title: Activatory and inhibitory Fcγ receptors augment rituximab-mediated internalization of CD20 independent of signaling via the cytoplasmic domain.
abstract: Type I anti-CD20 mAb such as rituximab and ofatumumab engage with the inhibitory FcγR, FcγRIIb on the surface of B cells, resulting in immunoreceptor tyrosine-based inhibitory motif (ITIM) phosphorylation. Internalization of the CD20·mAb·FcγRIIb complex follows, the rate of which correlates with FcγRIIb expression. In contrast, although type II anti-CD20 mAb such as tositumomab and obinutuzumab also interact with and activate FcγRIIb, this interaction fails to augment the rate of CD20·mAb internalization, raising the question of whether ITIM phosphorylation plays any role in this process. We have assessed the molecular requirements for the internalization process and demonstrate that in contrast to internalization of IgG immune complexes, FcγRIIb-augmented internalization of rituximab-ligated CD20 occurs independently of the FcγRIIb ITIM, indicating that signaling downstream of FcγRIIb is not required. In transfected cells, activatory FcγRI, FcγRIIa, and FcγRIIIa augmented internalization of rituximab-ligated CD20 in a similar manner. However, FcγRIIa mediated a slower rate of internalization than cells expressing equivalent levels of the highly homologous FcγRIIb. The difference was maintained in cells expressing FcγRIIa and FcγRIIb lacking cytoplasmic domains and in which the transmembrane domains had been exchanged. This difference may be due to increased degradation of FcγRIIa, which traffics to lysosomes independently of rituximab. We conclude that the cytoplasmic domain of FcγR is not required for promoting internalization of rituximab-ligated CD20. Instead, we propose that FcγR provides a structural role in augmenting endocytosis that differs from that employed during the endocytosis of immune complexes.
authors.affiliation: From the Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton, Faculty of Medicine, General Hospital, Southampton SO16 6YD, United Kingdom and.
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Vaughan
authors.affiliation: From the Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton, Faculty of Medicine, General Hospital, Southampton SO16 6YD, United Kingdom and.
authors.forename: Claude H T
authors.initials: CH
authors.lastname: Chan
authors.affiliation: the Roche Pharmaceutical Research and Early Development, Roche Innovation Center Zurich, CH-8952 Schlieren, Switzerland.
authors.forename: Christian
authors.initials: C
authors.lastname: Klein
authors.affiliation: From the Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton, Faculty of Medicine, General Hospital, Southampton SO16 6YD, United Kingdom and.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Glennie
authors.affiliation: From the Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton, Faculty of Medicine, General Hospital, Southampton SO16 6YD, United Kingdom and.
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Beers
authors.affiliation: From the Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton, Faculty of Medicine, General Hospital, Southampton SO16 6YD, United Kingdom and msc@soton.ac.uk.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Cragg
mesh_terms: D058846:Antibodies, Monoclonal, Murine-Derived / Q000378:metabolism*; D018951:Antigens, CD20 / Q000378:metabolism*; D019921:Biotinylation; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004705:Endocytosis; D006801:Humans; D018613:Microscopy, Confocal; D009154:Mutation; D017452:Receptors, IgG / Q000235:genetics / Q000378:metabolism*; D000069283:Rituximab; D015398:Signal Transduction
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; C499173:FCGR2B protein, human; C080568:Fc gamma receptor IIA; D017452:Receptors, IgG; D000069283:Rituximab
keywords: Antibody; B Cell; CD20; Cell Signaling; Fc-gamma Receptor; Lipid Raft; Lymphoma; Receptor Endocytosis