UC Berkeley

Macrophages comprise a compartment of immune cells capable of performing a variety of functions necessary for fighting disease. One of the key roles of macrophages, which exist throughout the body in both tissue-resident and monocyte-derived forms, is to act as sentinels able to detect the presence of pathogens. Once a pathogen is recognized, a macrophage can destroy it via a process of internalization and digestion known as phagocytosis. To recognize foreign objects and potential pathogens, macrophages rely on a diverse set of surface receptors that are able to bind to pathogen-associated ligands. Binding between macrophage surface receptors and their associated ligands on the surface of a pathogen leads to the formation of a cellular interface, which represents a critical checkpoint at which the decision whether to phagocytose or not is made. In this dissertation I will examine this phagocytic interface and highlight the importance of molecular biophysical properties – including size, density, and mobility – in forming such interfaces and determining outcomes of phagocytosis.

In order to precisely modulate the biophysical characteristics of target surface properties and record the resulting outcomes of phagocytosis, it is necessary to develop a robust in vitro phagocytosis assay. This is the focus of the first part of this dissertation, in which I outline a novel protocol for making reconstituted phagocytic target particles and measuring amounts of their phagocytosis by macrophages. Reconstituted target particles are made by coating glass beads with lipid bilayers and conjugated proteins of interest. The composition of the lipid bilayers can be varied to control the surface properties of the targets – including surface protein identity, density, and fluidity. Automated microscopy and analysis of multi-well plates containing macrophages and target particles allows me to easily quantify phagocytic efficiency for targets with different surface properties in a relatively high-throughput manner. As a demonstration of this assay, I compare rates of phagocytosis for antibody opsonized targets and find that targets coated with fluid lipid bilayers and antibody are more efficiently phagocytosed than targets coated with non-fluid bilayers and antibodies.

Next, I use the phagocytosis assay to determine the effect of antigen size on antibody-dependent phagocytosis. Macrophages are able to detect objects opsonized with antibody with a class of receptors known as Fc receptors (FcRs) that can bind to the Fc region of antibodies. There have a been a number of proposed mechanisms by which FcR binding is thought to lead to phagocytic signaling, one of which relies on dephosphorylation of the intracellular signaling motifs of FcRs by the phosphatase the CD45 phosphatase. The tall extracellular motif of CD45 (~20 nm) raises the question of whether it will be excluded from cellular interfaces with small intermembrane gaps. I find that the inclusion or exclusion of CD45 from the phagocytic interface varies with antigen size and that phagocytosis is more efficient for antibodies targeted to short antigens (< 10nm) than it is for antibodies targeted to long antigens. This finding has direct implications for designing antibodies for use in cancer immunotherapies.

The surface of target cells is composed of a dense array of proteins and only a small subset of these will act as antigens to which antibodies bind. The remaining proteins, which I refer to as ‘bystander proteins’, do not directly contribute to phagocytic signaling, but are, nevertheless, present on targets and at phagocytic interfaces. The final chapter of this dissertation uses the reconstituted in vitro phagocytosis assay to examine the effect of bystander proteins. I find that phagocytosis is inhibited for antibody opsonized targets with bystander proteins when compared to targets opsonized with the same density of antibody in the absence of bystander proteins. Using a combination of live cell microscopy and computational simulations, I find that phagocytic inhibition is caused by bystander proteins posing a steric barrier to interface formation. I demonstrate that this barrier can be partially overcome with the addition of a second, long binding protein (P-Selectin) to the target surface.

The biophysical insights presented in this dissertation add to our current understandings of the mechanisms that control macrophage target recognition and will be useful for designing more effective immune targeted therapies.