Macrophages can remove antigen from the surface of antibody-coated cells by a process termed trogocytosis. Using live cell microscopy and flow cytometry, we investigated the dynamics of trogocytosis by RAW264.7 macrophages of Ramos B cells opsonized with the anti-CD20 monoclonal antibody rituximab. Spontaneous and reversible formation of uropods was observed on Ramos cells, and these showed a strong enrichment in rituximab binding. RAW-Ramos conjugate interfaces were highly enriched in rituximab, and transfer of rituximab to the RAW cells in submicron-sized puncta occurred shortly after cell contact. Membrane from the target cells was concomitantly transferred along with rituximab to a variable extent. We established a flow cytometry-based approach to follow the kinetics of transfer and internalization of rituximab. Disruption of actin polymerization nearly eliminated transfer, while blocking phosphatidylinositol 3-kinase activity only resulted in a delay in its acquisition. Inhibition of Src family kinase activity both slowed acquisition and reduced the extent of trogocytosis. The effects of inhibiting these kinases are likely due to their role in efficient formation of cell-cell conjugates. Selective pre-treatment of Ramos cells with phenylarsine oxide blocked uropod formation, reduced enrichment of rituximab at cell-cell interfaces, and reduced the efficiency of trogocytic transfer of rituximab. Our findings highlight that dynamic changes in target cell shape and surface distribution of antigen may significantly influence the progression and extent of trogocytosis. Understanding the mechanistic determinants of macrophage trogocytosis will be important for optimal design of antibody therapies.

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Human myeloid cells express both activating and inhibitory receptors of the FcγRII family. FcγRIIA mediates processes associated with cell activation, including phagocytosis of IgG-opsonized particles, whereas coengagement of the inhibitory FcγRIIB downregulates such signaling. We analyzed the relative recruitment of these two receptors during phagocytosis of IgG-coated particles by ts20 Chinese hamster fibroblast cells cotransfected with both receptors carrying distinguishable fluorescent protein tags. We found that FcγRIIA is substantially enriched at sites of particle binding relative to its inhibitory counterpart, with a greater than 2-fold increase in the local ratio of activating to inhibitory receptor compared with that for the plasma membrane as a whole. Experiments with chimeric receptors revealed that the preferential enrichment of FcγRIIA results from differences between the extracellular domains of the receptors, and indicated that the lesser recruitment of FcγRIIB limits its ability to effectively inhibit FcγRIIA-mediated phagocytosis. Mutagenesis studies indicated that FcγRIIA residues leucine 132 and phenylalanine 160, which lie in IgG-binding regions of FcγRIIA and which differ in FcγRIIB, both contribute to the local relative enrichment of FcγRIIA by increasing its affinity for IgG1 relative to that of FcγRIIB. In human monocytes, engagement of approximately equal amounts of FcγRIIB was required to substantially inhibit FcγRIIA-mediated phagocytosis. These results demonstrate that differences in affinity for IgG between activating and inhibitory FcγR can result in substantial local changes in their relative concentrations during phagocytosis, with important functional consequences.