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Activatory and inhibitory Fcy receptors augment rituximab-mediated internalization of CD20 independent of signaling via the cytoplasmic domain

Activatory and inhibitory Fcy receptors augment rituximab-mediated internalization of CD20 independent of signaling via the cytoplasmic domain
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.
0021-9258
5424-5437
Vaughan, A.T.
bfb2ceab-a592-457e-89f9-00fcd1dddbdb
Chan, C.H.T.
b109c93f-7e9a-44ee-ad12-da757b1b11fc
Klein, C.
4546582b-512e-4725-b2ca-c160b1a967cd
Glennie, M.J.
9f6f0eff-4560-48c2-80cd-0ec116110ded
Beers, S.A.
a02548be-3ffd-41ab-9db8-d6e8c3b499a2
Cragg, M.
ec97f80e-f3c8-49b7-a960-20dff648b78c
Vaughan, A.T.
bfb2ceab-a592-457e-89f9-00fcd1dddbdb
Chan, C.H.T.
b109c93f-7e9a-44ee-ad12-da757b1b11fc
Klein, C.
4546582b-512e-4725-b2ca-c160b1a967cd
Glennie, M.J.
9f6f0eff-4560-48c2-80cd-0ec116110ded
Beers, S.A.
a02548be-3ffd-41ab-9db8-d6e8c3b499a2
Cragg, M.
ec97f80e-f3c8-49b7-a960-20dff648b78c

Vaughan, A.T., Chan, C.H.T., Klein, C., Glennie, M.J., Beers, S.A. and Cragg, M. (2015) Activatory and inhibitory Fcy receptors augment rituximab-mediated internalization of CD20 independent of signaling via the cytoplasmic domain. The Journal of Biological Chemistry, 290, 5424-5437. (doi:10.1074/jbc.M114.593806). (PMID:25568316)

Record type: Article

Abstract

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.

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More information

Accepted/In Press date: 24 December 2014
e-pub ahead of print date: 7 January 2015
Published date: 27 February 2015
Organisations: Cancer Sciences

Identifiers

Local EPrints ID: 390377
URI: http://eprints.soton.ac.uk/id/eprint/390377
ISSN: 0021-9258
PURE UUID: a6645acc-3b78-45c9-8659-a1a62a12a2a3
ORCID for A.T. Vaughan: ORCID iD orcid.org/0000-0001-6076-3649
ORCID for C.H.T. Chan: ORCID iD orcid.org/0000-0003-0530-9480
ORCID for S.A. Beers: ORCID iD orcid.org/0000-0002-3765-3342
ORCID for M. Cragg: ORCID iD orcid.org/0000-0003-2077-089X

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Date deposited: 01 Apr 2016 07:58
Last modified: 15 Mar 2024 03:08

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Contributors

Author: A.T. Vaughan ORCID iD
Author: C.H.T. Chan ORCID iD
Author: C. Klein
Author: M.J. Glennie
Author: S.A. Beers ORCID iD
Author: M. Cragg ORCID iD

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