Possible Implication of Fcγ Receptor-Mediated Trogocytosis in Susceptibility to Systemic Autoimmune Disease

Leukocytes can “gnaw away” the plasma membrane of other cells. This phenomenon, called trogocytosis, occurs subsequent to cell-to-cell adhesion. Currently, two mechanisms of trogocytosis, adhesion molecule-mediated trogocytosis and Fcγ receptor-(FcγR-) mediated trogocytosis, have been identified. In our earlier study, we established an in vitro model of FcγR-mediated trogocytosis, namely, CD8 translocation model from T cells to neutrophils. By using this model, we demonstrated that the molecules transferred to neutrophils via FcγR-mediated trogocytosis were taken into the cytoplasm immediately. This result suggests that the chance of molecules transferred via FcγR-mediated trogocytosis to play a role on the cell surface could be time-limited. Thus, we consider the physiological role of FcγR-mediated trogocytosis as a means to remove antibodies (Abs) that bind with self-molecules rather than to extract molecules from other cells. This concept means that FcγR-mediated trogocytosis can be a defense mechanism to Ab-mediated autoimmune response. Moreover, the activity of FcγR-mediated trogocytosis was revealed to be parallel to the endocytotic activity of neutrophils, which was critically related to the susceptibility to systemic autoimmune diseases. The collective findings suggest that FcγR-mediated trogocytosis could physiologically play a role in removal of Abs bound to self-antigens and prevent autoimmune diseases.


Introduction
Trogocytosis is the exchange of plasma membrane fragments between immune cells which form a conjugate [1,2]. The phenomenon was first described on CD8 + T cells [3]. The CD8 + T cells can take plasma membrane fragments of antigenpresenting cells (APCs) via T-cell receptor (TCR) and antigen (Ag)/class I major histocompatibility complex (MHC), when these cells form an immunological synapse. To date, it has been shown that not only CD8 + T cells but also other immune cells, including CD4 + T cells [4], T cells [5], B cells [6], natural killer cells [7], dendritic cells [8], monocytes [9], and macrophages [10], have potential for trogocytosis. These cells are able to accept plasma membrane fragments of other cells after recognition of cell surface Ags by the specific receptors.
This mechanism is called adhesion molecule-mediated trogocytosis.
Recently, another mechanism of trogocytosis mediated by Ag/antibody (Ab) immune complex and Fc receptor (Fc R), namely, Fc R-mediated trogocytosis, is advocated [11][12][13]. The major difference of Fc R-mediated trogocytosis from adhesion molecule-mediated trogocytosis is the intervention of Ab. This phenomenon is well characterized, wherein CD20 molecules on malignant B cells are lost after infusion of the humanized anti-CD20 monoclonal Ab, rituximab [14][15][16]. In this situation, Fc R + immune cells capture the plasma membrane fragments of B cells via the CD20/anti-CD20 immune complexes and Fc Rs. Similar phenomenon is seen when CD22 [17] and CD25 [18] are targeted by therapeutic Abs.  Figure 1: Schemas of CD8 translocation model from T cells to neutrophils CD8 molecules on T cells were transferred to neutrophils when these cells were incubated with anti-CD8 Ab in the serum. TCR and CD3 molecules on T cells were also transferred to neutrophils. Fc Rs (Fc RII and Fc RIII) were involved in the mechanism.
The physiological roles of trogocytosis have been discussed concerning T cells that acquired the Ag/MHC from the APCs in particular [24,25]. The acceptor CD8 + T cells for the Ag/MHC are prone to be killed by the fratricide mechanism. In another way, the acquisition of HLA-G1 from APCs via trogocytosis could change the T-cell phenotype from effector to regulatory phenotype [26,27]. It has been also demonstrated that CD4 CD8 double negative regulatory T cells, which accept the Ag/MHC from APCs via trogocytosis, can function as cytotoxic cells toward the Ag-specific CD8 + T cells [28]. These could be related to the convergence or suppression of the immune response. On the contrary, it is indicated that the T cells with the Ag/MHC can possibly function as new APCs resulting in the progression of the immune response. These controversial properties of T cells through adhesion molecule-mediated trogocytosis could be displayed in time and environment-dependent manners. In addition, recent studies have demonstrated that adhesion moleculemediated trogocytosis could be related to the exercise of naturally occurring FOXP3 + regulatory T cells [29]. However, the physiological role of Fc R-mediated trogocytosis remains unrevealed. In this perspective, the role of Fc R-mediated trogocytosis in the physiological immune system is discussed according to the current data.

Establishment of In Vitro Model of Fc R-Mediated Trogocytosis
Daubeuf et al. had established a simple method to detect adhesion molecule-mediated trogocytosis by flow cytometry (FCM) [30]. On the other hand, we earlier established an in vitro model of Fc R-mediated trogocytosis [23]. The protocol was simple and easy as follows: (1) [19,31]. Moreover, bystander molecules, such as TCR and CD3, were also transferred from T cells to neutrophils accompanied by CD8 molecules. Thus, this phenomenon was considered as Fc R-mediated trogocytosis ( Figure 1). The polymerization of actin was involved in the process of neutrophil Fc R-mediated trogocytosis similar to T cells [32]. By using this model, we demonstrated that human anti-mouse IgG Abs in serum accelerated Fc R-mediated trogocytosis. This is consistent with the finding that Fc R-mediated trogocytosis is prone to occur in arthritic patients positive for rheumatoid factor, which is an anti-IgG Ab [33].

Association between Fc R-Mediated Trogocytosis and Endocytosis
Next, the association between Fc R-mediated trogocytosis and endocytosis was examined using the CD8 translocation model from T cells to neutrophils. Endocytosis is a fundamental function of neutrophils. Two diverse mechanisms of endocytosis include phagocytosis and pinocytosis, wherein the difference between the two is the size of target molecules. Pinocytosis is used for the absorption of extracellular fluids, and in contrast to phagocytosis, the target molecules are very small. To determine the association between Fc R-mediated trogocytosis and endocytosis, experiments were performed using yellow-green carboxylate-modified latex beads and FITC-labeled ovalbumin (OVA). The process of intake of latex bead and OVA represents phagocytosis and pinocytosis, respectively. After enhancing Fc R-mediated trogocytosis by the CD8 translocation model from T cells to neutrophils, fluorescence-labeled latex beads and OVA were added to the cells. Subsequently, the engulfment of these molecules by CD8 + neutrophils that underwent Fc R-mediated trogocytosis was examined, and the data were compared with those by CD8 − neutrophils that did not perform Fc R-mediated trogocytosis. The activities of both phagocytosis and pinocytosis were higher in CD8 + neutrophils than those in CD8 − neutrophils ( Figure 3). These findings suggest that the activity of Fc R-mediated trogocytosis is parallel to the phagocytic and pinocytic activities of neutrophils.

Physiological Role of Fc R-Mediated Trogocytosis
It was revealed that the CD8 molecules transferred to neutrophils via Fc R-mediated trogocytosis were taken into the cytoplasm immediately. This evidence suggests that the chance of molecules transferred via Fc R-mediated trogocytosis to play a role on the cell surface could be time-limited. Thus, we consider the physiological role of Fc R-mediated trogocytosis as a means to remove Abs that bind with selfmolecules rather than to extract molecules from other cells. Essentially, a living body is always exposed to invading pathogens. The immune system produces Abs to compete with the pathogens; however, these Abs sometimes crossreact with self-Ags. In such situation, the presence of mechanism that can prevent Ab-mediated autoimmune response is beneficial. Fc R-mediated trogocytosis can be a defense mechanism to remove Abs, which unexpectedly bind to self-Ags ( Figure 4). In this study, the relationship between Fc R-mediated trogocytosis and endocytosis of neutrophils was examined using the established CD8 translocation model from T cells to neutrophils. Results clearly indicated that the activity of Fc R-mediated trogocytosis was closely linked to the endocytosis activity of neutrophils. The low activity of endocytosis of neutrophils is critically related to the high susceptibility to systemic autoimmune diseases including systemic lupus erythematosus [34]. Therefore, it is reasonable to consider that the low ability of Fc R-mediated trogocytosis could be also  Figure 3: Comparison of endocytotic activities between neutrophils with and without display of Fc R-medicated trogocytosis. Peripheral blood polymorphonuclear cells (0.5 × 10 6 ) and mononuclear cells (0.5 × 10 6 ) were mixed and preincubated in 100 L of the autologous serum for 20 min at 37 ∘ C. The cells were made to react with 0.1 g of PECy5-labeled anti-CD8 Ab for 1 hour at room temperature. After washing with PBS, the cells were resuspended in 1 mL of RPMI-1640 medium with 10% FBS. Subsequently, the cells were made to react with 2 L of yellow-green carboxylate-modified latex beads for 90 min at 37 ∘ C or with 100 g of FITC-labeled OVA for 30 min at 37 ∘ C. After washing 3 times with PBS, the cells were re-suspended in 100 L of PBS and then made to react with 0.1 g of PE-labeled anti-CD15 Ab, followed by serving for FCM. Experiments were repeated at least 3 times. Similar results were reproduced.

Neutrophil Antibody
Resistant to antibody-mediated autoimmune disease Fc R-mediated trogocytosis Fc RIII Fc RII

Self-antigen
Complement-dependent cytotoxicity Antibody-dependent cellular cytotoxicity Susceptible to antibody-mediated autoimmune disease Figure 4: Relation between Fc R-mediated trogocytosis and susceptibility to Ab-mediated autoimmune disease. Fc R-mediated trogocytosis can play a role in the removal of Abs that bind to self-Ags on the cell surface and prevent Ab-mediated autoimmune diseases.
linked to the high susceptibility to systemic autoimmune diseases. This is compatible with our concept that Fc Rmediated trogocytosis can play a role in the removal of autoantibodies and prevent autoimmune diseases. However, in autoimmune hemolytic anemia, trogocytosis mediated by anti-red blood cell (RBC) polymeric IgA Abs and Fc receptors is involved in the destruction of RBCs [35]. Thus, further prospective studies are needed to clarify if the ability of Fc R-mediated trogocytosis would be actually related to the susceptibility to systemic autoimmune diseases.