Background CD22, a transmembrane protein exclusively expressed on B cells, mediates migration by cell–cell interaction and negatively regulates B-cell receptor (BCR) signaling. CD22 acts as an inhibitory co-receptor of the BCR via de-phosphorylation of signaling molecules such as spleen tyrosine kinase (Syk) and subsequent phospholipase C (PLC)-γ2-triggered Ca2+ fluxes. The humanized anti-CD22 monoclonal antibody epratuzumab modulates adhesion molecule expression and B-cell migration in vitro. However, the potential of CD22 to regulate BCR signaling has not been fully delineated.
Objectives As toll-like receptor (TLR)-dependent activation of B cells represents an important pathway of B-cell hyperactivity in autoimmunity, our studies addressed whether epratuzumab might be able to modulate BCR signaling pathways in TLR9 pre-activated B cells or whether TLR9 activation abrogates the inhibitory effect of epratuzumab on the BCR signaling.
Methods Our study focused on the influence of epratuzumab on the B cells’ response to BCR alone or in combination with TLR9. The recruitment of CD22 to the BCR (CD79α) after epratuzumab incubation on B cells from healthy volunteers was analyzed by confocal microscopy. The in vitro effects of epratuzumab on BCR-induced signaling were evaluated by analyzing the phosphorylation status of BCR-signaling molecules, Syk and PLC-γ2 by flow cytometry with or without TLR9 pre-activation. The concentration of intracellular Ca2+ was monitored by flow cytometry after BCR stimulation. Finally, to evaluate the origin of the Ca2+ flux, extracellular Ca2+ was chelated with 1 mM ethylene glycol tetraacetic acid (EGTA).
Results B cells incubated with epratuzumab showed a specific co-localization of CD22 to the BCR-associated molecule CD79α on B cells. In addition, pre-treatment with epratuzumab led to a reduction in the MFI of phosphorylated Syk and PLC-γ2 induced by BCR stimulation compared with IgG1 isotype control. Similar results were observed when the cells were pre-incubated with F(ab’)2 fragment of epratuzumab, which excludes an inhibitory effect dependent on FcR signaling. Interestingly, the reduction of BCR induced kinase phosphorylation was demonstrated in both CD27- and CD27+ memory B cells. In addition, pre-activation of B cells by TLR9 did not circumvent the influence of epratuzumab on BCR signaling, as shown by a reduction in the MFI of phosphorylated Syk and PLC-γ2 in comparison to non-activated cells. Finally, a F(ab’)2 fragment of epratuzumab reduced BCR-induced calcium mobilization.
Conclusions Intracellular BCR signals can be modulated by CD22 ligation using epratuzumab, and pre-activation with TLR9 did not circumvent this effect. These data expand the potential mechanisms of action of epratuzumab.
Disclosure of Interest N. Sieger Grant/research support from: UCB Pharma, S. Fleischer Grant/research support from: UCB Pharma, K. Reiter Grant/research support from: UCB Pharma, H. Mei Grant/research support from: UCB Pharma, A. Shock Employee of: UCB Pharma, G. Burmester Grant/research support from: UCB Pharma, C. Daridon Grant/research support from: UCB Pharma, T. Dörner Grant/research support from: UCB Pharma, Consultant for: UCB Pharma
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