Rapid generation of human recombinant monoclonal antibodies from antibody-secreting cells using ferrofluid-based technology.

Monoclonal antibodies (mAbs) are one of the most important classes of biologics with high therapeutic and diagnostic value, but traditional methods for mAbs generation, such as hybridoma screening and phage display, have limitations, including low efficiency and loss of natural chain pairing. To overcome these challenges, novel single B cell antibody technologies have emerged, but they also have limitations such as in vitro differentiation of memory B cells and expensive cell sorters. In this study, we present a rapid and efficient workflow for obtaining human recombinant monoclonal antibodies directly from single antigen-specific antibody secreting cells (ASCs) in the peripheral blood of convalescent COVID-19 patients using ferrofluid technology. This process allows the identification and expression of recombinant antigen-specific mAbs in less than 10 days, using RT-PCR to generate linear Ig heavy and light chain gene expression cassettes, called "minigenes", for rapid expression of recombinant antibodies without cloning procedures. This approach has several advantages. First, it saves time and resources by eliminating the need for in vitro differentiation. It also allows individual antigen-specific ASCs to be screened for effector function prior to recombinant antibody cloning, enabling the selection of mAbs with desired characteristics and functional activity. In addition, the method allows comprehensive analysis of variable region repertoires in combination with functional assays to evaluate the specificity and function of the generated antigen-specific antibodies. Our approach, which rapidly generates recombinant monoclonal antibodies from single antigen-specific ASCs, could help to identify functional antibodies and deepen our understanding of antibody dynamics in the immune response through combined antibody repertoire sequence analysis and functional reactivity testing.

A nanowell platform to identify, sort and expand high antibody-producing cells.

Increased use of therapeutic monoclonal antibodies and the relatively high manufacturing costs fuel the need for more efficient production methods. Here we introduce a novel, fast, robust, and safe isolation platform for screening and isolating antibody-producing cell lines using a nanowell chip and an innovative single-cell isolation method. An anti-Her2 antibody producing CHO cell pool was used as a model. The platform; (1) Assures the single-cell origin of the production clone, (2) Detects the antibody production of individual cells and (3) Isolates and expands the individual cells based on their antibody production. Using the nanowell platform we demonstrated an 1.8-4.5 increase in anti-Her2 production by CHO cells that were screened and isolated with the nanowell platform compared to CHO cells that were not screened. This increase was also shown in Fed-Batch cultures where selected high production clones showed titers of 19-100 mg/L on harvest day, while the low producer cells did not show any detectable anti-Her2 IgG production. The screening of thousands of single cells is performed under sterile conditions and the individual cells were cultured in buffers and reagents without animal components. The time required from seeding a single cell and measuring the antibody production to fully expanded clones with increased Her-2 production was 4-6 weeks.

Safety and immunogenicity of a thermostable ID93 + GLA-SE tuberculosis vaccine candidate in healthy adults.

Adjuvant-containing subunit vaccines represent a promising approach for protection against tuberculosis (TB), but current candidates require refrigerated storage. Here we present results from a randomized, double-blinded Phase 1 clinical trial (NCT03722472) evaluating the safety, tolerability, and immunogenicity of a thermostable lyophilized single-vial presentation of the ID93 + GLA-SE vaccine candidate compared to the non-thermostable two-vial vaccine presentation in healthy adults. Participants were monitored for primary, secondary, and exploratory endpoints following intramuscular administration of two vaccine doses 56 days apart. Primary endpoints included local and systemic reactogenicity and adverse events. Secondary endpoints included antigen-specific antibody (IgG) and cellular immune responses (cytokine-producing peripheral blood mononuclear cells and T cells). Both vaccine presentations are safe and well tolerated and elicit robust antigen-specific serum antibody and Th1-type cellular immune responses. Compared to the non-thermostable presentation, the thermostable vaccine formulation generates greater serum antibody responses (p < 0.05) and more antibody-secreting cells (p < 0.05). In this work, we show the thermostable ID93 + GLA-SE vaccine candidate is safe and immunogenic in healthy adults.

Dysregulated B cell differentiation towards antibody-secreting cells in neuromyelitis optica spectrum disorder.

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.

Antibody secreting cells are critically dependent on integrin α4ß7/MAdCAM-1 for intestinal recruitment and control of the microbiota during chronic colitis.

T and B cells employ integrin α4ß7 to migrate to intestine under homeostatic conditions. Whether those cells differentially rely on α4ß7 for homing during inflammatory conditions has not been fully examined. This may have implications for our understanding of the mode of action of anti-integrin therapies in inflammatory bowel disease (IBD). Here, we examined the role of α4ß7 integrin during chronic colitis using IL-10-/- mice, ß7-deficient IL-10-/-, IgA-deficient IL-10-/- mice, and antibody blockade of MAdCAM-1. We found that α4ß7 was predominantly expressed by B cells. ß7 deficiency and MAdCAM-1 blockade specifically depleted antibody secreting cells (ASC) (not T cells) from the colonic LP, leading to a fecal pan-immunoglobulin deficit, severe colitis, and alterations of microbiota composition. Colitis was not due to defective regulation, as dendritic cells (DC), regulatory T cells, retinaldehyde dehydrogenase (RALDH) expression, activity, and regulatory T/B-cell cytokines were all comparable between the strains/treatment. Finally, an IgA deficit closely recapitulated the clinical phenotype and altered microbiota composition of ß7-deficient IL-10-/- mice. Thus, a luminal IgA deficit contributes to accelerated colitis in the ß7-deficient state. Given the critical/nonredundant dependence of IgA ASC on α4ß7:MAdCAM-1 for intestinal homing, B cells may represent unappreciated targets of anti-integrin therapies.

The presence of circulating antibody secreting cells and long-lived memory B cell responses to reticulocyte binding protein 1a in Plasmodium vivax patients.

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.

Diagnosis of Streptococcus pneumoniae infection using circulating antibody secreting cells.

BACKGROUND: Streptococcus pneumoniae infections cause morbidity and mortality worldwide. A rapid, simple diagnostic method could reduce the time needed to introduce definitive therapy potentially improving patient outcomes. METHODS: We introduce two new methods for diagnosing S. pneumoniae infections by measuring the presence of newly activated, pathogen-specific, circulating Antibody Secreting Cells (ASC). First, ASC were detected by ELISpot assays that measure cells secreting antibodies specific for signature antigens. Second, the antibodies secreted by isolated ASC were collected in vitro in a novel matrix, MENSA (media enriched with newly synthesized antibodies) and antibodies against S. pneumoniae antigens were measured using Luminex immunoassays. Each assay was evaluated using blood from S. pneumoniae and non-S. pneumoniae-infected adult patients. RESULTS: We enrolled 23 patients with culture-confirmed S. pneumoniae infections and 24 controls consisting of 12 non-S. pneumoniae infections, 10 healthy donors and two colonized with S. pneumoniae. By ELISpot assays, twenty-one of 23 infected patients were positive, and all 24 controls were negative. Using MENSA samples, four of five S. pneumoniae-infected patients were positive by Luminex immunoassays while all five non-S. pneumoniae-infected patients were negative. CONCLUSION: Specific antibodies produced by activated ASC may provide a simple diagnostic for ongoing S. pneumoniae infections. This method has the potential to diagnose acute bacterial infections.

The role of interferon regulatory factor 7 in the pathogenicity and immunogenicity of rabies virus in a mouse model.

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/ß 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.

Colloidal Manganese Salt Improves the Efficacy of Rabies Vaccines in Mice, Cats, and Dogs.

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.

Soluble FAS Ligand Enhances Suboptimal CD40L/IL-21-Mediated Human Memory B Cell Differentiation into Antibody-Secreting Cells.

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 importance of the Atlantic salmon peritoneal cavity B cell response: Local IgM secreting cells are predominant upon Piscirickettsia salmonis infection.

The intraperitoneal route is favored for administration of inactivated and attenuated vaccines in Atlantic salmon. Nevertheless, the immune responses in the teleost peritoneal cavity (PerC) are still incompletely defined. In this study, we investigated the B cell responses after intraperitoneal Piscirickettsia salmonis (P. salmonis) challenge of Atlantic salmon, focusing on the local PerC response versus responses in the lymphatic organs: spleen and head kidney. We observed a major increase of leukocytes, total IgM antibody secreting cells (ASC), and P. salmonis-specific ASC in the PerC at 3- and 6-weeks post infection (wpi). The increase in ASC frequency was more prominent in the spleen and PerC compared to the head kidney during the observed 6 wpi. The serum antibody response included P. salmonis-specific antibodies and non-specific antibodies recognizing the non-related bacterial pathogen Yersinia ruckeri and the model antigen TNP-KLH. Finally, we present evidence that supports a putative role for the adipose tissue in the PerC immune response.

The diversity of the plasmablast signature across species and experimental conditions: A meta-analysis.

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.

Extrafollicular IgD+ B cells generate IgE antibody secreting cells in the nasal mucosa.

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+ naïve-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 naïve 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+ naïve B cells locally undergo direct and indirect (through IgG and IgA), IgE class switch.

Prophylactic administration of fingolimod (FTY720) ameliorated experimental autoimmune myasthenia gravis by reducing the number of dendritic cells, follicular T helper cells and antibody-secreting cells.

Fingolimod (FTY720), a sphingosine 1-phosphate (S1P) receptor antagonist, possesses potent immunomodulatory activity via lymphocyte homing. The effects of FTY720 have been widely studied in various T-cell-mediated autoimmune diseases, while the immunomodulatory effects on experimental autoimmune myasthenia gravis (EAMG), a typical disease model for antibody-mediated autoimmunity, remain elusive. In the present study, FTY720 was administered to EAMG rats as prophylaxis. The clinical scores were recorded every other day, and serum antibodies at different time points were measured by enzyme-linked immunosorbent assay (ELISA). The immune cell subsets in the spleen, bone marrow, circulation, and thymus were determined by flow cytometry. The prophylactic administration alleviated EAMG symptoms by reducing the level of serum antibodies IgG and its isotype IgG2b on days 30 and 46 post immunization, as well as IgG and Ig kappa antibody-secreting cells in the spleen and bone marrow. The mitigated humoral immune response can be attributed to the decreased dendritic cells, follicular T help cells (Tfh) and Tfh subsets (Tfh1, Tfh2, and Tfh17), and T helper cell subsets (Th1, Th2, and Th17) in the spleen. The promotion of lymphocyte homing and inhibition of thymocyte egress contribute to the effects of FTY720 on these effector T cell subsets. Overall, the prophylactic administration of FTY720 ameliorated EAMG partially by regulating humoral immune response，suggesting that FTY720 could be part of a pharmacological strategy for managing myasthenia gravis.

DNA priming immunization is more effective than recombinant protein vaccine in eliciting antigen-specific B cell responses.

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.

Vibrio cholerae Sialidase-Specific Immune Responses Are Associated with Protection against Cholera.

Cholera remains a major public health problem in resource-limited countries. Vaccination is an important strategy to prevent cholera, but currently available vaccines provide only 3 to 5 years of protection. Understanding immune responses to cholera antigens in naturally infected individuals may elucidate which of these are key to longer-term protection seen following infection. We recently identified Vibrio cholerae O1 sialidase, a neuraminidase that facilitates binding of cholera toxin to intestinal epithelial cells, as immunogenic following infection in two recent high-throughput screens. Here, we present systemic, mucosal, and memory immune responses to sialidase in cholera index cases and evaluated whether systemic responses to sialidase correlated with protection using a cohort of household contacts. Overall, we found age-related differences in antisialidase immune response following cholera. Adults developed significant plasma anti-sialidase IgA, IgG, and IgM responses following infection, whereas older children (≥5 years) developed both IgG and IgM responses, and younger children only developed IgM responses. Neither older children nor younger children had a rise in IgA responses over the convalescent phase of infection (day 7/day 30). On evaluation of mucosal responses and memory B-cell responses to sialidase, we found adults developed IgA antibody-secreting cell (ASC) and memory B-cell responses. Finally, in household contacts, the presence of serum anti-sialidase IgA, IgG, and IgM antibodies at enrollment was associated with a decrease in the risk of subsequent infection. These data show cholera patients develop age-related immune responses against sialidase and suggest that immune responses that target sialidase may contribute to protective immunity against cholera.IMPORTANCE Cholera infection can result in severe dehydration that may lead to death within a short period of time if not treated immediately. Vaccination is an important strategy to prevent the disease. Oral cholera vaccines provide 3 to 5 years of protection, with 60% protective efficacy, while natural infection provides longer-term protection than vaccination. Understanding the immune responses after natural infection is important to better understand immune responses to antigens that mediate longer-term protection. Sialidase is a neuraminidase that facilitates binding of cholera toxin to intestinal epithelial cells. We show here that patients with cholera develop systemic, mucosal, and memory B-cell immune responses to the sialidase antigen of Vibrio cholerae O1 and that plasma responses targeting this antigen correlate with protection.

Breadth and function of antibody response to acute SARS-CoV-2 infection in humans.

Serological and plasmablast responses and plasmablast-derived IgG monoclonal antibodies (MAbs) have been analysed in three COVID-19 patients with different clinical severities. Potent humoral responses were detected within 3 weeks of onset of illness in all patients and the serological titre was elicited soon after or concomitantly with peripheral plasmablast response. An average of 13.7% and 3.5% of plasmablast-derived MAbs were reactive with virus spike glycoprotein or nucleocapsid, respectively. A subset of anti-spike (10 of 32) antibodies cross-reacted with other betacoronaviruses tested and harboured extensive somatic mutations, indicative of an expansion of memory B cells upon SARS-CoV-2 infection. Fourteen of 32 anti-spike MAbs, including five anti-receptor-binding domain (RBD), three anti-non-RBD S1 and six anti-S2, neutralised wild-type SARS-CoV-2 in independent assays. Anti-RBD MAbs were further grouped into four cross-inhibiting clusters, of which six antibodies from three separate clusters blocked the binding of RBD to ACE2 and five were neutralising. All ACE2-blocking anti-RBD antibodies were isolated from two recovered patients with prolonged fever, which is compatible with substantial ACE2-blocking response in their sera. Finally, the identification of non-competing pairs of neutralising antibodies would offer potential templates for the development of prophylactic and therapeutic agents against SARS-CoV-2.

Purification and Immunophenotypic Characterization of Murine Plasma Cells.

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.

Single-Cell Technologies for the Study of Antibody-Secreting Cells.

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.

Anti-centromere antibodies target centromere-kinetochore macrocomplex: a comprehensive autoantigen profiling.

OBJECTIVES: Anti-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. METHODS: A 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. RESULTS: A 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. CONCLUSIONS: We 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.