Sialic Acid Ligand Binding of CD22 and Siglec-G Determines Distinct B Cell Functions but Is Dispensable for B Cell Tolerance Induction.

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. Ca2+ 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.

Epratuzumab modulates B-cell signaling without affecting B-cell numbers or B-cell functions in a mouse model with humanized CD22.

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.

The ligand-binding domain of Siglec-G is crucial for its selective inhibitory function on B1 cells.

Siglec-G is an inhibitory receptor on B1 cells. Siglec-G-deficient mice show a large B1 cell expansion, owing to higher BCR-induced Ca(2+) 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 Ca(2+) 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.