Sasha Simakov

Actin Regulators Differentially Control FCR-Mediated Phagocytosis and Trogocytosis in Macrophages
 

Faculty PI: Meghan Morrissey

Macrophages are innate immune cells that clear pathogens, cancerous cells, and debris from the body utilizing phagocytosis (total engulfment of the target) and trogocytosis (cellular nibbling). Trogocytosis has been linked to decreased efficacy of antibody-based immunotherapies due to its stripping of antigen off of target cells without killing them. Antigen stripping makes it less likely for the next macrophage to recognize the target, ultimately leading to immune escape. Interestingly, these two cellular behaviors are activated by the same cellular receptor and ligand, the FcR and IgG, respectively. The signaling pathway for FcR-mediated phagocytosis has been well characterized. However, it remains unclear which proteins are utilized in trogocytosis. We investigated the roles of actin-regulating proteins Rac1, RhoA, and Arp 2/3 in macrophage trogocytosis. Bone Marrow Derived Macrophages (BMDMs) were treated with 4 incrementally increasing concentrations of pharmacological inhibitor, and then cocultured with Raji B cells opsonized with an anti-CD20 antibody. After coculture, we quantified the levels of phagocytosis and trogocytosis using a fluorescence-based flow cytometry assay. Upon inhibition of Rac1, levels of phagocytosis halved while levels of trogocytosis nearly doubled. This result suggests that Rac1 is not required during FcR-mediated trogocytosis. Inhibition of RhoA and Arp 2/3 yielded preliminary results, but these experiments are currently inconclusive and pending additional replicates. Characterizing the signaling pathway for macrophage trogocytosis will allow us to determine the point at which we can inhibit trogocytosis without inhibiting phagocytosis, allowing for more effective reduction of cancerous tumors in immunotherapies.