Physical mechanisms of immune-cell target recognition and point-of-care imaging diagnostics for neglected tropical diseases

Controlled activation of immune cells protects the body from pathogens and disease while limiting damage to healthy self-cells Immune cells collect information through pattern-recognition receptors (PRRs) displayed on the cell membrane. While some PRRs respond to soluble ligands – small molecules, proteins, nucleotides, and sugars – signals are often transduced through direct physical contact – surface to surface – between an immune-cell and a potential target cell. Here, the cell-membrane is more than a passive platform for receptor display. Rather, the physical environment of the cell membrane influences the two-dimensional spatial organization of PRRs, which in turn controls their activation./nThe goal of the first part of this dissertation is to describe a simple, protein size-dependent physical mechanism that can control protein organization at membrane-membrane interfaces. I begin by reconstituting a synthetic membrane-membrane interface in vitro using giant unilamellar vesicles decorated with adhesion proteins, and I use this model system to show that a size difference of ~5 nm relative to the membrane interface is sufficient to drive the exclusion of a tall non-binding protein from the interface. Combining in vitro measurements with Monte Carlo simulations, I find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and, to a lesser extent, thermally-driven membrane height fluctuations that transiently limit access to the interface. This simple, sensitive, and highly effective means of passively segregating proteins has implications for signaling at many cell-cell contacts./nNext, I explore the consequences of size-dependent protein segregation at the interface between a macrophage and a target cell. Phagocytosis is mediated through activation of Fc receptors upon ligation with a surface-bound antibody, but it remains unclear how receptor-antibody binding triggers phosphorylation of the receptor, or how Fc receptors can accommodate the variable binding geometries formed by antigens with diverse structure, shape, and size. To determine the influence of antigen height on antibody-dependent phagocytosis, I reconstitute a minimal model of a target cell surface using lipid-bilayer coated glass beads and protein antigens with height-specific antibody binding sites. I find that phagocytosis is dramatically impaired for antigens that position an antibody > 10 nm from the target surface. The finding that close contact (< 10 nm) between a macrophage and target cell is a requirement for triggering of Fc receptors suggests a new design principle for therapeutic monoclonal antibodies to stimulate immune effector cells against cancer and infectious disease./nNot all pathogens and diseases are recognized and cleared by the immune system. For those that result in chronic infections, especially in low-resource areas, advances in mobile technology can be used to aid in diagnosis and treatment. The last part of this dissertation describes the development of CellScope Loa, a mobile phone-based microscope for quantifying the blood-borne parasitic worm Loa loa at the point-of-care. Efforts to eliminate onchocerciasis and lymphatic filariasis in Central Africa through mass drug administration have been suspended because of ivermectin-associated serious adverse events, including death, in patients infected with the filarial parasite Loa loa. To safely administer ivermectin (IVM) for onchocerciasis or lymphatic filariasis in regions co-endemic with L. loa, a strategy termed “test and (not) treat” has been proposed whereby those with high levels of Loa loa microfilariae (>30,000/ml) that put them at risk for life-threatening serious adverse events are identified and excluded from mass drug administration. To address this, I worked on the development of an automated mobile-phone microscope for quantifying Loa loa directly in whole blood. The device has been used in Cameroon to identify and exclude individuals with >20,000 mf per milliliter of blood (at-risk for SAEs) from ivermectin treatment in a “test and treat” pilot, where IVM was safely delivered to more than 15,000 patients in a “test and treat” pilot.

Advisor: Fletcher, Daniel A