B-cell Antibody Production Research Articles

Sensory neurons regulate stimulus-dependent humoral immunity.

Sensory neurons sense pathogenic infiltration, serving to inform immune coordination of host defense. However, sensory neuron-immune interactions have been predominantly shown to drive innate immune responses. Humoral memory, whether protective or destructive, is acquired early in life - as demonstrated by both early exposure to streptococci and allergic disease onset. Our study further defines the role of sensory neuron influence on humoral immunity in the lung. Using a murine model of Streptococcus pneumonia pre-exposure and infection and a model of allergic asthma, we show that sensory neurons are required for B-cell and plasma cell recruitment and antibody production. In response to S. pneumoniae, sensory neuron depletion resulted in a larger bacterial burden, reduced B-cell populations, IgG release and neutrophil stimulation. Conversely, sensory neuron depletion reduced B-cell populations, IgE and asthmatic characteristics during allergen-induced airway inflammation. The sensory neuron neuropeptide released within each model differed. With bacterial infection, vasoactive intestinal polypeptide (VIP) was preferentially released, whereas substance P was released in response to asthma. Administration of VIP into sensory neuron-depleted mice suppressed bacterial burden and increased IgG levels, while VIP1R deficiency increased susceptibility to bacterial infection. Sensory neuron-depleted mice treated with substance P increased IgE and asthma, while substance P genetic ablation resulted in blunted IgE, similar to sensory neuron-depleted asthmatic mice. These data demonstrate that the immunogen differentially stimulates sensory neurons to release specific neuropeptides which specifically target B-cells. Targeting sensory neurons may provide an alternate treatment pathway for diseases involved with insufficient and/or aggravated humoral immunity.

Neuropeptide Y affects B-cell activation functions

Introduction: Sympathetic nerves secrete neurotransmitters in response to pathogenic organisms in various immunological contexts. Most studies have focused on norepinephrine (NE) which has shown numerous immunomodulatory effects involving pro- and anti-inflammatory responses. However, sympathetic neurons contain also varicosities where neuropeptides, mainly neuropeptide Y (NPY), are stored. NPY play roles in various physiological processes, including regulating metabolism, vascular function, and immune function but little is known on its action during bacterial respiratory infection. The regulation of its secretion and its impact on the immune system during infection remains unclear. We investigated the role of NPY in B-cell responses during bacterial infection. Methods: Naïve B-cells isolated from mice splenocytes (2M/2F) were stimulated with IL-4 and LPS, along with NE or NPY. After 96 hours, cells were stained and analyzed by flow cytometry and transcripts were isolated for quantitative real-time PCR. Media was collected until immunoglobulin analysis. To investigate NPY role during infection, we used a mice model of bacterial lung infection. Mice were intranasally exposed twice with streptococcus pneumoniae (serotype 19F on day 0: 104 CFU and on day 9: 108 CFU); experiments took place 16 hours later. Control animals were infused with vehicle only. Results: Apart from beta2-adrenergic receptor, isolated B-cells were found to express alpha2- and beta1-adrenergic receptor transcripts. In vitro stimulation of B-cell with IL-4 and LPS (T-cell independent activation) downregulated beta-adrenergic and upregulated alpha2-adrenergic, as well as NPY receptors (Npy1r, Npy2r, Npy4r, Npy5r). Unlike NE, NPY has shown to affect B-cell proliferation and drastically increases IgG positive-cells during stimulation. However, neither NE nor NPY seem to regulate IgG release in this condition. In vivo experiments indicate that NPY level increased in the lung in response to streptococcal challenge, contrasting with the unchanged levels of NE. Conclusion: Our findings contribute to our understanding of how sympathetic neurotransmitters regulates B-cell expansion, memory, and antibody production during infection. Furthermore, our research gives new insights into the function(s) and antimicrobial capacity of neuropeptide Y. These findings allow for the development of new and more effective therapeutic strategies for invasive bacterial infections. 1R21AI159221, UCOP TRDRP T32KT4708. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tfh cell-derived small extracellular vesicles exacerbate the severity of collagen-induced arthritis by enhancing B-cell responses

Soluble components secreted by Tfh cells are critical for the germinal center responses. In this study, we investigated whether Tfh cells could regulate the B-cell response by releasing small extracellular vesicles (sEVs). Our results showed that Tfh cells promote B-cell differentiation and antibody production through sEVs and that CD40L plays a crucial role in Tfh-sEVs function. In addition, increased Tfh-sEVs were found in mice with collagen-induced arthritis (CIA). Adoptive transfer of Tfh cells significantly exacerbated the severity of CIA; however, the effect of Tfh cells on exacerbating the CIA process was significantly diminished after inhibiting sEVs secretion. Moreover, the levels of plasma Tfh-like-sEVs and CD40L expression on Tfh-like-sEVs in RA patients were significantly higher than those in healthy subjects. In summary, Tfh cell-derived sEVs can enhance the B-cell response, and exacerbate the procession of autoimmune arthritis.

Regulatory T and CXCR3+ Circulating Tfh Cells Concordantly Shape the Neutralizing Antibody Responses in Individuals Who Have Recovered from Mild COVID-19.

Regulatory T (Treg) cells are involved in the antiviral immune response in patients with coronavirus disease 2019 (COVID-19); however, whether Treg cells are involved in the neutralizing antibody (nAb) response remains unclear. Here, we found that individuals who recovered from mild but not severe COVID-19 had significantly greater frequencies of Treg cells and lower frequencies of CXCR3+ circulating T follicular helper (cTfh) cells than healthy controls. Furthermore, the frequencies of Treg and CXCR3+ cTfh cells were negatively and positively correlated with the nAb responses, respectively, and Treg cells was inversely associated with CXCR3+ cTfh cells in individuals who recovered from mild COVID-19 but not in those with severe disease. Mechanistically, Treg cells inhibited memory B-cell differentiation and antibody production by limiting the activation and proliferation of cTfh cells, especially CXCR3+ cTfh cells, and functional molecule expression. This study provides novel insight showing that mild COVID-19 elicits concerted nAb responses, which are shaped by both Treg and Tfh cells.

Heterologous booster vaccination enhances antibody responses to SARS-CoV-2 by improving Tfh function and increasing B-cell clonotype SHM frequency.

Heterologous prime-boost has broken the protective immune response bottleneck of the COVID-19 vaccines. however, the underlying mechanisms have not been fully elucidated. Here, we investigated antibody responses and explored the response of germinal center (GC) to priming with inactivated vaccines and boosting with heterologous adenoviral-vectored vaccines or homologous inactivated vaccines in mice. Antibody responses were dramatically enhanced by both boosting regimens. Heterologous immunization induced more robust GC activation, characterized by increased Tfh cell populations and enhanced helper function. Additionally, increased B-cell activation and antibody production were observed in a heterologous regimen. Libra-seq was used to compare the differences of S1-, S2- and NTD-specific B cells between homologous and heterologous vaccination, respectively. S2-specific CD19+ B cells presented increased somatic hypermutations (SHMs), which were mainly enriched in plasma cells. Moreover, a heterologous booster dose promoted the clonal expansion of B cells specific to S2 and NTD regions. In conclusion, the functional role of Tfh and B cells following SARS-CoV-2 heterologous vaccination may be important for modulating antibody responses. These findings provide new insights for the development of SARS-CoV-2 vaccines that induce more robust antibody response.

Blockade of BLyS inhibits B-cell responses and antibody production for the prevention of chronic transplant rejection

BACKGROUNDChronic rejection, closely related to the activation of B cells and donor-specific antibody (DSA) production, has unsatisfactory therapeutic outcomes. B lymphocyte stimulator (BLyS) is a major regulatory factor that controls the activation and differentiation of B cells. However, it remains unclear whether BLyS blockade can regulate B and plasma cells in the transplantation setting and affect chronic rejection. Here, we investigated the efficacy of the BLyS inhibitors belimumab and telitacicept in controlling B-cell response and preventing chronic rejection. METHODSThe effects of belimumab and telitacicept on B-cell activation, differentiation and antibody production in vitro were determined. A chronic rejection model in mouse was established by allogeneic cardiac transplantation with CTLA4-Ig treatment. Allograft survival, histology, DSA levels, and B-cell responses were analyzed to evaluate the chronic rejection-preventive effects of belimumab and telitacicept. RESULTSIn vitro experiments confirmed that belimumab and telitacicept inhibited B-cell activation and differentiation and reduced antibody production. In vivo experiments indicated that they significantly prolonged allograft survival, attenuated chronic rejection through significant suppression of myocardial ischemic necrosis and interstitial fibrosis, and reduced DSA-IgG levels, C4d deposition, and inflammatory cell infiltration. Furthermore, the frequencies of B cells, plasma cells and IgG producing cells in the recipients’ spleen, lymph nodes, bone marrow, and blood were decreased after BLyS inhibitors treatment. CONCLUSIONSThis study demonstrated that belimumab and telitacicept inhibit B-cell responses and antibody production and alleviate chronic transplant rejection. Therefore, BLyS inhibitors are expected to be used for the prevention of chronic rejection in clinical practice.

Baricitinib in Combination with High-Dose Dexamethasone As First-Line Treatment for Newly Diagnosed Immune Thrombocytopenia: A Prospective, Multicenter, Randomized Trial

Introduction: High-dose dexamethasone is among the standard initial therapeutic options for patients with newly diagnosed immune thrombocytopenia (ITP). Although most patients achieve initial remission, optimal first-line therapy remains a challenge due to the low rates of sustained response and the high incidence of corticosteroid-dependency. Antiplatelet autoantibodies and splenic macrophages play central roles in the pathogenesis of ITP. Baricitinib, a Janus-associated kinase 1/2 (JAK1/2) inhibitor, can inhibit antigen presentation by innate immune cells and subsequent T-cell activation while also alleviating B-cell abnormalities and antibody production. The results of our single-arm pilot study suggested that baricitinib might increase the rate of durable response in patients with steroid-resistant or relapsed ITP (NCT05446831, presented at EHA2023, P1594). Accordingly, we further conducted a randomized, controlled trial to evaluate the efficacy and safety of baricitinib plus high-dose dexamethasone as first-line therapy for adulthood ITP. Methods: This multicenter, open-label, randomized, controlled, phase 2 trial was conducted in 10 different tertiary medical centers in China. Adult patients with newly diagnosed, treatment-naïve, confirmed primary ITP who had a platelet count <30×10⁹/L, or a platelet count <50×10⁹/L with clinically significant bleeding, were eligible for this study. Patients were randomly assigned at a 1:1 ratio to receive baricitinib (2 mg daily for 6 consecutive months) plus high-dose dexamethasone (40 mg daily for 4 consecutive days, repeated in the case of lack of response by Day 10) or high-dose dexamethasone alone. The primary endpoint was durable response at the 6-month follow-up. Efficacy endpoints were analyzed in the intention-to-treat population. Safety profiles were analyzed in all patients receiving at least one dose of study medications. The study protocol was approved by the ethics committee or institutional review board of each participating center and was registered with ClinicalTrials.gov (NCT 05932524). Results: A total of 172 patients were screened, of which 132 patients were eligible for this study. Patients were randomly assigned to receive baricitinib plus high-dose dexamethasone (n=66) or high-dose dexamethasone monotherapy (n=66). Four patients did not receive allocated treatment, leaving 128 patients in the safety analysis set. At the 6-month follow-up, a significantly higher proportion of participants in the baricitinib plus high-dose dexamethasone group (42/66, 63.6%) than in the high-dose dexamethasone monotherapy group (25/66, 37.9%) achieved durable response (OR 2.870, 95% CI 1.416-5.816; p=0.003). Initial response was achieved by 52 (78.8%) patients in the combination group and 47 (71.2%) patients in the monotherapy group. No significant difference was observed in the median time from initial treatment to response between the two groups. Complete response (at any time during the treatment) was achieved by 40 (60.6%) patients in the combination group and 35 (53.0%) patients in the monotherapy group. Patients responding to treatment in the combination group showed significantly longer durations of response than responders in the monotherapy group. Patients in both groups reported significantly improved health-related quality of life. The most common adverse events were infections (15/64, 23.4%) and insomnia (12/64, 18.8%) in the combination group, and insomnia (10/64, 15.6%) and anxiety or mood disorders (9/64, 14.1%) in the monotherapy group. No grade 4 or worse adverse events or treatment-related deaths were reported in either group. No thrombotic events, major adverse cardiovascular events (MACE), treatment-related bleeding or anemia were observed. Treatment discontinuation due to an adverse event was reported in 2 (3.1%) patients in the combination group and 3 (4.7%) patients in the monotherapy group. Conclusions: This is the first study reporting JAK inhibition as a first-line therapy for ITP. Baricitinib at 2 mg daily plus high-dose dexamethasone was well tolerated in patients with newly diagnosed ITP and might contribute to achieving a higher rate of durable response.

Quantifying the impact of PFOA Exposure on B cell development and antibody production.

Per- and polyfluoroalkyl substances (PFAS) are synthetic chemicals; the vast majority are environmentally and biologically persistent, and some have demonstrated toxicity, including cancer, effects on metabolism, endocrine disruption, and immune dysfunction. Suppression of T-cell dependent antibody responses (TDAR) has been observed in numerous studies of PFAS but mechanisms remain elusive. Evidence from our work suggests that B cells and how they use energy are impacted by PFAS exposure. We hypothesize that a well-studied and immunotoxic PFAS, perfluorooctanoic acid (PFOA), alters B cell subclasses and markers of their metabolism. Adult male and female C57BL/6 mice were given PFOA (0 or 7.5 mg/kg) via gavage for 15 days, a duration and dose sufficient to suppress the TDAR. After dosing and immunization of subgroups, spleens were prepared to quantify B cell subsets. Flow cytometric analysis revealed decreased numbers of plasmablasts, follicular, naïve, and overall B cell subclasses in female PFOA-exposed groups. Male PFOA-exposed groups had a significant increase in follicular B cells and other subsets had decreases, including in the overall number of B cells. Twenty-four hours after naïve B cell isolation and ex vivo activation, metabolic measurements revealed a five-fold increase in metabolic markers in response to stimulation in PFOA-exposed groups compared to controls. These findings suggest that B cell development and survival may be hindered by PFOA exposure, but that activation of the remaining B cells was not. Based on these findings, PFOA mediated suppression of the primary IgM antibody response results changes to specific subsets of B cells.

Aberrant pro-inflammatory responses of CD20+ T cells in experimental arthritis

CD20+ T cells comprise a highly inflammatory subset implicated in autoimmunity, including rheumatoid arthritis (RA). We sought to characterize the CD20+ T cell subset in the murine collagen-induced arthritis (CIA) model of RA and investigate the phenotype and functional relevance of CD3+CD20+ T cells in the lymph nodes and arthritic joints using flow cytometry and immunohistochemistry. We demonstrate that CD3+CD4+CD20+ and CD3+CD8+CD20+ T cells are expanded in the draining lymph nodes of CIA mice, produce increased levels of pro-inflammatory cytokines and are less susceptible to regulation by regulatory T cells. Notably, CD3+CD4+CD20+ and CD3+CD8+CD20+ T cells are enriched with CXCR5+PD-1+ T follicular helper cells and CXCR5-PD-1+ peripheral T helper cells, subsets of T cells implicated in promoting B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues in RA. Our findings suggest CD20+ T cells are associated with inflammatory responses and may exacerbate pathology by promoting inflammatory B-cell responses.

Structural Complementarity of Bruton’s Tyrosine Kinase and Its Inhibitors for Implication in B-Cell Malignancies and Autoimmune Diseases

Bruton's tyrosine kinase (BTK) is a critical component in B-cell receptor (BCR) signaling and is also expressed in haematogenic and innate immune cells. Inhibition of BTK hyperactivity is implicated in B-cell malignancies and autoimmune diseases. This review derives the structural complementarity of the BTK-kinase domain and its inhibitors from recent three-dimensional structures of inhibitor-bound BTK in the protein data bank (PDB). Additionally, this review analyzes BTK-mediated effector responses of B-cell development and antibody production. Covalent inhibitors contain an α, β-unsaturated carbonyl moiety that forms a covalent bond with Cys481, stabilizing αC-helix in inactive-out conformation which inhibits Tyr551 autophosphorylation. Asn484, located two carbons far from Cys481, influences the stability of the BTK-transition complex. Non-covalent inhibitors engage the BTK-kinase domain through an induced-fit mechanism independent of Cys481 interaction and bind to Tyr551 in the activation kink resulting in H3 cleft, determining BTK selectivity. Covalent and non-covalent binding to the kinase domain of BTK shall induce conformational changes in other domains; therefore, investigating the whole-length BTK conformation is necessary to comprehend BTK's autophosphorylation inhibition. Knowledge about the structural complementarity of BTK and its inhibitors supports the optimization of existing drugs and the discovery of drugs for implication in B-cell malignancies and autoimmune diseases.

Historical Review and Future of Cardiac Xenotransplantation.

Along with the development of immunosuppressive drugs, major advances on xenotransplantation were achieved by understanding the immunobiology of xenograft rejection. Most importantly, three predominant carbohydrate antigens on porcine endothelial cells were key elements provoking hyperacute rejection: α1,3-galactose, SDa blood group antigen, and N-glycolylneuraminic acid. Preformed antibodies binding to the porcine major xenoantigen causes complement activation and endothelial cell activation, leading to xenograft injury and intravascular thrombosis. Recent advances in genetic engineering enabled knock-outs of these major xenoantigens, thus producing xenografts with less hyperacute rejection rates. Another milestone in the history of xenotransplantation was the development of co-stimulation blockaded strategy. Unlike allotransplantation, xenotransplantation requires blockade of CD40-CD40L pathway to prevent T-cell dependent B-cell activation and antibody production. In 2010s, advanced genetic engineering of xenograft by inducing the expression of multiple human transgenes became available. So-called 'multi-gene' xenografts expressing human transgenes such as thrombomodulin and endothelial protein C receptor were introduced, which resulted in the reduction of thrombotic events and improvement of xenograft survival. Still, there are many limitations to clinical translation of cardiac xenotransplantation. Along with technical challenges, zoonotic infection and physiological discordances are major obstacles. Social barriers including healthcare costs also need to be addressed. Although there are several remaining obstacles to overcome, xenotransplantation would surely become the novel option for millions of patients with end-stage heart failure who have limited options to traditional therapeutics.

Human B cells.

The importance of B cells and their critical role in the maintenance of health through generation of antibody mediated immune protection is undoubted. However, the differences between the responses of B cells with different surface phenotypes in different microanatomical sites as well as diversity in B cell function outside antibody production are just starting to be acknowledged and resolved. This series of reviews and papers that focus on human B cells will be divided across two issues. The first part of the review series in this issue captures practical information on identifying B cell subtypes in blood in health and inflammatory diseases as well as describing aspects of B cell diversity depending on immunoglobulin isotype and microanatomical context. It also explores our current understanding of cytokine production by human B cells and the effect of obesity on the B cell response. The last review in this issue will reflect on the important lessons learned from the SARS-CoV-2 pandemic; in particular the role of antigen availability and its effect on B cell memory and antibody production.

A synthetic human 3D in vitro lymphoid model enhancing B-cell survival and functional differentiation

To investigate B-cell differentiation and maturation occurring in the germinal center (GC) using invitro culture systems, key factors and interactions of the GC reaction need to be accurately simulated. This study aims at improving invitro GC simulation using 3D culture techniques. Human B-cells were incorporated into PEG-4MAL hydrogels, to create a synthetic extracellular matrix, supported by CD40L cells, human tonsil-derived lymphoid stromal cells, and cytokines. The differentiation and antibody production of CD19+B-cells was best supported in a 5.0%-PEG-4MAL, 2.0mM-RGD-peptide composition. The 3D culture significantly increased plasmablast and plasma cell numbers as well as antibody production, with less B-cell death compared to 2D cultures. Class switching of naive CD19+IgD+B-cells toward IgG+ and IgA+B-cells was observed. The formation of large B-cell clusters indicates the formation of GC-like structures. In conclusion, a well-characterized and controllable hydrogel-based human 3D lymphoid model is presented that supports enhanced B-cell survival, proliferation, differentiation, and antibody production.

Considerations for Novel COVID-19 Mucosal Vaccine Development.

Mucosal surfaces are the first contact sites of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Most SARS-CoV-2 vaccines induce specific IgG responses but provide limited mucosal immunity. Cytokine B-cell activation factor (BAFF) and A proliferation-inducing ligand (APRIL) in the tumor necrosis factor (TNF) superfamily play key immunological functions during B cell development and antibody production. Furthermore, homeostatic chemokines, such as C-X-C motif chemokine ligand 13 (CXCL13), chemokine (C–C motif) ligand 19 (CCL19), and CCL21, can induce B- and T-cell responses to infection and promote the formation of inducible bronchus-associated lymphoid tissues (iBALT), where specific local immune responses and memory cells are generated. We reviewed the role of BAFF, APRIL, CXCL13, CCL19, and CCL21 in the activation of local B-cell responses and antibody production, and the formation of iBALT in the lung following viral respiratory infections. We speculate that mucosal vaccines may offer more efficient protection against SARS-CoV-2 infection than systematic vaccines and hypothesize that a novel SARS-CoV-2 mRNA mucosal vaccine using BAFF/APRIL or CXCL13 as immunostimulants combined with the spike protein-encoding mRNA may enhance the efficiency of the local immune response and prevent the early stages of SARS-CoV-2 replication and the rapid viral clearance from the airways.

Enriched Marine Oil Supplement Increases Specific Plasma Specialized Pro-Resolving Mediators in Adults with Obesity

Specialized pro-resolving mediators (SPMs), synthesized from PUFAs, resolve inflammation and return damaged tissue to homeostasis. Thus, increasing metabolites of the SPM biosynthetic pathway may have potential health benefits for select clinical populations, such as subjects with obesity who display dysregulation of SPM metabolism. However, the concentrations of SPMs and their metabolic intermediates in humans with obesity remains unclear. The primary objective of this study was to determine if a marine oil supplement increased specific metabolites of the SPM biosynthetic pathway in adults with obesity. The second objective was to determine if the supplement changed the relative abundance of key immune cell populations. Finally, given the critical role of antibodies in inflammation, we determined if ex vivo CD19+B-cell antibody production was modified by marine oil intervention. Twenty-three subjects [median age: 56 y; BMI (in kg/m2): 33.1] consumed 2g/d of a marine oil supplement for 28-30 d. The supplement was particularly enriched with 18-hydroxyeicosapentaenoic (HEPE), 14-hydroxydocosahexaenoic acid (14-HDHA), and 17-HDHA. Blood was collected pre- and postsupplementation for plasma mass spectrometry oxylipin and fatty acid analyses, flow cytometry, and B-cell isolation. Paired t-tests and Wilcoxon tests were used for statistical analyses. Relative to preintervention, the supplement increased 6 different HEPEs and HDHAs accompanied by changes in plasma PUFAs. Resolvin E1 and docosapentaenoic acid-derived maresin 1 concentrations were increased 3.5- and 4.7-fold upon intervention, respectively. The supplement did not increase the concentration of D-series resolvins and had no effect on the abundance of immune cells. Ex vivo B-cell IgG but not IgM concentrations were lowered postsupplementation. A marine oil supplement increased select SPMs and their metabolic intermediates in adults with obesity. Additional studies are needed to determine if increased concentrations of specific SPMs control the resolution of inflammation in humans with obesity. This trial was registered at clinicaltrials.gov as NCT04701138.

Fumarate suppresses B-cell activation and function through direct inactivation of LYN.

Activated B cells increase central carbon metabolism to fulfill their bioenergetic demands, yet the mechanistic basis for this, as well as metabolic regulation in B cells, remains largely unknown. Here, we demonstrate that B-cell activation reprograms the tricarboxylic acid cycle and boosts the expression of fumarate hydratase (FH), leading to decreased cellular fumarate abundance. Fumarate accumulation by FH inhibition or dimethyl-fumarate treatment suppresses B-cell activation, proliferation and antibody production. Mechanistically, fumarate is a covalent inhibitor of tyrosine kinase LYN, a key component of the BCR signaling pathway. Fumarate can directly succinate LYN at C381 and abrogate LYN activity, resulting in a block to B-cell activation and function in vitro and in vivo. Therefore, our findings uncover a previously unappreciated metabolic regulation of B cells, and reveal LYN is a natural sensor of fumarate, connecting cellular metabolism to B-cell antigen receptor signaling.

Innate stimulation of B cells ex vivo enhances antibody secretion and identifies tumour-reactive antibodies from cancer patients.

Human B cells and their expressed antibodies are crucial in conferring immune protection. Identifying pathogen-specific antibodies following infection is possible due to enhanced humoral immunity against well-described molecules on the pathogen surface. However, screening for cancer-reactive antibodies remains challenging since target antigens are often not identified a priori and the frequency of circulating B cells recognizing cancer cells is likely very low. We investigated whether combined ex vivo culture of human B cells with three innate stimuli, interleukin-17 (IL-17), B-cell activation factor (BAFF), and the toll-like receptor 9 (TLR-9) agonist DNA motif CpG ODN 2006 (CpG), each known to activate B cells through different signalling pathways, promote cell activation, proliferation, and antibody production. Combined IL-17+BAFF+CpG prolonged B-cell survival and increased proliferation compared with single stimuli. IL-17+BAFF+CpG triggered higher IgG secretion, likely by activating differentiated, memory and class-switched CD19+CD20+CD27+IgD- B cells. Regardless of anti-FOLR antibody seropositive status, IL-17+BAFF+CpG combined with a monovalent tumour-associated antigen (folate receptor alpha [FOLR]) led to secreted antibodies recognizing the antigen and the antigen-expressing IGROV1 cancer cells. In a seropositive individual, FOLR stimulation favoured class-switched memory B-cell precursors (CD27-CD38-IgD-), class-switched memory B cells and anti-FOLR antibody production, while IL-17+BAFF+CpG combined with FOLR, promoted class-switched memory B-cell precursors and antibody-secreting (CD138+IgD-) plasma cells. Furthermore, IL-17+BAFF+CpG stimulation of peripheral blood B cells from patients with melanoma revealed tumour cell-reactive antibodies in culture supernatants. These findings suggest that innate signals stimulate B-cell survival and antibody production and may help identify low-frequency antigen-reactive humoral responses.

Using Mass Cytometry to Address Tfh and Tfr Heterogeneity.

Recent advances in single-cell technologies have made it possible to gather increased amounts of information about even rare cell subtypes. In particular, mass cytometry is able to assess the expression of 30-50 proteins on millions of cells. Within CD4 T-cells, T-follicular helper cells (Tfh) and their regulatory counterpart, T-follicular regulatory cells (Tfr), localize to the B-cell follicle and have specialized roles in the maintenance and regulation of B-cell antibody production. The frequency of Tfh and Tfr in circulation has also been associated with ongoing antibody responses.In this chapter, we detail methods to analyze the frequency and phenotype of the populations of Tfh and Tfr found in humans by mass cytometry.

Specific antibody response of patients with common variable immunodeficiency to BNT162b2 coronavirus disease 2019 vaccination

Specific antibody response of patients with common variable immunodeficiency to BNT162b2 coronavirus disease 2019 vaccination

Up State of the SARS-COV-2 Spike Homotrimer Favors an Increased Virulence for New Variants.

The COVID-19 pandemic has spread worldwide. However, as soon as the first vaccines—the only scientifically verified and efficient therapeutic option thus far—were released, mutations combined into variants of SARS-CoV-2 that are more transmissible and virulent emerged, raising doubts about their efficiency. This study aims to explain possible molecular mechanisms responsible for the increased transmissibility and the increased rate of hospitalizations related to the new variants. A combination of theoretical methods was employed. Constant-pH Monte Carlo simulations were carried out to quantify the stability of several spike trimeric structures at different conformational states and the free energy of interactions between the receptor-binding domain (RBD) and angiotensin-converting enzyme II (ACE2) for the most worrying variants. Electrostatic epitopes were mapped using the PROCEEDpKa method. These analyses showed that the increased virulence is more likely to be due to the improved stability to the S trimer in the opened state, in which the virus can interact with the cellular receptor, ACE2, rather than due to alterations in the complexation RBD-ACE2, since the difference observed in the free energy values was small (although more attractive in general). Conversely, the South African/Beta variant (B.1.351), compared with the SARS-CoV-2 wild type (wt), is much more stable in the opened state with one or two RBDs in the up position than in the closed state with three RBDs in the down position favoring the infection. Such results contribute to understanding the natural history of disease and indicate possible strategies for developing new therapeutic molecules and adjusting the vaccine doses for higher B-cell antibody production.