A CD22-Shp1 phosphatase axis controls integrin ß7 display and B cell function in mucosal immunity.

The integrin α4ß7 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 α4ß7 surface expression and gut immunity. Shp1 selectively inhibited ß7 endocytosis, enhancing surface α4ß7 display and lymphocyte homing to GALT. In B cells, CD22 associated in a sialic acid-dependent manner with integrin ß7 on the cell surface to target intracellular Shp1 to ß7. Shp1 restrained plasma membrane ß7 phosphorylation and inhibited ß7 endocytosis without affecting ß1 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 α4ß7 and in homing to GALT. Consistent with the specialized role of α4ß7 in intestinal immunity, CD22 deficiency selectively inhibited intestinal antibody and pathogen responses.

LAG-3 Inhibitory Receptor Expression Identifies Immunosuppressive Natural Regulatory Plasma Cells.

B lymphocytes can suppress immunity through interleukin (IL)-10 production in infectious, autoimmune, and malignant diseases. Here, we have identified a natural plasma cell subset that distinctively expresses the inhibitory receptor LAG-3 and mediates this function in vivo. These plasma cells also express the inhibitory receptors CD200, PD-L1, and PD-L2. They develop from various B cell subsets in a B cell receptor (BCR)-dependent manner independently of microbiota in naive mice. After challenge they upregulate IL-10 expression via a Toll-like receptor-driven mechanism within hours and without proliferating. This function is associated with a unique transcriptome and epigenome, including the lowest amount of DNA methylation at the Il10 locus compared to other B cell subsets. Their augmented accumulation in naive mutant mice with increased BCR signaling correlates with the inhibition of memory T cell formation and vaccine efficacy after challenge. These natural regulatory plasma cells may be of broad relevance for disease intervention.

Role of inhibitory B cell co-receptors in B cell self-tolerance to non-protein antigens.

Antibodies to non-protein antigens such as nucleic acids, polysaccharides, and glycolipids play important roles in both host defense against microbes and development of autoimmune diseases. Although non-protein antigens are not recognized by T cells, antibody production to non-protein antigens involve T cell-independent mechanisms such as signaling through TLR7 and TLR9 in antibody production to nucleic acids. Although self-reactive B cells are tolerized by various mechanisms including deletion, anergy, and receptor editing, T cell tolerance is also crucial in self-tolerance of B cells to protein self-antigen because self-reactive T cells induce autoantibody production to these self-antigens. However, presence of T cell-independent mechanism suggests that T cell tolerance is not able to maintain B cell tolerance to non-protein self-antigens. Lines of evidence suggest that B cell response to non-protein self-antigens such as nucleic acids and gangliosides, sialic acid-containing glycolipids, are suppressed by inhibitory B cell co-receptors CD72 and Siglec-G, respectively. These inhibitory co-receptors recognize non-protein self-antigens and suppress BCR signaling induced by these antigens, thereby inhibiting B cell response to these self-antigens. Inhibitory B cell co-receptors appear to be involved in B cell self-tolerance to non-protein self-antigens that can activate B cells by T cell-independent mechanisms.

Neu5Gc-mediated high-affinity interaction is dispensable for CD22 cis-ligands to regulate B cell signaling.

CD22 (also known as Siglec-2) is an inhibitory receptor expressed in B cells. CD22 specifically recognizes α2,6 sialic acid and interacts with α2,6 sialylated membrane proteins expressed on the same cell (cis-ligands) and those derived from outside of the cell (trans-ligands). Previously, CD22 cis-ligands were shown to regulate the activity of CD22, thereby regulating both BCR ligation-induced signaling and low-level "tonic" signaling in the absence of BCR ligation that regulates the survival and differentiation of B cells. Mouse CD22 prefers Neu5Gc to Neu5Ac thereby binding to α2,6-linked Neu5Gc with high affinity. Although human CD22 binds to a distinct α2,6 sialylated glycan with high affinity, expression of high-affinity ligands is regulated in a conserved and stringent manner. However, how high- versus low-affinity CD22 ligands regulate B cells is poorly understood. Here we demonstrate that the interaction of CD22 with the endogenous ligands enhances BCR ligation-induced signaling but reduces tonic signaling in Cmah-/- mouse B cells deficient in Neu5Gc as well as wild-type B cells. Moreover, Cmah-/- B cells do not show alterations in the phenotypes correlated to tonic signaling. These results indicate that low-affinity interaction of the CD22 cis-ligands with CD22 is sufficient for the regulation of B cell signaling, and suggest that expression of high-affinity CD22 ligands might be involved in the regulation of B cells by competing for the binding of CD22 with exogenous trans-ligands of CD22.

LAPTM5 mediates immature B cell apoptosis and B cell tolerance by regulating the WWP2-PTEN-AKT pathway.

Elimination of autoreactive developing B cells is an important mechanism to prevent autoantibody production. However, how B cell receptor (BCR) signaling triggers apoptosis of immature B cells remains poorly understood. We show that BCR stimulation up-regulates the expression of the lysosomal-associated transmembrane protein 5 (LAPTM5), which in turn triggers apoptosis of immature B cells through two pathways. LAPTM5 causes BCR internalization, resulting in decreased phosphorylation of SYK and ERK. In addition, LAPTM5 targets the E3 ubiquitin ligase WWP2 for lysosomal degradation, resulting in the accumulation of its substrate PTEN. Elevated PTEN levels suppress AKT phosphorylation, leading to increased FOXO1 expression and up-regulation of the cell cycle inhibitor p27Kip1 and the proapoptotic molecule BIM. In vivo, LAPTM5 is involved in the elimination of autoreactive B cells and its deficiency exacerbates autoantibody production. Our results reveal a previously unidentified mechanism that contributes to immature B cell apoptosis and B cell tolerance.

CD72 is an inhibitory pattern recognition receptor that recognizes ribosomes and suppresses production of anti-ribosome autoantibody.

B cell responses to nucleic acid-containing self-antigens that involve intracellular nucleic acid sensors play a crucial role in autoantibody production in SLE. CD72 is an inhibitory B cell co-receptor that down-regulates BCR signaling, and prevents the development of SLE. We previously showed that CD72 recognizes the RNA-containing self-antigen Sm/RNP, a target of SLE-specific autoantibodies, and induces B cell tolerance to Sm/RNP by specifically inhibiting B cell response to this self-antigen. Here, we address whether CD72 inhibits B cell response to ribosomes because the ribosome is an RNA-containing self-antigen and is a target of SLE-specific autoantibodies as well as Sm/RNP. We demonstrate that CD72 recognizes ribosomes as a ligand, and specifically inhibits BCR signaling induced by ribosomes. Although conventional protein antigens by themselves do not induce proliferation of specific B cells, ribosomes induce proliferation of B cells reactive to ribosomes in a manner dependent on RNA. This proliferative response is down-regulated by CD72. These results suggest that ribosomes activate B cells by inducing dual signaling through BCR and intracellular RNA sensors and that CD72 inhibits B cell response to ribosomes. Moreover, CD72-/- but not CD72+/+ mice spontaneously produce anti-ribosome autoantibodies. Taken together, CD72 induces B cell self-tolerance to ribosomes by recognizing ribosomes and inhibiting RNA-dependent B cell response to this self-antigen. CD72 appears to prevent development of SLE by inhibiting autoimmune B cell responses to multiple RNA-containing self-antigens. Because these self-antigens but not protein self-antigens induce RNA-dependent B cell activation, self-tolerance to RNA-containing self-antigens may require a distinct tolerance mechanism mediated by CD72.

The ligand interactions of B cell Siglecs are involved in the prevention of autoimmunity to sialylated self-antigens and in the quality control of signaling-competent B cells.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of membrane molecules that recognize sialic acid. Most of them are inhibitory receptors that inhibit immune-cell activation by recognizing sialic acid as a self-motif. Human B cells express CD22 (also known as Siglec-2), Siglec-5, Siglec-6 and Siglec-10 whereas mouse B cells express CD22 and Siglec-G (ortholog of human Siglec-10). Siglecs recognize both sialylated molecules expressed on the same cell (cis-ligands) and those expressed by other cells (trans-ligands). In Guillain-Barré syndrome (GBS), antibody production to gangliosides (which are sialic acid-containing glycolipids) expressed by neurons plays a pathogenic role. A Siglec-10 variant deficient in recognition of gangliosides is genetically associated with GBS, suggesting that Siglec-10 induces self-tolerance to gangliosides by recognizing gangliosides as trans-ligands. Recognition of the BCR as a cis-ligand by Siglec-G and CD22 suppresses BCR signaling in B-1 cells and conventional B cells, respectively. This signal suppression prevents excess expansion of B-1 cells and is involved in the quality control of signaling-competent B cells by setting a threshold for tonic signaling during B cell development. CD22 recognizes other cis-ligands including CD22 and ß7 integrin. Interaction of CD22 with other CD22 molecules induces CD22 clustering that suppresses CD22-mediated signal inhibition upon BCR ligation, and interaction with ß7 integrin maintains its function in the gut-homing of B cells. Taken together, interactions of B cell Siglecs with multiple trans- and cis-ligands play important roles in B cell homeostasis and immune responses.

Siglec cis-ligands and their roles in the immune system.

Sialic acid-binding immunoglobulin-like lectins are a family of membrane molecules primarily expressed in immune cells. Most of them are inhibitory receptors containing immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic tail. On the cell surface, sialic acid-binding immunoglobulin-like lectins are mostly bound by sialylated glycans on membrane molecules expressed in the same cell (cis-ligands). Although ligands of sialic acid-binding immunoglobulin-like lectins are not efficiently identified by conventional methods such as immunoprecipitation, in situ labeling including proximity labeling is useful in identifying both cis-ligands and the sialylated ligands expressed by other cells (trans-ligands) of sialic acid-binding immunoglobulin-like lectins. Interaction of the inhibitory sialic acid-binding immunoglobulin-like lectins with cis-ligands including both those with and without signaling function modulates the inhibitory activity of sialic acid-binding immunoglobulin-like lectins by multiple different ways. This interaction also modulates signaling function of the cis-ligands. So far, little is known about the role of the interaction between sialic acid-binding immunoglobulin-like lectins and the cis-ligands. Nonetheless, recent studies showed that the inhibitory activity of CD22 (also known as Siglec-2) is regulated by endogenous ligands, most likely cis-ligands, differentially in resting B cells and those in which B-cell antigen receptor is ligated. This differential regulation plays a role in quality control of signaling-competent B cells and also partial restoration of B-cell antigen receptor signaling in immunodeficient B cells.

Glia maturation factor-γ is involved in S1P-induced marginal zone B-cell chemotaxis and optimal IgM production to type II T-independent antigen.

Marginal zone B cells (MZBs) represent a unique B-cell sub-population that rapidly differentiate into IgM-secreting plasma cells in response to T-independent (T-I) antigen. Sphingosine 1-phosphate (S1P) promotes MZB localization to the marginal zone. However, intracellular molecules involved in MZB localization and migration remain largely unknown. Here, we show that MZBs lacking the glia maturation factor-γ (GMFG) are impaired in chemotaxis toward S1P under both in vitro and in vivo conditions, suggesting that GMFG is an effector downstream of S1P receptors. GMFG undergoes serine phosphorylation upon S1P stimulation and is required for S1P-induced desensitization of S1P receptor 1 (S1PR1). Compared with wild-type mice, Gmfg-/- mice produce elevated levels of 4-hydroxy-3-nitrophenyl-acetyl (NP)-specific IgM against a T-I type II antigen, NP-Ficoll, accompanied by dysregulated MZB localization. These results identify GMFG as a regulator of S1P-induced MZB chemotaxis and reveal a role for MZB localization in the marginal zone for optimal IgM production against a T-I antigen.

Improved synthesis of CD22-binding sialosides and its application for further development of potent CD22 inhibitors.

CD22, one of the sialic acid-binding immunoglobulin-like lectins (Siglecs), regulates B lymphocyte signaling via its interaction with glycan ligands bearing the sequence Neu5Ac/Gcα(2→6)Gal. We have developed the synthetic sialoside GSC-718 as a ligand mimic for CD22 and identified it as a potent CD22 inhibitor. Although the synthesis of CD22-binding sialosides including GSC-718 has been reported by our group, the synthetic route was unfortunately not suitable for large-scale synthesis. In this study, we developed an improved scalable synthetic procedure for sialosides which utilized 1,5-lactam formation as a key step. The improved procedure yielded sialosides incorporating a series of aglycones at the C2 position. Several derivatives with substituted benzyl residues as aglycones were found to bind to mouse CD22 with affinity comparable to that of GSC-718. The new procedure developed in this study affords sialosides in sufficient quantities for cell-based assays, and will facilitate the search for promising CD22 inhibitors that have therapeutic potential.