Molecular mechanisms of macrophage phagocytic decision-making

Controlled and selective activation of immune cells is critical for ridding the body of disease-causing threats while limiting damage to healthy cells and maintaining normal tissue function. Though immune signaling can be initiated and mediated by soluble factors, many key immune responses require direct contact with a target to both evaluate the threat that it poses and initiate attack, if necessary. Macrophage phagocytosis is one such example of a contact-dependent effector function, in which macrophage Fc receptors (FcRs) directly engage with antigen-bound antibodies on a target cell. This drives FcR phosphorylation of intracellular ITAM motifs and initiates downstream signaling, ultimately leading to actin polymerization, membrane remodeling, and target engulfment. However, simultaneous to engagement of FcRs, inhibitory receptors—such as SIRPα—can bind their ligand on a target, shutting down phagocytosis through their intracellular ITIM motifs, despite the presence of activating signaling. While important for preventing auto-immune responses, these inhibitory checkpoints also reduce efficacy of tumor-targeting antibodies and other immunotherapies./nIn the first Chapter of this dissertation, I probe how macrophages integrate contradictory signals from activating FcR-antibody and inhibitory SIRPα-CD47 interactions. Using reconstituted tumor cell-like target particles, I conclude that macrophage phagocytosis decisions are dictated by the ratio of activating ligand to inhibitory ligand over a range of absolute molecular densities. Lowering the antibody:CD47 reduces FcR phosphorylation due to inhibitory phosphatases recruited to CD47-bound SIRPα, thus preventing downstream phagocytic signaling from proceeding. I demonstrate that this ratiometric signaling is important in tumor cell phagocytosis and that it can be manipulated by blocking SIRPα engagement, demonstrating that this decision-making paradigm may be important for controlling macrophage phagocytosis in cancer immunotherapy./nIn the next two Chapters, I discuss two strategies for probing and manipulating signaling at the macrophage-target interface. First, I probe the importance of ITAM and ITIM multiplicity on phagocytic receptors. By creating panels of synthetic receptors containing 0-4 ITAMs each, I conclude that increasing signaling motifs beyond two ITAMS does little to enhance phagocytosis and that additional ITAMs in series do not decrease inhibitory signaling. Second, I propose a novel therapeutic strategy that leverages spatial reorganization of the phagocytic interface to overcome inhibitory signaling. Using synthetic receptors, I show that tethering a protein with a short extracellular domain to one with a large extracellular domain can lead to exclusion of the short protein from a cell interface due to size-dependent segregation of the tall protein. I then propose a system for linking short SIRPα to tall CD45 to drive exclusion of SIRPα from a macrophage-target interface and reduce inhibitory signaling./nNext, I zoom out from the single macrophage-target interface to look more wholistically at macrophage signal integration. Here, I ask whether engagement with many inhibitory targets can impact phagocytosis of activating targets. I conclude that high levels of SIRPα-CD47 engagement does not significantly impact phagocytosis of other targets, highlighting how macrophage decisions are made locally./nLastly, in the final Chapter of this dissertation, I zoom into the phagocytic interface to examine phosphorylation of ITAM motifs at a single receptor level. ITAMs contain two tyrosines that become phosphorylated and subsequently engage with Syk kinase, and the phosphorylation state of the ITAM alters Syk binding kinetics. By using variation in these binding kinetics to readout ITAM state, I present a strategy for spatial mapping of ITAM phosphorylation. With ITAM phosphopeptides, I demonstrate the ability to kinetically distinguish between ITAM phosphorylation states. I then show how this kinetic variation can be implemented for mapping phosphorylation of endogenous FcRs in macrophages.

Advisor: Fletcher, Daniel A