Increased viral variants in children and young adults with impaired humoral immunity and persistent SARS-CoV-2 infection: A consecutive case series.
2021; 67: 103355
Shared B cell memory to coronaviruses and other pathogens varies in human age groups and tissues.
Science (New York, N.Y.)
BACKGROUND: There is increasing concern that persistent infection of SARS-CoV-2 within immunocompromised hosts could serve as a reservoir for mutation accumulation and subsequent emergence of novel strains with the potential to evade immune responses.METHODS: We describe three patients with acute lymphoblastic leukemia who were persistently positive for SARS-CoV-2 by real-time polymerase chain reaction. Viral viability from longitudinally-collected specimens was assessed. Whole-genome sequencing and serological studies were performed to measure viral evolution and evidence of immune escape.FINDINGS: We found compelling evidence of ongoing replication and infectivity for up to 162 days from initial positive by subgenomic RNA, single-stranded RNA, and viral culture analysis. Our results reveal a broad spectrum of infectivity, host immune responses, and accumulation of mutations, some with the potential for immune escape.INTERPRETATION: Our results highlight the potential need to reassess infection control precautions in the management and care of immunocompromised patients. Routine surveillance of mutations and evaluation of their potential impact on viral transmission and immune escape should be considered.
View details for DOI 10.1016/j.ebiom.2021.103355
View details for PubMedID 33915337
Plasma as an alternative COVID-19 diagnostic specimen in a hospitalized patient negative for SARS-CoV-2 by nasopharyngeal swab.
Diagnostic microbiology and infectious disease
2021; 100 (3): 115365
Vaccination and infection promote the formation, tissue distribution, and clonal evolution of B cells, which encode humoral immune memory. We evaluated convergent antigen-specific antibody genes of similar sequences shared between individuals in pediatric and adult blood, and deceased organ donor tissues. B cell memory varied for different pathogens. Polysaccharide antigen-specific clones were not exclusive to the spleen. Adults had higher clone frequencies and greater class-switching in lymphoid tissues than blood, while pediatric blood had abundant class-switched convergent clones. Consistent with reported serology, pre-pandemic children had class-switched convergent clones to SARS-CoV-2 with weak cross-reactivity to other coronaviruses, while adult blood or tissues showed few such clones. The results highlight the prominence of early childhood B cell clonal expansions and cross-reactivity for future responses to novel pathogens.
View details for DOI 10.1126/science.abf6648
View details for PubMedID 33846272
Efficient Identification of High-Titer Anti-Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antibody Plasma Samples by Pooling Method.
Archives of pathology & laboratory medicine
We present the case of an inpatient with pneumonia and repeatedly negative nasopharyngeal SARS-CoV-2 testing. In such challenging cases, alternative diagnostic options include lower respiratory tract and plasma SARS-CoV-2 RNA testing, of which the latter may be particularly useful where bronchoscopy is deferred due to clinical factors or transmission risk.
View details for DOI 10.1016/j.diagmicrobio.2021.115365
View details for PubMedID 33865070
mRNA vaccination compared to infection elicits an IgG-predominant response with greater SARS-CoV-2 specificity and similar decrease in variant spike recognition.
medRxiv : the preprint server for health sciences
The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has elicited a surge in demand for serological testing to identify previously infected individuals. In particular, antibody testing is crucial in identifying COVID-19 convalescent plasma (CCP), which has been approved by the Food and Drug Administration (FDA) under the Emergency Use Authorization (EUA) for use as passive immune therapy for hospitalized patients infected with COVID-19. Currently, high-titer CCP can be qualified by Ortho's Vitros COVID-19 IgG antibody test (VG).To explore the use of an efficient testing method to identify high-titer CCP for use in treating COVID-19 infected patients and track COVID-19 positivity over time.We evaluated an ELISA-based method that detects antibodies specific to the SARSCoV-2 receptor binding domain (RBD) with individual and pooled plasma samples and compared its performance against VG. Using the pooled RBD-ELISA (P-RE) method, we also screened over 10,000 longitudinal healthy blood donor samples to assess seroprevalence.P-RE demonstrates 100% sensitivity in detecting FDA-defined high-titer samples when compared to VG. Overall sensitivity of P-RE when compared to VG and our individual sample RBD-ELISA (I-RE) were 83% and 56%, respectively. When screening 10,218 healthy blood donor samples by P-RE, we found the seroprevalence correlated with the local infection rates with a correlation coefficient of 0.21 (P< .001).Pooling plasma samples can be used to efficiently screen large populations for individuals with high-titer anti-RBD antibodies, important for CCP identification.
View details for DOI 10.5858/arpa.2021-0215-SA
View details for PubMedID 34101801
Persistent SARS-CoV-2 infection and increasing viral variants in children and young adults with impaired humoral immunity.
medRxiv : the preprint server for health sciences
During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, new vaccine strategies including lipid nanoparticle delivery of antigen encoding RNA have been deployed globally. The BioNTech/Pfizer mRNA vaccine BNT162b2 encoding SARS-CoV-2 spike protein shows 95% efficacy in preventing disease, but it is unclear how the antibody responses to vaccination differ from those generated by infection. Here we compare the magnitude and breadth of antibodies targeting SARS-CoV-2, SARS-CoV-2 variants of concern, and endemic coronaviruses, in vaccinees and infected patients. We find that vaccination differs from infection in the dominance of IgG over IgM and IgA responses, with IgG reaching levels similar to those of severely ill COVID-19 patients and shows decreased breadth of the antibody response targeting endemic coronaviruses. Viral variants of concern from B.1.1.7 to P.1 to B.1.351 form a remarkably consistent hierarchy of progressively decreasing antibody recognition by both vaccinees and infected patients exposed to Wuhan-Hu-1 antigens.
View details for DOI 10.1101/2021.04.05.21254952
View details for PubMedID 33851181
View details for PubMedCentralID PMC8043478
Increased antiviral response in circulating lymphocytes from hypogammaglobulinemia patients.
There is increasing concern that persistent infection of SARS-CoV-2 within immunocompromised hosts could serve as a reservoir for mutation accumulation and subsequent emergence of novel strains with the potential to evade immune responses.We describe three patients with acute lymphoblastic leukemia who were persistently positive for SARS-CoV-2 by real-time polymerase chain reaction. Viral viability from longitudinally-collected specimens was assessed. Whole-genome sequencing and serological studies were performed to measure viral evolution and evidence of immune escape.We found compelling evidence of ongoing replication and infectivity for up to 162 days from initial positive by subgenomic RNA, single-stranded RNA, and viral culture analysis. Our results reveal a broad spectrum of infectivity, host immune responses, and accumulation of mutations, some with the potential for immune escape.Our results highlight the need to reassess infection control precautions in the management and care of immunocompromised patients. Routine surveillance of mutations and evaluation of their potential impact on viral transmission and immune escape should be considered.The work was partially funded by The Saban Research Institute at Children's Hospital Los Angeles intramural support for COVID-19 Directed Research (X.G. and J.D.B.), the Johns Hopkins Center of Excellence in Influenza Research and Surveillance HHSN272201400007C (A.P.), NIH/NIAID R01AI127877 (S.D.B.), NIH/NIAID R01AI130398 (S.D.B.), NIH 1U54CA260517 (S.D.B.), an endowment to S.D.B. from the Crown Family Foundation, an Early Postdoc.Mobility Fellowship Stipend to O.F.W. from the Swiss National Science Foundation (SNSF), and a Coulter COVID-19 Rapid Response Award to S.D.B. L.G. is a SHARE Research Fellow in Pediatric Hematology-Oncology.
View details for DOI 10.1101/2021.02.27.21252099
View details for PubMedID 33688673
View details for PubMedCentralID PMC7941650
A novel proangiogenic B cell subset is increased in cancer and chronic inflammation
2020; 6 (20): eaaz3559
BACKGROUND: B cells play a crucial role during rhinovirus (RV) infections by production of virus-neutralizing antibodies. A main feature of common variable immunodeficiency (CVID) is hypogammaglobulinemia (HG). HG patients have severely reduced levels of antibody-producing B cells and suffer from prolonged virus infections. Here, we addressed whether antiviral response of peripheral blood lymphocytes differs between HG patients and healthy individuals during natural RV infection.METHODS: Using fluorescence-activated cell sorting, B cell subsets were analyzed. Simultaneously, CD19+ B cells, CD14+ monocytes and CD3+ T cells were sorted from frozen peripheral blood mononuclear cells from 11 RV-infected hypogammaglobulinemia patients, 7 RV-infected control subjects and 14 non-infected control subjects. Real-time PCR was used to study expression of antiviral genes. A pan-RV PCR was used to detect RV genome in all samples.RESULTS: In HG patients, total B cell numbers, as well as IgA+ and IgG+ switched memory B cells were reduced while naive B cells and T cells were increased. STAT1 expression was increased in HG patients compared to controls in all lymphocyte subsets analyzed. The expression of antiviral genes IFITM1 and MX1 correlated with STAT1 expression in B cells and monocytes. RV RNA was found in 88.9 % of monocytes from infected HG patients, 85.7 % of monocytes from infected controls and 7.1 % of monocytes from uninfected controls.CONCLUSIONS: We demonstrate an increased antiviral response in B cells and monocytes in HG patients and their correlation with STAT1 expression. Monocytes of infected HG patients and infected non-HG controls carry RV RNA.
View details for DOI 10.1111/all.14445
View details for PubMedID 32533713
Defining the features and duration of antibody responses to SARS-CoV-2 infection associated with disease severity and outcome.
2020; 5 (54)
B cells contribute to immune responses through the production of immunoglobulins, antigen presentation, and cytokine production. Several B cell subsets with distinct functions and polarized cytokine profiles have been reported. In this study, we used transcriptomics analysis of immortalized B cell clones to identify an IgG4+ B cell subset with a unique function. These B cells are characterized by simultaneous expression of proangiogenic cytokines including VEGF, CYR61, ADM, FGF2, PDGFA, and MDK. Consequently, supernatants from these clones efficiently promote endothelial cell tube formation. We identified CD49b and CD73 as surface markers identifying proangiogenic B cells. Circulating CD49b+CD73+ B cells showed significantly increased frequency in patients with melanoma and eosinophilic esophagitis (EoE), two diseases associated with angiogenesis. In addition, tissue-infiltrating IgG4+CD49b+CD73+ B cells expressing proangiogenic cytokines were detected in patients with EoE and melanoma. Our results demonstrate a previously unidentified proangiogenic B cell subset characterized by expression of CD49b, CD73, and proangiogenic cytokines.
View details for DOI 10.1126/sciadv.aaz3559
View details for Web of Science ID 000533573300021
View details for PubMedID 32426497
View details for PubMedCentralID PMC7220305
Reinfection with SARS-CoV-2 and Failure of Humoral Immunity: a case report.
medRxiv : the preprint server for health sciences
SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals' SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection.
View details for DOI 10.1126/sciimmunol.abe0240
View details for PubMedID 33288645
SARS-CoV-2 RNAemia in a Healthy Blood Donor 40 Days After Respiratory Illness Resolution.
Annals of internal medicine
Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).
European journal of immunology
2019; 49 (10): 1457?1973
Recovery from COVID-19 is associated with production of anti-SARS-CoV-2 antibodies, but it is uncertain whether these confer immunity. We describe viral RNA shedding duration in hospitalized patients and identify patients with recurrent shedding. We sequenced viruses from two distinct episodes of symptomatic COVID-19 separated by 144 days in a single patient, to conclusively describe reinfection with a new strain harboring the spike variant D614G. With antibody and B cell analytics, we show correlates of adaptive immunity, including a differential response to D614G. Finally, we discuss implications for vaccine programs and begin to define benchmarks for protection against reinfection from SARS-CoV-2.
View details for DOI 10.1101/2020.09.22.20192443
View details for PubMedID 32995830
View details for PubMedCentralID PMC7523175
Comparison of regulatory B cells in asthma and allergic rhinitis.
2019; 74 (4): 815?18
Impaired memory B-cell development and antibody maturation with a skewing toward IgE in patients with STAT3 hyper-IgE syndrome.
These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
View details for DOI 10.1002/eji.201970107
View details for PubMedID 31633216
Role of Der p 1-specific B cells in immune tolerance during 2 years of house dust mite-specific immunotherapy.
The Journal of allergy and clinical immunology
2019; 143 (3): 1077?86.e10
Signal transducer and activator of transcription 3 hyper-IgE syndrome (STAT3-HIES) is caused by heterozygous mutations in the STAT3 gene and is associated with eczema, elevated serum IgE, and recurrent infections resembling severe atopic dermatitis, while clinically relevant specific IgE is almost absent.To investigate the impact of STAT3 signaling on B-cell responses, we assessed lymph node and bone marrow, blood B and plasma cell subsets, somatic hypermutations in Ig genes, and in vitro proliferation and antibody production in STAT3-HIES patients and healthy controls.Lymph nodes of STAT3-HIES patients showed normal germinal center architecture and CD138+ plasma cells residing in the paracortex, which expressed IgE, IgG, and IgM but not IgA. IgE+ plasma cells were abundantly present in STAT3-HIES bone marrow. Proliferation of naive B cells upon stimulation with CD40L and IL-4 was similar in patients and controls, while patient cells showed reduced responses to IL-21. IgE, IgG1, IgG3 and IgA1 transcripts showed reduced somatic hypermutations. Peripheral blood IgE+ memory B-cell frequencies were increased in STAT3-HIES, while other memory B-cell frequencies except for IgG4+ cells were decreased.Despite impaired STAT3 signaling, STAT3-HIES patients can mount in vivo T-cell-dependent B-cell responses, while circulating memory B cells, except for those expressing IgG4 and IgE, were reduced. Reduced molecular maturation demonstrated the critical need of STAT3 signaling for optimal affinity maturation and B-cell differentiation, supporting the need for immunoglobulin substitution therapy and explaining the high IgE serum level in the majority with absent allergic symptoms.
View details for DOI 10.1111/all.13969
View details for PubMedID 31269238
Exposure to nonmicrobial N-glycolylneuraminic acid protects farmers' children against airway inflammation and colitis.
The Journal of allergy and clinical immunology
2018; 141 (1): 382?90.e7
Long-term follow-up of allergen-specific B cells in terms of immunoglobulin isotype expression, plasmablast differentiation, and regulatory B (Breg) cell development during allergen-specific immunotherapy (AIT) has not been reported.Allergen-specific B-cell responses during 2 years of house dust mite AIT were compared between responder and nonresponder patients.B cells specific for Der p 1 were detected by using the fluorochrome-labeled allergen method. The frequency of IgA-, IgG1- and IgG4-switched Der p 1-specific B cells, plasmablasts, and IL-10- and IL-1 receptor antagonist (IL-1RA)-producing Breg cells were investigated and correlated to clinical response to AIT.Sixteen of 25 patients completed the 2-year study. Eleven responder patients showed a successful response to AIT, as measured by a decrease in symptom-medication scores from 13.23 ± 0.28 to 2.45 ± 0.24 (P = .001) and a decrease in skin prick test reactivity to house dust mite from 7.0 ± 1.3 to 2.7 ± 0.5 mm (P = .001). IgG4+ and IgA+ Der p 1-specific B cells showed a significant increase after AIT, with a significantly greater frequency in responders compared with nonresponders in the IgG4+ but not the IgA+ fraction. The frequency of plasmablasts and IL-10- and/or IL-1RA-producing Breg cells was greater among responders compared with nonresponders after 2 years. The increased frequency of Der p 1-specific IgG4+ B cells, plasmablasts, and IL-10+ and dual-positive IL-10+IL-1RA+ Breg cells significantly correlated with improved clinical symptoms over the course of AIT.Allergen-specific B cells in patients responding to AIT are characterized by increased numbers of IgA- and IgG4-expressing Der p 1-specific B cells, plasmablasts, and IL-10+ and/or IL-1RA+ Breg cells.
View details for DOI 10.1016/j.jaci.2018.10.061
View details for PubMedID 30529452
Two Distinct Pathways in Mice Generate Antinuclear Antigen-Reactive B Cell Repertoires.
Frontiers in immunology
2018; 9: 16
Childhood exposure to a farm environment has been shown to protect against the development of inflammatory diseases, such as allergy, asthma, and inflammatory bowel disease.We sought to investigate whether both exposure to microbes and exposure to structures of nonmicrobial origin, such as the sialic acid N-glycolylneuraminic acid (Neu5Gc), might play a significant role.Exposure to Neu5Gc was evaluated by quantifying anti-Neu5Gc antibody levels in sera of children enrolled in 2 farm studies: the Prevention of Allergy Risk factors for Sensitization in Children Related to Farming and Anthroposophic Lifestyle (PARSIFAL) study (n = 299) and the Protection Against Allergy Study in Rural Environments (PASTURE) birth cohort (cord blood [n = 836], 1 year [n = 734], 4.5 years [n = 700], and 6 years [n = 728]), and we associated them with asthma and wheeze. The effect of Neu5Gc was examined in murine airway inflammation and colitis models, and the role of Neu5Gc in regulating immune activation was assessed based on helper T-cell and regulatory T-cell activation in mice.In children anti-Neu5Gc IgG levels correlated positively with living on a farm and increased peripheral blood forkhead box protein 3 expression and correlated inversely with wheezing and asthma in nonatopic subjects. Exposure to Neu5Gc in mice resulted in reduced airway hyperresponsiveness and inflammatory cell recruitment to the lung. Furthermore, Neu5Gc administration to mice reduced the severity of a colitis model. Mechanistically, we found that Neu5Gc exposure reduced IL-17+ T-cell numbers and supported differentiation of regulatory T cells.In addition to microbial exposure, increased exposure to non-microbial-derived Neu5Gc might contribute to the protective effects associated with the farm environment.
View details for DOI 10.1016/j.jaci.2017.04.051
View details for PubMedID 28629745
Human rhinoviruses enter and induce proliferation of B lymphocytes.
2017; 72 (2): 232?43
The escape of anti-self B cells from tolerance mechanisms like clonal deletion, receptor editing, and anergy results in the production of autoantibodies, which is a hallmark of many autoimmune disorders. In this study, we demonstrate that both germline sequences and somatic mutations contribute to autospecificity of B cell clones. For this issue, we investigated the development of antinuclear autoantibodies (ANAs) and their repertoire in two different mouse models. First, in aging mice that were shown to gain several autoimmune features over time including ANAs. Second, in mice undergoing a chronic graft-versus-host disease (GVHD), thereby developing systemic lupus erythematosus-like symptoms. Detailed repertoire analysis revealed that somatic hypermutations (SHM) were present in all Vh and practically all Vl regions of ANAs generated in these two models. The ANA B cell repertoire in aging mice was restricted, dominated by clonally related Vh1-26/Vk4-74 antibodies. In the collection of GVHD-derived ANAs, the repertoire was less restricted, but the usage of the Vh1-26/Vk4-74 combination was still apparent. Germline conversion showed that the SHM in the 4-74 light chain are deterministic for autoreactivity. Detailed analysis revealed that antinuclear reactivity of these antibodies could be induced by a single amino acid substitution in the CDR1 of the Vk4-74. In both aging B6 and young GVHD mice, conversion of the somatic mutations in the Vh and Vl regions of non Vh1-26/Vk4-74 using antibodies showed that B cells with a germline-encoded V gene could also contribute to the ANA-reactive B cell repertoire. These findings indicate that two distinct pathways generate ANA-producing B cells in both model systems. In one pathway, they are generated by Vh1-26/Vk4-74 expressing B cells in the course of immune responses to an antigen that is neither a nuclear antigen nor any other self-antigen. In the other pathway, ANA-producing B cells are derived from progenitors in the bone marrow that express B cell receptors (BCRs), which bind to nuclear antigens and that escape tolerance induction, possibly as a result of crosslinking of their BCRs by multivalent determinants of nuclear antigens.
View details for DOI 10.3389/fimmu.2018.00016
View details for PubMedID 29403498
View details for PubMedCentralID PMC5786517
Novel mechanisms in immune tolerance to allergens during natural allergen exposure and allergen-specific immunotherapy.
Current opinion in immunology
2017; 48: 74?81
Human rhinoviruses (HRVs) are one of the main causes of virus-induced asthma exacerbations. Infiltration of B lymphocytes into the subepithelial tissue of the lungs has been demonstrated during rhinovirus infection in allergic individuals. However, the mechanisms through which HRVs modulate the immune responses of monocytes and lymphocytes are not yet well described.To study the dynamics of virus uptake by monocytes and lymphocytes, and the ability of HRVs to induce the activation of in vitro-cultured human peripheral blood mononuclear cells.Flow cytometry was used for the enumeration and characterization of lymphocytes. Proliferation was estimated using 3 H-thymidine or CFSE labeling and ICAM-1 blocking. We used bead-based multiplex assays and quantitative PCR for cytokine quantification. HRV accumulation and replication inside the B lymphocytes was detected by a combination of in situ hybridization (ISH), immunofluorescence, and PCR for positive-strand and negative-strand viral RNA. Cell images were acquired with imaging flow cytometry.By means of imaging flow cytometry, we demonstrate a strong and quick binding of HRV types 16 and 1B to monocytes, and slower interaction of these HRVs with CD4+ T cells, CD8+ T cells, and CD19+ B cells. Importantly, we show that HRVs induce the proliferation of B cells, while the addition of anti-ICAM-1 antibody partially reduces this proliferation for HRV16. We prove with ISH that HRVs can enter B cells, form their viral replication centers, and the newly formed virions are able to infect HeLa cells. In addition, we demonstrate that similar to epithelial cells, HRVs induce the production of pro-inflammatory cytokines in PBMCs.Our results demonstrate for the first time that HRVs enter and form viral replication centers in B lymphocytes and induce the proliferation of B cells. Newly formed virions have the capacity to infect other cells (HeLa). These findings indicate that the regulation of human rhinovirus-induced B-cell responses could be a novel approach to develop therapeutics to treat the virus-induced exacerbation of asthma.
View details for DOI 10.1111/all.12931
View details for PubMedID 27170552
High-dose bee venom exposure induces similar tolerogenic B-cell responses in allergic patients and healthy beekeepers.
2017; 72 (3): 407?15
Allergen-specific immunotherapy (AIT) has been used for more than 100 years as a clinical tolerance-inducing and immune tolerance-inducing therapy for allergic diseases and represents a potentially curative method of treatment. AIT functions through multiple mechanisms including early desensitization of basophils and mast cells, regulating T-cell and B-cell responses, changing antibody isotypes, and decreasing activation, mediator release and affected tissue migration of eosinophils, basophils, and mast cells. Similar molecular and cellular mechanisms have been observed in subcutaneous AIT, sublingual AIT and peptide immunotherapy as well as natural tolerance to high doses of allergen exposure in beekeepers and cat owners.
View details for DOI 10.1016/j.coi.2017.08.012
View details for PubMedID 28888176
A novel, dual cytokine-secretion assay for the purification of human Th22 cells that do not co-produce IL-17A.
2016; 71 (1): 47?57
The involvement of B cells in allergen tolerance induction remains largely unexplored. This study investigates the role of B cells in this process, by comparing B-cell responses in allergic patients before and during allergen immunotherapy (AIT) and naturally exposed healthy beekeepers before and during the beekeeping season.Circulating B cells were characterized by flow cytometry. Phospholipase A2 (PLA)-specific B cells were identified using dual-color staining with fluorescently labeled PLA. Expression of regulatory B-cell-associated surface markers, interleukin-10, chemokine receptors, and immunoglobulin heavy-chain isotypes, was measured. Specific and total IgG1, IgG4, IgA, and IgE from plasma as well as culture supernatants of PLA-specific cells were measured by ELISA.Strikingly, similar responses were observed in allergic patients and beekeepers after venom exposure. Both groups showed increased frequencies of plasmablasts, PLA-specific memory B cells, and IL-10-secreting CD73- CD25+ CD71+ BR 1 cells. Phospholipase A2-specific IgG4-switched memory B cells expanded after bee venom exposure. Interestingly, PLA-specific B cells showed increased CCR5 expression after high-dose allergen exposure while CXCR4, CXCR5, CCR6, and CCR7 expression remained unaffected.This study provides the first detailed characterization of allergen-specific B cells before and after bee venom tolerance induction. The observed B-cell responses in both venom immunotherapy-treated patients and naturally exposed beekeepers suggest a similar functional immunoregulatory role for B cells in allergen tolerance in both groups. These findings can be investigated in other AIT models to determine their potential as biomarkers of early and successful AIT responses.
View details for DOI 10.1111/all.12966
View details for PubMedID 27341567
Role of regulatory B cells in immune tolerance to allergens and beyond.
The Journal of allergy and clinical immunology
2016; 138 (3): 654?65
Interleukin-22 is produced by certain T helper cells subsets (Th17, Th22) and at lower levels by ?-? T cells, NKT and innate lymphoid cells. Th22 cells are unique immune cells that regulate tissue responses by IL-22 production. The exact discrimination between Th17 cells that co-produce IL-22 and single IL-22-producing Th22 cells has not been possible until the present study. Isolation of pure Th22 cells without co-expression of cytokines of other T-cell subsets is essential to better understand their function in humans. The aim of this study is the isolation and characterization of viable, human IL-22-producing CD4+ T cells that do not produce IL-17A.Isolation of viable Th22 cells was performed with the combination of two cytokine secretion assays detecting IL-17A- and IL-22-producing cells in a single purification step.The newly developed cytokine secretion assay consists of anti-IL-22 and anti-IL-17A catch antibodies, which via biotin-streptavidin interaction are bound to the biotinylated surface of the target cell, and anti-IL-22 and IL-17A detection antibody labelled with a fluorescent dye, which detects cytokines bound to these catch antibodies. A unique population of human Th22 cells, which do not produce IL-17A, was sorted, and cytokine expression pattern was confirmed by quantitative PCR analysis and ELISA. The presented technique allows the detection and isolation of pure human Th22 cells.This technique may allow the purification of any single cytokine-producing cell subset, and the combination of several different cytokine secretion assays can be used to purify and characterize novel and unique cell subsets.
View details for DOI 10.1111/all.12768
View details for PubMedID 26392196
Interleukins (from IL-1 to IL-38), interferons, transforming growth factor ?, and TNF-?: Receptors, functions, and roles in diseases.
The Journal of allergy and clinical immunology
2016; 138 (4): 984?1010
Immune tolerance to both self-antigens and innocuous non-self-antigens is essential to protect the host against chronic inflammatory diseases and tissue damage. A wide range of cell types and suppressive molecules are involved in induction and maintenance of tolerance. In addition to their key function in the production of immunoglobulins, B cells can regulate immune responses through their surface molecules and secretion of cytokines. Regulatory B (Breg) cells are characterized by their immunosuppressive capacity, which is often mediated through IL-10 secretion. However, IL-35 and TGF-? have also been associated with B cell-mediated immunosuppression. Several types of murine and human Breg cells have been described, such as mouse CD5(+)CD1d(hi) B10 cells, CD21(hi)CD23(hi)CD24(hi) transitional stage 2-like B cells, and CD138(+) plasma cells and plasmablasts. Human Breg cell types include CD27(+)CD24(high) B10 cells, CD24(hi)CD38(hi) immature transitional B cells, and CD73(-)CD25(+)CD71(+) BR1 cells and a subset of plasma cells. Support for the in vivo existence of allergen-specific human Breg cells comes from direct detection of their increase during the course of allergen-specific immunotherapy, as well as their increased expression in nonallergic but high-dose allergen-exposed beekeepers. Human BR1 cells selectively upregulate IgG4 antibodies on differentiation to plasma cells. This suggests an additional immune regulatory role because of the noninflammatory and blocking antibody function of IgG4. Taken together, Breg cells appear to be involved in mediating allergen tolerance, but many open questions remain to be answered.
View details for DOI 10.1016/j.jaci.2016.07.006
View details for PubMedID 27596706
IL-10-overexpressing B cells regulate innate and adaptive immune responses.
The Journal of allergy and clinical immunology
2015; 135 (3): 771?80.e8
There have been extensive developments on cellular and molecular mechanisms of immune regulation in allergy, asthma, autoimmune diseases, tumor development, organ transplantation, and chronic infections during the last few years. Better understanding the functions, reciprocal regulation, and counterbalance of subsets of immune and inflammatory cells that interact through interleukins, interferons, TNF-?, and TGF-? offer opportunities for immune interventions and novel treatment modalities in the era of development of biological immune response modifiers particularly targeting these molecules or their receptors. More than 60 cytokines have been designated as interleukins since the initial discoveries of monocyte and lymphocyte interleukins (called IL-1 and IL-2, respectively). Studies of transgenic or gene-deficient mice with altered expression of these cytokines or their receptors and analyses of mutations and polymorphisms in human genes that encode these products have provided essential information about their functions. Here we review recent developments on IL-1 to IL-38, TNF-?, TGF-?, and interferons. We highlight recent advances during the last few years in this area and extensively discuss their cellular sources, targets, receptors, signaling pathways, and roles in immune regulation in patients with allergy and asthma and other inflammatory diseases.
View details for DOI 10.1016/j.jaci.2016.06.033
View details for PubMedID 27577879
Pollen-derived nonallergenic substances enhance Th2-induced IgE production in B cells.
2015; 70 (11): 1450?60
Distinct human IL-10-producing B-cell subsets with immunoregulatory properties have been described. However, the broader spectrum of their direct cellular targets and suppressive mechanisms has not been extensively studied, particularly in relation to direct and indirect IL-10-mediated functions.The aim of the study was to investigate the effects of IL-10 overexpression on the phenotype and immunoregulatory capacity of B cells.Primary human B cells were transfected with hIL-10, and IL-10-overexpressing B cells were characterized for cytokine and immunoglobulin production by means of specific ELISA and bead-based assays. Antigen presentation, costimulation capacity, and transcription factor signatures were analyzed by means of flow cytometry and quantitative RT-PCR. Effects of IL-10-overexpresing B cells on Toll-like receptor-triggered cytokine release from PBMCs, LPS-triggered maturation of monocyte-derived dendritic cells, and tetanus toxoid-induced PBMC proliferation were assessed in autologous cocultures.IL-10-overexpressing B cells acquired a prominent immunoregulatory profile comprising upregulation of suppressor of cytokine signaling 3 (SOCS3), glycoprotein A repetitions predominant (GARP), the IL-2 receptor ? chain (CD25), and programmed cell death 1 ligand 1 (PD-L1). Concurrently, their secretion profile was characterized by a significant reduction in levels of proinflammatory cytokines (TNF-?, IL-8, and macrophage inflammatory protein 1?) and augmented production of anti-inflammatory IL-1 receptor antagonist and vascular endothelial growth factor. Furthermore, IL-10 overexpression was associated with a decrease in costimulatory potential. IL-10-overexpressing B cells secreted less IgE and potently suppressed proinflammatory cytokines in PBMCs, maturation of monocyte-derived dendritic cells (rendering their profile to regulatory phenotype), and antigen-specific proliferation in vitro.Our data demonstrate an essential role for IL-10 in inducing an immunoregulatory phenotype in B cells that exerts substantial anti-inflammatory and immunosuppressive functions.
View details for DOI 10.1016/j.jaci.2014.07.041
View details for PubMedID 25240783
The development of autoimmune features in aging mice is closely associated with alterations of the peripheral CD4? T-cell compartment.
European journal of immunology
2014; 44 (10): 2893?2902
B cells play a central role in IgE-mediated allergies. In damaged airway epithelium, they are exposed directly to aeroallergens. We aimed to assess whether direct exposure of B cells to pollen constituents affects allergic sensitization.B cells from murine splenocytes and from blood samples of healthy donors were incubated for 8 days under Th2-like conditions with aqueous ragweed pollen extracts (Amb-APE) or its constituents. Secreted total IgM, IgG, and IgE was quantified by ELISA. Additionally, birch, grass, or pine-pollen extracts were tested. The number of viable cells was evaluated by ATP measurements. B-cell proliferation was measured by CFSE staining. IgE class switch was analyzed by quantitation of class switch transcripts. In an OVA/Alum i.p.-sensitization mouse model, Amb-APE was intranasally instilled for 11 consecutive days.Upon Th2 priming of murine B cells, ragweed pollen extract caused a dose-dependent increase in IgE production, while IgG and IgM were not affected. The low-molecular-weight fraction and phytoprostane E1 (PPE1) increased IgE production, while Amb a 1 did not. PPE1 enhanced IgE also in human memory B cells. Under Th1 conditions, Amb-APE did not influence immunoglobulin secretion. The IgE elevation was not ragweed specific. It correlated with proliferation of viable B cells, but not with IgE class switch. In vivo, Amb-APE increased total IgE and showed adjuvant activity in allergic airway inflammation.Aqueous pollen extracts, the protein-free fraction of Amb-APE, and the pollen-contained substance PPE1 specifically enhance IgE production in Th2-primed B cells. Thus, pollen-derived nonallergenic substances might be responsible for B-cell-dependent aggravation of IgE-mediated allergies.
View details for DOI 10.1111/all.12707
View details for PubMedID 26214762
Some signs of potential autoimmunity, such as the appearance of antinuclear antibodies (ANAs) become prevalent with age. In most cases, elderly people with ANAs remain healthy. Here, we investigated whether the same holds true for inbred strains of mice. Indeed, we show that most mice of the C57BL/6 (B6) strain spontaneously produced IgG ANA at 8-12 months of age, showed IgM deposition in kidneys and lymphocyte infiltrates in submandibular salivary glands. Despite all of this, the mice remained healthy. ANA production is likely CD4(+) T-cell dependent, since old (40-50 weeks of age) B6 mice deficient for MHC class II do not produce IgG ANAs. BM chimeras showed that ANA production was not determined by age-related changes in radiosensitive, hematopoietic progenitor cells, and that the CD4(+) T cells that promote ANA production were radioresistant. Thymectomy of B6 mice at 5 weeks of age led to premature alterations in T-cell homeostasis and ANA production, by 15 weeks of age, similar to that in old mice. Our findings suggest that a disturbed T-cell homeostasis may drive the onset of some autoimmune features.
View details for DOI 10.1002/eji.201344408
View details for PubMedID 25044476