Modulation of FcεRI-dependent mast cell response by OX40L.

OX40L is expressed by many cell types, including antigen presenting cells (APCs), T cells, vascular endothelial cells, mast cells (MCs), and natural killer cells. The importance of OX40L:OX40 interactions and the OX40L signaling is crucial for the homeostasis and for the modulation of the effector functions of the immune system. However, the lack of non-murine/non-IgG commercially available OX40L-triggering antibodies and the potential signal cross-contamination caused by the binding to the FcγRs co-expressed by several immune cells have limited the study of the OX40L-signaling cascade. We recently characterized the functions and described the molecular events, which follow the engagement of OX40L in MCs, by the use of the soluble OX40 molecule, able to mimic the regulatory T cell-driven engagement of MC-OX40L. This molecule enables signaling studies in MCs with any requirement for OX40-expressing cells. Using this unique reagent, we determined the modality and the extent by which the engagement of OX40L in MCs influences the IgE-dependent MC degranulation. This tool may find a potential application for signaling studies of other OX40L-expressing populations other than MCs, mainly APCs, with similar approaches we reported for the study of OX40L cascade.

Activity of soluble OX40 ligand is enhanced by oligomerization and cell surface immobilization.

OX40 ligand (OX40L) and OX40 are typical members of the tumor necrosis factor ligand family and the tumor necrosis factor receptor superfamily, respectively, and are involved in the costimulation and differentiation of T cells. Like other tumor necrosis factor ligands, OX40L is a type II transmembrane protein. Recombinant soluble human OX40L assembles into trimers and is practically inactive despite binding to OX40. However, oligomerization of soluble OX40L trimers by cross-linking with antibodies or by expression as a hexameric fusion protein strongly increased the activity of the ligand. Moreover, a fusion protein of OX40L with a single chain fragment recognizing the tumor stroma antigen fibroblast activation protein showed a cell surface antigen-dependent increase in the activity of the ligand domain of the molecule and thus mimicked the activity of membrane OX40L upon antigen binding. Trimeric single chain OX40L fusion proteins therefore represent a novel type of OX40L-derived immunostimulatory molecule with potentially reduced systemic side effects.