Overcoming steric inhibition of antibody-dependent phagocytosis with tall adhesions

Macrophages recognize and phagocytose opsonized target cells, including those coated with IgG antibodies. This process relies on binding of IgG to Fcγ receptors (FcγR) expressed on the macrophage surface, resulting in formation of a phagocytic synapse. Since the surface of both macrophages and target cells are densely packed with macromolecules of diverse sizes, most of which are not directly involved in phagocytic signaling, it is possible for tall ‘bystander’ proteins to sterically interfere with FcγR engagement. Here, we use cell-like target particles to show that bystander proteins can inhibit phagocytosis by blocking synapse formation. We then demonstrate that adding a tall binding protein to the target particle can overcome inhibition by the crowded environment and substantially recover phagocytosis, a process we call kinetic enhancement. Using a cell-free system of giant unilamellar vesicles and synthetic binders, we demonstrate that kinetic enhancement is a tunable feature of interface formation that can determine whether short binders engage, and we present theory and computer simulations to explain the nonmonotonic dependence of phagocytosis on tall binding protein surface density. These findings point to a strategy for overcoming surface crowding on phagocytic targets by re-engineering transition states with tall adhesion proteins, one that could be used to promote short receptor binding at other cell-cell junctions. Significance Statement Macrophages contribute to our immune defenses by phagocytosing pathogens and diseased cells. To accomplish this, they must first establish close contacts between receptors on their membranes and antibodies or other ligands decorating target cells. However, macrophage binding to the target can be disrupted by the presence of tall neighboring proteins and glycans—‘bystander’ molecules—that sterically prevent the two surfaces from coming into close contact. Counterintuitively, this inhibition can be overcome by the addition of even taller binding proteins between the macrophage and target cell, albeit at low concentrations. Using live cell and in vitro experiments, theory, and computer simulations, we show that tall binders can promote close contact that enables phagocytosis, even in the presence of bystander proteins that would normally block close contact.