title: Engagement of CD22 on B cells with the monoclonal antibody epratuzumab stimulates the phosphorylation of upstream inhibitory signals of the B cell receptor.
abstract: The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events that ultimately drive B cell activation. Uncontrolled B cell activation is regulated by negative feedback loops that involve inhibitory co-receptors such as CD22 and CD32B that exert their functions following phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). The CD22-targeted antibody epratuzumab has previously been shown to inhibit BCR-driven signalling events, but its effects on ITIM phosphorylation of CD22 and CD32B have not been properly evaluated. The present study therefore employed both immunoprecipitation and flow cytometry approaches to elucidate the effects of epratuzumab on direct phosphorylation of key tyrosine (Tyr) residues on both these proteins, using both transformed B cell lines and primary human B cells. Epratuzumab induced the phosphorylation of Tyr(822) on CD22 and enhanced its co-localisation with SHP-1. Additionally, in spite of high basal phosphorylation of other key ITIMs on CD22, in primary human B cells epratuzumab also enhanced phosphorylation of Tyr(807), a residue involved in the recruitment of Grb2. Such initiation events could explain the effects of epratuzumab on downstream signalling in B cells. Finally, we were able to demonstrate that epratuzumab stimulated the phosphorylation of Tyr(292) on the low affinity inhibitory Fc receptor CD32B which would further attenuate BCR-induced signalling. Together, these data demonstrate that engagement of CD22 with epratuzumab leads to the direct phosphorylation of key upstream inhibitory receptors of BCR signalling and may help to explain how this antibody modulates B cell function.
authors.affiliation: UCB Pharma, 216 Bath Road, Slough, Berkshire, SL1 4EN, UK. Simon.Lumb@ucb.com.
authors.forename: Simon
authors.initials: S
authors.lastname: Lumb
authors.affiliation: CC12, Department Medicine/Rheumatology and Clinical Immunology and German Rheumatism Research Center Berlin (DRFZ), a Leibniz Institute, Charité University Medicine Berlin, Berlin, Germany.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Fleischer
authors.affiliation: CC12, Department Medicine/Rheumatology and Clinical Immunology and German Rheumatism Research Center Berlin (DRFZ), a Leibniz Institute, Charité University Medicine Berlin, Berlin, Germany.
authors.forename: Annika
authors.initials: A
authors.lastname: Wiedemann
authors.affiliation: CC12, Department Medicine/Rheumatology and Clinical Immunology and German Rheumatism Research Center Berlin (DRFZ), a Leibniz Institute, Charité University Medicine Berlin, Berlin, Germany.|, 47 rue Forestou Huella, 29200, Brest, France.
authors.forename: Capucine
authors.initials: C
authors.lastname: Daridon
authors.affiliation: UCB Pharma, 216 Bath Road, Slough, Berkshire, SL1 4EN, UK.
authors.forename: Alison
authors.initials: A
authors.lastname: Maloney
authors.affiliation: UCB Pharma, 216 Bath Road, Slough, Berkshire, SL1 4EN, UK.
authors.forename: Anthony
authors.initials: A
authors.lastname: Shock
authors.affiliation: CC12, Department Medicine/Rheumatology and Clinical Immunology and German Rheumatism Research Center Berlin (DRFZ), a Leibniz Institute, Charité University Medicine Berlin, Berlin, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Dörner
keywords: B cell receptor; CD22; CD32B; Epratuzumab; Phosphorylation