Search

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Post-exposure prophylaxis (PEP) is highly effective in preventing disease progression of rabies when used in timely and appropriate manner. The key treatment for PEP is infiltration of rabies immune globulin (RIG) into lesion site after bite exposure, besides wound care and vaccination. Unfortunately, however, RIG is expensive and its supply is limited. Currently, several anti-rabies virus monoclonal antibody (mAb) products are under development as alternatives to RIG, and two recently received regulatory approval in India. In this study, fully human mAbs that recognize different rabies virus glycoprotein conformational antigenic site (II and III) were created from peripheral blood mononuclear cells of heathy vaccinated subjects. These mAbs neutralized a diverse range of lyssavirus types. As at least two anti-rabies virus mAbs are recommended for use in human PEP to ensure broad coverage against diverse lyssaviruses and to minimize possible escape variants, two most potent mAbs, NP-19-9 and 11B6, were selected to be used as cocktail treatment. These two mAbs were broadly reactive to different types of lyssaviruses isolates, and were shown to have no interference with each other. These results suggest that NP-19-9 and 11B6 are potent candidates to be used for PEP, suggesting further studies involving clinical studies in human.

The development of B and T lymphocytes critically depends on RAG1/2 endonuclease activity to mediate antigen receptor gene assembly by V(D)J recombination. Although control of RAG1/2 activity through cell cycle- and ubiquitin-dependent degradation of RAG2 has been studied in detail, relatively little is known about mechanisms regulating RAG1 stability. We recently demonstrated that VprBP/DCAF1, a substrate adaptor for the CRL4 E3 ubiquitin ligase complex, is required to maintain physiological levels of RAG1 protein in murine B cells by facilitating RAG1 turnover. Loss of VprBP/DCAF1 in vivo results in elevated RAG1 expression, excessive V(D)J recombination, and immunoglobulin light chain repertoire skewing. Here we show that RAG1 is constitutively degraded when ectopically expressed in a human fibroblast cell line. Consistent with our findings in murine B cells, RAG1 turnover under these conditions is sensitive to loss of VprBP, as well as CRL4 or proteasome inhibition. Further evidence indicates that RAG1 degradation is ubiquitin-dependent and that RAG1 association with the CRL4VPRBP/DCAF1 complex is independent of CUL4 activation status. Taken together, these findings suggest V(D)J recombination co-opts an evolutionarily conserved and constitutively active mechanism to ensure rapid RAG1 turnover to restrain excessive RAG activity.

AbstractImmune homeostasis is critically regulated by the balance between activating and inhibitory receptors expressed on various immune cells such as T and B lymphocytes, and myeloid cells. The inhibitory receptors play a fundamental role in the immune checkpoint pathway, thus maintaining peripheral tolerance. We recently found that expression of leukocyte immunoglobulin-like receptor (LILR)B4, an inhibitory member of the human LILR family, is augmented in auto-antibody-producing plasmablasts/plasma cells of systemic lupus erythematosus (SLE) patients. However, the mechanism behind the ‘paradoxical’ up-regulation of this inhibitory receptor upon pathogenic antibody-secreting cells is yet to be known. To this end, in this study, we examined if glycoprotein 49B (gp49B), the murine counterpart of human LILRB4, is also elevated in auto-antibody-producing cells in several SLE mouse models, and tried to clarify the underlying mechanism. We found that gp49B is expressed on plasma cells of lupus-prone models but not of healthy C57BL/6 mice, and the level was positively correlated to the anti-double-stranded DNA IgG titer in serum. Gp49B genetic deletion, however, did not abolish the serum auto-antibodies or fully ameliorate the lethal glomerulonephritis, indicating that gp49B is not the sole regulator of lupus but a pathogenic element in the disease. We conclude that the elevated expression of this inhibitory receptor on pathogenic plasma cells was also relevant upon the murine SLE model. The mechanism of gp49B underlying the disease progression in lupus-prone mice has been discussed.

Protection against encapsulated bacteria can be elicited using polysaccharide vaccines. These antigens often behave as T-cell-independent type 2 antigens (TI-2 Ags). However, TI-2 Ags, including pneumococcal polysaccharides, often elicit weak immunoglobulin G (IgG) responses and are refractive to boosting. Conjugate vaccines have not completely overcome this challenge and hence, alternative strategies are required to enhance polysaccharide vaccine responses. Herein, we describe an adjuvant consisting of a Toll-like receptor and C-type lectin receptor agonist pairing that significantly increases primary immunoglobulin M and IgG responses to TI-2 Ags as well as enables significant boosting when coadministered with polysaccharide vaccines. Consistent with this, the adjuvant significantly increased the generation of both TI-2 memory B cells and long-lived antibody secreting cells. Adjuvant effects were highly dependent on B-cell-intrinsic MyD88, but not Trif expression. Importantly, coadministration of the adjuvant with the Pneumovax vaccine significantly increased the protective efficacy of vaccination in a lethal challenge mouse model of pneumococcal respiratory infection. Collectively, these data provide evidence that B-cell-directed adjuvants have promise in significantly improving the quality and quantity of serologic and B-cell memory responses to clinically relevant polysaccharide vaccines.

Salivarian trypanosomes are extracellular protozoan parasites causing infections in a wide range of mammalian hosts, with Trypanosoma evansi having the widest geographic distribution, reaching territories far outside Africa and occasionally even Europe. Besides causing the animal diseases, T. evansi can cause atypical Human Trypanosomosis. The success of this parasite is attributed to its capacity to evade and disable the mammalian defense response. To unravel the latter, we applied here for the first time a scRNA-seq analysis on splenocytes from trypanosome infected mice, at two time points during infection, i.e. just after control of the first parasitemia peak (day 14) and a late chronic time point during infection (day 42). This analysis was combined with flow cytometry and ELISA, revealing that T. evansi induces prompt activation of splenic IgM+CD1d+ Marginal Zone and IgMIntIgD+ Follicular B cells, coinciding with an increase in plasma IgG2c Ab levels. Despite the absence of follicles, a rapid accumulation of Aicda+ GC-like B cells followed first parasitemia peak clearance, accompanied by the occurrence of Xbp1+ expressing CD138+ plasma B cells and Tbx21+ atypical CD11c+ memory B cells. Ablation of immature CD93+ bone marrow and Vpreb3+Ly6d+Ighm+ expressing transitional spleen B cells prevented mature peripheral B cell replenishment. Interestingly, AID-/- mice that lack the capacity to mount anti-parasite IgG responses, exhibited a superior defense level against T. evansi infections. Here, elevated natural IgMs were able to exert in vivo and in vitro trypanocidal activity. Hence, we conclude that in immune competent mice, trypanosomosis associated B cell activation and switched IgG production is rapidly induced by T. evansi, facilitating an escape from the detrimental natural IgM killing activity, and resulting in increased host susceptibility. This unique role of IgM and its anti-trypanosome activity are discussed in the context of the dilemma this causes for the future development of anti-trypanosome vaccines., Author summary Trypanosoma evansi parasites can infect mammals, occasionally also humans, by evading the humoral immune response. In this study, cellular and transcriptomic profiling reveals that T. evansi induces rapid activation of mature splenic B cells, followed by differentiation into Aicda+ GC-like B cells, Tbx21+ atypical memory B cells and Sdc1+Xbp1+ plasma B cells. The process triggers early-stage Ighg2c expression in Follicular B cells. Simultaneous ablation of the bone marrow early B cell lineage prevents B cell replenishment, causing loss of the host’s parasitemia control capacity. Surprisingly, AID-/- mice lacking anti-parasite IgGs, exhibit a superior defense level against T. evansi infections, with elevated natural IgMs being able to exert trypanocidal activity. Hence, we conclude that in immune competent mice, trypanosomosis associated B cell Aicda activation and IgG2c production is rapidly induced by T. evansi in order to evade natural IgM mediated killing, resulting in increased host susceptibility.

Intravenous Ig (IVIG) is a treatment option for intractable cases of pemphigus vulgaris (PV), an autoimmune blistering disease caused by autoantibodies against desmoglein 3 (DSG3). To investigate the efficacy of IVIG on autoantibody secretion, we produced PV model mice by adoptive transfer of immunized Dsg3

During cellular differentiation chromosome conformation is intricately remodelled to support the lineage-specific transcriptional programs required for initiating and maintaining lineage identity. When these changes occur in relation to cell cycle, division and time in response to cellular activation and differentiation signals has yet to be explored, although it has been proposed to occur during DNA synthesis or after mitosis. Here, we elucidate the chromosome conformational changes in B lymphocytes as they differentiate and expand from a naive, quiescent state into antibody secreting plasma cells. We find gene-regulatory chromosome reorganization in late G1 phase before the first division, and that this configuration is remarkably stable as the cells massively and rapidly clonally expand. A second wave of conformational change occurs as cells terminally differentiate into plasma cells, coincident with increased time in G1 phase. These results provide further explanation for how lymphocyte fate is imprinted prior to the first division. They also suggest that chromosome reconfiguration occurs prior to DNA replication and mitosis, and is linked to a gene expression program that controls the differentiation process required for the generation of immunity., During differentiation, chromosome conformation is remodelled to support lineage-specific transcriptional programs. Here, the authors characterise chromosome conformational changes in B lymphocytes as they differentiate into plasma cells, and provide evidence that chromosome reconfiguration occurs prior to DNA replication and mitosis and guides gene expression that controls differentiation.

Immunity against malaria depends on germinal center (GC)-derived antibody responses that are orchestrated by T follicular helper (TFH) cells. Emerging data show that the regulatory cytokine IL-10 plays an essential role in promoting GC B cell responses during both experimental malaria and virus infections. Here we investigated the cellular source and temporal role of IL-10, and whether IL-10 additionally signals to CD4 T-cells to support anti-Plasmodium humoral immunity. Distinct from reports of virus infection, we found that IL-10 was expressed by conventional, Foxp3-negative effector CD4 T cells and functioned in a B cell-intrinsic manner only during the first 96 hours of Plasmodium infection to support humoral immunity. The critical functions of IL-10 manifested only before the orchestration of GC responses and were primarily localized outside of B cell follicles. Mechanistically, our studies showed that the rapid and transient provision of IL-10 promoted B cell expression of anti-apoptotic factors, MHC class II, CD83, and cell-cell adhesion proteins that are essential for B cell survival and interaction with CD4 T cells. Together, our data reveal temporal features and mechanisms by which IL-10 critically supports humoral immunity during blood-stage Plasmodium infection, information that may be useful for developing new strategies designed to lessen the burden of malaria., Author summary Malaria is associated with the expression of both inflammatory and anti-inflammatory cytokines that together regulate disease severity and the function of parasite-specific immune cells subsets. Emerging data show that the anti-inflammatory cytokine Interleukin (IL)-10 can function to sustain an already established humoral immune response during acute and chronic virus infections. Unexpectedly, our results show that IL-10 functions only within the first 72–96 hours of Plasmodium blood-stage infection to promote initial helper T cell and B cell interactions, as well as B cell differentiation and survival. Our studies of the temporal features, spatial relationships, critical cellular sources and mechanisms by which IL-10 promotes humoral immunity during malaria highlight the distinct mechanisms governing anti-malarial immunity. This new information may lead to the identification of novel pathways and approaches for eliciting durable protection against malaria.

B cells are primarily known for their capacity to differentiate into antibody-secreting cells (ASCs). ASCs are usually viewed as terminally differentiated cells sharing a unique phenotype. However, it lately became evident that ASCs exist in a variety of subsets differing by their lifespan, anatomic location, and immunological function. Thus, ASCs can exist as long-lived plasma cells (LLPC) that can persist for years in a nonproliferating state within particular niches in the bone marrow (BM), or as short-lived plasma cells (SLPC) that are primarily found in secondary lymphoid organs or inflamed tissues and wane upon the termination of the associated immune response. Another layer of ASC diversity was uncovered with the discovery of their capacity to produce various pro- or anti-inflammatory cytokines. Notably, a subset of natural regulatory plasma cells characterized by the distinctive expression of the inhibitory receptor lymphocyte activation gene (LAG)-3 and a unique capacity to produce interleukin (IL)-10 upon stimulation was recently identified. Here, we describe how to immunophenotypically characterize murine plasma cells as well as how to isolate them using cell sorting, with a special focus on these recently described natural regulatory plasma cells.

DNA topoisomerase II-β (TOP2B) is fundamental to remove topological problems linked to DNA metabolism and 3D chromatin architecture, but its cut-and-reseal catalytic mechanism can accidentally cause DNA double-strand breaks (DSBs) that can seriously compromise genome integrity. Understanding the factors that determine the genome-wide distribution of TOP2B is therefore not only essential for a complete knowledge of genome dynamics and organization, but also for the implications of TOP2-induced DSBs in the origin of oncogenic translocations and other types of chromosomal rearrangements. Here, we conduct a machine-learning approach for the prediction of TOP2B binding using publicly available sequencing data. We achieve highly accurate predictions, with accessible chromatin and architectural factors being the most informative features. Strikingly, TOP2B is sufficiently explained by only three features: DNase I hypersensitivity, CTCF and cohesin binding, for which genome-wide data are widely available. Based on this, we develop a predictive model for TOP2B genome-wide binding that can be used across cell lines and species, and generate virtual probability tracks that accurately mirror experimental ChIP-seq data. Our results deepen our knowledge on how the accessibility and 3D organization of chromatin determine TOP2B function, and constitute a proof of principle regarding the in silico prediction of sequence-independent chromatin-binding factors., Author summary Type II DNA topoisomerases (TOP2) are a double-edged sword. They solve topological problems in the form of supercoiling, knots and tangles that inevitably accompany genome metabolism, but they do so at the cost of transiently cleaving DNA, with the risk that this entails for genome integrity, and the serious consequences for human health, such as neurodegeneration, developmental disorders or predisposition to cancer. A comprehensive analysis of TOP2 distribution throughout the genome is therefore essential for a deep understanding of its function and regulation, and how this can affect genome dynamics and stability. Here, we use machine learning to thoroughly explore genome-wide binding of TOP2B, a vertebrate TOP2 paralog that has been linked to genome organization and cancer-associated translocations. Our analysis shows that TOP2B-DNA binding can be accurately predicted exclusively using information on DNA accessibility and binding of genome-architecture factors. We show that such information is enough to generate virtual maps of TOP2B binding along the genome, which we validate with de novo experimental data. Our results highlight the importance of TOP2B for accessibility and 3D organization of chromatin, and show that computationally predicted TOP2 maps can be accurately obtained using minimal publicly available datasets, opening the door for their use in different organisms, cell types and conditions with experimental and/or clinical relevance.

Children with Down syndrome have an approximately 10-fold increased risk of developing acute lymphoblastic leukemia and this risk is influenced by inherited genetic variation. Genome-wide association studies have identified IKZF1 as a strong acute lymphoblastic leukemia susceptibility locus in children both with and without Down syndrome, with association signals reported at rs4132601 in non-Down syndrome and rs58923657 in individuals with Down syndrome (r2 = 0.98 for these two loci). Expression quantitative trait locus analysis in non-Down syndrome lymphoblastoid cell lines has demonstrated an association between the rs4132601 risk allele and decreased IKZF1 mRNA levels. In this study, we provide further mechanistic evidence linking the region encompassing IKZF1-associated polymorphisms to pro-leukemogenic effects in both human lymphoblastoid cell lines and murine hematopoietic stem cells. CRISPR/Cas9-mediated deletion of the region encompassing the rs17133807 major allele (r2 with rs58923657 = 0.97) resulted in significant reduction of IKZF1 mRNA levels in lymphoblastoid cell lines, with a greater effect in Down syndrome versus non-Down syndrome cells. Since rs17133807 is highly conserved in mammals, we also evaluated the orthologous murine locus at rs263378223, in hematopoietic stem cells from the Dp16(1)Yey mouse model of Down syndrome as well as non-Down syndrome control mice. Homozygous deletion of the region encompassing rs263378223 resulted in significantly reduced Ikzf1 mRNA, confirming that this polymorphism maps to a strong murine Ikzf1 enhancer, and resulted in increased B-lymphoid colony growth and decreased B-lineage differentiation. Our results suggest that both the region encompassing rs17133807 and its conserved orthologous mouse locus have functional effects that may mediate increased leukemia susceptibility in both the Down syndrome and non-Down syndrome genetic backgrounds.

Copyright: © 2021 Machado et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., Adipose tissue is one of the major reservoirs of Trypanosoma brucei parasites, the causative agent of sleeping sickness, a fatal disease in humans. In mice, the gonadal adipose tissue (AT) typically harbors 2-5 million parasites, while most solid organs show 10 to 100-fold fewer parasites. In this study, we tested whether the AT environment responds immunologically to the presence of the parasite. Transcriptome analysis of T. brucei infected adipose tissue revealed that most upregulated host genes are involved in inflammation and immune cell functions. Histochemistry and flow cytometry confirmed an increasingly higher number of infiltrated macrophages, neutrophils and CD4+ and CD8+ T lymphocytes upon infection. A large proportion of these lymphocytes effectively produce the type 1 effector cytokines, IFN-γ and TNF-α. Additionally, the adipose tissue showed accumulation of antigen-specific IgM and IgG antibodies as infection progressed. Mice lacking T and/or B cells (Rag2-/-, Jht-/-), or the signature cytokine (Ifng-/-) displayed a higher parasite load both in circulation and in the AT, demonstrating the key role of the adaptive immune system in both compartments. Interestingly, infections of C3-/- mice showed that while complement system is dispensable to control parasite load in the blood, it is necessary in the AT and other solid tissues. We conclude that T. brucei infection triggers a broad and robust immune response in the AT, which requires the complement system to locally reduce parasite burden., This work was supported by the European Research Council (FatTryp, ref. 771714) awarded to LMF, by Fundação para a Ciência e Tecnologia (CEECIND/03322/2018) awarded to LMF, (PTDC/MED-IMU/30948/2017 and CEECIND/00697/2018) awarded to KS, (PD/BD/128286/2017) awarded to HM, (SFRH/BPD/89833/2012) awarded to ST, (IMM/BI/83-2017 through PTDC/BIM-MET/4471/2014) awarded to TB-R and by the National Institutes of Health (NIGMS K99GM132557) awarded to FR-F. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Regulation of the anti-apoptotic BCL2 protein determines cell survival and is frequently abnormal in B cell lymphomas. An evolutionarily conserved post-translational mechanism for over-expression of BCL2 in human B cell lymphomas and the BCL2 paralogue CED-9 in Caenorhabditis elegans results from loss-of-function mutations in human FBXO10 and its C.elegans paralogue DRE-1, a BCL2/CED-9-binding subunit of the SKP-CULLIN-FBOX (SCF) ubiquitin ligase. Here, we tested the role of FBXO10 in BCL2 regulation by producing mice with two different CRISPR/Cas9-engineered Fbxo10 mutations: an Asp54Lys (E54K) missense mutation in the FBOX domain and a Cys55SerfsTer55 frameshift (fs) truncating mutation. Mice homozygous for either mutant allele were born at the expected Mendelian frequency and appeared normal in body weight and appearance as adults. Spleen B cells from homozygous mutant mice did not have increased BCL2 protein, nor were the numbers of mature B cells or germinal centre B cells increased as would be expected if BCL2 was increased. Other lymphocyte subsets that are also regulated by BCL2 levels also displayed no difference in frequency in homozygous Fbxo10 mutant mice. These results support one of two conclusions: either FBXO10 does not regulate BCL2 in mice, or it does so redundantly with other ubiquitin ligase complexes. Possible candidates for the latter include FBXO11 or ARTS-XIAP. The difference between the role of FBXO10 in regulating BCL2 protein levels in C. elegans and in human DLBCL, relative to single-gene deficient mouse leukocytes, should be further investigated.

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease representing a serious unmet medical need. The disease is associated with the loss of self-tolerance and exaggerated B cell activation, resulting in autoantibody production and the formation of immune complexes that accumulate in the kidney, causing glomerulonephritis. TLR7, an important mediator of the innate immune response, drives the expression of type-1 interferon (IFN), which leads to expression of type-1 IFN induced genes and aggravates lupus pathology. Because the lysosomal peptide symporter slc15a4 is critically required for type-1 interferon production by pDC, and for certain B cell functions in response to TLR7 and TLR9 signals, we considered it as a potential target for pharmacological intervention in SLE. We deleted the slc15a4 gene in C57BL/6, NZB, and NZW mice and found that pristane-challenged slc15a4-/- mice in the C57BL/6 background and lupus prone slc15a4-/- NZB/W F1 mice were both completely protected from lupus like disease. In the NZB/W F1 model, protection persisted even when disease development was accelerated with an adenovirus encoding IFNα, emphasizing a broad role of slc15a4 in disease initiation. Our results establish a non-redundant function of slc15a4 in regulating both innate and adaptive components of the immune response in SLE pathobiology and suggest that it may be an attractive drug target.

Mice lacking B cells are more susceptible to S. typhimurium infection. How B cells contribute to protective immunity against Salmonella and what signals drive their activation are still unclear. Neutrophils (Nphs), monocytes (MOs), and dendritic cells (DCs) are involved in early immune responses to control the initial replication of S. typhimurium. These cells can produce B cell activating factor (BAFF) required for mature B cell survival and may help regulate B cell responses during Salmonella infection. Using BAFF reporter mice (BAFF-RFP+/-), we discovered that an i.p. infection with a virulent strain of S. typhimurium increased BAFF expression in splenic conventional DCs (cDC) and inflammatory Ly6Chi MOs/DCs four days post-infection. S. typhimurium infection induced the release of BAFF from Nphs, a decrease of BAFF-RFP expression and expansion of BAFF-RFP+ Nphs in the spleen and peritoneal cavity. After S. typhimurium infection, serum BAFF levels and immature and mature B cell subsets and plasma cells increased substantially. Conditional knockout (cKO) mice lacking BAFF in either Nphs or cDCs compared to control Bafffl/fl mice had reduced up-regulation of systemic BAFF levels and reduced expansion of mature and germinal center B cell subsets after infection. Importantly, the cKO mice lacking BAFF from either Nphs or cDCs had impaired induction of Salmonella-specific IgM Abs, and were more susceptible to S. typhimurium infection. Thus, Nphs and cDCs are major cellular sources of BAFF driving B cell responses, required for mounting optimal protective immunity against lethal Salmonella infection.