Session Title: B-cell Biology and Targets in Autoimmune Disease
Session Type: Abstract Submissions (ACR)
CD22, a member of the sialic acid-binding immunoglobulin-like lectins (Siglec) family, is exclusively expressed on B cells at mature stage and is lost upon plasma cells differentiation. CD22 mediates migration by modulating cell-cell interaction and negatively regulates B-cell receptor (BCR) signaling. By recruiting a tyrosine phosphatase to its intracellular tail, 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 ligand of CD22 is α-2,6-sialic acid residues present on many proteins, including CD22. Sialo-interactions appear to be crucial for optimal CD22 function and can occur in cis (to ligands on the same cell surface) or trans (to ligands on neighboring cells). The humanized anti-CD22 monoclonal antibody epratuzumab, currently in phase III clinical trials in SLE, modulates adhesion molecule expression and B cell migration in vitro; however, the potential of CD22-ligation with sialic acid to regulate BCR signaling has not been delineated.
To investigate the impact of epratuzumab on the B cells response, the recruitment of CD22 to the BCR after epratuzumab incubation on peripheral blood B cells from healthy volunteers was studied by confocal microscopy. The In vitro effects of epratuzumab on BCR-induced signaling was evaluated by analyzing the phosphorylation status of the BCR-signaling molecules Syk and PLC-g2 by flow cytometry. To assess the importance of sialo-interactions on epratuzumab-induced effects, peripheral blood mononuclear cells were treated with neuraminidase (to remove sialic acid) and BCR-induced phosphorylation (Syk and PLC-γ2) was monitored with or without epratuzumab incubation. Finally, to monitor B cell activation after epratuzumab incubation, the concentration of intracellular Ca2+ was monitored by flow cytometry after BCR stimulation.
We have shown by confocal microscopy that incubation with epratuzumab or a F(ab’)2 fragment of epratuzumab specifically induced the recruitment of CD22 to the CD79a molecule associated to the BCR on B cells. When B cells are pre-treated with epratuzumab, we observed a reduction of the phosphorylated Syk and PLC-γ2 induced by BCR stimulation. This reduction was observed in the same manner when the cells were pre-incubated with F(ab’)2 fragment of epratuzumab which excludes an inhibitory effect dependent on FcR signaling. The reduction of BCR-induced kinase phosphorylation was demonstrated in both CD27– naïve and CD27+ memory B cells. Interestingly, preventing sialo-interactions of CD22 partially reduced the inhibitory effect of epratuzumab on Syk and PLC-γ2 phosphorylation. In addition, a F(ab’)2fragment of epratuzumab reduced the BCR-induced calcium flux.
Taking these data together, these data show that targeting CD22 with epratuzumab potentially raises the threshold for BCR activation and therefore would provide additional control of B cell function. Furthermore, the analysis of CD22 ligation by epratuzumab would provide further understanding of CD22 biology in human autoimmune diseases.
Funding: Sonderforschungsbereich 650 and DFG491/7-1
S. J. Fleischer,
H. E. Mei,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/targeting-of-cd22-by-epratuzumab-potentially-raises-the-threshold-of-b-cell-receptor-activation/