Cell-dependent Humoral Responses Research Articles

A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation

Plasma cells (PC) are antibody-secreting cells and terminal effectors in humoral responses. PCs differentiate directly from activated B cells in response to T cell-independent (TI) antigens or from germinal center B (GCB) cells in T cell-dependent (TD) antigen-induced humoral responses, both of which pathways are essentially regulated by the transcription factor BLIMP1. The p38 mitogen-activated protein kinase isoforms have already been implicated in B cell development, but the precise role of p38α in B cell differentiation is still largely unknown. Here we show that PC differentiation and antibody responses are severely impaired in mice with B cell-specific deletion of p38α, while B cell development and the GCB cell response are spared. By utilizing a Blimp1 reporter mouse model, we show that p38α-deficiency results in decreased BLIMP1 expression. p38α-driven BLIMP1 up-regulation is required for both TI and TD PCs differentiation. By combining CRISPR/Cas9 screening and other approaches, we identify TCF3, TCF4 and IRF4 as downstream effectors of p38α to control PC differentiation via Blimp1 transcription. This study thus identifies an important signalling pathway underpinning PC differentiation upstream of BLIMP1, and points to a highly specialized and non-redundant role for p38α among p38 isoforms.

Exploring the mechanism of berberine-mediated Tfh cell immunosuppression

BackgroundOur previous research revealed a novel function of berberine (BBR), a clinically relevant plant-derived alkaloid, as a suppressor of follicular T helper (Tfh) cell proliferation in secondary lymphoid organs of BBR-treated mice that underwent immunization for collagen-induced arthritis (CIA) in DBA1/J mice. Due to the importance of Tfh cell and B cell interactions in the generation of T cell-dependent humoral responses, the suppression of Tfh cell activity may have implications for the general safety of BBR as a prophylactic dietary supplement, and its potential use in antibody-driven autoimmune and hypersensitivity disorders. PurposeThis research aims to characterize BBR's impact on the activation, differentiation, and proliferation of Tfh cells by examining the expression of key extracellular signaling molecules, as well as the activity of intracellular signaling molecules involved in the Ca2+-calcineurin-NFAT pathway and STAT3 phosphorylation, following activation. Study designIn vitro experimental study using primary tissues. MethodsTo explore the direct effects of BBR on the proliferation and differentiation of Tfh cells, isolated naïve CD4+ T cells (>95% pure) were activated and differentiated into pre- Tfh cells in the presence or absence of BBR. The resulting Tfh cell populations and the expression of the key extracellular molecules CXCR5, ICOS, and PD-1 were measured. In addition, we examined the impact of BBR treatment on the activity of key intracellular signaling molecules involved in Tfh cell activation and differentiation following TCR ligation and/or CD28 signaling (p-ZAP-70, p-Lck, p-PLCγ1, NFATc1 and intracellular calcium, Ca2+, concentrations), as well as IL-6 signaling (p-STAT3). ResultsTreatment with BBR significantly reduced the expression of both CXCR5 (p < 0.01) and ICOS (p < 0.005), but not PD-1, and reduced the percentage of Tfh cells within the total CD4+ T cell population. BBR treatment also led to a reduction in intracellular Ca2+ flux, activation of p-STAT3, and IL-21 production. ConclusionOur observations provide insight into the mechanism of BBR-mediated Tfh cell suppression and suggest that BBR treatment can directly inhibit Tfh cell activity, perhaps through interfering with cytokine receptor or downstream signaling.

Assessment of SARS-CoV-2 Immunity in Convalescent Children and Adolescents.

Persistence of protective immunity for SARS-CoV-2 is important against reinfection. Knowledge on SARS-CoV-2 immunity in pediatric patients is currently lacking. We opted to assess the SARS-CoV-2 adaptive immunity in recovered children and adolescents, addressing the pediatrics specific immunity towards COVID-19. Two independent assays were performed to investigate humoral and cellular immunological memory in pediatric convalescent COVID-19 patients. Specifically, RBD IgG, CD4+, and CD8+ T cell responses were identified and quantified in recovered children and adolescents. SARS-CoV-2-specific RBD IgG detected in recovered patients had a half-life of 121.6 days and estimated duration of 7.9 months compared with baseline levels in controls. The specific T cell response was shown to be independent of days after diagnosis. Both CD4+ and CD8+ T cells showed robust responses not only to spike (S) peptides (a main target of vaccine platforms) but were also similarly activated when stimulated by membrane (M) and nuclear (N) peptides. Importantly, we found the differences in the adaptive responses were correlated with the age of the recovered patients. The CD4+ T cell response to SARS-CoV-2 S peptide in children aged <12 years correlated with higher SARS-CoV-2 RBD IgG levels, suggesting the importance of a T cell-dependent humoral response in younger children under 12 years. Both cellular and humoral immunity against SARS-CoV-2 infections can be induced in pediatric patients. Our important findings provide fundamental knowledge on the immune memory responses to SARS-CoV-2 in recovered pediatric patients.

Regeneration of immunocompetent B lymphopoiesis from pluripotent stem cells guided by transcription factors

Regeneration of functional B lymphopoiesis from pluripotent stem cells (PSCs) is challenging, and reliable methods have not been developed. Here, we unveiled the guiding role of three essential factors, Lhx2, Hoxa9, and Runx1, the simultaneous expression of which preferentially drives B lineage fate commitment and in vivo B lymphopoiesis using PSCs as a cell source. In the presence of Lhx2, Hoxa9, and Runx1 expression, PSC-derived induced hematopoietic progenitors (iHPCs) immediately gave rise to pro/pre-B cells in recipient bone marrow, which were able to further differentiate into entire B cell lineages, including innate B-1a, B-1b, and marginal zone B cells, as well as adaptive follicular B cells. In particular, the regenerative B cells produced adaptive humoral immune responses, sustained antigen-specific antibody production, and formed immune memory in response to antigen challenges. The regenerative B cells showed natural B cell development patterns of immunoglobulin chain switching and hypermutation via cross-talk with host T follicular helper cells, which eventually formed T cell-dependent humoral responses. This study exhibits de novo evidence that B lymphopoiesis can be regenerated from PSCs via an HSC-independent approach, which provides insights into treating B cell-related deficiencies using PSCs as an unlimited cell resource.

Exopolysaccharide from Lactobacillus rhamnosus KL37 Inhibits T Cell-dependent Immune Response in Mice

Exopolysaccharides (EPSs), major components of the bacterial biofilm, display strong strain-specific immunomodulatory properties. Previously, we have shown that crude EPS derived from Lactobacillus rhamnosus KL37 depresses the production of arthritogenic anti-collagen IgG and ameliorates collagen-induced arthritis (CIA) in DBA/1 mice, when lipopolysaccharide (LPS) was used as adjuvant. In this study, we used highly purified EPS from L. rhamnosus KL37 (EPS-37) to verify its anti-inflammatory properties and the ability to suppress T cell-dependent humoral response. We have employed the model of active CIA, in which mice immunized with type II collagen (CII) along with LPS were treated with pure EPS-37. Intravenous administration of purified EPS-37 markedly ameliorated arthritis and reduced CII-specific antibody production. EPS-37 injected subcutaneously reduced the clinical symptoms of CIA but without the reduction of arthritogenic antibodies. In addition, the effect of EPS-37 on T-cell functions was tested ex vivo and in vitro. EPS-37 inhibited the in vitro proliferation of T cells activated both in vivo (CII immunization) and in vitro (antigen/mitogen), and markedly reduced the production of interferon (IFN)-γ. These results together with other reports suggest that anti-inflammatory potential of EPS-37 depends on its ability to inhibit either one or the other or both possible inflammatory signaling pathways. Namely, Th1 → IFN-γ → M1 inflammatory macrophages → arthritis and/or Th1 → IFN-γ → B cells → arthritogenic antibodies → arthritis. We suggest that L. rhamnosus KL37 EPS might be utilized to control T cell-dependent immune responses in various inflammatory diseases. However, the most effective route of EPS-37 administration needs to be tailored for a given disorder.

Role of TRAFs in Signaling Pathways Controlling T Follicular Helper Cell Differentiation and T Cell-Dependent Antibody Responses.

Follicular helper T (TFH) cells represent a highly specialized CD4+ T cell subpopulation that supports the generation of germinal centers (GC) and provides B cells with critical signals promoting antibody class switching, generation of high affinity antibodies, and memory formation. TFH cells are characterized by the expression of the chemokine receptor CXCR5, the transcription factor Bcl-6, costimulatory molecules ICOS, and PD-1, and the production of cytokine IL-21. The acquisition of a TFH phenotype is a complex and multistep process that involves signals received through engagement of the TCR along with a multitude of costimulatory molecules and cytokines receptors. Members of the Tumor necrosis factor Receptor Associated Factors (TRAF) represent one of the major classes of signaling mediators involved in the differentiation and functions of TFH cells. TRAF molecules are the canonical adaptor molecules that physically interact with members of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF) and actively modulate their downstream signaling cascades through their adaptor function and/or E3 ubiquitin ligase activity. OX-40, GITR, and 4-1BB are the TRAF-dependent TNFRSF members that have been implicated in the differentiation and functions of TFH cells. On the other hand, emerging data demonstrate that TRAF proteins also participate in signaling from the TCR and CD28, which deliver critical signals leading to the differentiation of TFH cells. More intriguingly, we recently showed that the cytoplasmic tail of ICOS contains a conserved TANK-binding kinase 1 (TBK1)-binding motif that is shared with TBK1-binding TRAF proteins. The presence of this TRAF-mimicking signaling module downstream of ICOS is required to mediate the maturation step during TFH differentiation. In addition, JAK-STAT pathways emanating from IL-2, IL-6, IL-21, and IL-27 cytokine receptors affect TFH development, and crosstalk between TRAF-mediated pathways and the JAK-STAT pathways can contribute to generate integrated signals required to drive and sustain TFH differentiation. In this review, we will introduce the molecular interactions and the major signaling pathways controlling the differentiation of TFH cells. In each case, we will highlight the contributions of TRAF proteins to these signaling pathways. Finally, we will discuss the role of individual TRAF proteins in the regulation of T cell-dependent humoral responses.

Correction: Anti-TNF treatment blocks the induction of T cell-dependent humoral responses.

Correction: Anti-TNF treatment blocks the induction of T cell-dependent humoral responses.

X-Box Binding Protein-1 Dependent Plasma Cell Responses Limit the Development of Atherosclerosis.

Diverse B cell responses and functions may be involved in atherosclerosis. Protective antibody responses, such as those against oxidized lipid epitopes, are thought to mainly derive from T cell-independent innate B cell subsets. In contrast, both pathogenic and protective roles have been associated with T cell-dependent antibodies, and their importance in both humans and mouse models is still unclear. To specifically target antibody production by plasma cells and determine the impact on atherosclerotic plaque development in mice with and without CD4+ T cells. We combined a model of specific antibody deficiency, B cell-specific CD79a-Cre x XBP1 (X-box binding protein-1) floxed mice (XBP1-conditional knockout), with antibody-mediated depletion of CD4+ T cells. Ldlr knockout mice transplanted with XBP1-conditional knockout (or wild-type control littermate) bone marrow were fed western diet for 8 weeks with or without anti-CD4 depletion. All groups had similar levels of serum cholesterol. In Ldlr/XBP1-conditional knockout mice, serum levels of IgG, IgE, and IgM were significantly attenuated, and local antibody deposition in atherosclerotic plaque was absent. Antibody deficiency significantly accelerated atherosclerosis at both the aortic root and aortic arch. T cell and monocyte responses were not modulated, but necrotic core size was greater, even when adjusting for plaque size, and collagen deposition significantly lower. Anti-CD4 depletion in Ldlr/wild-type mice led to a decrease of serum IgG1 and IgG2c but not IgG3, as well as decreased IgM, associated with increased atherosclerosis and necrotic cores, and a decrease in plaque collagen. The combination of antibody deficiency and anti-CD4 depletion has no additive effects on aortic root atherosclerosis. The endogenous T cell-dependent humoral response can be protective. This has important implications for novel vaccine strategies for atherosclerosis and in understanding the impacts of immunotherapies used in patients at high risk for cardiovascular disease.

Generation of a soluble recombinant trimeric form of bovine CD40L and its potential use as a vaccine adjuvant in cows

Vaccination is the most cost-effective way to control infectious diseases in cattle. However, many infectious diseases leading to severe economical losses worldwide still remain for which a really effective and safe vaccine is not available. These diseases are most often due to intracellular pathogens such as bacteria or viruses, which are, by their localization, protected from antibiotics and/or CD4+ T cell-dependent humoral responses. We therefore postulated that strategies leading to induction of not only CD4+ T cell responses but also CD8+ cytotoxic T lymphocyte (CTL) responses against infected cells should be privileged in the development of new vaccines against problematic intracellular pathogens in bovines. CD40 signaling in antigen-presenting cells may lead to the induction of robust CD4-independent CTL responses and several studies, especially in mice, have used CD40 stimulation to promote CD8+ T cell-mediated immunity. For example, we have recently shown that immunization of mice with heat-killed Staphylococcus aureus (HKSA) and agonistic anti-CD40 monoclonal antibodies elicits strong CTL responses capable of protecting mice from subsequent staphylococcal mastitis. Unfortunately, there is at present no tool available to efficiently stimulate CD40 in cattle. In this study, we therefore first produced a soluble recombinant trimeric form of the natural bovine CD40 ligand (sboCD40LT). We then observed that sboCD40LT was able to potently stimulate bovine cells in vitro. Finally, we provide evidence that immunization of cows with sboCD40LT combined with HKSA was able to significantly increase the number of both HKSA-specific CD4+ and CD8+ T cells in the draining lymph nodes. In conclusion, we suggest that this new molecular tool could help in the development of vaccine strategies against bovine diseases caused by intracellular pathogens.

Hybrid flagellin as a T cell independent vaccine scaffold.

BackgroundTo extend the potency of vaccines against infectious diseases, vaccines should be able to exploit multiple arms of the immune system. One component of the immune system that is under-used in vaccine design is the subset of B cells known to be capable of responding to repetitive antigenic epitopes and differentiate into plasma cells even in the absence of T cell help (T-independent, TI).ResultsTo target vaccine responses from T-independent B cells, we reengineered a bacterial Flagellin (FliC) by replacing its exposed D3 domain with a viral envelope protein from Dengue virus (DENV2). The resulting hybrid FliC protein (hFliC) was able to form stable filaments decorated with conformationally intact DENV2 envelope domains. These filaments were not only capable of inducing a T cell-dependent (TD) humoral antibody response, but also significant IgM and IgG3 antibody response in a helper T cell repertoire-restricted transgenic mouse model.ConclusionsOur results provide proof-of-principle demonstration that a reengineered hybrid FliC could be used as a platform for polymeric subunit vaccines, enhancing T cell-dependent and possibly inducing T-independent antibody responses from B-1 B cells as well.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0194-0) contains supplementary material, which is available to authorized users.

Aryl hydrocarbon receptor modulates the development and activity of innate-like B cells (HEM1P.226)

Abstract The Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that recognizes polyaromatic hydrocarbons. While the contributions of AhR ligands to the differentiation and functions of Th17/Treg cells are relatively well understood, little is known about AhR in B cells. Microarray and quantitative PCR analysis of B cell subsets revealed that B1 and marginal zone (MZ) B cells expressed higher levels of AhR compared to follicular (FO) B cells. We found ~ two-fold more B1 B cells, but fewer MZ B cells in AhR-/- mice compared to AhR+/+ mice. Following immunization with Streptococcus pneumonia, AhR-/- mice had significantly higher titers of anti-phosphorylcholine IgM antibodies. In contrast, after immunization with a model T cell-dependent antigen, Trinitrophenyl Keyhole Limpet Hemocyanin (TNP-KLH), AhR-/- mice had anti-TNP titers that were comparable to those in AhR+/+ mice. Further, we found significantly fewer B cells in C57BL/6 mice raised on AhR ligand free diet, and an increase in titers of IgM and IgG3 in mice following a switch to an AhR ligand free diet when compared to littermates raised and maintained on conventional diet. Taken together, these findings suggests that AhR plays a role in the development of B1 and MZ B cells and regulates T cell-independent but not T cell-dependent humoral response. Further studies using mice with a B cell specific deletion of AhR are underway to elucidate the mechanism by which AhR ligands influence humoral immunity.

Role of CD154 in cancer pathogenesis and immunotherapy

Many factors and molecules have been investigated as potential players in the pathogenesis or immunosurveillance of cancer. Among these, CD154 has been recognized as a co-stimulatory molecule with high potential for treating cancer, in addition to its contribution in the development of the disease. CD154 was initially described for its pivotal role in T cell-dependent humoral responses via an interaction with its classical receptor, CD40. Subsequent studies showed that CD154 is also implicated in cell-mediated immunity and inflammation via an interaction with CD40 alone or in combination with newly identified receptors, members of the integrin family, leading to the development of chronic inflammatory and autoimmune diseases. In the current article, we present an overview of the role of CD154 as a potential etiological factor in tumors inducing proliferation of malignant cells, their rescue from apoptosis and their invasiveness. In addition, this review describes the immuno-regulatory functions of CD154 against cancer reflected by its stimulation of antigen-presenting cells and the subsequent activation of effector cells, its enhancement of malignant cells' immunogenicity, its modulation of immune settings around tumors, and its initiation of proliferation inhibiting effects in malignant cells. In vitro as well as in vivo studies are outlined and a particular attention is given to clinical studies and progress reached at this point. Findings reviewed herein will improve our knowledge of the role of the CD154 system in cancers from causative to immunotherapeutic functions, paving the way for the identification of new targets for prevention and/or treatment of malignant disorders.

AB0093 Study of B-cell activation factor (BAFF) and proliferation-inducing ligand (april) in rheumatoid arthritis patients

BackgroundThe TNF superfamily member B lymphocyte stimulator (BLyS), referred to as BAFF, is known to be an effective modulator of peripheral B cell homeostasis that promotes B cell survival and...

The TNF family member APRIL dampens collagen-induced arthritis

BackgroundThe tumour necrosis factor (TNF)-family members B cell activating factor (BAFF) and A PRoliferation-Inducing Ligand (APRIL) play important roles in B cell biology, and share binding to B cell maturation...

Anti-TNF treatment blocks the induction of T cell-dependent humoral responses

ObjectiveExperimental and human data suggest that tumour necrosis factor (TNF) blockade may affect B cell responses, in particular the induction of T cell-dependent (TD) humoral immunity. This study aimed to...

TNF blockade abrogates the induction of T cell-dependent humoral responses in an allotransplantation model

TNF blockade modulates many aspects of the immune response and is commonly used in a wide array of immune-mediated inflammatory diseases. As anti-TNF induces anti-dsDNA IgM antibodies but not other antinuclear reactivities in human arthritis, we investigated here the effect of TNF blockade on the induction of TD humoral responses using cardiac allograft and xenograft models. A single injection of an anti-rat TNF antibody in LEW.1A recipients grafted with congenic LEW.1W hearts almost completely abrogated the induction of IgM and IgG alloantibodies. This was associated with decreased Ig deposition and leukocyte infiltration in the graft at Day 5. TNF blockade did not affect germinal-center formation in the spleen or expression of Th1/Th2 cytokines, costimulatory and regulatory molecules, and TLRs in spleen and graft of the recipient animals. Clinically, the abrogation of the induction of the alloantibodies was associated with a marked prolongation of graft survival. In contrast, anti-TNF did not alter acute xenograft rejection mediated by TI antibodies in a hamster-to-rat model. Taken together, these data indicate that TNF blockade abrogates the induction of TD humoral responses and accordingly, may have a beneficial effect in antibody-mediated inflammatory pathologies.

Modulation of humoral immune responses as a novel mechanism of action of tumour necrosis factor blockade

Based on our observations of IgM but not IgG anti-dsDNA antibody induction in patients treated with tumour necrosis factor (TNF) blockers, we proposed the hypothesis that TNF blockade interferes with...

Induction of Autoantibody-Mediated Spontaneous Arthritis Critically Depends on Follicular Dendritic Cells

Follicular dendritic cells (FDCs) are important for the induction of protective T cell-dependent humoral responses, but their contribution to autoimmunity remains elusive. Here, gene-targeted interruption of FDC development was combined with the K/BxN mouse model of arthritis. We found that FDCs were essential for autoantibody production through two distinct but cooperative functions. In a T cell-independent fashion, FDCs loaded with autoantigen-containing immune complexes supported germinal center (GC) B cell development. Additionally, the integrity of FDC networks was required for the recruitment of arthritogenic follicular helper T cells, a process that drove T-B cell interactions and productive GC reactivity. Importantly, pharmacological interference in the maintenance of FDCs ameliorated disease development, suggesting the FDC as a potential target for dampening autoimmunity.

Prevention of Autoimmunity and Control of Recall Response to Exogenous Antigen by Fas Death Receptor Ligand Expression on T Cells

Mice with mutations in the gene encoding Fas ligand (FasL) develop lymphoproliferation and systemic autoimmune diseases. However, the cellular subset responsible for the prevention of autoimmunity in FasL-deficient mice remains undetermined. Here, we show that mice with FasL loss on either B or T cells had identical life span as littermates, and both genotypes developed signs of autoimmunity. In addition, we show that T cell-dependent death was vital for the elimination of aberrant T cells and for controlling the numbers of B cells and dendritic cells that dampen autoimmune responses. Furthermore, we show that the loss of FasL on T cells affected the follicular dentritic cell network in the germinal centers, leading to an impaired recall response to exogenous antigen. These results disclose the distinct roles of cellular subsets in FasL-dependent control of autoimmunity and provide further insight into the role of FasL in humoral immunity.

Random outcomes of allergen-specific responses in atopic families.

Allergens are common non-infectious antigens to which people will mount T cell dependent humoral responses. Among genetically susceptible individuals, an antigen-specific response results involving the production of allergen-specific IgE (atopy). Determine if this susceptibility is manifested as an inherited, allergen-specific trait or a random response to allergens among susceptible people. We evaluated allergen-specific outcomes in 1099 members of families with positive atopic history (26 multi-generation and 112 nuclear families). Each was tested for sensitivity to 14 common allergens by standardized skin prick test (SPT), a marker of specific IgE production. Over 15,000 individual SPT's were evaluated. Among five randomly selected multi-generation families (N=163), semi-quantitative determinations of Amb a 1-specific IgA1,2 and IgG1-4 were determined in three groups: (A) Amb a SPT(+)/Amb a 1-IgE(+), (B) Amb a SPT(-)/Amb a 1-IgE(+), (C) Amb a SPT(-)/Amb a 1-IgE(-). By rank correlation statistics, there were no discernible 'patterns' of specific SPT outcomes among any of the multi-generation families, suggesting that environmental exposure rather than allergen-specific inheritance determined the responses. This was confirmed among the nuclear families since the conditional SPT outcomes among children were independent of the SPT responses of their parents. Among five randomly selected multi-generation families, the relative proportionate concentrations of the Amb a 1-specific IgA and IgG subclasses were comparable, regardless of atopic sensitization to the ragweed allergen Amb a. While the general propensity for atopy may be inherited, an individual's specific atopic outcome is a random variable independent of familial sensitization patterns.