Siglec-G is an inhibitory receptor on B1 cells. Siglec-G–deficient mice show a large B1 cell expansion, owing to higher BCR-induced Ca2+ signaling and enhanced cellular survival. It was unknown why Siglec-G shows a B1 cell–restricted inhibitory function. With a new mAb we could show a comparable Siglec-G expression on B1 cells and conventional B2 cells. However, Siglec-G has a different ligand sialic acid–binding pattern on peritoneal B1 cells than on splenic B cells, and its sialic acid ligands are expressed differentially on these two B cell populations, suggesting that cis-ligand binding plays a crucial role on B1 cells. This observation was further studied by generation of Siglec-G knockin mice with a mutated ligand-binding domain. These mice show increased B1 cell numbers, increased B1 cell Ca2+ signaling, better B1 cell survival, and changes in the B1 cell Ig repertoire. These phenotypes are very similar to Siglec-G–deficient mice. The mutation of the ligand-binding domain of Siglec-G strongly reduces the Siglec-G–IgM association on the B cell surface. Thus, Siglec-G sialic acid–dependent binding to the BCR is crucial for the B1 cell–restricted inhibitory function of Siglec-G and is regulated in an opposite way to that of the related protein CD22 (Siglec-2) on B cells.

Siglec-G and CD22 are inhibitory receptors on B cells and play an important role in the maintenance of tolerance. Although both molecules are expressed on all B cell populations at a similar level, Siglec-G was found to regulate exclusively B1a cells, whereas CD22 functions as an inhibitory receptor specifically on B2 cells. It is known that the mechanistic function of both Siglecs is regulated by sialic acid binding in a reciprocal manner, although it was not known until now how B cells would act when both Siglec-G and CD22 lack their ability to bind sialic acids. We answered this question by analyzing Siglec-G R120E x CD22 R130E mice. These mice show decreased numbers of mature recirculating B cells in the bone marrow similar to mice with mutations in CD22. Also, they show an increased B1a cell population in peritoneal cavity and a skewed BCR repertoire in peritoneal B1a cells, which is characteristic for mice with mutated Siglec-G. Ca mobilization was strongly reduced in B2 cells and was altered in peritoneal B1a cells, whereas B cell survival was neither affected in B2 cells nor in B1a cells. Also, aging Siglec-G R120E x CD22 R130E mice do neither develop a general hyperactivated immune status nor autoimmunity. This demonstrates that Siglec binding to sialic acids as abundant self-ligands cannot be a dominant mechanism for the Siglec-mediated B cell tolerance induction.

Treatment of systemic lupus erythematosus patients with epratuzumab (Emab), a humanized monoclonal antibody targeting CD22, leads to moderately reduced B-cell numbers but does not completely deplete B cells. Emab appears to induce immunomodulation of B cells, but the exact mode of action has not been defined. In the present study, we aimed to understand the effects of Emab on B cells using a humanized mouse model (Huki CD22), in which the B cells express human instead of murine CD22. Emab administration to Huki CD22 mice results in rapid and long-lasting CD22 internalization. There was no influence on B-cell turnover, but B-cell apoptosis ex vivo was increased. Emab administration to Huki CD22 mice had no effect on B-cell numbers in several lymphatic organs, nor in blood. In vitro exposure of B cells from Huki CD22 mice to Emab resulted in decreased B-cell receptor (BCR) induced Ca 2+ mobilization, whereas B-cell proliferation after Toll-like receptor (TLR) stimulation was not affected. In addition, IL-10 production was slightly increased after TLR and anti-CD40 stimulation, whereas IL-6 production was unchanged. In conclusion, Emab appears to inhibit BCR signaling in a CD22-dependent fashion without strong influence on B-cell development and B-cell populations.Keywords: Antibody therapy r B-cell differentiation r B-cell signaling r Systemic lupus erythematosus Additional supporting information may be found in the online version of this article at the publisher's web-site

Background Epratuzumab is a humanized monoclonal antibody that targets the B cell-specific protein CD22 and is currently in Phase 3 clinical trials in patients (pts) with systemic lupus erythematosus (SLE). Epratuzumab does not deplete B cells and appears to act in an immunomodulatory fashion, eg. by inhibiting activation through the B cell receptor (BCR).1 It has also been shown to modulate adhesion molecule expression on B cells and their responsiveness to chemokines.2 Objectives The present study aimed to understand the effect of epratuzumab on B cells in vivo using human CD22 knock-in (Huki) mice, in which the B cells express the human CD22 gene instead of the murine gene.3 Methods Huki mice received a single intravenous injection (0.5mg) of epratuzumab and, at various time points up to Week (Wk) 12, B cell sub-populations in blood, spleen, bone marrow and lymph nodes were measured, along with B cell activation and homing markers. Ex vivo functional assays were also performed: Ca+ flux, apoptosis and CD22 internalization on B cells were measured by flow cytometry and the proliferation of B cells was assessed after in vivo administration of BrdU. Results Epratuzumab-treated mice showed rapid and long-lasting human CD22 internalization on B cells from all organs up to Wk8 and an increase of B cell apoptosis (an increase of cells in sub G1 phase) ex vivo. Furthermore, decreased BCR-activated Ca+-flux was demonstrated after epratuzumab treatment in vitro. However, BrdU incorporation in several B cell subsets was unchanged at early time points and there were no consistent effects demonstrated on the numbers or proportions of such subsets in various organs at any time point up to Wk12. Conclusions Epratuzumab treatment of Huki mice induces functional effects on B cells assessed ex vivo but does not seem to strongly influence B cell development and B cell populations in various organs. These data have implications for understanding the effects of epratuzumab treatment on B cell function in SLE pts. References Sieger N. Arth Rheum 2013;65:770. Daridon C. Arth Res Ther 2010;12:R204. Wöhner M. Eur J Immunol 2012;42:3009. Acknowledgements C. Brandl and L. Özgör contributed equally to the work. The authors acknowledge Costello Medical Consulting for editorial assistance which was funded by UCB Pharma. Disclosure of Interest C. Brandl Grant/research support: UCB Pharma, L. Özgör Grant/research support: UCB Pharma, M. Wöhner: None declared, A. Shock Employee of: UCB Pharma, L. Nitschke Grant/research support: UCB Pharma DOI 10.1136/annrheumdis-2014-eular.1751

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