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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.

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.

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.

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.

CD22 is an inhibitory B cell coreceptor that regulates B cell development and activation by downregulating BCR signaling through activation of SH2-containing protein tyrosine phosphatase-1 (SHP-1). CD22 recognizes α2,6 sialic acid as a specific ligand and interacts with α2,6 sialic acid-containing membrane molecules, such as CD45, IgM, and CD22, expressed on the same cell. Functional regulation of CD22 by these endogenous ligands enhances BCR ligation-induced signaling and is essential for normal B cell responses to Ags. In this study, we demonstrate that CD45 plays a crucial role in CD22-mediated inhibition of BCR ligation-induced signaling. However, disruption of ligand binding of CD22 enhances CD22 phosphorylation, a process required for CD22-mediated signal inhibition, upon BCR ligation in CD45−/− as well as wild-type mouse B cells but not in mouse B cells expressing a loss-of-function mutant of SHP-1. This result indicates that SHP-1 but not CD45 is required for ligand-mediated regulation of CD22. We further demonstrate that CD22 is a substrate of SHP-1, suggesting that SHP-1 recruited to CD22 dephosphorylates nearby CD22 as well as other substrates. CD22 dephosphorylation by SHP-1 appears to be augmented by homotypic CD22 clustering mediated by recognition of CD22 as a ligand of CD22 because CD22 clustering increases the number of nearby CD22. Our results suggest that CD22 but not CD45 is an endogenous ligand of CD22 that enhances BCR ligation-induced signaling through SHP-1–mediated dephosphorylation of CD22 in CD22 clusters.

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.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) expressed in T cells may regulate immune responses in the gut. In addition to T cells, B cells are also an important population in the gut-associated lymphoid tissues that orchestrate mucosal homeostasis. However, the role of CEACAM1 in B cells has not been elucidated. We herein analyzed mature B cells to determine the functions of CEACAM1. Flow cytometry revealed high expression of CEACAM1 on B cells in secondary lymphoid tissues. Cytokine production induced by activation of B cell receptor (BCR) signaling was suppressed by CEACAM1 signaling in contrast to that associated with either Toll-like receptor 4 or CD40 signaling. Confocal microscopy revealed co-localization of CEACAM1 and BCR when activated with anti-Igμ F(ab’)2 fragment. Overexpression of CEACAM1 in a murine B cell line, A20, resulted in reduced expressions of activation surface markers with decreased Ca2+ influx after BCR signal activation. Overexpression of CEACAM1 suppressed BCR signal cascade in A20 cells in association with decreased spontaneous proliferation. Our results suggest that CEACAM1 can regulate BCR-mediated mature B cell activation in lymphoid tissues. Therefore, further studies of this molecule may lead to greater insights into the mechanisms of immune responses within peripheral tissues and the potential treatment of inflammatory diseases.

The protein tyrosine phosphatase CD45 plays a crucial role in B cell antigen receptor (BCR) signaling by activating Src family kinases.

Carriers that augment delivery, immunogenicity or both are crucial in the development of vaccines especially component vaccines as components of pathogens are often poorly immunogenic. Cholesteryl pullulan (CHP) that forms nano-sized hydrogel (nanogel) and encapsulates proteins was shown to be useful in the delivery of vaccines. Here we demonstrate that subcutaneous immunization of mice with bovine serum albumin (BSA) chemically conjugated to NH2-CHP nanogel induces strong antibody production. This augmented antibody production requires covalent conjugation between BSA and CHP, but does not require nanogel formation. Conjugation of NH2-CHP nanogel induces persistence of BSA in dendritic cells (DCs) in vivo. As resistance to lysosomal degradation was previously shown to augment antigen presentation by DCs, conjugation of antigens with CHP nanogel may enhance antibody production to antigens by delaying lysosomal degradation. Therefore, delayed degradation of antigens by covalent conjugation with nanoparticles may be a good strategy for the development of effective vaccines.

B-cell novel protein 1 (BCNP1) has recently been identified as a new B-cell receptor (BCR) signaling molecule but its physiological function remains unknown. Here, we demonstrate that mice deficient in BCNP1 exhibit impaired B-cell maturation and a reduction of B-1a cells. BCNP1-deficient spleen B cells show enhanced survival, proliferation and Ca2+ influx in response to BCR cross-linking as compared with wild-type spleen B cells. Consistently, mutant B cells show elevated phosphorylation of SYK, B-cell linker protein (BLNK) and PLCγ2 upon BCR cross-linking. In vivo, BCNP1-deficient mice exhibit enhanced humoral immune responses to T-independent and T-dependent antigens. Moreover, aged mutant mice contain elevated levels of serum IgM and IgG3 antibodies and exhibit polyclonal and monoclonal B-cell expansion in lymphoid organs. These results reveal distinct roles for BCNP1 in B-cell development, activation and homeostasis.

IgA is the most abundantly produced antibody in the body and plays a crucial role in gut homeostasis and mucosal immunity. IgA forms a dimer that covalently associates with the joining (J) chain, which is essential for IgA transport into the mucosa. Here, we demonstrate that the marginal zone B and B-1 cell-specific protein (MZB1) interacts with IgA through the α-heavy-chain tailpiece dependent on the penultimate cysteine residue and prevents the intracellular degradation of α-light-chain complexes. Moreover, MZB1 promotes J-chain binding to IgA and the secretion of dimeric IgA. MZB1-deficient mice are impaired in secreting large amounts of IgA into the gut in response to acute inflammation and develop severe colitis. Oral administration of a monoclonal IgA significantly ameliorated the colitis, accompanied by normalization of the gut microbiota composition. The present study identifies a molecular chaperone that promotes J-chain binding to IgA and reveals an important mechanism that controls the quantity, quality, and function of IgA.

The BCR plays a central role in B cell development, survival, activation, and differentiation. We have identified the B cell novel protein 1 (BCNP1) as a new regulator of BCR signaling. BCNP1 contains a pleckstrin homology domain, three proline-rich motifs, and a potential SH2 binding site, and is predominantly expressed by B cells. We found that BCNP1 overexpression in WEHI231 immature B cells potentiated α-IgM-induced apoptosis. Conversely, BCNP1-deficient WEHI231 cells, generated by CRISPR-Cas9-mediated genome editing, exhibited reduced apoptosis after BCR crosslinking. Biochemical analyses revealed that BCNP1 physically interacted with the B cell linker protein (BLNK), Grb2, and PLCγ2. Moreover, absence of BCNP1 resulted in accelerated dephosphorylation of BLNK, reduced phosphorylation of SYK and PLCγ2, and decreased Ca2+ influx after BCR crosslinking. These results demonstrate that BCNP1 promotes BCR signaling by modulating the phosphorylation of BLNK, SYK, and PLCγ2.

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.

Guillain-Barre syndrome (GBS), including its variant Miller Fisher syndrome (MFS), is an acute peripheral neuropathy that involves autoimmune mechanisms leading to the production of autoantibodies to gangliosides; sialic acid-containing glycosphingolipids. Although association with various genetic polymorphisms in the major histocompatibility complex (MHC) is shown in other autoimmune diseases, GBS is an exception, showing no such link. No significant association was found by genome wide association studies, suggesting that GBS is not associated with common variants. To address the involvement of rare variants in GBS, we analyzed Siglec-10, a sialic acid-recognizing inhibitory receptor expressed on B cells. Here we demonstrate that two rare variants encoding R47Q and A108V substitutions in the ligand-binding domain are significantly accumulated in patients with GBS. Because of strong linkage disequilibrium, there was no patient carrying only one of them. Recombinant Siglec-10 protein containing R47Q but not A108V shows impaired binding to gangliosides. Homology modeling revealed that the R47Q substitution causes marked alteration in the ligand-binding site. Thus, GBS is associated with a rare variant of the SIGLEC10 gene that impairs ligand binding of Siglec-10. Because Siglec-10 regulates antibody production to sialylated antigens, our finding suggests that Siglec-10 regulates development of GBS by suppressing antibody production to gangliosides, with defects in its function predisposing to disease.

Reactive oxygen species (ROS) are not only toxic substances inducing oxidative stress but also play a role in receptor signaling as a second messenger, which augments signaling through various receptors by oxidizing ROS-sensitive signaling molecules. Among ROS, H

Siglecs are known to be bound and regulated by membrane molecules that display specific sialic acid-containing ligands and are present on the same cell (cis-ligands). Because of the low-affinity binding of Siglecs to the glycan ligands, conventional methods such as immunoprecipitation are not suitable for identification of Siglec cis-ligands. Here we describe efficient and specific labeling of cis-ligands of CD22 (also known as Siglec-2) on B lymphocytes by proximity labeling using tyramide. This method may also be applicable to labeling of cis-ligands of other Siglecs.

The regulation of integrin expression and function controls interactions of immune cells and targets their trafficking locally and systemically. We show here that the tyrosine phosphatase SHP-1 is required for lymphocyte surface expression of the intestinal immune response-associated integrin β7, but not for β1 or β2 integrins. Viable motheaten mice deficient for SHP-1 have less β7 on T cells and lack β7 on B cells. SHP-1 function is targeted in B cells by the B cell specific lectin CD22 (Siglec-2), suggesting a potential role for CD22 in β7 expression. CD22-deficiency on B cells phenocopies the effects of SHP-1 haplodeficiency. Mechanistically, we show that SHP-1 suppresses β7 endocytosis: internalization of β7 but not β1 integrin is accelerated in SHP-1+/− and CD22−/− B cells. Moreover, mutations in CD22 cytoplasmic SHP1-binding ITIM sequences reduce α4β7 comparably, and loss of CD22 lectin activity has an intermediate effect suggesting a model in which the CD22 ITIM sequences recruit SHP-1 to control β7 expression. Integrin α4β7 selectively contributes to cell interactions in intestinal immunity. Consistent with this, CD22 deficient and SHP-1+/− B cells display reduced β7-dependent homing to gut associated Peyer’s patches (PP); and CD22-deficiency impairs intestinal but not systemic antibody responses and delays clearance of the gut pathogen rotavirus. The results define a novel role for SHP-1 in the differential control of leukocyte integrins and an unexpected integrin β7-specific role for CD22-SHP-1 interplay in mucosal immunity.

Reactive oxygen species (ROS) are not only toxic substances inducing oxidative stress but also play a role in receptor signaling as a second messenger, which augments signaling through various receptors by oxidizing ROS-sensitive signaling molecules. Among ROS, H2O2 is suggested to be an important second messenger because of its relative stability. H2O2 is generated by superoxide dismutase (SOD)-mediated conversion of superoxide produced by membrane-localized NADPH oxidases (NOXes). Superoxide and H2O2 are also produced as a by-product of mitochondrial respiratory chain and various other metabolic reactions. BCR ligation induces ROS production in two phases. ROS production starts immediately after BCR ligation and ceases in 1 h, then re-starts 2 h after BCR ligation and lasts 4–6 h. ROS production in the early phase is mediated by NOX2, a NOX isoform, but does not regulate BCR signaling. In contrast, ROS production at the late phase augments BCR signaling. Although the involvement of mitochondrial respiration was previously suggested in prolonged BCR ligation-induced ROS production, we recently demonstrated that NOX3, another NOX isoform, plays a central role in ROS production at the late phase. NOXes are shown to be a component of ROS-generating signaling endosome called redoxosome together with endocytosed receptors and receptor-associated signaling molecules. In redoxosome, ROS generated by NOXes augment signaling through the endocytosed receptor. The role of NOXes and redoxosome in BCR signaling needs to be further elucidated.

The integrin α4β7 selectively regulates lymphocyte trafficking and adhesion in the gut and gut-associated lymphoid tissues (GALT). Here we describe unexpected involvement of tyrosine phosphatase Shp1 and the B cell lectin CD22 (Siglec 2) in 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, B cells lacking CD22, or B cells 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.

The antibody response to RNA-related antigens such as Sm/RNP requires the endosomal RNA sensor TLR7, and this process is crucial in the development of systemic lupus erythematosus at least in animal models. The inhibitory B cell receptor CD72 is unique because it recognizes Sm/RNP and specifically inhibits the activation of Sm/RNP-reactive B cells by activating SH2-containing protein tyrosine phosphatase 1 (SHP-1). In the normal immune system, Sm/RNP-reactive B cells are tolerized by a unique mechanism that probably involves SHP-1. These self-reactive B cells appear in the peripheral lymphoid organs, differentiate into marginal zone B cells, and then undergo apoptosis without further maturation into plasma cells. Thus, CD72 is involved in the suppression of TLR7-mediated response to RNA in complexes with nuclear proteins that are resistant to nucleases, whereas free RNAs are degraded by nucleases before they encounter the endosomal RNA sensor.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) expressed in T cells may regulate immune responses in the gut. In addition to T cells, B cells are also an important population in the gut-associated lymphoid tissues that orchestrate mucosal homeostasis. However, the role of CEACAM1 in B cells has not been elucidated. We herein analyzed mature B cells to determine the functions of CEACAM1. Flow cytometry revealed high expression of CEACAM1 on B cells in secondary lymphoid tissues. Cytokine production induced by activation of B cell receptor (BCR) signaling was suppressed by CEACAM1 signaling in contrast to that associated with either Toll-like receptor 4 or CD40 signaling. Confocal microscopy revealed co-localization of CEACAM1 and BCR when activated with anti-Igμ F(ab')

Systemic lupus erythematosus (SLE) is the prototypic systemic autoimmune disease characterized by production of autoantibodies to various nuclear antigens and overexpression of genes regulated by IFN-I called IFN signature. Genetic studies on SLE patients and mutational analyses of mouse models demonstrate crucial roles of nucleic acid (NA) sensors in development of SLE. Although NA sensors are involved in induction of anti-microbial immune responses by recognizing microbial NAs, recognition of self NAs by NA sensors induces production of autoantibodies to NAs in B cells and production of IFN-I in plasmacytoid dendritic cells. Among various NA sensors, the endosomal RNA sensor TLR7 plays an essential role in development of SLE at least in mouse models. CD72 is an inhibitory B cell co-receptor containing an immunoreceptor tyrosine-based inhibition motif (ITIM) in the cytoplasmic region and a C-type lectin like-domain (CTLD) in the extracellular region. CD72 is known to regulate development of SLE because CD72 polymorphisms associate with SLE in both human and mice and CD72-/- mice develop relatively severe lupus-like disease. CD72 specifically recognizes the RNA-containing endogenous TLR7 ligand Sm/RNP by its extracellular CTLD, and inhibits B cell responses to Sm/RNP by ITIM-mediated signal inhibition. These findings indicate that CD72 inhibits development of SLE by suppressing TLR7-dependent B cell response to self NAs. CD72 is thus involved in discrimination of self-NAs from microbial NAs by specifically suppressing autoimmune responses to self-NAs.

Akatsu and colleagues show that CD72 specifically recognizes Sm/RNP, a lupus-related self-antigen and an endogenous TLR7 ligand, and inhibits B cell responses to Sm/RNP. In mice, CD72 prevents production of anti-Sm/RNP antibodies crucial for lupus development., Toll-like receptor 7 (TLR7) plays an essential role in development of systemic lupus erythematosus by co-stimulating B cells reactive to the endogenous TLR7 ligand Sm/ribonucleoprotein (RNP), a crucial lupus self-antigen. However, how the TLR7-mediated autoimmune response is regulated is not yet known. In this study, we demonstrate that CD72, an inhibitory B cell co-receptor known to prevent development of lupus, recognizes Sm/RNP at the extracellular C-type lectin-like domain (CTLD) and specifically inhibits B cell response to Sm/RNP. Moreover, the CTLD of CD72c, a lupus-susceptible allele, binds to Sm/RNP less strongly than that of lupus-resistant CD72a. Reduced binding of CD72c is supported by x-ray crystallographic analysis that reveals a considerable alteration in charge at the putative ligand-binding site. Thus, CD72 appears to specifically inhibit B cell response to the endogenous TLR7 ligand Sm/RNP through CTLD-mediated recognition of Sm/RNP, thereby preventing production of anti-Sm/RNP antibody crucial for development of lupus.

Reactive oxygen species (ROS) are not only toxic substances inducing oxidative stress but also play a role as a second messenger in signal transduction through various receptors. Previously, B cell activation was shown to involve prolonged ROS production induced by ligation of BCR. However, the mechanisms for ROS production and ROS-mediated activation in B cells are still poorly understood. In this study, we demonstrate that BCR ligation induces biphasic ROS production in both mouse spleen B cells and the mouse B cell line BAL17; transient and modest ROS production is followed by sustained and robust ROS production at 2–6 h after BCR ligation. ROS production in the late phase but not in the early phase augments activation of signaling pathways, such as the NF-κB and PI3K pathways, and is essential for B cell proliferation. ROS production in the late phase appears to be mediated by NADPH oxidases (NOXes) because prolonged ROS production is inhibited by various NOX inhibitors, including the specific inhibitor VAS2870. BCR ligation–induced ROS production is also inhibited by CRISPR/Cas9-mediated deletion of either the Cyba gene encoding p22phox, the regulator of NOX1-4 required for their activation, or NOX3, whereas ROS production is not affected by double deficiency of the DUOXA1 and DUOXA2 genes essential for the activation of the NOX isoforms DUOX1 and DUOX2. These results indicate that NOXes play a crucial role in sustained but not early BCR signaling and suggest an essential role of NOX-dependent sustained BCR signaling in B cell activation.

The marginal zone B cells (MZB) are located at the interface between the circulation and lymphoid tissue and as a gatekeeper play important roles in both innate and adaptive immune responses. We have previously found that MZB are significantly reduced in mice deficient in the IgM Fc receptor (FcμR) but how FcμR regulates the development and function of MZB remains unknown. In the present study, we found that both marginal zone precursor (MZP) and MZB were decreased in FcμR-/- mice. The reduction of MZP and MZB was not due to impaired proliferation of these cells but rather due to their increased death. Further analysis revealed that FcμR-/- MZB had reduced tonic BCR signal, as evidenced by their decreased levels of phosphorylated SYK and AKT relative to WT MZB. MZB in FcμR-/- mice responded poorly to LPS in vivo as compared to MZB in WT mice. Consistent with the reduced proportion of MZB and their impaired response to LPS, antibody production against the type 1 T-independent Ag, NP-LPS, was significantly reduced in FcμR-/- mice. Moreover, FcμR-/- mice were highly susceptible to Citrobacter rodentium-induced sepsis. These results reveal a critical role for FcμR in the survival and activation of MZB and in protection against acute bacterial infection.

Summary 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., Graphical Abstract, Highlights • LAG-3 expression identifies natural regulatory plasma cells • LAG-3+CD138hi plasma cells express IL-10 within hours of stimulation • LAG-3+CD138hi plasma cells have a unique epigenome poised to express IL-10 • LAG-3+CD138hi plasma cells develop via an antigen-specific mechanism, Plasma cells secrete antibodies and play a key role in host defense against infection. Lino et al. identify a novel subset of natural regulatory plasma cells characterized by the expression of LAG-3 that develops at steady state independently of microbiota, and respond to innate stimulation by producing immunosuppressive IL-10.

B-1 cells are innate-like B-cell population and produce natural antibodies that contribute to the first line of host defense. There are two subsets of B-1 cells: B-1a and B-1b. B-1a cells are the main producer of poly-reactive and autoreactive natural IgM antibodies, whereas B-1b cells can respond specifically to T-cell-independent antigens. Despite the functional significance of B-1a and B-1b cells, little information is available about what regulates the development of these two subsets. We found that Kelch-like protein 14 (KLHL14) was expressed at high levels in B cells but only at low levels in a few non-lymphoid tissues. Although mice lacking KLHL14 died right after birth, the heterozygotes developed normally with no gross abnormalities by appearance. B-cell development in the bone marrow and maturation and activation in the spleen were not affected in the heterozygous mice. However, the number of peritoneal B-1a cells was significantly reduced while B-1b cells were increased in Klhl14 heterozygous mice compared with wild-type (WT) mice. Consistently, Rag1−/− mice reconstituted with Klhl14−/− fetal liver cells had a more severe reduction of B-1a and an increase of B-1b cells in the peritoneal cavity. KLHL14 did not affect the turnover or apoptosis of B-1a and B-1b cells in vivo. Moreover, Klhl14−/− fetal liver contained a similar proportion and absolute numbers of the B-1 progenitor cells as did WT fetal liver. These results suggest that KLHL14 promotes B-1a development in mice.

Lectins expressed on the cell surface are often bound and regulated by the membrane molecules containing the glycan ligands on the same cell (cis-ligands). However, molecular nature and function of cis-ligands are generally poorly understood partly because of weak interaction between lectins and glycan ligands. Cis-ligands are most extensively studied in CD22 (also known as Siglec-2), an inhibitory B lymphocyte receptor specifically recognizing α2,6 sialic acids. CD22, CD45 and IgM are suggested to be ligands of CD22. Here we labeled molecules in the proximity of CD22 in situ on B cell surface using biotin-tyramide. Molecules including CD22, CD45 and IgM were labeled in wild-type but not ST6GalI-/- B cells that lack α2,6 sialic acids, indicating that these molecules associate with CD22 by lectin-glycan interaction, and are therefore cis-ligands. In ST6GalI-/- B cells, these cis-ligands are located in a slightly more distance from CD22. Thus, the lectin-glycan interaction recruits cis-ligands already located in the relative proximity of CD22 through non-lectin-glycan interaction to the close proximity. Moreover, cis-ligands are labeled in Cmah-/- B cells that lack Neu5Gc preferred by mouse CD22 as efficiently as in wild-type B cells, indicating that very low affinity lectin-glycan interaction is sufficient for recruiting cis-ligands, and can be detected by proximity labeling. Thus, proximity labeling with tyramide appears to be a useful method to identify cis-ligands and to analyze their interaction with the lectins.

Activation-induced cytidine deaminase (AID) initiates both somatic hypermutation (SHM) and class switch recombination (CSR) of Ig genes. How AID is targeted to the Ig V gene and switch region to trigger SHM and CSR remains elusive. Primary B cells stimulated with CD40L plus IL-4 or LPS plus IL-4 undergo efficient CSR, but it has been difficult to induce SHM in these cells. In the current study, we used B cells from B1-8hi mice carrying a prerecombined VH186.2DFL16.1JH2 Ab gene to investigate the induction of SHM under in vitro culture conditions. B1-8hi splenic B cells stimulated with CD40L plus IL-4 or LPS plus IL-4 underwent robust CSR to IgG1, but failed to generate SHM in the VH186.2 gene. Remarkably, ectopic expression of AID in AID-deficient, but not wild-type, B1-8hi B cells induced efficient SHM at a rate close to that observed in germinal center B cells. We further established an AID-deficient CH12 B lymphoma line and found that ectopic expression of AID in the mutant line, but not in AID-sufficient CH12 cells, induced efficient point mutations and deletions in the V gene. These results demonstrate that the endogenous AID in ex vivo–activated primary B and B lymphoma cells not only cannot induce SHM but also inhibit the induction of SHM by the exogenous AID. Our results further suggest that the spatiotemporal distribution and/or posttranslational modification of AID strongly affects the induction of SHM in ex vivo–activated primary B cells.

The lysosomal protein LAPTM5 has been shown to negatively regulate cell surface T cell receptor (TCR) expression and T-cell activation by promoting CD3ζ degradation in lysosomes, but the mechanism remains largely unknown. Here we show that LAPTM5 promotes lysosomal translocation of intracellular CD3ζ but not of the cell surface CD3ζ associated with the mature TCR complex. Kinetic analysis of the subcellular localization of the newly synthesized CD3ζ suggests that LAPTM5 targets CD3ζ in the Golgi apparatus and promotes its lysosomal translocation. Consistently, a Golgi-localizing mutant CD3ζ can be transported to and degraded in the lysosome by LAPTM5. A CD3ζ YF mutant in which all six tyrosine residues in the immunoreceptor tyrosine-based activation motif are mutated to phenylalanines is degraded as efficiently as is wild type CD3ζ, further suggesting that TCR signaling-triggered tyrosine phosphorylation of CD3ζ is dispensable for LAPTM5-mediated degradation. Previously, Src-like adapter protein (SLAP) and E3 ubiquitin ligase c-Cbl have been shown to mediate the ubiquitination of CD3ζ in the internalized TCR complex and its subsequent lysosomal degradation. We show that LAPTM5 and SLAP/c-Cbl function in distinct genetic pathways to negatively regulate TCR expression. Collectively, these results suggest that CD3ζ can be degraded by two pathways: SLAP/c-Cbl, which targets internalized cell surface CD3ζ dependent on TCR signaling, and LAPTM5, which targets intracellular CD3ζ independent of TCR signaling.

Persistent cell shrinkage is a major hallmark of apoptotic cell death. The early-phase shrinkage, which starts within 30−120 min after apoptotic stimulation and is called apoptotic volume decrease (AVD), is known to be accomplished by activation of K+ channels and volume-sensitive outwardly rectifying (VSOR) Cl− channels in a manner independent of caspase-3 activation. However, it is controversial whether AVD depends on apoptotic dysfunction of mitochondria and activation of initiator caspases. Here, we observed that AVD is induced not only by a mitochondrial apoptosis inducer, staurosporine (STS), in mouse B lymphoma WEHI-231 cells, but also by ligation of the death receptor Fas in human B lymphoblastoid SKW6.4 cells, which undergo Fas-mediated apoptosis without involving mitochondria. Overexpression of Bcl-2 failed to inhibit the STS-induced AVD in WEHI-231 cells. These results indicate that AVD does not require the mitochondrial pathway of apoptosis. In human epithelial HeLa cells stimulated with anti-Fas antibody or STS, the AVD induction was found to precede activation of caspase-8 and caspase-9 and to be resistant to pan-caspase blockers. Thus, it is concluded that the AVD induction is an early event independent of the mitochondrial apoptotic signaling pathway and initiator caspase activation.

CD40L is excessively produced in both human and murine lupus and plays a role in lupus pathogenesis. To address how excess CD40L induces autoantibody production, we crossed CD40L-transgenic mice with the anti-DNA H-chain transgenic mouse lines 3H9 and 56R, well-characterized models for studying B-cell tolerance to nuclear antigens. Excess CD40L did not induce autoantibody production in 3H9 mice in which anergy maintains self-tolerance, nor did it perturb central tolerance, including deletion and receptor editing, of anti-DNA B cells in 56R mice. In contrast, CD40L/56R mice restored a large number of marginal zone (MZ) B cells reactive to Sm/ribonucleoprotein (RNP) and produced autoantibody, whereas these B cells were deleted by apoptosis in MZ of 56R mice. Thus, excess CD40L efficiently blocked tolerance of Sm/RNP-reactive MZ B cells, leading to production of anti-Sm/RNP antibody implicated in the pathogenesis of lupus. These results suggest that self-reactive B cells such as anti-Sm/RNP B cells, which somehow escape tolerance in the bone marrow and migrate to MZ, are tolerized by apoptotic deletion in MZ and that a break in this tolerance may play a role in the pathogenesis of lupus.