Activatory and Inhibitory Fcγ Receptors Augment Rituximab-mediated Internalization of CD20 Independent of Signaling via the Cytoplasmic Domain

Type I anti-CD20 mAb such as rituximab and ofatumumab engage with the inhibitory Fc gamma R, Fc gamma RIIb on the surface of B cells, resulting in immunoreceptor tyrosine-based inhibitory motif (ITIM) phosphorylation. Internalization of the CD20 center dot mAb center dot Fc gamma RIIb complex follows, the rate of which correlates with Fc gamma RIIb expression. In contrast, although type II anti-CD20 mAb such as tositumomab and obinutuzumab also interact with and activate Fc gamma RIIb, this interaction fails to augment the rate of CD20 center dot 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, Fc gamma RIIb-augmented internalization of rituximab-ligated CD20 occurs independently of the Fc gamma RIIb ITIM, indicating that signaling downstream of Fc gamma RIIb is not required. In transfected cells, activatory Fc gamma RI, Fc gamma RIIa, and Fc gamma RIIIa augmented internalization of rituximab-ligated CD20 in a similar manner. However, Fc gamma RIIa mediated a slower rate of internalization than cells expressing equivalent levels of the highly homologous Fc gamma RIIb. The difference was maintained in cells expressing Fc gamma RIIa and Fc gamma RIIb lacking cytoplasmic domains and in which the transmembrane domains had been exchanged. This difference may be due to increased degradation of Fc gamma RIIa, which traffics to lysosomes independently of rituximab. We conclude that the cytoplasmic domain of Fc gamma R is not required for promoting internalization of rituximab-ligated CD20. Instead, we propose that Fc gamma R provides a structural role in augmenting endocytosis that differs from that employed during the endocytosis of immune complexes.