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1. Anti-S-layer monoclonal antibodies impact Clostridioides difficile physiology

Author

Lise Hunault, Emile Auria, Patrick England, Julien Deschamps, Romain Briandet, Vanessa Kremer, Bruno Iannascoli, Léo Vidal-Maison, Chunguang Guo, Lynn Macdonald, Séverine Péchiné, Cécile Denève-Larrazet, Bruno Dupuy, Guy Gorochov, Pierre Bruhns, and Delphine Sterlin

Subjects
Clostridioides difficile, monoclonal antibodies, S-layer, growth, biofilms, toxins, Diseases of the digestive system. Gastroenterology, RC799-869
Abstract

ABSTRACTClostridioides difficile (C. difficile), a gram-positive anaerobic and spore-forming bacterium, is the leading cause of nosocomial antibiotic-associated diarrhea in adults which is characterized by high levels of recurrence and mortality. Surface (S)-layer Protein A (SlpA), the most abundantly expressed protein on the bacterial surface, plays a crucial role in the early stages of infection although the nature of its involvement in C. difficile physiology is yet to be fully understood. Anti-S-layer antibodies have been identified in the sera of convalescent patients and have been correlated with improved outcomes of C. difficile infection (CDI). However, the precise mechanisms by which anti-S-layer antibodies confer protection to the host remain unknown. In this study, we report the first monoclonal antibodies (mAbs) targeting the S-layer of reference strain 630. Characterization of these mAbs unraveled important roles for the S-layer protein in growth, toxin secretion, and biofilm formation by C. difficile, with differential and even opposite effects of various anti-SlpA mAbs on these functions. Moreover, one anti-SlpA mAb impaired C. difficile growth and conferred sensitivity to lysozyme-induced lysis. The results of this study show that anti-S-layer antibody responses can be beneficial or harmful for the course of CDI and provide important insights for the development of adequate S-layer-targeting therapeutics.

Published
2024
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2. A monoclonal antibody collection for C. difficile typing ?

Author

Lise Hunault, Patrick England, Frédéric Barbut, Bruno Iannascoli, Ophélie Godon, François Déjardin, Christophe Thomas, Bruno Dupuy, Chunguang Guo, Lynn Macdonald, Guy Gorochov, Delphine Sterlin, and Pierre Bruhns

Subjects
Clostridioides difficile, Monoclonal antibodies, S-layer, Hybridomas, Ribotypes, Diseases of the digestive system. Gastroenterology, RC799-869
Abstract

Abstract Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in adults. Various C. difficile strains circulate currently, associated with different outcomes and antibiotic resistance profiles. However, most studies still focus on the reference strain 630 that does not circulate anymore, partly due to the lack of immunological tools to study current clinically important C. difficile PCR ribotypes. The goal of this study was to generate monoclonal antibodies recognizing various epidemic ribotypes of C. difficile. To do so, we immunized mice expressing human variable antibody genes with the Low Molecular Weight (LMW) subunit of the surface layer protein SlpA from various C. difficile strains. Monoclonal antibodies purified from hybridomas bound LMW with high-affinity and whole bacteria from current C. difficile ribotypes with different cross-specificities. This first collection of anti-C. difficile mAbs represent valuable tools for basic and clinical research.

Published
2024
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3. Qualitative monitoring of SARS-CoV-2 mRNA vaccination in humans using droplet microfluidics

Author

Matteo Broketa, Aurélien Sokal, Michael Mor, Pablo Canales-Herrerias, Angga Perima, Annalisa Meola, Ignacio Fernández, Bruno Iannascoli, Guilhem Chenon, Alexis Vandenberghe, Laetitia Languille, Marc Michel, Bertrand Godeau, Sébastien Gallien, Giovanna Melica, Marija Backovic, Felix A. Rey, Jean Baudry, Natalia T. Freund, Matthieu Mahévas, and Pierre Bruhns

Subjects
Immunology, Vaccines, Medicine
Abstract

SARS-CoV-2 mRNA vaccination generates protective B cell responses targeting the SARS-CoV-2 spike glycoprotein. Whereas anti-spike memory B cell responses are long lasting, the anti-spike humoral antibody response progressively wanes, making booster vaccinations necessary for maintaining protective immunity. Here, we qualitatively investigated the plasmablast responses by measuring from single cells within hours of sampling the affinity of their secreted antibody for the SARS-CoV-2 spike receptor binding domain (RBD) in cohorts of BNT162b2-vaccinated naive and COVID-19–recovered individuals. Using a droplet microfluidic and imaging approach, we analyzed more than 4,000 single IgG-secreting cells, revealing high interindividual variability in affinity for RBD, with variations over 4 logs. High-affinity plasmablasts were induced by BNT162b2 vaccination against Hu-1 and Omicron RBD but disappeared quickly thereafter, whereas low-affinity plasmablasts represented more than 65% of the plasmablast response at all time points. Our droplet-based method thus proves efficient at fast and qualitative immune monitoring and should be helpful for optimization of vaccination protocols.

Published
2023
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4. High-affinity autoreactive plasma cells disseminate through multiple organs in patients with immune thrombocytopenic purpura

Author

Pablo Canales-Herrerias, Etienne Crickx, Matteo Broketa, Aurélien Sokal, Guilhem Chenon, Imane Azzaoui, Alexis Vandenberghe, Angga Perima, Bruno Iannascoli, Odile Richard-Le Goff, Carlos Castrillon, Guillaume Mottet, Delphine Sterlin, Ailsa Robbins, Marc Michel, Patrick England, Gael A. Millot, Klaus Eyer, Jean Baudry, Matthieu Mahevas, and Pierre Bruhns

Subjects
Autoimmunity, Therapeutics, Medicine
Abstract

The major therapeutic goal for immune thrombocytopenic purpura (ITP) is to restore normal platelet counts using drugs to promote platelet production or by interfering with mechanisms responsible for platelet destruction. Eighty percent of patients with ITP possess anti–integrin αIIbβ3 IgG autoantibodies that cause platelet opsonization and phagocytosis. The spleen is considered the primary site of autoantibody production by autoreactive B cells and platelet destruction. The immediate failure in approximately 50% of patients to recover a normal platelet count after anti-CD20 rituximab-mediated B cell depletion and splenectomy suggests that autoreactive, rituximab-resistant, IgG-secreting B cells (IgG-SCs) reside in other anatomical compartments. We analyzed more than 3,300 single IgG-SCs from spleen, bone marrow, and/or blood of 27 patients with ITP, revealing high interindividual variability in affinity for αIIbβ3, with variations over 3 logs. IgG-SC dissemination and range of affinities were, however, similar for each patient. Longitudinal analysis of autoreactive IgG-SCs upon treatment with the anti-CD38 mAb daratumumab demonstrated variable outcomes, from complete remission to failure with persistence of high-affinity anti–αIIbβ3 IgG-SCs in the bone marrow. This study demonstrates the existence and dissemination of high-affinity autoreactive plasma cells in multiple anatomical compartments of patients with ITP that may cause the failure of current therapies.

Published
2022
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6. Conditional neutrophil depletion challenges their contribution to mouse models of anaphylaxis

Author

Julien Stackowicz, Caitlin M. Gillis, Ophélie Godon, Bruno Iannascoli, Eva Conde, Edouard Leveque, William P. M. Worrall, Stephen J. Galli, Pierre Bruhns, Laurent L. Reber, Friederike Jönsson, Anticorps en thérapie et pathologie - Antibodies in Therapy and Pathology, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Collège Doctoral, Sorbonne Université (SU), Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Stanford School of Medicine [Stanford], Stanford Medicine, Stanford University-Stanford University, This work was supported by a Jeunes Chercheuses/Jeunes Chercheurs grant from the Agence National de la Recherche (ANR-16-CE15-0012, to FJ). The Phenomin project receives funds from the ANR under the framework 'Investissements d'Avenir' labelled ANR-10-INBS-07 PHENOMIN. PB acknowledges support from the European Research Council (ERC)–Seventh Framework Program (ERC-2013-CoG 616,050), Institut Pasteur and INSERM (Institut National de la Santé et de la Recherche Médicale), SJG from NIH grant R01 AI165373, CMG was supported by a stipend from the Pasteur – Paris University (PPU) International PhD program and by the Institut Carnot Pasteur Maladies Infectieuses, and LLR from an ATIP-Avenir grant from the INSERM and the Fondation pour la Recherche Médicale (FRM EQU202103012566)., ANR-16-CE15-0012,PlanA,Rôle des plaquettes et leurs interactions dans les réactions anaphylactiques(2016), ANR-10-INBS-0007,PHENOMIN,INFRASTRUCTURE NATIONALE EN PHENOGENOMIQUE SOURIS(2010), and European Project: 616050,EC:FP7:ERC,ERC-2013-CoG,MYELOSHOCK(2014)

Subjects
[SDV]Life Sciences [q-bio], Immunology, Immunology and Allergy
Abstract

International audience; No abstract available

Published
2023

7. IgG Subclass-Dependent Pulmonary Antigen Retention during Acute IgG-Dependent Systemic Anaphylaxis in Mice

Author

Biliana Todorova, Ophélie Godon, Eva Conde, Caitlin M. Gillis, Bruno Iannascoli, Odile Richard-Le Goff, Daniel Fiole, Lubka T. Roumenina, Jeanette H. W. Leusen, Andrew J. Murphy, Lynn E. Macdonald, Laurent L. Reber, Friederike Jönsson, and Pierre Bruhns

Subjects
Complement C1q, Immunology, Receptors, IgG, Antigen-Antibody Complex, Allergens, Receptors, Complement, Mice, Inbred C57BL, Disease Models, Animal, Mice, 1107 Immunology, Immunoglobulin G, Immunology and Allergy, Animals, Anaphylaxis, Lung
Abstract

Mouse models of active systemic anaphylaxis rely predominantly on IgG Abs forming IgG–allergen immune complexes that induce IgG receptor–expressing neutrophils and monocytes/macrophages to release potent mediators, leading to systemic effects. Whether anaphylaxis initiates locally or systemically remains unknown. In this study, we aimed at identifying the anatomical location of IgG–allergen immune complexes during anaphylaxis. Active systemic anaphylaxis was induced following immunization with BSA and i.v. challenge with fluorescently labeled BSA. Ag retention across different organs was examined using whole-body fluorescence imaging, comparing immunized and naive animals. Various mouse models and in vivo deletion strategies were employed to determine the contribution of IgG receptors, complement component C1q, myeloid cell types, and anaphylaxis mediators. We found that following challenge, Ag diffused systemically, but specifically accumulated in the lungs of mice sensitized to that Ag, where it formed large Ab-dependent aggregates in the vasculature. Ag retention in the lungs did not rely on IgG receptors, C1q, neutrophils, or macrophages. IgG2a-mediated, but neither IgG1- nor IgG2b-mediated, passive systemic anaphylaxis led to Ag retention in the lung. Neutrophils and monocytes significantly accumulated in the lungs after challenge and captured high amounts of Ag, which led to downmodulation of surface IgG receptors and triggered their activation. Thus, within minutes of systemic injection in sensitized mice, Ag formed aggregates in the lung and liver vasculature, but accumulated specifically and dose-dependently in the lung. Neutrophils and monocytes recruited to the lung captured Ag and became activated. However, Ag aggregation in the lung vasculature was not necessary for anaphylaxis induction.

Published
2022

8. High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics

Author

Andrew D. Griffiths, Alexei Godina, Guillaume Mottet, Gaël A. Millot, Bingqing Shen, Samantha N. Stewart, Vera Menrath, Sosthene Essono, Baptiste Saudemont, Clément Nizak, Annabelle Gérard, Antoine Sautel-Caillé, Scott McNamara, Matthieu Delince, Sami Ellouze, Jean Baudry, Marcel Reichen, Pablo Canales-Herrerias, Charina Ortega, Patrick England, Carlos Castrillon, Odile Richard-Le Goff, Colin Brenan, Klaus Eyer, Pascaline Mary, Luis Briseño-Roa, Allan Jensen, Pierre Bruhns, Raphael Clement Li-Ming Doineau, Adam Woolfe, Roshan Kumar, Bin Jia, Bruno Iannascoli, Yannick Pousse, Kevin Grosselin, Gregory Rose, Adeline Poitou, HiFiBiO Therapeutics SAS [Paris], Anticorps en thérapie et pathologie - Antibodies in Therapy and Pathology, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Ecole Doctorale Frontières du Vivant - Programme Bettencourt (FdV), Université Paris Descartes - Paris 5 (UPD5)-PRES Sorbonne Paris Cité, Chimie-Biologie-Innovation (UMR 8231) (CBI), Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Colloïdes et Matériaux Divisés (LCMD), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS), HiFiBiO Therapeutics Inc. [Cambridge], Abbvie Bioresearch Center [Worcester], Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Biophysique Moléculaire (Plate-forme), Biophysique des macromolécules et leurs interactions, This work was supported by the French Agence Nationale de la Recherche (ANR-14-CE16-0011 project DROPmAbs), by the Centre d’Innovation et Recherche Technologique (Citech) through the Institut Carnot Pasteur Microbes & Santé (ANR 16 CARN 0023-01), by BPI France (OSIRIS and CELLIGO projects) and by the French ‘Investissements d’Avenir’ program via the CELLIGO project and grant agreements ANR-10-NANO-02, ANR-10-IDEX-0001-02 PSL, ANR-10-LABX-31 and ANR-10-EQPX-34. NGS was performed by the ICGex NGS platform of the Institut Curie and the Institut de Biologie Intégrative de la Cellule (I2BC) platform (Gif-sur-Yvette) supported by the grants ANR-10-EQPX-03 and ANR-10-INBS-09-08 (France Génomique Consortium), by the Canceropole Ile-de-France and by the SiRIC-Curie program (SiRIC Grant INCa-DGOS- 4654). C.C. acknowledges financial support from CONCYTEC, Peru. P.C.H. is a scholar in the Pasteur - Paris University (PPU) International PhD program and was also supported by the Fondation pour la Recherche Médicale (FRM, FDT201904008240). K.E. acknowledges financial support from the Swiss National Science Foundation and The Branco Weiss Fellowship - Society in Science., We would like to thank M. Holsti and W. Somers (Pfizer, BioMedicine Design) for advice on the technology development and critical reading of the manuscript, R. Nicol (Whitehead Institute, MIT Center for Genome Research) for advice on barcoded sequencing, at the Institut Pasteur, H. Mouquet for advice on antibody sequence selection, and F. Jönsson for advice on flow cytometry analysis, T. Kirk and S. Foulon (ESPCI Paris) for their help developing the barcoded hydrogel beads, S. N. Stewart (HiFiBio Therapeutics Inc.) for her help producing and analysing recombinant antibodies, the Institut Pierre-Gilles de Gennes (IPGG) for use of clean room facilities and the laser engraver (CII08, Axyslaser)., ANR-14-CE16-0011,DROP-mAbs,Analyse en profondeur de répertoires d'anticorps par microfluidique en gouttelettes couplé à du séquençage haut-débit pour le diagnostic et la découverte d'anticorps thérapeutiques(2014), ANR-10-IDEX-0001,PSL,Paris Sciences et Lettres(2010), ANR-10-EQPX-0034,IPGG,Institut Pierre Gilles de Gennes pour la microfluidique(2010), ANR-10-EQPX-0003,ICGex,Equipement de biologie intégrative du cancer pour une médecine personnalisée(2010), ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Centre National de la Recherche Scientifique (CNRS)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Bruhns, Pierre, Appel à projets générique - Analyse en profondeur de répertoires d'anticorps par microfluidique en gouttelettes couplé à du séquençage haut-débit pour le diagnostic et la découverte d'anticorps thérapeutiques - - DROP-mAbs2014 - ANR-14-CE16-0011 - Appel à projets générique - VALID, Initiative d'excellence - Paris Sciences et Lettres - - PSL2010 - ANR-10-IDEX-0001 - IDEX - VALID, Equipements d'excellence - Institut Pierre Gilles de Gennes pour la microfluidique - - IPGG2010 - ANR-10-EQPX-0034 - EQPX - VALID, Equipements d'excellence - Equipement de biologie intégrative du cancer pour une médecine personnalisée - - ICGex2010 - ANR-10-EQPX-0003 - EQPX - VALID, and Organisation et montée en puissance d'une Infrastructure Nationale de Génomique - - France-Génomique2010 - ANR-10-INBS-0009 - INBS - VALID

Subjects
[SDV.IMM] Life Sciences [q-bio]/Immunology, Biomedical Engineering, Somatic hypermutation, Bioengineering, Cancer Vaccines, Applied Microbiology and Biotechnology, Antibodies, Mice, 03 medical and health sciences, 0302 clinical medicine, Antibody Repertoire, Antigen, Animals, Humans, Antigens, 030304 developmental biology, chemistry.chemical_classification, B-Lymphocytes, 0303 health sciences, biology, High-Throughput Nucleotide Sequencing, DNA, Microfluidic Analytical Techniques, Molecular biology, Reverse transcriptase, 3. Good health, Enzyme, chemistry, Immunization, Immunoglobulin G, biology.protein, [SDV.IMM]Life Sciences [q-bio]/Immunology, Molecular Medicine, Single-Cell Analysis, Antibody, 030217 neurology & neurosurgery, Ex vivo, Biotechnology
Abstract

International audience; Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays2, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100–1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450–900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility.

Published
2020

9. The anti-IgE mAb omalizumab induces adverse reactions by engaging Fcγ receptors

Author

Bianca Balbino, Julien Stackowicz, Kari C. Nadeau, Pauline Herviou, Ophélie Godon, Sébastien Brûlé, Delphine Sterlin, Andrew J. Murphy, Vera Voronina, Bruno Iannascoli, Odile Richard-Le Goff, Gaël A. Millot, Macdonald Lynn, Laurent L. Reber, Harris Faith, Pierre Bruhns, Anticorps en thérapie et pathologie - Antibodies in Therapy and Pathology, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Sorbonne Université (SU), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Biophysique Moléculaire (Plate-forme), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Regeneron Pharmaceuticals [Tarrytown], Stanford University, Centre de Physiopathologie Toulouse Purpan (CPTP), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), European Commission (Marie Skłodowska-Curie Individual Fellowship H2020-MSCA-IF-2014 656086)European 235Research Council (ERC)–Seventh Framework Program (ERC-2013-CoG 616050)Institut Pasteur initiative for valorizing the applications of research (ValoExpress 2017)INSERM, ATIP-Avenir program'Investissement d’Avenir'(grant ANR-11-INBS-0006)Pasteur -Paris University (PPU) International Ph.DprogramFrench 'Fondation pour la Recherche Médicale FRMAP-HP, ANR-11-INBS-0006,FLI,France Life Imaging(2011), European Project: 616050,EC:FP7:ERC,ERC-2013-CoG,MYELOSHOCK(2014), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Service d'Immunologie [CHU Pitié-Salpétrière], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Allergy, MESH: Asthma / immunology, Omalizumab, MESH: Anaphylaxis / immunology, Immunoglobulin E, MESH: Mice, Knockout, Mice, MESH: Asthma / pathology, 0302 clinical medicine, Medicine, MESH: Animals, MESH: Drug Eruptions / pathology, Mice, Knockout, biology, Concise Communication, General Medicine, MESH: Drug Eruptions / genetics, 3. Good health, MESH: Anaphylaxis / pathology, MESH: Omalizumab / genetics, 030220 oncology & carcinogenesis, MESH: Drug Eruptions / immunology, [SDV.IMM]Life Sciences [q-bio]/Immunology, Drug Eruptions, Antibody, medicine.symptom, Anaphylaxis, medicine.drug, MESH: Mutation, MESH: Anaphylaxis / chemically induced, medicine.drug_class, Immunology, Immunoglobulins, Inflammation, Monoclonal antibody, MESH: Receptors, IgG / immunology, 03 medical and health sciences, Immune system, MESH: Omalizumab / adverse effects, Animals, MESH: Receptors, IgG / genetics, MESH: Mice, MESH: Omalizumab / pharmacology, business.industry, Receptors, IgG, MESH: Asthma / drug therapy, medicine.disease, Asthma, 030104 developmental biology, Mutation, Mast cells, MESH: Anaphylaxis / genetics, biology.protein, business
Abstract

International audience; Omalizumab is an anti-IgE monoclonal antibody (mAb) approved for the treatment of severe asthma and chronic spontaneous urticaria. Use of Omalizumab is associated with reported side effects, ranging from local skin inflammation at the injection site to systemic anaphylaxis. To date, the mechanisms through which Omalizumab induces adverse reactions are still unknown. Here, we demonstrated that immune complexes formed between Omalizumab and IgE can induce both skin inflammation and anaphylaxis through engagement of IgG receptors (FcγRs) in FcγR-humanized mice. We further developed an Fc-engineered mutant version of Omalizumab, and demonstrated that this mAb is equally potent as Omalizumab at blocking IgE-mediated allergic reactions, but does not induce FcγR-dependent adverse reactions. Overall, our data indicate that Omalizumab can induce skin inflammation and anaphylaxis by engaging FcγRs, and demonstrate that Fc-engineered versions of the mAb could be used to reduce such adverse reactions.

Published
2020

10. Specificity of mouse and human Fcgamma receptors and their polymorphic variants for IgG subclasses of different species

Author

Yu Wang, Vanessa Krémer, Bruno Iannascoli, Odile Richard‐Le Goff, David A. Mancardi, Leoni Ramke, Luc Chaisemartin, Pierre Bruhns, Friederike Jönsson, Anticorps en thérapie et pathologie - Antibodies in Therapy and Pathology, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Université Paris Cité (UPCité), AP-HP - Hôpital Bichat - Claude Bernard [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Centre National de la Recherche Scientifique (CNRS), This work was supported by a Jeunes Chercheuses/Jeunes Chercheurs grant from the Agence National de la Recherche (ANR- 16-CE15-0012), Institut National de la Recherche Médicale (INSERM) and Institut Pasteur. YW and VK are part of the Pasteur–Paris University (PPU) International PhD Program. This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 665807, and from the Labex Milieu Intérieur, Institut Pasteur. For the purpose of open access, the author has applied a CC BY public copyright license to any author accepted manuscript version arising from this submission., ANR-16-CE15-0012,PlanA,Rôle des plaquettes et leurs interactions dans les réactions anaphylactiques(2016), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), European Project: 665807,H2020,H2020-MSCA-COFUND-2014,PASTEURDOC(2015), Jönsson, Friederike, Rôle des plaquettes et leurs interactions dans les réactions anaphylactiques - - PlanA2016 - ANR-16-CE15-0012 - AAPG2016 - VALID, Laboratoires d'excellence - GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE - - MILIEU INTERIEUR2010 - ANR-10-LABX-0069 - LABX - VALID, and Institut Pasteur International Docotal Program - PASTEURDOC - - H20202015-10-01 - 2020-10-01 - 665807 - VALID

Subjects
Hybridomas, [SDV.IMM] Life Sciences [q-bio]/Immunology, Fcγ receptors, cross-binding, IgG, Receptors, IgG, Immunology, Mice, Transgenic, 3. Good health, immune complexes, Mice, Immunoglobulin G, Animals, Humans, Immunology and Allergy, [SDV.IMM]Life Sciences [q-bio]/Immunology, Carrier Proteins
Abstract

International audience; Immunoglobulin G (IgG) is the predominant antibody class generated during infections and used for the generation of therapeutic antibodies. Antibodies are mainly characterized in or generated from animal models that support particular infections, respond to particular antigens or allow the generation of hybridomas. Due to the availability of numerous transgenic mouse models and the ease of performing bioassays with human blood cells in vitro, most antibodies from species other than mice and humans are tested in vitro using human cells and/or in vivo using mice. In this process, it is expected, but not yet systematically documented, that IgG from these species interact with human or mouse IgG receptors (FcRs). In this study, we undertook a systematic assessment of binding specificities of IgG from various species to the families of mouse and human FcRs, including their polymorphic variants. Our results document the specific binding patterns for each of these IgG (sub)classes, reveal possible caveats of antibody-based immunoassays, and will be a useful reference for the transition from one animal model to preclinical mouse models or human cell-based bioassays.

Published
2022

11. Single-cell analyses of immune thrombocytopenic patients reveal multiorgan dissemination of high-affinity autoreactive plasma cells

Author

Pablo Canales-Herrerias, Etienne Crickx, Matteo Broketa, Aurélien Sokal, Guilhem Chenon, Imane Azzaoui, Alexis Vandenberghe, Angga Perima, Bruno Iannascoli, Odile Richard-Le Goff, Carlos Castrillon, Guillaume Mottet, Delphine Sterlin, Ailsa Robbins, Marc Michel, Patrick England, Gael A. Millot, Klaus Eyer, Jean Baudry, Matthieu Mahevas, and Pierre Bruhns

Subjects
business.industry, medicine.drug_class, medicine.medical_treatment, Splenectomy, Autoantibody, Spleen, Monoclonal antibody, Antibody opsonization, medicine.anatomical_structure, Immune system, Immunology, medicine, Platelet, Bone marrow, business
Abstract

The major therapeutic goal for immune thrombocytopenia (ITP) is to restore normal platelet counts using drugs to promote platelet production or by interfering with mechanisms responsible for platelet destruction. 80% of patients possess anti-integrin αIIbβ3 (GPIIbIIIa) IgG autoantibodies causing platelet opsonization and phagocytosis. The spleen is considered the primary site of autoantibody production by autoreactive B cells and platelet destruction. The immediate failure in ~50% of patients to recover a normal platelet count after anti-CD20 Rituximab-mediated B cell depletion and splenectomy suggest that autoreactive, rituximab-resistant, IgG-secreting B cells (IgG-SC) reside in other anatomical compartments. We analyzed >3,300 single IgG-SC from spleen, bone marrow and/or blood of 27 patients with ITP revealing high inter-individual variability in affinity for GPIIbIIIa with variations over 3 logs. IgG-SC dissemination and range of affinities were however similar per patient. Longitudinal analysis of autoreactive IgG-SC upon treatment with anti-CD38 mAb daratumumab demonstrated variable outcomes, from complete remission to failure with persistence of high-affinity anti-GPIIbIIIa IgG-SC in the bone marrow. This study demonstrates the existence and dissemination of high-affinity autoreactive plasma cells in multiple anatomical compartments of patients with ITP that may cause the failure of current therapies.

Published
2021

12. Author Correction: High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics

Author

Samantha N. Stewart, Annabelle Gérard, Bingqing Shen, Colin Brenan, Pascaline Mary, Matthieu Delince, Scott McNamara, Klaus Eyer, Guillaume Mottet, Sami Ellouze, Pierre Bruhns, Adam Woolfe, Pablo Canales-Herrerias, Charina Ortega, Antoine Sautel-Caillé, Carlos Castrillon, Raphael Clement Li-Ming Doineau, Jean Baudry, Allan Jensen, Odile Richard-Le Goff, Clément Nizak, Bruno Iannascoli, Vera Menrath, Gregory Rose, Adeline Poitou, Luis Briseño-Roa, Yannick Pousse, Bin Jia, Roshan Kumar, Marcel Reichen, Baptiste Saudemont, Alexei Godina, Patrick England, Kevin Grosselin, Gaël A. Millot, Sosthene Essono, and Andrew D. Griffiths

Subjects
Cell activity, Chemistry, Biomedical Engineering, Molecular Medicine, Bioengineering, Droplet microfluidics, Computational biology, Applied Microbiology and Biotechnology, Throughput (business), Biotechnology
Abstract

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Published
2020

13. Evidence that neutrophils do not promote Echis carinatus venom-induced tissue destruction

Author

Friederike Jönsson, Bruno Iannascoli, Bianca Balbino, Laurent L. Reber, Julien Stackowicz, Ophélie Godon, Pierre Bruhns, Biliana Todorova, Anticorps en thérapie et pathologie - Antibodies in Therapy and Pathology, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Pierre et Marie Curie - Paris 6 (UPMC), Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), This work was supported by the Institut Pasteur, the French 'Institut National de la Santé et de la Recherche Médicale' (INSERM) and the European Research Council Seventh Framework Programme (ERC-2013-CoG 616050). J.S. acknowledges support from École Normale Supérieure Paris-Saclay, B.B. is supported by a stipend from the Pasteur—Paris University (PPU) International Ph.D. Program, F.J. is an employee of the Centre National de La Recherche Scientifique (CNRS), L.L.R. acknowledges support from the European Commission Marie Skłodowska-Curie Individual Fellowship (H2020-MSCA-IF-2014 656086)., European Project: 616050,EC:FP7:ERC,ERC-2013-CoG,MYELOSHOCK(2014), European Project: 656086,H2020,H2020-MSCA-IF-2014,AllergyBLOCK(2015), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], HAL-SU, Gestionnaire, Role of myeloid cells, their mediators and their antibody receptors in allergic shock (anaphylaxis) using humanized mouse models and clinical samples - MYELOSHOCK - - EC:FP7:ERC2014-09-01 - 2019-08-31 - 616050 - VALID, and Blocking peanut allergy through protective IgG antibodies - AllergyBLOCK - - H20202015-10-01 - 2017-09-30 - 656086 - VALID

Subjects
0301 basic medicine, Science, 030231 tropical medicine, Immunology, General Physics and Astronomy, Inflammation, Venom, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, 0302 clinical medicine, Viperidae, biology.animal, medicine, lcsh:Science, [SDV.IMM.II] Life Sciences [q-bio]/Immunology/Innate immunity, ComputingMilieux_MISCELLANEOUS, Innate immunity, Multidisciplinary, Innate immune system, biology, General Chemistry, biology.organism_classification, 030104 developmental biology, Echis carinatus, lcsh:Q, medicine.symptom, Leukocyte Reduction Procedures
Abstract

International audience

Published
2018

14. Neutrophils mediate antibody-induced antitumor effects in mice

Author

James P. Di Santo, Pierre Bruhns, Laurence Fiette, Friederike Jönsson, David A. Mancardi, Clifford A. Lowell, Bruno Iannascoli, Marcello Albanesi, Anticorps en Thérapie et Pathologie, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Pierre et Marie Curie - Paris 6 (UPMC), Histopathologie humaine et Modèles animaux, Institut Pasteur [Paris], Immunité Innée, University of California [San Francisco] (UCSF), University of California, This work was supported by the Institut Pasteur and the Institut National de la Santé et de la Recherche Médicale, by Agence Nationale de la Recherche (grant ANR-09-GENO-014-01), Fondation ARC pour la Recherche sur le Cancer and Ligue Nationale contre le Cancer (Comité de Paris) grants (P.B.), through an 'Equipe Labellisée Ligue Contre le Cancer' grant (J.P.D.S.), and by National Institutes of Health, National Institute of Allergy and Infectious Diseases grants AI065495 and AI068150 (C.A.L.)., The authors are thankful to our colleagues at Institut Pasteur (Paris, France): P. Bousso (Dynamics of Immune Responses Unit) for discussions and advice, M.-A. Nicola (Plate-Forme d'Imagerie Dynamique) for help with bioluminescence experiments, X. Montagutelli, Q. Mille, D. Montean, and G. Labas for help with mouse colony management (Central Animal Facility), C. Detchepare and C. Nizak for administrative help (Laboratoire Anticorps en Thérapie et Pathologie), and at University of California, San Francisco (San Francisco, CA): Y. Hu for animal husbandry. The authors are grateful to their colleagues for providing mice or reagents: R. Coffman (DNAX, Palo Alto, CA), B. Hann (University of California, San Francisco), J. J. Lee (Mayo Clinic, Scottsdale, AZ), H. Karasuyama (Tokyo Medical and Dental University Graduate School, Tokyo, Japan), M.P. Reilly (Jefferson Medical College, Philadelphia, PA), and M. Tarik (Montreal University, Montreal, QC, Canada)., ANR-09-GENO-0014,InflammAbs,Contrôle de l'inflammation par les RFc humains dans des modèles murins d'allergie et d'autoimmunité.(2009), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP), University of California [San Francisco] (UC San Francisco), University of California (UC), Martin, Marie, and Physiopathologie moléculaire: des maladies rares aux maladies communes - Contrôle de l'inflammation par les RFc humains dans des modèles murins d'allergie et d'autoimmunité. - - InflammAbs2009 - ANR-09-GENO-0014 - GENO - VALID

Subjects
MESH: Tumor Burden, Neutrophils, medicine.medical_treatment, Melanoma, Experimental, MESH: Neutrophils, MESH: Mice, Knockout, Biochemistry, Immunoglobulin G, MESH: Antibodies, Monoclonal, Antibodies, Monoclonal, Murine-Derived, Mice, 0302 clinical medicine, Conditional gene knockout, MESH: Animals, MESH: Trastuzumab, Mice, Knockout, 0303 health sciences, biology, Melanoma, Antibodies, Monoclonal, Hematology, Tumor Burden, 3. Good health, MESH: Melanoma, Experimental, 030220 oncology & carcinogenesis, [SDV.IMM]Life Sciences [q-bio]/Immunology, MESH: Rituximab, [SDV.IMM.IMM] Life Sciences [q-bio]/Immunology/Immunotherapy, Antibody, Rituximab, MESH: Xenograft Model Antitumor Assays, MESH: Cell Line, Tumor, [SDV.IMM] Life Sciences [q-bio]/Immunology, MESH: Mice, Transgenic, medicine.drug_class, Immunology, Mice, Nude, Breast Neoplasms, Mice, Transgenic, MESH: Receptors, IgG, Antibodies, Monoclonal, Humanized, Monoclonal antibody, 03 medical and health sciences, Antigen, MESH: Mice, Inbred C57BL, Cell Line, Tumor, MESH: Mice, Nude, MESH: Antibody-Dependent Cell Cytotoxicity, medicine, Animals, Humans, MESH: Mice, 030304 developmental biology, MESH: Humans, Receptors, IgG, Antibody-Dependent Cell Cytotoxicity, Cancer, [SDV.IMM.IMM]Life Sciences [q-bio]/Immunology/Immunotherapy, Cell Biology, Immunotherapy, Trastuzumab, medicine.disease, Xenograft Model Antitumor Assays, Mice, Inbred C57BL, MESH: Antibodies, Monoclonal, Humanized, MESH: Antibodies, Monoclonal, Murine-Derived, biology.protein, MESH: Breast Neoplasms
Abstract

International audience; Tumor engraftment followed by monoclonal antibody (mAb) therapy targeting tumor antigens represents a gold standard for assessing the efficiency of mAbs to eliminate tumor cells. Mouse models have demonstrated that receptors for the Fc portion of immunoglobulin G (FcγRs) are critical determinants of mAb therapeutic efficacy, but the FcγR-expressing cell populations responsible remain elusive. We show that neutrophils are responsible for mAb-induced therapy of both subcutaneous syngeneic melanoma and human breast cancer xenografts. mAb-induced tumor reduction, abolished in neutropenic mice, could be restored in FcγR-deficient hosts upon transfer of FcγR+ neutrophils or upon human FcγRIIA/CD32A transgenic expression. Finally, conditional knockout mice unable to perform FcγR-mediated activation and phagocytosis specifically in neutrophils were resistant to mAb-induced therapy. Our work suggests that neutrophils are necessary and sufficient for mAb-induced therapy of subcutaneous tumors, and represent a new and critical focal point for optimizing mAb-induced immunotherapies that will impact on human cancer treatment.

Published
2013

15. Cutting Edge: FcγRIII (CD16) and FcγRI (CD64) Are Responsible for Anti-Glycoprotein 75 Monoclonal Antibody TA99 Therapy for Experimental Metastatic B16 Melanoma

Author

Pierre Bruhns, Andrew J. Murphy, Marcello Albanesi, Laurence Zitvogel, Jeanette H. W. Leusen, Bruno Iannascoli, David A. Mancardi, Macdonald Lynn, Anticorps en Thérapie et Pathologie, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Allergologie Moléculaire et Cellulaire, Regeneron Pharmaceuticals [Tarrytown], Immunologie des tumeurs et immunothérapie (UMR 1015), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), University Medical Center [Utrecht], This work was supported by the Institut Pasteur, the INSERM, the Agence Nationale de la Recherche (Grant 09-GENO-014-01), the Fondation Association pour la Recherche sur le Cancer, and the Ligue Nationale contre le Cancer (Comité de Paris). M.A. is a scholar from the Pasteur Paris University International Doctoral Program., ANR-09-GENO-0014,InflammAbs,Contrôle de l'inflammation par les RFc humains dans des modèles murins d'allergie et d'autoimmunité.(2009), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Lung Neoplasms, Cyclophosphamide, Combination therapy, medicine.drug_class, MESH: Hybridomas, Immunology, Melanoma, Experimental, MESH: Receptors, IgG, CD16, Monoclonal antibody, MESH: Mice, Knockout, MESH: Antibodies, Monoclonal, Mice, Viral Proteins, 03 medical and health sciences, 0302 clinical medicine, MESH: Mice, Inbred C57BL, MESH: Arboviruses, MESH: Antibodies, Blocking, medicine, Animals, Immunology and Allergy, MESH: Animals, Antibodies, Blocking, MESH: Mice, 030304 developmental biology, Mice, Knockout, CD64, chemistry.chemical_classification, 0303 health sciences, Hybridomas, business.industry, Receptors, IgG, Antibodies, Monoclonal, MESH: Viral Proteins, MESH: Lung Neoplasms, 3. Good health, Mice, Inbred C57BL, MESH: Melanoma, Experimental, chemistry, Tumor reduction, [SDV.IMM]Life Sciences [q-bio]/Immunology, Glycoprotein, business, Arboviruses, B16 melanoma, 030215 immunology, medicine.drug
Abstract

Erratum in J Immunol. 2013 Feb 1;190(3):1381. Daëron, Marc [added].; International audience; mAb therapy for experimental metastatic melanoma relies on activating receptors for the Fc portion of IgG (FcγR). Opposing results on the respective contribution of mouse FcγRI, FcγRIII, and FcγRIV have been reported using the gp75-expressing B16 melanoma and the protective anti-gp75 mAb TA99. We analyzed the contribution of FcγRs to this therapy model using bioluminescent measurement of lung metastases loads, novel mouse strains, and anti-FcγR blocking mAbs. We found that the TA99 mAb-mediated effects in a combination therapy using cyclophosphamide relied on activating FcγRs. The combination therapy, however, was not more efficient than mAb therapy alone. We demonstrate that FcγRI and, unexpectedly, FcγRIII contributed to TA99 mAb therapeutic effects, whereas FcγRIV did not. Therefore, FcγRIII and FcγRI are, together, responsible for anti-gp75 mAb therapy of B16 lung metastases. Our finding that mouse FcγRIII contributes to Ab-induced tumor reduction correlates with clinical data on its human functional equivalent human FcγRIIIA (CD16A).

Published
2012

16. IgG subclasses determine pathways of anaphylaxis in mice

Author

Mark S. Cragg, Caitlin M. Gillis, Patrick England, Nico van Rooijen, Héloïse Beutier, Stephen J. Galli, Riccardo Sibilano, Laurent L. Reber, Pierre Bruhns, Friederike Jönsson, Bruno Iannascoli, Ophélie Godon, David A. Mancardi, Université Pierre et Marie Curie - Paris 6 (UPMC), Anticorps en thérapie et pathologie - Antibodies in Therapy and Pathology, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Biophysique Moléculaire (Plate-forme), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Stanford School of Medicine [Stanford], Stanford Medicine, Stanford University-Stanford University, University of Southampton, VU University Medical Center [Amsterdam], We thank our colleagues at Institut Pasteur, Paris: D. Sinnaya for administrative help, Stéphane Petres for help with antibody purifications, and Laurence Fiette for help with histological analyses. We thank our colleagues for their generous gifts: T. Moroy (Montreal University, Montreal, Quebec, Canada), C. Lowell (University of California at San Francisco, San Francisco, Calif), J. V. Ravetch (Rockefeller University, New York, NY, USA), and J. Leusen (University Medical Center, Utrecht, The Netherlands) for mice and R. Coffman (DNAX, Palo Alto, Calif), R. Good (USFCM, Tampa, Fla), B. Heyman (Uppsala Universitet, Uppsala, Sweden), H. Karasuyama (Tokyo Medical and Dental University Graduate School, Tokyo, Japan), and D. Voehringer (Universitätsklinikum, Erlangen, Germany) for antibodies. Cl2MDP was a gift of Roche Diagnostics GmbH., Bidault, Floran, Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Allergy, Basophil, chemistry.chemical_compound, basophil, 0302 clinical medicine, Immunology and Allergy, Macrophage, Myeloid Cells, Receptor, Chemistry, Antibodies, Monoclonal, neutrophil, Serum Albumin, Bovine, hemic and immune systems, 3. Good health, medicine.anatomical_structure, 1107 Immunology, monocyte, [SDV.IMM]Life Sciences [q-bio]/Immunology, Female, Anaphylaxis, Histamine, platelet-activating factor, [SDV.IMM] Life Sciences [q-bio]/Immunology, IgG, mouse model, Immunology, Mice, Transgenic, chemical and pharmacologic phenomena, IgG Fc receptor, macrophage, Article, 03 medical and health sciences, Downregulation and upregulation, medicine, Animals, Platelet-activating factor, Monocyte, Receptors, IgG, Immunoglobulin E, medicine.disease, histamine, Mice, Inbred C57BL, Protein Subunits, 030104 developmental biology, Immunoglobulin G, Haptens, 030215 immunology
Abstract

Background: Animal models have demonstrated that allergen-specific IgG confers sensitivity to systemic anaphylaxis that relies on IgG Fc receptors (Fc?Rs). Mouse IgG2a and IgG2b bind activating Fc?RI, Fc?RIII, and Fc?RIV and inhibitory Fc?RIIB; mouse IgG1 binds only Fc?RIII and Fc?RIIB. Although these interactions are of strikingly different affinities, these 3 IgG subclasses have been shown to enable induction of systemic anaphylaxis.Objective: We sought to determine which pathways control the induction of IgG1-, IgG2a-, and IgG2b-dependent passive systemic anaphylaxis.Methods: Mice were sensitized with IgG1, IgG2a, or IgG2b anti-trinitrophenyl mAbs and challenged with trinitrophenyl-BSA intravenously to induce systemic anaphylaxis that was monitored by using rectal temperature. Anaphylaxis was evaluated in mice deficient for Fc?Rs injected with mediator antagonists or in which basophils, monocytes/macrophages, or neutrophils had been depleted. Fc?R expression was evaluated on these cells before and after anaphylaxis.Results: Activating Fc?RIII is the receptor primarily responsible for all 3 models of anaphylaxis, and subsequent downregulation of this receptor was observed. These models differentially relied on histamine release and the contribution of mast cells, basophils, macrophages, and neutrophils. Strikingly, basophil contribution and histamine predominance in mice with IgG1- and IgG2b-induced anaphylaxis correlated with the ability of inhibitory Fc?RIIB to negatively regulate these models of anaphylaxis.Conclusion: We propose that the differential expression of inhibitory Fc?RIIB on myeloid cells and its differential binding of IgG subclasses controls the contributions of mast cells, basophils, neutrophils, and macrophages to IgG subclass–dependent anaphylaxis. Collectively, our results unravel novel complexities in the involvement and regulation of cell populations in IgG-dependent reactions in vivo.

Published
2016

17. Human FcγRIIA induces anaphylactic and allergic reactions

Author

Nico van Rooijen, Friederike Jönsson, Lawrence B. Schwartz, Takao Shimizu, Wei Zhao, Marc Daëron, David A. Mancardi, Yoshihiro Kita, Bruno Iannascoli, Pierre Bruhns, Huot Khun, Allergologie Moléculaire et Cellulaire, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Virginia Commonwealth University (VCU), The University of Tokyo (UTokyo), Histopathologie humaine et Modèles animaux, Institut Pasteur [Paris], Department of Molecular Cell Biology [Amsterdam UMC], Vrije Universiteit Medical Centre (VUMC), Vrije Universiteit Amsterdam [Amsterdam] (VU)-Vrije Universiteit Amsterdam [Amsterdam] (VU), This work was supported by the Institut Pasteur, Inserm, the Agence Nationale de la Recherche (ANR, grant GENOPAT-09-GENO-014-01), the Société Française d'Allergologie (SFA, Soutien de la recherche en allergologie 2010), and the Balsan company. F.J. is a recipient of a fellowship from the Fondation pour la Recherche Médicale (FRM). D.A.M. is a recipient of a fellowship from the Institut Pasteur (Bourse Roux). W.Z. and L.B.S. were funded in part by U19AI077435 from the National Institutes of Health., ANR-09-GENO-0014,InflammAbs,Contrôle de l'inflammation par les RFc humains dans des modèles murins d'allergie et d'autoimmunité.(2009), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP), Molecular cell biology and Immunology, and CCA - Immuno-pathogenesis

Subjects
MESH: Inflammation, Allergy, Respiratory System, mast cells, airway inflammation, MESH: Neutrophils, Immunoglobulin E, MESH: Mice, Knockout, Biochemistry, 0302 clinical medicine, neutrophils, MESH: Animals, [SDV.IMM.ALL]Life Sciences [q-bio]/Immunology/Allergology, Receptor, Cells, Cultured, MESH: Respiratory System, Mice, Knockout, 0303 health sciences, biology, Passive Cutaneous Anaphylaxis, Airway inflammation, MESH: Mast Cells, Hematology, immunoglobulin e, macrophages, 3. Good health, immunoglobulin g, [SDV.IMM]Life Sciences [q-bio]/Immunology, hypersensitivity, monocytes, MESH: Passive Cutaneous Anaphylaxis, MESH: Cells, Cultured, mice, MESH: Mice, Transgenic, MESH: Hypersensitivity, Immunology, Mice, Transgenic, MESH: Receptors, IgG, 03 medical and health sciences, MESH: Eosinophils, MESH: Mice, Inbred C57BL, In vivo, anaphylaxis, medicine, Animals, Humans, MESH: Mice, 030304 developmental biology, Inflammation, MESH: Humans, Passive anaphylaxis, business.industry, Receptors, IgG, Anaphylactic reactions, Cell Biology, medicine.disease, Eosinophils, Mice, Inbred C57BL, biology.protein, business, 030215 immunology
Abstract

IgE and IgE receptors (FcϵRI) are well-known inducers of allergy. We recently found in mice that active systemic anaphylaxis depends on IgG and IgG receptors (FcγRIIIA and FcγRIV) expressed by neutrophils, rather than on IgE and FcϵRI expressed by mast cells and basophils. In humans, neutrophils, mast cells, basophils, and eosinophils do not express FcγRIIIA or FcγRIV, but FcγRIIA. We therefore investigated the possible role of FcγRIIA in allergy by generating novel FcγRIIA-transgenic mice, in which various models of allergic reactions induced by IgG could be studied. In mice, FcγRIIA was sufficient to trigger active and passive anaphylaxis, and airway inflammation in vivo. Blocking FcγRIIA in vivo abolished these reactions. We identified mast cells to be responsible for FcγRIIA-dependent passive cutaneous anaphylaxis, and monocytes/macrophages and neutrophils to be responsible for FcγRIIA-dependent passive systemic anaphylaxis. Supporting these findings, human mast cells, monocytes and neutrophils produced anaphylactogenic mediators after FcγRIIA engagement. IgG and FcγRIIA may therefore contribute to allergic and anaphylactic reactions in humans.

Published
2012

18. Peritoneal Cell-Derived Mast Cells: An In Vitro Model of Mature Serosal-Type Mouse Mast Cells

Author

Michel Arock, Antoine Ribadeau Dumas, Marc Daëron, Karine Roget, Bruno Iannascoli, Cécile Schiffer, Odile Malbec, Allergologie Moléculaire et Cellulaire, Institut Pasteur [Paris] - Institut National de la Santé et de la Recherche Médicale (INSERM), Cytokines, hématopoïèse et réponse immune (CHRI), Université Paris Descartes - Paris 5 (UPD5) - Institut National de la Santé et de la Recherche Médicale (INSERM) - Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Chemokine, MESH : Phosphoric Monoester Hydrolases, Cellular differentiation, Cell, Cell Count, MESH : Cell Count, MESH : Models, Immunological, Immunoglobulin E, MESH: Mice, Knockout, MESH: Down-Regulation, MESH : Down-Regulation, Mice, Serous Membrane, 0302 clinical medicine, Immunology and Allergy, MESH: Animals, Mast Cells, Cells, Cultured, Mice, Knockout, MESH: Immunoglobulin G, 0303 health sciences, education.field_of_study, biology, Inositol Polyphosphate 5-Phosphatases, MESH: Immunoglobulin E, Degranulation, MESH: Peritoneum, Cell Differentiation, MESH: Mast Cells, Mast cell, Cell biology, medicine.anatomical_structure, Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases, MESH : Serous Membrane, MESH: Phosphoric Monoester Hydrolases, [SDV.IMM]Life Sciences [q-bio]/Immunology, MESH : Mast Cells, Peritoneum, MESH : Cell Differentiation, MESH: Cells, Cultured, MESH: Cell Differentiation, MESH : Immunoglobulin G, [SDV.IMM] Life Sciences [q-bio]/Immunology, Immunology, Population, Down-Regulation, MESH: Serous Membrane, MESH : Mice, Inbred C57BL, MESH: Models, Immunological, MESH : Immunoglobulin E, 03 medical and health sciences, Immune system, MESH: Mice, Inbred C57BL, MESH : Cells, Cultured, MESH : Mice, medicine, Animals, education, MESH: Mice, MESH : Peritoneum, 030304 developmental biology, MESH: Cell Count, Models, Immunological, Phosphoric Monoester Hydrolases, Mice, Inbred C57BL, Immunoglobulin G, biology.protein, MESH : Mice, Knockout, MESH : Animals, 030215 immunology
Abstract

Bone marrow-derived mast cells (BMMC) have been used extensively as a mast cell model. BMMC, however, are immature cells that have no known physiological equivalent in tissues. They do not respond to IgG immune complexes. They may therefore not be appropriate for studying the physiopathology of IgE-induced allergies or IgG-induced tissue-specific inflammatory diseases which both depend on mature mast cells. Resident peritoneal mast cells are a minor population of differentiated cells that are not readily purified. They, however, can be expanded in culture to generate large numbers of homogeneous cells. We show here that these peritoneal cell-derived mast cells (PCMC) are mature serosal-type mouse mast cells which retain most morphological, phenotypic, and functional features of peritoneal mast cells. Like peritoneal mast cells, PCMC respond to IgG Abs. IgG immune complex-induced responses depended on FcγRIIIA and were negatively regulated by FcγRIIB. We found that a moderate FcγRIIB-dependent negative regulation, due not to a higher FcγRIIIA/FcγRIIB ratio, but to a relatively inefficient use of the lipid phosphatase SHIP1, determines this property of PCMC. PCMC also respond to IgE Abs. IgE-induced PCMC responses, however, differed quantitatively and qualitatively from BMMC responses. PCMC secreted no or much lower amounts of lipid mediators, chemokines, and cytokines, but they contained and released much higher amounts of preformed granular mediators. PCMC, but not BMMC, also contained and, upon degranulation, released molecules with a potent proteolytic activity. These properties make PCMC a useful new model for understanding the physiopathology of mast cells in IgE- and IgG-dependent tissue inflammation.

Published
2007

19. Evidence of a role for Munc18-2 and microtubules in mast cell granule exocytosis

Author

Sophie Martin-Verdeaux, Nadine Varin-Blank, Juan Rivera, Bruno Iannascoli, Michèle Roa, Isabel Pombo, and Ulrich Blank

Subjects
Vesicular Transport Proteins, Nerve Tissue Proteins, Biology, Membrane Fusion, Microtubules, Exocytosis, Mice, chemistry.chemical_compound, Munc18 Proteins, Microtubule, Animals, Protein Isoforms, Syntaxin, Mast Cells, Pseudopodia, Cytoskeleton, Inflammation, Qa-SNARE Proteins, Secretory Vesicles, Cell Membrane, Granule (cell biology), Membrane Proteins, Proteins, Lipid bilayer fusion, Cell Biology, Immunoglobulin E, Actins, Rats, Cell biology, Protein Transport, Nocodazole, chemistry, COS Cells, Cell Surface Extensions, Lamellipodium, Protein Binding
Abstract

Compound exocytosis of inflammatory mediators from mast cells requires SNARE and a series of accessory proteins. However, the molecular steps that regulate secretory granule movement and membrane fusion as well as the role of the cytoskeleton are still poorly understood. Here, we report on our investigation of the role of syntaxin-binding Munc18 isoforms and the microtubule network in this process. We found that mast cells express Munc18-2, which interacts with target SNAREs syntaxin 2 or 3, as well as Munc18-3, which interacts with syntaxin 4. Munc18-2 was localised to secretory granules, whereas Munc18-3 was found on the plasma membrane. Increased expression of Munc18-2 and derived peptides containing an interfering effector loop inhibited IgE-triggered exocytosis, while increased expression of Munc18-3 showed no effect. Munc18-2 localisation on granules is polarised; however, upon stimulation Munc18-2 redistributed into forming lamellipodia and persisted on granules that were aligned along microtubules, but was excluded from F-actin ruffles. Disruption of the microtubule network with nocodazole provoked Munc18-2 redistribution and affected mediator release. These findings suggest a role for Munc18-2 and the microtubule network in the regulation of secretory granule dynamics in mast cells.

Published
2003

20. Proteomic Analysis of the SH2 Domain-containing Leukocyte Protein of 76 kDa (SLP76) Interactome in Resting and Activated Primary Mast Cells

Author

Luca Grieco, Marc Daëron, Anna Niarakis, Yohann Couté, Yacine Bounab, Bruno Iannascoli, Jérôme Garin, Denis Thieffry, Anne-Marie-Hesse, Eunkyung Lie, Kong-Peng Lam, Caroline Demangel, Bernard Malissen, and Romain Roncagalli

Subjects
Cell signaling, Cell type, T cell, Degranulation, Biology, Mast cell, SH2 domain, Biochemistry, Interactome, Analytical Chemistry, Cell biology, Interleukin 33, medicine.anatomical_structure, medicine, Molecular Biology, Additions & Corrections
Abstract

We report the first proteomic analysis of the SLP76 interactome in resting and activated primary mouse mast cells. This was made possible by a novel genetic approach used for the first time here. It consists in generating knock-in mice that express signaling molecules bearing a C-terminal tag that has a high affinity for a streptavidin analog. Tagged molecules can be used as molecular baits to affinity-purify the molecular complex in which they are engaged, which can then be studied by mass spectrometry. We examined first SLP76 because, although this cytosolic adapter is critical for both T cell and mast cell activation, its role is well known in T cells but not in mast cells. Tagged SLP76 was expressed in physiological amounts and fully functional in mast cells. We unexpectedly found that SLP76 is exquisitely sensitive to mast cell granular proteases, that Zn(2+)-dependent metalloproteases are especially abundant in mast cells and that they were responsible for SLP76 degradation. Adding a Zn(2+) chelator fully protected SLP76 in mast cell lysates, thereby enabling an efficient affinity-purification of this adapter with its partners. Label-free quantitative mass spectrometry analysis of affinity-purified SLP76 interactomes uncovered both partners already described in T cells and novel partners seen in mast cells only. Noticeably, molecules inducibly recruited in both cell types primarily concur to activation signals, whereas molecules recruited in activated mast cells only are mostly associated with inhibition signals. The transmembrane adapter LAT2, and the serine/threonine kinase with an exchange factor activity Bcr were the most recruited molecules. Biochemical and functional validations established the unexpected finding that Bcr is recruited by SLP76 and positively regulates antigen-induced mast cell activation. Knock-in mice expressing tagged molecules with a normal tissue distribution and expression therefore provide potent novel tools to investigate signalosomes and to uncover novel signaling molecules in mast cells.

Published
2014

21. IgE Receptor Type I-dependent Regulation of a Rab3D-associated Kinase

Author

Ulrich Blank, Juan Rivera, Joëlle Le Mao, Bruno Iannascoli, Isabel Pombo, Sophie Martin-Verdeaux, and Ludovic Deriano

Subjects
Degranulation, Cell Biology, Biology, Mast cell, Biochemistry, Syntaxin 3, Cell biology, EGTA, chemistry.chemical_compound, medicine.anatomical_structure, chemistry, SNAP23, medicine, Syntaxin, Rab, Kinase activity, Molecular Biology
Abstract

Following activation through high affinity IgE receptors (FceRI), mast cells release, within a few minutes, their granule content of inflammatory and allergic mediators. FceRI-induced degranulation is a SNARE (soluble N -ethylmaleimide attachment proteinreceptors)-dependent fusion process. It is regulated by Rab3D, a subfamily member of Rab GTPases. Evidence exists showing that Rab3 action is calcium-regulated although the molecular mechanisms remain unclear. To obtain an understanding of Rab3D function we have searched for Rab3D-associated effectors that respond to allergic triggering through FceRI. Using the RBL-2H3 mast cell line we detected a Ser/Thr kinase activity, termed here Rak3D (fromRab3D-associatedkinase), because it was specifically co-immunoprecipitated with anti-Rab3D antibody. Rak3D activity, as measured by its auto- or transphosphorylation, was maximal in resting cells and decreased upon stimulation. The down-regulation of the observed activity was blocked with EGTA, but not with other degranulation inhibitors, suggesting that its activity functions downstream of calcium influx. We found that Rak3D phosphorylates the NH2-terminal regulatory domain of the t-SNARE syntaxin 4, but not syntaxin 2 or 3. The phosphorylation of syntaxin 4 decreased its binding to its partner SNAP23. Thus, we propose a novel phosphorylation-dependent mechanism by which Rab3D controls SNARE assembly in a calcium-dependent manner.

Published
2001

22. Specific signaling pathways in the regulation of TNF-α mRNA synthesis and TNF-α secretion in RBL-2H3 mast cells stimulated through the high affinity IgE receptor

Author

Claudine Guerin-Marchand, A. Weyer, F. Marchand, Bruno Iannascoli, Ulrich Blank, Bernard David, and C. Pelletier

Subjects
Indoles, Immunology, Gene Expression, Biology, Maleimides, Wortmannin, chemistry.chemical_compound, Ribonucleases, Gene expression, Tumor Cells, Cultured, Animals, Secretion, Mast Cells, RNA, Messenger, Enzyme Inhibitors, Receptor, Egtazic Acid, Protein Kinase C, Protein kinase C, Phosphoinositide-3 Kinase Inhibitors, Pharmacology, Receptors, IgE, Reverse Transcriptase Polymerase Chain Reaction, Tumor Necrosis Factor-alpha, Molecular biology, beta-N-Acetylhexosaminidases, Rats, Androstadienes, EGTA, Leukemia, Basophilic, Acute, chemistry, Tetradecanoylphorbol Acetate, Tumor necrosis factor alpha, Signal transduction, Signal Transduction
Abstract

In the present study, we investigated signal transduction pathways involved in TNF-alpha gene expression and TNF-alpha secretion by mast cells stimulated through the high affinity IgE receptor (FcepsilonRI).TNF-alpha mRNA steady state levels and TNF-alpha secretion in the presence of specific pharmacological agents were monitored using rat basophilic leukemia cells (RBL-2H3) stimulated through FcepsilonRI. Relative amounts of TNF-alpha mRNA versus beta-actin levels were quantified by RNase protection and RT-PCR assays. TNF-alpha secretion was measured by a current ELISA test.We show that EGTA (5 mM) prevented TNF-alpha mRNA expression and TNF-alpha secretion in antigen-stimulated cells. The protein kinase C (PKC) inhibitor bisindolylmaleimide I substantially blocked TNF-alpha secretion at 2 microM but had only a marginal effect on TNF-alpha mRNA expression. The results were similar when PKC isoforms were depleted by long-term exposure to 100 nM phorbol ester (PMA). The PI 3-kinase inhibitor wortmannin blocked TNF-alpha secretion at low doses (EC50= 13 nM), but only partially affected mRNA expression.Our results show that in FcepsilonRI-stimulated RBL-2H3 cells calcium mobilization, activation of PKC and PI 3-kinase are necessary for TNF-alpha secretion while for the increased TNF-alpha mRNA expression PKC activity is dispensable and PI 3-kinase activity only partially required.

Published
1998

23. Proteomic Analysis of the SH2Domain-containing Leukocyte Protein of 76 kDa (SLP76) Interactome

Author

Luca Grieco, Anne-Marie Hesse, Jérôme Garin, Yacine Bounab, Eunkyung Lie, Caroline Demangel, Romain Roncagalli, Kong-Peng Lam, Denis Thieffry, Anna Niarakis, Bruno Iannascoli, Marc Daëron, Bernard Malissen, Yohann Couté, Allergologie Moléculaire et Cellulaire, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie de Marseille - Luminy (CIML), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Immunobiologie de l'Infection, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie du Développement de Marseille (IBDM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de biologie de l'ENS Paris (IBENS), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), École normale supérieure - Paris (ENS Paris)-École normale supérieure - Paris (ENS Paris)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Collège de France (CdF)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Proteomics, Cell type, Cell signaling, T cell, Bone Marrow Cells, Mice, Transgenic, Biology, Biochemistry, Interactome, Analytical Chemistry, Mice, 03 medical and health sciences, 0302 clinical medicine, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], medicine, Animals, Mast Cells, Protein Interaction Maps, Molecular Biology, Cells, Cultured, Adaptor Proteins, Signal Transducing, 030304 developmental biology, 0303 health sciences, Receptors, IgE, Kinase, Research, T-cell receptor, Phosphoproteins, Mast cell, Cell biology, Mice, Inbred C57BL, Interleukin 33, medicine.anatomical_structure, Proto-Oncogene Proteins c-bcr, 030215 immunology
Abstract

International audience; We report the first proteomic analysis of the SLP76 interactome in resting and activated primary mouse mast cells. This was made possible by a novel genetic approach used for the first time here. It consists in generating knock-in mice that express signaling molecules bearing a C-terminal tag that has a high affinity for a streptavidin analog. Tagged molecules can be used as molecular baits to affinity-purify the molecular complex in which they are engaged, which can then be studied by mass spectrometry. We examined first SLP76 because, although this cytosolic adapter is critical for both T cell and mast cell activation, its role is well known in T cells but not in mast cells. Tagged SLP76 was expressed in physiological amounts and fully functional in mast cells. We unexpectedly found that SLP76 is exquisitely sensitive to mast cell granular proteases, that Zn2+-dependent metalloproteases are especially abundant in mast cells and that they were responsible for SLP76 degradation. Adding a Zn2+ chelator fully protected SLP76 in mast cell lysates, thereby enabling an efficient affinity-purification of this adapter with its partners. Label-free quantitative mass spectrometry analysis of affinity-purified SLP76 interactomes uncovered both partners already described in T cells and novel partners seen in mast cells only. Noticeably, molecules inducibly recruited in both cell types primarily concur to activation signals, whereas molecules recruited in activated mast cells only are mostly associated with inhibition signals. The transmembrane adapter LAT2, and the serine/threonine kinase with an exchange factor activity Bcr were the most recruited molecules. Biochemical and functional validations established the unexpected finding that Bcr is recruited by SLP76 and positively regulates antigen-induced mast cell activation. Knock-in mice expressing tagged molecules with a normal tissue distribution and expression therefore provide potent novel tools to investigate signalosomes and to uncover novel signaling molecules in mast cells.

Published
2013

24. Mouse and human neutrophils induce anaphylaxis

Author

Hajime Karasuyama, Nico van Rooijen, David A. Mancardi, Marc Daëron, Takao Shimizu, Yoshihiro Kita, Bruno Iannascoli, Pierre Bruhns, Friederike Jönsson, Allergologie Moléculaire et Cellulaire, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), The University of Tokyo (UTokyo), Department of Immune Regulation [Tokyo, Japan], Graduate School of Medical and Dental Sciences [Tokyo, Japan]-Tokyo Medical and Dental University [Japan] (TMDU), Department of Molecular Cell Biology [Amsterdam UMC], Vrije Universiteit Medical Centre (VUMC), Vrije Universiteit Amsterdam [Amsterdam] (VU)-Vrije Universiteit Amsterdam [Amsterdam] (VU), This work was supported by the Institut Pasteur, the Institut National de la Santé et de la Recherche Médicale (INSERM), the Agence Nationale de la Recherche (ANR) (grants 05-JCJC-0236-01 and GENOPAT-09-GENO-014-01), the Fondation pour la Recherche Médicale (FRM) (Programme Allergie 2007), by funding under the Sixth Research Framework Programme of the European Union, Project MUGEN (MUGEN LSHG-CT-2005-005203), by the Société Française d’Allergologie (SFA) (Soutien de la recherche en allergologie 2010) and the Balsan company. F. Jönsson was financially supported by ANR, MUGEN, and is currently a recipient of a fellowship from the FRM. D.A. Mancardi was financially supported by FRM and is currently a recipient of a fellowship from the Institut Pasteur (Bourse Roux). T. Shimizu and Y. Kita are supported in part by Grants-in-Aid from the Ministry of Education, Science, Culture, Sports and Technology of Japan., ANR-05-JCJC-0236,FcR & Allergy,Roles and interactions of human FcR in allergic symptoms induced by human immunoglobulins and allergens: construction of a ' humanized ' mouse model.(2005), ANR-09-GENO-0014,InflammAbs,Contrôle de l'inflammation par les RFc humains dans des modèles murins d'allergie et d'autoimmunité.(2009), European Project: 39516,MUGEN, Martin, Marie, Jeunes chercheuses et jeunes chercheurs - Roles and interactions of human FcR in allergic symptoms induced by human immunoglobulins and allergens: construction of a ' humanized ' mouse model. - - FcR & Allergy2005 - ANR-05-JCJC-0236 - JCJC - VALID, Physiopathologie moléculaire: des maladies rares aux maladies communes - Contrôle de l'inflammation par les RFc humains dans des modèles murins d'allergie et d'autoimmunité. - - InflammAbs2009 - ANR-09-GENO-0014 - GENO - VALID, INTEGRATED FUNCTIONAL GENOMICS IN MUTANT MOUSE MODELS AS TOOLS TO INVESTIGATE THE COMPLEXITY OF HUMAN IMMUNOLOGICAL DISEASE - MUGEN - 39516 - OLD, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Molecular cell biology and Immunology, and CCA - Immuno-pathogenesis

Subjects
Male, MESH: Mice, Inbred NOD, Neutrophils, MESH: Neutrophils, Immunoglobulin E, MESH: Mice, Knockout, Immunoglobulin G, Neutrophil Activation, MESH: Basophils, chemistry.chemical_compound, Mice, 0302 clinical medicine, Mice, Inbred NOD, Medicine, MESH: Animals, [SDV.IMM.ALL]Life Sciences [q-bio]/Immunology/Allergology, Receptor, Mice, Knockout, MESH: Immunoglobulin G, 0303 health sciences, biology, MESH: Neutrophil Activation, General Medicine, 3. Good health, Basophils, [SDV.IMM]Life Sciences [q-bio]/Immunology, Antibody, Histamine, Anaphylaxis, [SDV.IMM.ALL] Life Sciences [q-bio]/Immunology/Allergology, Research Article, [SDV.IMM] Life Sciences [q-bio]/Immunology, MESH: Mice, Transgenic, Mice, Transgenic, MESH: Receptors, IgG, 03 medical and health sciences, Species Specificity, MESH: Mice, Inbred C57BL, Animals, Humans, MESH: Species Specificity, Platelet Activating Factor, MESH: Mice, 030304 developmental biology, MESH: Platelet Activating Factor, MESH: Humans, Platelet-activating factor, business.industry, Receptors, IgG, medicine.disease, MESH: Male, Mice, Inbred C57BL, MESH: Anaphylaxis, chemistry, Immunology, biology.protein, IgG deficiency, business, 030215 immunology
Abstract

International audience; Anaphylaxis is a life-threatening hyperacute immediate hypersensitivity reaction. Classically, it depends on IgE, FcεRI, mast cells, and histamine. However, anaphylaxis can also be induced by IgG antibodies, and an IgG1-induced passive type of systemic anaphylaxis has been reported to depend on basophils. In addition, it was found that neither mast cells nor basophils were required in mouse models of active systemic anaphylaxis. Therefore, we investigated what antibodies, receptors, and cells are involved in active systemic anaphylaxis in mice. We found that IgG antibodies, FcγRIIIA and FcγRIV, platelet-activating factor, neutrophils, and, to a lesser extent, basophils were involved. Neutrophil activation could be monitored in vivo during anaphylaxis. Neutrophil depletion inhibited active, and also passive, systemic anaphylaxis. Importantly, mouse and human neutrophils each restored anaphylaxis in anaphylaxis-resistant mice, demonstrating that neutrophils are sufficient to induce anaphylaxis in mice and suggesting that neutrophils can contribute to anaphylaxis in humans. Our results therefore reveal an unexpected role for IgG, IgG receptors, and neutrophils in anaphylaxis in mice. These molecules and cells could be potential new targets for the development of anaphylaxis therapeutics if the same mechanism is responsible for anaphylaxis in humans.

Published
2011

25. Specificity and affinity of human Fc{gamma} receptors and their polymorphic variants for human IgG subclasses

Author

Nadine Fernandez, Marc Daëron, Patrick England, Bruno Iannascoli, David A. Mancardi, Pierre Bruhns, Sylvie Jorieux, Allergologie Moléculaire et Cellulaire, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Biophysique des Macromolécules et de leurs Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Laboratoire Français du fractionnement et des Biotechnologies, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Immunology, Plasma protein binding, CHO Cells, FCGR2A, Biochemistry, Immunoglobulin G, 03 medical and health sciences, Mice, 0302 clinical medicine, Cricetulus, Cricetinae, parasitic diseases, Animals, Humans, [SDV.IMM.ALL]Life Sciences [q-bio]/Immunology/Allergology, Receptor, 030304 developmental biology, 0303 health sciences, biology, Receptors, IgG, Antibodies, Monoclonal, Cell Biology, Hematology, Molecular biology, 3. Good health, Polyclonal antibodies, Humoral immunity, biology.protein, Fc-Gamma Receptor, Antibody, 030215 immunology, Protein Binding
Abstract

International audience; Distinct genes encode six human receptors for IgG (hFcgammaRs), three of which have two or three polymorphic variants. The specificity and affinity of individual hFcgammaRs for the four human IgG subclasses is unknown. This information is critical for antibody-based immunotherapy which has been increasingly used in the clinics. We investigated the binding of polyclonal and monoclonal IgG1, IgG2, IgG3 and IgG4 to FcgammaRI, FcgammaRIIA, IIB and IIC, FcgammaRIIIA and IIIB and all known polymorphic variants. Wt and low-fucosylated IgG1 anti-CD20 and anti-RhD mAbs were also examined. We found: 1) that IgG1 and IgG3 bind to all hFcgammaRs; 2) that IgG2 bind not only to FcgammaRIIAH131, but also, with a lower affinity, to FcgammaRIIAR131 and FcgammaRIIIAV158; 3) that IgG4 bind to FcgammaRI, FcgammaRIIA, IIB and IIC and FcgammaRIIIAV158; 4) that the inhibitory receptor FcgammaRIIB has a lower affinity for IgG1, IgG2 and IgG3 than all other hFcgammaRs. We also identified parameters which determine the specificity and affinity of hFcgammaRs for IgG subclasses. These results document how hFcgammaR specificity and affinity may account for the biological activities of antibodies. They therefore highlight the role of specific hFcgammaRs in the therapeutic and pathogenic effects of antibodies in disease.

Published
2008

26. The engagement of activating FcgammaRs inhibits primate lentivirus replication in human macrophages

Author

Florence Herschke, Odile Malbec, Annie David, Bruno Iannascoli, Gianfranco Pancino, Marianne Lucas, Asier Sáez-Cirión, Françoise Barré-Sinoussi, Jean-François Mouscadet, Marc Daëron, Pierre Versmisse, Régulation des Infections Rétrovirales, Institut Pasteur [Paris] (IP), Allergologie Moléculaire et Cellulaire, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Interactions Moléculaires Flavivirus-Hôtes, Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), and Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Cachan (ENS Cachan)

Subjects
Primates, Chemokine, DNA, Complementary, Immunology, Stimulation, MESH: Receptors, IgG, Virus Replication, Article, MESH: Proviruses, MESH: HIV-1, 03 medical and health sciences, 0302 clinical medicine, Immune system, Proviruses, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Gene expression, Animals, Humans, Immunology and Allergy, Secretion, MESH: Animals, Cells, Cultured, 030304 developmental biology, MESH: Lentivirus, 0303 health sciences, MESH: Humans, biology, Macrophages, Lentivirus, Receptors, IgG, MESH: Virus Replication, virus diseases, MESH: Macrophage Activation, MESH: Macrophages, MESH: DNA, Complementary, Macrophage Activation, biology.organism_classification, Virology, Reverse transcriptase, 3. Good health, MESH: DNA, Viral, MESH: Primates, Viral replication, DNA, Viral, HIV-1, biology.protein, 030215 immunology, MESH: Cells, Cultured
Abstract

We previously reported that the stimulation of monocyte-derived macrophages (MDM) by plate-bound i.v. Igs inhibits HIV-1 replication. In this study, we show that IgG immune complexes also suppress HIV-1 replication in MDMs and that activating receptors for the Fc portion of IgG–FcγRI, FcγRIIA, and FcγRIII–are responsible for the inhibition. MDM stimulation through FcγRs induces activation signals and the secretion of HIV-1 modulatory cytokines, such as M-CSF, TNF-α, and macrophage-derived chemokine. However, none of these cytokines contribute to HIV-1 suppression. HIV-1 entry and postintegration steps of viral replication are not affected, whereas reduced levels of reverse transcription products and of integrated proviruses, as determined by real-time PCR analysis, account for the suppression of HIV-1 gene expression in FcγR-activated MDMs. We found that FcγR-dependent activation of MDMs also inhibits the replication of HIV-2, SIVmac, and SIVagm, suggesting a common control mechanism for primate immunodeficiency lentiviruses in activated macrophages.

Published
2006

27. Human serum IgE-mediated mast cell degranulation shows poor correlation to allergen-specific IgE content

Author

A. Weyer, Salaheddine Mécheri, F. Marchand, L. Guilloux, Ulrich Blank, and Bruno Iannascoli

Subjects
Hypersensitivity, Immediate, Allergy, Dermatophagoides pteronyssinus, Immunology, Dose-Response Relationship, Immunologic, Biology, Immunoglobulin E, medicine.disease_cause, Transfection, Cell Degranulation, Cell Line, Allergen, medicine, Immunology and Allergy, Animals, Humans, Mast Cells, Receptor, Dactylis, Sensitization, Cells, Cultured, Degranulation, Allergens, Mast cell, medicine.disease, beta-N-Acetylhexosaminidases, Rats, medicine.anatomical_structure, biology.protein
Abstract

Background: Although allergen-specific IgE content in serum can be determined immunochemically, little is known about the relationship between this parameter and the strength of the degranulation response upon allergen triggering. Objectives: Analyse the degranulation capacity of immunochemically defined purified and serum IgE after challenge with anti-IgE or allergen using a rat mast cell line (RBL) transfected with the α-chain of the human high-affinity IgE receptor (FcɛRI). Methods: Purified IgE specific for 4-hydroxy-3nitrophenylacetyl, purified IgE of unknown specificity, and sera from allergic patients sensitive to Dermatophagoides pteronyssinus and Dactylis glomerata were assessed. Degranulation was measured by a β-hexosaminidase release assay after anti-IgE or allergen-specific challenge. Results: For purified monoclonal IgE a significant correlation (r = 0.97) was found between the proportion of bound allergen-specific IgE and the strength of the degranulation response. In contrast, no correlation (r = 0.27) was detected after sensitization with serum IgE. Conclusion: Our studies demonstrate that mast cell activation mediated through IgE from allergic patients is a result of complex relationships that are not only dependent on allergen-specific IgE content but also relate to the capacity to efficiently sensitize and trigger the signalling responses that lead to degranulation.

Published
2003

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