No abstract

We have developed a method exploiting the phenomenon of trogocytosis to detect lymphocytes reacting specifically with target cells by flow cytometry. Trogocytosis is a process by which lymphocytes capture fragments of the plasma membrane from the antigen-presenting cells (APCs) expressing their cognate antigen. For this method, a label (such as a fluorescent lipid or biotin) is first incorporated in the membrane of APCs. These labeled cells are then co-cultured for a few hours with a population of cells containing the lymphocytes to be detected. After this period of stimulation, lymphocytes that have performed trogocytosis are identified by their acquisition of the label initially present on the APC membrane using flow cytometry. A major advantage of this method is its compatibility with the simultaneous detection of phenotypic and/or functional markers on the lymphocytes. Furthermore, cells can be recovered alive and active after detection of trogocytosis, and are therefore available for further characterization or even conceivably for therapeutic purposes.

Exchange of plasma membrane fragments, including cell-surface proteins and lipids, in conjugates formed between lymphocytes and their cellular partners is a field of intense investigation. Apart from its natural occurrence during Ag recognition, the process of membrane transfer can be triggered in experimental or therapeutic settings when lymphocytes targeted by Abs are conjugated to FcγR-expressing accessory cells. The direction of membrane capture (i.e., which of the two cells is going to donate or accept plasma membrane fragments) can have important functional consequences, such as insensitivity of tumor cells to treatment by therapeutic mAbs. This effect, called antigenic modulation or shaving, occurs as a result of a process in which the FcγR-expressing cells remove the mAb and its target protein from the tumor cells. We therefore analyzed this process in conjugates formed between various FcγR-expressing cells and a series of normal or tumor T and B cells opsonized with different Abs capable of triggering membrane exchange (including the therapeutic Ab rituximab). Our results show that the direction of membrane capture is dictated by the identity of the FcγR-expressing cell, much more so than the type of lymphocyte or the Ab used. We found that monocytes and macrophages are prone to be involved in bidirectional trogocytosis with opsonized target cells, a process they can perform in parallel to phagocytosis. Our observations open new perspectives to understand the mechanisms involved in trogocytosis and may contribute to optimization of Ab-based immunotherapeutic approaches.