Transcriptional Analysis of the Human IgE-Expressing Plasma Cell Differentiation Pathway

IgE is secreted by plasma cells (PCs) and is central to allergic disease. Using an ex vivo tonsil B cell culture system, which mimics the Th2 responses in vivo, we have recently characterized the development pathway of human IgE-expressing PCs. In this system, as in mice, we reported the predisposition of IgE-expressing B cells to differentiate into PCs. To gain a comprehensive understanding of the molecular events involved in the differentiation of human IgE+ B cells into PCs we have used the Illumina HumanHT-12 v4 Expression BeadChip array to analyse the gene expression profile of ex vivo generated human IgE+ B cells at various stages of their differentiation into PCs. We also compared the transcription profiles of IgE+ and IgG1+ cells to discover isotype-specific patterns. Comparisons of IgE+ and IgG1+ cell transcriptional profiles revealed molecular signatures specific for IgE+ cells, which diverge from their IgG1+ cell counterparts upon differentiation into PCs. At the germinal center (GC) stage of development, unlike in some mouse studies of IgE biology, we observed similar rates of apoptosis and no significant differences in the expression of apoptosis-associated genes between the IgE+ and IgG1+ B cells. We identified a gene interaction network associated with early growth response 1 (EGR1) that, together with the up-regulated IRF4, may account for the predisposition of IgE+ B cells to differentiate into PCs. However, despite their swifter rates of PC differentiation, the transcription profile of IgE+ PCs is more closely related to IgE+ and IgG1+ plasmablasts (PBs) than to IgG1+ PCs, suggesting that the terminal differentiation of IgE+ cells is impeded. We also show that IgE+ PCs have increased levels of apoptosis suggesting that the IgE+ PCs generated in our in vitro tonsil B cell cultures, as in mice, are short-lived. We identified gene regulatory networks as well as cell cycle and apoptosis signatures that may explain the diverging PC differentiation programme of these cells. Overall, our study provides a detailed analysis of the transcriptional pathways underlying the differentiation of human IgE-expressing B cells and points to molecular signatures that regulate IgE+ PC differentiation and function

Local Clonal Diversification and Dissemination of B Lymphocytes in the Human Bronchial Mucosa

The efficacy of the adaptive humoral immune response likely requires diverse, yet focused regional B cell antibody production throughout the body. Here we address, in the first study of its kind, the B cell repertoire in the bronchial mucosa, an important barrier to antigens inhaled from the atmosphere. To accomplish this, we have applied high-throughput Adaptive Immune Receptor Repertoire Sequencing (AIRR-Seq) to 10 bronchial biopsies from altogether four different sites in the right lungs from an asthmatic patient and a healthy subject. While the majority of identified B cell clones were restricted to a single site, many were disseminated in multiple sites. Members of a clone were shared more between adjacent biopsies than between distal biopsies, suggesting local mucosal migration and/or a homing mechanism for B cells through the blood or lymph. A smaller fraction of clones spanned the bronchial mucosa and peripheral blood, suggesting ongoing trafficking between these compartments. The bronchial mucosal B cell repertoire in the asthmatic patient was geographically more variable but less diverse compared to that of the healthy subject, suggesting an ongoing, antigen-driven humoral immune response in atopic asthma. Whether this is a feature of atopy or disease status remains to be clarified in future studies. We observed a subset of highly mutated and antigen-selected IgD-only cells in the bronchial mucosa. These cells were found in relative high abundance in the asthmatic individual but also, albeit at lower abundance, in the healthy subject. This novel finding merits further exploration using a larger cohort of subjects

Ontogeny of Human IgE-expressing B Cells and Plasma Cells

BACKGROUND: IgE‐expressing (IgE(+)) plasma cells (PCs) provide a continuous source of allergen‐specific IgE that is central to allergic responses. The extreme sparsity of IgE(+) cells in vivo has confined their study almost entirely to mouse models. OBJECTIVE: To characterize the development pathway of human IgE(+) PCs and to determine the ontogeny of human IgE(+) PCs. METHODS: To generate human IgE(+) cells, we cultured tonsil B cells with IL‐4 and anti‐CD40. Using FACS and RT‐PCR, we examined the phenotype of generated IgE(+) cells, the capacity of tonsil B‐cell subsets to generate IgE(+) PCs and the class switching pathways involved. RESULTS: We have identified three phenotypic stages of IgE(+) PC development pathway, namely (i) IgE(+)germinal centre (GC)‐like B cells, (ii) IgE(+) PC‐like ‘plasmablasts’ and (iii) IgE(+) PCs. The same phenotypic stages were also observed for IgG1(+) cells. Total tonsil B cells give rise to IgE(+) PCs by direct and sequential switching, whereas the isolated GC B‐cell fraction, the main source of IgE(+) PCs, generates IgE(+) PCs by sequential switching. PC differentiation of IgE(+) cells is accompanied by the down‐regulation of surface expression of the short form of membrane IgE (mIgE(S)), which is homologous to mouse mIgE, and the up‐regulation of the long form of mIgE (mIgE(L)), which is associated with an enhanced B‐cell survival and expressed in humans, but not in mice. CONCLUSION: Generation of IgE(+) PCs from tonsil GC B cells occurs mainly via sequential switching from IgG. The mIgE(L)/mIgE(S) ratio may be implicated in survival of IgE(+) B cells during PC differentiation and allergic disease

A tool kit for rapid cloning and expression of recombinant antibodies

Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. However, cost-effective tools for engineering antibodies of different specificities, isotypes and species are still needed for many research and clinical applications in academia. Here, we report a method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated. As a proof of principle we have cloned, expressed and characterized functional recombinant tumor-associated antigen-specific chimeric IgE/κ and IgG(1)/κ, as well as recombinant grass pollen allergen Phl p 7 specific fully human IgE/λ and IgG(4)/λ antibodies. This method utilizing the antibody expression vectors, available at Addgene, has many applications, including the potential to support simultaneous processing of antibody panels, to facilitate mechanistic studies of antigen-antibody interactions and to conduct early evaluations of antibody functions

IgE to epitopes of Ara h 2 enhance the diagnostic accuracy of Ara h 2‐specific IgE

© 2020 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Background: Understanding the discrepancy between IgE sensitization and allergic reactions to peanut could facilitate diagnosis and lead to novel means of treating peanut allergy. Objective: To identify differences in IgE and IgG4 binding to peanut peptides between peanut-allergic (PA) and peanut-sensitized but tolerant (PS) children. Methods: PA (n = 56), PS (n = 42) and nonsensitized nonallergic (NA, n = 10) patients were studied. Synthetic overlapping 15-mer peptides of peanut allergens (Ara h 1-11) were spotted onto microarray slides, and patients' samples were tested for IgE and IgG4 binding using immunofluorescence. IgE and IgG4 levels to selected peptides were quantified using ImmunoCAP. Diagnostic model comparisons were performed using likelihood-ratio tests between each specified nominal logistic regression models. Results: Seven peptides on Ara h 1, Ara h 2, and Ara h 3 were bound more by IgE of PA compared to PS patients on the microarray. IgE binding to one peptide on Ara h 5 and IgG4 binding to one Ara h 9 peptide were greater in PS than in PA patients. Using ImmunoCAP, IgE to the Ara h 2 peptides enhanced the diagnostic accuracy of Ara h 2-specific IgE. Ratios of IgG4/IgE to 4 out of the 7 peptides were higher in PS than in PA subjects. Conclusions: Ara h 2 peptide-specific IgE added diagnostic value to Ara h 2-specific IgE. Ability of peptide-specific IgG4 to surmount their IgE counterpart seems to be important in established peanut tolerance.This work was supported by the Medical Research Council (MRC Clinical Research Training Fellowship G090218, MRC Centenary Early Career Award and MRC Clinician Scientist Fellowship MR/M008517/1, all awarded to A. F. Santos), the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy's & St Thomas' NHS Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust; the US Department of Agriculture, Agricultural Research Service (USDA-ARS6054-43440-046-00D; USDA-NIFA) and the National Peanut Board (GRANT12229460).info:eu-repo/semantics/publishedVersio

Interplay between Affinity and Valency in Effector Cell Degranulation: A Model System with Polcalcin Allergens and Human Patient-Derived IgE Antibodies.

This is the accepted, uncopyedited version of the manuscript. The definitive version was published in The Journal of Immunology August 28, 2019, ji1900509; DOI: https://doi.org/10.4049/jimmunol.1900509Originally published in The Journal of Immunology. Bucaite G, Kang-Pettinger T, Moreira J, et al. Interplay between Affinity and Valency in Effector Cell Degranulation: A Model System with Polcalcin Allergens and Human Patient-Derived IgE Antibodies. J Immunol. 2019;203(7):1693-1700. doi:10.4049/jimmunol.1900509 The American Association of Immunologists, Inc.An allergic reaction is rapidly generated when allergens bind and cross-link IgE bound to its receptor FcεRI on effector cells, resulting in cell degranulation and release of proinflammatory mediators. The extent of effector cell activation is linked to allergen affinity, oligomeric state, valency, and spacing of IgE-binding epitopes on the allergen. Whereas most of these observations come from studies using synthetic allergens, in this study we have used Timothy grass pollen allergen Phl p 7 and birch pollen allergen Bet v 4 to study these effects. Despite the high homology of these polcalcin family allergens, Phl p 7 and Bet v 4 display different binding characteristics toward two human patient-derived polcalcin-specific IgE Abs. We have used native polcalcin dimers and engineered multimeric allergens to test the effects of affinity and oligomeric state on IgE binding and effector cell activation. Our results indicate that polcalcin multimers are required to stimulate high levels of effector cell degranulation when using the humanized RBL-SX38 cell model and that multivalency can overcome the need for high-affinity interactions.This work was supported by Medical Research Council Grant G1100090. G.B. was supported by a studentship from the King’s Bioscience Institute and by the Guy’s and St. Thomas’ Charity Prize Ph.D. Program in Biomedical and Translational Science. We acknowledge the support of the Centre for Biomolecular Spectroscopy, King’s College London, established with a Capital Award from the Wellcome Trust (Grant 085944)

IgG4 inhibits peanut-induced basophil and mast cell activation in peanut-tolerant children sensitized to peanut major allergens

BackgroundMost children with detectable peanut-specific IgE (P-sIgE) are not allergic to peanut. We addressed 2 non–mutually exclusive hypotheses for the discrepancy between allergy and sensitization: (1) differences in P-sIgE levels between children with peanut allergy (PA) and peanut-sensitized but tolerant (PS) children and (2) the presence of an IgE inhibitor, such as peanut-specific IgG4 (P-sIgG4), in PS patients.MethodsTwo hundred twenty-eight children (108 patients with PA, 77 PS patients, and 43 nonsensitized nonallergic subjects) were studied. Levels of specific IgE and IgG4 to peanut and its components were determined. IgE-stripped basophils or a mast cell line were used in passive sensitization activation and inhibition assays. Plasma of PS subjects and patients submitted to peanut oral immunotherapy (POIT) were depleted of IgG4 and retested in inhibition assays.ResultsBasophils and mast cells sensitized with plasma from patients with PA but not PS patients showed dose-dependent activation in response to peanut. Levels of sIgE to peanut and its components could only partially explain differences in clinical reactivity between patients with PA and PS patients. P-sIgG4 levels (P = .023) and P-sIgG4/P-sIgE (P < .001), Ara h 1–sIgG4/Ara h 1–sIgE (P = .050), Ara h 2–sIgG4/Ara h 2–sIgE (P = .004), and Ara h 3–sIgG4/Ara h 3–sIgE (P = .016) ratios were greater in PS children compared with those in children with PA. Peanut-induced activation was inhibited in the presence of plasma from PS children with detectable P-sIgG4 levels and POIT but not from nonsensitized nonallergic children. Depletion of IgG4 from plasma of children with PS (and POIT) sensitized to Ara h 1 to Ara h 3 partially restored peanut-induced mast cell activation (P = .007).ConclusionsDifferences in sIgE levels and allergen specificity could not justify the clinical phenotype in all children with PA and PS children. Blocking IgG4 antibodies provide an additional explanation for the absence of clinical reactivity in PS patients sensitized to major peanut allergens

Characterisation of GLUT4 trafficking in HeLa cells: comparable kinetics and orthologous trafficking mechanisms to 3T3-L1 adipocytes

Insulin-stimulated glucose transport is a characteristic property of adipocytes and muscle cells and involves the regulated delivery of glucose transporter (GLUT4)-containing vesicles from intracellular stores to the cell surface. Fusion of these vesicles results in increased numbers of GLUT4 molecules at the cell surface. In an attempt to overcome some of the limitations associated with both primary and cultured adipocytes, we expressed an epitope- and GFP-tagged version of GLUT4 (HA–GLUT4–GFP) in HeLa cells. Here we report the characterisation of this system compared to 3T3-L1 adipocytes. We show that insulin promotes translocation of HA–GLUT4–GFP to the surface of both cell types with similar kinetics using orthologous trafficking machinery. While the magnitude of the insulin-stimulated translocation of GLUT4 is smaller than mouse 3T3-L1 adipocytes, HeLa cells offer a useful, experimentally tractable, human model system. Here, we exemplify their utility through a small-scale siRNA screen to identify GOSR1 and YKT6 as potential novel regulators of GLUT4 trafficking in human cells