CD22-Binding Synthetic Sialosides Regulate B Lymphocyte Proliferation Through CD22 Ligand-Dependent and Independent Pathways, and Enhance Antibody Production in Mice

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.

Coordinated roles for glycans in regulating the inhibitory function of CD22 on B cells

“…Additional evidence for CD22 being largely maintained away from the BCR comes from studies that force CD22 with the BCR, showing how profoundly this can inhibit BCR signaling relative to the modest difference in BCR signaling comparing WT to CD22 −/− B cells. A recent study using a nanomolar affinity soluble CD22 ligand also reported that this compound was capable of causing hypo-responsiveness of B cells in an St6gal1-dependent manner, which is consistent with this model [82]. Therefore, pharmacological, enzymatic, and genetic approaches to ablate CD22 ligands on the cell surface all demonstrate the same phenomenon, which is that they increase association of CD22 with the BCR, leading to hypo-responsive BCR signaling [Fig.…”

“…Earlier studies from CD22 −/− mice revealed an altered response in B cells following stimulation with a variety of TLR ligands [91], where CD22 appears to act as a negative regulator. More detailed analysis of WT versus CD22 −/− B cell responses to a variety of TLR ligands have solidified these findings [82], [111]. It is intriguing that up to a 5-10-fold increase in responsiveness to TLR ligands in CD22 −/− B cells is significantly more than the 50% increase in BCR stimulation.…”

Glycosylation in health and disease

“…Examples of such glycan-containing molecules include bacterial lipopolysaccharides, peptidoglycans, teichoic acids, capsular polysaccharides and fungal mannans. The recognition of these glycosylated microbial patterns by the immune system has been exploited for the development of vaccines 45,46 ; pneumococcal vaccines, for example, are formulated using a mixture of capsular polysaccharides 47 . The recent progress in H IV-1 vaccine development has also been driven by a better understanding of the HIV-1 envelope (Env) glycoprotein and the effects of its glycan composition on immune responses and immune evasion 48–53 .…”

CD22: A Regulator of Innate and Adaptive B Cell Responses and Autoimmunity

“…Thus, CD22 normally may play a role in the direct inhibition of TLR signaling in B cells. The natural ligands for CD22 apparently do not play a direct role in regulating proliferative responses to TLR agonists since CD22 ligand-deficient ST6Gal1 −/− B cells have normal responses to LPS and CpG ( 40 ). CD22 is an endocytic receptor that recycles between the cell surface and the endosomes, where endosomal TLRs resides ( 41 ).…”