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1. Generation of Immunodeficient Rats With Rag1 and Il2rg Gene Deletions and Human Tissue Grafting Models

Author

Jean-Paul Concordet, Carine Giovannangeli, Véronique Nerrière-Daguin, Frédérique Bellier-Waast, Laurent Tesson, Vanessa Chenouard, Delphine Garnier, Tuan H. Nguyen, Alexandre Fraichard, Séverine Ménoret, Emmanuel Merieau, Ignacio Anegon, Lucas Brusselle, Séverine Remy, Franck Duteille, Frédéric Delbos, Laure-Hélène Ouisse, Claire Usal, Giovannangeli, Carine, Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), 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), Genetic and Cellular Engineering in Immunology and Regenerative Medicine (Team 2 - U1064 Inserm - CRTI), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Structure et Instabilité des Génomes (STRING), Muséum national d'Histoire naturelle (MNHN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut de transplantation urologie-néphrologie (ITUN), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), Chirurgie Plastique Reconstructrice et Esthétique [CHU Nantes], Centre hospitalier universitaire de Nantes (CHU Nantes), and genOway, Lyon

Subjects
Male, 0301 basic medicine, Genotype, Rodent, [SDV]Life Sciences [q-bio], Transplantation, Heterologous, Transplants, Heterologous, medicine.disease_cause, Recombination-activating gene, Animals, Genetically Modified, Rats, Sprague-Dawley, 03 medical and health sciences, Immune system, biology.animal, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, medicine, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Crosses, Genetic, ComputingMilieux_MISCELLANEOUS, Homeodomain Proteins, Transplantation, Mutation, biology, Exons, Skin Transplantation, Phenotype, Rats, 3. Good health, Genetically modified organism, [SDV] Life Sciences [q-bio], Disease Models, Animal, 030104 developmental biology, Liver, Immune System, Immunology, Hepatocytes, Female, Gene Deletion, Interleukin Receptor Common gamma Subunit
Abstract

Background Immunodeficient mice are invaluable tools to analyze the long-term effects of potentially immunogenic molecules in the absence of adaptive immune responses. Nevertheless, there are models and experimental situations that would beneficiate of larger immunodeficient recipients. Rats are ideally suited to perform experiments in which larger size is needed and are still a small animal model suitable for rodent facilities. Additionally, rats reproduce certain human diseases better than mice, such as ankylosing spondylitis and Duchenne disease, and these disease models would greatly benefit from immunodeficient rats to test different immunogenic treatments. Methods We describe the generation of Il2rg-deficient rats and their crossing with previously described Rag1-deficient rats to generate double-mutant RRG animals. Results As compared with Rag1-deficient rats, Il2rg-deficient rats were more immunodeficient because they partially lacked not only T and B cells but also NK cells. RRG animals showed a more profound immunossuppressed phenotype because they displayed undetectable levels of T, B, and NK cells. Similarly, all immunoglobulin isotypes in sera were decreased in Rag1- or Il2rg-deficient rats and undetectable in Rats Rag1 and Il2rg (RRG) animals. Rag1- or Il2rg-deficient rats rejected allogeneic skin transplants and human tumors, whereas animals not only accepted allogeneic rat skin but also xenogeneic human tumors, skin, and hepatocytes. Immune humanization of RRG animals was unsuccessful. Conclusions Thus, immunodeficient RRG animals are useful recipients for long-term studies in which immune responses could be an obstacle, including tissue humanization of different tissues.

Published
2018

2. Comparative Study of the Immunoregulatory Capacity of In Vitro Generated Tolerogenic Dendritic Cells, Suppressor Macrophages, and Myeloid-Derived Suppressor Cells

Author

Régis Josien, Cédric Louvet, Laura Carretero-Iglesia, Elise Chiffoleau, Laurence Bouchet-Delbos, Emmanuel Merieau, Lucile Drujont, Marcelo Hill, Maria-Cristina Cuturi, Mercedes Segovia, Aurélie Moreau, Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), 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), Institut de transplantation urologie-néphrologie (ITUN), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), Faculté de Médecine - Université de Nantes, Fondation Progreffe, IMBIO-DC, The ONE Study (FP7-260687)., ANR: ANR-10IBHU-005, ANR: ANR-11-LABX-0016-01, European Project, Le Bihan, Sylvie, 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, Laboratoires d'excellence - Immunothérapies Grand Ouest - - IGO2011 - ANR-11-LABX-0016 - LABX - VALID, BIODRIM (FP7-305147) European Union 7th Framework Programs - INCOMING, ANR-10-IBHU-0005,CESTI (TSI-IHU),Centre Européen des Sciences de la Transplantation et de l'Immunothérapie (TSI-IHU)(2010), and ANR-11-LABX-0016,IGO,Immunothérapies Grand Ouest(2011)

Subjects
Male, 0301 basic medicine, Adoptive cell transfer, Myeloid, T-Lymphocytes, Cell, Lymphocyte Activation, law.invention, Mice, 03 medical and health sciences, 0302 clinical medicine, law, medicine, Animals, Autoimmune disease, Mice, Inbred BALB C, Transplantation, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, business.industry, Macrophages, Myeloid-Derived Suppressor Cells, Graft Survival, Dendritic Cells, medicine.disease, Adoptive Transfer, 3. Good health, Transplant rejection, Mice, Inbred C57BL, 030104 developmental biology, medicine.anatomical_structure, Immunology, Myeloid-derived Suppressor Cell, Suppressor, Bone marrow, business, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, 030215 immunology
Abstract

International audience; Background. Regulatory myeloid cell (RMC) therapy is a promising strategy for the treatment of immunological disorders such as autoimmune disease and allograft transplant rejection. Various RMC subsets can be derived from total bone marrow using different protocols, but their phenotypes often overlap, raising questions about whether they are truly distinct.Methods. In this study, we directly compared the phenotype and function of 3 types of RMCs, tolerogenic dendritic cells, suppressor macrophages, and myeloid-derived suppressor cells, generated in vitro from the same mouse strain in a single laboratory.Results. We show that the 3 RMC subsets tested in this study share some phenotypic markers, suppress T cell proliferation in vitro and were all able to prolong allograft survival in a model of skin transplantation. However, our results highlight distinct mechanisms of action that are specific to each cell population.Conclusions. This study shows for the first time a side-by-side comparison of 3 types of RMCs using the same phenotypic and functional assays, thus providing a robust analysis of their similarities and differences.

Published
2016

3. Successful Percutaneous Thrombolysis and Aspiration Thrombectomy for Graft Salvage After Pancreas Transplant Venous Thrombosis

Author

Frédéric Douane, Georges Karam, Christophe Perret, Diego Cantarovich, Julien Branchereau, Arthur David, Eric Frampas, Le Bihan, Sylvie, Service de Radiologie Centrale [CHU Nantes], Centre hospitalier universitaire de Nantes (CHU Nantes), Institut de transplantation urologie-néphrologie (ITUN), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), 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)

Subjects
Transplantation, medicine.medical_specialty, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, business.industry, Aspiration Thrombectomy, medicine.disease, [SDV.MHEP.UN] Life Sciences [q-bio]/Human health and pathology/Urology and Nephrology, [SDV.MHEP.UN]Life Sciences [q-bio]/Human health and pathology/Urology and Nephrology, Surgery, Venous thrombosis, medicine.anatomical_structure, medicine, Pancreas, Percutaneous thrombolysis, business, ComputingMilieux_MISCELLANEOUS, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

International audience

Published
2019

4. DHRS9 Is a Stable Marker of Human Regulatory Macrophages

Author

Paloma Riquelme, Diana Metes, Tuija Kekarainen, Fred Fändrich, Cristina Cuturi, Angus W. Thomson, Silvia Gregori, Giada Amodio, Norbert Ahrens, Nataša Obermajer, James A. Hutchinson, Edward K. Geissler, Camila Macedo, Christoph Brochhausen, Hans J. Schlitt, Aurélie Moreau, Department of Surgery, Section of Experimental Surgery [Regensburg, Germany], University Hospital Regensburg, Division of Regenerative Medicine, Stem Cells and Gene Therapy [Milan, Italy], IRCCS Ospedale San Raffaele [Milan, Italy]-S. Raffaele Scientific Institute-San Raffaele Telethon Institute for Gene Therapy [Milan, Italy] (SR-Tiget), Department of Surgery [Pittsburgh, PA, USA] (Thomas E. Starzl Transplantation Institute), University of Pittsburgh School of Medicine, Pennsylvania Commonwealth System of Higher Education (PCSHE)-Pennsylvania Commonwealth System of Higher Education (PCSHE), Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), 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), Institut de transplantation urologie-néphrologie (ITUN), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), Faculté de Médecine - Université de Nantes, Division of Surgical Oncology [Pittsburgh, PA, USA] (Hillman Cancer Center), University of Pittsburgh (PITT), Institute of Pathology [Regensburg, Germany], Department of Transfusion Medicine [Regensburg, Germany], Department of Biotechnology and Molecular Medicine [Kuopio, Finland] (A.I. Virtanen Institute for Molecular Sciences), University of Eastern Finland-A.I. Virtanen Institute for Molecular Sciences [Kuopio, Finland], FinVector Vision Therapies Oy [Kuopio, Finland], Institute for Applied Cell Therapy [Kiel, Germany] (Campus Kiel), University Hospital of Schleswig-Holstein [Kiel, Germany], The Regensburg Center for Interventional Immunology., European Project: 30042,RISET, European Project, Le Bihan, Sylvie, Reprogramming the immune System for the Establishment of Tolerance - RISET - 30042 - OLD, and EU-funded ONE Study Consortium - INCOMING

Subjects
0301 basic medicine, Lipopolysaccharides, Cell type, 3-Hydroxysteroid Dehydrogenases, Time Factors, Cell, Biology, Reductase, Regulatory macrophages, Gene Expression Regulation, Enzymologic, 03 medical and health sciences, Interferon-gamma, Mice, 0302 clinical medicine, Cog, Species Specificity, medicine, Macrophage, Animals, Humans, RNA, Messenger, Cells, Cultured, Regulation of gene expression, Transplantation, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Macrophages, Macrophage Activation, Phenotype, 3. Good health, Mice, Inbred C57BL, 030104 developmental biology, medicine.anatomical_structure, Immunology, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, Biomarkers, 030215 immunology
Abstract

International audience; Background. The human regulatory macrophage (Mreg) has emerged as a promising cell type for use as a cell-based adjunct immunosuppressive therapy in solid organ transplant recipients. In this brief report, dehydrogenase/reductase 9 (DHRS9) is identified as a robust marker of human Mregs.Methods. The cognate antigen of a mouse monoclonal antibody raised against human Mregs was identified as DHRS9 by immunoprecipitation and MALDI-MS sequencing. Expression of DHRS9 within a panel of monocyte-derived macrophages was investigated by quantitative PCR, immunoblotting and flow cytometry.Results. DHRS9 expression discriminated human Mregs from a panel of in vitro derived macrophages in other polarisation states. Likewise, DHRS9 expression distinguished Mregs from a variety of human monocyte-derived tolerogenic antigen-presenting cells in current development as cell-based immunotherapies, including Tol-DC, Rapa-DC, DC-10, and PGE 2-induced myeloid-derived suppressor cells. A subpopulation of DHRS9-expressing human splenic macrophages was identified by immunohistochemistry. Expression of DHRS9 was acquired gradually during in vitro development of human Mregs from CD14 + monocytes and was further enhanced by IFN-γ treatment on day 6 of culture. Stimulating Mregs with 100 ng/mL lipopolysaccharide for 24 hours did not extinguish DHRS9 expression. Dhrs9 was not an informative marker of mouse Mregs. Conclusion. DHRS9 is a specific and stable marker of human Mregs.

Published
2017

5. Efficacy of Myeloid Derived Suppressor Cells on Transplant Survival

Author

Carole Guillonneau, Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), 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), Institut de transplantation urologie-néphrologie (ITUN), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), and Le Bihan, Sylvie

Subjects
Transplantation, Adoptive cell transfer, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, business.industry, medicine.medical_treatment, Adoptive Transfer, Corneal Transplantation, Myeloid cells, Cancer research, Myeloid-derived Suppressor Cell, Animals, Medicine, Female, Myeloid Cells, business, ComputingMilieux_MISCELLANEOUS, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, Corneal transplantation
Abstract

International audience

Published
2015

6. Differential modulation of donor-specific antibodies after B-cell depleting therapies to cure chronic antibody mediated rejection

Author

Magali Giral, Stéphanie Castagnet, Maxime Touzot, Diego Cantarovich, Karine Renaudin, Grégoire Couvrat-Desvergnes, Anne Cesbron, Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), 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), Institut de transplantation urologie-néphrologie (ITUN), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), Etablissement Français du Sang [Nantes], Service d'Anatomie Pathologique [CHU Nantes], Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre Hospitalier Universitaire G. R. Laennec (HGRL Saint-Herblain), CIC biothérapies CBT 0503 [Nantes], Hôtel-Dieu de Nantes-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre hospitalier universitaire de Nantes (CHU Nantes), and Le Bihan, Sylvie

Subjects
Graft Rejection, Male, Time Factors, medicine.medical_treatment, Biopsy, Bortezomib, Antibodies, Monoclonal, Murine-Derived, HLA Antigens, Child, B-Lymphocytes, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, biology, Graft Survival, Immunosuppression, Plasmapheresis, Middle Aged, Boronic Acids, Treatment Outcome, Pyrazines, Monoclonal, Rituximab, Female, France, Antibody, Immunosuppressive Agents, medicine.drug, Adult, medicine.medical_specialty, Adolescent, Urology, Renal function, Young Adult, medicine, Humans, Aged, Autoantibodies, Retrospective Studies, Transplantation, business.industry, Kidney Transplantation, body regions, Concomitant, Immunology, biology.protein, business, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, Biomarkers
Abstract

International audience; Background. Donor-specific antibodies (DSA) are considered as reliable biomarkers for antibody-mediated rejection (ABMR) diagnosis. However, it is unclear whether DSA monitoring is necessary and could predict graft outcome after antire-jection treatment.Methods. We analyzed 28 non-sensitized kidney transplant patients with ABMR associated with de novo anti-human leukocyte antigen (HLA) DSA. Donor-specific antibody levels were measured by single antigen bead assays 12 months after antirejection therapy onset. Patients were placed in three groups according to their antirejection treatment: group I (n = 10), plasma exchange-Rituximab; group II (n = 8), Bortezomib; and group III (n = 10), optimization of maintenance immunosuppression. Half of the patients in group I demonstrated concomitant acute cellular rejection (ACR+).Results. De novo DSA were mainly anti-DQ (60%). Anti-class I and anti-DR DSA disappeared after treatment in group I and remained negative during follow-up, whereas anti-DQ DSA persisted without any modulation. In contrast, class I-II HLA-DSA mean fluorescence intensity remained unchanged in groups II and III. Graft loss was observed in 80% and 20% of patients from group I (ACR+) and group III, respectively. One year after the ABMR treatment, a 16-mL/min decline in estimated glomerular filtration rate was observed in patients from group I (ACR−) and group III. Group II showed better outcomes with a mean estimated glomerular filtration rate decline of 6.4 mL/min. Conclusion. Modulation of DSA at and after treatment of ABMR did not correlate with graft outcome over a 12-month period.

Published
2014

7. Increased soluble Flt-1 correlates with delayed graft function and early loss of peritubular capillaries in the kidney graft

Author

Anne Moreau, Fadi Fakhouri, Maryvonne Hourmant, Emanuelle Meffray, Florent Le Borgne, Yohann Foucher, Marion Chapal, Mélanie Néel, Odette Carceles, Magalie Giral, Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), 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), Institut de transplantation urologie-néphrologie (ITUN), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), Biostatistique, Recherche Clinique et Mesures Subjectives en Santé, Université de Nantes (UN), Département de Pathologie [CHU Nantes], Centre hospitalier universitaire de Nantes (CHU Nantes), An ATIP-AVENIR funding from INSERM., and Le Bihan, Sylvie

Subjects
Adult, Male, medicine.medical_specialty, Vascular repair, 030232 urology & nephrology, Urology, Endothelial repair, Delayed Graft Function, Peritubular capillaries, Monocytes, 03 medical and health sciences, 0302 clinical medicine, Postoperative Complications, Risk Factors, medicine, Living Donors, Humans, Prospective Studies, Risk factor, Tyrosine, 030304 developmental biology, Aged, 0303 health sciences, Transplantation, Kidney, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Vascular Endothelial Growth Factor Receptor-1, business.industry, Ischemia-reperfusion, Plasma levels, Anatomy, Middle Aged, VEGF, Kidney Transplantation, Capillaries, medicine.anatomical_structure, Kidney Tubules, Solubility, Reperfusion Injury, embryonic structures, Female, business, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

International audience; Background. Ischemia-reperfusion induces tubular and endothelial damage in the renal graft and leads to delayed graft function (DGF) and to an early loss of peritubular capillaries (PTC). Few, if any, clinical studies have assessed the impact of proangiogenic and antiangiogenic factors on endothelial repair during renal transplantation (RT)Yrelated ischemia-reperfusion. Methods. We prospectively assessed the kinetics of the antiangiogenic factor soluble Fms-like tyrosine kinase-1 (sFlt-1) in 136 consecutive RT patients and analyzed sFlt-1 impact on DGF and PTC loss.Results. sFlt-1 plasma levels increased by twofold to threefold throughout the first week after RT. This increase was more marked in recipients of grafts from deceased donors compared with living donors. Patients with DGF had higher sFlt-1 levels at all time points during the first 7 days after RT and a higher peak sFlt-1 compared with those without DGF. In multivariate analysis, a peak plasma sFlt-1 of 250 pg/mL or higher was associated with 2.5-fold increase in the risk of DGF (P=0.04). Similarly, patients with a peak plasma sFlt-1 of 250 pg/mL or higher had a more pronounced early decrease in PTC compared with those with a peak sFlt-1 less than 250 pg/mL.Conclusions. sFlt-1 is a new nonimmunologic independent risk factor for DGF and PTC loss. Its inhibition may help improve the outcome of RT.

Published
2013

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