Inhibiting phagocytosis with CD47: From the effects of red cell rigidity and shape to display on lentivirus -- Implications for aging and gene therapy

A macrophage engulfs another cell, or foreign particle, via phagocytosis, an engulfment process crucial not only to innate and adaptive immunity, but also to the maintenance of homeostasis. Phagocytosis is a receptormediated process that is dependent on Myosin-IIA motors, among other cytoskeletal proteins. Adhesion processes of both hematopoietic and mesenchymal derived cells can activate Myosin, and increasingly so on rigid substrates. Macrophage engulfment becomes inefficient if the macrophage also engages `Marker of Self ' CD47 that inhibits Myosin accumulation to the phagocytic synapse. CD47 is a ubiquitously expressed transmembrane cell surface protein that binds to signal regulatory protein alpha (SIRPA) that is highly expressed by macrophages. CD47's role in downregulating macrophage phagocytosis was first discovered in murine erythrocytes (RBCs), where wild-type RBCs are long-lived in circulation, while RBCs derived from a CD47 knockout mouse are rapidly cleared. More recently CD47 has been found to inhibit clearance of a variety of viable cell types including stem cells, leukocytes, platelets, and cancers. However, the limitations of CD47 as a `marker of self ' on apoptotic and experimentally oxidized cells are beginning to be realized, as CD47 surface expression does not effectively inhibit the phagocytosis of these aged cells. Furthermore, while it is well known that macrophage-mediated clearance controls the removal of aged RBCs, that are reported to become rigid, from the bloodstream, the role that rigidification plays in countering CD47 inhibitory signals remains to be clarified. To study the effects of RBC rigidity on the regulation of phagocytosis by CD47 expression, RBCs were controllably stiffened in different shapes without compromising CD47-SIRPA interactions. Uptake of antibody-opsonized human-RBC was accelerated, as expected, by blocking CD47, but was fastest with rigid RBC-Discocytes that mediated maximum levels of Myosin-IIA accumulation at the phagocytic synapse. Attenuation of the antibody-driven `eat me' signal partially recovered `Self ' signaling by rigid RBC-Discocytes, and more rounded but rigid RBC-Stomatocytes also signaled `Self ' more efficiently. These results highlight the biophysical nature of the CD47-SIRPA inhibitory mechanism that can be overpowered by rigidity and can be rescued by target shape. Resident tissue macrophages are adept in capturing non-self particles from the extracellular environment, including HIV-derived lentiviral gene therapy vectors, thus limiting efforts at therapeutic vector delivery. CD47-SIRPA inhibition has just recently been shown to inhibit in vivo macrophage clearance of nano-sized synthetic particles that are significantly smaller than previously studied mammalian cells. A novel lentiviral vector was engineered here to present an oriented human CD47-GFP fusion protein on the vector envelope. Using Total internal reflection microscopy (TIRFM), atomic force microscopy (AFM), and immunoblotting, we have demonstrated that engineered lentivectors display the CD47-GFP protein. In vivo results show that lentiviral vector display of CD47 increases circulation of the vector in the bloodstream, and reduces off-target transgene expression in splenic and liver macrophages. Use of this CD47-displaying lentiviral vector, further shows increased delivery to solid tumors. CD47 on the surface of lentiviral gene therapy vectors was found to attenuate off-target macrophage uptake of the vector, and thereby increase therapeutic efficiency. The results here examine the boundaries of CD47's role as a `marker of self '; CD47 inhibition is diminished on the surface of rigid self-cells, while an enveloped virus shows the potential to masquerade as self by taking its envelope from a cell with sufficient CD47. The observed role of red cell shape and rigidity in CD47s ability This dissertation is available at ScholarlyCommons: http://repository.upenn. edu/edissertations/1451 to signal self highlights the biophysical aspect of phagocytosis with implications for CD47 inhibition of clearance of aged, diseased, and cancerous cells that differ from viable cells in their physical properties. Study of the novel CD47-lenti reveals the first evidence of CD47 being able to limit macrophage engulfment of a non-mammalian physiological entity, as current studies have as of yet been limited to mammalian cells and synthetic particles. This finding may have potential application in the understanding of disease progression and vaccine development for enveloped viruses such as HIV-1. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Chemical and Biomolecular Engineering First Advisor Dennis E. Discher