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1. Rational modification, synthesis and biological evaluation of N-substituted phthalazinone derivatives designed to target interleukine-15 protein

Author

Monique Mathé-Allainmat, Jacques Lebreton, Jimmy Smadja, Laurence Arzel, Agnès Quéméner, Erwan Mortier, Benoit Sicard, Didier Dubreuil, Aurélien Leray, Mike Maillasson, Centre de Recherche en Cancérologie et Immunologie Nantes-Angers (CRCINA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA), Immunobiology of Human αβ and γδ T Cells and Immunotherapeutic Applications (CRCINA-ÉQUIPE 1), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), LabEx IGO 'Immunotherapy, Graft, Oncology' [Nantes], Chimie Et Interdisciplinarité : Synthèse, Analyse, Modélisation (CEISAM), Centre National de la Recherche Scientifique (CNRS)-Université de Nantes - UFR des Sciences et des Techniques (UN UFR ST), Université de Nantes (UN)-Université de Nantes (UN), Plateforme IMPACT Nantes (CRCINA-IMPACT), Centre de Recherche en Cancérologie Nantes-Angers (CRCNA), Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM)-PRES Université Nantes Angers Le Mans (UNAM)-Hôtel-Dieu de Nantes-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Laennec-Centre National de la Recherche Scientifique (CNRS)-Faculté de Médecine d'Angers-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM)-PRES Université Nantes Angers Le Mans (UNAM)-Hôtel-Dieu de Nantes-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Laennec-Centre National de la Recherche Scientifique (CNRS)-Faculté de Médecine d'Angers-Centre hospitalier universitaire de Nantes (CHU Nantes), Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes), Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes), LabEX IGO Immunothérapie Grand Ouest, Nantes Université (Nantes Univ), Université de Nantes - UFR des Sciences et des Techniques (UN UFR ST), Université de Nantes (UN)-Université de Nantes (UN)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Mortier, Erwan, and Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)

Subjects
[SDV.IMM] Life Sciences [q-bio]/Immunology, medicine.medical_treatment, T cell, Clinical Biochemistry, Pharmaceutical Science, autoimmune disease, 01 natural sciences, Biochemistry, Cell Line, Structure-Activity Relationship, Drug Discovery, medicine, Humans, Receptor, Molecular Biology, Interleukin-15, Virtual screening, 010405 organic chemistry, Chemistry, Spectrum Analysis, Organic Chemistry, Interleukin, Protein-protein interaction PPI, inflammatory disease, 0104 chemical sciences, 3. Good health, Cell biology, inhibitor, 010404 medicinal & biomolecular chemistry, Cytokine, medicine.anatomical_structure, Interleukin 15, Phthalazines, [SDV.IMM]Life Sciences [q-bio]/Immunology, Molecular Medicine, IL-15Rbeta, Pharmacophore, CD8
Abstract

International audience; Interleukin (IL)-15 is a pleiotropic cytokine structurally close to IL-2 and sharing with the IL-2Rβ and γc receptor (R) subunits. IL-15 plays important roles in innate and adaptative immunity, supporting the activation and proliferation of NK, NK-T, and CD8 + T cells. Over-expression of IL-15 has been shown to participate to the development of inflammatory and autoimmune diseases and diverse T cell malignancies. This study is in continuity of our previous work through which a family of small-molecule inhibitors impeding IL-15/IL-2Rβ interaction with sub-micromolar activity has been identified using pharmacophore-based virtual screening and hit optimization methods. With the aim to improve the efficacy and selectivity of our lead inhibitor, specific modifications have been introduced on the basis of optimized SAR and modelisation. The new series of compounds generated have been evaluated for their capacity to inhibit the proliferation as well as the downstream signaling of IL-15-dependent cells and to bind to IL-15.

Published
2021

2. Discovery of a Small-Molecule Inhibitor of Interleukin 15: Pharmacophore-Based Virtual Screening and Hit Optimization

Author

Yannick Jacques, Monique Mathé-Allainmat, Laurence Arzel, Mike Maillasson, Erwan Mortier, Jacques Lebreton, Romy Vomiandry, Didier Dubreuil, Agnès Quéméner, Benoit Sicard, Centre de Recherche en Cancérologie et Immunologie Nantes-Angers (CRCINA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA), Chimie Et Interdisciplinarité : Synthèse, Analyse, Modélisation (CEISAM), Université de Nantes - UFR des Sciences et des Techniques (UN UFR ST), Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), This work was funded by the programs CIMATH2 (Ciblage Moléculaire et Applications Thérapeutiques 2) and PIRAMID (Protein‐protein Interactions in Rational Approaches for Medicinal Innovative Drugs) supported by La Région Pays de la Loire. R.V. was supported by a fellowship from La Région Pays de la Loire. We thank CHEM)Symbiose and IMPACT facilities for technical assistance., Bernardo, Elizabeth, Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes), and Université de Nantes (UN)-Université de Nantes (UN)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, medicine.medical_treatment, [SDV.CAN]Life Sciences [q-bio]/Cancer, 01 natural sciences, Cell Line, Small Molecule Libraries, Mice, Structure-Activity Relationship, 03 medical and health sciences, [SDV.CAN] Life Sciences [q-bio]/Cancer, Drug Discovery, medicine, Animals, Humans, Receptor, Cell Proliferation, Interleukin-15, Virtual screening, 010405 organic chemistry, Chemistry, Interleukin, Stereoisomerism, Triazoles, Small molecule, 0104 chemical sciences, 3. Good health, Cell biology, Interleukin-2 Receptor beta Subunit, Molecular Docking Simulation, 030104 developmental biology, Cytokine, Interleukin 15, Immunology, Phthalazines, Molecular Medicine, Pharmacophore, Databases, Chemical, CD8
Abstract

International audience; Interleukin (IL)-15 is a pleiotropic cytokine, which is structurally close to IL-2 and shares with it the IL-2 β and γ receptor (R) subunits. By promoting the activation and proliferation of NK, NK-T and CD8+ T cells, IL-15 plays important roles in innate and adaptative immunity. Moreover, the association of high levels of IL-15 expression with inflammatory and autoimmune diseases has led to the development of various antagonistic approaches targeting IL- 15. This study is an original approach aimed at discovering small-molecule inhibitors impeding IL-15/IL-15R interaction. A pharmacophore and docking-based virtual screening of compound libraries led to the selection of 240 high-scoring compounds, 36 of which were found to bind IL-15, to inhibit the binding of IL-15 to the IL-2Rβ chain and/or the proliferation of IL-15-dependent cells. One of them was selected as a hit, optimized by a structure-activity relationship approach, leading to the first small-molecule IL-15 inhibitor with sub-micromolar activity.

Published
2017

3. IL-15Rα membrane anchorage in either

Author

Agnès, Quéméner, Sébastien, Morisseau, Rui P, Sousa, Kilian, Trillet, Mike, Maillasson, Isabelle, Leray, Yannick, Jacques, Johann, Dion, Isabelle, Barbieux, Marie, Frutoso, Adèle D, Laurent, Jean-Yves, Le Questel, and Erwan, Mortier

Subjects
Interleukin-15, Interleukin-2 Receptor beta Subunit, Models, Molecular, Epitopes, Binding Sites, Receptors, Interleukin-15, Multiprotein Complexes, Humans, Crystallography, X-Ray, Cell Line, Signal Transduction
Abstract

Interleukin (IL)-15 plays an important role in the communication between immune cells. It delivers its signal through different modes involving three receptor chains: IL-15Rα, IL-2Rβ and IL-2Rγc. The combination of the different chains result in the formation of IL-15Rα/IL-2Rβ/γc trimeric or IL-2Rβ/γc dimeric receptors. In this study, we have investigated the role of the IL-15Rα chain in stabilizing the cytokine in the IL-2Rβ/γc dimeric receptor. By analyzing the key amino acid residues of IL-15 facing IL-2Rβ, we provide evidence of differential interfaces in the presence or in the absence of membrane-anchored IL-15Rα. Moreover, we found that the anchorage of IL-15Rα to the cell surface regardless its mode of presentation - i.e.

Published
2019

4. Mechanistic and Structural Insights on the IL-15 System through Molecular Dynamics Simulations

Author

Agnès Quéméner, Erwan Mortier, Adèle D. Laurent, Jean-Yves Le Questel, Rui P. Sousa, Université de Nantes - UFR Sciences et Techniques (UN UFR ST), Université de Nantes (UN), Chimie Et Interdisciplinarité : Synthèse, Analyse, Modélisation (CEISAM), Université de Nantes - Faculté des Sciences et des Techniques, Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche en Cancérologie et Immunologie Nantes-Angers (CRCINA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA), Immunobiology of Human αβ and γδ T Cells and Immunotherapeutic Applications (CRCINA-ÉQUIPE 1), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes - UFR des Sciences et des Techniques (UN UFR ST), Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), LabEX IGO Immunothérapie Grand Ouest, Structure fédérative de recherche François Bonamy (SFR François Bonamy), Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche en Santé de l'Université de Nantes (IRS-UN), This research was funded by the Région des Pays de la Loire (PIRAMID project), through a 'Dynamiques Scientifiques' grant. This work was also supported by the CNRS, Inserm, and realized in the context of the IHU-Cesti (ANR-10-IBHU-005) project which received French government financial support managed by the National Research Agency and supported by Nantes Métropole and Pays de la Loire Région., ANR-10-IBHU-0005,CESTI (TSI-IHU),Centre Européen des Sciences de la Transplantation et de l'Immunothérapie (TSI-IHU)(2010), Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes), Université de Nantes (UN)-Université de Nantes (UN)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Nantes Université (Nantes Univ), Bernardo, Elizabeth, and Instituts Hospitalo-Universitaires B - Centre Européen des Sciences de la Transplantation et de l'Immunothérapie (TSI-IHU) - - CESTI (TSI-IHU)2010 - ANR-10-IBHU-0005 - IBHU - VALID

Subjects
[SDV.IMM] Life Sciences [q-bio]/Immunology, Cellular functions, Pharmaceutical Science, [SDV.CAN]Life Sciences [q-bio]/Cancer, Computational biology, Molecular Dynamics Simulation, Crystallography, X-Ray, IL-15 interfaces, Article, Analytical Chemistry, Protein–protein interaction, lcsh:QD241-441, 03 medical and health sciences, Molecular dynamics, 0302 clinical medicine, [SDV.CAN] Life Sciences [q-bio]/Cancer, lcsh:Organic chemistry, Interleukin-15 Receptor alpha Subunit, protein protein interactions, Drug Discovery, Humans, Physical and Theoretical Chemistry, Receptor, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Interleukin-15, 0303 health sciences, Chemistry, Organic Chemistry, molecular dynamics simulations, 3. Good health, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, Interleukin-2 Receptor beta Subunit, Chemistry (miscellaneous), Interleukin 15, 030220 oncology & carcinogenesis, Multiprotein Complexes, Molecular Medicine, [SDV.IMM]Life Sciences [q-bio]/Immunology, Interleukin-2, interleukin 15, Protein Binding, Signal Transduction
Abstract

Interleukin 15 (IL-15), a four-helix bundle cytokine, is involved in a plethora of different cellular functions and, particularly, plays a key role in the development and activation of immune responses. IL-15 forms receptor complexes by binding with IL-2R&beta, and common &gamma, (&gamma, c)-signaling subunits, which are shared with other members of the cytokines family (IL-2 for IL-2R&beta, and all other &gamma, c- cytokines for &gamma, c). The specificity of IL-15 is brought by the non-signaling &alpha, subunit, IL-15R&alpha, Here we present the results of molecular dynamics simulations carried out on four relevant forms of IL-15: its monomer, IL-15 interacting individually with IL-15R&alpha, (IL-15/IL-15R&alpha, ), with IL-2R&beta, /&gamma, c subunits (IL-15/IL-2R&beta, c) or with its three receptors simultaneously (IL-15/IL-15R&alpha, /IL-2R&beta, c). Through the analyses of the various trajectories, new insights on the structural features of the interfaces are highlighted, according to the considered form. The comparison of the results with the experimental data, available from X-ray crystallography, allows, in particular, the rationalization of the importance of IL-15 key residues (e.g. Asp8, Lys10, Glu64). Furthermore, the pivotal role of water molecules in the stabilization of the various protein-protein interfaces and their H-bonds networks are underlined for each of the considered complexes.

Published
2019

5. Emergence of NK Cell Hyporesponsiveness after Two IL-15 Stimulation Cycles

Author

Fella Tamzalit, Isabelle Leray, Dihia Meghnem, Yannick Jacques, Sébastien Morisseau, Marie Frutoso, Agnès Quéméner, Erwan Mortier, Immunobiology of Human αβ and γδ T Cells and Immunotherapeutic Applications (CRCINA-ÉQUIPE 1), Centre de Recherche en Cancérologie et Immunologie Nantes-Angers (CRCINA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA), This work was supported by CNRS, INSERM, the University of Nantes, Cytune Pharma, Agence Nationale de la Recherche Grant ANR-15-CE-17-0023-02, and by the Agence Nationale de la Recherche et de la Technologie., Bernardo, Elizabeth, and Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes)

Subjects
0301 basic medicine, CD4-Positive T-Lymphocytes, Adoptive cell transfer, medicine.medical_treatment, Immunology, Melanoma, Experimental, Mice, Nude, Stimulation, [SDV.CAN]Life Sciences [q-bio]/Cancer, Biology, CD8-Positive T-Lymphocytes, Lymphocyte Activation, T-Lymphocytes, Regulatory, 03 medical and health sciences, Mice, 0302 clinical medicine, Immune system, [SDV.CAN] Life Sciences [q-bio]/Cancer, Cell Line, Tumor, medicine, Immunology and Allergy, Cytotoxic T cell, Animals, Cell Proliferation, Interleukin-15, Adoptive Transfer, Cell biology, Killer Cells, Natural, Mice, Inbred C57BL, 030104 developmental biology, Cytokine, Hyaluronan Receptors, Interleukin 15, Cell culture, Female, CD8, 030215 immunology, Signal Transduction
Abstract

IL-15 is a cytokine playing a crucial role in the function of immune cells, including NK and CD8 T cells. In this study, we demonstrated that in vivo, in mice, IL-15–prestimulated NK cells were no longer able to respond to a second cycle of IL-15 stimulation. This was illustrated by defects in cell maturation, proliferation, and activation, seemingly linked to the environment surrounding NK cells but not related to the presence of CD4 regulatory T cells, TGF-β, or IL-10. Moreover, NK cells from immunodeficient mice could respond to two cycles of IL-15 stimulation, whereas an adoptive transfer of CD44+CD8+ cells impaired their responsiveness to the second cycle. Conversely, in immunocompetent mice, NK cell responsiveness to a second IL-15 stimulation was restored by the depletion of CD8+ cells. These biological findings refine our understanding of the complex mode of action of NK cells in vivo, and they should be taken into consideration for IL-15–based therapy.

Published
2018

6. Cutting Edge: Differential Fine-Tuning of IL-2- and IL-15- Dependent Functions by targeting their Common IL-2/15Rβ/γc Receptor

Author

Sarah Khaddage, Markus Hildinger, Dihia Meghnem, Mike Maillasson, Isabelle Barbieux, Marie Frutoso, Isabelle Leray, Yannick Jacques, Sébastien Morisseau, Kilian Trillet, Erwan Mortier, Agnès Quéméner, Bernardo, Elizabeth, Instituts Hospitalo-Universitaires B - Centre Européen des Sciences de la Transplantation et de l'Immunothérapie (TSI-IHU) - - CESTI (TSI-IHU)2010 - ANR-10-IBHU-0005 - IBHU - VALID, Centre de Recherche en Cancérologie et Immunologie Nantes-Angers (CRCINA), Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes), Centre hospitalier universitaire de Nantes (CHU Nantes), Evitria [Schlieren, Suisse], Infors AG [Bottmingen, Suisse], This work was supported by CNRS, Inserm, Inserm Transfert (R13079NS) and was realized in the context of the Institut-Hospitalo Universitaire–Centre Europeen des Sciences de la Transplantation et Immunotherapies (ANR-10-IBHU-005) project, which received French government financial support managed by the National Research Agency and supported by Nantes Metropole and Pays de la Loire Region. D.M. was supported by a specific thesis allocation from the Institut-Hospitalo Universitaire–Centre Europeen des Sciences de la Transplantation et Immunotherapies., ANR-10-IBHU-0005,CESTI (TSI-IHU),Centre Européen des Sciences de la Transplantation et de l'Immunothérapie (TSI-IHU)(2010), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), and Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA)

Subjects
0301 basic medicine, Interleukin 2, Regulatory T cell, Immunology, [SDV.CAN]Life Sciences [q-bio]/Cancer, Biology, CD8-Positive T-Lymphocytes, Lymphocyte Activation, T-Lymphocytes, Regulatory, Cell Line, 03 medical and health sciences, Mice, Immune system, Interleukin-15 Receptor alpha Subunit, [SDV.CAN] Life Sciences [q-bio]/Cancer, medicine, Immunology and Allergy, Animals, Humans, Interleukin-7 receptor, Receptor, Common gamma chain, Cell Proliferation, Interleukin-15, Interleukin-2 Receptor alpha Subunit, Cell biology, Interleukin-2 Receptor beta Subunit, Killer Cells, Natural, 030104 developmental biology, medicine.anatomical_structure, Interleukin 15, Interleukin-2, CD8, medicine.drug, Interleukin Receptor Common gamma Subunit, Protein Binding, Signal Transduction
Abstract

Interleukin 2 and IL-15 are two closely related cytokines, displaying important functions in the immune system. They share the heterodimeric CD122/CD132 receptor to deliver their signals within target cells. Their specificity of action is conferred by their α receptor chains, IL-2Rα and IL-15Rα. By combining an increased affinity for CD122 and an impaired recruitment of CD132, we have generated an original molecule named IL-2Rβ/γ (CD122/CD132) inhibitor (BiG), targeting the CD122/CD132 receptor. BiG efficiently inhibited IL-15– and IL-2–dependent functions of primary cells, including CD8 T and NK cells, in vitro and in vivo. We also report a differential dynamic of action of these cytokines by highlighting a major role played by the IL-2Rα receptor. Interestingly, due to the presence of IL-2Rα, BiG had no impact on IL-2–dependent regulatory T cell proliferation. Thus, by acting as a fine switch in the immune system, BiG emphasizes the differential roles of these two cytokines.

Published
2017

7. IL-15 trans-presentation promotes human NK cell development and differentiation in vivo

Author

Yannick Jacques, Hergen Spits, James P. Di Santo, Nicholas D. Huntington, Nicolas Legrand, Kees Weijer, Erwan Corcuff, Erwan Mortier, Ariane Plet, Nuno L. Alves, Barbara Jaron, Cytokines et Développement Lymphoïde, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Régulation Immunitaire et Vaccinologie, Academic Medical Center - Academisch Medisch Centrum [Amsterdam] (AMC), University of Amsterdam [Amsterdam] (UvA), Centre de Recherche en Cancérologie Nantes-Angers (CRCNA), Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM)-PRES Université Nantes Angers Le Mans (UNAM)-Hôtel-Dieu de Nantes-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Laennec-Centre National de la Recherche Scientifique (CNRS)-Faculté de Médecine d'Angers-Centre hospitalier universitaire de Nantes (CHU Nantes), This work is supported by grants from Institut Pasteur, Institut National de la Santé et de la Recherche Médicale, Ligue National Contre le Cancer, National Health and Medical Research Council of Australia, The Menzies Foundation, and a Grand Challenges in Global Health grant from the Bill and Melinda Gates Foundation. Authors from the Institut Pasteur and Academic Medical Center are part of the Human Vaccine Consortium 'Grand Challenges in Global Health: Devise Reliable Testing Systems for New Vaccines:' (http://www.hv-consortium.org)., We acknowledge the Bloemenhove Clinic (Heemstede, The Netherlands) for providing fetal tissues. We would like to thank Allison Bordack, Dr. Jean-Jacques Mention, Dr. Ferenc Scheeren, Joran Volmer, and Jenny Meerding for reagents and technical assistance and Prof. Andreas Strasser for critiques on the manuscript., Other departments, AII - Amsterdam institute for Infection and Immunity, Cell Biology and Histology, AGEM - Amsterdam Gastroenterology Endocrinology Metabolism, Mortier, Erwan, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Faculteit der Geneeskunde

Subjects
Cellular differentiation, [SDV]Life Sciences [q-bio], Mice, Interleukin 21, 0302 clinical medicine, Transduction, Genetic, Immunology and Allergy, Cytotoxic T cell, Interleukin-15, Mice, Knockout, Mice, Inbred BALB C, 0303 health sciences, Receptors, Interleukin-15, Hematopoietic Stem Cell Transplantation, Cell Differentiation, hemic and immune systems, Cell biology, DNA-Binding Proteins, Killer Cells, Natural, [SDV] Life Sciences [q-bio], Interleukin 15, Interleukin 12, [SDV.IMM]Life Sciences [q-bio]/Immunology, Stem cell, Interleukin Receptor Common gamma Subunit, [SDV.IMM] Life Sciences [q-bio]/Immunology, Cell Survival, Lymphoid Tissue, Recombinant Fusion Proteins, Transplantation, Heterologous, Immunology, bcl-X Protein, Mice, Nude, chemical and pharmacologic phenomena, Biology, CD16, 03 medical and health sciences, Animals, Humans, Cell Proliferation, 030304 developmental biology, Lymphokine-activated killer cell, Brief Definitive Report, Hematopoietic Stem Cells, Antigens, Differentiation, Mice, Inbred C57BL, Retroviridae, Animals, Newborn, Brief Definitive Reports, 030215 immunology
Abstract

The in vivo requirements for human natural killer (NK) cell development and differentiation into cytotoxic effectors expressing inhibitory receptors for self-major histocompatibility complex class I (MHC-I; killer Ig-like receptors [KIRs]) remain undefined. Here, we dissect the role of interleukin (IL)-15 in human NK cell development using Rag2(-/-)gamma c(-/-) mice transplanted with human hematopoietic stem cells. Human NK cell reconstitution was intrinsically low in this model because of the poor reactivity to mouse IL-15. Although exogenous human IL-15 (hIL-15) alone made little improvement, IL-15 coupled to IL-15 receptor alpha (IL-15R alpha) significantly augmented human NK cells. IL-15-IL-15R alpha complexes induced extensive NK cell proliferation and differentiation, resulting in accumulation of CD16(+)KIR(+) NK cells, which was not uniquely dependent on enhanced survival or preferential responsiveness of this subset to IL-15. Human NK cell differentiation in vivo required hIL-15 and progressed in a linear fashion from CD56(hi)CD16(-)KIR(-) to CD56(lo)CD16(+)KIR(-), and finally to CD56(lo)CD16(+)KIR(+). These data provide the first evidence that IL-15 trans-presentation regulates human NK cell homeostasis. Use of hIL-15 receptor agonists generates a robust humanized immune system model to study human NK cells in vivo. IL-15 receptor agonists may provide therapeutic tools to improve NK cell reconstitution after bone marrow transplants, enhance graft versus leukemia effects, and increase the pool of IL-15-responsive cells during immunotherapy strategies.

Published
2009

8. IL-15Rα chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation

Author

Tammy Woo, Erwan Mortier, Averil Ma, Sara Gozalo, Rommel Advincula, Mortier, Erwan, Department of Medicine (Dep Med - San Francisco), University of California [San Francisco] (UC San Francisco), University of California (UC)-University of California (UC), University of California [San Francisco] (UCSF), and University of California-University of California

Subjects
[SDV.IMM] Life Sciences [q-bio]/Immunology, [SDV]Life Sciences [q-bio], Immunology, Enzyme-Linked Immunosorbent Assay, Cell Communication, Biology, Lymphocyte Activation, Article, Mice, 03 medical and health sciences, Interleukin 21, 0302 clinical medicine, Immune system, Interleukin-15 Receptor alpha Subunit, Animals, Immunology and Allergy, Antigen-presenting cell, 030304 developmental biology, Interleukin-15, Mice, Knockout, 0303 health sciences, Innate immune system, Lymphokine-activated killer cell, Cell Membrane, Dendritic Cells, Articles, Dendritic cell, Adoptive Transfer, Molecular biology, Cell biology, Killer Cells, Natural, Mice, Inbred C57BL, [SDV] Life Sciences [q-bio], Interleukin 15, Interleukin 12, [SDV.IMM]Life Sciences [q-bio]/Immunology, 030215 immunology
Abstract

Natural killer (NK) cells are innate immune effectors that mediate rapid responses to viral antigens. Interleukin (IL)-15 and its high affinity IL-15 receptor, IL-15Ralpha, support NK cell homeostasis in resting animals via a novel trans presentation mechanism. To better understand how IL-15 and IL-15Ralpha support NK cell activation during immune responses, we have used sensitive assays for detecting native IL-15 and IL-15Ralpha proteins and developed an assay for detecting complexes of these proteins. We find that IL-15 and IL-15Ralpha are preassembled in complexes within the endoplasmic reticulum/Golgi of stimulated dendritic cells (DCs) before being released from cells. IL-15Ralpha is required for IL-15 production by DCs, and IL-15 that emerges onto the cell surface of matured DCs does not bind to neighboring cells expressing IL-15Ralpha. We also find that soluble IL-15-IL-15Ralpha complexes are induced during inflammation, but membrane-bound IL-15-IL-15Ralpha complexes, rather than soluble complexes, support NK cell activation in vitro and in vivo. Finally, we provide in vivo evidence that expression of IL-15Ralpha specifically on DCs is critical for trans presenting IL-15 and activating NK cells. These studies define an unprecedented cytokine-receptor biosynthetic pathway in which IL-15Ralpha serves as a chaperone for IL-15, after which membrane-bound IL-15Ralpha-IL-15 complexes activate NK cells via direct cell-cell contact.

Published
2008

9. Docking of human interleukin-15 to its specific receptor α chain: Correlation between molecular modeling and mutagenesis experimental data

Author

Erwan Mortier, Ariane Plet, Yannick Jacques, Jérôme Bernard, Agnès Quéméner, and V.H. Tran

Subjects
Models, Molecular, Sushi domain, Molecular model, Protein Conformation, Stereochemistry, Molecular Sequence Data, Computational biology, Biology, Models, Biological, Biochemistry, Protein–protein interaction, Protein structure, Structural Biology, Humans, Computer Simulation, Amino Acid Sequence, Homology modeling, Site-directed mutagenesis, Molecular Biology, Interleukin-15, Binding Sites, Interleukin-2 Receptor alpha Subunit, Recombinant Proteins, Protein Structure, Tertiary, Searching the conformational space for docking, Docking (molecular), Mutagenesis, Site-Directed, Sequence Alignment
Abstract

A structural model of the sushi domain of IL-15Ralpha was first obtained by homology modeling to study its interactions with IL-15 by means of molecular modeling, peptide scanning, and site-directed mutagenesis. From these experimental data, a putative interacting surface of IL-15Ralpha with a previously published IL-15 model was inferred: Leu25, Leu44, and Glu46 of IL-15 and Arg35 of IL-15Ralpha were found to be key interfacial residues and were subsequently used as filters for the construction of docking solutions. Human IL-15/IL-15Ralpha complexes were constructed in two stages, with a preliminary docking procedure, treating the two partners as rigid bodies and using these filters. In this first stage, two classes of docking solutions were characterized. From a topological point of view, each solution could be derived from the other by reverse orientation of one partner in relation to the other. In a second stage, several further energy refinements clearly favored one solution. Moreover, this unique docking solution was confirmed by molecular modeling of IL-15 mutants previously built and tested in our laboratory. Finally, this complex model, which is a useful tool to study the IL-15/IL-15Ralpha interface, was topologically compared to IL-2/IL-2Ralpha complexes (previous model in the literature and recent crystal structure).

Published
2006

10. IL-15.IL-15Rα complex shedding following trans-presentation is essential for the survival of IL-15 responding NK and T cells

Author

Y. Jacques, Steven Nedellec, Isabelle Barbieux, Fella Tamzalit, Sébastien Morisseau, Ariane Plet, Julie Heim, Erwan Mortier, Mortier, Erwan, Centre de Recherche en Cancérologie Nantes-Angers (CRCNA), Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM)-PRES Université Nantes Angers Le Mans (UNAM)-Hôtel-Dieu de Nantes-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Laennec-Centre National de la Recherche Scientifique (CNRS)-Faculté de Médecine d'Angers-Centre hospitalier universitaire de Nantes (CHU Nantes), Structure fédérative de recherche François Bonamy (SFR François Bonamy), and Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche en Santé de l'Université de Nantes (IRS-UN)

Subjects
[SDV.IMM] Life Sciences [q-bio]/Immunology, Cell Survival, medicine.medical_treatment, Lymphocyte, [SDV]Life Sciences [q-bio], T-Lymphocytes, Antigen presentation, Cell Communication, lymphocyte, IL-2Rb, Cell Line, Cell–cell interaction, Interleukin-15 Receptor alpha Subunit, synapse, Cell Line, Tumor, cell-cell interaction, medicine, STAT5 Transcription Factor, Cytotoxic T cell, Humans, IL-2 receptor, Cell Proliferation, Interleukin-15, Antigen Presentation, Multidisciplinary, CD40, biology, autocrine, Dendritic Cells, Biological Sciences, Flow Cytometry, Coculture Techniques, Cell biology, [SDV] Life Sciences [q-bio], Killer Cells, Natural, Protein Transport, Cytokine, medicine.anatomical_structure, HEK293 Cells, Microscopy, Fluorescence, Interleukin 15, biology.protein, [SDV.IMM]Life Sciences [q-bio]/Immunology, HeLa Cells, Protein Binding, Signal Transduction
Abstract

International audience; Interleukin (IL)-15 and its specific receptor chain, IL-15Rα, support the development of various effector cells, including NK and CD8 T cells via a mechanism called trans-presentation. Whereas the dynamic of trans-presentation has been shown to involve the recycling of IL-15Rα by presenting cells, the way responding cells integrate, or take advantage of this process has not been evaluated yet. To address this question, we set up a trans-presentation model using a membrane-bound IL-15.IL-15Rα fusion protein, and found that IL-15 is detectable within responding cells following IL-15 trans-presentation. The role of the proteolytic cleavage of IL-15Rα in this process was investigated by generating an uncleavable form of IL-15Rα. We showed that IL-15 entry into responding cells necessitates the cleavage of IL-15.IL-15Rα complex from the surface of IL-15 presenting cells, and observed that IL-15Rα cleav-age is associated with a decrease of the duration of Stat5 signaling. Once separated from presenting cells, responding cells are able to recycle IL-15.IL-15Rα complexes via intracellular compartments , for residual proliferation in a time-limited manner. These studies define an unprecedented cytokine pathway in which the IL-15.IL-15Rα complex cleaved from presenting cells allows responding cells to internalize, store and use IL-15.IL-15Rα complex for their own proliferation and survival.

Published
2014

11. Regulation of CD4+NKG2D+ Th1 Cells in Patients with Metastatic Melanoma Treated with Sorafenib: Role of IL-15Rα and NKG2D Triggering

Author

Sylvie Rusakiewicz, Sophie Caillat-Zucman, Christine Mateus, Yannick Jacques, Caroline Flament, Caroline Robert, Vichnou Poirier-Colame, Nathalie Chaput, Danila Valmori, Philippe Dessen, Philippe Vielh, Erwan Mortier, Nicolas Jacquelot, Maha Ayyoub, Camillo Porta, Laurence Zitvogel, Alexander M.M. Eggermont, Ana I. Romero, Anne Auperin, Anne Caignard, Meriem Messaoudene, Kariman Chaba, Institut Gustave Roussy (IGR), Immunologie des tumeurs et immunothérapie (UMR 1015), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre d'Investigation Clinique en Biotherapie des cancers (CIC 1428 , CBT 507 ), Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche en Cancérologie Nantes-Angers (CRCNA), Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM)-PRES Université Nantes Angers Le Mans (UNAM)-Hôtel-Dieu de Nantes-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Laennec-Centre National de la Recherche Scientifique (CNRS)-Faculté de Médecine d'Angers-Centre hospitalier universitaire de Nantes (CHU Nantes), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Service de biostatistique et d'épidémiologie (SBE), Direction de la recherche clinique [Gustave Roussy], Institut Gustave Roussy (IGR)-Institut Gustave Roussy (IGR), Laboratoire de recherche translationnelle, Direction de la recherche [Gustave Roussy], Hématopoïèse normale et pathologique (U1170 Inserm), Stabilité Génétique et Oncogenèse (UMR 8200), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), IRCCS San Matteo Hospital Foundation & University of Pavia, Service de dermatologie, Département de médecine oncologique [Gustave Roussy], Institut de Cancérologie de l'Ouest [Angers/Nantes] (UNICANCER/ICO), UNICANCER, Université Paris-Sud - Paris 11 (UP11), This work was supported by Schering-Plough legacy, Bayer Schering Pharma, Institut National du Cancer INCa, ANR, Ligue contre le cancer (équipe labellisée de LZ) and INFLACARE EU grant, ISREC Foundation, SIRIC SOCRATES, LABEX OncoImmunology, PACRI network. S. Rusakiewicz and V. Poirier-Colame were supported by the Fondation pour la Recherche Medicale (FRM) and the Fondation de France respectively. A.I. Romero was supported by the Swedish Research Council., Institut Gustave Roussy (IGR)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Università degli Studi di Pavia = University of Pavia (UNIPV), and Mortier, Erwan

Subjects
Male, Cancer Research, medicine.medical_treatment, [SDV]Life Sciences [q-bio], Ligands, 0302 clinical medicine, Enzymes -- Regulation, Vemurafenib, Melanoma, Interleukin-15, 0303 health sciences, Interleukin, Middle Aged, Sorafenib, 3. Good health, [SDV] Life Sciences [q-bio], Oncology, NK Cell Lectin-Like Receptor Subfamily K, 030220 oncology & carcinogenesis, CD4 Antigens, [SDV.IMM]Life Sciences [q-bio]/Immunology, Female, CD4 antigen, Adjuvant, medicine.drug, Adult, Niacinamide, [SDV.IMM] Life Sciences [q-bio]/Immunology, Killer cells, Cells -- Growth, chemical and pharmacologic phenomena, [SDV.CAN]Life Sciences [q-bio]/Cancer, Cell Growth Processes, Young Adult, 03 medical and health sciences, Immune system, Interleukin-15 Receptor alpha Subunit, Antigen, [SDV.CAN] Life Sciences [q-bio]/Cancer, Th1 cells, medicine, Humans, Antigens, Aged, 030304 developmental biology, business.industry, Phenylurea Compounds, Th1 Cells, medicine.disease, NKG2D, Cancer research, business
Abstract

Beyond cancer-cell intrinsic factors, the immune status of the host has a prognostic impact on patients with cancer and influences the effects of conventional chemotherapies. Metastatic melanoma is intrinsically immunogenic, thereby facilitating the search for immune biomarkers of clinical responses to cytotoxic agents. Here, we show that a multi-tyrosine kinase inhibitor, sorafenib, upregulates interleukin (IL)-15Rα in vitro and in vivo in patients with melanoma, and in conjunction with natural killer (NK) group 2D (NKG2D) ligands, contributes to the Th1 polarization and accumulation of peripheral CD4+NKG2D+ T cells. Hence, the increase of blood CD4+NKG2D+ T cells after two cycles of sorafenib (combined with temozolomide) was associated with prolonged survival in a prospective phase I/II trial enrolling 63 patients with metastatic melanoma who did not receive vemurafenib nor immune checkpoint-blocking antibodies. In contrast, in metastatic melanoma patients treated with classical treatment modalities, this CD4+NKG2D+ subset failed to correlate with prognosis. These findings indicate that sorafenib may be used as an "adjuvant" molecule capable of inducing or restoring IL-15Rα/IL-15 in tumors expressing MHCclass I-related chain A/B (MICA/B) and on circulating monocytes of responding patients, hereby contributing to the bioactivity of NKG2D+ Th1 cells., peer-reviewed

Published
2014

12. Interleukin-15 Plays a Central Role in Human Kidney Physiology and Cancer through the γc Signaling Pathway

Author

Hélène François, Julien Giron-Michel, Bruno Azzarone, Krystel Khawam, Aurore Devocelle, Bernard Charpentier, Anne Caignard, Erwan Mortier, Salem Chouaib, Silvano Ferrini, Lola Lecru, Michela Croce, Pierre Eid, Sandy Azzi, Régulation de la survie cellulaire et des allogreffes, Université Paris-Sud - Paris 11 (UP11)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Paul Brousse-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche en Cancérologie Nantes-Angers (CRCNA), Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM)-PRES Université Nantes Angers Le Mans (UNAM)-Hôtel-Dieu de Nantes-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Laennec-Centre National de la Recherche Scientifique (CNRS)-Faculté de Médecine d'Angers-Centre hospitalier universitaire de Nantes (CHU Nantes), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratory of Immunotherapy [Genova, Italy], Instituto Nazionale per la Ricerca sul Cancro [Genova, Italy], Cytokines et Immunologie des Tumeurs Humaines (U753), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM), This work was supported by grants from Agence Française de Biomédecine, ARC (Association pour la Recherche sur le Cancer) (Nu RAC11005LLA), InCa, NRB-Vaincre le Cancer, AIRC (Associazione Italiana per la Ricerca sul Cancro) IG project 5642 and Italian Ministry of Health. S.A. was a recipient of ARC and NRB-Vaincre le Cancer doctoral fellowships. K.K. was a recipient of doctoral fellowships from Société Française de Néphrologie, ARC and NRB-Vaincre le Cancer. M.C. was a recipient of a fellowship from Fondazione Italiana per la Lotta al Neuroblastoma., and Mortier, Erwan

Subjects
Anatomy and Physiology, medicine.medical_treatment, [SDV]Life Sciences [q-bio], lcsh:Medicine, urologic and male genital diseases, Biochemistry, Kidney Tubules, Proximal, 0302 clinical medicine, Renal cell carcinoma, Immune Physiology, Molecular Cell Biology, Basic Cancer Research, lcsh:Science, STAT5, Interleukin-15, 0303 health sciences, Kidney, Multidisciplinary, biology, Cadherins, Immunohistochemistry, Kidney Neoplasms, 3. Good health, Up-Regulation, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, Cytokine, Oncology, Interleukin 15, 030220 oncology & carcinogenesis, Medicine, [SDV.IMM]Life Sciences [q-bio]/Immunology, Signal transduction, Cell Division, Interleukin Receptor Common gamma Subunit, Signal Transduction, Research Article, Epithelial-Mesenchymal Transition, Tumor suppressor gene, [SDV.IMM] Life Sciences [q-bio]/Immunology, Urology, Immunology, [SDV.CAN]Life Sciences [q-bio]/Cancer, Cell Line, 03 medical and health sciences, [SDV.CAN] Life Sciences [q-bio]/Cancer, medicine, Cell Adhesion, Humans, Epithelial–mesenchymal transition, Carcinoma, Renal Cell, Biology, 030304 developmental biology, lcsh:R, Epithelial Cells, Molecular Development, Immunologic Subspecialties, medicine.disease, Solubility, Immune System, biology.protein, Cancer research, lcsh:Q, Clinical Immunology, Developmental Biology
Abstract

International audience; The ability of Interleukin-15 (IL-15) to activate many immune antitumor mechanisms renders the cytokine a good candidate for the therapy of solid tumors, particularly renal cell carcinoma. Although IL-15 is being currently used in clinical trials, the function of the cytokine on kidney's components has not been extensively studied; we thus investigated the role of IL-15 on normal and tumor renal epithelial cells. Herein, we analyzed the expression and the biological functions of IL-15 in normal renal proximal tubuli (RPTEC) and in their neoplastic counterparts, the renal clear cell carcinomas (RCC). This study shows that RPTEC express a functional heterotrimeric IL-15Rαβγc complex whose stimulation with physiologic concentrations of rhIL-15 is sufficient to inhibit epithelial mesenchymal transition (EMT) commitment preserving E-cadherin expression. Indeed, IL-15 is not only a survival factor for epithelial cells, but it can also preserve the renal epithelial phenotype through the cc-signaling pathway, demonstrating that the cytokine possess a wide range of action in epithelial homeostasis. In contrast, in RCC in vitro and in vivo studies reveal a defect in the expression of γc-receptor and JAK3 associated kinase, which strongly impacts IL-15 signaling. Indeed, in the absence of the cc/JAK3 couple we demonstrate the assembly of an unprecedented functional high affinity IL-15Rαβ heterodimer, that in response to physiologic concentrations of IL-15, triggers an unbalanced signal causing the down-regulation of the tumor suppressor gene E-cadherin, favoring RCC EMT process. Remarkably, the rescue of IL-15/γc-dependent signaling (STAT5), by co-transfecting γc and JAK3 in RCC, inhibits EMT reversion. In conclusion, these data highlight the central role of IL-15 and γc-receptor signaling in renal homeostasis through the control of E-cadherin expression and preservation of epithelial phenotype both in RPTEC (up-regulation) and RCC (down-regulation).

Published
2012

13. Interleukin-15 and Its Soluble Receptor Mediate the Response to Infliximab in Patients With Crohn's Disease

Author

Grégory Bouchaud, Isabelle Barbieux, Ariane Plet, Yannick Jacques, Arnaud Bourreille, Erwan Mortier, Mathurin Flamant, Jean Paul Galmiche, Centre de Recherche en Cancérologie Nantes-Angers (CRCNA), Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM)-PRES Université Nantes Angers Le Mans (UNAM)-Hôtel-Dieu de Nantes-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Laennec-Centre National de la Recherche Scientifique (CNRS)-Faculté de Médecine d'Angers-Centre hospitalier universitaire de Nantes (CHU Nantes), Institut de Recherche Thérapeutique [Nantes], Neuropathies du système nerveux entérique et pathologies digestives, implication des cellules gliales entériques, Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre d'Investigation Clinique [Nantes] (CIC004), Centre hospitalier universitaire de Nantes (CHU Nantes), and Mortier, Erwan

Subjects
Male, medicine.medical_treatment, [SDV]Life Sciences [q-bio], Inflammatory bowel disease, Etanercept, 0302 clinical medicine, Interleukin-15 Receptor Alpha Subunit, Crohn Disease, immune system diseases, skin and connective tissue diseases, Interleukin-15, 0303 health sciences, Receptors, Interleukin-15, Gastroenterology, Antibodies, Monoclonal, Interleukin, Middle Aged, Crohn’s Disease, Infliximab, Epithelial Cell, 3. Good health, [SDV] Life Sciences [q-bio], C-Reactive Protein, Cytokine, Interleukin 15, [SDV.IMM]Life Sciences [q-bio]/Immunology, Female, 030211 gastroenterology & hepatology, Tumor necrosis factor alpha, medicine.drug, Adult, musculoskeletal diseases, [SDV.IMM] Life Sciences [q-bio]/Immunology, Colon, ADAM17 Protein, 03 medical and health sciences, medicine, Adalimumab, Humans, 030304 developmental biology, Hepatology, Interleukin-6, Tumor Necrosis Factor-alpha, business.industry, [SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, medicine.disease, [SDV.MHEP.HEG] Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, ADAM Proteins, Immunology, business
Abstract

International audience; BACKGROUND & AIMS: Infliximab is a monoclonal antibody against tumor necrosis factor that is used to treat patients with inflammatory bowel disease. We investigated serum levels and cellular expression of inter-leukin (IL)-15 and its receptor (sIL-15Ra) in patients with Crohn's disease (CD) treated with infliximab; and the effect on sIL-15Ra secretion by epithelial cells. METHODS: CD patients were given infliximab (n = 40; 3 infusions); 37 healthy controls were studied. Serum levels of IL-15, sIL-15Ra, and complex were determined by radioimmunoassay and cytokine levels by enzyme-linked immunosorbent assay. IL-15Ra and A Desintegrin and Metalloproteinase 17 levels were assessed by immunohis-tochemistry. Epithelial cell lines (HT-29 and Caco-2) were cultured with infliximab, adalimumab, or etanercept. Patients were classified as responders and nonresponders according to their Crohn's Disease Activity Index and clinical observations. RESULTS: Before infliximab, IL-15 was higher in responders than in controls and nonre-sponders. After infliximab, IL-15 decreased in responders while remaining stable in nonresponders. sIL-15Ra and IL-15/sIL-15Ra complex levels were higher in CD than in controls and increased only in responders after inflix-imab. IL-15Ra and A Desintegrin and Metalloproteinase 17 colocalized in epithelial cells and were higher in CD patients. In vitro, infliximab but not adalimumab and etanercept induced sIL-15Ra secretion by epithelial cells. CONCLUSIONS: Serum level of sIL-15Ra and the IL-15/sIL-15Ra complex increased in responder patients and the response was associated with a decrease of IL-15. Infliximab induced the release of the IL-15 receptor alpha, suggesting a specific modulation of IL-15 and its soluble receptor by reverse signaling through transmembrane tumor necrosis factor alpha.

Published
2010

14. The exon-3-encoded domain of IL-15ralpha contributes to IL-15 high-affinity binding and is crucial for the IL-15 antagonistic effect of soluble IL-15Ralpha

Author

Yvan Boublik, Ariane Plet, Véronique Solé, Yannick Jacques, Laure Garrigue-Antar, Agnès Quéméner, Erwan Mortier, Grégory Bouchaud, Harmonie Perdreau, Centre de Recherche en Cancérologie Nantes-Angers (CRCNA), Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM)-PRES Université Nantes Angers Le Mans (UNAM)-Hôtel-Dieu de Nantes-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Laennec-Centre National de la Recherche Scientifique (CNRS)-Faculté de Médecine d'Angers-Centre hospitalier universitaire de Nantes (CHU Nantes), Centre de Recherche en Cancérologie et Immunologie Nantes-Angers (CRCINA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA), Centre de recherche en Biologie Cellulaire (CRBM), Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1), Université de Nantes (UN), Université Montpellier 1 (UM1)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Université Montpellier 2 - Sciences et Techniques (UM2), Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), and QUEMENER, Agnès

Subjects
Sushi domain, receptor, [SDV]Life Sciences [q-bio], Recombinant Fusion Proteins, Peptide, Plasma protein binding, Biology, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, Binding, Competitive, Models, Biological, Protein Structure, Secondary, 03 medical and health sciences, Structure-Activity Relationship, 0302 clinical medicine, Protein structure, Interleukin-15 Receptor alpha Subunit, Structural Biology, Cell Line, Tumor, fusion protein, Humans, Receptor, sushi domain, [SDV.IMM.II] Life Sciences [q-bio]/Immunology/Innate immunity, Molecular Biology, 030304 developmental biology, Cell Proliferation, Interleukin-2 Receptor beta Subunit, chemistry.chemical_classification, Interleukin-15, 0303 health sciences, Cell Membrane, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, antagonist, Exons, Fusion protein, Protein Structure, Tertiary, [SDV] Life Sciences [q-bio], Ectodomain, Biochemistry, chemistry, [SDV.IMM.IA]Life Sciences [q-bio]/Immunology/Adaptive immunology, Solubility, [SDV.IMM.IA] Life Sciences [q-bio]/Immunology/Adaptive immunology, Biophysics, Mutant Proteins, 030215 immunology, Protein Binding
Abstract

We previously showed that a natural soluble form of interleukin-15 (IL-15) Ralpha corresponding to the full-length ectodomain of IL-15Ralpha behaved as a potent antagonist of IL-15 action through IL-15Ralpha/beta/gamma, whereas a recombinant soluble IL-15Ralpha sushi domain did not, but instead acted as an agonist of IL-15 action through IL-15Rbeta/gamma. In order to determine precisely the molecular basis governing these antagonistic versus agonistic actions, we compared the binding properties and biological effects of recombinant soluble IL-15Ralpha (sIL-15Ralpha) species containing the sushi domain and different remaining parts of the ectodomain. We first demonstrate that the exon-3-encoded domain and, more particularly, its N-terminal 13-amino-acid (aa) peptide are important, in addition to the adjacent exon-2-encoded sushi domain, for the stabilization of the high-affinity IL-15.IL-15Ralpha complex by slowing down its dissociation rate and by contributing to about 10-20% of the free energy of interaction. We next show that all sushi-containing sIL-15Ralpha are agonists on IL-15Rbeta/gamma, coordinately increasing IL-15 binding and IL-15-induced proliferation. Their agonistic potencies are proportional to their respective affinities for IL-15. We then show that the antagonistic effect of sIL-15Ralpha in the context of IL-15Ralpha/beta/gamma is due to the 13-aa peptide that creates a sterical constraint impeding the binding of the sIL-15Ralpha.IL-15 complex to the membrane-anchored IL-15Ralpha/beta/gamma. In the frame of the soluble IL-15Ralpha sushi domain-IL-15 fusion protein that contains the 13-aa peptide, this constraint is alleviated as a result of a conformational effect due to the covalent linking of the 13-aa peptide to the N-terminus of IL-15. The soluble IL-15Ralpha sushi domain-IL-15 fusion protein is therefore able to bind and activate both the IL-15Rbeta/gamma and the IL-15Ralpha/beta/gamma receptors.

Published
2008

15. The Soluble α Chain of Interleukin-15 Receptor: A Proinflammatory Molecule Associated with Tumor Progression in Head and Neck Cancer

Author

Françoise Quintin-Colonna, Wolf H. Fridman, Yannick Jacques, Ariane Plet, Daniel Brasnu, Patrick Bruneval, Laure Garrigue-Antar, Fahima Fernani, Xavier Sastre, Eric Tartour, Alain Gey, Stéphane Hans, Erwan Mortier, Cécile Badoual, Séverine Peyrard, Pierre Laurent Puig, Grégory Bouchaud, Nour El Houda Agueznay, Immunothérapie des cancers (EA 4054), École nationale vétérinaire - Alfort (ENVA)-Université Paris Descartes - Paris 5 (UPD5), École nationale vétérinaire - Alfort (ENVA), Service d'anatomo-pathologie [CHU HEGP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Centre de Recherche en Cancérologie Nantes-Angers (CRCNA), Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM)-PRES Université Nantes Angers Le Mans (UNAM)-Hôtel-Dieu de Nantes-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Laennec-Centre National de la Recherche Scientifique (CNRS)-Faculté de Médecine d'Angers-Centre hospitalier universitaire de Nantes (CHU Nantes), Service d'oto-rhino-laryngologie et chirurgie cervico-faciale [CHU HEGP], Service d'immunologie [HEGP, Paris], Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Centre d'Investigation Clinique - Epidemiologie Clinique/essais Cliniques [CHU HEGP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Institut National de la Santé et de la Recherche Médicale (INSERM), Bases moléculaires de la réponse aux xénobiotiques (U775 (IFR95)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Service d'oncologie médicale [CHU HEGP], Service d’Anatomie Pathologique [Institut Curie, Paris], Institut Curie [Paris], European Community under the Sixth Framework Programme (grant Nj LSHC-2005-518234, ‘‘Cancerimmunotherapy’’), Institut National du Cancer, Cancéropole, e Ligue contre le Cancer (comité du Val de Marne), Pôle de Compétitivité Medicen (Immucan project), and CICBT AP-HP/Institut National de la Santé et de la Recherche Médicale (E. Tartour) and Région Pays de Loire (grant CIMATH) and Cancéropole e Grand-Ouest (grant MabImpact, Y. Jacques)., European Project: 518234,LSHC-CT, Mortier, Erwan, Integrated project Cancerimmunotherapy LSHC-CT-2006-518234 to A-MS-V and to BVdE - LSHC-CT - 518234 - INCOMING, Unité de Biotechnologie, Biocatalyse et Biorégulation (U3B), Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Géomatériaux (DGCB-LGM), École Nationale des Travaux Publics de l'État (ENTPE)-Centre National de la Recherche Scientifique (CNRS), Hlth Sci Ctr, Dept Med, Cardiol Sect, Louisiana State University (LSU), LPP - Laboratoire de Phonétique et Phonologie - UMR 7018 (LPP), Université Sorbonne Nouvelle - Paris 3-Centre National de la Recherche Scientifique (CNRS), Paris-Centre de Recherche Cardiovasculaire (PARCC - UMR-S U970), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), École nationale vétérinaire d'Alfort (ENVA)-Université Paris Descartes - Paris 5 (UPD5), École nationale vétérinaire d'Alfort (ENVA), Université Paris Descartes - Paris 5 (UPD5)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Européen Georges Pompidou [APHP] (HEGP)

Subjects
Cancer Research, [SDV.IMM] Life Sciences [q-bio]/Immunology, [SDV]Life Sciences [q-bio], Radioimmunoassay, Inflammation, [SDV.CAN]Life Sciences [q-bio]/Cancer, ADAM17 Protein, Biology, Models, Biological, Proinflammatory cytokine, 03 medical and health sciences, 0302 clinical medicine, Interleukin-15 Receptor alpha Subunit, [SDV.CAN] Life Sciences [q-bio]/Cancer, medicine, Humans, Protein Isoforms, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Interleukin-15, 0303 health sciences, Head and neck cancer, Interleukin, Cancer, Prognosis, medicine.disease, 3. Good health, Gene Expression Regulation, Neoplastic, [SDV] Life Sciences [q-bio], ADAM Proteins, Interleukin-15 receptor, Cross-Linking Reagents, Oncology, Head and Neck Neoplasms, Tumor progression, 030220 oncology & carcinogenesis, Multivariate Analysis, Immunology, Disease Progression, Cancer research, [SDV.IMM]Life Sciences [q-bio]/Immunology, Tumor necrosis factor alpha, medicine.symptom
Abstract

Interleukin (IL)-15 is a proinflammatory cytokine, as it induces the production of inflammatory cytokines [IL-6, tumor necrosis factor α (TNFα), IL-17, etc.]. A correlation between high intratumoral IL-15 concentrations and poor clinical outcome in lung and head and neck cancer patients has been recently reported. The purpose of this study was to investigate the role of the soluble α chain of IL-15 receptor (sIL-15Rα), a natural regulator of IL-15, in head and neck cancer. Fifty-three newly diagnosed untreated head and neck cancer patients were included in this study. Quantification of sIL-15Rα was performed with a newly developed RIA. Increased serum sIL-15Rα concentrations were found in head and neck cancer patients and were closely correlated with poor clinical outcome both in terms of locoregional control and survival even on multivariate analysis. sIL-15Rα was mainly produced by tumor cells via proteolytic cleavage of IL-15Rα mediated by ADAM-17. A correlation was observed between ADAM-17 expression in tumor cells and serum sIL-15Rα concentrations. Surprisingly, sIL-15Rα did not act in vitro as an IL-15 antagonist but rather as an enhancer of IL-15–induced proinflammatory cytokines (IL-6, TNFα, and IL-17) that may promote tumor progression. This new tumor evasion mechanism based on amplification of the intratumoral inflammatory reaction is probably not restricted to head and neck cancer, as other tumors have been shown to release sIL-15Rα. Overall, these results support for the first time an original protumor role of sIL-15Rα in cancer. [Cancer Res 2008;68(10):3907–14]

Published
2008

16. Soluble interleukin-15 receptor alpha (IL-15R alpha)-sushi as a selective and potent agonist of IL-15 action through IL-15R beta/gamma. Hyperagonist IL-15 x IL-15R alpha fusion proteins

Author

Erwan, Mortier, Agnès, Quéméner, Patricia, Vusio, Inken, Lorenzen, Yvan, Boublik, Joachim, Grötzinger, Ariane, Plet, and Yannick, Jacques

Subjects
Interleukin-15, Models, Molecular, Receptors, Interleukin-15, Recombinant Fusion Proteins, Receptors, Interleukin-2, CHO Cells, Receptors, Interleukin, Transfection, Protein Structure, Secondary, Interleukin-2 Receptor beta Subunit, Kinetics, Cell Line, Tumor, Cricetinae, Animals, Humans, Leukemia, Erythroblastic, Acute, Dimerization, Interleukin Receptor Common gamma Subunit, Protein Binding
Abstract

Interleukin-15 (IL-15) is crucial for the generation of multiple lymphocyte subsets (natural killer (NK), NK-T cells, and memory CD8 T cells), and transpresentation of IL-15 by monocytes and dendritic cells has been suggested to be the dominant activating process of these lymphocytes. We have previously shown that a natural soluble form of IL-15R alpha chain corresponding to the entire extracellular domain of IL-15R alpha behaves as a high affinity IL-15 antagonist. In sharp contrast with this finding, we demonstrate in this report that a recombinant, soluble sushi domain of IL-15R alpha, which bears most of the binding affinity for IL-15, behaves as a potent IL-15 agonist by enhancing its binding and biological effects (proliferation and protection from apoptosis) through the IL-15R beta/gamma heterodimer, whereas it does not affect IL-15 binding and function of the tripartite IL-15R alpha/beta/gamma membrane receptor. Our results suggest that, if naturally produced, such soluble sushi domains might be involved in the IL-15 transpresentation mechanism. Fusion proteins (RLI and ILR), in which IL-15 and IL-15R alpha-sushi are attached by a flexible linker, are even more potent than the combination of IL-15 plus sIL-15R alpha-sushi. After binding to IL-15R beta/gamma, RLI is internalized and induces a biological response very similar to the IL-15 high affinity response. Such hyper-IL-15 fusion proteins appear to constitute potent adjuvants for the expansion of lymphocyte subsets.

Published
2005

17. Natural, Proteolytic Release of a Soluble Form of Human IL-15 Receptor a-Chain That Behaves as a Specific, High Affinity IL-15 Antagonist

Author

Ariane Plet, Erwan Mortier, Jérôme Bernard, Yannick Jacques, Recherches en cancérologie, Université de Nantes (UN)-IFR26-Institut National de la Santé et de la Recherche Médicale (INSERM), This work was supported by Grants P00/3/5692 and A03/1/3311 from the Association pour la Recherche sur le Cancer (ARC), Institut National de la Santé et de la Recherche Médicale, and Centre National de la Recherche Scientifique. E.M. is a recipient of a fellowship from the Ministère de la Recherche et des Nouvelles Technologies. J.B. is a recipient of a fellowship from the Ligue Nationale Contre le Cancer (LNCC, Comité de Vendée)., and Mortier, Erwan

Subjects
DNA, Complementary, [SDV.IMM] Life Sciences [q-bio]/Immunology, Glycosylphosphatidylinositols, [SDV]Life Sciences [q-bio], Immunology, Biology, Lymphoma, T-Cell, Transfection, chemistry.chemical_compound, Cell surface receptor, Cell Line, Tumor, Chlorocebus aethiops, Animals, Humans, Immunology and Allergy, Protease Inhibitors, Receptor, Interleukin-15, COS cells, U937 cell, Receptors, Interleukin-15, Tumor Necrosis Factor-alpha, Ionomycin, Receptors, Interleukin-2, Dipeptides, U937 Cells, Molecular biology, Molecular Weight, [SDV] Life Sciences [q-bio], Protein Subunits, Interleukin-15 receptor, Solubility, chemistry, Ectodomain, Interleukin 15, Culture Media, Conditioned, COS Cells, Tetradecanoylphorbol Acetate, [SDV.IMM]Life Sciences [q-bio]/Immunology, Glioblastoma, Cell Division, Interleukin-1, Protein Binding
Abstract

IL-15 and IL-2 are two structurally and functionally related cytokines whose high affinity receptors share the IL-2R beta-chain and gamma-chain in association with IL-15R alpha-chain (IL-15R alpha) or IL-2R alpha-chain, respectively. Whereas IL-2 action seems restricted to the adaptative T cells, IL-15 appears to be crucial for the function of the innate immune responses, and the pleiotropic expression of IL-15 and IL-15R alpha hints at a much broader role for the IL-15 system in multiple cell types and tissues. In this report, using a highly sensitive radioimmunoassay, we show the existence of a soluble form of human IL-15R alpha (sIL-15R alpha) that arises from proteolytic shedding of the membrane-anchored receptor. This soluble receptor is spontaneously released from IL-15R alpha-expressing human cell lines as well as from IL-15R alpha transfected COS-7 cells. This release is strongly induced by PMA and ionomycin, and to a lesser extent by IL-1 beta and TNF-alpha. The size of sIL-15R alpha (42 kDa), together with the analysis of deletion mutants in the ectodomain of IL-15R alpha, indicates the existence of cleavage sites that are proximal to the plasma membrane. Whereas shedding induced by PMA was abrogated by the synthetic matrix metalloproteinases inhibitor GM6001, the spontaneous shedding was not, indicating the occurrence of at least two distinct proteolytic mechanisms. The sIL-15R alpha displayed high affinity for IL-15 and behaved as a potent and specific inhibitor of IL-15 binding to the membrane receptor, and of IL-15-induced cell proliferation (IC(50) in the range from 3 to 20 pM). These results suggest that IL-15R alpha shedding may play important immunoregulatory functions.

Published
2004

18. Identification of an Interleukin-15 α Receptor-binding Site on Human Interleukin-15

Author

Jérôme Bernard, Catherine Harb, John Wijdeness, Peter van Dijken, Ariane Plet, Yannick Jacques, Claudine Vermot-Desroches, Erwan Mortier, Rob H. Meloen, Joachim Grötzinger, Jerry W. Slootstra, Agnès Quéméner, Mortier, Erwan, Recherches en cancérologie, Université de Nantes (UN)-IFR26-Institut National de la Santé et de la Recherche Médicale (INSERM), Pepscan Systems [Lelystad, The Netherlands], Diaclone Laboratories [Besançon, France], Christian-Albrechts-Universität zu Kiel (CAU), and This work was supported in part by Grant P00/3/5692 from the Association pour la Recherche sur le Cancer, INSERM, and CNRS.

Subjects
Models, Molecular, [SDV]Life Sciences [q-bio], alpha-chain, Peptide, Ligands, in-vivo, Biochemistry, Epitope, law.invention, Epitopes, Mice, law, Cricetinae, Receptor, Interleukin-15, chemistry.chemical_classification, biology, Receptors, Interleukin-15, beta-chain, Antibodies, Monoclonal, Recombinant Proteins, [SDV] Life Sciences [q-bio], 3-dimensional structure, Databases as Topic, sequence alignment, Recombinant DNA, [SDV.IMM]Life Sciences [q-bio]/Immunology, Antibody, Cell Division, Protein Binding, [SDV.IMM] Life Sciences [q-bio]/Immunology, medicine.drug_class, Recombinant Fusion Proteins, Molecular Sequence Data, cell growth-factor, Sequence alignment, CHO Cells, natural-killer, Monoclonal antibody, Cell Line, medicine, Animals, Humans, Amino Acid Sequence, Molecular Biology, Binding Sites, Dose-Response Relationship, Drug, Sequence Homology, Amino Acid, Granulocyte-Macrophage Colony-Stimulating Factor, Receptors, Interleukin-2, Cell Biology, Molecular biology, Protein Structure, Tertiary, chemistry, Mutation, Mutagenesis, Site-Directed, biology.protein, Interleukin-2, Interleukin-3, il-15r-alpha chain, Peptides, protein, ID - Dier en Omgeving, il-2 receptor, Alpha chain
Abstract

To identify the epitopes in human interleukin-15 (IL-15) that are responsible for binding to the interleukin-15 receptor alpha chain, antibody and receptor mapping by peptide scanning and site-directed mutagenesis was used. By using peptide scanning, we identified four regions in IL-15. The first region ((85)CKECEELEEKN(95)) is located in the C-D loop and is recognized by a set of non-inhibitory antibodies. The second region ((102)SFVHIVQMFIN(112)) is located in helix D and is recognized by two antibodies that are inhibitory of IL-15 bio-activity but not of IL-15 binding to IL-15Ralpha. The two remaining regions react with a recombinant soluble form of the IL-15Ralpha; the first ((44)LLELQVISL(52), peptide 1) corresponds to a sequence located in the B-helix and the second ((64)ENLII(68), peptide 2) to a sequence located in helix C. The latter is also contained in the epitope recognized by an antibody (monoclonal antibody B-E29) that prevents IL-15 binding to IL-15Ralpha. By site-directed mutagenesis, we confirmed that residues present in peptide 1 (Leu-45, Glu-46, Val-49, Ser-51, and Leu-52) and peptide 2 (Leu-66 and Ile-67) are involved in the binding of IL-15 to IL-15Ralpha. Furthermore, the results presented indicate that residues in the second peptide (Glu-64, Asn-65, and Ile-68) participate in IL-2Rbeta recruitment. This finding could have implications for the dynamics of receptor assembly. These results also indicate that the modes of interaction of IL-15 and IL-2 with their respective alpha chains are not completely analogous. Finally, some of the IL-15 mutants generated in this study displayed agonist or antagonist properties and may be useful as therapeutic agents.

Published
2004

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