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