Dynamics of Macrophage Trogocytosis of Rituximab-Coated B Cells

Abstract

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.

Nanoscale Imaging and Mechanical Analysis of Fc Receptor-Mediated Macrophage Phagocytosis against Cancer Cells

“…In future studies, we can improve the experiments by using a temperature-controlled stage 14 that will allow us to investigate cell behavior better during cellular biological functions. Besides, in antibody-dependent macrophage phagocytosis, researchers have found that trogocytosis (trogocytosis is the fast uptake of membranes and associated molecules from one cell by another 24 ) can occur simultaneously, 25 and this may also influence phagocytosis.…”

“…When the rituximab-coated cancer cells and macrophages were coincubated, cancer cells could come into contact with adherent macrophages simply by landing on or near them under the influence of gravity. 25 After cancer cells came into contact with macrophages, the Fc domains of rituximab on the cancer cells then bound to the Fc receptors on the surface of macrophages, and this activated macrophage phagocytosis. The ability of eukaryotic cells to establish their asymmetrical shapes, to transport intracellular constituents, and to drive their motility depends on the cytoskeleton, 51 an interconnected network of filamentous polymers and regulatory proteins.…”

Imaging and measuring the biophysical properties of Fc gamma receptors on single macrophages using atomic force microscopy

“…Binding of FccRs to the Fc portion of the antibody triggers effector functions that are important in the antibody-based immunotherapy of cancer [2]. RAW264.7 cell is a mouse macrophage cell line that has been used extensively for studying FccR-mediated phagocytosis [29]. Here we used RAW 264.7 cells for the SMFS experiments.…”

Clinical severity in adult warm autoimmune hemolytic anemia and its relationship to antibody specificity

“…11, 12 The testing of phagocytic reactivity of monocytes in vitro with Ab-coated RBCs is a useful parameter for evaluating in vivo immune RBC destruction in the diagnosis of AIHA or evaluation of the patient response to treatment. 13 For this test, normal adherent monocytes were overlaid onto RBCs, sensitized, or not, with eluted autoAbs and incubated for 2 hours at 37°C.…”

“…This assay is thus more sensitive than the phagocytosis assay with monocytes, but less stringent. 4,11,12 Although the trogocytosis assay may have some limitations in assessing functional properties of Abs, it has been reported to predict the clinical consequences of immune blood cell destruction in vivo. 5 We confirmed significant membrane transfer between non-sensitized donor RBCs and monocytes incubated together for 60 min (MFI of 2,311 for non-sensitized RBCs at T60 vs. MFI of 56 for T0, Figure 3A).…”