Search

T cell independent type II (TI-II) antigens, such as capsular polysaccharides, have multivalent epitopes, which induce B cell activation, plasma cell differentiation and antibody production by strongly cross-linking B cell receptors. However, the mechanism of B cell activation by TI-II antigens remains unclear. In this study, we demonstrate that DNA endonuclease DNase1L3 (also termed DNase γ) is required for the TI-II response. The production of antigen-specific antibodies was severely diminished in DNase1L3-deficient mice upon immunization with TI-II antigens, but not with T cell dependent (TD) antigens. Bone marrow chimeric mice and B cell transfer experiments revealed that B cell-intrinsic DNase1L3 was required for the TI-II response. DNase1L3-deficient B cells were defective in cell proliferation and plasma cell differentiation in the TI-II response in vivo as well as in vitro, which was not rescued by co-culture with DNase1L3-sufficient B cells in vitro, disproving an involvement of a secretory DNase1L3. In vitro stimulation with TI-II antigen transiently increased expression of DNase1L3 and its translocation into the nucleus. RNA-seq analysis of ex vivo B cells that had responded to TI-II antigen in vivo revealed a marked reduction of Myc-target gene sets in DNase1L3-deficient B cells. Expression of IRF4, a gene that Myc targets, was diminished in the ex vivo DNase1L3-deficient B cells, in which forced expression of IRF4 restored the TI-II response in vivo. These data revealed an unexpected role of DNase1L3 in a missing link between B cell receptor signaling and B cell activation in the TI-II response, giving a valuable clue to molecularly dissect this response.

IgE Abs are a common mediator of allergic responses and are generally produced in type 2 immune responses to allergens. Allergen stimulation of IgE-bound FcεRI on mast cells or basophils induces the production of chemical mediators and cytokines. In addition, IgE binding to FcεRI without allergen promotes the survival or proliferation of these and other cells. Thus, spontaneously produced natural IgE can increase an individual’s susceptibility to allergic diseases. Mice deficient in MyD88, a major TLR signaling molecule, have high serum levels of natural IgE, the mechanism for which remains unknown. In this study, we demonstrated that the high serum IgE levels were maintained from weaning by memory B cells (MBCs). IgE from plasma cells and sera from most Myd88−/− mice, but none of the Myd88+/− mice, recognized Streptococcus azizii, a commensal bacterium overrepresented in the lungs of Myd88−/− mice. IgG1+ MBCs from the spleen also recognized S. azizii. The serum IgE levels declined with the administration of antibiotics and were boosted by challenge with S. azizii in Myd88−/− mice, indicating the contribution of S. azizii–specific IgG1+ MBCs to the natural IgE production. Th2 cells were selectively increased in the lungs of Myd88−/− mice and were activated upon addition of S. azizii in the lung cells ex vivo. Finally, lung nonhematopoietic cells, and CSF1 overproduced therefrom, were responsible for natural IgE production in Myd88−/− mice. Thus, some commensal bacteria may prime the Th2 response and natural IgE production in the MyD88-defective lung environment in general.

IgA is the most abundant isotype of antibodies and provides a first line of defense at the mucosa against pathogens invading the host. It has been widely accepted that the mucosal IgA response provided by vaccination requires mucosal inoculation, and intranasal inoculation has been proposed for vaccines against influenza virus. Considering the difficulty of intranasal vaccination in infants or elderly people, however, parenteral vaccination that provides the mucosal IgA response is desirable. Here, we demonstrate that subcutaneous immunization with zymosan, a yeast cell wall constituent known to be recognized by Dectin-1 and TLR2, potentiates the production of antigen-specific IgA antibodies in the sera and airway mucosa upon intranasal antigen challenge. We confirmed that the antigen-specific IgA-secreting cells accumulated in the lung and nasal-associated lymphoid tissues after the antigen challenge. Such an adjuvant effect of zymosan in the primary immunization for the IgA response depended on Dectin-1 signaling, but not on TLR2. The IgA response to the antigen challenge required both antigen-specific memory B and T cells, and the generation of memory T cells, but not memory B cells, depended on zymosan as an adjuvant. Finally, we demonstrated that subcutaneous inoculation of inactivated influenza virus with zymosan, but not with alum, mostly protected the mice from infection with a lethal dose of a heterologous virus strain. These data suggest that zymosan is a possible adjuvant for parenteral immunization that generates memory IgA responses to respiratory viruses such as influenza virus.

The immunopathogenic mechanisms underlying immunoglobulin A nephropathy (IgAN) are poorly understood, yet it is one of the most common causes of kidney failure globally. The commonly referenced syndrome of synpharyngitic gross hematuria as a presenting feature of IgAN has led to a logical association between infections and development of IgAN, however no pathogenic organism has been clearly linked to IgAN. Advances in sequencing technology have enabled more detailed characterization of host microbial communities, and highlighted the interrelationship between microbiota and immune responses in health and disease. This review will summarize current thinking on the relationship between microbiota and development of IgAN with a focus on recent studies relating aberrant mucosal IgA-biased immune responses to microbiota and how this may be related to the immunopathogenesis of IgAN.

Foxp3+ regulatory T (Treg) cells prevent excessive immune responses against dietary antigens and commensal bacteria in the intestine. Moreover, Treg cells contribute to the establishment of a symbiotic relationship between the host and gut microbes, partly through immunoglobulin A. However, the mechanism by which Treg cell dysfunction disturbs the balanced intestinal microbiota remains unclear. In this study, we used Foxp3 conditional knockout mice to conditionally ablate the Foxp3 gene in adult mice and examine the relationship between Treg cells and intestinal bacterial communities. Deletion of Foxp3 reduced the relative abundance of Clostridia, suggesting that Treg cells have a role in maintaining Treg-inducing microbes. Additionally, the knockout increased the levels of fecal immunoglobulins and immunoglobulin-coated bacteria. This increase was due to immunoglobulin leakage into the gut lumen as a result of loss of mucosal integrity, which is dependent on the gut microbiota. Our findings suggest that Treg cell dysfunction leads to gut dysbiosis via aberrant antibody binding to the intestinal microbes.

Upon antigen stimulation, IgG+ B cells rapidly proliferate and differentiate into plasma cells, which has been attributed to the characteristics of membrane-bound IgG (mIgG), but the underlying molecular mechanisms remain elusive. We have found that a part of mouse mIgG1 is ubiquitinated through the two responsible lysine residues (K378 and K386) in its cytoplasmic tail and this ubiquitination is augmented upon antigen stimulation. The ubiquitination of mIgG1 involves its immunoglobulin tail tyrosine (ITT) motif, Syk/Src-family kinases and Cbl proteins. Analysis of a ubiquitination-defective mutant of mIgG1 revealed that ubiquitination of mIgG1 facilitates its ligand-induced endocytosis and intracellular trafficking from early endosome to late endosome, and also prohibits the recycling pathway, thus attenuating the surface expression level of mIgG1. Accordingly, ligation-induced activation of B-cell receptor (BCR) signalling molecules is attenuated by the mIgG1 ubiquitination, except MAP kinase p38 whose activation is up-regulated due to the ubiquitination-mediated prohibition of mIgG1 recycling. Adaptive transfer experiments demonstrated that ubiquitination of mIgG1 facilitates expansion of germinal centre B cells. These results indicate that mIgG1-mediated signalling and cell activation is regulated by ubiquitination of mIgG1, and such regulation may play a role in expansion of germinal centre B cells.

The immunopathogenic mechanisms underlying immunoglobulin A nephropathy (IgAN) are poorly understood, yet it is one of the most common causes of kidney failure globally. The commonly referenced syndrome of synpharyngitic gross hematuria as a presenting feature of IgAN has led to a logical association between infections and development of IgAN, however no pathogenic organism has been clearly linked to IgAN. Advances in sequencing technology have enabled more detailed characterization of host microbial communities, and highlighted the interrelationship between microbiota and immune responses in health and disease. This review will summarize current thinking on the relationship between microbiota and development of IgAN with a focus on recent studies relating aberrant mucosal IgA-biased immune responses to microbiota and how this may be related to the immunopathogenesis of IgAN.

Antigens (Ags) with multivalent and repetitive structure elicit IgG production in a T cell-independent manner. However, the mechanisms by which such T cell-independent type-2 (TI-2) Ags induce IgG responses remain obscure. Here we report that BCR engagement with a TI-2 Ag but not with a T cell-dependent (TD) Ag was able to induce the transcription ofactivation-induced cytidine deaminase(AID) and efficient class switching to IgG3 upon co-stimulation with IL-1 or IFN-α. TI-2 Ags strongly induced the phosphorylation of protein kinase C (PKC)δ and PKCδ mediated theAIDtranscription through the induction of BATF, the key transcriptional regulator of AID expression. In PKCδ-deficient mice, production of IgG was intact against TD Ag but abrogated against typical TI-2 Ags as well as commensal bacteria, and experimental disruption of the gut epithelial barrier resulted in fatal bacteremia. Thus, our results revealed novel molecular requirements for class-switching in the TI-2 response and highlighted its importance in homeostatic commensal-specific IgG production.

Although eosinophils are important contributors to mucosal immune responses, mechanisms that regulate their accumulation in mucosal-associated lymphoid tissues remain ill-defined. Combining bone marrow chimeras and pharmacological inhibition approaches, here we find that lymphotoxin-beta receptor (LTβR) signaling during the neonatal period is required for the accumulation of eosinophils in the mesenteric lymph nodes (MLN) during an enteric viral infection in adult male and female mice. We demonstrate that MLN stromal cells express genes that are important for eosinophil migration and survival, such as Ccl-11 (eotaxin-1), Ccl7, Ccl9, and Cxcl2, and that expression of most of these genes is downregulated as a consequence of neonatal LTβR blockade. We also find that neonatal LTβR signaling is required for the generation of a rotavirus-specific IgA antibody response in the adult MLN, but eosinophils are dispensable for this response. Collectively, our studies reveal a role for neonatal LTβR signaling in regulating eosinophil numbers in the adult MLN.

Memory B cells (Bmem cells) are the basis of long-lasting humoral immunity. They respond to re-encountered antigens by rapidly producing specific antibodies and forming germinal centers (GCs), a recall response that has been known for decades but remains poorly understood. We found that the receptor for the cytokine IL-9 (IL-9R) was induced selectively on Bmem cells after primary immunization and that IL-9R-deficient mice exhibited a normal primary antibody response but impaired recall antibody responses, with attenuated population expansion and plasma-cell differentiation of Bmem cells. In contrast, there was augmented GC formation, possibly due to defective downregulation of the ligand for the co-stimulatory receptor ICOS on Bmem cells. A fraction of Bmem cells produced IL-9. These findings indicate that IL-9R signaling in Bmem cells regulates humoral recall responses.

The germinal center (GC) reaction is essential for long-lived humoral immunity. However, molecular requirements for the induction of Bcl6, the master regulator for GC B cell differentiation, remain unclear. Through screening for cytokines and other stimuli that regulate Bcl6 expression, we identified IL-4 as the strongest inducer. IL-4-signaling altered the metabolomic profile in activated B cells and induced accumulation of the TCA cycle intermediate α-ketoglutarate (αKG), which was required for epigenetic activation of the Bcl6 gene locus. Mechanistically, after IL-4-treatment, STAT6 bound to the known enhancers in the Bcl6 locus and recruited UTX, a demethylase for the repressive histone mark H3K27me3 that requires αKG as a cofactor. In turn, the H3K27me3 demethylation activated the enhancers and transcription of the Bcl6 gene. We propose that IL-4-medicated metabolic reprogramming in B cells is the key factor that regulates epigenetic activation of the master regulator of GC B cell differentiation.

The germinal center (GC) is the site where B cells undergo clonal expansion, affinity-based selection, and differentiation into memory B cells or plasma cells. It has been difficult to elucidate regulatory mechanisms for the dynamic GC B cell maturation and differentiation, partly because experimental manipulation of GC B cells in vivo has been limited and no in vitro system has been available that resembles B cell reaction in GC. Here we describe the protocol for a culture system named "induced GC B (iGB) culture system" which can induce massive expansion of B cells that exhibit GC B cell-like phenotype, and thus it mimics the GC reaction. This protocol can be useful to elucidate the molecular mechanisms of GC B cell differentiation.

In germinal centers (GCs), B cells undergo repeated cycles of proliferation and affinity-based selection, and differentiate into memory B cells or long-lived plasma cells. It has been difficult to elucidate regulatory mechanisms for the dynamic GC B cell maturation and differentiation, partly because experimental manipulation of GC B cells has been limited. Here we describe a culture system in which we can induce massive expansion of naive B cells that exhibit GC B cell-like phenotype and acquire abilities to differentiate into memory B cells or bone marrow plasma cells depending on cytokine conditions. This system will allow us to elucidate the molecular mechanisms of GC B cell differentiation.

The rapid Ab responses observed after primary and secondary immunizations are mainly derived from marginal zone (MZ) and memory B cells, respectively, but it is largely unknown how these responses are negatively regulated. Several inhibitory receptors have been identified and their roles have been studied, but mainly on follicular B cells and much less so on MZ B, and never on memory B cells. gp49B is an Ig superfamily member that contains two ITIMs in its cytoplasmic tail, and it has been shown to negatively regulate mast cell, macrophage, and NK cell responses. In this study, we demonstrate that gp49B is preferentially expressed on memory and MZ B cells. We show that gp49B−/− mice produce more IgM after a primary immunization and more IgM and IgG1 after a secondary immunization than gp49B+/+ mice in T cell–dependent immune responses. Memory and MZ B cells from gp49B−/− mice also produce more Abs upon in vitro stimulation with CD40 than those from gp49B+/+ mice. The in vitro IgM production by MZ B cells from gp49B+/+, but not gp49B−/−, mice is suppressed by interaction with a putative gp49B ligand, the integrin αvβ3 heterodimer. In addition, gp49B−/− mice exhibited exaggerated IgE production in the memory recall response. These results suggest that plasma cell development from memory and MZ B cells, as well as subsequent Ab production, are suppressed via gp49B. In memory B cells, this suppression also prevents excessive IgE production, thus curtailing allergic diseases.

In germinal centers (GCs), B cells undergo repeated cycles of proliferation and affinity-based selection, and differentiate into memory B cells or long-lived plasma cells. It has been difficult to elucidate regulatory mechanisms for the dynamic GC B cell maturation and differentiation, partly because experimental manipulation of GC B cells has been limited. Here we describe a culture system in which we can induce massive expansion of naive B cells that exhibit GC B cell-like phenotype and acquire abilities to differentiate into memory B cells or bone marrow plasma cells depending on cytokine conditions. This system will allow us to elucidate the molecular mechanisms of GC B cell differentiation.

Memory B cells generated during a T cell-dependent immune response rapidly respond to a secondary immunization by producing abundant IgG Abs that bind cognate Ag with high affinity. It is currently unclear whether this heightened recall response by memory B cells is due to augmented IgG-BCR signaling, which has only been demonstrated in the context of naive transgenic B cells. To address this question, we examined whether memory B cells can respond in vivo to Ags that stimulate only through BCR, namely T cell-independent type II (TI-II) Ags. In this study, we show that the TI-II Ag (4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll cannot elicit the recall response in mice first immunized with the T cell-dependent Ag NP-chicken γ-globulin. Moreover, the NP-Ficoll challenge in vivo as well as in vitro significantly inhibits a subsequent recall response to NP-chicken γ-globulin in a B cell-intrinsic manner. This NP-Ficoll–mediated tolerance is caused by the preferential elimination of IgG+ memory B cells binding to NP with high affinity. These data indicate that BCR cross-linking with a TI-II Ag does not activate IgG+ memory B cells, but rather tolerizes them, identifying a terminal checkpoint of memory B cell differentiation that may prevent autoimmunity.

Aberrant production of IgE antibodies can lead to allergic diseases. Normally, IgE(+) B cells rarely differentiate into memory B cells (Bmem) or long-lived plasma cells (LLPCs), as they only transiently participate in the germinal center (GC), but the mechanism behind this remains elusive. We found that membrane IgE (mIgE) autonomously triggered rapid plasma-cell differentiation and apoptosis independently of antigen or cellular context, predominantly through the mutually independent CD19-PI3K-Akt-IRF4 and BLNK-Jnk/p38 pathways, respectively, and we identified the ectodomains of mIgE as being responsible. Accordingly, deregulated GC IgE(+) B cell proliferation and prolonged IgE production with exaggerated anaphylaxis were observed in CD19- and BLNK-deficient mice. Our findings reveal an autonomous mIgE signaling mechanism that normally prevents IgE(+) Bmem and LLPC formation, providing insights into the molecular pathogenesis of allergic diseases.

In response to T cell-dependent antigens, B cells proliferate extensively to form germinal centres (GC), and then differentiate into memory B (B(mem)) cells or long-lived plasma cells (LLPCs) by largely unknown mechanisms. Here we show a new culture system in which mouse naive B cells undergo massive expansion and isotype switching, and generate GC-phenotype B (iGB) cells. The iGB cells expressing IgG1 or IgM/D, but not IgE, differentiate into B(mem) cells in vivo after adoptive transfer and can elicit rapid immune responses with the help of cognate T cells. Secondary culture with IL-21 maintains the proliferation of the iGB cells, while shifting their in vivo developmental fate from B(mem) cells to LLPCs, an outcome that can be reversed by withdrawal of IL-21 in tertiary cultures. Thus, this system enables in vitro manipulation of B-cell fate, into either B(mem) cells or LLPCs, and will facilitate dissection of GC-B cell differentiation programs.