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1. 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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2. An Engineered Human Fc variant With Exquisite Selectivity for FcγRIIIaV158 Reveals That Ligation of FcγRIIIa Mediates Potent Antibody Dependent Cellular Phagocytosis With GM-CSF-Differentiated Macrophages

Author

Tae Hyun Kang, Chang-Han Lee, George Delidakis, Jiwon Jung, Odile Richard-Le Goff, Jiwon Lee, Jin Eyun Kim, Wissam Charab, Pierre Bruhns, and George Georgiou

Subjects
Fc engineering, ADCP, FcgR, macrophage, monocyte, Immunologic diseases. Allergy, RC581-607
Abstract

IgG antibodies mediate the clearance of target cells via the engagement of Fc gamma receptors (FcγRs) on effector cells by eliciting antibody-dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP, respectively). Because (i) the IgG Fc domain binds to multiple FcγRs with varying affinities; (ii) even low Fc:FcγRs affinity interactions can play a significant role when antibodies are engaged in high avidity immune complexes and (iii) most effector cells express multiple FcγRs, the clearance mechanisms that can be mediated by individual FcγR are not well-understood. Human FcγRIIIa (hFcγRIIIa; CD16a), which exists as two polymorphic variants at position 158, hFcγRIIIaV158 and hFcγRIIIaF158, is widely considered to only trigger ADCC, especially with natural killer (NK) cells as effectors. To evaluate the role of hFcγRIIIa ligation in myeloid-derived effector cells, and in particular on macrophages and monocytes which express multiple FcγRs, we engineered an aglycosylated engineered human Fc (hFc) variant, Fc3aV, which binds exclusively to hFcγRIIIaV158. Antibodies formatted with the Fc3aV variant bind to the hFcγRIIIaV158 allotype with a somewhat lower KD than their wild type IgG1 counterparts, but not to any other hFcγR. The exceptional selectivity for hFcγRIIIaV158 was demonstrated by SPR using increased avidity, dimerized GST-fused versions of the ectodomains of hFcγRs and from the absence of binding of large immune complex (IC) to CHO cells expressing each of the hFcγRs, including notably, the FcγRIIIaF158 variant or the highly homologous FcγRIIIb. We show that even though monocyte-derived GM-CSF differentiated macrophages express hFcγRIIIa at substantially lower levels than the other two major activating receptors, namely hFcγRI or hFcγRIIa, Fc3aV-formatted Rituximab and Herceptin perform ADCP toward CD20- and Her2-expressing cancer cells, respectively, at a level comparable to that of the respective wild-type antibodies. We further show that hFcγRIIIa activation plays a significant role on ADCC by human peripheral monocytes. Our data highlight the utility of Fc3aV and other similarly engineered exquisitely selective, aglycosylated Fc variants toward other hFcγRs as tools for the detailed molecular understanding of hFcγR biology.

Published
2019
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3. 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
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4. 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

5. 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
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6. 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
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7. 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
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8. High seroprevalence but short‐lived immune response to SARS‐CoV‐2 infection in Paris

Author

Pierre Charneau, Philippe Souque, Evelyne Dufour, Marion Gransagne, Stéphane Petres, Ana Ines Lalanne, Thierry Rose, Isabelle Turbiez, François Dejardin, François Anna, Odile Richard-Le Goff, Olivier Helynck, Maude Guillot-Delost, Sophie Goyard, Alexia Savignoni, François-Clément Bidard, Zaineb Choucha, Fabien Nevo, Franck Perez, Delphine Louis, Véronique Gillon, Yves L. Janin, A Gobillion, Nicolas Escriou, Olivier Lantz, Laboratoire commun Pasteur-TheraVectys, Institut Pasteur [Paris]-TheraVectys, Biologie Cellulaire des Lymphocytes - Lymphocyte Cell Biology, Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], Institut Curie [Paris], Virologie Moléculaire et Vaccinologie / Molecular Virology and Vaccinology, Institut Pasteur [Paris], Laboratoire d’innovation : vaccins – Innovation lab : vaccines, Risques cliniques et sécurité en santé des femmes et en santé périnatale (RISCQ), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Immunité et cancer (U932), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Plateforme technologique Production et purification de protéines recombinantes – Production and Purification of Recombinant Proteins Technological Platform (PPR), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Anticorps en thérapie et pathologie - Antibodies in Therapy and Pathology, Chimie et Biocatalyse, This work was made possible thanks to the financial support obtained through the URGENCE nouveau coronavirus fundraising campaign of Institut Pasteur and the financial support of the Fondation Total. This study was funded in part by a grant from Fondation de France and by Institut Curie Institutional funding. The luciferin synthesis development has been supported by DARRI (ValoExpress 2016-2018). LuLISA development has been supported by IARP Pasteur-Carnot MI (2019-2020)., Institut Pasteur [Paris] (IP)-TheraVectys, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP), Université Paris-Saclay, Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Lassailly-Bondaz, Anne, Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Curie [Paris], Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), and Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]

Subjects
0301 basic medicine, Male, Time Factors, Antibodies, Viral, SARS‐CoV‐2, MESH: Antibodies, Neutralizing, Serology, 0302 clinical medicine, Seroepidemiologic Studies, Epidemiology, MESH: COVID-19, Immunology and Allergy, 030212 general & internal medicine, Research Articles, MESH: Immunoglobulin G, 0303 health sciences, education.field_of_study, MESH: Paris, 3. Good health, Titer, Research Article|Basic, ELISA, Female, medicine.symptom, Bioluminescence, Adult, MESH: Pandemics, medicine.medical_specialty, Paris, Population, Immunology, Anosmia, Immunity to infection, Biology, Asymptomatic, Virus, 03 medical and health sciences, COVID‐19, Internal medicine, medicine, Humans, Basic, education, Pandemics, 030304 developmental biology, LuLISA, MESH: Seroepidemiologic Studies, MESH: Humans, SARS-CoV-2, business.industry, MESH: Time Factors, COVID-19, MESH: Adult, Ageusia, Antibodies, Neutralizing, MESH: Male, 030104 developmental biology, Immunization, [SDV.SPEE] Life Sciences [q-bio]/Santé publique et épidémiologie, Immunoglobulin G, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, business, MESH: Female, MESH: Antibodies, Viral, 030215 immunology
Abstract

International audience; Background Although the COVID-19 pandemic peaked in March/April 2020 in France, the prevalence of infection is barely known. Herein, we assessed the serological response against the SARS-CoV-2 virus in a large population working in one institution of the Paris conurbation. We set up two high-throughput and sensitive methods to assess SARS CoV-2 Nucleoprotein and Spike protein-specific IgG response along with a pseudo-neutralization assay in sera. We studied 1847 participants who also answered a web-based survey on clinical symptoms.Methods and Results In May-July 2020, 11% (95% CI: 9.7-12.6) of serums were positive for IgG against the SARS-CoV-2 N and S protein and 9.5% (CI:8.2-11.0) were pseudo-neutralizer. The prevalence of immunization was 11.6% (CI:10.2-13.2) considering positivity in at least one assays. In 5% (CI:3.9-7.1) of RT-qPCR positive individuals, no systemic IgGs were detected. Among immune individuals, 21% had been asymptomatic.Anosmia and ageusia occurred in 52% of the IgG-positive individuals and in 3% of the negative ones. In contrast, 30% of the anosmia-ageusia cases were seronegative suggesting that the true prevalence of infection may reach 16.6%. In sera obtained 4-8 weeks after the first sampling anti-N and anti-S IgG titers and pseudo-neutralization activity declined by 31%, 17% and 53%, respectively with half-life of 35, 87 and 28 days, respectively.Conclusions The population studied being not particularly exposed to SARS-CoV-2 infection is representative of active workers in the Paris conurbation, suggesting that the current epidemiological models may underestimate the true prevalence of infection. The short lifespan of the serological systemic responses hinders retrospective assessment of the epidemic extent.

Published
2020
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9. 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

10. IgG Fc domains that bind C1q but not effector Fcγ receptors delineate the importance of complement-mediated effector functions

Author

Bianca Balbino, George Delidakis, Tae Hyun Kang, Makiko Watanabe, Kendra Triplett, Anja Lux, Navin Varadarajan, Odile Richard-Le Goff, Oana I. Lungu, Pierre Bruhns, George Georgiou, Corrine Alford, Gabrielle Romain, Margaret A. Lindorfer, Wissam Charab, Falk Nimmerjahn, Yan Jessie Zhang, Hidetaka Tanno, Wupeng Yan, Nicholas M. Marshall, Moses Donkor, Chang-Han Lee, Jiwon Lee, Ronald P. Taylor, Biliana Todorova, University of Texas at Austin [Austin], University of Houston, Anticorps en Thérapie et Pathologie, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), University of Erlangen-Nürnberg [Germany], University of Virginia School of Medicine [US], Université Pierre et Marie Curie - Paris 6 - UFR de Médecine Pierre et Marie Curie (UPMC), Université Pierre et Marie Curie - Paris 6 (UPMC), University of Virginia, Department of Biochemistry and Molecular Genetics (xxx), University of Virginia [Charlottesville], We thank Y. Tanno for assistance with protein expression, A. Bui for assistance with liquid chromatography–tandem mass spectrometry, P. Tucker (University of Texas at Austin) for cancer cell lines, D. Lee (MD Anderson Cancer Center) for patient-derived primary acute lymphocytic leukemia cells, the Macromolecular Crystallography Facility of the University of Texas at Austin, the Berkeley Center for Structural Biology, the Advanced Light Source (supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under contract DE-AC02-05CH11231), the Proteomics Facility at the University of Texas at Austin (supported by grant RP110782 from the Cancer Prevention Research Training Program), and A. Nicola and the Plate-Forme d'Imagerie Dynamique (Institut Pasteur, Paris) for help with the bioluminescence experiments. Supported by the Clayton Foundation, the Institut Pasteur (P.B. laboratory), the Institut National de la Santé et de la Recherche Médicale (P.B. laboratory), the European Research Council Seventh Frame-work Program (ERC-2013-CoG 616050 for the P.B. laboratory), the Pasteur–Paris University International PhD program (B.B.), the Cancer Prevention Research Training Program (RP140108 to M.D., RP160015 to H.T., and RP130570 to N.V.), the American Cancer Society (123506-PF-13-354-01-CDD to N.M.), Uehara Memorial Foundation (H.T.), Japan Society for the Promotion of Science (H.T.), Deutsche Forschungsgemeinschaft (CRC1181-A07 to F.N.), the US National Institutes of Health (R01CA174385 to N.V., and R01 GM104896 to Y.J.Z.) and the Welch Foundation (F-1778 to Y.J.Z.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the Cancer Prevention and Research Institute of Texas., European Project: 616050,EC:FP7:ERC,ERC-2013-CoG,MYELOSHOCK(2014), Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), University of Virginia, Martin, Marie, and 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

Subjects
Cytotoxicity, Immunologic, 0301 basic medicine, Cellular immunity, MESH: Complement C1q, MESH: Immunotherapy, Crystallography, X-Ray, Mass Spectrometry, MESH: Antibodies, Monoclonal, Mice, Tandem Mass Spectrometry, Neoplasms, Immunology and Allergy, MESH: Animals, MESH: Neoplasms, Cytotoxicity, Receptor, MESH: Phagocytosis, MESH: Crystallization, MESH: Immunoglobulin G, biology, Effector, Antibodies, Monoclonal, MESH: Enzyme-Linked Immunosorbent Assay, MESH: Chromatography, Gel, Precursor Cell Lymphoblastic Leukemia-Lymphoma, Burkitt Lymphoma, 3. Good health, Cell biology, MESH: Surface Plasmon Resonance, Chromatography, Gel, [SDV.IMM]Life Sciences [q-bio]/Immunology, Immunotherapy, Lymphoma, Large B-Cell, Diffuse, Antibody, Crystallization, Lymphoma, B-Cell, MESH: Cell Line, Tumor, [SDV.IMM] Life Sciences [q-bio]/Immunology, Immunology, Enzyme-Linked Immunosorbent Assay, In Vitro Techniques, MESH: Receptors, IgG, MESH: Immunoglobulin Fc Fragments, 03 medical and health sciences, Classical complement pathway, Immune system, Phagocytosis, Cell Line, Tumor, Animals, Humans, MESH: Cytotoxicity, Immunologic, MESH: Mice, MESH: Lymphoma, B-Cell, MESH: In Vitro Techniques, MESH: Mass Spectrometry, MESH: Precursor Cell Lymphoblastic Leukemia-Lymphoma, MESH: Humans, Complement C1q, Receptors, IgG, MESH: Tandem Mass Spectrometry, Surface Plasmon Resonance, MESH: Crystallography, X-Ray, Immunoglobulin Fc Fragments, Complement system, MESH: Burkitt Lymphoma, 030104 developmental biology, Immunoglobulin G, biology.protein, MESH: Lymphoma, Large B-Cell, Diffuse, MESH: Chromatography, Liquid, Chromatography, Liquid
Abstract

International audience; Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.

Published
2017
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11. Characterization of the thymic IL-7 niche in vivo

Author

Ana Cumano, Odile Richard-Le Goff, Ana Patricia Sousa, Nuno L. Alves, Richard L. Boyd, Vera S. G. Ribeiro, Allison Bordack, Gérard Eberl, Lucie Peduto, James P. Di Santo, Ann P. Chidgey, Francina Langa Vives, Nicholas D. Huntington, Cytokines et Développement Lymphoïde, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Développement des Lymphocytes, Centre d'Ingénierie génétique murine - Mouse Genetics Engineering Center (CIGM), Institut Pasteur [Paris] (IP), Développement des Tissus Lymphoïdes, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Monash Immunology and Stem Cell Laboratories (MISCL), Monash University [Clayton], Institut Pasteur [Paris]-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
Genetically modified mouse, Chromosomes, Artificial, Bacterial, Chemokine, MESH: Chromosomes, Artificial, Bacterial, MESH: Mice, Transgenic, medicine.medical_treatment, T cell, Mice, Transgenic, Thymus Gland, Biology, Polymerase Chain Reaction, Mice, 03 medical and health sciences, 0302 clinical medicine, Genes, Reporter, In vivo, medicine, Animals, MESH: Animals, RNA, Messenger, MESH: Mice, Cells, Cultured, MESH: RNA, Messenger, 030304 developmental biology, 0303 health sciences, Thymic involution, Multidisciplinary, Interleukin-7, MESH: Genes, Reporter, CCL19, Epithelial Cells, MESH: Polymerase Chain Reaction, MESH: Thymus Gland, Biological Sciences, Molecular biology, MESH: Interleukin-7, Luminescent Proteins, Cytokine, medicine.anatomical_structure, MESH: Epithelial Cells, biology.protein, [SDV.IMM]Life Sciences [q-bio]/Immunology, MESH: Luminescent Proteins, CCL25, MESH: Cells, Cultured, 030215 immunology
Abstract

The thymus represents the “cradle” for T cell development, with thymic stroma providing multiple soluble and membrane cues to developing thymocytes. Although IL-7 is recognized as an essential factor for thymopoiesis, the “environmental niche” of thymic IL-7 activity remains poorly characterized in vivo. Using bacterial artificial chromosome transgenic mice in which YFP is under control of IL-7 promoter, we identify a subset of thymic epithelial cells (TECs) that co-express YFP and high levels of Il7 transcripts (IL-7 hi cells). IL-7 hi TECs arise during early fetal development, persist throughout life, and co-express homeostatic chemokines ( Ccl19 , Ccl25 , Cxcl12 ) and cytokines ( Il15 ) that are critical for normal thymopoiesis. In the adult thymus, IL-7 hi cells localize to the cortico-medullary junction and display traits of both cortical and medullary TECs. Interestingly, the frequency of IL-7 hi cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining IL-7 levels. Our temporal-spatial analysis of IL-7-producing cells in the thymus in vivo suggests that thymic IL-7 levels are dynamically regulated under distinct physiological conditions. This IL-7 reporter mouse provides a valuable tool to further dissect the mechanisms that govern thymic IL-7 expression in vivo.

Published
2009
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12. Erratum: Corrigendum: IgG Fc domains that bind C1q but not effector Fcγ receptors delineate the importance of complement-mediated effector functions

Author

Corrine Alford, Gabrielle Romain, Margaret A. Lindorfer, Falk Nimmerjahn, Wissam Charab, Moses Donkor, Navin Varadarajan, George Delidakis, Bianca Balbino, Anja Lux, Hidetaka Tanno, Wupeng Yan, Ronald P. Taylor, Kendra Triplett, Pierre Bruhns, Odile Richard-Le Goff, Chang-Han Lee, Tae Hyun Kang, George Georgiou, Oana I. Lungu, Biliana Todorova, Jiwon Lee, Nicholas M. Marshall, Makiko Watanabe, and Yan Jessie Zhang

Subjects
0301 basic medicine, 03 medical and health sciences, 030104 developmental biology, Nat, Effector, Immunology, Immunology and Allergy, Effector functions, Biology, Receptor, Complement (complexity), Cell biology
Abstract

Nat. Immunol.; doi:10.1038/ni.3770; corrected online 27 June 2017 In the version of this article initially published online, the labels identifying each plot in Figure 1b were missing. The labels are as follows (left to right): CHO, FcγRI, FcγRIIaR131, FcγRIIaH131, FcγRIIb, FcγRIIIaF158 and FcγRIIIaV158.

Published
2017
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13. GATA-3 promotes T-cell specification by repressing B-cell potential in pro–T cells in mice

Author

Ana Cumano, Odile Richard-Le Goff, Roel G. J. Klein Wolterink, Rudolf W. Hendriks, Fabrice Lemaître, Milena Hasan, James P. Di Santo, Marcos E. García-Ojeda, Immunité Innée, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Développement des Lymphocytes, Erasmus University Medical Center [Rotterdam] (Erasmus MC), Cellule Pasteur, Université Paris Diderot - Paris 7 (UPD7)-PRES Sorbonne Paris Cité, Dynamiques des Réponses Immunes, Centre d'Immunologie Humaine (CIH), This work was supported by a fellowship from the Pasteur Foundation (M.E.G.-O.), by grants from the Huygens Scholarship Program and the Royal Netherlands Academy for Arts and Sciences (both the Netherlands) and by the Ligue Nationale Contre le Cancer (France, R.G.J.K.W.). This work was also supported by grants from the Institut Pasteur, the Inserm, and the Ligue Nationale Contre le Cancer (all in France, J.P.D.)., Pulmonary Medicine, Immunology, and Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Male, MESH: Signal Transduction, Receptors, Antigen, T-Cell, alpha-beta, MESH: Flow Cytometry, MESH: Receptor, Notch1, Biochemistry, MESH: Mice, Knockout, Mice, 0302 clinical medicine, Inside Blood, b-lymphocytes, MESH: Animals, Receptor, Notch1, Cells, Cultured, Mice, Knockout, 0303 health sciences, MESH: Receptors, Antigen, T-Cell, alpha-beta, Cell Differentiation, Hematology, MESH: Transcription Factors, Flow Cytometry, Cell biology, medicine.anatomical_structure, embryonic structures, [SDV.IMM]Life Sciences [q-bio]/Immunology, Female, biological phenomena, cell phenomena, and immunity, medicine.symptom, Signal Transduction, MESH: Cells, Cultured, MESH: Cell Differentiation, T cell, Immunology, gata3 gene, GATA3 Transcription Factor, Thymus Gland, Biology, 03 medical and health sciences, MESH: B-Lymphocytes, MESH: GATA3 Transcription Factor, medicine, Animals, Notch1 signaling, Cell Lineage, Transcription factor, t-lymphocytes, MESH: Mice, B cell, 030304 developmental biology, Cell Biology, MESH: Thymus Gland, MESH: Cell Lineage, MESH: Male, MESH: T-Lymphocytes, Mechanism of action, MESH: Female, 030215 immunology, Transcription Factors
Abstract

International audience; Transcription factors orchestrate T-lineage differentiation in the thymus. One critical checkpoint involves Notch1 signaling that instructs T-cell commitment at the expense of the B-lineage program. While GATA-3 is required for T-cell specification, its mechanism of action is poorly understood. We show that GATA-3 works in concert with Notch1 to commit thymic progenitors to the T-cell lineage via 2 distinct pathways. First, GATA-3 orchestrates a transcriptional “repertoire” that is required for thymocyte maturation up to and beyond the pro–T-cell stage. Second, GATA-3 critically suppresses a latent B-cell potential in pro–T cells. As such, GATA-3 is essential to sealing in Notch-induced T-cell fate in early thymocyte precursors by promoting T-cell identity through the repression of alternative developmental options.

Published
2013
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14. Characterization of the role of dendritic cells in prion transfer to primary neurons

Author

Chiara Zurzolo, Maddalena Costanzo, Christelle Langevin, Odile Richard-Le Goff, Karine Gousset, Trafic membranaire et Pathogénèse, Institut Pasteur [Paris], Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli 'Federico II', Università degli studi di Napoli Federico II, KG is supported by the Pasteur Foundation Fellowship Program and MC is supported by a fellowship from the Ministère de Education Nationale et de la Recherche., ANR-09-BLAN-0122,Priontraf(2009), European Project: 34002,STRAINBARRIER, European Project: 222887,EC:FP7:KBBE,FP7-KBBE-2007-2A,PRIORITY(2009), Institut Pasteur [Paris] (IP), University of Naples Federico II = Università degli studi di Napoli Federico II, Langevin, C., Gousset, K., Costanzo, M., Richard Le Goff, O, and Zurzolo, Chiara

Subjects
Gene isoform, Nervous system, Time Factors, PrPSc Proteins, Prions, animal diseases, Central nervous system, Bone Marrow Cells, Biology, Biochemistry, 03 medical and health sciences, Mice, Prion infection, Prion infectivity, medicine, Animals, Molecular Biology, Cells, Cultured, 030304 developmental biology, Neurons, 0303 health sciences, Transmissible spongiform encephalopathy, Catabolism, 030302 biochemistry & molecular biology, Life Sciences, Cell Biology, Dendritic cell, Dendritic Cells, medicine.disease, Virology, Endocytosis, Cell biology, nervous system diseases, Mice, Inbred C57BL, Protein Transport, medicine.anatomical_structure, nervous system, [SDV.IMM]Life Sciences [q-bio]/Immunology, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Protein Processing, Post-Translational
Abstract

International audience; Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases caused by pathogenic isoforms (PrPSc) of the host-encoded cellular prion protein (PrPc). After consumption of contaminated food, PrPSc deposits rapidly accumulate in lymphoid tissues before invasion of the central nervous system (CNS). However the mechanisms of prion spreading from the periphery to the nervous system are still unclear. In this study, we investigated the role of dendritic cells (DCs) in the spreading of prion infection to neuronal cells. First, we determined that bone-marrow derived dendritic cells (BMDCs) rapidly uptake PrPSc after exposure to infected brain homogenate. Next, we observed a progressive catabolism of the internalized prion aggregates. Similar experiments performed with BMDCs isolated from knock-out (KO) or mice over-expressing PrP (tga20) indicate that both PrPSc uptake and catabolism are independent of PrPc expression in these cells. Finally, using co-cultures of prion-loaded BMDCs and cerebellar neurons, we characterized the transfer of the prion protein and the resulting infection of the neuronal cultures. Interestingly, the transfer of PrPSc was triggered by direct cell-to-cell contact. As a consequence, BMDCs kept the prion protein when cultured alone and no transfer to the recipient neurons was observed when a filter separated the two cultures or when neurons were exposed to the BMDCs conditioned media. Additionally, fixed BMDCs also failed to transfer prion infectivity to neurons suggesting an active transport of prion aggregates, in accordance with a role of tunnelling nanotubes (TNTs) observed in the co-cultures.

Published
2010
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15. Cutting Edge: A Thymocyte-Thymic Epithelial Cell Cross-Talk Dynamically Regulates Intrathymic IL-7 Expression In Vivo

Author

Nuno L. Alves, Jean-Jacques Mention, Nicholas D. Huntington, Odile Richard-Le Goff, James P. Di Santo, Cytokines et Développement Lymphoïde, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Instituto de Biologia Molecular e Celular - institute for molecular and cell biology [Porto, Portugal] (IBMC), Universidade do Porto = University of Porto, This work was supported in part by Institut Pasteur, Institut National de la Santé et de la Recherche Médicale, the Fondation pour le Recherche Médicale (SPF20081214865), the Ligue Nationale Contre le Cancer, and the Foundation for Science and Technology (SFRH/45932/2008 and Program Ciência 2008)., and RICHARD-LE GOFF, ODILE

Subjects
Yellow fluorescent protein, Cellular differentiation, T-Lymphocytes, Gene Expression, MESH: Flow Cytometry, Cell Separation, MESH: T-Lymphocyte Subsets, MESH: Mice, Knockout, Mice, T-Lymphocyte Subsets, Immunology and Allergy, MESH: Animals, Thymopoietin, Regulation of gene expression, Mice, Knockout, medicine.diagnostic_test, biology, Cell Differentiation, hemic and immune systems, Flow Cytometry, Immunohistochemistry, MESH: Gene Expression Regulation, Cell biology, Thymocyte, Haematopoiesis, MESH: Epithelial Cells, [SDV.IMM]Life Sciences [q-bio]/Immunology, tissues, MESH: Cell Differentiation, medicine.medical_specialty, MESH: Gene Expression, [SDV.IMM] Life Sciences [q-bio]/Immunology, Immunology, education, MESH: Receptor Cross-Talk, Thymus Gland, MESH: Cell Separation, Flow cytometry, Internal medicine, medicine, Animals, Progenitor cell, MESH: Mice, Interleukin-7, Epithelial Cells, MESH: Immunohistochemistry, Receptor Cross-Talk, MESH: Thymus Gland, MESH: Interleukin-7, Endocrinology, MESH: T-Lymphocytes, Gene Expression Regulation, biology.protein
Abstract

Thymic epithelial cells (TECs) are the predominant intrathymic source of the essential thymopoietin IL-7. Whether thymocyte-TEC interactions have a role in the regulation of IL-7 expression is not known. By exploiting IL-7 reporter mice in which yellow fluorescent protein expression identifies TECs expressing high levels of IL-7 (Il7+ TECs), we show that Il7+ TECs segregate from emerging medullary TECs during thymic organogenesis. Although Il7+ TECs normally diminish with age, we found that Il7+ TECs are markedly retained in alymphoid Rag2−/−Il2rg−/− IL-7 reporter mice that manifest a profound thymopoietic arrest. Transfer of Tcra−/− or wild-type (but not Rag2−/−) hematopoietic progenitors to alymphoid IL-7 reporter recipients normalizes the frequency of Il7+ TECs and re-establishes cortical TEC/medullary TEC segregation. Although thymocyte-derived signals are often considered stimulatory for TEC maturation, our findings identify a negative feedback mechanism in which signals derived from TCRβ-selected thymocytes modulate TEC-dependent IL-7 expression.

Published
2010
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16. CD11cloB220+ interferon-producing killer dendritic cells are activated natural killer cells

Author

Ofer Mandelboim, Odile Richard-Le Goff, Christian A. J. Vosshenrich, Sarah Lesjean-Pottier, Milena Hasan, Erwan Corcuff, James P. Di Santo, Cytokines et Développement Lymphoïde, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Lautenberg Center for General and Tumor Immunology, Hebrew University-Hadassah Medical School, This work was supported by grants from the Institut Pasteur and the Institut National de la Santé et de la Recherche Médicale, and as an Equipe Labelisé of the Ligue National Contre le Cancer., and RICHARD-LE GOFF, ODILE

Subjects
MESH: Spleen, MESH: Interleukin-15, Lymphocyte Activation, MESH: Mice, Knockout, Interleukin 21, Mice, Interferon, Immunology and Allergy, MESH: Animals, Interleukin-15, Mice, Knockout, Immunity, Cellular, MESH: Dendritic Cells, Interleukin, hemic and immune systems, Natural killer T cell, MESH: Crosses, Genetic, Killer Cells, Natural, MESH: Leukocyte Common Antigens, Interleukin 15, Interleukin 12, [SDV.IMM]Life Sciences [q-bio]/Immunology, MESH: Immunity, Innate, medicine.drug, MESH: Killer Cells, Natural, [SDV.IMM] Life Sciences [q-bio]/Immunology, Immunology, MESH: Lymphocyte Subsets, CD11c, chemical and pharmacologic phenomena, Biology, Major histocompatibility complex, MESH: Immunity, Cellular, MESH: Mice, Inbred C57BL, medicine, Animals, MESH: Lymphocyte Activation, MESH: Mice, Crosses, Genetic, Histocompatibility Antigens Class II, Brief Definitive Report, Dendritic Cells, Molecular biology, Immunity, Innate, Lymphocyte Subsets, CD11c Antigen, Mice, Inbred C57BL, biology.protein, MESH: CD11c Antigen, MESH: Histocompatibility Antigens Class II, Leukocyte Common Antigens, Brief Definitive Reports, Spleen
Abstract

Interferon-producing killer dendritic cells (IKDCs) are a recently described subset of CD11c(lo)B220(+) cells that share phenotypic and functional properties of DCs and natural killer (NK) cells (Chan, C.W., E. Crafton, H.N. Fan, J. Flook, K. Yoshimura, M. Skarica, D. Brockstedt, T.W. Dubensky, M.F. Stins, L.L. Lanier, et al. 2006. Nat. Med. 12:207-213; Taieb, J., N. Chaput, C. Menard, L. Apetoh, E. Ullrich, M. Bonmort, M. Pequignot, N. Casares, M. Terme, C. Flament, et al. 2006. Nat. Med. 12:214-219). IKDC development appears unusual in that cytokines using the interleukin (IL)-2 receptor beta (IL-2Rbeta) chain but not those using the common gamma chain (gamma(c)) are necessary for their generation. By directly comparing Rag2(-/-)gamma(c)(-/y), Rag2(-/-)IL-2Rbeta(-/-), Rag2(-/-)IL-15(-/-), and Rag2(-/-)IL-2(-/-) mice, we demonstrate that IKDC development parallels NK cell development in its strict IL-15 dependence. Moreover, IKDCs uniformly express NK-specific Ncr-1 transcripts (encoding NKp46), whereas NKp46(+) cells are absent in Ncr1(gfp/+)gamma(c)(-/y) mice. Distinguishing features of IKDCs (CD11c(lo)B220(+)MHC-II(+)) were carefully examined on developing NK cells in the bone marrow and on peripheral NK cells. As B220 expression was heterogeneous, defining B220(lo) versus B220(hi) NK1.1(+) NK cells could be considered as arbitrary, and few phenotypic differences were noted between NK1.1(+) NK cells bearing different levels of B220. CD11c expression did not correlate with B220 or major histocompatibility complex (MHC) class II (MHC-II) expression, and most MHC-II(+) NK1.1(+) cells did not express B220 and were thus not IKDCs. Finally, CD11c, MHC-II, and B220 levels were up-regulated on NK1.1(+) cells upon activation in vitro or in vivo in a proliferation-dependent fashion. Our data suggest that the majority of CD11c(lo)B220(+) "IKDC-like" cells represent activated NK cells.

Published
2007
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17. A thymic pathway of mouse natural killer cell development characterized by expression of GATA-3 and CD127

Author

Benedita Rocha, Ana Cumano, Sophie Ezine, Erwan Corcuff, Lars Rogge, Marcos E. García-Ojeda, Odile Richard-Le Goff, Christian A. J. Vosshenrich, Sandrine I. Samson-Villeger, Delphine Guy-Grand, James P. Di Santo, Valérie Pasqualetto, Laurence Enault, Cytokines et Développement Lymphoïde, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Paris 5 (UPD5), Differenciation Thymique et Physiologie des Lymphocytes T (U591), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Développement des Lymphocytes, Immunorégulation, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-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 ), Differenciation Thymique et Physiologie des Lymphocytes T ( U591 ), 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]-Centre National de la Recherche Scientifique ( CNRS )

Subjects
Cytotoxicity, Immunologic, MESH: Signal Transduction, MESH : GATA3 Transcription Factor, MESH : Cytokines, MESH : Cytotoxicity, Immunologic, MESH : Interleukin-7 Receptor alpha Subunit, Interleukin 21, Mice, 0302 clinical medicine, [ SDV.IMM ] Life Sciences [q-bio]/Immunology, Immunology and Allergy, MESH: Animals, 0303 health sciences, MESH: Cytokines, MESH : Lymphocyte Subsets, MESH : Mice, Nude, Interleukin, virus diseases, Cell Differentiation, hemic and immune systems, Natural killer T cell, MESH : Mice, Transgenic, Cell biology, Killer Cells, Natural, MESH : Immunophenotyping, Interleukin 12, Cytokines, [SDV.IMM]Life Sciences [q-bio]/Immunology, MESH : Killer Cells, Natural, MESH : Cell Differentiation, Signal Transduction, MESH : Interleukin-7, MESH: Cell Differentiation, MESH: Killer Cells, Natural, MESH: Immunophenotyping, MESH: Mice, Transgenic, Immunology, MESH: Lymphocyte Subsets, Mice, Nude, Mice, Transgenic, chemical and pharmacologic phenomena, MESH : Mice, Inbred C57BL, GATA3 Transcription Factor, Thymus Gland, Biology, CD16, Immunophenotyping, Interleukin-7 Receptor alpha Subunit, 03 medical and health sciences, MESH: Mice, Inbred C57BL, MESH : Mice, MESH: GATA3 Transcription Factor, MESH: Mice, Nude, Animals, Humans, MESH : Thymus Gland, MESH: Cytotoxicity, Immunologic, MESH: Mice, 030304 developmental biology, MESH : Signal Transduction, Lymphokine-activated killer cell, MESH: Humans, Cell growth, Interleukin-7, MESH : Humans, MESH: Interleukin-7 Receptor alpha Subunit, MESH: Thymus Gland, Lymphocyte Subsets, MESH: Interleukin-7, Mice, Inbred C57BL, Myeloid-derived Suppressor Cell, MESH : Animals, 030215 immunology
Abstract

International audience; Natural killer (NK) cell development is thought to occur in the bone marrow. Here we identify the transcription factor GATA-3 and CD127 (IL-7R alpha) as molecular markers of a pathway of mouse NK cell development that originates in the thymus. Thymus-derived CD127+ NK cells repopulated peripheral lymphoid organs, and their homeostasis was strictly dependent on GATA-3 and interleukin 7. The CD127+ NK cells had a distinct phenotype (CD11b(lo) CD16- CD69(hi) Ly49(lo)) and unusual functional attributes, including reduced cytotoxicity but considerable cytokine production. Those characteristics are reminiscent of human CD56(hi) CD16- NK cells, which we found expressed CD127 and had more GATA-3 expression than human CD56+ CD16+ NK cells. We propose that bone marrow and thymic NK cell pathways generate distinct mouse NK cells with properties similar to those of the two human CD56 NK cell subsets.

Published
2006
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18. GATA-3 seals Notch-induced T cell commitment (46.1)

Author

Marcos E García-Ojeda, Fabrice Lemaître, Odile Richard-Le Goff, Rudi W Hendriks, Ana Cumano, and James P Di Santo

Subjects
Immunology, Immunology and Allergy
Abstract

Transcription factors orchestrate T-lineage differentiation in the thymus. One early checkpoint involves Notch-1 stimulation of thymic immigrants that promotes T-cell commitment at the expense of the B-lineage program. The zinc-finger transcription factor GATA-3 is required for T cell specification, although its mechanism of action is unknown. Here we identify molecular targets of GATA-3 in early thymocytes and show that conditional ablation of GATA-3 expression in committed pro-T-cells unmasks a latent B-cell potential that is undetectable in normal T-cell precursors. Thus, GATA-3 delimits the developmental options in early thymocyte precursors by sealing T-cell fate induced by Notch signals. M.E. García-Ojeda was funded by a Pasteur Foundation Fellowship. This work was supported by grants from the Institut Pasteur, the Inserm and the Ligue Nationale contre le Cancer. M.E. Garcia-Ojeda and F. Lemaître contributed equally to this work.

Published
2009
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20. Characterization of the role of dendritic cells in prion transfer to primary neurons.

Author

Christelle Langevin, Karine Gousset, Maddalena Costanzo, and Odile Richard‑Le Goff

Subjects
DENDRITIC cells, PRIONS, NEURONS, CHRONIC wasting disease, NEURODEGENERATION, PATHOGENIC microorganisms, BONE marrow
Abstract

TSEs (transmissible spongiform encephalopathies) are neurodegenerative diseases caused by pathogenic isoforms (PrPSc) of the host-encoded PrPc (cellular prion protein). After consumption of contaminated food, PrPSc deposits rapidly accumulate in lymphoid tissues before invasion of the CNS (central nervous system). However, the mechanisms of prion spreading from the periphery to the nervous system are still unclear. In the present study, we investigated the role of DCs (dendritic cells) in the spreading of prion infection to neuronal cells. First, we determined that BMDCs (bone-marrow-derived DCs) rapidly uptake PrPSc after exposure to infected brain homogenate. Next, we observed a progressive catabolism of the internalized prion aggregates. Similar experiments performed with BMDCs isolated from KO (knockout) mice or mice overexpressing PrP (tga20) indicate that both PrPSc uptake and catabolism are independent of PrPc expression in these cells. Finally, using co-cultures of prion-loaded BMDCs and cerebellar neurons, we characterized the transfer of the prion protein and the resulting infection of the neuronal cultures. Interestingly, the transfer of PrPSc was triggered by direct cell–cell contact. As a consequence, BMDCs retained the prion protein when cultured alone, and no transfer to the recipient neurons was observed when a filter separated the two cultures or when neurons were exposed to the BMDC-conditioned medium. Additionally, fixed BMDCs also failed to transfer prion infectivity to neurons, suggesting an active transport of prion aggregates, in accordance with a role of TNTs (tunnelling nanotubes) observed in the co-cultures. [ABSTRACT FROM AUTHOR]

Published
2010
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