Measurement of Trogocytosis: Quantitative Analyses Validated with Rigorous Controls

Abstract

AbstractTrogocytosis is a process in which receptors on acceptor cells remove and internalize cognate ligands from donor cells. Trogocytosis has a profound and negative impact on mAb‐based cancer immunotherapy, as seen in the treatment of chronic lymphocytic leukemia (CLL) with CD20 mAbs, such as rituximab (RTX) and ofatumumab (OFA). Our clinical observations of RTX/OFA‐mediated loss of the CD20 target from circulating CLL cells have been replicated in our in vitro studies. Here we describe flow cytometry and fluorescence microscopy experiments, which demonstrate that acceptor cells, such as monocytes/macrophages that express FcγR, remove and internalize both antigen and donor cell‐bound cognate IgG mAbs for several different mAb‐donor cell pairs. Fluorescent mAbs and portions of the plasma cell membrane are transferred from donor cells to acceptor cells, which include the THP‐1 monocytic cell line as well as freshly isolated monocytes. We describe rigorous controls to validate the reactions and eliminate dissociation or internalization as alternative mechanisms. Trogocytosis is likely to contribute to neutropenia, thrombocytopenia, and liver damage associated with use of antibody‐drug conjugates. The methods we have described should allow for examination of strategies focused on blocking trogocytosis and its adverse effects. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Trogocytosis of mAb‐opsonized donor cells mediated by adherent THP‐1 cellsAlternate Protocol: Application of fluorescence microscopy to examine THP‐1 cell‐mediated trogocytosisSupport Protocol 1: Alexa labeling of mAbs and determination of F/P ratiosSupport Protocol 2: Standard washing procedureSupport Protocol 3: Labeling and opsonization of cellsBasic Protocol 2: Trogocytosis mediated by human monocytes as acceptor cellsSupport Protocol 4: Isolation of human monocytesBasic Protocol 3: Trogocytosis mediated by THP‐1 cells in solutionSupport Protocol 5: Retinoic acid treatment of THP‐1 cellsSupport Protocol 6: Culturing of SCC‐25, BT‐474, MOLT‐4 and THP‐1 cell lines