Extrafollicular Responses Research Articles

Early IgE Production Is Linked with Extrafollicular B- and T-Cell Activation in Low-Dose Allergy Model.

Despite its paramount importance, the predominant association of early IgE production with harmless antigens, via germinal-center B- and T-cell subpopulations or extrafollicular activation, remains unresolved. The aim of this work was to clarify whether the reinforced IgE production following the subcutaneous immunization of BALB/c mice with low antigen doses in withers adipose tissue might be linked with intensified extrafollicular or germinal-center responses. The mice were immunized three times a week for 4 weeks in the withers region, which is enriched in subcutaneous fat and tissue-associated B cells, with high and low OVA doses and via the intraperitoneal route for comparison. During long-term immunization with both low and high antigen doses in the withers region, but not via the intraperitoneal route, we observed a significant accumulation of B220-CD1d-CD5-CD19+ B-2 extrafollicular plasmablasts in the subcutaneous fat and regional lymph nodes but not in the intraperitoneal fat. Only low antigen doses induced a significant accumulation of CXCR4+ CXCR5- CD4+ extrafollicular T helpers in the withers adipose tissue but not in the regional lymph nodes or abdominal fat. Only in subcutaneous fat was there a combination of extrafollicular helper accumulation. In conclusion, extrafollicular B- and T-cell activation are necessary for early IgE class switching.

B-cell receptor physical properties affect relative IgG1 and IgE responses in mouse egg allergy

Mutated and unmutated IgE and IgG play different and partly opposing roles in allergy development, but the mechanisms controlling their relative production are incompletely understood. Here, we analyzed the IgE-response in murine food allergy. Deep sequencing of the complementary-determining region (CDR) repertoires indicated that an ongoing unmutated extrafollicular IgE response coexists with a germinal center response, even after long-lasting allergen challenges. Despite overall IgG1-dominance, a significant proportion of clonotypes contained several-fold more IgE than IgG1. Clonotypes with differential bias to either IgE or IgG1 showed distinct hypermutation and clonal expansion. Hypermutation rates were associated with different physiochemical binding properties of individual B-cell receptors (BCR). Increasing BCR signaling strength inhibited class switching from IgG1 to IgE in vitro, preferentially constraining IgE formation. These data indicate that antigen-binding properties of individual BCRs determine differential IgE hypermutation and IgE versus IgG1 production on the level of single B-cell clones.

Up-Regulated Interleukin-10 Induced by E2F Transcription Factor 2-MicroRNA-17-5p Circuitry in Extrafollicular Effector B Cells Contributes to Autoantibody Production in Systemic Lupus Erythematosus.

Elevated interleukin-10 (IL-10) levels in patients with systemic lupus erythematosus (SLE) have B cell-promoting effects, contributing to autoantibody production and tissue damage. We aimed to characterize up-regulated IL-10+ B cell subsets and dysregulated IL10 expression in SLE B cells for new therapeutic options. Proportions of Th10 and IL-10+ B cell subsets in peripheral blood mononuclear cells (PBMCs) were assessed using flow cytometry. The IL10 3'-untranslated region (3'-UTR) dual-luciferase vector was constructed and cotransfected with small interfering RNA (siRNA), microRNA (miRNA) mimics, or miRNA inhibitors into Raji cells. Transcript levels were quantified using TaqMan assays. Culture conditions that induced IL-10+ Breg cells in healthy controls resulted in expansion of IL-10+ double-negative 2 (DN2; IgD-CD27-CD21-CD11c+) B cells in SLE PBMCs. Proportions of IL-10+ DN2, but not those of IL-10- DN2, correlated with disease activity and levels of antibodies to double-stranded DNA (dsDNA) (r = 0.60, P = 0.03 for cohort 1; r = 0.38, P = 0.03 for cohort 2), and were associated with high levels or seropositivity of anti-Sm (P = 0.03 for cohort 1; P = 0.01 for cohort 2) and IgG anticardiolipin (P < 0.01 for cohort 1; P = 0.02 for cohort 2) in SLE patients from 2 cohorts, of mainly African American subjects (cohort 1) and of Asian subjects (cohort 2). Proportions of Th10 (CD45RA-CXCR5-CXCR3+PD-1high CD4+) cells correlated with IL-10+ DN2 frequencies (r = 0.60, P < 0.01 for cohort 2), antinuclear antibody titers (r = 0.52, P = 0.01 for cohort 2), and proteinuria levels (r = 0.72, P < 0.01 for cohort 2) in SLE patients. Screening of predicted IL10 3'-UTR-targeting miRNAs in SLE B cells identified miRNA-17-5p (miR-17-5p) and miR-20a-5p, with their levels inversely correlated with IL10 (r = -0.47, P < 0.01 for miR-17-5p; r = -0.37, P = 0.03 for miR-20-5p) and transcription factor E2F2 (r = -0.48, P = 0.04 for miR-17-5p; r = -0.45, P = 0.05 for miR-20-5p). In Raji cells, knockdown of E2F2 expression resulted in increased levels of miR-17-5p and miR-20a-5p and decreased IL10 messenger RNA (mRNA) and protein levels, and overexpression and inhibition of miR-17-5p down-regulated and up-regulated, respectively, IL10 mRNA levels, suggesting regulation of IL10 expression by an E2F2-miR-17-5p loop. IL-10 promotes extrafollicular autoimmune responses in patients with active SLE, which might be dampened by targeting the E2F2-miR-17-5p circuitry.

SARS-Cov2 acute and post-active infection in the context of autoimmune and chronic inflammatory diseases

The clinical and immunological spectrum of acute and post-active COVID-19 syndrome overlaps with criteria used to characterize autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Indeed, following SARS-Cov2 infection, the innate immune response is altered with an initial delayed production of interferon type I (IFN–I), while the NF-kappa B and inflammasome pathways are activated. In lung and digestive tissues, an alternative and extrafollicular immune response against SARS-Cov2 takes place with, consequently, an altered humoral and memory T cell response leading to breakdown of tolerance with the emergence of autoantibodies. However, the risk of developing severe COVID-19 among SLE and RA patients did not exceed the general population except in those having pre-existing neutralizing autoantibodies against IFN-I. Treatment discontinuation rather than COVID-19 infection or vaccination increases the risk of developing flares. Last but not least, a limited number of case reports of individuals having developed SLE or RA following COVID-19 infection/vaccination have been reported. Altogether, the SARS-Cov2 pandemic represents an unique opportunity to investigate the dangerous interplay between the immune response against infectious agents and autoimmunity, and to better understand the triggering role of infection as a risk factor in autoimmune and chronic inflammatory disease development.

Plasma cell generation during T-cell-dependent immune responses.

Plasma cells are terminally differentiated from activated B cells and are specialized for secreting antibodies, which are essential effector molecules in humoral immunity to neutralize invading pathogens. Upon challenge with T-cell-dependent antigens, plasma cells can be generated during the primary extrafollicular response, the germinal center (GC) response or the secondary memory response. Recent studies have revealed that plasma cell generation is regulated not only by several key transcription factors but also by epigenetic modifications. In addition, the differentiation of GC B cells toward a plasma cell fate is associated with affinity for antigens and is determined by the strength of contact with T follicular helper cells.

BCR Affinity Influences T-B Interactions and B Cell Development in Secondary Lymphoid Organs.

B cells produce high-affinity immunoglobulins (Igs), or antibodies, to eliminate foreign pathogens. Mature, naïve B cells expressing an antigen-specific cell surface Ig, or B cell receptor (BCR), are directed toward either an extrafollicular (EF) or germinal center (GC) response upon antigen binding. B cell interactions with CD4+ pre-T follicular helper (pre-Tfh) cells at the T-B border and effector Tfh cells in the B cell follicle and GC control B cell development in response to antigen. Here, we review recent studies demonstrating the role of B cell receptor (BCR) affinity in modulating T-B interactions and the subsequent differentiation of B cells in the EF and GC response. Overall, these studies demonstrate that B cells expressing high affinity BCRs preferentially differentiate into antibody secreting cells (ASCs) while those expressing low affinity BCRs undergo further affinity maturation or differentiate into memory B cells (MBCs).

Short- and Long-Lived Autoantibody-Secreting Cells in Autoimmune Neurological Disorders.

As B cells differentiate into antibody-secreting cells (ASCs), short-lived plasmablasts (SLPBs) are produced by a primary extrafollicular response, followed by the generation of memory B cells and long-lived plasma cells (LLPCs) in germinal centers (GCs). Generation of IgG4 antibodies is T helper type 2 (Th2) and IL-4, -13, and -10-driven and can occur parallel to IgE, in response to chronic stimulation by allergens and helminths. Although IgG4 antibodies are non-crosslinking and have limited ability to mobilize complement and cellular cytotoxicity, when self-tolerance is lost, they can disrupt ligand-receptor binding and cause a wide range of autoimmune disorders including neurological autoimmunity. In myasthenia gravis with predominantly IgG4 autoantibodies against muscle-specific kinase (MuSK), it has been observed that one-time CD20+ B cell depletion with rituximab commonly leads to long-term remission and a marked reduction in autoantibody titer, pointing to a short-lived nature of autoantibody-secreting cells. This is also observed in other predominantly IgG4 autoantibody-mediated neurological disorders, such as chronic inflammatory demyelinating polyneuropathy and autoimmune encephalitis with autoantibodies against the Ranvier paranode and juxtaparanode, respectively, and extends beyond neurological autoimmunity as well. Although IgG1 autoantibody-mediated neurological disorders can also respond well to rituximab induction therapy in combination with an autoantibody titer drop, remission tends to be less long-lasting and cases where titers are refractory tend to occur more often than in IgG4 autoimmunity. Moreover, presence of GC-like structures in the thymus of myasthenic patients with predominantly IgG1 autoantibodies against the acetylcholine receptor and in ovarian teratomas of autoimmune encephalitis patients with predominantly IgG1 autoantibodies against the N‐methyl‐d‐aspartate receptor (NMDAR) confers increased the ability to generate LLPCs. Here, we review available information on the short-and long-lived nature of ASCs in IgG1 and IgG4 autoantibody-mediated neurological disorders and highlight common mechanisms as well as differences, all of which can inform therapeutic strategies and personalized medical approaches.

Lupus-like extrafollicular B cell responses and autoreactivity are hallmarks of severe COVID-19

Abstract In late 2019, the emergence of the coronavirus SARS-CoV-2 resulted in a global pandemic, costing over 2 million lives. A hallmark of the disease resulting from SARS-CoV-2 infection, COVID-19, has been it’s heterogeneity in presentation. To understand the immunological underpinnings of this variability in symptoms, we performed a deep characterization of the B cell responses generated in patients within the NIH-defined mild/moderate or severe/critical categories. Somewhat surprisingly, patients with severe disease had higher neutralizing antibody titers than their mild/moderate counterparts, but showed strong evidence of extrafollicular B cell activation by flow cytometry – previously described in humans primarily in autoimmune disease. Further analysis of the B cell repertoire through single cell VDJ sequencing revealed low (or fully absent) levels of somatic hypermutation in the antibody secreting cell compartment, phenocopying the low selective pressures previously associated with extrafollicular responses. Together, these findings suggested the possible relaxation of peripheral tolerance in severe patients. This was confirmed through clinical autoreactivity testing, revealing more than 70% of the most severe COVID-19 patients, despite no history of autoimmunity, test positive for anti-nuclear antibodies or rheumatoid factor. Altogether, these findings provide the immunological context for an emerging consensus in the literature whereby severe COVID-19 is characterized by robust extrafollicular B cell activation, resulting not only in neutralizing antibody production, but also autoreactive B cell targeting with the potential for direct involvement in severe disease pathology.

CD27hiCD38hi plasmablasts are activated B cells of mixed origin with distinct function

SummaryClinically important broadly reactive B cells evolve during multiple infections, with B cells re-activated after secondary infection differing from B cells activated after a primary infection. Here we studied CD27highCD38high plasmablasts from patients with a primary or secondary dengue virus infection. Three transcriptionally and functionally distinct clusters were identified. The largest cluster 0/1 was plasma cell-related, with cells coding for serotype cross-reactive antibodies of the IgG1 isotype, consistent with memory B cell activation during an extrafollicular response. Cells in clusters 2 and 3 expressed low levels of antibody genes and high levels of genes associated with oxidative phosphorylation, EIF2 pathway, and mitochondrial dysfunction. Clusters 2 and 3 showed a transcriptional footprint of T cell help, in line with activation from naive B cells or memory B cells. Our results contribute to the understanding of the parallel B cell activation events that occur in humans after natural primary and secondary infection.

Single-Cell Transcriptomic Analyses Define Distinct Peripheral B Cell Subsets and Discrete Development Pathways.

Separation of B cells into different subsets has been useful to understand their different functions in various immune scenarios. In some instances, the subsets defined by phenotypic FACS separation are relatively homogeneous and so establishing the functions associated with them is straightforward. Other subsets, such as the “Double negative” (DN, CD19+CD27-IgD-) population, are more complex with reports of differing functionality which could indicate a heterogeneous population. Recent advances in single-cell techniques enable an alternative route to characterize cells based on their transcriptome. To maximize immunological insight, we need to match prior data from phenotype-based studies with the finer granularity of the single-cell transcriptomic signatures. We also need to be able to define meaningful B cell subsets from single cell analyses performed on PBMCs, where the relative paucity of a B cell signature means that defining B cell subsets within the whole is challenging. Here we provide a reference single-cell dataset based on phenotypically sorted B cells and an unbiased procedure to better classify functional B cell subsets in the peripheral blood, particularly useful in establishing a baseline cellular landscape and in extracting significant changes with respect to this baseline from single-cell datasets. We find 10 different clusters of B cells and applied a novel, geometry-inspired, method to RNA velocity estimates in order to evaluate the dynamic transitions between B cell clusters. This indicated the presence of two main developmental branches of memory B cells. A T-independent branch that involves IgM memory cells and two DN subpopulations, culminating in a population thought to be associated with Age related B cells and the extrafollicular response. The other, T-dependent, branch involves a third DN cluster which appears to be a precursor of classical memory cells. In addition, we identify a novel DN4 population, which is IgE rich and closely linked to the classical/precursor memory branch suggesting an IgE specific T-dependent cell population.

Hematologic disorder–associated Cxcr4 gain-of-function mutation leads to uncontrolled extrafollicular immune response

The extrafollicular immune response is essential to generate a rapid but transient wave of protective antibodies during infection. Despite its importance, the molecular mechanisms controlling this first response are poorly understood. Here, we demonstrate that enhanced Cxcr4 signaling caused by defective receptor desensitization leads to exacerbated extrafollicular B-cell response. Using a mouse model bearing a gain-of-function mutation of Cxcr4 described in 2 human hematologic disorders, warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome and Waldenström macroglobulinemia, we demonstrated that mutant B cells exhibited enhanced mechanistic target of rapamycin signaling, cycled more, and differentiated more potently into plasma cells than wild-type B cells after Toll-like receptor (TLR) stimulation. Moreover, Cxcr4 gain of function promoted enhanced homing and persistence of immature plasma cells in the bone marrow, a phenomenon recapitulated in WHIM syndrome patient samples. This translated in increased and more sustained production of antibodies after T-independent immunization in Cxcr4 mutant mice. Thus, our results establish that fine-tuning of Cxcr4 signaling is essential to limit the strength and length of the extrafollicular immune response.

Human antiviral B cell responses: Emerging lessons from hepatitis B and COVID-19.

Humoral immunity is a critical component of the coordinated response required to resolve viral infections and mediate protection following pathogen clearance or vaccination. A better understanding of factors shaping the memory B cell response will allow tailored development of efficient preventative vaccines against emerging acute viral infections, therapeutic vaccines, and immunotherapies for chronic viral infections. Here, we use recent data obtained by profiling antigen‐specific B cell responses in hepatitis B as a framework to explore lessons that can be learnt from different viral infections about the diverse influences on humoral immunity. Hepatitis B provides a paradigm where successful B cell responses in resolved or vaccinated individuals can be contrasted to the failed response in chronic infection, while also exemplifying the degree to which B cell responses within infected individuals can differ to two antigens from the same virus. Drawing on studies in other human and murine infections, including emerging data from COVID‐19, we consider the influence of antigen quantity and structure on the quality of the B cell response, the role of differential CD4 help, the importance of germinal center vs extrafollicular responses and the emerging concept that responses residing in non‐lymphoid organs can participate in B cell memory.

In silico immune infiltration profiling combined with functional enrichment analysis reveals a potential role for naïve B cells as a trigger for severe immune responses in the lungs of COVID-19 patients.

COVID-19, caused by SARS-CoV-2, has rapidly spread to more than 160 countries worldwide since 2020. Despite tremendous efforts and resources spent worldwide trying to explore antiviral drugs, there is still no effective clinical treatment for COVID-19 to date. Approximately 15% of COVID-19 cases progress to pneumonia, and patients with severe pneumonia may die from acute respiratory distress syndrome (ARDS). It is believed that pulmonary fibrosis from SARS-CoV-2 infection further leads to ARDS, often resulting in irreversible impairment of lung function. If the mechanisms by which SARS-CoV-2 infection primarily causes an immune response or immune cell infiltration can be identified, it may be possible to mitigate excessive immune responses by modulating the infiltration and activation of specific targets, thereby reducing or preventing severe lung damage. However, the extent to which immune cell subsets are significantly altered in the lung tissues of COVID-19 patients remains to be elucidated. This study applied the CIBERSORT-X method to comprehensively evaluate the transcriptional estimated immune infiltration landscape in the lung tissues of COVID-19 patients and further compare it with the lung tissues of patients with idiopathic pulmonary fibrosis (IPF). We found a variety of immune cell subtypes in the COVID-19 group, especially naïve B cells were highly infiltrated. Comparison of functional transcriptomic analyses revealed that non-differentiated naïve B cells may be the main cause of the over-active humoral immune response. Using several publicly available single-cell RNA sequencing data to validate the genetic differences in B-cell populations, it was found that the B-cells collected from COVID-19 patients were inclined towards naïve B-cells, whereas those collected from IPF patients were inclined towards memory B-cells. Further differentiation of B cells between COVID-19 mild and severe patients showed that B cells from severe patients tended to be antibody-secreting cells, and gene expression showed that B cells from severe patients were similar to DN2 B cells that trigger extrafollicular response. Moreover, a higher percentage of B-cell infiltration seems associated with poorer clinical outcome. Finally, a comparison of several specific COVID-19 cases treated with targeted B-cell therapy suggests that appropriate suppression of naïve B cells might potentially be a novel strategy to alleviate the severe symptoms of COVID-19.

Polyreactivity and Somatic Hypermutation Analysis Reveals the Innate B Cell Origin of Human PF4/Heparin Reactive Antibodies

Heparin-induced thrombocytopenia (HIT) is a serious reaction to heparin treatment characterized by antibodies that recognize a complex formed between heparin and platelet factor 4 (PF4/H) and are capable of activating platelets and inducing a pro-thrombotic state. Although a high percentage of heparin-treated patients produce antibodies to PF4/H, only a subset of these antibodies are platelet-activating (pathogenic) and capable of causing HIT. We previously reported that we cloned B cells from six patients experiencing HIT and identified two types of PF4/H-binding antibodies: seven platelet-activating (PA) and 48 non-activating (NA). Comparison of the structural features in the PA, NA, and non PF4/H-binding (NB) clones showed that the length and the number of basic amino acid and tyrosine residue in the heavy chain complementarity determining region 3 (HCDR3) were significantly different, and was in the order of PA&amp;gt;NA&amp;gt;NB. Most significantly, the seven platelet-activating antibodies each have one of the two pathogenic motifs: RX1-2 R/KX1-2 R/H and YYYYY in an unusually long HCDR3 (≥ 20 residues). In the current study, we attempt to understand the origin of the B cells that produce the PA and NA antibodies and the nature of the immune response in HIT through analyzing somatic hypermutation and biological property of such antibodies. Longer HCDR3 and more basic Aas and Tyr residues in the HCDR3 are features of autoreactive and polyreactive antibodies. With this in mind, we tested PA and NA clones in a standard antinuclear antibody (ANA) assay and found that these clones were significantly more reactive than NB antibodies, and the plasma of HIT patients were significantly more reactive than normal plasma (Figure1). We then compared reactions of PA, NA and NB clones against a group of self and foreign antigens commonly used in polyreactivity assays: dsDNA, ssDNA, LPS, insulin, and keyhole limpet hemocyanin (KLH). About 90% of PA and NA clones were reactive to at least two antigens, this was true of only 20% of the NB clones, and the latter is consistent with the frequency of polyreactive clones in the IgG+ B cells (Figure2). Taken together, these data indicate that PA and NA antibodies are largely polyreactive. We then investigated the development of the PA and NA B cells through analyzing somatic hypermutation in the antibodies. Through analyzing the HCDR3 nucleotide insertion, trimming and VDJ segment usage, we found that longer HCDR3 typical of PF4/H-binding clones and the RKH and Y5 motifs identified in PA clones were the result of original recombination not somatic hypermutation. Consistently, the average number of nucleotide mutations in the VH genes of the binding clones was lower (PA and NA, 9.4 ± 9.5) compared to that of peripheral blood IgG+ memory B cells in healthy subjects (~18) (Figure3). Total mutation frequency in the VH and Vk CDRs of the PF4/H-binding PA and NA clones was comparable to that of the framework regions. This finding contrasts with findings made in peripheral blood IgG+ memory B cells of healthy subjects showing that the mutation frequencies are much higher in the CDRs than in the FRs of VH. Taken together, these findings suggest that affinity maturation plays a limited role in the evolution PF4/H-binding antibodies during the immune response that leads to HIT. In this study, we showed thay PF4/H-binding PA and NA IgGs are largely polyreactive antibodies and contain lower levels of mutations compared to IgG+ memory B cells. B1 and MZ B cells are innate B cells that are main producers of polyreactive natural antibodies and can respond to toll-like receptor signaling, quickly differentiate into antibody-secreting cells, and undergo IgG class switch extrafollicularly. Polyreactivity identified in the PF4/H-binding PA and NA IgGs supports the possibility that human B cells producing PF4/H-binding antibodies are innate B cells akin to MZ B cells shown to be a source of PF4/H antibodies in mice. A mutation rate lower than that of IgG+ memory cells in the PF4/H-binding IgGs is also consistent with an extrafollicular response typical of innate B cells. These observations would help to improve our understanding of the immunological responses and B cell origin in HIT patients. Disclosures Padmanabhan: Retham Technologies: Current equity holder in private company; Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees; Versiti Blood Research Institute: Patents &amp; Royalties.

PIK3IP1 Promotes Extrafollicular Class Switching in T-Dependent Immune Responses

PI3K plays multiple roles throughout the life of a B cell. As such, its signaling is tightly regulated. The importance of this is illustrated by the fact that both loss- and gain-of-function mutations in PI3K can cause immunodeficiency in humans. PIK3IP1, also known as TrIP, is a transmembrane protein that has been shown to inhibit PI3K in T cells. Results from the ImmGen Consortium indicate that PIK3IP1 expression fluctuates throughout B cell development in a manner inversely correlated with PI3K activity; however, its role in B cells is poorly understood. In this study, we define the consequences of B cell-specific deletion of PIK3IP1. B cell development, basal Ig levels, and T-independent responses were unaffected by loss of PIK3IP1. However, there was a significant delay in the production of IgG during T-dependent responses, and secondary responses were impaired. This is likely due to a role for PIK3IP1 in the extrafollicular response because germinal center formation and affinity maturation were normal, and PIK3IP1 is not appreciably expressed in germinal center B cells. Consistent with a role early in the response, PIK3IP1 was downregulated at late time points after B cell activation, in a manner dependent on PI3K. Increased activation of the PI3K pathway was observed in PIK3IP1-deficient B cells in response to engagement of both the BCR and CD40 or strong cross-linking of CD40 alone. Taken together, these observations suggest that PIK3IP1 promotes extrafollicular responses by limiting PI3K signaling during initial interactions between B and T cells.

Antigen valency dictates the affinity of B cells recruited to germinal centers and the magnitude of extrafollicular B cell responses

Abstract While it is widely agreed that antigen valency is a factor in immunogenicity, how antigen valency contributes to B cell selection during antigen-specific responses is poorly understood. Here, using HIV immunogens with a defined valency ranging from 1 to 60, we investigated the role of antigen valency during different phases of the antigen-specific B cell response. We show that highly multimerized immunogens rapidly activated cognate B cells in vivo with little affinity discrimination, initially. This subsequently led to strong extrafollicular and germinal center responses where B cells within a broad affinity range were efficiently recruited. Low-valency (1–8 copy) antigens induced less diverse effector B cell responses, with limited recruitment of low-affinity B cells. The data reveal roles of antigen valency in dictating affinity thresholds required for recruitment of B cells to germinal centers or rapid extrafollicular plasma cell responses, with important implications for vaccine design.

Anti-dsDNA specific antibody secreting cells are increased in both frequency and abundance in Chronic Rhinosinusitis with Nasal Polyps

Abstract Background We have previously found that anti-dsDNA IgG autoantibodies are increased in Chronic Rhinosinusitis with Nasal Polyps (CRSwNP) patients. Recent studies find that B cells with the Epstein-Barr virus-induced protein 2(EBI2), is critical to the extrafollicular response. We have described elevated numbers of EBI2+ B cells in NP and they were more frequently antibody secreting cells (ASCs). In this study we evaluate whether dsDNA-specific ASCs are increased in NP and further compared the EBI2+ and EBI2− compartments. Methods To investigate this problem, flow cytometry and ELISpot assays were utilized to compare the frequency of total IgG and dsDNA specific IgG ASCs and cells were sorted into EBI2+ and EBI2− B cells from human NP and tonsils. Results Flow cytometry results (n=10 tonsil, 4 NP) found that although tonsils had significantly more B cells than NP, a significantly smaller proportion were CD27+CD38+ plasmablasts (PB). Further ELISpot analysis found NP B cells were more frequently IgG ASCs than tonsil (0.06% and 0.43% in tonsil (n=9) and NP (n=5) respectively, p&amp;lt;0.001). Anti-dsDNA specific ASCs were two-fold more in NP representing 0.95% of IgG ASCs compared with 0.4% in tonsil ASCs (p&amp;lt;0.05). In analysis of ASCs sorted from tonsils (n=3) and NP (n=2), we found anti-dsDNA IgG ASCs were more frequent among EBI2+ compared to EBI2− cells with 10- and 14-fold increases in dsDNA reactivity (p&amp;lt;0.01). Conclusions Although B-cells are significantly more abundant in tonsils, NP derived B-cells more frequently secrete total- and dsDNA-specific IgG. These differences appear to be driven by extrafollicular EBI2+ B-cells suggesting that EBI2+ B cells represent the major pool of autoreactive B-cells observed in CRSwNP and tonsils.

B Cell Activation and Response Regulation During Viral Infections.

Acute viral infections are characterized by rapid increases in viral load, leading to cellular damage and the resulting induction of complex innate and adaptive antiviral immune responses that cause local and systemic inflammation. Successful antiviral immunity requires the activation of many immune cells, including T cells, natural killer cells, and macrophages. B cells play a unique part through their production of antibodies that can both neutralize and clear viral particles before virus entry into a cell. Protective antibodies are produced even before the first exposure of a pathogen, through the regulated secretion of so-called natural antibodies that are generated even in the complete absence of prior microbial exposure. An early wave of rapidly secreted antibodies from extrafollicular (EF) responses draws on the preexisting naive or memory repertoire of B cells to induce a strong protective response that in kinetics tightly follows the clearance of acute infections, such as with influenza virus. Finally, the generation of germinal centers (GCs) provides long-term protection through production of long-lived plasma cells and memory B cells, which shape and broaden the B cell repertoire for more effective responses following repeat exposures. In this study, we review B cell responses to acute viral infections, primarily influenza virus, from the earliest nonspecific B-1 cell to early, antigen-specific EF responses and finally to GC responses. Throughout, we address known factors that lead to distinct B cell response outcomes and discuss how their functions effect viral clearance, highlighting the critical contributions of each response type to the induction of highly protective antiviral humoral immunity.

CD27 &lt;sup&gt;hi&lt;/sup&gt;CD38 &lt;sup&gt;hi&lt;/sup&gt;&amp;nbsp;Plasmablasts are Activated B Cells of Mixed Origin with Distinct Function

Clinically important broadly reactive B cells evolve during multiple infections. The phenotype and function of B cells re-activated during secondary infection is different compared to B cells activated after a primary infection. Here we studied CD27high CD38high plasmablasts from patients with a primary or secondary dengue virus infection. Three transcriptionally and functionally distinct plasmablast clusters were identified. The largest cluster 0/1 was plasma cell-related and contained cells coding for virus serotype cross-reactive antibodies of the IgG1 isotype, consistent with memory B cell activation during an extrafollicular response. In contrast, cells in clusters 2 and 3 expressed low levels of antibody genes and high levels of genes associated with oxidative phosphorylation, EIF2 pathway and mitochondrial dysfunction. Clusters 2 and 3 also showed a transcriptional footprint of T cell help, in line with activation from either naive B cells or memory B cells in the context of a germinal center reaction.

Regulation of Plasma Cell Differentiation.

Humoral immunity provides protection from pathogen infection, and this is mediated by antibodies that are produced by plasma cells. Plasma cells are terminally differentiated from activated B cells and are specialized for secreting antibodies. Plasma cells are generated during extrafollicular or germinal center (GC) responses, but GC-derived plasma cells are thought to be the major precursors of long-lived plasma cells, which confer long-term protection. Here, we review recent progress in our understanding of the cellular and molecular basis for plasma cell differentiation from GC B cells.