Maf expression in B cells restricts reactive plasmablast and germinal center B cell expansion.

Precise regulation of B cell differentiation is essential for an effective adaptive immune response. Here, we show that B cell development in mice with B cell-specific Maf deletion is unaffected, but marginal zone B cells, germinal centre B cells, and plasmablasts are significantly more frequent in the spleen of naive Maf-deficient mice compared to wild type controls. In the context of a T cell-dependent immunization, Maf deletion causes increased proliferation of germinal centre B cells and extrafollicular plasmablasts. This is accompanied by higher production of antigen-specific IgG1 antibodies with minimal modification of early memory B cells, but a reduction in plasma cell numbers. Single-cell RNA sequencing shows upregulation of genes associated with DNA replication and cell cycle progression, confirming the role of Maf in cell proliferation. Subsequent pathway analysis reveals that Maf influences cellular metabolism, transporter activity, and mitochondrial proteins, which have been implicated in controlling the germinal centre reaction. In summary, our findings demonstrate that Maf acts intrinsically in B cells as a negative regulator of late B cell differentiation, plasmablast proliferation and germinal centre B cell formation.

Long-termin vitromaintenance of plasma cells in a hydrogel-enclosed human bone marrow microphysiological 3D model system.

Plasma cells (PCs) in bone marrow (BM) play an important role in both protective and pathogenic humoral immune responses, e.g. in various malignant and non-malignant diseases such as multiple myeloma, primary and secondary immunodeficiencies and autoimmune diseases. Dedicated microenvironmental niches in the BM provide PCs with biomechanical and soluble factors that support their long-term survival. There is a high need for appropriate and robust model systems to better understand PCs biology, to develop new therapeutic strategies for PCs-related diseases and perform targeted preclinical studies with high predictive value. Most preclinical data have been derived fromin vivostudies in mice, asin vitrostudies of human PCs are limited due to restricted survival and functionality in conventional 2D cultures that do not reflect the unique niche architecture of the BM. We have developed a microphysiological, dynamic 3D BM culture system (BM-MPS) based on human primary tissue (femoral biopsies), mechanically supported by a hydrogel scaffold casing. While a bioinert agarose casing did not support PCs survival, a photo-crosslinked collagen-hyaluronic acid (Col-HA) hydrogel preserved the native BM niche architecture and allowed PCs survivalin vitrofor up to 2 weeks. Further, the Col-HA hydrogel was permissive to lymphocyte migration into the microphysiological system´s circulation. Long-term PCs survival was related to the stable presence in the culture of soluble factors, as APRIL, BAFF, and IL-6. Increasing immunoglobulins concentrations in the medium confirm their functionality over culture time. To the best of our knowledge, this study is the first report of successful long-term maintenance of primary-derived non-malignant PCsin vitro. Our innovative model system is suitable for in-depthin vitrostudies of human PCs regulation and exploration of targeted therapeutic approaches such as CAR-T cell therapy or biologics.

Intracellular Flow Cytometry ("Phosphoflow") to Assess Signal Transduction in Rare Populations Such As Memory B Cell Subsets and Plasma Cells.

Intracellular flow cytometry is a powerful technique that can be used to interrogate signalling in rare cellular populations. The strengths of the technique are that massively parallel readouts can be gained from thousands of single cells simultaneously, and the assay is fast and relatively straightforward. This plate-based protocol enables different doses and different timepoints of stimulation to be assessed and has been optimized for rare B cell populations. Combining this technique with high-dimensional flow cytometry enables multiple signalling proteins to be measured with high confidence.

Assessing the Survival of Human Plasma Cells Ex Vivo.

One way memory B cells provide protection is by rapidly differentiating into plasma cells. Plasma cells are vital in providing long-term protection against pathogens; however, they can also be detrimental to health in the case of antibody-mediated autoimmunity. Therefore, compounds which modulate the survival of plasma cells have been of interest for therapeutic intervention. Investigation of ex vivo plasma cell survival has previously been limited by the low frequency of plasma cells in the blood. Here we describe a novel ex vivo culture system that only requires 3000-5000 cells per condition. This method permits the assessment of human plasma cell survival derived from blood and can assess the impact of small molecule inhibitors on plasma cell viability.

Analysis of Normal Plasma Cell Distribution Across Distinct Age Cohorts Reveals Age-Dependent Changes.

Hematopoietic and stromal cells within the bone marrow (BM) provide membrane-bound and/or soluble factors that are vital for the survival of plasma cells (PCs). Recent reports in murine BM demonstrated the dynamic formation and dispersion of PC clusters. To date, PC clustering in normal human BM has yet to be thoroughly examined. The goal of this study was to determine whether PC clusters are present in human BM and whether clustering changes as a function of age. Quantification of PCs and clustering in BM sections across six different age groups revealed that fewer PCs and PC clusters were observed in the youngest and oldest age groups. PC clustering increased with age until the sixth decade and then began to decrease. A positive correlation between the number of PCs and PC clusters was observed across all age groups. PC clusters were typically heterogeneous for immunoglobulin heavy- and light-chain expression. Taken together, these data demonstrate that PC clusters are present in human BM and that PC clustering increases until middle adulthood and then begins to diminish. These results suggest the spatial distribution of BM PC-supportive stromal cells changes with age.

New insights into the mechanisms regulating plasma cell survival and longevity.

Plasma cells correspond to the last stage of B cell differentiation and are professional antibody-secreting cells. While most persist for only few days, some may survive for weeks to years in dedicated survival niches. The determination of plasma cell survival rate seems to rely both on intrinsic and extrinsic factors. Although often opposed, the deterministic and environmental models for plasma cell longevity are certainly overlapping. Understanding the contribution and the regulation of these different factors is paramount to develop better vaccines but also to target malignant plasma cells. Here, we review recent literature highlighting new findings pertaining to plasma cell survival rate, intrinsic regulation of plasma cell persistence and function, as well as the plasma cell/niche dialogue. Moreover, the now well-recognised heterogeneity observed among plasma cells is also discussed.

Turbinate-homing IgA-secreting cells originate in the nasal lymphoid tissues.

Nasal vaccination elicits a humoral immune response that provides protection from airborne pathogens1, yet the origins and specific immune niches of antigen-specific IgA-secreting cells in the upper airways are unclear2. Here we define nasal glandular acinar structures and the turbinates as immunological niches that recruit IgA-secreting plasma cells from the nasal-associated lymphoid tissues (NALTs)3. Using intact organ imaging, we demonstrate that nasal vaccination induces B cell expansion in the subepithelial dome of the NALT, followed by invasion into commensal-bacteria-driven chronic germinal centres in a T cell-dependent manner. Initiation of the germinal centre response in the NALT requires pre-expansion of antigen-specific T cells, which interact with cognate B cells in interfollicular regions. NALT ablation and blockade of PSGL-1, which mediates interactions with endothelial cell selectins, demonstrated that NALT-derived IgA-expressing B cells home to the turbinate region through the circulation, where they are positioned primarily around glandular acinar structures. CCL28 expression was increased in the turbinates in response to vaccination and promoted homing of IgA+ B cells to this site. Thus, in response to nasal vaccination, the glandular acini and turbinates provide immunological niches that host NALT-derived IgA-secreting cells. These cellular events could be manipulated in vaccine design or in the treatment of upper airway allergic responses.

Immunological memory diversity in the human upper airway.

The upper airway is an important site of infection, but immune memory in the human upper airway is poorly understood, with implications for COVID-19 and many other human diseases1-4. Here we demonstrate that nasal and nasopharyngeal swabs can be used to obtain insights into these challenging problems, and define distinct immune cell populations, including antigen-specific memory B cells and T cells, in two adjacent anatomical sites in the upper airway. Upper airway immune cell populations seemed stable over time in healthy adults undergoing monthly swabs for more than 1 year, and prominent tissue resident memory T (TRM) cell and B (BRM) cell populations were defined. Unexpectedly, germinal centre cells were identified consistently in many nasopharyngeal swabs. In subjects with SARS-CoV-2 breakthrough infections, local virus-specific BRM cells, plasma cells and germinal centre B cells were identified, with evidence of local priming and an enrichment of IgA+ memory B cells in upper airway compartments compared with blood. Local plasma cell populations were identified with transcriptional profiles of longevity. Local virus-specific memory CD4+ TRM cells and CD8+ TRM cells were identified, with diverse additional virus-specific T cells. Age-dependent upper airway immunological shifts were observed. These findings provide new understanding of immune memory at a principal mucosal barrier tissue in humans.

Plasma cell differentiation is regulated by the expression of histone variant H3.3.

The differentiation of B cells into plasma cells is associated with substantial transcriptional and epigenetic remodeling. H3.3 histone variant marks active chromatin via replication-independent nucleosome assembly. However, its role in plasma cell development remains elusive. Herein, we show that during plasma cell differentiation, H3.3 is downregulated, and the deposition of H3.3 and chromatin accessibility are dynamically changed. Blockade of H3.3 downregulation by enforced H3.3 expression impairs plasma cell differentiation in an H3.3-specific sequence-dependent manner. Mechanistically, enforced H3.3 expression inhibits the upregulation of plasma cell-associated genes such as Irf4, Prdm1, and Xbp1 and maintains the expression of B cell-associated genes, Pax5, Bach2, and Bcl6. Concomitantly, sustained H3.3 expression prevents the structure of chromatin accessibility characteristic for plasma cells. Our findings suggest that appropriate H3.3 expression and deposition control plasma cell differentiation.

Integrative single-cell chromatin and transcriptome analysis of human plasma cell differentiation.

ABSTRACT: Plasma cells (PCs) are highly specialized cells representing the end stage of B-cell differentiation. We have shown that PC differentiation can be reproduced in vitro using elaborate culture systems. The molecular changes occurring during PC differentiation are recapitulated in this in vitro differentiation model. However, a major challenge exists to decipher the spatiotemporal epigenetic and transcriptional programs that drive the early stages of PC differentiation. We combined single cell (sc) RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with high throughput sequencing (scATAC-seq) to decipher the trajectories involved in PC differentiation. ScRNA-seq experiments revealed a strong heterogeneity of the preplasmablastic and plasmablastic stages. Among genes that were commonly identified using scATAC-seq and scRNA-seq, we identified several transcription factors with significant stage specific potential importance in PC differentiation. Interestingly, differentially accessible peaks characterizing the preplasmablastic stage were enriched in motifs of BATF3, FOS and BATF, belonging to activating protein 1 (AP-1) transcription factor family that may represent key transcriptional nodes involved in PC differentiation. Integration of transcriptomic and epigenetic data at the single cell level revealed that a population of preplasmablasts had already undergone epigenetic remodeling related to PC profile together with unfolded protein response activation and are committed to differentiate in PC. These results and the supporting data generated with our in vitro PC differentiation model provide a unique resource for the identification of molecular circuits that are crucial for early and mature PC maturation and biological functions. These data thus provide critical insights into epigenetic- and transcription-mediated reprogramming events that sustain PC differentiation.

Accelerated plasma-cell differentiation in Bach2-deficient mouse B cells is caused by altered IRF4 functions.

Transcription factors BACH2 and IRF4 are both essential for antibody class-switch recombination (CSR) in activated B lymphocytes, while they oppositely regulate the differentiation of plasma cells (PCs). Here, we investigated how BACH2 and IRF4 interact during CSR and plasma-cell differentiation. We found that BACH2 organizes heterochromatin formation of target gene loci in mouse splenic B cells, including targets of IRF4 activation such as Aicda, an inducer of CSR, and Prdm1, a master plasma-cell regulator. Release of these gene loci from heterochromatin in response to B-cell receptor stimulation was coupled to AKT-mTOR pathway activation. In Bach2-deficient B cells, PC genes' activation depended on IRF4 protein accumulation, without an increase in Irf4 mRNA. Mechanistically, a PU.1-IRF4 heterodimer in activated B cells promoted BACH2 function by inducing gene expression of Bach2 and Pten, a negative regulator of AKT signaling. Elevated AKT activity in Bach2-deficient B cells resulted in IRF4 protein accumulation. Thus, BACH2 and IRF4 mutually modulate the activity of each other, and BACH2 inhibits PC differentiation by both the repression of PC genes and the restriction of IRF4 protein accumulation.

New insights into the ontogeny, diversity, maturation and survival of long-lived plasma cells.

Plasma cells are unique immune effectors, capable of producing large amounts of high-affinity antibodies that protect against pathogenic infections. Although most plasma cells have short lifespans, certain conditions or vaccinations can give rise to long-lived plasma cells (LLPCs) that provide individuals with lifelong protection against pathogen exposure. The nature of these LLPCs is poorly understood; however, recent studies have shed new light on the ontogeny, diversity, maturation and survival of these unique cells. Whereas LLPCs had been thought to arise preferentially from germinal centres, novel genetic tools have revealed that they can originate from various stages throughout the humoral response. Furthermore, new single-cell analyses have shown that mouse and human plasma cells are heterogeneous and may undergo further maturation in situ in the bone marrow niche. Finally, plasma cells were previously considered to be sessile cells maintained in fixed survival niches, but new data show that plasma cell subsets can differentially migrate and organize into clusters that may be associated with survival niches. These descriptive findings provide new insights into how cell-intrinsic programmes and extrinsic factors may regulate the longevity of plasma cells in various contexts, which suggest new research avenues for their functional validation.

Bone marrow plasma cells require P2RX4 to sense extracellular ATP.

Plasma cells produce large quantities of antibodies and so play essential roles in immune protection1. Plasma cells, including a long-lived subset, reside in the bone marrow where they depend on poorly defined microenvironment-linked survival signals1. We show that bone marrow plasma cells use the ligand-gated purinergic ion channel P2RX4 to sense extracellular ATP released by bone marrow osteoblasts through the gap-junction protein pannexin 3 (PANX3). Mutation of Panx3 or P2rx4 each caused decreased serum antibodies and selective loss of bone marrow plasma cells. Compared to their wild-type counterparts, PANX3-null osteoblasts secreted less extracellular ATP and failed to support plasma cells in vitro. The P2RX4-specific inhibitor 5-BDBD abrogated the impact of extracellular ATP on bone marrow plasma cells in vitro, depleted bone marrow plasma cells in vivo and reduced pre-induced antigen-specific serum antibody titre with little posttreatment rebound. P2RX4 blockade also reduced autoantibody titre and kidney disease in two mouse models of humoral autoimmunity. P2RX4 promotes plasma cell survival by regulating endoplasmic reticulum homeostasis, as short-term P2RX4 blockade caused accumulation of endoplasmic reticulum stress-associated regulatory proteins including ATF4 and B-lineage mutation of the pro-apoptotic ATF4 target Chop prevented bone marrow plasma cell demise on P2RX4 inhibition. Thus, generating mature protective and pathogenic plasma cells requires P2RX4 signalling controlled by PANX3-regulated extracellular ATP release from bone marrow niche cells.

SARS-CoV-2 Omicron boosting induces de novo B cell response in humans.

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses and the development of vaccines aimed at the new variants1-4. SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells5-9. However, it remains unclear whether the additional doses induce germinal centre reactions whereby re-engaged B cells can further mature, and whether variant-derived vaccines can elicit responses to variant-specific epitopes. Here we show that boosting with an mRNA vaccine against the original monovalent SARS-CoV-2 mRNA vaccine or the bivalent B.1.351 and B.1.617.2 (Beta/Delta) mRNA vaccine induced robust spike-specific germinal centre B cell responses in humans. The germinal centre response persisted for at least eight weeks, leading to significantly more mutated antigen-specific bone marrow plasma cell and memory B cell compartments. Spike-binding monoclonal antibodies derived from memory B cells isolated from individuals boosted with either the original SARS-CoV-2 spike protein, bivalent Beta/Delta vaccine or a monovalent Omicron BA.1-based vaccine predominantly recognized the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted sorting approach, we isolated monoclonal antibodies that recognized the BA.1 spike protein but not the original SARS-CoV-2 spike protein from individuals who received the mRNA-1273.529 booster; these antibodies were less mutated and recognized novel epitopes within the spike protein, suggesting that they originated from naive B cells. Thus, SARS-CoV-2 booster immunizations in humans induce robust germinal centre B cell responses and can generate de novo B cell responses targeting variant-specific epitopes.

Isotype-specific plasma cells express divergent transcriptional programs.

Antibodies are produced across multiple isotypes with distinct properties that coordinate initial antigen clearance and confer long-term antigen-specific immune protection. Here, we interrogate the molecular programs of isotype-specific murine plasma cells (PC) following helper T cell-dependent immunization and within established steady-state immunity. We developed a single-cell-indexed and targeted molecular strategy to dissect conserved and divergent components of the rapid effector phase of antigen-specific IgM+ versus inflammation-modulating programs dictated by type 1 IgG2a/b+ PC differentiation. During antibody affinity maturation, the germinal center (GC) cycle imparts separable programs for post-GC type 2 inhibitory IgG1+ and type 1 inflammatory IgG2a/b+ PC to direct long-term cellular function. In the steady state, two subsets of IgM+ and separate IgG2b+ PC programs clearly segregate from splenic type 3 IgA+ PC programs that emphasize mucosal barrier protection. These diverse isotype-specific molecular pathways of PC differentiation control complementary modules of antigen clearance and immune protection that could be selectively targeted for immunotherapeutic applications and vaccine design.

Persistence of Virus-Specific Antibody after Depletion of Memory B Cells.

Humoral immunity is a major component of the adaptive immune response against viruses and other pathogens with pathogen-specific antibody acting as the first line of defense against infection. Virus-specific antibody levels are maintained by continual secretion of antibody by plasma cells residing in the bone marrow. This raises the important question of how the virus-specific plasma cell population is stably maintained and whether memory B cells are required to replenish plasma cells, balancing their loss arising from their intrinsic death rate. In this study, we examined the longevity of virus-specific antibody responses in the serum of mice following acute viral infection with three different viruses: lymphocytic choriomeningitis virus (LCMV), influenza virus, and vesicular stomatitis virus (VSV). To investigate the contribution of memory B cells to the maintenance of virus-specific antibody levels, we employed human CD20 transgenic mice, which allow for the efficient depletion of B cells with rituximab, a human CD20-specific monoclonal antibody. Mice that had resolved an acute infection with LCMV, influenza virus, or VSV were treated with rituximab starting at 2 months after infection, and the treatment was continued for up to a year postinfection. This treatment regimen with rituximab resulted in efficient depletion of B cells (>95%), with virus-specific memory B cells being undetectable. There was an early transient drop in the antibody levels after rituximab treatment followed by a plateauing of the curve with virus-specific antibody levels remaining relatively stable (half-life of 372 days) for up to a year after infection in the absence of memory B cells. The number of virus-specific plasma cells in the bone marrow were consistent with the changes seen in serum antibody levels. Overall, our data show that virus-specific plasma cells in the bone marrow are intrinsically long-lived and can maintain serum antibody titers for extended periods of time without requiring significant replenishment from memory B cells. These results provide insight into plasma cell longevity and have implications for B cell depletion regimens in cancer and autoimmune patients in the context of vaccination in general and especially for COVID-19 vaccines. IMPORTANCE Following vaccination or primary virus infection, virus-specific antibodies provide the first line of defense against reinfection. Plasma cells residing in the bone marrow constitutively secrete antibodies, are long-lived, and can thus maintain serum antibody levels over extended periods of time in the absence of antigen. Our data, in the murine model system, show that virus-specific plasma cells are intrinsically long-lived but that some reseeding by memory B cells might occur. Our findings demonstrate that, due to the longevity of plasma cells, virus-specific antibody levels remain relatively stable in the absence of memory B cells and have implications for vaccination.

Features of B Cell Responses Relevant to Allergic Disease.

This Brief Review delves into B cell responses in the context of allergy. The primary contribution of B cells to allergy is the production of IgE, the Ab isotype that triggers immediate hypersensitivity reactions through the release of mediators from mast cells and basophils. B cells may also have protective roles in allergy, such as through the production of IgG or as regulatory B cells. In this review, I focus on the basic principles of B cell differentiation and discuss features relevant to allergic immune responses. In particular, I discuss: (1) class-switch recombination; (2) plasma cell differentiation; (3) germinal centers and affinity maturation; and (4) memory B cells and recall responses, with an emphasis on IgE, IgG1, and IgG4. I also consider how B cells may contribute to allergic responses independent of Ab production-for example, by serving as APCs.