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 Tyr822 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 Tyr807, 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 Tyr292 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.
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Abbreviations
- BCR:
-
B cell receptor
- HD:
-
Healthy donor
- FcR:
-
Fc receptor
- ITIM:
-
Immunoreceptor tyrosine-based inhibition motif
- PBMC:
-
Peripheral blood mononuclear cells
- SLE:
-
Systemic lupus erythematosus
- Tyr:
-
Tyrosine
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Acknowledgments
The authors acknowledge Jennifer Timoshanko, PhD, UCB Pharma, UK, for publication coordination and Helen Chambers, DPhil, Costello Medical Consulting, UK, for editorial assistance, which was funded by UCB Pharma. The authors also thank Helen Brand, BSc, UCB Pharma, UK, for preparing epratuzumab F(ab’)2 and Fab batches.
Author contributions
SL designed the study, performed the experiments, analyzed the data, wrote the manuscript and was involved in the interpretation of the data. SJF and AW designed the study, performed the experiments, analyzed the data and were involved in the interpretation of the data. AS designed the study, analyzed the data, wrote the manuscript and was involved in the interpretation of the data. AM, CD and TD designed the study, analyzed the data and were involved in the interpretation of the data. All authors critically reviewed the manuscript for important intellectual content and approved the final version.
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Lumb, S., Fleischer, S.J., Wiedemann, A. et al. Engagement of CD22 on B cells with the monoclonal antibody epratuzumab stimulates the phosphorylation of upstream inhibitory signals of the B cell receptor. J. Cell Commun. Signal. 10, 143–151 (2016). https://doi.org/10.1007/s12079-016-0322-1
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DOI: https://doi.org/10.1007/s12079-016-0322-1