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1. Human Apoptotic Cells, Generated by Extracorporeal Photopheresis, Modulate Allogeneic Immune Response

Author

Caroline Pilon, Thomas Stehlé, Asma Beldi-Ferchiou, Marie Matignon, Allan Thiolat, Aude Burlion, Cynthia Grondin, Brigitte Birebent, France Pirenne, Hélène Rouard, Philippe Lang, Gilles Marodon, Philippe Grimbert, and José L. Cohen

Subjects
tolerance, NSG mice, immunomodulation, transplantation, apoptotic cell, Immunologic diseases. Allergy, RC581-607
Abstract

The induction of specific and sustainable tolerance is a challenging issue in organ transplantation. The discovery of the immunosuppressive properties of apoptotic cells in animal models has paved the way for their use in human transplantation. In this work, we aimed to define a stable, reproducible, and clinically compatible production procedure of human apoptotic cells (Apo-cells). Using a clinically approved extracorporeal photopheresis technique, we have produced and characterized phenotypically and functionally human apoptotic cells. These Apo-cells have immunosuppressive properties proved in vitro and in vivo in NOD/SCID/γC mice by their capacity to modulate an allogeneic response following both a direct and an indirect antigen presentation. These results brought the rationale for the use of Apo-cells in tolerance induction protocol for organ transplantation.

Published
2019
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2. A novel combination of chemotherapy and immunotherapy controls tumor growth in mice with a human immune system

Author

Aude Burlion, Rodrigo N. Ramos, Pukar KC, Kélhia Sendeyo, Aurélien Corneau, Christine Ménétrier-Caux, Eliane Piaggio, Daniel Olive, Christophe Caux, and Gilles Marodon

Subjects
icos, humanized mice, regulatory t cells, chemotherapy, immunotherapy, cyclophosphamide, Immunologic diseases. Allergy, RC581-607, Neoplasms. Tumors. Oncology. Including cancer and carcinogens, RC254-282
Abstract

Mice reconstituted with a human immune system and bearing human tumors represent a promising model for developing novel cancer immunotherapies. Here, we used mass cytometry and multi-parametric flow cytometry to characterize human leukocytes infiltrating a human breast cancer tumor model in immunocompromised NOD.SCID.γc-null mice reconstituted with a human immune system and compared it to samples of breast cancer patients. We observed highly activated human CD4+ and CD8+ T cells in the tumor, as well as minor subsets of innate immune cells in both settings. We also report that ICOS+ CD4+ regulatory T cells (Treg) were enriched in the tumor relative to the periphery in humanized mice and patients, providing a target to affect Treg and tumor growth. Indeed, administration of a neutralizing mAb to human ICOS reduced Treg proportions and numbers and improved CD4 + T cell proliferation in humanized mice. Moreover, a combination of the anti-ICOS mAb with cyclophosphamide reduced tumor growth, and that was associated with an improved CD8 to Treg ratio. Depletion of human CD8+ T cells or of murine myeloid cells marginally affected the effect of the combination therapy. Altogether, our results indicate that a combination of anti-ICOS mAb and chemotherapy controls tumor growth in humanized mice, opening new perspectives for the treatment of breast cancer. One sentence summary: Targeting ICOS in combination with chemotherapy is a promising strategy to improve tumor immunity in humans.

Published
2019
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3. Targeting the Human T-Cell Inducible COStimulator Molecule with a Monoclonal Antibody Prevents Graft-vs-Host Disease and Preserves Graft vs Leukemia in a Xenograft Murine Model

Author

Aude Burlion, Simon Brunel, Nicolas Y. Petit, Daniel Olive, and Gilles Marodon

Subjects
graft-vs-host disease, graft vs leukemia effect, antibodies, monoclonal, mice, NSG, Immunologic diseases. Allergy, RC581-607
Abstract

BackgroundGraft-vs-host disease (GVHD) is a major complication of allogenic bone marrow transplantation (BMT). Targeting costimulatory molecules with antagonist antibodies could dampen the excessive immune response that occurs, while preserving the beneficial graft vs leukemia (GVL) of the allogeneic response. Previous studies using a mouse model of GVHD have shown that targeting the T-cell Inducible COStimulator (ICOS, CD278) molecule is beneficial, but it is unclear whether the same applies to human cells.MethodsHere, we assessed whether a monoclonal antibody (mAb) to human ICOS was able to antagonize the costimulatory signal delivered in vivo to human T cells. To test this hypothesis, we used a xenogeneic model of GVHD where human peripheral blood mononuclear cells were adoptively transferred in immunocompromised NOD.SCID.gc-null mice (NSG).ResultsIn this model, control mice invariably lost weight and died by day 50. In contrast, 65% of the mice receiving a single injection of the anti-hICOS mAb survived beyond 100 days. Moreover, a significant improvement in survival was obtained in a curative xeno-GVHD setting. Mechanistically, administration of the anti-hICOS mAb was associated with a strong reduction in perivascular infiltrates in liver and lungs and reduction in frequencies and numbers of human T cells in the spleen. In addition, the mAb prevented T-cell expansion in the blood during xeno-GVHD. Importantly, GVHD-protected mice retained the ability to control the P815 mastocytoma cell line, mimicking GVL in humans.ConclusionA mAb-targeting human ICOS alleviated GVHD without impairing GVL in a xenograft murine model. Thus, ICOS represents a promising target in the management of BMT, preventing GVHD while preserving GVL.

Published
2017
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4. Inhibition of the JAK/STAT Signaling Pathway in Regulatory T Cells Reveals a Very Dynamic Regulation of Foxp3 Expression.

Author

Jérémie D Goldstein, Aude Burlion, Bruno Zaragoza, Kélhia Sendeyo, Julia K Polansky, Jochen Huehn, Eliane Piaggio, Benoit L Salomon, and Gilles Marodon

Subjects
Medicine, Science
Abstract

The IL-2/JAK3/STAT-5 signaling pathway is involved on the initiation and maintenance of the transcription factor Foxp3 in regulatory T cells (Treg) and has been associated with demethylation of the intronic Conserved Non Coding Sequence-2 (CNS2). However, the role of the JAK/STAT pathway in controlling Foxp3 in the short term has been poorly investigated. Using two different JAK/STAT pharmacological inhibitors, we observed a detectable loss of Foxp3 after 10 min. of treatment that affected 70% of the cells after one hour. Using cycloheximide, a general inhibitor of mRNA translation, we determined that Foxp3, but not CD25, has a high turnover in IL-2 stimulated Treg. This reduction was correlated with a rapid reduction of Foxp3 mRNA. This loss of Foxp3 was associated with a loss in STAT-5 binding to the CNS2, which however remains demethylated. Consequently, Foxp3 expression returns to normal level upon restoration of basal JAK/STAT signaling in vivo. Reduced expression of several genes defining Treg identity was also observed upon treatment. Thus, our results demonstrate that Foxp3 has a rapid turn over in Treg partly controlled at the transcriptional level by the JAK/STAT pathway.

Published
2016
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5. A novel combination of chemotherapy and immunotherapy controls tumor growth in mice with a human immune system

Author

Christine Ménétrier-Caux, Rodrigo Nalio Ramos, Pukar Kc, Gilles Marodon, Eliane Piaggio, Aude Burlion, Christophe Caux, Kelhia Sendeyo, Daniel Olive, Aurélien Corneau, Centre d'Immunologie et de Maladies Infectieuses (CIMI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Immunité et cancer (U932), Université Paris Descartes - Paris 5 (UPD5)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Curie [Paris], Unité Mixte de Service Production et Analyse de données en Sciences de la vie et en Santé (PASS), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Plateforme Cytométrie Pitié-Salpêtrière (LUMIC-CYPS), Unité Mixte de Service d'Imagerie et de Cytométrie [CHU Saint-Antoine] (UMS LUMIC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Centre Léon Bérard [Lyon], Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, Centre de Recherche en Cancérologie de Marseille (CRCM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU), Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC), Aix Marseille Université (AMU), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centro de Informatica UFPE [Recife] (CIn), Universidade Federal de Pernambuco [Recife] (UFPE), Plateforme Cytométrie Pitié-Salpêtrière (CyPS), Unité Mixte de Service d'Imagerie et de Cytométrie (UMS LUMIC), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP), Centre de Recherche en Cancérologie de Lyon (CRCL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Curie, Aix Marseille Université (AMU)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Schering-Plough Laboratories for Immunological Research, Schering Plough Laboratories, Immunologie - Immunopathologie - Immunothérapeutique (I3), Centre d'Immunologie et des Maladies Infectieuses (CIMI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Unité Mixte de Service d'Imagerie et de Cytométrie [CHU Saint-Antoine] (UMS LUMIC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Unité Mixte de Service d'Imagerie et de Cytométrie (UMS LUMIC), Gestionnaire, Hal Sorbonne Université, Université de Lyon-Université de Lyon-Centre Léon Bérard [Lyon]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), and Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
0301 basic medicine, lcsh:Immunologic diseases. Allergy, Cyclophosphamide, T cell, medicine.medical_treatment, [SDV]Life Sciences [q-bio], Immunology, [SDV.CAN]Life Sciences [q-bio]/Cancer, chemotherapy, lcsh:RC254-282, regulatory T cells, Flow cytometry, 03 medical and health sciences, 0302 clinical medicine, Immune system, [SDV.CAN] Life Sciences [q-bio]/Cancer, medicine, Immunology and Allergy, Mass cytometry, Tumor growth, 030304 developmental biology, Original Research, Chemotherapy, 0303 health sciences, Innate immune system, medicine.diagnostic_test, business.industry, Cancer, Immunotherapy, [SDV.IMM.IMM]Life Sciences [q-bio]/Immunology/Immunotherapy, medicine.disease, lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens, 3. Good health, 030104 developmental biology, medicine.anatomical_structure, humanized mice, Oncology, ICOS, 030220 oncology & carcinogenesis, Cancer research, Immunogenic cell death, cyclophosphamide, immunotherapy, [SDV.IMM.IMM] Life Sciences [q-bio]/Immunology/Immunotherapy, business, lcsh:RC581-607, CD8, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, medicine.drug
Abstract

Mice reconstituted with a human immune system and bearing human tumors represent a promising model for developing novel cancer immunotherapies. Here, we used mass cytometry and multi-parametric flow cytometry to characterize human leukocytes infiltrating a human breast cancer tumor model in immunocompromised NOD.SCID.γc-null mice reconstituted with a human immune system and compared it to samples of breast cancer patients. We observed highly activated human CD4+ and CD8+ T cells in the tumor, as well as minor subsets of innate immune cells in both settings. We also report that ICOS+ CD4+ regulatory T cells (Treg) were enriched in the tumor relative to the periphery in humanized mice and patients, providing a target to affect Treg and tumor growth. Indeed, administration of a neutralizing mAb to human ICOS reduced Treg proportions and numbers and improved CD4+ T cell proliferation in humanized mice. Moreover, a combination of the anti-ICOS mAb with cyclophosphamide reduced tumor growth, and that was associated with an improved CD8 to Treg ratio. However, depletion of human CD8+ T cells only marginally affected tumor control whereas depletion of murine myeloid cells abrogated the effect of the combination therapy. Altogether, our results indicate that a combination of anti-ICOS mAb and chemotherapy controls tumor growth in humanized mice and highlight the crucial implication of innate immunity in treatment efficacy, opening new perspectives for the treatment of breast cancer.One sentence summaryICOS expressed on Tregs is a promising target to improve tumor immunity in humansAbbreviationsICOSInducible CostimulatoryNSGNOD.SCID.gc-nullTregregulatory T cellsCTXcyclophosphamideHuMicehumanized miceCyTOFcytometry time-of-flighttSNEtdistributed stochastic neighbor embeddingpDCsplasmacytoid dendritic cellsDCdendritic cellsICDimmunogenic cell death

Published
2019

6. Targeting the Human T-Cell Inducible COStimulator Molecule with a Monoclonal Antibody Prevents Graft-vs-Host Disease and Preserves Graft vs Leukemia in a Xenograft Murine Model

Author

Daniel Olive, Gilles Marodon, Aude Burlion, Nicolas Petit, S. Brunel, Centre d'Immunologie et de Maladies Infectieuses (CIMI), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche en Cancérologie de Marseille (CRCM), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), ANR-11-EMMA-0045,VICIT,Validation de l'anticorps anti-ICOS comme un candidat-médicament pour l'immunothérapie anti-tumorale par inhibition de l'activité suppressive des lymphocytes T régulateurs(2011), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU), Centre d'Immunologie et des Maladies Infectieuses (CIMI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Service de Médecine nucléaire [CHU Pitié-Salpétrière], CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Service de médecine nucléaire [CHU Pitié-Salpétrière], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), HAL-UPMC, Gestionnaire, and Emergence de produits, technologies, ou services à fort potentiel de valorisation - Validation de l'anticorps anti-ICOS comme un candidat-médicament pour l'immunothérapie anti-tumorale par inhibition de l'activité suppressive des lymphocytes T régulateurs - - VICIT2011 - ANR-11-EMMA-0045 - Emergence - VALID

Subjects
0301 basic medicine, lcsh:Immunologic diseases. Allergy, CD278, ICOS, NSG, Antibodies, Graft vs leukemia effect, Graft-vs-host disease, Mice, Monoclonal, [SDV.IMM] Life Sciences [q-bio]/Immunology, Graft-vs-Leukemia Effect, medicine.drug_class, T cell, Immunology, [SDV.CAN]Life Sciences [q-bio]/Cancer, chemical and pharmacologic phenomena, Monoclonal antibody, Antibodies, 03 medical and health sciences, Graft vs leukemia effect, Mice, 0302 clinical medicine, Immune system, CD278, immune system diseases, Monoclonal, medicine, Immunology and Allergy, NSG, Original Research, biology, business.industry, [SDV.IMM.IMM]Life Sciences [q-bio]/Immunology/Immunotherapy, medicine.disease, 3. Good health, Leukemia, 030104 developmental biology, medicine.anatomical_structure, surgical procedures, operative, ICOS, Graft-vs-host disease, biology.protein, [SDV.IMM]Life Sciences [q-bio]/Immunology, Antibody, business, lcsh:RC581-607, 030215 immunology
Abstract

International audience; Background: Graft-vs-host disease (GVHD) is a major complication of allogenic bone marrow transplantation (BMT). Targeting costimulatory molecules with antagonist antibodies could dampen the excessive immune response that occurs, while preserving the beneficial graft vs leukemia (GVL) of the allogeneic response. Previous studies using a mouse model of GVHD have shown that targeting the T-cell Inducible COStimulator (ICOS, CD278) molecule is beneficial, but it is unclear whether the same applies to human cells.Methods: Here, we assessed whether a monoclonal antibody (mAb) to human ICOS was able to antagonize the costimulatory signal delivered in vivo to human T cells. To test this hypothesis, we used a xenogeneic model of GVHD where human peripheral blood mononuclear cells were adoptively transferred in immunocompromised NOD.SCID.gc-null mice (NSG).Results: In this model, control mice invariably lost weight and died by day 50. In contrast, 65% of the mice receiving a single injection of the anti-hICOS mAb survived beyond 100 days. Moreover, a significant improvement in survival was obtained in a curative xeno-GVHD setting. Mechanistically, administration of the anti-hICOS mAb was associated with a strong reduction in perivascular infiltrates in liver and lungs and reduction in frequencies and numbers of human T cells in the spleen. In addition, the mAb prevented T-cell expansion in the blood during xeno-GVHD. Importantly, GVHD-protected mice retained the ability to control the P815 mastocytoma cell line, mimicking GVL in humans.Conclusion: A mAb-targeting human ICOS alleviated GVHD without impairing GVL in a xenograft murine model. Thus, ICOS represents a promising target in the management of BMT, preventing GVHD while preserving GVL.

Published
2017

7. Inhibition of the JAK/STAT Signaling Pathway in Regulatory T Cells Reveals a Very Dynamic Regulation of Foxp3 Expression

Author

Kelhia Sendeyo, Jérémie D. Goldstein, Julia K. Polansky, Gilles Marodon, Benoît L. Salomon, Bruno Zaragoza, Aude Burlion, Jochen Huehn, Eliane Piaggio, Centre d'Immunologie et de Maladies Infectieuses (CIMI), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Dpt of Experimental Immunology [Braunschweig], Helmholtz Centre for Infection Research (HZI), Institut Curie [Paris], Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany., and HAL UPMC, Gestionnaire

Subjects
0301 basic medicine, Gene Expression, lcsh:Medicine, 600 Technik, Medizin, angewandte Wissenschaften::610 Medizin und Gesundheit, Biochemistry, T-Lymphocytes, Regulatory, White Blood Cells, Mice, 0302 clinical medicine, Cell Signaling, Animal Cells, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Cellular types, Medicine and Health Sciences, STAT5 Transcription Factor, Transcriptional regulation, IL-2 receptor, lcsh:Science, STAT4, Staining, Multidisciplinary, Messenger RNA, Cell Staining, JAK-STAT signaling pathway, FOXP3, Forkhead Transcription Factors, hemic and immune systems, Regulatory T cells, Cell biology, Nucleic acids, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.IMM]Life Sciences [q-bio]/Immunology, Signal transduction, Research Article, Signal Transduction, Blood cells, Signal Inhibition, [SDV.IMM] Life Sciences [q-bio]/Immunology, Immune Cells, Immunology, T cells, chemical and pharmacologic phenomena, Biology, Research and Analysis Methods, stat, 03 medical and health sciences, DNA-binding proteins, Genetics, Animals, Humans, Gene Regulation, RNA, Messenger, Molecular Biology Techniques, Molecular Biology, Protein Kinase Inhibitors, Transcription factor, Biology and life sciences, lcsh:R, Proteins, Janus Kinase 3, DNA Methylation, Molecular biology, Introns, Regulatory Proteins, 030104 developmental biology, Gene Expression Regulation, Specimen Preparation and Treatment, RNA, lcsh:Q, Cloning, Transcription Factors, 030215 immunology
Abstract

International audience; The IL-2/JAK3/STAT-5 signaling pathway is involved on the initiation and maintenance of the transcription factor Foxp3 in regulatory T cells (Treg) and has been associated with demethylation of the intronic Conserved Non Coding Sequence-2 (CNS2). However, the role of the JAK/STAT pathway in controlling Foxp3 in the short term has been poorly investigated. Using two different JAK/STAT pharmacological inhibitors, we observed a detectable loss of Foxp3 after 10 min. of treatment that affected 70% of the cells after one hour. Using cycloheximide, a general inhibitor of mRNA translation, we determined that Foxp3, but not CD25, has a high turnover in IL-2 stimulated Treg. This reduction was correlated with a rapid reduction of Foxp3 mRNA. This loss of Foxp3 was associated with a loss in STAT-5 binding to the CNS2, which however remains demethylated. Consequently, Foxp3 expression returns to normal level upon restoration of basal JAK/STAT signaling in vivo. Reduced expression of several genes defining Treg identity was also observed upon treatment. Thus, our results demonstrate that Foxp3 has a rapid turn over in Treg partly controlled at the transcriptional level by the JAK/STAT pathway.

Published
2016

8. Caspase-3 triggers a TPCK-sensitive protease pathway leading to degradation of the BH3-only protein puma

Author

Aude Burlion, Cyril Clybouw, Marie-Thérèse Auffredou, Konstantinos Poalas, Gérald Leca, Aimé Vazquez, Abbas Hadji, Olivier Feraud, and Catherine Alexia

Subjects
Cancer Research, Cellular differentiation, Clinical Biochemistry, Regulator, Pharmaceutical Science, Caspase 3, TNF-Related Apoptosis-Inducing Ligand, Mice, Transforming Growth Factor beta, Cell Line, Tumor, Proto-Oncogene Proteins, hemic and lymphatic diseases, Puma, Transcriptional regulation, Animals, Humans, Gene Silencing, Enzyme Inhibitors, Embryonic Stem Cells, Caspase, Pharmacology, biology, Effector, Tosylphenylalanyl Chloromethyl Ketone, Biochemistry (medical), Cell Differentiation, Cell Biology, biology.organism_classification, Molecular biology, Peptide Fragments, Protein Structure, Tertiary, Cell biology, Apoptosis, biology.protein, Serine Proteases, biological phenomena, cell phenomena, and immunity, Apoptosis Regulatory Proteins, Anisomycin, Signal Transduction
Abstract

The protein Puma (p53-upregulated modulator of apoptosis) belongs to the BH3-only group of the Bcl-2 family and is a major regulator of apoptosis. Although the transcriptional regulation of Puma is clearly established, little is known about the regulation of its expression at the protein levels. We show here that various signals--including the cytokine TGFβ, the death effector TRAIL or chemical drugs such as anisomycin--downregulate Puma protein levels via a novel pathway based on the sequential activation of caspase-3 and a protease inhibited by the serpase inhibitor N-tosyl-L-phenylalanine chloromethyl ketone. This pathway is specific for Puma because (1) the levels of other BH3-only proteins, such as Bim and Noxa were not modified by these stimuli and (2) this caspase-mediated degradation was dependent on both the BH3 and C-terminal domains of Puma. Our data also show that Puma is regulated during the caspase-3-dependent differentiation of murine embryonic stem cells and suggest that this pathway may be relevant and important during caspase-mediated cell differentiation not associated with apoptosis.

Published
2010

9. Gene transfer of two entry inhibitors protects CD4+ T cell from HIV-1 infection in humanized mice

Author

Nicolas Petit, Aude Burlion, Claude Baillou, Gilles Marodon, Karim Dorgham, V Schneider, François M. Lemoine, Guy Gorochov, C Amiel, Béatrice Levacher, Centre d'Immunologie et de Maladies Infectieuses (CIMI), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), Immunologie - Immunopathologie - Immunothérapie (I3), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), CHU Tenon [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Service d'Immunologie [CHU Pitié-Salpétrière], CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), and HAL-UPMC, Gestionnaire

Subjects
CD4-Positive T-Lymphocytes, 0301 basic medicine, Receptors, CCR5, Recombinant Fusion Proteins, Genetic enhancement, Transgene, Genetic Vectors, Antigens, CD34, HIV Infections, [SDV.GEN] Life Sciences [q-bio]/Genetics, Mice, SCID, Biology, Gene delivery, Viral vector, Mice, 03 medical and health sciences, Immune system, Mice, Inbred NOD, Viral entry, Genetics, Animals, Humans, Chemokine CCL5, Molecular Biology, [SDV.GEN]Life Sciences [q-bio]/Genetics, Lentivirus, Gene Transfer Techniques, Virus Internalization, Virology, 3. Good health, Genetically modified organism, 030104 developmental biology, CCR5 Receptor Antagonists, HIV-1, Molecular Medicine, Female, Viral load
Abstract

International audience; Targeting viral entry is the most likely gene therapy strategy to succeed in protecting the immune system from pathogenic HIV-1 infection. Here, we evaluated the efficacy of a gene transfer lentiviral vector expressing a combination of viral entry inhibitors, the C46 peptide (an inhibitor of viral fusion) and the P2-CCL5 intrakine (a modulator of CCR5 expression), to prevent CD4+ T-cell infection in vivo. For this, we used two different models of HIV-1-infected mice, one in which ex vivo genetically modified human T cells were grafted into immunodeficient NOD.SCID.γc−/− mice before infection and one in which genetically modified T cells were derived from CD34+ hematopoietic progenitors grafted few days after birth. Expression of the transgenes conferred a major selective advantage to genetically modified CD4+ T cells, the frequency of which could increase from 10 to 90% in the blood following HIV-1 infection. Moreover, these cells resisted HIV-1-induced depletion, contrary to non-modified cells that were depleted in the same mice. Finally, we report lower normalized viral loads in mice having received genetically modified progenitors. Altogether, our study documents that targeting viral entry in vivo is a promising avenue for the future of HIV-1 gene therapy in humans.

Published
2015

10. Targeting Both Viral and Host Determinants of Human Immunodeficiency Virus Entry, Using a New Lentiviral Vector Coexpressing the T20 Fusion Inhibitor and a Selective CCL5 Intrakine

Author

Karim Dorgham, Béatrice Levacher, Gilles Marodon, Aude Burlion, Guy Gorochov, Nicolas Petit, Marodon, Gilles, Centre d'Immunologie et de Maladies Infectieuses (CIMI), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Immunologie - Immunopathologie - Immunothérapeutique (I3), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), This work was funded by the French National Agencyfor AIDS Research (ANRS). N.P. is a recipient for adoctoral fellowship from the SIDAction/Fond de DotationPierre Bergé.A.B.istherecipientofadoctoralfellowshipfrom the Ministe`re de la Recherche et de l’EnseignementSupérieur. The authors thank Dr. D. von Laer for the kindgift of the C46 cassette, Dr. A. Moris for the kind gift ofreagents, and Prof. D. Klatzmann for initial support of thisproject., Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), and Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)

Subjects
[SDV.BIO]Life Sciences [q-bio]/Biotechnology, [SDV.IMM] Life Sciences [q-bio]/Immunology, Recombinant Fusion Proteins, T-Lymphocytes, Genetic enhancement, Genetic Vectors, HIV Infections, Virus Replication, Applied Microbiology and Biotechnology, Virus, Viral vector, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Genetics, Humans, Vector (molecular biology), Chemokine CCL5, Cells, Cultured, Genetics (clinical), Pharmacology, biology, Lentivirus, HEK 293 cells, virus diseases, Genetic Therapy, Virus Internalization, biology.organism_classification, Virology, 3. Good health, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, HEK293 Cells, Viral replication, Cell culture, HIV-1, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Molecular Medicine, [SDV.IMM]Life Sciences [q-bio]/Immunology
Abstract

This is a copy of an article published in Human Gene Therapy Methods © [2014 [copyright Mary Ann Liebert, Inc.]; [Human Gene Therapy Methods] is available online at: http://online.liebertpub.com.; International audience; Numerous strategies targeting early and late steps of the HIV life cycle have been proposed for gene therapy. However, targeting viral and host determinants of HIV entry is the only strategy that would prevent viral DNA-mediated CD4 + cell death while diminishing the possibility for the virus to escape. To this end, we devised a bicistronic lentiviral vector expressing the membrane-bound form of the T20 fusion inhibitor, referred to as the C46 peptide, and a CCR5 superagonist, modified to sequester CCR5 away from the cell surface, referred to as the P2-CCL5 intrakine. We tested the effects of the vector on HIV infection and replication, using the human CEMR5 cell line expressing CD4 and CCR5, and primary human T cells. Transduced cells expressed the C46 peptide, detected with the 2F5 monoclonal antibody by flow cytometry. Expression of the P2-CCL5 intrakine correlates with lower levels of cell surface CCR5. Complete protection against HIV infection could be observed in cells expressing the protective transgenes. Importantly, we show that the combination of the transgenes was more potent than either transgene alone, showing the interest of expressing two entry inhibitors to inhibit HIV infection. Last, genetically modified cells possessed a selective advantage over nonmodified cells on HIV challenge in vitro, showing that modified cells were protected from HIV-induced cell death. Our results demonstrate that lentiviral vectors coexpressing the T20 fusion inhibitor and the P2-CCL5 intrakine represent promising tools for HIV gene therapy.

Published
2014

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