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The integrin α4β7 selectively regulates lymphocyte trafficking and adhesion in the gut and gut-associated lymphoid tissue (GALT). Here, we describe unexpected involvement of the tyrosine phosphatase Shp1 and the B cell lectin CD22 (Siglec-2) in the regulation of α4β7 surface expression and gut immunity. Shp1 selectively inhibited β7 endocytosis, enhancing surface α4β7 display and lymphocyte homing to GALT. In B cells, CD22 associated in a sialic acid-dependent manner with integrin β7 on the cell surface to target intracellular Shp1 to β7. Shp1 restrained plasma membrane β7 phosphorylation and inhibited β7 endocytosis without affecting β1 integrin. B cells with reduced Shp1 activity, lacking CD22 or expressing CD22 with mutated Shp1-binding or carbohydrate-binding domains displayed parallel reductions in surface α4β7 and in homing to GALT. Consistent with the specialized role of α4β7 in intestinal immunity, CD22 deficiency selectively inhibited intestinal antibody and pathogen responses.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are expressed in various immune cells and most of them carry signaling functions. High-affinity synthetic sialoside ligands have been developed for various Siglecs. Therapeutic potentials of the nanoparticles and compounds that contain multiple numbers of these sialosides and other reagents such as toxins and antigens have been demonstrated. However, whether immune responses can be regulated by monomeric sialoside ligands has not yet been known. CD22 (also known as Siglec-2) is an inhibitory molecule preferentially expressed in B lymphocytes (B cells) and is constitutively bound and functionally regulated by α2,6 sialic acids expressed on the same cell (cis-ligands). Here, we developed synthetic sialosides GSC718 and GSC839 that bind to CD22 with high affinity (IC50 ~100 nM), and inhibit ligand binding of CD22. When B cells are activated by B cell antigen receptor (BCR) ligation, both GSC718 and GSC839 downregulate proliferation of B cells, and this regulation requires both CD22 and α2,6 sialic acids. This result suggests that these sialosides regulate BCR ligation-induced B cell activation by reversing endogenous ligand-mediated regulation of CD22. By contrast, GSC718 and GSC839 augment B cell proliferation induced by TLR ligands or CD40 ligation, and this augmentation requires CD22 but not α2,6 sialic acids. Thus, these sialosides appear to enhance B cell activation by directly suppressing the inhibitory function of CD22 independently of endogenous ligand-mediated regulation. Moreover, GSC839 augments B cell proliferation that depends on both BCR ligation and CD40 ligation as is the case for in vivo B cell responses to antigens, and enhanced antibody production to the extent comparable to CpG oligonuleotides or a small amount of alum. Although these known adjuvants induce production of the inflammatory cytokines or accumulation of inflammatory cells, CD22-binding sialosides do not. Thus, synthetic sialosides that bind to CD22 with high-affinity modulate B cell activation through endogenous ligand-dependent and independent pathways, and carry an adjuvant activity without inducing inflammation.

The protein tyrosine phosphatase CD45 plays a crucial role in B cell antigen receptor (BCR) signaling by activating Src family kinases. Cd45−/− mice show altered B cell development and a phenotype likely due to reduced steady-state signaling; however, Cd45−/− B cells show relatively normal BCR ligation–induced signaling. In our investigation of how BCR signaling was restored in Cd45−/− cells, we found that the coreceptor CD22 switched from an inhibitory to a stimulatory function in these cells. We disrupted the ability of CD22 to interact with its ligands in Cd45−/− B cells by generating Cd45−/−St6galI−/− mice, which cannot synthesize the glycan ligand of CD22, or by treating Cd45−/− B cells in vitro with the sialoside GSC718, which inhibits ligand binding to CD22. BCR ligation–induced signaling was reduced by ST6GalI deficiency, but not by GSC718 treatment, suggesting that CD22 restored BCR ligation–induced signaling in Cd45−/− mature B cells by altering cellular phenotypes during development. CD22 was required for the increase in the surface amount of IgM-BCR on Cd45−/− B cells, which augmented signaling. Because B cell survival depends on steady-state BCR signaling, IgM-BCR abundance was likely increased by the selective survival of IgM-BCRhiCd45−/− B cells because of CD22-mediated signaling under conditions of substantially reduced steady-state signaling. Because the amount of surface IgM-BCR is increased on B cells from patients with other BCR signaling deficiencies, including X-linked agammaglobulinemia, our findings suggest that CD22 may contribute to the partial restoration of B cell function in these patients. Restoring B cell function: The protein tyrosine phosphatase CD45 activates Src family kinases to mediate signaling downstream of the B cell antigen receptor (BCR) in response to antigens. In addition, antigen-independent, steady-state signaling by the BCR is important for B cell survival and development. Loss of CD45 alters the development and function of B cells in mice, but BCR ligation–induced signaling in these cells is normal. Akatsu et al. found that the inhibitory coreceptor CD22, which normally inhibits BCR ligation–induced signaling, paradoxically enhanced this pathway in B cells lacking CD45. In mice, CD22 signaling in CD45-deficient B cells resulted in an increase in the surface abundance of the IgM class of the BCR during their development. Such changes in IgM-BCR abundance are also seen in patients with immunodeficiencies. Together, these data suggest that CD22 stimulates a compensatory mechanism by which BCR ligation–induced signaling is partially restored in immunodeficient B cells. [ABSTRACT FROM AUTHOR]