Abstract
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.
Highlights
Sialic acid-binding immunoglobulin-like lectins (Siglecs) are type I membrane proteins expressed in various cell types, especially those of immune cells [1]
We demonstrated that the synthetic sialoside GSC718 and GSC839 bind to both human and mouse recombinant CD22 proteins with high affinity (IC50 ~100 nM)
Both GSC718 and GSC839 modulate in vitro activation of mouse B cells and enhance antibody production in mice. These sialosides do not regulate activation of CD22−/− B cells or enhance antibody production in CD22−/− mice, suggesting that these sialosides regulate CD22. Treatment with these synthetic sialosides down-modulates B cell proliferation induced by B cell antigen receptor (BCR) ligation, whereas the same treatment does not alter BCR ligation-induced proliferation of ST6GalI−/− B cells, suggesting that this effect of the sialosides depends on endogenous CD22 ligands
Summary
Sialic acid-binding immunoglobulin-like lectins (Siglecs) are type I membrane proteins expressed in various cell types, especially those of immune cells [1]. We demonstrated that modification of the C9-modified sialoside 9-(4′-hydroxy-4-biphenyl)acetamido-9-deoxy-Neu5Gc (hydroxy-BPAc-Neu5Gc) at C2 position by hydrophobic groups such as benzyl and biphenyl groups augmented affinity to human CD22 by 12-fold (70 nM) and mouse CD22 by 38-fold (100 nM) [6]. This finding suggested that introduction of multiple hydrophobic groups in different positions of the sialic acid backbone enhances affinity to CD22. Multimers of high-affinity sialoside ligands and nanoparticles carrying these sialosides were developed to target various molecules such as toxins and antigens to Siglecs. Whether immune responses can be regulated by monomeric sialoside ligands has not yet been known
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