Patients with chronic lymphocytic leukemia (CLL) undergoing ibrutinib treatment often experience incomplete response, yet the molecular level underlying clonal inertia remains to be explored. We investigated the molecular and clinical dynamics of CLL during 16 months of ibrutinib monotherapy by analyzing blood samples from two patients who continued having CLL cells in the peripheral blood during treatment. At diagnosis, the clonal burden within the B cell compartment was found to be 55% (pt1) and 86% (pt2) for the dominant clones. At 16 months following treatment these clones still constituted 66% and 89%, respectively. Utilizing multi-omic methodologies at the DNA and RNA levels, including single-cell transcriptomics, we aimed to establish a comprehensive framework for multi-omics analysis for longitudinal ibrutinib response evaluation. The presented study revealed genomically stable disease during ibrutinib treatment, but with intensified expression of genes involved in pathways related to apoptosis, cellular stress response, and canonical NF-κB signaling from diagnosis to 16 months of treatment.

It would be advantageous if a strategy could be found to accelerate vaccine induction of anti-influenza adaptive immunity to more rapidly protect frontline workers and other vulnerable individuals during virus outbreaks. This study asked whether Advax® delta inulin adjuvant could accelerate the kinetics of protection afforded by a single dose of inactivated influenza vaccine (IIV) in two different strains of mice. A single dose of Advax®-adjuvanted IIV, but not IIV alone, gave complete protection if given 7 or 3 days before, and even when given at the same time as virus challenge (same day protection). Co-administration of both IIV and Advax® were critical to obtaining robust same day protection which was not seen with the individual vaccine components. Same day protection was lost in B-cell deficient μMT mice but was still evident in T cell-depleted mice, confirming its dependence on humoral but not T cell immunity. Day 6 post-challenge serum from protected mice demonstrated elevated influenza-binding IgM which had hemagglutination inhibition activity not seen in mice that received IIV alone. This confirmed that Advax® accelerated the kinetics of anti-influenza IgM production in response to IIV. Influenza protection could be transferred to naïve mice using day 6 sera or purified IgM from Advax®-adjuvanted IIV-immunised mice. Draining lymph nodes from protected mice showed increased CD138+ B220+ migratory and IgM+ extrafollicular, antibody secreting cells. These results show Advax® adjuvant accelerates the kinetics of anti-influenza IgM production in response to IIV thereby enabling the vaccine to protect against an infection acquired at the same time as the immunisation. While further studies are required to confirm that this ability to accelerate humoral immune kinetics extends to other species, including non-human primates, the phenomenon of adjuvant-accelerated IIV protection offers promise as a strategy for development of faster-acting vaccines.

Newly generated plasma cells in secondary lymphoid organs migrate to niches in the bone marrow, wherein they survive as long-lived plasma cells (LLPCs). Although LLPCs have been extensively characterized, it is still unclear what the key determinant(s) are for plasma cell longevity. One model postulates that plasma cell heterogeneity is established at the induction site, thereby instructing their longevity. Here, we found that, among newly generated IgG plasma cells, integrin β7hi marks plasma cells predisposed to home to the bone marrow, whereas integrin β7lo cells remain in secondary lymphoid organs. Mechanistically, this egress-prone fraction had a higher expression of the KLF2 transcription factor, the loss of which resulted in defective egress by downregulating S1PR1 and CD11b. Disruption of plasma cell egress results in defective antibody durability, thereby making mice more susceptible to influenza reinfection. Thus, the migration program of plasma cells established at the induction site plays a critical role in determining antibody durability.

Exploring the impact of persistent mutations in SARS-CoV-2 variants and reduced immunity on breakthrough infections (BTIs) is crucial, particularly in understanding how antigen-specific memory B cells (MBCs) respond to new variants. We followed 107 participants who received the ancestral inactivated vaccine and experienced one or two Omicron BTIs over six months. Using flow cytometry, SARS-CoV-2 antigen probes, single-cell RNA sequencing, and B cell receptor (BCR) profiling, we assessed MBCs and immune diversity. Our findings revealed that although neutralizing antibody levels decreased over time, the number of specific MBCs remained stable and matured progressively. Notably, pre-existing Omicron-specific MBCs played a key role in preventing secondary Omicron infections. Differential gene analysis showed enrichment in antigen processing and immune regulation pathways, while clonal lineage analysis revealed more B cell expansion and V(D)J gene-specific rearrangements in high neutralization samples. These results emphasize MBCs' critical role in long-term immunity and inform future vaccination strategies.

The antibody-mediated immune response is crucial for the development of protective immunity against SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Understanding the interaction between SARS-CoV-2 and the immune system is critical because new variants emerge as a result of the virus's ongoing evolution. Understanding the function of B cells in the SARS-CoV-2 infection process is critical for developing effective and long-lasting vaccines against this virus. Triggered by the innate immune response, B cells transform into memory B cells (MBCs). It is fascinating to observe how MBCs provide enduring immune defence, not only eradicating the infection but also safeguarding against future reinfection. If there is a lack of B cell activation or if the B cells are not functioning properly, it can lead to a serious manifestation of the disease and make immunisation less effective. Individuals with disruptions in the B cells have shown increased production of cytokines and chemokines, resulting in a poor prognosis for the disease. Therefore, we have developed an updated review article to gain insight into the involvement of B cells in SARS-CoV-2 infection. The discussion has covered the generation, functioning, and dynamics of neutralising antibodies (nAbs). Furthermore, we have emphasised immunotherapeutics that rely on nAbs.

Plasma cells provide protective antibodies following an infection or vaccination. A network of intrinsic and extrinsic factors fine-tunes the generation of a heterogenous plasma cell pool with varying metabolic requirements, transcriptional profiles and lifespans. Among these, the B cell maturation antigen (BCMA) has been implicated in the APRIL-mediated survival of long-lived plasma cells. To characterize the terminal maturation of plasma cells, we constructed a BCMA reporter mouse (BCMA:Tom) that exclusively labeled antibody-secreting cells and revealed that BCMA:Tom expression varied by IgH isotype and increased with plasma cell maturity. The BCMA reporter, used alongside the Blimp1-GFP reporter, also allowed detailed tracking of plasma cell development and highlighted the importance of the in vivo microenvironment to complete plasma cell maturation. Therefore, the BCMA:Tom reporter mouse provides a valuable tool for tracking plasma cell development and maturation with flow cytometry or advanced imaging techniques, enabling a deeper understanding of the mechanisms regulating plasma cell heterogeneity and longevity.

Plasma cell subsets vary in their lifespans and ability to sustain humoral immunity. We conducted a genome-wide CRISPR-Cas9 screen in a myeloma cell line for factors that promote surface expression of CD98, a marker of longevity in primary mouse plasma cells. A large fraction of genes found to promote CD98 expression in this screen are involved in secretory and other vesicles, including many subunits of the V-type ATPase complex. Chemical inhibition or genetic ablation of V-type ATPases in myeloma cells reduced antibody secretion. Primary mouse and human long-lived plasma cells had greater numbers of acidified vesicles than did their short-lived counterparts, and this correlated with increased secretory capacity of IgM, IgG, and IgA. The screen also identified PI4KB, which promoted acidified vesicle numbers and secretory capacity, and DDX3X, an ATP-dependent RNA helicase, the deletion of which reduced immunoglobulin secretion independently of vesicular acidification. Finally, we report a plasma-cell intrinsic function of the signaling adapter MYD88 in both antibody secretion and plasma cell survival in vivo. These data reveal novel regulators of plasma cell secretory capacity, including those that also promote lifespan.

The gut microbiota enhances systemic immunoglobulin G (IgG) responses to vaccines, but it is unknown whether this effect involves IgA, which coats intestinal microbes. That IgA may amplify postimmune IgG production is suggested by the impaired IgG response to pneumococcal vaccines in some IgA-deficient patients. Here, we found that antipneumococcal but not total IgG production was impaired in mice with IgA deficiency. The positive effect of gut IgA on antipneumococcal IgG responses started very early in life and could implicate gut bacteria, as these responses were attenuated in germ-free mice recolonized with gut microbes from IgA-deficient donors. IgA could exert this effect by constraining the systemic translocation of gut antigens, which was associated with chronic immune activation, including T cell overexpression of programmed cell death protein 1 (PD-1). This inhibitory receptor may attenuate antipneumococcal IgG production by causing B cell hyporesponsiveness, which improved upon anti-PD-1 treatment. Thus, gut IgA functionally interacts with systemic IgG to enhance antipneumococcal vaccine responses.

Recombinant influenza viruses are promising vectors that can bolster antibody and resident lymphocyte responses within mucosal sites. This study evaluates recombinant influenza viruses with SARS-CoV-2 RBD genes in eliciting mucosal and systemic responses. Using reverse genetics, we generated replication-competent recombinant influenza viruses carrying heterologous RBD genes in monomeric, trimeric, or ferritin-based nanoparticle forms. Following intranasal immunisation, mice developed potent serological anti-RBD responses, with ferritin nanoparticles superseding monomeric or trimeric RBD responses. While parenteral and mucosal immunisation elicited robust anti-RBD IgG in serum, mucosal immunisation seeded respiratory IgA, RBD-specific lung-resident memory and germinal centre (GC) B cells. In animals with prior intramuscular vaccination, intranasal boosting with recombinant influenza vectors augmented mucosal IgG, IgA, GC and memory B cells, and SARS-CoV-2 lung neutralising titres. Recall of RBD-specific memory B cells via antigen re-exposure in the lung increased antibody-secreting cells in the lung-draining lymph nodes, with maintenance of lung GC B cells. Recombinant influenza-based vaccines effectively deliver highly immunogenic self-assembling nanoparticles, generating antibodies and B cells in the respiratory mucosa. This strategy provides a tractable pathway to augment lung-localised responses against recurrent respiratory viral infections.

The establishment of protective immune responses relies on the ability of terminally differentiated B cells to secrete a broad variety of antigen-specific antibodies with different effector functions. RIF1 is a multifunctional protein that promotes antibody isotype diversification via its DNA end protection activity during class switch recombination. In this study, we showed that RIF1 ablation resulted in increased plasmablast formation ex vivo and enhanced terminal differentiation into plasma cells upon immunization. Mechanistically, this phenotype is independent from RIF1's role in DNA repair and class switch recombination, and reflects its ability to modulate the transcriptional status of a subset of BLIMP1 target genes. Therefore, here we show that, in addition to promoting antibody diversification, RIF1 fine-tunes the kinetics of late B cell differentiation, thus providing an additional layer of control in the establishment of humoral immunity.

Mouse models are an indispensable tool in lymphoma research. Here, we focus on the utilization of genetically engineered mouse models as preclinical avatars in lymphoma research. We describe lymphoma-relevant alleles and allele combinations, as well as general considerations for model selection. We further illustrate concepts of gene targeting and model design and provide guidelines for breeding strategies and colony maintenance.

Combining mathematical modeling with experiments enables quantitative understanding of cell signaling, transcriptional regulation, and cell fate decisions. Here, we provide a systems biology approach to link signal transduction with B cells fate decisions, to enable quantitative prediction of B-cell proliferation, and differentiation. We describe methodology to run simulations that reveal how signal transduction regulates gene expression and predicts cell fate decision. We describe how to quantitively validate modeling predictions with wet-lab experiments.

Diverse antibody repertoires spanning multiple lymphoid organs (i.e., bone marrow, spleen, lymph nodes) form the foundation of protective humoral immunity. Changes in their composition across lymphoid organs are a consequence of B-cell selection and migration events leading to a highly dynamic and unique physiological landscape of antibody repertoires upon antigenic challenge (e.g., vaccination). However, to what extent B cells encoding identical or similar antibody sequences (clones) are distributed across multiple lymphoid organs and how this is shaped by the strength of a humoral response remains largely unexplored. Here, we performed an in-depth systems analysis of antibody repertoires across multiple distinct lymphoid organs of immunized mice and discovered that organ-specific antibody repertoire features (i.e., germline V-gene usage and clonal expansion profiles) equilibrated upon a strong humoral response (multiple immunizations and high serum titers). This resulted in a surprisingly high degree of repertoire consolidation, characterized by highly connected and overlapping B-cell clones across multiple lymphoid organs. Finally, we revealed distinct physiological axes indicating clonal migrations and showed that antibody repertoire consolidation directly correlated with antigen specificity. Our study uncovered how a strong humoral response resulted in a more uniform but redundant physiological landscape of antibody repertoires, indicating that increases in antibody serum titers were a result of synergistic contributions from antigen-specific B-cell clones distributed across multiple lymphoid organs. Our findings provide valuable insights for the assessment and design of vaccine strategies.

The efficacy of anti-cancer therapies depends on the genomic composition of the tumor, its microenvironment, spatial organization, and intra-tumor heterogeneity. B-cell lymphomas are a heterogeneous group of tumors emerging from B-cells at different stages of differentiation and exhibiting tumor-specific interactions with the tumor microenvironment. Thus, the effect of drug treatments can be influenced by the tumor composition and functional interactions among immune cells. Here, we develop a platform to maintain small fragments of human lymphoma tissue in culture for several days, and use them to test response to small molecules. We collect 27 patient samples representative of different lymphoma subtypes, and establish ex vivo tissue fragments that retain histological, cellular, and molecular characteristics of the original tissue, here referred to as lymphomoids. Using lymphomoids, we test sensitivity to several clinically approved drugs in parallel and examine tissue remodeling upon treatment. Moreover, when this information is available, we show that the effect of the inhibitors observed in lymphomoids is consistent with the patients' response in the clinic. Thus, lymphomoids represent an innovative ex vivo model to assess the effect of anti-cancer therapies while preserving the tissue structure and its components.

Immune dysregulation is a hallmark of autoimmune diseases of the central nervous system (CNS), characterized by an excessive immune response, and primary CNS tumors (pCNS-tumors) showing a highly immunosuppressive parenchymal microenvironment.

Aiming to provide novel insights into the pathogenesis of CNS autoimmunity and cerebral tumor immunity, we analyzed the peripheral blood (PB) and cerebrospinal fluid (CSF) of 81 autoimmune limbic encephalitis (ALE), 148 relapsing-remitting multiple sclerosis (RRMS), 33 IDH-wildtype glioma, 9 primary diffuse large B cell lymphoma of the CNS (CNS-DLBCL), and 110 controls by flow cytometry (FC). Additionally, an in-depth immunophenotyping of the PB from an independent cohort of 20 RRMS and 18 IDH-wildtype glioblastoma patients compared to 19 controls was performed by FC combined with unsupervised computational approaches.

We identified alterations in peripheral and intrathecal adaptive immunity, mainly affecting the T cell (Tc) but also the B cell (Bc) compartment in ALE, RRMS, and pCNS-tumors compared to controls. ALE, RRMS, and pCNS-tumors featured higher expression of the T cell activation marker HLA-DR, which was even more pronounced in pCNS-tumors than in ALE or RRMS. Glioblastoma patients showed signs of T cell exhaustion that were not visible in RRMS patients. In-depth characterization of the PB revealed differences mainly in the T effector and memory compartment between RRMS and glioblastoma patients and similar alterations in the Bc compartment, including atypical Bc, CD19+CD20- double negative Bc, and plasma cells. PB and CSF mFC together with CSF routine parameters could reliably differentiate ALE and RRMS from pCNS-tumors facilitating early diagnosis and treatment.

ALE, RRMS, and pCNS-tumors show distinct but partially overlapping changes mainly in HLA-DR+ Tc, memory Tc, exhausted Tc, and Bc subsets providing insights into disease pathogenesis. Moreover, mFC shows diagnostic potential facilitating early diagnosis and treatment.

The establishment of long-lasting immunity against pathogens is facilitated by the germinal center (GC) reaction, during which B cells increase their antibody affinity and differentiate into antibody-secreting cells (ASC) and memory cells. These events involve modifications in chromatin packaging that orchestrate the profound restructuring of gene expression networks that determine cell fate. While several chromatin remodelers were implicated in lymphocyte functions, less is known about SMARCA5. Here, using ribosomal pull-down for analyzing translated genes in GC B cells, coupled with functional experiments in mice, we identified SMARCA5 as a key chromatin remodeler in B cells. While the naive B cell compartment remained unaffected following conditional depletion of Smarca5, effective proliferation during B cell activation, immunoglobulin class switching, and as a result GC formation and ASC differentiation were impaired. Single-cell multiomic sequencing analyses revealed that SMARCA5 is crucial for facilitating the transcriptional modifications and genomic accessibility of genes that support B cell activation and differentiation. These findings offer novel insights into the functions of SMARCA5, which can be targeted in various human pathologies.

Gastrointestinal (GI) B cells and plasma cells (PCs) are critical to mucosal homeostasis and the host response to HIV-1 infection. Here, high-resolution mapping of human B cells and PCs sampled from the colon and ileum during both viremic and suppressed HIV-1 infection identified a reduction in germinal center (GC) B cells and follicular dendritic cells (FDCs) during HIV-1 viremia. Immunoglobulin A-positive (IgA+) PCs are the major cellular output of intestinal GCs and were significantly reduced during viremic HIV-1 infection. PC-associated transcriptional perturbations, including type I interferon signaling, persisted in antiretroviral therapy (ART)-treated individuals, suggesting ongoing disruption of the intestinal immune milieu during ART. GI humoral immune perturbations were associated with changes in the intestinal microbiome composition and systemic inflammation. These findings highlight a key immune defect in the GI mucosa due to HIV-1 viremia.

Plasma cells (PCs) constitute a small proportion of B cells, limiting their biochemical characterization. An in vitro culture system that reliably generates PCs could provide an alternative method to obtain PCs for further analysis and manipulation. To date, most in vitro PC differentiation methods rely on B cell receptor (BCR)-independent stimulants, including TLR ligands, CD40L, and cytokines. However, these methods do not fully recapitulate the natural T cell-dependent PC differentiation process, in which BCR activation is the initial events. In this study, we established an efficient in vitro PC differentiation method incorporating BCR stimulation. Naïve B cells were first stimulated with anti-IgM and anti-CD40 Abs, followed by stimulation with various cytokines. By screening cytokines known to participate in PC differentiation in vivo, we identified that the combination of IL-4 and IL-5 induced the most efficient PC differentiation. The in vitro generated PCs highly expressed PC-associated surface markers and regulatory genes. Additionally, they secreted high amounts of IgM and IgG Abs. Moreover, retroviral transduction of B cells resulted in efficient target gene expression in PCs. Our new method closely mimics natural PC differentiation and effectively generates a large quantity of PCs for various applications, including elucidating the molecular mechanisms underlying PC differentiation.

Bats are reservoirs of many zoonotic viruses that are fatal in humans but do not cause disease in bats. Moreover, bats generate low neutralizing antibody titers in response to experimental viral infection, although more robust antibody responses have been observed in wild-caught bats during times of food stress. Here, we compared the antibody titers and B cell receptor (BCR) diversity of Jamaican fruit bats (Artibeus jamaicensis; JFBs) and BALB/c mice generated in response to T-dependent and T-independent antigens. We then manipulated the diet of JFBs and challenged them with H18N11 influenza A-like virus or a replication incompetent Nipah virus VSV (Nipah-riVSV). Under standard housing conditions, JFBs generated a lower avidity antibody response and possessed more BCR mRNA diversity compared to BALB/c mice. However, withholding protein from JFBs improved serum neutralization in response to Nipah-riVSV and improved serum antibody titers specific to H18 but reduced BCR mRNA diversity.

During the perinatal period, the immune system sets the threshold to select either response or tolerance to environmental Ags, which leads to the potential to provide a lifetime of protection and health. B-1a B cells have been demonstrated to develop during this perinatal time window, showing a unique and restricted BCR repertoire, and these cells play a major role in natural Ab secretion and immune regulation. In the current study, we developed a highly efficient temporally controllable RAG2-based lymphoid lineage cell labeling and tracking system and applied this system to understand the biological properties and contribution of B-1a cells generated at distinct developmental periods to the adult B-1a compartments. This approach revealed that B-1a cells with a history of RAG2 expression during the embryonic and neonatal periods dominate the adult B-1a compartment, including those in the bone marrow (BM), peritoneal cavity, and spleen. Moreover, the BCR repertoire of B-1a cells with a history of RAG2 expression during the embryonic period was restricted, becoming gradually more diverse during the neonatal period, and then heterogeneous at the adult stage. Furthermore, more than half of plasmablasts/plasma cells in the adult BM had embryonic and neonatal RAG2 expression histories. Moreover, BCR analysis revealed a high relatedness between BM plasmablasts/plasma cells and B-1a cells derived from embryonic and neonatal periods, suggesting that these cell types have a common origin. Taken together, these findings define, under native hematopoietic conditions, the importance in adulthood of B-1a cells generated during the perinatal period.

Durable serological memory following vaccination is critically dependent on the production and survival of long-lived plasma cells (LLPCs). Yet, the factors that control LLPC specification and survival remain poorly resolved. Using intravital two-photon imaging, we find that in contrast to most plasma cells (PCs) in the bone marrow (BM), LLPCs are uniquely sessile and organized into clusters that are dependent on APRIL, an important survival factor. Using deep, bulk RNA sequencing, and surface protein flow-based phenotyping, we find that LLPCs express a unique transcriptome and phenotype compared to bulk PCs, fine-tuning expression of key cell surface molecules, CD93, CD81, CXCR4, CD326, CD44, and CD48, important for adhesion and homing. Conditional deletion of Cxcr4 in PCs following immunization leads to rapid mobilization from the BM, reduced survival of antigen-specific PCs, and ultimately accelerated decay of antibody titer. In naïve mice, the endogenous LLPCs BCR repertoire exhibits reduced diversity, reduced somatic mutations, and increased public clones and IgM isotypes, particularly in young mice, suggesting LLPC specification is non-random. As mice age, the BM PC compartment becomes enriched in LLPCs, which may outcompete and limit entry of new PCs into the LLPC niche and pool.

Affinity-matured plasma cells (PCs) of varying lifespans are generated through a germinal center (GC) response. The developmental dynamics and genomic programs of antigen-specific PC precursors remain to be elucidated. Here, using a model antigen in mice, we demonstrate biphasic generation of PC precursors, with those generating long-lived bone marrow PCs preferentially produced in the late phase of GC response. Clonal tracing using single-cell RNA sequencing and B cell antigen receptor sequencing in spleen and bone marrow compartments, coupled with adoptive transfer experiments, reveals a new PC transition state that gives rise to functionally competent PC precursors. The latter undergo clonal expansion, dependent on inducible expression of TIGIT. We propose a model for the proliferation and programming of precursors of long-lived PCs, based on extended antigen encounters in the GC.

Hypertension is a principal risk factor for cardiovascular morbidity and mortality, with its severity exacerbated by high sodium intake, particularly in individuals with salt-sensitive blood pressure. However, the mechanisms underlying hypertension and salt sensitivity are only partly understood. Herein, we review potential interactions in hypertension pathophysiology involving the immune system, endoplasmic reticulum (ER) stress, the unfolded protein response (UPR), and proteostasis pathways; identify knowledge gaps; and discuss future directions.

Recent advancements by our research group and others reveal interactions within and between adaptive and innate immune responses in hypertension pathophysiology. The salt-immune-hypertension axis is further supported by the discovery of the role of dendritic cells in hypertension, marked by isolevuglandin (IsoLG) formation. Alongside these broadened understandings of immune-mediated salt sensitivity, the contributions of T cells to hypertension have been recently challenged by groups whose findings did not support increased resistance of Rag-1-deficient mice to Ang II infusion. Hypertension has also been linked to ER stress and the UPR. Notably, a holistic approach is needed because the UPR engages in crosstalk with autophagy, the ubiquitin proteasome, and other proteostasis pathways, that may all involve hypertension. There is a critical need for studies to establish cause and effect relationships between ER stress and the UPR in hypertension pathophysiology in humans and to determine whether the immune system and ER stress function mainly to exacerbate or initiate hypertension and target organ injury. This review of recent studies proposes new avenues for future research for targeted therapeutic interventions.

We investigated the biological function of the mould allergen Alt a 1 as a carrier of micronutrients, such as the vitamin A metabolite retinoic acid (RA) and the influence of RA binding on its allergenicity in vitro and in vivo.

Alt a 1-RA complex formation was analyzed in silico and in vitro. PBMCs from Alternaria-allergic donors were stimulated with Alt a 1 complexed with RA (holo-Alt a 1) or empty apo-Alt a 1 and analyzed for cytokine production and CD marker expression. Serum IgE-binding and crosslinking assays to apo- and holo-protein were correlated to B-cell epitope analysis. Female BALB/c mice already sensitized to Alt a 1 were intranasally treated with apo-Alt a 1, holo-Alt a 1 or RA alone before measuring anaphylactic response, serum antibody levels, splenic cytokines and CD marker expression.

In silico docking calculations and in vitro assays showed that the extent of RA binding depended on the higher quaternary state of Alt a 1. Holo-Alt a 1 loaded with RA reduced IL-13 released from PBMCs and CD3+CD4+CRTh2 cells. Complexing Alt a 1 to RA masked its IgE B-cell epitopes and reduced its IgE-binding capacity. In a therapeutic mouse model of Alternaria allergy nasal application of holo-Alt a 1, but not of apo-Alt a 1, significantly impeded the anaphylactic response, impaired splenic antigen-presenting cells and induced IL-10 production.

Holo-Alt a 1 binding to RA was able to alleviate Th2 immunity in vitro, modulate an ongoing Th2 response and prevent anaphylactic symptoms in vivo, presenting a novel option for improving allergen-specific immunotherapy in Alternaria allergy.

Mechanisms behind the impaired response of antigen-specific B cells to therapeutic vaccination in chronic hepatitis B virus (HBV) infection remain unclear. The development of vaccines or strategies to overcome this obstacle is vital for advancing the management of chronic hepatitis B.

A mouse model, denominated as E6F6-B, was engineered to feature a knock-in of a B-cell receptor (BCR) that specifically recognizes HBsAg. This model served as a valuable tool for investigating the temporal and spatial dynamics of humoral responses following therapeutic vaccination under continuous antigen exposure. Using a suite of immunological techniques, we elucidated the differentiation trajectory of HBsAg-specific B cells post-therapeutic vaccination in HBV carrier mice.

Utilizing the E6F6-B transfer model, we observed a marked decline in antibody-secreting cells 2 weeks after vaccination. A dysfunctional and atypical pre-plasma cell population (BLIMP-1+ IRF4+ CD40- CD138- BCMA-) emerged, manifested by sustained BCR signaling. By deploying an antibody to purge persistent HBsAg, we effectively prompted the therapeutic vaccine to provoke conventional plasma cell differentiation. This resulted in an enhanced anti-HBs antibody response and facilitated HBsAg clearance.

Sustained high levels of HBsAg limit the ability of therapeutic hepatitis B vaccines to induce the canonical plasma cell differentiation necessary for anti-HBs antibody production. Employing a strategy combining antibodies with vaccines can surmount this altered humoral response associated with atypical pre-plasma cells, leading to improved therapeutic efficacy in HBV carrier mice.

Therapeutic vaccines aimed at combatting HBV encounter suboptimal humoral responses in clinical settings, and the mechanisms impeding their effectiveness have remained obscure. Our research, utilizing the innovative E6F6-B mouse transfer model, reveals that the persistence of HBsAg can lead to the emergence of an atypical pre-plasma cell population, which proves to be relevant to the potency of therapeutic HBV vaccines. Targeting the aberrant differentiation process of these atypical pre-plasma cells stands out as a critical strategy to amplify the humoral response elicited by HBV therapeutic vaccines in carrier mouse models. This discovery suggests a compelling avenue for further study in the context of human chronic hepatitis B. Encouragingly, our findings indicate that synergistic therapy combining HBV-specific antibodies with vaccines offers a promising approach that could significantly advance the pursuit of a functional cure for HBV.

While cellular metabolism was proposed to be a driving factor of the activation and differentiation of B cells and the function of the resulting antibody-secreting cells (ASCs), the study of correlations between cellular metabolism and functionalities has been difficult due to the absence of technologies enabling the parallel measurement. Herein, we performed single-cell transcriptomics and introduced a direct concurrent functional and metabolic flux quantitation of individual murine B cells. Our transcriptomic data identified lactate metabolism as dynamic in ASCs, but antibody secretion did not correlate with lactate secretion rates (LSRs). Instead, our study of all splenic B cells during an immune response linked increased lactate metabolism with acidic intracellular pH and the upregulation of apoptosis. T cell-dependent responses increased LSRs, and added TLR4 agonists affected the magnitude and boosted LSRhigh B cells in vivo, while resulting in only a few immunoglobulin-G secreting cells (IgG-SCs). Therefore, our observations indicated that LSRhigh cells were not differentiating into IgG-SCs, and were rather removed due to apoptosis.

Durable serological memory following vaccination is critically dependent on the production and survival of long-lived plasma cells (LLPCs). Yet, the factors that control LLPC specification and survival remain poorly resolved. Using intra-vital two-photon imaging, we find that in contrast to most plasma cells in the bone marrow, LLPCs are uniquely sessile and organized into clusters that are dependent on April, an important survival factor. Using deep, bulk RNA sequencing, and surface protein flow-based phenotyping, we find that LLPCs express a unique transcriptome and proteome compared to bulk PCs, fine tuning expression of key cell surface molecules, CD93, CD81, CXCR4, CD326, CD44 and CD48, important for adhesion and homing, and phenotypically label LLPCs within mature PC pool. Conditional deletion of Cxcr4 in PCs following immunization leads to rapid mobilization from the BM, reduced survival of antigen-specific PCs, and ultimately accelerated decay of antibody titer. In naive mice, the endogenous LLPCs BCR repertoire exhibits reduced diversity, reduced somatic mutations, and increased public clones and IgM isotypes, particularly in young mice, suggesting LLPC specification is non-random. As mice age, the BM PC compartment becomes enriched in LLPCs, which may outcompete and limit entry of new PC into the LLPC niche and pool.

Malaria remains a global health challenge, necessitating the development of effective vaccines. The RTS,S vaccination prevents Plasmodium falciparum (Pf) malaria but is ineffective against Plasmodium vivax (Pv) disease. Herein, we evaluated the murine immunogenicity of a recombinant PvCSP incorporating prevalent polymorphisms, adjuvanted with Alhydrogel or Poly I:C. Both formulations induced prolonged IgG responses, with IgG1 dominance by the Alhydrogel group and high titers of all IgG isotypes by the Poly I:C counterpart. Poly I:C-adjuvanted vaccination increased splenic plasma cells, terminally-differentiated memory cells (MBCs), and precursors relative to the Alhydrogel-combined immunization. Splenic B-cells from Poly I:C-vaccinated mice revealed an antibody-secreting cell- and MBC-differentiating gene expression profile. Biological processes such as antibody folding and secretion were highlighted by the Poly I:C-adjuvanted vaccination. These findings underscore the potential of Poly I:C to strengthen immune responses against Pv malaria.

The activation and differentiation of B cells into plasma cells (PCs) play critical roles in the immune response to infections and autoimmune diseases. Toll-like receptor 9 (TLR9) responds to bacterial and viral DNA containing unmethylated CpG motifs and triggers immune responses in B cells; however, abnormal recognition of self-DNA by TLR9 can cause autoimmune diseases. When stimulated with TLR9 agonists, follicular (FO) B cells, a subset of B cells residing in the FO regions of secondary lymphoid organs, exhibit a propensity for activation but fail to give rise to PCs. The factors that enable the transition of TLR9-activated FO B cells from activation to differentiation into PCs remain unclear. In this study, we show that type I interferon-alpha (IFNα) signaling causes FO B cells activated by CpG stimulation to differentiate into PCs. Although CpG stimulation alone only temporarily increased interferon regulatory factor 4 (IRF4) expression in FO B cells, co-stimulation with both CpG and IFNα enhanced and maintained high IRF4 expression levels, ultimately enabling the cells to differentiate into PCs. Overexpression of IRF4 in FO B cells results in CpG-induced PC transition without IFN signaling. Furthermore, co-stimulation of TLR9 and IFNα receptors significantly enhanced mammalian target of rapamycin (mTOR) signaling, which regulates IRF4 expression and PC generation. These findings suggest that IFNα may play a key role in promoting the fate of PC differentiation in FO B cells activated by TLR9 stimulation.

Our current understanding of whether B cell involvement in the tumor microenvironment benefits the patient or the tumor - in distinct cancers, subcohorts and individual patients - is quite limited. Both statements are probably true in most cases: certain clonal B cell populations contribute to the antitumor response, while others steer the immune response away from the desired mechanics. To step up to a new level of understanding and managing B cell behaviors in the tumor microenvironment, we need to rationally discern these roles, which are cumulatively defined by B cell clonal functional programs, specificities of their B cell receptors, specificities and isotypes of the antibodies they produce, and their spatial interactions within the tumor environment. Comprehensive analysis of these characteristics of clonal B cell populations is now becoming feasible with the development of a whole arsenal of advanced technical approaches, which include (1) methods of single-cell and spatial transcriptomics, genomics, and proteomics; (2) methods of massive identification of B cell specificities; (3) methods of deep error-free profiling of B cell receptor repertoires. Here we overview existing techniques, summarize their current application for B cells studies and propose promising future directions in advancing B cells exploration.

Antibody-secreting plasma cells (PCs) are generated in secondary lymphoid organs but are reported to reside in an emerging range of anatomical sites. Analysis of the transcriptome of different tissue-resident (Tr)PC populations revealed that they each have their own transcriptional signature indicative of functional adaptation to the host tissue environment. In contrast to expectation, all TrPCs were extremely long-lived, regardless of their organ of residence, with longevity influenced by intrinsic factors like the immunoglobulin isotype. Analysis at single-cell resolution revealed that the bone marrow is unique in housing a compendium of PCs generated all over the body that retain aspects of the transcriptional program indicative of their tissue of origin. This study reveals that extreme longevity is an intrinsic property of TrPCs whose transcriptome is imprinted by signals received both at the site of induction and within the tissue of residence.