CD22 is a B cell–specific transmembrane protein of the Siglec family. It binds specifically to α2,6-linked sialic acid (Sia) residues, which are also present on glycoproteins on the B cell surface. CD22 acts as a negative regulator in B cell receptor–mediated signaling by recruitment of Src homology 2 domain–containing tyrosine phosphatase (SHP)-1 to its intracellular tail. To analyze how ligand-binding of CD22 influences its intracellular signaling domain, we designed synthetic sialosides as inhibitors for the lectin domain of CD22. One of these compounds inhibited binding of human CD22-Fc to target cells over 200-fold better than Sia and was highly selective for human CD22. When Daudi cells or primary B cells were stimulated with anti-immunoglobulin (Ig)M in presence of this sialoside inhibitor, a higher Ca2+ response was observed, similar to CD22-deficient B cells. Accordingly, a lower tyrosine-phosphorylation of CD22 and SHP-1 recruitment was demonstrated in presence of the sialoside. Thus, by interfering with ligand binding of CD22 on the B cell surface, we have shown for the first time that the lectin domain of CD22 has a direct, positive influence on its intracellular inhibitory domain. Also, we have developed a novel low molecular weight compound which can enhance the response of human B cells.

CD22 is a B cell‐specific member of the immunoglobulin superfamily and binds to sialic acid. CD22 inhibits B cell receptor signaling. Mice deficient for CD22 show a largely normal B cell development. Here, we have performed a detailed analysis of the splenic B cell population and found that the subset of marginal zone (MZ) B cells was selectively reduced in CD22‐deficient mice. CD22‐deficient mice showed a lack of TNP‐ficoll capturing cells in the MZ and a reduced response to TNP‐ficoll, particularly when the antigen was applied intravenously. CD22‐deficient B cells showed both enhanced motility as well as enhanced chemotaxis to certain chemokines. The altered chemokine responsiveness or the higher signaling capacity of CD22‐deficient B cells may lead to the compromised MZ B cell compartment, as both processes have previously been shown to affect MZ composition.

BOB.1/OBF.1 (also called OCA‐B), a B lymphocyte‐specific transcriptional coactivator, is recruited to octamer‐containing promoters by interacting with the Oct‐1 or Oct‐2 proteins. BOB.1/OBF.1‐deficient mice show impaired secondary immunoglobulin isotype secretion and complete absence of germinal centers. Furthermore, numbers of splenic B cells are reduced due to a developmental block at the transitional B cell stage in the bone marrow. We found that surface expression of CD22 is selectively increased on B lineage cells in the bone marrow of BOB.1/OBF.1‐deficient mice. CD22 is knownas a negative regulator of B cell receptor signaling. We therefore investigated whether defects in B cell development in the BOB.1/OBF.1‐deficient mice might be due to CD22 up‐regulation. Mice weregenerated lacking both genes. In BOB.1/OBF.1×CD22 double‐deficient mice, numbers of transitional B cells in the bone marrow were normal. Consequently, double‐deficient mice also had normal B to T cell ratios in the spleen. We show that BOB.1/OBF.1–/– B cells were incapable to induce BCR‐triggered Ca2+ mobilization. This Ca2+‐signalling defect was restored in BOB.1/OBF.1×CD22 double‐deficient B cells. Nevertheless, double‐deficient animals were unable to mount humoral immune responses and to form germinal centers. Finally, we demonstrate that CD22–/– splenic B cells proliferate independently of BOB.1/OBF.1 upon stimulation with LPS. These studies suggest that the B cell differentiation defect observed in BOB.1/OBF.1–/– mice is BCR‐signal dependent. However, the impairment in germinal center formation is caused by a different mechanism.

CD22 is an inhibitory coreceptor for B cell receptor (BCR) signaling. The inhibition is most likely mediated by activation of SHP-1. We found that SLP65/BLNK reaches maximal tyrosine-phosphorylation at earlier time points in CD22 -/-than in wild type B cells upon BCR cross-linking, suggesting that SLP65/BLNK is a substrate of SHP-1. However, in contrast to the defective Ca 2+ mobilization of SLP65/BLNK -/-B cells, there was a clear Ca 2+ response in SLP65/BLNK×CD22 double-deficient B cells. This implies that SLP65/BLNK is not the sole target of SHP-1 in the regulation of the Ca 2+ signaling strength. While SLP65 -/-mice show several blocks of B cell differentiation, in SLP65/BLNK×CD22 double-deficient mice the maturation block of B cells in the spleen was partially rescued. However, the proliferative responses of B cells from both SLP65/BLNK -/-and double-deficient mice were defective after IgM-or CD40-stimulation. These results show that SLP65/BLNK is not absolutely essential for Ca 2+ induction in B cells, because the deficiency of this adapter can be bypassed by the additional deletion of an inhibitory receptor. Furthermore, these experiments suggest that B cell maturation in the spleen is directly dependent on the strength of BCRderived Ca 2+ signals.

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