Search

Differentiation of B cells into antibody-secreting cells (ASCs) is a key process to generate protective humoral immunity. A detailed understanding of the cues controlling ASC differentiation is important to devise strategies to modulate antibody formation. Here, we dissected differentiation trajectories of human naive B cells into ASCs using single-cell RNA sequencing. By comparing transcriptomes of B cells at different stages of differentiation from an in vitro model with ex vivo B cells and ASCs, we uncovered a novel pre-ASC population present ex vivo in lymphoid tissues. For the first time, a germinal-center-like population is identified in vitro from human naive B cells and possibly progresses into a memory B cell population through an alternative route of differentiation, thus recapitulating in vivo human GC reactions. Our work allows further detailed characterization of human B cell differentiation into ASCs or memory B cells in both healthy and diseased conditions.

Understanding the development and sustainability of the virus-specific protective immune response to infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains incomplete with respect to the appearance and disappearance of virus-specific antibody-secreting cells (ASCs) in circulation. Therefore, we performed cross-sectional and longitudinal analyses of peripheral blood mononuclear cells and plasma collected from 55 hospitalized patients up to 4 months after onset of COVID-19 symptoms. Spike (S)- and nucleocapsid (N)-specific IgM and IgG ASCs appeared within 2 weeks accompanied by flow cytometry increases in double negative plasmablasts consistent with a rapid extrafollicular B cell response. Total and virus-specific IgM and IgG ASCs peaked at 3–4 weeks and were still being produced at 3–4 months accompanied by increasing antibody avidity consistent with a slower germinal center B cell response. N-specific ASCs were produced for longer than S-specific ASCs and avidity maturation was greater for antibody to N than S. Patients with more severe disease produced more S-specific IgM and IgG ASCs than those with mild disease and had higher levels of N- and S-specific antibody. Women had more B cells in circulation than men and produced more S-specific IgA and IgG and N-specific IgG ASCs. Flow cytometry analysis of B cell phenotypes showed an increase in circulating B cells at 4–6 weeks with decreased percentages of switched and unswitched memory B cells. These data indicate ongoing antigen-specific stimulation, maturation, and production of ASCs for several months after onset of symptoms in patients hospitalized with COVID-19.

Background Memory B cells are an antigen-experienced B-cell population with the ability to rapidly differentiate into antibody-producing cells by recall responses. We recently found that dedicator of cytokinesis 11 (DOCK11) contributes to the expansion of antigen-specific populations among germinal center B cells upon immunization. In comparison, limited information is available on the contribution of DOCK11 to secondary humoral immune responses. Results In this study, effects of the DOCK11 deficiency in B cells were examined on secondary immune responses to protein antigen. The lack of DOCK11 in B cells resulted in the impaired induction of antibody-producing cells upon secondary immunization with protein antigen. DOCK11 was dispensable for the recall responses of antigen-experienced B cells, as demonstrated by the comparable induction of antibody-producing cells in mice given transfer of antigen-experienced B cells with no DOCK11 expression. Instead, the lack of DOCK11 in B cells resulted in the impaired secondary immune responses in a B cell-extrinsic manner, which was recovered by the adoptive transfer of cognate T cells. Conclusions We addressed that intrinsic and extrinsic effects of DOCK11 expression in B cells may contribute to secondary humoral immune responses in manner of the induction of cognate T-cell help.

A rare antibody that is able to tolerate physio-chemical factors is preferred and highly demanded in diagnosis and therapy. Rabbit monoclonal antibodies (RmAbs) are distinguished owing to their high affinity and stability. However, the efficiency and availability of traditional methods for RmAb discovery are limited, particularly for small molecules. Here, we present an indirect competitive screening method in nanowells, named CSMN, for single rabbit antibody-secreting cells (ASCs) selection with 20.6 h and propose an efficient platform for RmAb production against small molecules within 5.8 days for the first time. Chloramphenicol (CAP) as an antibacterial agent poses a great threat to public health. We applied CSMN to select CAP-specific ASCs and produced one high-affinity RmAb, surprisingly showed extremely halophilic properties with an IC

Central to protective humoral immunity is the activation of B cells and their terminal differentiation into antibody-secreting plasma cells. Long-lived plasma cells (LLPC) may survive for years to decades. Such long-lived plasma cells are also responsible for producing pathogenic antibodies that cause a variety of challenges such as autoimmunity, allograft rejection, and drug neutralization. Up to now, various therapeutic strategies aimed at durably eliminating pathogenic antibodies have failed, in large part due to their inability to efficiently target LLPCs. Several antibody-based therapies have recently gained regulatory approval or are in clinical phases of development for the treatment of multiple myeloma, a malignancy of plasma cells. We discuss the exciting potential of using these emerging cancer immunotherapies to solve the antibody problem.

OBJECTIVE: This study was undertaken to characterize kidney and urine antibody-secreting cells (ASCs) from patients with active lupus nephritis, before and after induction therapy. METHODS: We included patients with biopsy-proven active lupus nephritis and performed anti-CD138 staining of kidney biopsy samples to visualize ASCs. We performed single-cell gene expression profiling on sorted ASCs from fresh biopsy samples using multiplex reverse transcriptase-polymerase chain reaction. We used a gene set that allowed for the study of ASC maturation from plasmablasts to long-lived plasma cells. We quantified urine ASCs from untreated patients with lupus nephritis at diagnosis and after 6 months of prospective follow-up during induction therapy. RESULTS: The number of kidney CD138+ ASCs in 46 untreated patients with lupus nephritis was correlated with a low estimated glomerular filtration rate and with tubulointerstitial damage. Most kidney ASCs from 3 untreated patients had a plasmablast molecular signature; in contrast, in 4 patients with refractory lupus nephritis, the kidney ASCs were mainly long-lived plasma cells, representing an ASC transcriptional profile similar to that in the bone marrow of 2 healthy donors. Some urine ASCs with a plasmablast signature were detected in patients with untreated active lupus nephritis. The presence of urine ASCs at 6 months was associated with treatment failure. CONCLUSION: Our results suggest potential for ASC-directed therapy in refractory lupus nephritis.

Antibody secreting cells (ASCs) are considered work horses of the humoral immune response for their tireless effort to produce large amounts of antibodies that fulfill an array of functions in host defense, inflammation, and maintenance of homeostasis. While traditionally considered largely senescent cells, surprising recent findings demonstrate that subsets of ASCs downmodulate ongoing immune responses independent of antibody formation. Such regulatory ASCs produce IL-10 or IL-35 and are implicated in maintaining tissue and immune homeostasis. They also serve to suppress pathogenic leukocytes in infection, allergy, and inflammatory diseases that affect tissues, such as the central nervous system and the respiratory tract. Additionally, regulatory ASCs infiltrate various cancer types and restrict effective anti-tumor T cell responses. While incompletely understood, there is significant overlap in factors that control ASC differentiation, IL-10 expression by B cells and the generation of ASCs that secrete both antibodies and IL-10. In this review, we will cover the biology, phenotype, generation, maintenance and function of regulatory ASCs in various tissues under pathological and steady states. An improved understanding of the development of regulatory ASCs and their biological roles will be critical for generating novel ASC-targeted therapies for the treatment of inflammatory diseases, infection, and cancer.

Platelet transfusion and transplantation of allogeneic stem cells and solid organs are life-saving therapies. Unwanted alloantibodies to nonself human leukocyte antigens (HLAs) on donor cells increase the immunological barrier to these therapies and are important causes of platelet transfusion refractoriness and graft rejection. Although the specificities of anti-HLA antibodies can be determined at the allelic level, traditional treatments for antibody-mediated rejection nonselectively suppress humoral immunity and are not universally successful. We designed HLA-Fc fusion proteins with a bivalent targeting module derived from extracellular domains of HLA and an Fc effector module from mouse IgG2a. We found that HLA-Fc with A2 (A2Fc) and B7 (B7Fc) antigens lowered HLA-A2− and HLA-B7−specific reactivities, respectively, in sera from HLA-sensitized patients. A2Fc and B7Fc bound to B-cell hybridomas bearing surface immunoglobulins with cognate specificities and triggered antigen-specific and Fc-dependent cytotoxicity in vitro. In immunodeficient mice carrying HLA-A2–specific hybridoma cells, A2Fc treatment lowered circulating anti−HLA-A2 levels, abolished the outgrowth of hybridoma cells, and prolonged survival compared with control groups. In an in vivo anti-HLA-A2−mediated platelet transfusion refractoriness model, A2Fc treatment mitigated refractoriness. These results support HLA-Fc being a novel strategy for antigen-specific humoral suppression to improve transfusion and transplantation outcomes. With the long-term goal of targeting HLA-specific memory B cells for desensitization, further studies of HLA-Fc’s efficacy in immune-competent animal models are warranted.

Vaccines typically protect against (re)infections by generating pathogen-neutralising antibodies. However, as we age, antibody-secreting cell formation and vaccine-induced antibody titres are reduced. Antibody-secreting plasma cells differentiate from B cells either early post-vaccination through the extrafollicular response or from the germinal centre (GC) reaction, which generates long-lived antibody-secreting cells. As the formation of both the extrafollicular antibody response and the GC requires the interaction of multiple cell types, the impaired antibody response in ageing could be caused by B cell intrinsic or extrinsic factors, or a combination of the two. Here, we show that B cells from older people do not have intrinsic defects in their proliferation and differentiation into antibody-secreting cells in vitro compared to those from the younger donors. However, adoptive transfer of B cells from aged mice to young recipient mice showed that differentiation into extrafollicular plasma cells was favoured at the expense of B cells entering the GC during the early stages of GC formation. In contrast, by the peak of the GC response, GC B cells derived from the donor cells of aged mice had expanded to the same extent as those from the younger donors. This indicates that age-related intrinsic B cell changes delay the GC response but are not responsible for the impaired antibody-secreting response or smaller peak GC response in ageing. Collectively, this study shows that B cells from aged individuals are not intrinsically defective in responding to stimulation and becoming antibody-secreting cells, implicating B cell-extrinsic factors as the primary cause of age-associated impairment in the humoral immunity.

International audience; Antibody-secreting cells (ASC) are divided into two principal subsets, including the long-lived plasma cell (PC) subset residing in the bone marrow and the short-lived subset, also called plasmablast (PB). PB are described as a proliferating subset circulating through the blood and ending its differentiation in tissues. Due to their inherent heterogeneity, the molecular signature of PB is not fully established. The purpose of this study was to decipher a specific PB signature in humans and mice through a comprehensive meta-analysis of different data sets exploring the PB differentiation in both species and across different experimental conditions. The present study used recent analyses using whole RNA sequencing in prdm1-GFP transgenic mice to define a reliable and accurate PB signature. Next, we performed similar analysis using current data sets obtained from human PB and PC. The PB-specific signature is composed of 155 and 113 genes in mouse and human being, respectively. Although only nine genes are shared between the human and mice PB signature, the loss of B-cell identity such as the down-regulation of PAX5, MS4A1, (CD20) CD22 and IL-4R is a conserved feature across species and across the different experimental conditions. Additionally, we observed that the IRF8 and IRF4 transcription factors have a specific dynamic range of expression in human PB. We thus demonstrated that IRF4/IRF8 intranuclear staining was useful to define PB in vivo and in vitro and able to discriminate between atypical PB populations and transient states.

Humoral immunity provides protection from pathogenic infection and is mediated by antibodies following the differentiation of naive B cells (nBs) to antibody-secreting cells (ASCs). This process requires substantial epigenetic and transcriptional rewiring to ultimately repress the nB program and replace it with one conducive to ASC physiology and function. Notably, these reprogramming events occur within the framework of cell division. Efforts to understand the relationship of cell division with reprogramming and ASC differentiation in vivo have uncovered the timing and scope of reprogramming, as well as key factors that influence these events. Herein, we discuss the unique physiology of ASC and how nBs undergo epigenetic and genome architectural reorganization to acquire the necessary functions to support antibody production. We also discuss the stage-wise manner in which reprogramming occurs across cell divisions and how key molecular determinants can influence B cell fate outcomes.

Increased IgE is a typical feature of allergic rhinitis. Local class-switch recombination has been intimated but B cell precursors and mechanisms remain elusive. Here we describe the dynamics underlying the generation of IgE-antibody secreting cells (ASC) in human nasal polyps (NP), mucosal tissues rich in ASC without germinal centers (GC). Using VH next generation sequencing, we identified an extrafollicular (EF) mucosal IgD+ naive-like intermediate B cell population with high connectivity to the mucosal IgE ASC. Mucosal IgD+ B cells, express germline epsilon transcripts and predominantly co-express IgM. However, a small but significant fraction co-express IgG or IgA instead which also show connectivity to ASC IgE. Phenotypically, NP IgD+ B cells display an activated profile and molecular evidence of BCR engagement. Transcriptionally, mucosal IgD+ B cells reveal an intermediate profile between naive B cells and ASC. Single cell IgE ASC analysis demonstrates lower mutational frequencies relative to IgG, IgA, and IgD ASC consistent with IgE ASC derivation from mucosal IgD+ B cell with low mutational load. In conclusion, we describe a novel mechanism of GC-independent, extrafollicular IgE ASC formation at the nasal mucosa whereby activated IgD+ naive B cells locally undergo direct and indirect (through IgG and IgA), IgE class switch.

While DNA prime-protein boost vaccination approach has been widely used in preclinical and clinical studies especially in the field of HIV vaccine development, the exact role of DNA immunization has not been fully identified. Our previous work demonstrated that DNA immunization was able to elicit T follicular helper (Tfh) cell responses and germinal center (GC) B cell development in a mouse model. In the current report, a mouse immunogenicity study was conducted to further ask whether DNA immunization is able to elicit antigen-specific B cell responses. Using HIV-1 Env as model antigen delivered in the form of DNA prime-protein boost, our data demonstrated that DNA prime was able to enhance the antigen-specific B cell responses for both Env-specific antibody secreting cells (ASC) and memory B cells. Furthermore, the DNA priming can greatly reduce the need of including an adjuvant as part of the recombinant protein vaccine boost formulation. Our findings revealed one mechanism that supports the value of DNA priming in assisting the inductin of high affinity and long lasting antigen specific antibody responses.

The stability and plasticity of B cell-mediated immune memory ensures the ability to respond to the repeated challenges. We have analyzed the longitudinal dynamics of immunoglobulin heavy chain repertoires from memory B cells, plasmablasts, and plasma cells from the peripheral blood of generally healthy volunteers. We reveal a high degree of clonal persistence in individual memory B cell subsets, with inter-individual convergence in memory and antibody-secreting cells (ASCs). ASC clonotypes demonstrate clonal relatedness to memory B cells, and are transient in peripheral blood. We identify two clusters of expanded clonal lineages with differing prevalence of memory B cells, isotypes, and persistence. Phylogenetic analysis revealed signs of reactivation of persisting memory B cell-enriched clonal lineages, accompanied by new rounds of affinity maturation during proliferation and differentiation into ASCs. Negative selection contributes to both persisting and reactivated lineages, preserving the functionality and specificity of BCRs to protect against current and future pathogens.

ObjectivesAnti-centromere antibodies (ACAs) are detected in patients with various autoimmune diseases such as Sjögren’s syndrome (SS), systemic sclerosis (SSc) and primary biliary cholangitis (PBC). However, the targeted antigens of ACAs are not fully elucidated despite the accumulating understanding of the molecular structure of the centromere. The aim of this study was to comprehensively reveal the autoantigenicity of centromere proteins.MethodsA centromere antigen library including 16 principal subcomplexes composed of 41 centromere proteins was constructed. Centromere protein/complex binding beads were used to detect serum ACAs in patients with SS, SSc and PBC. ACA-secreting cells in salivary glands obtained from patients with SS were detected with green fluorescent protein-fusion centromere antigens and semiquantified with confocal microscopy.ResultsA total of 241 individuals with SS, SSc or PBC and healthy controls were recruited for serum ACA profiling. A broad spectrum of serum autoantibodies was observed, and some of them had comparative frequency as anti-CENP-B antibody, which is the known major ACA. The prevalence of each antibody was shared across the three diseases. Immunostaining of SS salivary glands showed the accumulation of antibody-secreting cells (ASCs) specific for kinetochore, which is a part of the centromere, whereas little reactivity against CENP-B was seen.ConclusionsWe demonstrated that serum autoantibodies target the centromere–kinetochore macrocomplex in patients with SS, SSc and PBC. The specificity of ASCs in SS salivary glands suggests kinetochore complex-driven autoantibody selection, providing insight into the underlying mechanism of ACA acquisition.

Marginal zone (MZ) B cells are innate‐like B cells that produce polyreactive antibodies with an affinity for microbial molecular patterns and carbohydrate ligands. MZ B cells have been shown to be important in mediating immunity to various bacteria including Streptococcus pneumoniae and are also implicated in inflammatory syndromes including lupus erythematosus. The intestinal microbiota is responsible for producing short‐chain fatty acids, which can regulate immune cell function by several mechanisms including ligation of the G‐protein‐coupled receptor (GPR)43. Herein, we show that MZ B cells express Gpr43 messenger RNA and that the absence of this receptor impacts on MZ B‐cell surface marker expression and antibody production. In T‐cell‐independent responses to the hapten 4‐hydroxy‐3‐nitrophenylacetic acid (NP), mice deficient in GPR43 displayed higher serum titers of NP‐specific antibodies. Moreover, in response to a pneumococcal polysaccharide vaccine, GPR43‐deficient mice developed robust serum antibody responses and had markedly increased numbers of splenic antibody‐secreting cells, compared with control mice. Finally, serum immunoglobulin M autoantibodies to double‐stranded DNA and phosphatidylcholine were increased in resting 10–15‐week‐old mice lacking GPR43. Taken together, mice lacking GPR43 have heightened antibody responses to T‐cell‐independent antigens, which may be a result of impaired regulation of MZ B cells., Marginal zone B cells express the short‐chain fatty acid receptor GPR43. Mice lacking GPR43 have heightened responses to T‐cell‐independent antigens and have elevated levels of immunoglobulin M specific for double‐stranded DNA and phosphatidylcholine. Thus, short‐chain fatty acids may regulate marginal zone B‐cell responses to several antigens.

Antibody-secreting cells (ASCs) contribute to immunity through production of antibodies and cytokines. Identification of specific markers of ASC would allow selective targeting of these cells in several disease contexts. Here, we performed an unbiased, large-scale protein screening, and identified twelve new molecules that are specifically expressed by murine ASCs. Expression of these markers, particularly CD39, CD81, CD130, and CD326, is stable and offers an improved resolution for ASC identification. We accessed their expression in germ-free conditions and in T cell deficient mice, showing that at least in part their expression is controlled by microbial- and T cell-derived signals. Further analysis of lupus mice revealed the presence of a subpopulation of LAG-3–plasma cells, co-expressing high amounts of CD39 and CD326 in the bone marrow. This population was IgM+and correlated with IgM anti-dsDNA autoantibodies in sera. Importantly, we found that CD39, CD81, CD130, and CD326 are also expressed by human peripheral blood and bone marrow ASCs. Our data provide innovative insights into ASC biology and function in mice and human, and identify an intriguing BM specific CD39++CD326++ASC subpopulation in autoimmunity.

International audience; Antibody-secreting cells (ASC), plasmablasts and plasma cells, are terminally differentiated B cells responsible for large-scale production and secretion of antibodies. ASC are derived from activated B cells, which may differentiate extrafollicularly or form germinal center (GC) reactions within secondary lymphoid organs. ASC therefore consist of short-lived, poorly matured plasmablasts that generally secrete lower-affinity antibodies, or long-lived, highly matured plasma cells that generally secrete higher-affinity antibodies. The ASC population is responsible for producing an immediate humoral B cell response, the polyclonal antibody repertoire, as well as in parallel building effective humoral memory and immunity, or potentially driving pathology in the case of autoimmunity. ASC are phenotypically and transcriptionally distinct from other B cells and further distinguishable by morphology, varied lifespans, and anatomical localization. Single cell analyses are required to interrogate the functional and transcriptional diversity of ASC and their secreted antibody repertoire and understand the contribution of individual ASC responses to the polyclonal humoral response. Here we summarize the current and emerging functional and molecular techniques for high-throughput characterization of ASC with single cell resolution, including flow and mass cytometry, spot-based and microfluidic-based assays, focusing on functional approaches of the secreted antibodies: specificity, affinity, and secretion rate.

Inappropriate expansion of antibody-secreting cells (ASCs) is typical of systemic lupus erythematosus (SLE), but the regulatory signaling of pathogenic ASCs is unclear. The present study shows that brain-derived neurotrophic factor precursor (proBDNF) and its high-affinity pan-75 neurotrophin receptor (p75NTR) are highly expressed in CD19+CD27hiCD38hi ASCs in patients with SLE and in CD19+CD44hiCD138+ ASCs in lupus-like mice. The increased proBDNF+ ASCs were positively correlated with clinical symptoms and higher titers of autoantibodies in SLE. Administration of monoclonal antibodies against proBDNF or specific knockout of p75NTR in CD19+ B cells exerted a therapeutic effect on lupus mice by limiting the proportion of ASCs, reducing the production of autoantibodies and attenuating kidney injury. Blocking the biological function of proBDNF or p75NTR also inhibits ASC differentiation and antibody production in vitro. Together, these findings suggest that proBDNF-p75NTR signaling plays a critical pathogenic role in SLE through promoting ASC dysfunction., Description, ProBDNF/p75NTR signaling drives systemic lupus erythematosus by dysregulating antibody-secreting cells.

In Japan, there is a long history of consumption of miso, a fermented soybean paste, which possesses beneficial effects on human health. However, the mechanism behind these effects is not fully understood. To clarify the effects of miso on immune cells, we evaluated its immunomodulatory activity in mice. Miso did not alter the percentage of B and T cells in the spleen; however, it increased CD69+ B cells, germinal center B cells and regulatory T cells. Anti-DNA immunoglobulin M antibodies, which prevent autoimmune disease, were increased following ingestion of miso. Transcriptome analysis of mouse spleen cells cultured with miso and its raw material revealed that the expression of genes, including interleukin (IL)-10, IL-22 and CD86, was upregulated. Furthermore, intravital imaging of the small intestinal epithelium using a calcium biosensor mouse line indicated that miso induced Ca2+ signaling in a manner similar to that of probiotics. Thus, ingestion of miso strengthened the immune response and tolerance in mice. These results appear to account, at least in part, to the salubrious effects of miso.

Background Anti-aquaporin 4 (AQP4) antibody (AQP4-Ab) is involved in the pathogenesis of neuromyelitis optica spectrum disorder (NMOSD). However, the mechanism involved in AQP4-Ab production remains unclear. Methods We analyzed the immunophenotypes of patients with NMOSD and other neuroinflammatory diseases as well as healthy controls (HC) using flow cytometry. Transcriptome analysis of B cell subsets obtained from NMOSD patients and HCs was performed. The differentiation capacity of B cell subsets into antibody-secreting cells was analyzed. Results The frequencies of switched memory B (SMB) cells and plasmablasts were increased and that of naïve B cells was decreased in NMOSD patients compared with relapsing–remitting multiple sclerosis patients and HC. SMB cells from NMOSD patients had an enhanced potential to differentiate into antibody-secreting cells when cocultured with T peripheral helper cells. Transcriptome analysis revealed that the profiles of B cell lineage transcription factors in NMOSD were skewed towards antibody-secreting cells and that IL-2 signaling was upregulated, particularly in naïve B cells. Naïve B cells expressing CD25, a receptor of IL-2, were increased in NMOSD patients and had a higher potential to differentiate into antibody-secreting cells, suggesting CD25+ naïve B cells are committed to differentiate into antibody-secreting cells. Conclusions To the best of our knowledge, this is the first study to demonstrate that B cells in NMOSD patients are abnormally skewed towards antibody-secreting cells at the transcriptome level during the early differentiation phase, and that IL-2 might participate in this pathogenic process. Our study indicates that CD25+ naïve B cells are a novel candidate precursor of antibody-secreting cells in autoimmune diseases.

The sympathetic nervous system (SNS) contributes to immune balance by promoting anti-inflammatory B cells. However, whether B cells possess a self-regulating mechanism by which they modulate regulatory B cell (Breg) function is not well understood. In this study, we investigated the ability of B cells to synthesize their own catecholamines upon stimulation with different B cell activators and found that expression of the enzyme tyrosine hydroxylase (TH), required to generate catecholamines, is up-regulated by Toll-like receptor (TLR)9. This TLR9-dependent expression of TH correlated with up-regulation of adrenergic receptors (ADRs), enhanced interleukin (IL)-10 production, and overexpression of the co-inhibitory ligands programmed death ligand 1 (PD-L1) and Fas ligand (FasL). Moreover, concomitant stimulation of ß1-3-ADRs together with a B cell receptor (BCR)/TLR9 stimulus clearly enhances the anti-inflammatory potential of Bregs to suppress CD4 T cells, a crucial population in the pathogenesis of autoimmune diseases, like rheumatoid arthritis (RA). Furthermore, TH up-regulation was also demonstrated in B cells during the course of collagen-induced arthritis (CIA), a mouse model for the investigation of RA. In conclusion, our data show that B cells possess an autonomous mechanism to modulate their regulatory function in an autocrine and/or paracrine manner. These findings help to better understand the function of B cells in the regulation of autoimmune diseases and the interplay of SNS., The sympathetic nervous system produces neurotransmitters such as catecholamines which contribute to immune balance by promoting anti-inflammatory B cells. This study shows that mouse B cells can themselves synthesize, sense, and transport catecholamines, which in turn modulate regulatory B cell function in an autocrine and/or paracrine manner to suppress T cell proliferation.

It has been estimated that more than 390 million people are infected with Dengue virus every year; around 96 millions of these infections result in clinical pathologies. To date, there is only one licensed viral vector-based Dengue virus vaccine CYD-TDV approved for use in dengue endemic areas. While initially approved for administration independent of serostatus, the current guidance only recommends the use of this vaccine for seropositive individuals. Therefore, there is a critical need for investigating the influence of Dengue virus serostatus and immunological mechanisms that influence vaccine outcome. Here, we provide comprehensive evaluation of sero-status and host immune factors that correlate with robust immune responses to a Dengue virus vector based tetravalent vaccine (TV003) in a Phase II clinical cohort of human participants. We observed that sero-positive individuals demonstrate a much stronger immune response to the TV003 vaccine. Our multi-layered immune profiling revealed that sero-positive subjects have increased baseline/pre-vaccination frequencies of circulating T follicular helper (cTfh) cells and the Tfh related chemokine CXCL13/BLC. Importantly, this baseline/pre-vaccination cTfh profile correlated with the vaccinees’ ability to launch neutralizing antibody response against all four sero-types of Dengue virus, an important endpoint for Dengue vaccine clinical trials. Overall, we provide novel insights into the favorable cTfh related immune status that persists in Dengue virus sero-positive individuals that correlate with their ability to mount robust vaccine specific immune responses. Such detailed interrogation of cTfh cell biology in the context of clinical vaccinology will help uncover mechanisms and targets for favorable immuno-modulatory agents., Author summary Dengue virus (DENV) is a worldwide threat that causes significant health and economic burden. Currently, there are several challenges in the development of a DENV vaccine including the existence of four different serotypes, which are all capable of causing disease and antibody dependent enhancement (ADE). For complete protection, a vaccine must be able to generate neutralizing antibodies against all 4 serotypes to avoid ADE. Currently, there is one licensed DENV vaccine, CYD-TDV (DENGVAXIA). However, this vaccine is only efficacious in protecting against severe disease in DENV seropositive individuals therefore serostatus effect must be further studied for optimal vaccine design. A subset of CD4+ T cells called T-follicular helper (Tfh) cells have been well known to play a major role in aiding high affinity antibody production. Therefore, we chose to look at subsets of Tfh and the cytokines they produce in human blood that can serve as biomarkers for effective vaccine design. We found that DENV sero-positive participants had increased pre-vaccination frequencies of Tfh cells and higher levels of the Tfh related chemokine CXCL13/BLC that plays a role in directing antigen-specific responses. This pre-vaccination Tfh profile and CXCL13/BLC are then correlated positively with the vaccinees’ ability to produce neutralizing antibody against all four sero-types (breadth of the Response) of DENV, an important goal for all DENV vaccine trials.

Prophylactic, serological memory relies on maintaining stable reservoirs of plasma cells, capable of constitutively-secreting high-affinity, anti-pathogen antibody for a lifetime. Although antibody titers generated by some vaccines (e.g. measles) can last a lifetime, other vaccinations (e.g. tetanus) need repeated boosting because long-lived plasma cells are not produced or maintained. Moreover, in old age, the ability to generate long-lived humoral responses diminishes. Despite their importance to health, it is unknown how long-lived plasma cells survive over years, whereas most antibody secreting cells die off within weeks after vaccination. In this review, we focus on how known factors regulate the longevity of plasma cell fitness and survival, and how that landscape is shaped by environmental influences, such as inflammation, infection and aging. In addition, we highlight newly discovered cellular dynamics in the bone marrow that may reframe the mechanisms supporting long-lived plasma cell survival and function.

Background Development of an effective vaccine against blood-stage malaria requires the induction of long-term immune responses. Plasmodium vivax Reticulocyte Binding Protein 1a (PvRBP1a) is a blood-stage parasite antigen which is associated with invasion of red blood cells and induces antibody responses. Thus, PvRBP1a is considered as a target for design of a blood-stage vaccine against vivax malaria. Methods Both cross-sectional and cohort studies were used to explore the development and persistence of long-lived antibody and memory B cell responses to PvRBP1a in individuals who lived in an area of low malaria endemicity. Antibody titers and frequency of memory B cells specific to PvRBP1a were measured during infection and following recovery for up to 12 months. Results IgG antibody responses against PvRBP1a were prevalent during acute vivax malaria, predominantly IgG1 subclass responses. High responders to PvRBP1a had persistent antibody responses for at least 12-month post-infection. Further analysis of high responder found a direct relation between antibody titers and frequency of activated and atypical memory B cells. Furthermore, circulating antibody secreting cells and memory B cells specific to PvRBP1a were generated during infection. The PvRBP1a-specific memory B cells were maintained for up to 3-year post-infection, indicating the ability of PvRBP1a to induce long-term humoral immunity. Conclusion The study revealed an ability of PvRBP1a protein to induce the generation and maintenance of antibody and memory B cell responses. Therefore, PvRBP1a could be considered as a vaccine candidate against the blood-stage of P. vivax.

Epstein Barr Virus (EBV) infects more than 95% of the population whereupon it establishes a latent infection of B-cells that persists for life under immune control. Primary EBV infection can cause infectious mononucleosis (IM) and long-term viral carriage is associated with several malignancies and certain autoimmune diseases. Current efforts developing EBV prophylactic vaccination have focussed on neutralising antibodies. An alternative strategy, that could enhance the efficacy of such vaccines or be used alone, is to generate T-cell responses capable of recognising and eliminating newly EBV-infected cells before the virus initiates its growth transformation program. T-cell responses against the EBV structural proteins, brought into the newly infected cell by the incoming virion, are prime candidates for such responses. Here we show the structural EBV capsid proteins BcLF1, BDLF1 and BORF1 are frequent targets of T-cell responses in EBV infected people, identify new CD8+ and CD4+ T-cell epitopes and map their HLA restricting alleles. Using T-cell clones we demonstrate that CD4+ but not CD8+ T-cell clones specific for the capsid proteins can recognise newly EBV-infected B-cells and control B-cell outgrowth via cytotoxicity. Using MHC-II tetramers we show a CD4+ T-cell response to an epitope within the BORF1 capsid protein epitope is present during acute EBV infection and in long-term viral carriage. In common with other EBV-specific CD4+ T-cell responses the BORF1-specific CD4+ T-cells in IM patients expressed perforin and granzyme-B. Unexpectedly, perforin and granzyme-B expression was sustained over time even when the donor had entered the long-term infected state. These data further our understanding of EBV structural proteins as targets of T-cell responses and how CD4+ T-cell responses to EBV change from acute disease into convalescence. They also identify new targets for prophylactic EBV vaccine development., Author summary Epstein-Barr virus is a widespread herpesvirus carried by most individuals. Whilst infection is usually asymptomatic, development of a prophylactic vaccine against EBV is desirable because of the virus’s association with infectious mononucleosis in primary infection and several cancers and autoimmune diseases during long-term virus carriage. Identifying T-cell responses that can recognise newly infected B-cells at very early stages of infection may provide novel targets for T-cell vaccination. Here we characterise T-cell responses against three virus proteins, BcLF1, BDLF1 and BORF1 that, as structural proteins of the virus particle, are delivered into the cell by the infecting virus. We find that all three proteins are recognised by T-cells from infected individuals. Moreover, isolated structural antigen-specific CD4+ T-cells rapidly recognise newly infected B-cells and prevent their outgrowth in vitro. As reported for CD4+ T-cells against other EBV proteins, structural antigen-specific CD4+ T-cells induced by primary EBV infection have cytotoxic function. However, we also demonstrate that, unusually, this cytotoxic function is retained in memory T-cells present in long-term infected individuals. Structural antigens may therefore represent useful targets for prophylactic EBV vaccine development to induce CD4+ T-cells able to rapidly eliminate virus-infected cells.

Protective immunity to parasitic infections has been difficult to elicit by vaccines. Among parasites that evade vaccine-induced immunity is Toxoplasma gondii, which causes lethal secondary infections in chronically infected mice. Here we report that unlike susceptible C57BL/6J mice, A/J mice were highly resistant to secondary infection. To identify correlates of immunity, we utilized forward genetics to identify Nfkbid, a nuclear regulator of NF-κB that is required for B cell activation and B-1 cell development. Nfkbid-null mice (“bumble”) did not generate parasite-specific IgM and lacked robust parasite-specific IgG, which correlated with defects in B-2 cell maturation and class-switch recombination. Though high-affinity antibodies were B-2 derived, transfer of B-1 cells partially rescued the immunity defects observed in bumble mice and were required for 100% vaccine efficacy in bone marrow chimeric mice. Immunity in resistant mice correlated with robust isotype class-switching in both B cell lineages, which can be fine-tuned by Nfkbid gene expression. We propose a model whereby humoral immunity to T. gondii is regulated by Nfkbid and requires B-1 and B-2 cells for full protection., Author summary Eukaryotic parasitic diseases account for approximately one fifth of all childhood deaths, yet no highly protective vaccine exists for any human parasite. More research must be done to discover how to elicit protective vaccine-induced immunity to parasitic pathogens. We used an unbiased genetic screen to find key genes responsible for immunity to the eukaryotic parasite Toxoplasma gondii. Our screen found Nfkbid, a transcription factor regulator, which controls B cell activation and innate-like B-1 cell development. Mice without Nfkbid were not protected against T. gondii and were deficient at making antibodies against the parasite. Our survival studies of vaccinated mice with and without B-1 compartments found that B-1 cells improved survival, suggesting that B-1 cells act in conjunction with B-2 cells to provide vaccine-induced immunity. Nfkbid and other loci identified in our unbiased screen represent potential targets for vaccines to elicit protective immune responses against parasitic pathogens.

Mammalian cells release different types of vesicles, collectively termed extracellular vesicles (EVs). EVs contain cellular microRNAs (miRNAs) with an apparent potential to deliver their miRNA cargo to recipient cells to affect the stability of individual mRNAs and the cells’ transcriptome. The extent to which miRNAs are exported via the EV route and whether they contribute to cell-cell communication are controversial. To address these issues, we defined multiple properties of EVs and analyzed their capacity to deliver packaged miRNAs into target cells to exert biological functions. We applied well-defined approaches to produce and characterize purified EVs with or without specific viral miRNAs. We found that only a small fraction of EVs carried miRNAs. EVs readily bound to different target cell types, but EVs did not fuse detectably with cellular membranes to deliver their cargo. We engineered EVs to be fusogenic and document their capacity to deliver functional messenger RNAs. Engineered fusogenic EVs, however, did not detectably alter the functionality of cells exposed to miRNA-carrying EVs. These results suggest that EV-borne miRNAs do not act as effectors of cell-to-cell communication., Author summary The majority of metazoan cells release vesicles of different types and origins, such as exosomes and microvesicles, now collectively termed extracellular vesicles (EVs). EVs have gained much attention because they contain microRNAs (miRNAs) and thus could regulate their specific mRNA targets in recipient or acceptor cells that take up EVs. Using a novel fusion assay with superior sensitivity and specificity, we revisited this claim but found no convincing evidence for an efficient functional uptake of EVs in many different cell lines and primary human blood cells. Even EVs engineered to fuse and deliver their miRNA cargo to recipient cells had no measurable effect on target mRNAs in very carefully controlled, quantitative experiments. Our negative results clearly indicate that EVs do not act as vehicles for miRNA-based cell-to-cell communication.

Background Pemphigus vulgaris (PV) is a rare autoimmune blistering disease characterized by the development of autoantibodies targeting desmoglein (Dsg) 3, but also against Dsg1 in mucocutaneous disease. Given that existing PV animal models only recapitulate aspects of the disease, we aimed to establish a more comprehensive disease model based on the immunization of mice with PV autoantigen(s). Methods The following immunization strategies were tested: (i) C57Bl/6J, B6.SJL-H2s C3c/1CyJ, DBA2/J, or SJL/J mice were immunized with recombinant murine Dsg3 (mDsg3), (ii) DBA2/J and SJL/J mice were immunized with mDsg3 and additionally injected a single non-blister inducing dose of exfoliative toxin A (ETA), and (iii) DBA2/J and SJL/J mice were immunized with human Dsg (hDsg) 1 and 3. Results Despite the induction of autoantibodies in each immunization protocol, the mice did not develop a clinical phenotype. Tissue-bound autoantibodies were not detected in the skin or mucosa. Circulating autoantibodies did not bind to the native antigen in indirect immunofluorescence microscopy using monkey esophagus as a substrate. Conclusion Immunization with PV autoantigens induced non-pathogenic Dsg1/3 antibodies, but did not cause skin/mucous membrane disease in mice. These findings, confirmed by failure of binding of the induced autoantibodies to their target in the skin, suggest that the autoantibodies which were formed were unable to bind to the conformational epitope present in vivo.

The Dengue pathophysiology has had several aspects determined over the years. However, some points remain elusive, such as the metabolic factors that regulate the massive B cell differentiation into antibody-secreting cells observed in Dengue patients. In this chapter, we describe an in vitro method capable of mimicking this Dengue-induced cell expansion. More specifically, this approach allows dengue virus-stimulated peripheral blood mononuclear cells (PBMCs) from healthy individuals to enhance the frequency of phenotypical and functional antibody-secreting cells (ASCs) after 7 days of culture. A manuscript recently published by Bonezi and colleagues displays results generated through this methodology.

Rabies is a zoonotic disease caused by the rabies virus (RABV). RABV can lead to fatal encephalitis and is still a serious threat in most parts of the world. Interferon regulatory factor 7 (IRF7) is the main transcriptional regulator of type I IFN, and it is crucial for the induction of IFNα/β and the type I IFN-dependent immune response. In this study, we focused on the role of IRF7 in the pathogenicity and immunogenicity of RABV using an IRF7-/- mouse model. The results showed that the absence of IRF7 made mice more susceptible to RABV, because IRF7 restricted the replication of RABV in the early stage of infection. IRF7 deficiency affected the recruitment of plasmacytoid dendritic cells to the draining lymph nodes (dLNs), reduced the production of type I IFN and expression of IFN-stimulated genes. Furthermore, we found that the ability to produce specific RABV-neutralizing antibody was impaired in IRF7-/- mice. Consistently, IRF7 deficiency affected the recruitment of germinal-centre B cells to dLNs, and the generation of plasma cells and RABV-specific antibody secreting cells. Moreover, the absence of IRF7 downregulated the induction of IFN-γ and reduced type 1 T helper cell (Th1)-dependent antibody production. Collectively, our findings demonstrate that IRF7 promotes humoral immune responses and compromises the pathogenicity of RABV in a mouse model.

HIV-1 vaccine immunofocusing strategies may be able to induce broadly-reactive neutralizing antibodies (NAbs). Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets—the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus (PV) assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and eliminate other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-NAbs, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the C-strand. Notably, a D167N mutation improved V2-sensitivity in several cases. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing glycans at other positions frequently had global "ripple" effects on glycan maturation and sequon occupation throughout the gp120 outer domain and gp41. V2 MAb CH01 selectively bound to trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a rare N49 glycan was found to perturb gp41 glycans, increasing FP NAb sensitivity—and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30–40 spikes. Taken together, we identified 7 diverse trimers with a range of sensitivities to two targets to allow rigorous testing of immunofocusing vaccine concepts., Author summary Despite almost 40 years of innovation, a vaccine to induce antibodies that block HIV infection remains elusive. Challenges include the unparalleled sequence diversity of HIV’s surface spikes and its dense sugar coat that limits antibody access. A growing number of monoclonal antibodies from HIV infected donors provide vaccine blueprints, but have been difficult to induce by vaccination, due to their unusual features. However, two targets, one at the viral spike apex and another at the side of the spikes are more forgiving in their ’demands’ for unusual antibodies. Here, we made a diverse panel of HIV spikes vulnerable at these two sites to be used as vaccines to try to focus antibodies on these targets. Our selection criteria for these spikes were: i) that when expressed on particles, they are infectious, allowing us to evaluate immunogens and vaccine sera using particles made with the same trimers, ii) that spikes are easy to produce by cells in quantities sufficient for vaccine use. Ultimately, we selected 7 trimers that will allow us to explore concepts that could bring us closer to an HIV vaccine.

Nucleoside modified mRNA combined with Acuitas Therapeutics’ lipid nanoparticles (LNPs) has been shown to support robust humoral immune responses in many preclinical animal vaccine studies and later in humans with the SARS-CoV-2 vaccination. We recently showed that this platform is highly inflammatory due to the LNPs’ ionizable lipid component. The inflammatory property is key to support the development of potent humoral immune responses. However, the mechanism by which this platform drives T follicular helper (Tfh) cells and humoral immune responses remains unknown. Here we show that lack of Langerhans cells or cDC1s neither significantly affected the induction of PR8 HA and SARS-CoV-2 RBD-specific Tfh cells and humoral immune responses, nor susceptibility towards the lethal challenge of influenza and SARS-CoV-2. However, the combined deletion of these two DC subsets led to a significant decrease in the induction of PR8 HA and SARS-CoV-2 RBD-specific Tfh cell and humoral immune responses. Despite these observed defects, these mice remained protected from lethal influenza and SARS-CoV-2 challenges. We further found that IL-6, unlike neutrophils, was required to generate normal Tfh cells and antibody responses, but not for protection from influenza challenge. In summary, here we bring evidence that the mRNA-LNP platform can support the induction of protective immune responses in the absence of certain innate immune cells and cytokines., Author summary The mRNA-LNP vaccine platform has gained much attention with the ongoing SARS-CoV-2 pandemic. Millions of people are exposed to these vaccines daily, but their immune mechanism driving the antibody responses and side effects remains unexplored. Therefore, we tested this vaccine platform in the context of influenza and SARS-CoV-2 infections in mouse models that lack specific innate immune cells or cytokines known to play a role in inducing antibody responses. We show that the combined deletion of antigen-presenting innate immune cells and one of the inflammatory cytokines significantly inhibited adaptive immune cell responses triggered by this platform. However, despite the observed defects, the immune responses driven by the mRNA-LNP vaccine were still able to protect the animals from lethal influenza or SARS-CoV-2 challenges. This high degree of redundancy might be associated with the highly inflammatory nature of this platform.

Here, we assessed the efficacy of a short-course multimodal therapy (enrofloxacin, azithromycin, fenbendazole, and paromomycin) to eliminate common macaque endemic pathogens (EPs) and evaluated its impact on gastrointestinal (GI) microbiota, mucosal integrity, and local and systemic inflammation in sixteen clinically healthy macaques. Treatment combined with expanded practices resulted in successful maintenance of rhesus macaques (RM) free of common EPs, with no evidence of overt microbiota diversity loss or dysbiosis and instead resulted in a more defined luminal microbiota across study subjects. Creation of a GI pathogen free (GPF) status resulted in improved colonic mucosal barrier function (histologically, reduced colonic MPO+, and reduced pan-bacterial 16s rRNA in the MLN), reduced local and systemic innate and adaptive inflammation with reduction of colonic Mx1 and pSTAT1, decreased intermediate (CD14+CD16+) and non-classical monocytes (CD14-CD16+), reduced populations of peripheral dendritic cells, Ki-67+ and CD38+ CD4+ T cells, Ki-67+IgG+, and Ki-67+IgD+ B cells indicating lower levels of background inflammation in the distal descending colon, draining mesenteric lymph nodes, and systemically in peripheral blood, spleen, and axillary lymph nodes. A more controlled rate of viral acquisition resulted when untreated and treated macaques were challenged by low dose intrarectal SIVmac239x, with an ~100 fold increase in dose required to infect 50% (AID50) of the animals receiving treatment compared to untreated controls. Reduction in and increased consistency of number of transmitted founder variants resulting from challenge seen in the proof of concept study directly correlated with post-treatment GPF animal’s improved barrier function and reduction of key target cell populations (Ki-67+ CD4+T cells) at the site of viral acquisition in the follow up study. These data demonstrate that a therapeutic and operational strategy can successfully eliminate varying background levels of EPs and their associated aberrant immunomodulatory effects within a captive macaque cohort, leading to a more consistent, better defined and reproducible research model., Author summary Simian Immunodeficiency virus in macaques remains the most translational model for HIV. Macaques are also key models for other infectious diseases and colitis, where background colon health and inflammation could confound experimental results. To address these issues SPF breeding colonies were established to exclude specific viruses detrimental to mucosal and systemic health. To further improve macaque models, we established a gastrointestinal pathogen free (GPF) colony by administration of a multimodal therapeutic regimen for common endemic pathogens (EPs) and established expanded operational practices for continued exclusion. Through extensive longitudinal microbiota, parasitology, microbiology, and tissue sampling we demonstrated that our treatment and exclusion practices successfully eliminated common EPs, improved mucosal barriers, and reduced mucosal and systemic inflammation resulting in less inter-animal variation without overt evidence of microbiota disruption. Finally, compared to treatment-naïve controls, GPF animals challenged with SIV intrarectally demonstrated a more controlled and consistent rate of SIV acquisition, further indicating underlying EPs even at subclinical levels may cause deleterious variations between study subjects. Collectively the GPF macaque represents a model that eliminates a significant source of morbidity, reduces inter-animal variability, and thus has the potential to improve both animal welfare and research outcomes.

SARS-CoV-2 infection elicits a robust B cell response, resulting in the generation of long-lived plasma cells and memory B cells. Here, we aimed to determine the effect of COVID-19 severity on the memory B cell response and characterize changes in the memory B cell compartment between recovery and five months post-symptom onset. Using high-parameter spectral flow cytometry, we analyzed the phenotype of memory B cells with reactivity against the SARS-CoV-2 spike protein or the spike receptor binding domain (RBD) in recovered individuals who had been hospitalized with non-severe (n = 8) or severe (n = 5) COVID-19. One month after symptom onset, a substantial proportion of spike-specific IgG+ B cells showed an activated phenotype. In individuals who experienced non-severe disease, spike-specific IgG+ B cells showed increased expression of markers associated with durable B cell memory, including T-bet and FcRL5, as compared to individuals who experienced severe disease. While the frequency of T-bet+ spike-specific IgG+ B cells differed between the two groups, these cells predominantly showed an activated switched memory B cell phenotype in both groups. Five months post-symptom onset, the majority of spike-specific memory B cells had a resting phenotype and the percentage of spike-specific T-bet+ IgG+ memory B cells decreased to baseline levels. Collectively, our results highlight subtle differences in the B cells response after non-severe and severe COVID-19 and suggest that the memory B cell response elicited during non-severe COVID-19 may be of higher quality than the response after severe disease.

Rabies, caused by rabies virus (RABV), remains a serious threat to public health in most countries worldwide. At present, the administration of rabies vaccines has been the most effective strategy to control rabies. Herein, we evaluate the effect of colloidal manganese salt (Mn jelly [MnJ]) as an adjuvant of rabies vaccine in mice, cats, and dogs. The results showed that MnJ promoted type I interferon (IFN-I) and cytokine production in vitro and the maturation of dendritic cells (DCs) in vitro and in vivo. Besides, MnJ serving as an adjuvant for rabies vaccines could significantly facilitate the generation of T follicular helper (Tfh) cells, germinal center (GC) B cells, plasma cells (PCs), and RABV-specific antibody-secreting cells (ASCs), consequently improve the immunogenicity of rabies vaccines, and provide better protection against virulent RABV challenge. Similarly, MnJ enhanced the humoral immune response in cats and dogs as well. Collectively, our results suggest that MnJ can facilitate the maturation of DCs during rabies vaccination, which can be a promising adjuvant candidate for rabies vaccines. IMPORTANCE Extending the humoral immune response by using adjuvants is an important strategy for vaccine development. In this study, a novel adjuvant, MnJ, supplemented in rabies vaccines was evaluated in mice, cats, and dogs. Our results in the mouse model revealed that MnJ increased the numbers of mature DCs, Tfh cells, GC B cells, PCs, and RABV-specific ASCs, resulting in enhanced immunogenicity and protection rate of rabies vaccines. We further found that MnJ had the same stimulative effect in cats and dogs. Our study provides the first evidence that MnJ serving as a novel adjuvant of rabies vaccines can boost the immune response in both a mouse and pet model.

After allogeneic hematopoietic stem cell transplantation (HSCT), recovery of humoral immunity is essential to protect from life-threatening infections. However, monitoring the humoral immune system after transplantation with standard techniques in the clinical routine is imprecise. Here, we performed sequencing of mononuclear bone marrow cells to characterize the VH1-repertoire of switched B cells of healthy volunteers and patients undergoing HSCT. Analysis of healthy bone marrow donors and patients showed virtually no clonally related sequences between individuals. Interestingly, clonally related sequences were present in pre- and post-transplantation bone marrow of patients undergoing HSCT for acute myeloid leukemia treatment. We consistently observed such related B cell clones, irrespective of conditioning regimen, donor source or time post transplantation. In general, repertoire diversity was lower in post-HSCT as compared to pre-HSCT samples. However, post-HSCT repertoires retained highly mutated sequences, despite immunosuppressive therapy and presence of T cell deficiency after HSCT. These observations identify key properties of the recovering B cell compartment and provide a conceptual framework for the surveillance of humoral immunity after allogeneic transplantation.

Bayesian networks can be used to identify possible causal relationships between variables based on their conditional dependencies and independencies, which can be particularly useful in complex biological scenarios with many measured variables. Here we propose two improvements to an existing method for Bayesian network analysis, designed to increase the power to detect potential causal relationships between variables (including potentially a mixture of both discrete and continuous variables). Our first improvement relates to the treatment of missing data. When there is missing data, the standard approach is to remove every individual with any missing data before performing analysis. This can be wasteful and undesirable when there are many individuals with missing data, perhaps with only one or a few variables missing. This motivates the use of imputation. We present a new imputation method that uses a version of nearest neighbour imputation, whereby missing data from one individual is replaced with data from another individual, their nearest neighbour. For each individual with missing data, the subsets of variables to be used to select the nearest neighbour are chosen by sampling without replacement the complete data and estimating a best fit Bayesian network. We show that this approach leads to marked improvements in the recall and precision of directed edges in the final network identified, and we illustrate the approach through application to data from a recent study investigating the causal relationship between methylation and gene expression in early inflammatory arthritis patients. We also describe a second improvement in the form of a pseudo-Bayesian approach for upweighting certain network edges, which can be useful when there is prior evidence concerning their directions., Author summary Data analysis using Bayesian networks can help identify possible causal relationships between measured biological variables. Here we propose two improvements to an existing method for Bayesian network analysis. Our first improvement relates to the treatment of missing data. When there is missing data, the standard approach is to remove every individual with any missing data before performing analysis, even if only one or a few variables are missing. This is undesirable as it can reduce the ability of the approach to infer correct relationships. We propose a new method to instead fill in (impute) the missing data prior to analysis. We show through computer simulations that our method improves the reliability of the results obtained, and we illustrate the proposed approach by applying it to data from a recent study in early inflammatory arthritis. We also describe a second improvement involving the upweighting of certain network edges, which can be useful when there is prior evidence concerning their directions.

Anemia caused by trypanosome infection is poorly understood. Autoimmunity during Trypanosoma brucei infection was proposed to have a role during anemia, but the mechanisms involved during this pathology have not been elucidated. In mouse models and human patients infected with malaria parasites, atypical B-cells promote anemia through the secretion of autoimmune anti-phosphatidylserine (anti-PS) antibodies that bind to uninfected erythrocytes and facilitate their clearance. Using mouse models of two trypanosome infections, Trypanosoma brucei and Trypanosoma cruzi, we assessed levels of autoantibodies and anemia. Our results indicate that acute T. brucei infection, but not T. cruzi, leads to early increased levels of plasma autoantibodies against different auto antigens tested (PS, DNA and erythrocyte lysate) and expansion of atypical B cells (ABCs) that secrete these autoantibodies. In vitro studies confirmed that a lysate of T. brucei, but not T. cruzi, could directly promote the expansion of these ABCs. PS exposure on erythrocyte plasma membrane seems to be an important contributor to anemia by delaying erythrocyte recovery since treatment with an agent that prevents binding to it (Annexin V) ameliorated anemia in T. brucei-infected mice. Analysis of the plasma of patients with human African trypanosomiasis (HAT) revealed high levels of anti-PS antibodies that correlated with anemia. Altogether these results suggest a relation between autoimmunity against PS and anemia in both mice and patients infected with T. brucei., Author summary African trypanosomes are relevant pathogens and a cause of great morbidity and economic burden in endemic countries. Anemia during infection by African Trypanosomes is one of the most common pathologies associated with the disease, but the mechanisms leading to it are poorly understood. Here, we report evidence of autoimmunity, particularly autoantibodies against the lipid phosphatidylserine that are secreted by autoimmune/atypical B-cells, in Trypanosoma brucei infected mice and that are also found in Human African Trypanosomiasis patients and correlate with levels of anemia. These findings point to a relation between autoimmunity and anemia during infection with African Trypanosomes, possibly through the secretion of autoantibodies against phosphatidylserine.

ilustraciones, fotografías, gráficas, tablas Actualmente, los anticuerpos monoclonales (mAbs) corresponden al segmento más importante en el mercado de productos biotecnológicos con efecto terapéutico. La síntesis a escala industrial de este tipo de proteínas recombinantes se lleva a cabo en biorreactores empleando líneas celulares provenientes de un mamífero, las cuales son modificadas genéticamente para incluir dentro de su metabolismo la síntesis del mAb. Para comprender mejor la manera en la que estas células deben ser cultivadas, se realizan ensayos a escala laboratorio, en las cuales se determinan las condiciones más apropiadas para la producción en el biorreactor a escala industrial. Dentro de los retos más desafiantes en términos técnicos a replicar en reactores a escalas mayores a las del laboratorio se encuentra la velocidad de transferencia de masa de oxígeno. Este gas es vital para el cultivo de las células de mamífero dado su rol en la respiración, por lo tanto, en la obtención de energía para la vida celular. No obstante, dada la baja solubilidad del oxígeno en el agua, es necesario agitar y/o burbujear el medio de cultivo, y así, el gas se solubilizará más rápido en el líquido. La presente tesis de maestría evaluó la cinética del crecimiento, consumo de sustratos y síntesis de coproductos por una línea parental celular de Ovario de Hámster Chino en dos geometrías de cultivo, Erlenmeyer y Spinner. Con el fin de comparar los resultados en las dos configuraciones de biorreactores, se definieron las condiciones de operación (agitación y burbujeo) de manera tal que la velocidad de transferencia de oxígeno fuera igual en las dos geometrías. (Texto tomado de la fuente) Nowadays, monoclonal antibodies (mAbs) correspond to the most important segment in the market for biotechnological products with therapeutic effects. The synthesis of this type of recombinant proteins on an industrial scale is carried out in bioreactors using mammalian cell lines, which are genetically modified to include mAb synthesis within their metabolism. To better understand the way in which these cells should be cultured, tests are carried out on a laboratory scale, in which the conditions for production in the bioreactor on an industrial scale are determined rather. Assuring an adequate oxygen mass transfer rate is one of the most demanding challenges in technical terms to be replicated in reactors on larger scales than laboratory vessels. This gas is vital for the cultivation of mammalian cells given its role in respiration, and thus, in obtaining energy for cellular life. Nonetheless, since oxygen is sparingly soluble in water, it is necessary to stir and/or bubble the culture medium, for increasing the solubilization rate of the gas in the liquid. In this master's thesis, the kinetics of growth, consumption of substrates and synthesis of co-products by a parental cell line of Chinese Hamster Ovary were evaluated in two culture geometries, Erlenmeyer and Spinner. In order to compare the results in the two bioreactor configurations, the operating conditions (stirring and bubbling) were defined in such a way that the oxygen transfer rate was the same in the two geometries. Doctorado Magíster en Ingeniería - Ingeniería Química Bioprocesos

This study was aimed at exploring whether latent tuberculosis infection (LTBI) contributes to the pathogenesis of immune-mediated inflammatory diseases in a TB endemic setting. We screened 198 rheumatoid arthritis (RA) patients with tuberculin skin test (TST) and studied 61 (median DAS28-ESR = 6.3) who were positive. Whole blood T cell proliferative responses to Mycobacterium tuberculosis (Mtb) membrane (MtM) antigens, including the latency-induced protein alpha crystallin (Acr), were determined by flow cytometry using Ki67 expression as the marker for nuclear proliferation. Serum antibody levels were determined by ELISA. Follow-up investigations (at 3–6, 9–12 and 15–18 months after baseline) were performed in 41 patients who were classified empirically as ‘high’ (HR-T/HR-B) or ‘low’ (LR-T/LR-B) responders based on their dynamic T cell or antibody responses. Significant correlations were seen between baseline T cell responses to MtM and Acr, and between IgG, IgA and IgM antibody responses to MtM. However, no correlation was seen between T and B cell responses. At all time points during the follow-up, T cell responses to both antigens (except for MtM at one point) were significantly higher in HR-T (n = 25) than LR-T (n = 16) patients. Levels of IgA and IgM (but not IgG) antibodies to MtM were also significantly higher in HR-B (n = 13) than LR-B (n = 28) at all time points. Importantly, HR-T patients exhibited significantly higher baseline and follow-up DAS28 scores than LR-T. Ten (of 61) patients had a history of TB and developed RA 6 years (median) after contracting TB. Three new TB cases (1 from TST-positive and 2 from TST-negative groups) emerged during the follow-up. Our results suggest that persistently elevated T cell responses to Mtb antigens may contribute to disease activity in RA.

In humans, orthohantaviruses can cause hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). An earlier study reported that acute Andes virus HPS caused a massive and transient elevation in the number of circulating plasmablasts with specificity towards both viral and host antigens suggestive of polyclonal B cell activation. Immunoglobulins (Igs), produced by different B cell populations, comprise heavy and light chains; however, a certain amount of free light chains (FLCs) is constantly present in serum. Upregulation of FLCs, especially clonal species, associates with renal pathogenesis by fibril or deposit formations affecting the glomeruli, induction of epithelial cell disorders, or cast formation in the tubular network. We report that acute orthohantavirus infection increases the level of Ig FLCs in serum of both HFRS and HPS patients, and that the increase correlates with the severity of acute kidney injury in HFRS. The fact that the kappa to lambda FLC ratio in the sera of HFRS and HPS patients remained within the normal range suggests polyclonal B cell activation rather than proliferation of a single B cell clone. HFRS patients demonstrated increased urinary excretion of FLCs, and we found plasma cell infiltration in archival patient kidney biopsies that we speculate to contribute to the observed FLC excreta. Analysis of hospitalized HFRS patients’ peripheral blood mononuclear cells showed elevated plasmablast levels, a fraction of which stained positive for Puumala virus antigen. Furthermore, B cells isolated from healthy donors were susceptible to Puumala virus in vitro, and the virus infection induced increased production of Igs and FLCs. The findings propose that hantaviruses directly activate B cells, and that the ensuing intense production of polyclonal Igs and FLCs may contribute to acute hantavirus infection-associated pathological findings., Author summary Orthohantaviruses are globally spread zoonotic pathogens, which can cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) with significant burden to human health. The pathogenesis mechanisms of orthohantavirus-caused diseases are not known in detail; however, excessive immune response towards the virus with concomitant pathological effects against host tissues appears to be a contributing factor. Here we report an increase of free immunoglobulin (Ig) light chains (FLCs), components required to make complete Ig molecules, in blood of acute HFRS and HPS. Samples collected during acute HFRS demonstrated increased FLCs levels in the urine and blood of patients hospitalized due the disease. Furthermore, the FLC levels positively correlated with markers of acute kidney injury. In addition, our results show that orthohantaviruses can infect and activate B cells to produce FLCs as well as whole Igs, which provides a mechanistic explanation of the increased FLC levels in patients. Taken together, our results suggest that aberrant antibody responses might play a role in the pathogenesis of orthohantavirus infections.

Background Brucellosis, as a serious zoonotic infectious disease, has been recognized as a re-emerging disease in the developing countries worldwide. In china, the incidence of brucellosis is increasing each year, seriously threatening the health of humans as well as animal populations. Despite a quite number of diagnostic methods currently being used for brucellosis, innovative technologies are still needed for its rapid and accurate diagnosis, especially in area where traditional diagnostic is unavailable. Methodology/Principal findings In this study, a total of 22 B cell linear epitopes were predicted from five Brucella outer membrane proteins (OMPs) using an immunoinformatic approach. These epitopes were then chemically synthesized, and with the method of indirect ELISA (iELISA), each of them displayed a certain degree of capability in identifying human brucellosis positive sera. Subsequently, a fusion protein consisting of the 22 predicted epitopes was prokaryotically expressed and used as diagnostic antigen in a newly established brucellosis testing method, nano-ZnO modified paper-based ELISA (nano-p-ELISA). According to the verifying test using a collection of sera collected from brucellosis and non-brucellosis patients, the sensitivity and specificity of multiepitope based nano-p-ELISA were 92.38% and 98.35% respectively. The positive predictive value was 98.26% and the negative predictive value was 91.67%. The multiepitope based fusion protein also displayed significantly higher specificity than Brucella lipopolysaccharide (LPS) antigen. Conclusions B cell epitopes are important candidates for serologically testing brucellosis. Multiepitope fusion protein based nano-p-ELISA displayed significantly sensitivity and specificity compared to Brucella LPS antigen. The strategy applied in this study will be helpful to develop rapid and accurate diagnostic method for brucellosis in human as well as animal populations., Author summary Brucellosis is one of the most important zoonosis in the world and has caused tremendous economic losses in agriculture and animal husbandry in many countries. Developing rapid, sensitive and specific diagnostic methods is very important for early detection and treatment of brucellosis patients. In this study, a novel diagnostic technique, nano-ZnO modified paper ELISA, was established. The antigen used in this technique was a fusion protein containing multiple B cell epitopes, which were predicted from Brucella major outer membrane proteins such as Bp26, Omp31, Omp16, Omp2b and Omp25. Comparing to traditional LPS antigen, this multiepitope based antigen displayed considerably higher sensitivity and higher specificity in laboratory. With the strategy described in this paper, more efficient epitopes and protein antigen can be identified in the future. Currently, LPS antigen is only prepared from live Brucella, while protein antigen can be produced in large quantities in prokaryotic expression system. In addition to nano-p-ELISA, this protein antigen can also be used for development other methods such as fluorescent polarization assay (FPA) and immunochromatographic assay (ICA) to meet the varied demand for brucellosis testing.

The Epstein-Barr virus (EBV) human herpesvirus is associated with B-cell and epithelial-cell malignancies, and both the latent and lytic forms of viral infection contribute to the development of EBV-associated tumors. Here we show that the Hippo signaling effectors, YAP and TAZ, promote lytic EBV reactivation in epithelial cells. The transcriptional co-activators YAP/TAZ (which are inhibited by Hippo signaling) interact with DNA-binding proteins, particularly TEADs, to induce transcription. We demonstrate that depletion of either YAP or TAZ inhibits the ability of phorbol ester (TPA) treatment, cellular differentiation or the EBV BRLF1 immediate-early (IE) protein to induce lytic EBV reactivation in oral keratinocytes, and show that over-expression of constitutively active forms of YAP and TAZ reactivate lytic EBV infection in conjunction with TEAD family members. Mechanistically, we find that YAP and TAZ interact with, and activate, the EBV BZLF1 immediate-early promoter. Furthermore, we demonstrate that YAP, TAZ, and TEAD family members are expressed at much higher levels in epithelial cell lines in comparison to B-cell lines, and find that EBV infection of oral keratinocytes increases the level of activated (dephosphorylated) YAP and TAZ. Finally, we have discovered that lysophosphatidic acid (LPA), a known YAP/TAZ activator that plays an important role in inflammation, induces EBV lytic reactivation in epithelial cells through a YAP/TAZ dependent mechanism. Together these results establish that YAP/TAZ are powerful inducers of the lytic form of EBV infection and suggest that the ability of EBV to enter latency in B cells at least partially reflects the extremely low levels of YAP/TAZ and TEADs in this cell type., Author summary EBV causes infectious mononucleosis and persists in latently infected B cells for life. The virus periodically reactivates to a lytic form that produces infectious virus particles, allowing the virus to infect new cells and be transmitted to new hosts. Oral epithelial cells are an important site of lytic viral infection, promoting secretion of infectious virus into saliva. Rarely, EBV infection results in B-cell and epithelial-cell human tumors, and both the latent and lytic forms of infection contribute to these malignancies. However, the viral and cellular factors that determine whether the virus remains latent or lytic are still incompletely understood. Here we have discovered that the Hippo signaling pathway is an important regulator of lytic EBV reactivation, particularly in epithelial cells. We show that transcriptional co-activators YAP and TAZ (which are turned off by Hippo signaling) are strong inducers of lytic EBV reactivation, collaborating with DNA-binding TEAD proteins to activate EBV immediate-early genes. Furthermore, we demonstrate that epithelial cells express much higher levels of YAP/TAZ/TEADs in comparison to B cells, helping to explain why B cells support latent infection. Finally, we find that lysophosphatidic acid, a YAP/TAZ activator, induces lytic reactivation through a YAP/TAZ-dependent mechanism.

Epstein Barr virus (EBV) causes a highly prevalent and lifelong infection contributing to the development of some malignancies. In addition to the key role played by T cells in controlling this pathogen, NK cells mediate cytotoxicity and IFNγ production in response to EBV-infected B cells in lytic cycle, both directly and through antibody (Ab)-dependent activation. We recently described that EBV-specific Ab-dependent NK cell interaction with viral particles (VP) bound to B cells triggered degranulation and TNFα secretion but not B cell lysis nor IFNγ production. In this report we show that NK cell activation under these conditions reduced B cell transformation by EBV. NK cells eliminated VP from the surface of B cells through a specific and active process which required tyrosine kinase activation, actin polymerization and Ca2+, being independent of proteolysis and perforin. VP were displayed at the NK cell surface before being internalized and partially shuttled to early endosomes and lysosomes. VP transfer was encompassed by a trogocytosis process including the EBV receptor CD21, together with CD19 and CD20. Our study reveals a novel facet of the antibody-dependent NK cell mediated response to this viral infection., Author summary Epstein-Barr virus (EBV) is a member of the herpesvirus family which causes a frequent and lifelong infection. The immune system is unable to fully eliminate the virus, which remains dormant in infected B lymphocytes. EBV reactivation leads to the production of new infective particles, spreading to other cells and favoring its transmission. EBV infection goes generally unnoticed in healthy individuals, though it may occasionally cause a disease termed Infectious Mononucleosis, as well as severe disorders in patients with a defective immune response. Remarkably, EBV has oncogenic potential contributing to the development of some tumors, and has been associated to autoimmune diseases. T lymphocytes and Natural Killer (NK) cells play an essential role in the defense against EBV, killing infected cells when the virus reactivates. Antiviral NK cell functions may be also triggered by antibodies (Ab) recognizing infected cells. In this report we provide the first evidence supporting that NK cells in combination with anti-EBV Ab are able to eliminate the virus attached to the surface of B cells, reducing their infection without killing them.

Differentiation of Ag-specific B cells into class-switched, high-affinity, Ab-secreting cells provides protection against invading pathogens but is undesired when Abs target self-tissues in autoimmunity, beneficial non–self-blood transfusion products, or therapeutic proteins. Essential T cell factors have been uncovered that regulate T cell–dependent B cell differentiation. We performed a screen using a secreted protein library to identify novel factors that promote this process and may be used to combat undesired Ab formation. We tested the differentiating capacity of 756 secreted proteins on human naive or memory B cell differentiation in a setting with suboptimal T cell help in vitro (suboptimal CD40L and IL-21). High-throughput flow cytometry screening and validation revealed that type I IFNs and soluble FAS ligand (sFASL) induce plasmablast differentiation in memory B cells. Furthermore, sFASL induces robust secretion of IgG1 and IgG4 Abs, indicative of functional plasma cell differentiation. Our data suggest a mechanistic connection between elevated sFASL levels and the induction of autoreactive Abs, providing a potential therapeutic target in autoimmunity. Indeed, the modulators identified in this secretome screen are associated with systemic lupus erythematosus and may also be relevant in other autoimmune diseases and allergy.

Antibody secreting cells (ASC) are the effectors of protective humoral immunity and the only cell type that produces antibodies or immunoglobulins in mammals. In addition to their formidable capacity to secrete massive quantities of proteins, ASC are terminally-differentiated and have unique features to become long-lived plasma cells (LLPC). Upon antigen encounter, B cells are activated through a complex multistep process to undergo fundamental morphological, subcellular, and molecular transformation to become an efficient protein factory with life-long potential. The ASC survival potential is determined by factors at the time of induction, capacity to migration from induction to survival sites, and ability to mature in the specialized bone marrow microenvironments. In the past decade, considerable progress has been made in identifying factors regulating ASC longevity. Here, we review the intrinsic drivers, trafficking signals, and extrinsic regulators with particular focus on how they impact the survival potential to become a LLPC.

The main avenue for the development of an HIV-1 vaccine remains the induction of protective antibodies. A rationale approach is to target antigen to specific receptors on dendritic cells (DC) via fused monoclonal antibodies (mAb). In mouse and non-human primate models, targeting of skin Langerhans cells (LC) with anti-Langerin mAbs fused with HIV-1 Gag antigen drives antigen-specific humoral responses. The development of these immunization strategies in humans requires a better understanding of early immune events driven by human LC. We therefore produced anti-Langerin mAbs fused with the HIV-1 gp140z Envelope (αLC.Env). First, we show that primary skin human LC and in vitro differentiated LC induce differentiation and expansion of naïve CD4+ T cells into T follicular helper (Tfh) cells. Second, when human LC are pre-treated with αLC.Env, differentiated Tfh cells significantly promote the production of specific IgG by B cells. Strikingly, HIV-Env-specific Ig are secreted by HIV-specific memory B cells. Consistently, we found that receptors and cytokines involved in Tfh differentiation and B cell functions are upregulated by LC during their maturation and after targeting Langerin. Finally, we show that subcutaneous immunization of mice by αLC.Env induces germinal center (GC) reaction in draining lymph nodes with higher numbers of Tfh cells, Env-specific B cells, as well as specific IgG serum levels compared to mice immunized with the non-targeting Env antigen. Altogether, we provide evidence that human LC properly targeted may be licensed to efficiently induce Tfh cell and B cell responses in GC., Author summary In recent years, the place of innovative vaccines based on the induction/regulation and modulation of the immune response with the aim to elicit an integrated T- and B cell immune responses against complex antigens has emerged besides “classical” vaccine vectors. Targeting antigens to dendritic cells is a vaccine technology concept supported by more than a decade of animal models and human pre-clinical experimentation. Recent investigations in animals underscored that Langerhans cells (LC) are an important target to consider for the induction of antibody responses by DC targeting vaccine approaches. Nonetheless, the development of these immunization strategies in humans remains elusive. We therefore developed and produced an HIV vaccine candidate targeting specifically LC through the Langerin receptor. We tested the ability of our vaccine candidate of targeting LC from skin explant and of inducing in vitro the differentiation of T follicular helper (Tfh) cells. Using complementary in vitro models, we demonstrated that Tfh cells induced by human LC are functional and the targeting of LC by our vaccine candidate promotes the secretion of anti-HIV IgG by memory B cells from HIV-infected individuals. In this study human LC exhibit key cellular functions able to drive potent anti-HIV-1 humoral responses providing mechanistic evidence of the Tfh- and B cell stimulating functions of primary skin targeted LC. Finally, we demonstrated in Xcr1DTA mice the significant advantage of LC targeting for inducing Tfh and germinal center (GC)-B cells and anti-HIV-1 antibodies. Therefore, the targeting of the human Langerin receptor appears to be a promising strategy for developing efficient HIV-1 vaccine.