Peripheral Plasmablasts Research Articles

Anti-HLA serologic response to CD38-targeting desensitization therapy is challenged by peripheral memory B cells in highly sensitized kidney transplant candidates

High human leukocyte antigen (HLA) sensitization limits access to compatible transplantation. New CD38-targeting agents have been shown to reduce anti-HLA antibodies, although with important interpatient variability. Thus, pretreatment identification of responder and nonresponder (NR) patients is needed for treatment decision-making. We analyzed 26 highly sensitized (HS) patients from 2 desensitization trials using anti-CD38 monoclonal antibodies. Hierarchical clustering identified 3 serologic responder groups: high responders, low responders, and NR. Spectral flow cytometry and functional HLA-specific memory B cell (mBC) assessment were first conducted on peripheral blood mononuclear cells and bone marrow samples from 16 patients treated with isatuximab (NCT04294459). Isatuximab effectively depleted bone marrow plasma cells, peripheral CD38-expressing plasmablasts, plasma cells, transitional B cells, and class-switch mBCs, ultimately reducing frequencies of HLA-specific immunoglobulin G (IgG)-producing mBCs. Multidimensional spectral flow cytometry with partial least squares discriminant analysis revealed that pretreatment abundance of specific circulating mBC phenotypes, especially CD38neg class-switch mBCs, accurately distinguished between high serologic responders and low responders or NR (AUC 0.958, 0.860-1.000, P = .009), who also displayed significantly lower frequencies of HLA-specific IgG-producing mBCs (P < .0001). This phenotypical mBC signature predicting response to therapy was validated in an external HS patient cohort (n = 10) receiving daratumumab (NCT04204980). This study identifies critical circulating mBC subset phenotypes that distinguish HS patients with successful serologic responses to CD38-targeting desensitization therapies, potentially guiding treatment decision-making.

Astrocytic stress response is induced by exposure to astrocyte-binding antibodies expressed by plasmablasts from pediatric patients with acute transverse myelitis

BackgroundPediatric acute transverse myelitis (ATM) accounts for 20–30% of children presenting with a first acquired demyelinating syndrome (ADS) and may be the first clinical presentation of a relapsing ADS such as multiple sclerosis (MS). B cells have been strongly implicated in the pathogenesis of adult MS. However, little is known about B cells in pediatric MS, and even less so in pediatric ATM. Our lab previously showed that plasmablasts (PB), the earliest B cell subtype producing antibody, are expanded in adult ATM, and that these PBs produce self-reactive antibodies that target neurons. The goal of this study was to examine PB frequency and phenotype, immunoglobulin selection, and B cell receptor reactivity in pediatric patients presenting with ATM to gain insight to B cell involvement in disease.MethodsWe compared the PB frequency and phenotype of 5 pediatric ATM patients and 10 pediatric healthy controls (HC) and compared them to previously reported adult ATM patients using cytometric data. We purified bulk IgG from the plasma samples and cloned 20 recombinant human antibodies (rhAbs) from individual PBs isolated from the blood. Plasma-derived IgG and rhAb autoreactivity was measured by mean fluorescence intensity (MFI) in neurons and astrocytes of murine brain or spinal cord and primary human astrocytes. We determined the potential impact of these rhAbs on astrocyte health by measuring stress and apoptotic response.ResultsWe found that pediatric ATM patients had a reduced frequency of peripheral blood PB. Serum IgG autoreactivity to neurons in EAE spinal cord was similar in the pediatric ATM patients and HC. However, serum IgG autoreactivity to astrocytes in EAE spinal cord was reduced in pediatric ATM patients compared to pediatric HC. Astrocyte-binding strength of rhAbs cloned from PBs was dependent on somatic hypermutation accumulation in the pediatric ATM cohort, but not HC. A similar observation in predilection for astrocyte binding over neuron binding of individual antibodies cloned from PBs was made in EAE brain tissue. Finally, exposure of human primary astrocytes to these astrocyte-binding antibodies increased astrocytic stress but did not lead to apoptosis.ConclusionsDiscordance in humoral immune responses to astrocytes may distinguish pediatric ATM from HC.

Single-cell RNA sequencing reveals the expansion of circulating tissue-homing B cell subsets in secondary acute dengue viral infection

The roles of antibodies secreted by subsets of B cells in dengue virus (DENV) infection have been extensively studied, yet, the contribution of tissue-homing B cells to antiviral immunity remains unclear. In this study, we performed a comprehensive analysis of B cell subpopulations in peripheral blood samples from DENV-infected patients using single-cell RNA-sequencing (scRNA-seq) datasets and flow cytometry. We showed that plasma cells (PCs) and plasmablasts (PBs) were the predominant B cell populations during the acute phase of secondary natural DENV infection, but not in convalescent phase nor in healthy controls. Interestingly, these cells expressed proliferation, adhesion, and tissue-homing genes, including SELPLG, a homing marker of the skin, the initial infected site of DENV. Flow cytometry analysis confirmed a significant upregulation of cell surface expression of a cutaneous lymphocyte-associated antigen (CLA) encoded by SELPLG in PCs and PBs, compared to naive and memory B cells from the same patients. The analysis of an independent single-cell B-cell receptor sequencing (scBCR-seq) dataset of DENV-infected patients revealed that the peripheral blood PCs and PBs exhibited the highest clonal expansion in secondary DENV infection compared to other B cell subsets. These clonally expanded cells also expressed the highest levels of tissue-homing genes, including SELPLG. In addition, by utilizing a public scRNA-seq dataset of SARS-CoV2 infection, we demonstrated the upregulation of several tissue-homing genes in PCs and PBs. Our study provides evidence for the potential roles of tissue-homing B cell subsets in the context of immune responses against viral infections in humans.

The Janus face of proliferating plasmablasts in dengue and COVID-19 infections.

B cells play an integral role in the immune response to both dengue fever and COVID-19. Prior scRNAseq analyses of peripheral plasmablasts in COVID-19 have revealed a heterogeneous population with distinct cell subsets associated with proliferation; prior studies in patients with dengue fever have likewise shown the presence of proliferative pre-plasmablasts in the circulation. These findings may have implications for disease severity. In this study, we sought to gain a mechanistic understanding of the intracellular processes in naive and memory B cells that are associated with and may lead to an expanded proliferative plasmablast population in the circulation. We analyzed age-controlled (pediatric and adult), peripheral blood mononuclear cell scRNAseq datasets from patients infected with either dengue (primary or secondary) or COVID-19 (non-severe or severe) from previously published studies. Our preliminary analysis showed that pediatric patients with dengue and adults with COVID-19 had an expanded proliferative plasmablast (p-PB) population. By contrast, neither the adults with dengue nor the children with COVID-19 in our dataset had p-PBs. We used this distinctive preliminary signature to guide our analyses design and expanded our analyses to naive and memory B cells. In age/disease conditions with and without p-PBs, we found differences in cell sensing and activation, including via the B cell receptor and downstream signal transduction. Likewise, inflammation was mediated differently: relative to groups without p-PBs, those with p-PBs had increased expression of interferon response and S100 genes (particularly severe COVID-19). Furthermore, several transcription factors at the nexus of activation, inflammation, and cell fate decisions were expressed differently in groups with and without p-PBs. We used dengue and COVID-19 infections in adult and pediatric patients (focusing on naive B, memory B, and plasmablast cells) as a model to better understand the mechanisms that may give rise to p-PB populations in the circulation. Our results indicate that a more pro-inflammatory state in naive and memory B cells correlated with - and could influence the generation of- proliferating plasmablasts. Further exploration of these mechanisms will have implications for immune memory, vaccine development, and post-viral autoimmune syndromes.

Peripheral CD23hi/IgE+ Plasmablasts Secrete IgE and Correlate with Allergic Disease Severity.

Production and secretion of IgE by B cells, plasmablasts, and plasma cells is a central step in the development and maintenance of allergic diseases. IgE can bind to one of its receptors, the low-affinity IgE receptor CD23, which is expressed on activated B cells. As a result, most B cells bind IgE through CD23 on their surface. This makes the identification of IgE producing cells challenging. In this study, we report an approach to clearly identify live IgE+ plasmablasts in peripheral blood for application by both flow cytometry analysis and in vitro assay. These IgE+ plasmablasts readily secrete IgE, upregulate specific mRNA transcripts (BLIMP-1 IRF4, XBP1, CD138, and TACI), and exhibit highly differentiated morphology all consistent with plasmablast differentiation. Most notably, we compared the presence of IgE+ plasmablasts in peripheral blood of allergic and healthy individuals using a horse model of naturally occurring seasonal allergy, Culicoides hypersensitivity. The model allows the comparison of immune cells both during periods of clinical allergy and when in remission and clinically healthy. Allergic horses had significantly higher percentages of IgE+ plasmablasts and IgE secretion while experiencing clinical allergy compared with healthy horses. Allergy severity and IgE secretion were both positively correlated to the frequency of IgE+ plasmablasts in peripheral blood. These results provide strong evidence for the identification and quantification of peripheral IgE-secreting plasmablasts and provide a missing cellular link in the mechanism of IgE secretion and upregulation during allergy.

Clonal expansion and markers of directed mutation of IGHV4-34 B cells in plasmablasts during Kawasaki disease

Kawasaki disease (KD) is the leading cause of acquired heart disease in children. The cause remains unknown; however, epidemiologic and demographic data support a single preceding infectious agent may lead to KD. A variety of pathophysiologic responses have been proposed, including direct invasion of the coronary arteries, a superantigen response, and a post-infectious autoimmune phenomenon. A role for B cell responses during KD are supported by numerous findings including B cell specific markers identified in genome wide association studies. We have recently published data showing children with KD have similar plasmablast (PB) responses to children with infections. Since during other infections, cells expressing antibodies against the preceding infection are enriched in PBs, we sought to explore the specific antibodies encoded by PBs during KD. In one child we see a massive expansion in IGHV4–34 utilizing antibodies, which has been associated with autoimmunity in the past. We further explored this expansion of IGHV4–34 utilization during the peripheral PB rise with next generation sequencing (NGS) analysis and utilizing newer techniques of chromium chip single cell separation (10x Genomics®). We also utilized peptide array screening to attempt to identify an antigen to the most prolific clones.

Expanded CD4+CXCR5-PD-1+ peripheral T helper like cells and clinical significance in IgG4-related disease

To identify the phenotypic features and clinical significance of peripheral T helper (Tph) like cells in IgG4-RD, 54 untreated IgG4-RD patients and 57 healthy controls (HCs) were enrolled. Flow cytometry analysis, ELISA and correlation analysis were performed. Results indicated that percentages of CD4 + CXCR5-PD-1+ Tph like cells in the peripheral blood of IgG4-RD patients were significantly higher than those of HCs (2.27% ± 1.99% vs 1.12% ± 0.98%, P < 0.001). Expression of CD38, CD25, and TIGIT was higher, whereas that of CCR7, CD127 was lower in the Tph like cells from the IgG4-RD patients than in those from the HCs. The IgG4-RD patients with affected internal organs had higher circulating Tph like cell levels than those without (2.69% ±1.99% vs 1.23% ± 0.93%, respectively, P = 0.003). In addition, Tph like cells correlated with serum IgG and IgG4 and peripheral plasmablast levels which could be a promising biomarker for disease activity monitoring.

SARS-CoV-2 in severe COVID-19 induces a TGF-\u03b2-dominated chronic immune response that does not target itself

The pathogenesis of severe COVID-19 reflects an inefficient immune reaction to SARS-CoV-2. Here we analyze, at the single cell level, plasmablasts egressed into the blood to study the dynamics of adaptive immune response in COVID-19 patients requiring intensive care. Before seroconversion in response to SARS-CoV-2 spike protein, peripheral plasmablasts display a type 1 interferon-induced gene expression signature; however, following seroconversion, plasmablasts lose this signature, express instead gene signatures induced by IL-21 and TGF-β, and produce mostly IgG1 and IgA1. In the sustained immune reaction from COVID-19 patients, plasmablasts shift to the expression of IgA2, thereby reflecting an instruction by TGF-β. Despite their continued presence in the blood, plasmablasts are not found in the lungs of deceased COVID-19 patients, nor does patient IgA2 binds to the dominant antigens of SARS-CoV-2. Our results thus suggest that, in severe COVID-19, SARS-CoV-2 triggers a chronic immune reaction that is instructed by TGF-β, and is distracted from itself.

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

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

SAT0166 BIOMARKERS OF B-CELL DEPLETION AND RESPONSE IN A RANDOMIZED, CONTROLLED TRIAL OF OBINUTUZUMAB FOR PROLIFERATIVE LUPUS NEPHRITIS

Background:Incomplete B-cell and plasmablast depletion, as measured using highly sensitive flow cytometry (HSFC), is associated with lower response rates following rituximab in SLE [1]. Enhanced B-cell depletion with the type II anti-CD20 mAb obinutuzumab resulted in increased renal responses in proliferative lupus nephritis (LN) in the NOBILITY trial (NCT02550652) and will be further evaluated in the Phase 3 REGENCY trial (NCT04221477).Objectives:To measure peripheral B-cells, B-cell subsets (naïve, memory and plasmablast) and B-cell activating factor (BAFF) levels and to assess associations between B-cell depletion and renal response in LN patients in a clinical trial of obinutuzumab.Methods:126 patients with active Class III/IV LN were randomized to obinutuzumab or placebo infusions in combination with mycophenolate and glucocorticoids. Peripheral B-cells were measured using a HSFC method with a lower limit of quantitation of 0.441 cells/μL. Serum levels of BAFF were evaluated using ELISA. Sustained depletion was defined by total B-cells below the limit of detection at both weeks 24 and 52. Renal response definitions from Phase 2 NOBILITY and Phase 3 REGENCY trials were used.Results:Obinutuzumab resulted in rapid and complete depletion of total B-cells, memory and naïve B-cells, and plasmablasts from peripheral blood, with 88% of obinutuzumab patients depleted to &lt; 0.441 total B-cells/μL at week 2 (Figure). Mean serum BAFF increased from 4,585 pg/mL at baseline to 14,601 pg/mL at week 52 in the obinutuzumab group. Sustained B-cell depletion was achieved in 32/52 (62%) of patients with complete data and was associated with higher renal response rate at week 76 (Table), although patients who achieved sustained depletion also had lower baseline proteinuria and serum creatinine.Conclusion:Obinutuzumab, a type II anti-CD20 mAb, mediated rapid, complete and sustained depletion of peripheral B-cells and plasmablasts and large increases in serum BAFF. Similar to previous reports, sustained B-cell depletion was associated with increased renal response though there may be confounding factors. REGENCY is being conducted to further evaluate the therapeutic hypothesis with obinutuzumab in LN.

Clinical and immunological differences between MOG associated disease and anti AQP4 antibody-positive neuromyelitis optica spectrum disorders: Blood–brain barrier breakdown and peripheral plasmablasts

BackgroundPatients with anti-aquaporin-4 (AQP4) water channel antibody-positive neuromyelitis optica spectrum disorders (AQP4-NMOSD) and myelin oligodendrocyte glycoprotein (MOG) associated disease (MOGAD) often present with similar clinical symptoms, and some cases are hard to differentiate at the time of onset. In this study, we compared the clinical characteristics, cerebrospinal fluid (CSF) analysis parameters, and peripheral T/B lymphocyte subsets during the active and chronic phases in AQP4-NMOSD and MOGAD. MethodsA total of 17 MOGAD cases and 24 AQP4-NMOSD cases were studied. The clinical characteristics in both groups were summarized, including disease duration, total number of attacks, lesions, prevention of relapse during remission, and CSF analysis results during the active phase. T/B lymphocyte subsets were further investigated in the active and chronic phases. ResultsIn the comparative study on clinical symptoms, a large proportion of optic neuritis was unilateral in MOGAD. In the comparative study on CSF analysis, protein level was significantly lower in MOGAD compared with AQP4-NMOSD (p = 0.006); myelin basic protein was significantly lower in MOGAD compared with AQP4-NMOSD (p = 0.04); albumin quotient was significantly lower in MOGAD compared with AQP4-NMOSD (p = 0.02); and IgG Quotient was significantly lower in MOGAD compared with AQP4-NMOSD (p = 0.05). In the analysis of T/B lymphocyte subsets, plasmablasts of the B cell subset in the active phase were significantly lower in MOGAD (2.1 ± 2.4) compared to AQP4-NMOSD (7.8 ± 7.2) (p < 0.05). In the chronic phase, transitional B cells were significantly higher in MOGAD (2.1 ± 1.8) compared to AQP4-NMOSD (0.6 ± 0.4) (p < 0.01). ConclusionClinical characteristics of MOGAD were similar to those of AQP4-NMOSD, but increased blood brain barrier permeability was suggested to be less severe in MOGAD compared to AQP4-NMOSD from CSF analysis. Furthermore, the pathogenesis of the two diseases was clearly distinct as plasmablasts in the active phase were not elevated in MOGAD, but were increased in AQP4-NMOSD.

Association between peripheral plasmablasts and postoperative exacerbation in patients with thymoma‐associated myasthenia gravis

AbstractObjectiveThymoma‐associated myasthenia gravis (TAMG) is a type of myasthenia gravis (MG) that has a higher likelihood of worsening in comparison with non‐TAMG, and MG symptoms often worsen after thymectomy. We measured peripheral T/B lymphocyte subsets before and after thymectomy in TAMG patients, and investigated the relationships with the occurrence of postoperative exacerbation of MG symptoms.MethodsParticipants were 10 TAMG patients, 40 healthy controls and five other surgical control patients. Peripheral lymphocyte subsets were measured using FACS‐Canto flow cytometry before, and 1, 7, and 28 days after thymectomy in the TAMG, and before and 7 days after thymectomy in the other surgical control patients.ResultsNatural killer cells modestly decreased, and plasmablasts increased significantly (P = 0.005) after 7 days postoperatively in comparison with preoperative values. Conversely, no obvious changes were observed in plasmablasts or natural killer cells in T/B lymphocyte subsets. From evaluation of postoperative clinical course and plasmablasts, exacerbation of MG symptoms was observed between 2 and 3 weeks after thymectomy in five of 10 patients (50%) of TAMG. In these five patients, plasmablasts markedly increased after thymectomy in three patients, among whom two patients had originally shown high plasmablast levels before thymectomy. There were also two patients who exacerbated without changes in plasmablast levels.ConclusionsIn patients with exacerbation of neurological symptoms after thymectomy for TAMG, the plasmablast levels were originally high and/or became high postoperatively. Thus, elevated plasmablast levels could potentially be predictive of postoperative exacerbation of MG.

Suppressed plasmablast responses in febrile infants, including children with Kawasaki disease.

Abstract Kawasaki disease (KD), the leading cause of acquired heart disease in children, primarily affects infants and toddlers. Future studies on KD immune responses are hampered by a limited understanding of normal immune responses in these ages. Plasmablasts are a transitional form of B cells that lead to long-term plasma cells, and their levels rise in the peripheral blood after exposure to a foreign antigen. In adults, these responses are both temporally and functionally well characterized. To date, there have been few studies on plasmablasts in the predominant age range of KD. This study is focused on characterizing the normal plasmablast responses in the predominant age range of KD presentation. Children presenting to an urban pediatric emergency room who had fever and symptoms overlapping those of KD, were recruited. Peripheral blood mononuclear cells were isolated and evaluated utilizing flow cytometry with specific B cell markers from 18 KD subjects and 69 febrile controls. Plasmablast numbers and timing of response are similar between infectious disease control and KD subjects. In both groups, infants have diminished plasmablast responses compared to older children. Using deep sequencing, immunoglobluin variable gene usage and sequences will be compared between infants and older children. In this single-time point survey, we demonstrate that infants have a blunted peripheral plasmablast response. Overall, similar plasmablast responses in KD and controls support an infectious disease relationship to KD. Future time-course studies of plasmablasts in infants are warranted as this phenomenon may contribute to observed poor serum immune responses and maintenance of such responses in this age group.

Suppressed plasmablast responses in febrile infants, including children with Kawasaki disease

BackgroundKawasaki disease (KD), the leading cause of acquired heart disease in children, primarily affects infants and toddlers. Investigations on immune responses during KD are hampered by a limited understanding of normal immune responses in these ages. It’s well known that Infants have poorer vaccine responses and difficulty with maintaining prolonged serum immunity, but there are few studies on human infants detailing immune deficiencies. Limited studies propose an inability to maintain life-long bone marrow plasma cells. Plasmablasts are a transitional cell form of B cells that lead to long-term Plasma cells. Plasmablasts levels rise in the peripheral blood after exposure to a foreign antigen. In adult studies, these responses are both temporally and functionally well characterized. To date, there have been few studies on plasmablasts in the predominant age range of KD.MethodsChildren presenting to an urban pediatric emergency room undergoing laboratory evaluation, who had concern of KD or had fever and symptoms overlapping those of KD, were recruited. Peripheral blood mononuclear cells were isolated and evaluated utilizing flow cytometry with specific B cell markers from 18 KD subjects and 69 febrile controls.ResultsPlasmablast numbers and temporal formation are similar between infectious disease controls and KD subjects. In both groups, infants have diminished plasmablast responses compared to older children.ConclusionIn this single-time point survey, infants have a blunted peripheral plasmablast response. Overall, similar plasmablast responses in KD and controls support an infectious disease relationship to KD. Future time-course studies of plasmablasts in infants are warranted as this phenomenon may contribute to observed immune responses in this age group.

Peripheral VH4+plasmablasts demonstrate autoreactive B cell expansion toward brain antigens in early multiple sclerosis patients.

Plasmablasts are a highly differentiated, antibody secreting B cell subset whose prevalence correlates with disease activity in Multiple Sclerosis (MS). For most patients experiencing partial transverse myelitis (PTM), plasmablasts are elevated in the blood at the first clinical presentation of disease (known as a clinically isolated syndrome or CIS). In this study we found that many of these peripheral plasmablasts are autoreactive and recognize primarily gray matter targets in brain tissue. These plasmablasts express antibodies that over-utilize immunoglobulin heavy chain V-region subgroup 4 (VH4) genes, and the highly mutated VH4+ plasmablast antibodies recognize intracellular antigens of neurons and astrocytes. Most of the autoreactive, highly mutated VH4+ plasmablast antibodies recognize only a portion of cortical neurons, indicating that the response may be specific to neuronal subgroups or layers. Furthermore, CIS-PTM patients with this plasmablast response also exhibit modest reactivity toward neuroantigens in the plasma IgG antibody pool. Taken together, these data indicate that expanded VH4+ peripheral plasmablasts in early MS patients recognize brain gray matter antigens. Peripheral plasmablasts may be participating in the autoimmune response associated with MS, and provide an interesting avenue for investigating the expansion of autoreactive B cells at the time of the first documented clinical event.

The auto-reactive profile of B cells from early MS patients

Abstract My lab’s recent work has led to the discovery that antibodies expressed by memory B cells in the cerebrospinal fluid (CSF) of patients with Multiple Sclerosis recognize neurons and astrocytes in the brain. Genetic analysis further revealed that only certain heavy chain genes accommodate binding to these brain targets, and that memory B cells from control cohorts rarely express these antibody genes. In fact, in a recent clinical trial, we demonstrated that the prevalence of these antibody genes expressed by memory B cells in the CSF support identification of patients who either have MS or will develop MS in the future with 89–94% accuracy. This Antibody Gene Signature (AGS) is a novel biomarker that heralds the advent of antibody genetics as a means to support categorization of patients early in their disease course. Our goal for this project was to determine whether antibodies expressed by activated memory B cells that have initiated antibody production (called “plasmablasts”) in the peripheral blood of MS patients also recognize neurons and astrocytes. We cloned and expressed more than 50 antibodies expressed by peripheral plasmablasts of early MS patients and control cohorts. We found that antibodies expressed by peripheral plasmablasts from patients who develop MS bind to neurons and astrocytes in the brain. Those plasmablasts that did not express antibody heavy chains associated with the AGS did not bind to brain targets. Interestingly, only those patients in our cohort who have an elevated frequency of peripheral plasmablasts continue to accumulate brain MRI activity. It is possible that brain-reactive peripheral plasmablasts contribute to the brain damage associated with MS.

Ongoing activation of autoantigen-specific B cells in primary biliary cirrhosis.

The serologic hallmark of primary biliary cirrhosis (PBC), the antimitochondrial response to the E2 component of the pyruvate dehydrogenase complex (PDC-E2), has unique features, including continuous high titers of immunoglobulin M (IgM) and IgG reactivity throughout all stages of disease, capable not only of target enzyme inhibition, but also crossreactive with chemical xenobiotics that share molecular homology with the inner lipoyl domain of PDC-E2; such chemicals have been proposed as potential etiological agents. We used flow cytometry and enzyme-linked immunospot assay (ELISPOT) to examine B-cell subsets in 59 subjects, including 28 with PBC, 13 with primary sclerosing cholangitis (PSC), and 18 healthy controls. Strikingly, in PBC, although there were no significant differences in B-cell phenotype subpopulations, 10% of the total IgG and IgA plasmablast population and 23% of the IgM plasmablast population were uniquely reactive with PDC-E2, detected in the CXCR7+ CCR10low plasmablast population. In contrast, plasmablast reactivity to a control antigen, tetanus toxoid, was minimal and similar in all groups. Additionally, we isolated plasmablast-derived polyclonal antibodies and compared reactivity with plasma-derived antibodies and noted a distinct noncirculating tissue source of xenobiotic crossreacting antibodies. The high levels of autoantigen specific peripheral plasmablasts indicate recent activation of naive or memory B cells and a continuous and robust activation. The presence of CXCR7+ CCR10low PDC-E2-specific ASCs suggests a mechanistic basis for the migration of circulating antigen specific plasmablasts to the mucosal epithelial ligands CXCL12 and CCL28. Our findings suggest a sustained rigorous B-cell response in PBC, likely activated and perpetuated by cognate autoantigen.

Circulating B-lymphocytes as potential biomarkers of tuberculosis infection activity.

Accurate biomarkers of Mycobacterium tuberculosis infection activity would significantly improve early diagnosis, treatment and management of M. tuberculosis infection. We hypothesised that circulating B-lymphocytes may be useful biomarkers of tuberculosis (TB) infection status in highly TB-endemic settings. Ex-vivo and in-vitro mycobacteria-specific B-cell ELISPOT assays were used to examine the plasmablast (PB) and memory B-cell (MBC) responses in the peripheral blood of adult, healthy, community controls (n = 151) and of active TB patients (n = 48) living in Uganda. Frequencies of mycobacteria-specific PBs were markedly higher in active TB patients compared to healthy controls, and, conversely, MBCs were markedly higher in the healthy controls compared to active TB patients. In addition, the community controls with evidence of latent TB infection had higher peripheral blood PB and MBC responses than those without evidence of TB infection. These data demonstrate that peripheral blood B-cell responses are differentially modulated during latent and active M. tuberculosis infection, and suggest that the PB to MBC ratio may be a useful biomarker of TB infection activity.

Distinct Cross-reactive B-Cell Responses to Live Attenuated and Inactivated Influenza Vaccines

The immunological bases for the efficacies of the 2 currently licensed influenza vaccines, live attenuated influenza vaccine (LAIV) and inactivated influenza vaccine (IIV), are not fully understood. The goal of this study was to identify specific B-cell responses correlated with the known efficacies of these 2 vaccines. We compared the B-cell and antibody responses after immunization with 2010/2011 IIV or LAIV in young adults, focusing on peripheral plasmablasts 6-8 days after vaccination. The quantities of vaccine-specific plasmablasts and plasmablast-derived polyclonal antibodies (PPAbs) in IIV recipients were significantly higher than those in LAIV recipients. No significant difference was detected in the avidity of vaccine-specific PPAbs between the 2 vaccine groups. Proportionally, LAIV induced a greater vaccine-specific immunoglobulin A plasmablast response, as well as a greater plasmablast response to the conserved influenza nuclear protein, than IIV. The cross-reactive plasmablast response to heterovariant strains, as indicated by the relative levels of cross-reactive plasmablasts and the cross-reactive PPAb binding reactivity, was also greater in the LAIV group. Distinct quantitative and qualitative patterns of plasmablast responses were induced by LAIV and IIV in young adults; a proportionally greater cross-reactive response was induced by LAIV.

SAT0178 IGA+ plasmablasts contribute to the expansion of peripheral plasmablasts in SLE

BackgroundActive systemic lupus erythematosus (SLE) is associated with increased levels of peripheral blood antibody-secreting plasmablasts which correlate with autoantibody production and disease activity. Plasmablasts which circulate in healthy individuals at...