293T cells. The structure of rat CD4 domains 3 and 4 has been determined (41), and they have previously been found to be expressed at high levels when truncated (42) or expressed as a fusion protein (43). Levels of expression were quantified by CD4 enzyme-linked immunosorbent assay.…”

Triggering of TNFRSF25 promotes CD8+ T‐cell responses and anti‐tumor immunity

“…Soluble recombinant TL1A (sTL1A) was produced as a fusion protein with domains 3 and 4 of rat CD4 in CHO cells using previously described methods [17]. Briefly, DNA encoding the extracellular domain of mouse TL1A (amino acids 77-252) was cloned downstream of the sequence encoding the leader peptide and domains 3 and 4 of rat CD4 in the expression vector pEE14 [17]. Anti-TL1A mAb (TAN2-2) was described previously [16], and biotinylated anti-TNFRSF25 Ab was obtained from R&D Systems.…”

CD134L Expression on Dendritic Cells in the Mesenteric Lymph Nodes Drives Colitis in T Cell-Restored SCID Mice

“…Soluble (s)CD4.CD134 protein contains the TNFR-like repeat region of rat CD134 that binds to mouse CD134L plus domains 3 and 4 of rat CD4, and was produced and purified, as described (26). A multimeric form of the protein was generated by binding CD4.CD134 recombinant protein to streptavidin-coated fluorescent Sphero beads (Spherotech, Libertyville, IL) via a biotinylated OX68 Ab, as described previously (42,43).…”

“…The OX40 molecule (CD134) is expressed transiently on activated CD4 ϩ T cells (23,24) and on some CD8 ϩ cells. It interacts with OX40L (CD134L) (25,26), which is a type II membrane protein with amino acid sequence similarity to TNF. CD134L has been reported to be present after activation on B cells (27,28), dendritic cells (DC) (29), microglia (30), and human endothelium (31).…”