The Immunomodulatory Effects of Arsenic Trioxide in

The Immunomodulatory Effects of Arsenic Trioxide in Autoimmunity and Alloreactivity
yishan ye
To cite this version:
yishan ye. The Immunomodulatory Effects of Arsenic Trioxide in Autoimmunity and Alloreactivity. Immunology. Sorbonne Université, 2019. English. ￿NNT : 2019SORUS426￿. ￿tel-03233559￿

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Submitted on 25 May 2021

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Sorbonne Université
Ecole doctorale 394: Physiologie, Physiopathologie et Thérapeutique
Centre de recherche Saint Antoine / Equipe Mohty
The Immunomodulatory Effects of Arsenic Trioxide in Autoimmunity and Alloreactivity
Par Yishan YE Thèse de doctorat de biologie
Spécialité : Immunologie
Dirigée par Mohamad Mohty
Présentée et soutenue publiquement le 23 Mai 2019
Devant un jury composé de : AUCOUTURIER Pierre, PU-PH, Président du Jury
BAZARBACHI Ali, Professeur, Rapporteur SAAS Philippe, Professeur, Rapporteur HERMINE Olivier, PU-PH, Examinateur
MALARD Florent, MCU-PH, Co-directeur de thèse MOHTY Mohamad, PU-PH, Directeur de thèse
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Acknowledgments
First I would like to thank the jury members, Professor Pierre Aucouturier, Professor Ali Bazarbachi, Professor Philippe Saas, Professor Olivier Hermine for having accepted to evaluate my work.
I can still remember the day in 2014 when I asked my distinguished guest from France, the president of the European Society for Blood and Marrow Transplantation, to write me a recommendation letter for a future PhD. The answer was ‘Perhaps you can come to my lab’, which opened the door to a long and fascinating journey.
To Professor Mohty, despite tons of work that you are dealing with on a daily basis, you are always the first to respond to my requirements, and the first to congratulate me when something good happens. Your global vision, leadership nature, passion towards work, great care and patience for me make you a role model that I will always follow. Thank you Professor Mohty, with my greatest appreciation.
To Dr. Florent Malard, you are really the most outstanding young physician scientist that I have ever had the privilege to meet. You are in charge of all the details of my project, and always help me make correct choice when problem happens. It is my honor to have you, a man with sharp mind, accurate choice, impartial judgement as my co-director. Thank you Florent.
To Dr. Béatrice Gaugler, you were the first person who gave me greetings during my first visit to the hospital, the moment that I will always remember. As the scientific leader of the lab, you offered me the first idea for my project, and later became the main body of this thesis. I can’t finish this thesis without the ideas, and the continuous and unselfish intellectual supports from you. Thank you Béatrice.
To Dr. Laure Ricard and Dr. Nicolas Stocker, my greatest colleagues, for fighting together, for creating a really welcoming and harmonious atmosphere, for helping and encouraging me during the hard time of research. Thank you! To Prof. Arsène
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Mekinian, for great support on the clinical samples and suggestions offered during my PhD study. Thank you! To Dr. Lama Siblany, an excellent start and I am sure that you will do great academic work in the near future. Good luck! Nevertherless, to Dr. Charlotte Laurent, Mr. Maxime Tenon, Mr. Christophe de Vassoigne, and all the other people that I have had the chance to meet in the lab, thank you so much. To Dr. Baptiste Lamarthée, Mr. Frédéric de Vassoigne, and Dr. Ruoping Tang, you were the first who offered me, a nervous foreigner without any lab techniques, the warmest welcome and the orientation courses with great patience. Thank you so much. It is impossible to list all the people who have helped and supported me during the three years. France has become so warm and lovely because of you. To Prof. He Huang and all the colleagues in Hangzhou, for offering me the best clinical training, and continuous care during my PhD study. Thank you. To China Scholarship Council for the financial support. Finally, I would like to extend my most sincere thanks to my parents, my girlfriend, my friends, and all the other people who cared and supported me continuously, far away in China. Love you forever, and see you soon.
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List of abbreviations
ABT-199: chemical name of venetoclax aGVHD: acute graft-versus-host disease AHR: airway hyperresponsiveness AP3: adapter protein-3 APCs: antigen presenting cells APL: acute promyelocytic leukemia As(III): trivalent arsenicals As2O3: arsenic trioxide ATG: antithymocyte globulin ATL: adult T-cell leukemia/lymphoma ATRA: all-trans retinoic acid BALF: broncho-alveolar lavage fluid BST2: bone marrow stromal antigen 2 BU: busulfan cDC: conventional dendritic cell CDP: common DC progenitor cGAMP: cyclic guanosine monophosphate-adenosine monophosphate CLP: common lymphoid precursor CMP: common myeloid precursor CNI: calcineurin inhibitor CpG-ODN: CpG oligodeoxyribonucleotides CsA: cyclosporine G-CSF: granulocyte-colony stimulating factor M-CSFR: macrophage colony-stimulating factor receptor CX3CR1: CX3C chemokine receptor 1 CY: cyclophosphomide DAMPs: damage-associated molecular patterns DC: dendritic cell EC50: half maximal effective concentration ER: endoplasmic reticulum FKBP12: FK506-binding protein 12 Flt3L: Fms-like tyrosine kinase 3 ligand GC: glucocorticoid GI: gastrointestinal tract

G-CSF: granulocyte-colony stimulating factor GM-CSF: granulocyte-macrophage colony-stimulating factor GRFS: GVHD-free/relapse-free survival GVHD: graft-versus-host disease GVL: graft-versus-leukemia H2O2: hydrogen peroxide HEVs: high endothelial venules HLA: human leukocyte antigen HMGB1: high mobility group box 1 HSC: hematopoietic stem cell HCT: hematopoietic cell transplantation iAs: inorganic arsenic compounds IBD: inflammatory bowel disease ID2: Inhibitor Of DNA Binding 2 IDO: indoleamine 2,3-dioxygenase IFNAR: IFN-I receptor IFN-I: type-I interferons IKK: IĸB kinase IRF7: interferon regulatory factor 7 LAK: lymphokine activated killer LAP: LC3-associated phagocytosis LC3: microtubule-associated protein 1A/1B-light chain 3 LFA-1: Lymphocyte function-associated antigen 1 Lin: lineage markers LMPP: lymphoid-primed multi-potent progenitor LP: lymphoid precursor LPS: lipopolysaccharide MA: myeloablative mAb: monoclonal antibodies MAPKs: mitogen-activated protein kinases MCMV: mouse cytomegalovirus MDDC: monocyte derived dendritic cell MDP: macrophage and DC precursor MHC: major histocompatibility complex miHA: minor histocompatibility antigen

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miRNA: microRNA MM: multiple myeloma MMF: mycophenolate mofetil MP: myeloid precursor mRNA: messenger RNA mTOR: mammalian target of rapamycin MTX: methotrexate MYD88: myeloid differentiation primary response protein 88 NaAsO2: sodium arsenite NADPH: nicotinamide adenine dinucleotide phosphate NBs: nuclear bodies NETs: neutrophil extracellular traps Nf-κB: nuclear factor-κB NK cell: natural killer cell NOD: non-obese diabetic Nrf2: nuclear factor erythroid 2-related factor 2 NRM: non-relapse mortality PAMPs: pathogen-associated molecular patterns PBMC: peripheral blood mononuclear cell pDC: plasmacytoid dendritic cell PML: promyelocytic leukemia PTCy: posttransplantation cyclophosphomide RA: rheumatoid arthritis RARE: retinoic acid response elements RARα: retinoic acid receptor-α RIC: reduced-intensity conditioning RNS: reactive nitrogen species ROR: retinoic-related orphan receptor ROS: reactive oxygen species RXR: retinoid X receptor Sca-1: stem cells antigen-1 Siglec-H: sialic acid-binding immunoglobulin-like lectin H SLE: systemic lupus erythematosus SPF: specific pathogen-free SSc: systemic sclerosis

STAT3: signal transducer and activator of transcription 3 Syk: spleen tyrosine kinase TBI: total body irradiation TCF4: transcription factor 4 Th: T helper cell TLR: toll-like receptor TNF: tumor necrosis factor Treg: regulatory T cell UPR: unfolded protein response

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List of Figures
Figure 1 . Reaction of vicinal sulfhydryl groups in a protein structure with trivalent arsenic (Emadi et al., Blood Rev, 2010)...............................................................10
Figure 2 . Dual targeting of PML-RARα and PML by As2O3 lead to APL cell differentiation and loss of self-renewal (de Thé., Nat Rev Cancer, 2018) .........11
Figure 3 . Diverse functions of As(III) on immune cells............................................. 15 Figure 4 . Mechanisms of action: As2O3 effects on CD4+ T cell............................... 20 Figure 5 . The proposed spectrum between pDC and cDC (Reizis, Immunity, 2019) 30 Figure 6 . Possible pathways of plasmacytoid dendritic cell development (modified
from Shortman et al, Adv Immunol, 2013).......................................................... 34 Figure 7 . Factors involved in pDC trafficking (modified from Swiecki et al, Nat Rev
Immunol, 2015).................................................................................................... 36 Figure 8 . TLR9 signaling (Swiecki et al, Nat Rev Immunol, 2015)........................... 40 Figure 9 . Cell-intrinsic and cooperative sensing of pDCs (modified from Reizis,
Immunity, 2019)...................................................................................................41 Figure 10 . aGVHD pathophysiology (Ghimire et al, Front Immunol, 2017)............. 51 Figure 11 . Initiation phase of aGVHD (Ghimire et al, Front Immunol, 2017)........... 52 Figure 12 . Chemotherapy and HCT protocol ............................................................97 Figure 13 . GVHD Clinical manifestations.................................................................. 98 Figure 14 . Donor engraftment and lymphoid subpopulations...................................100 Figure 15 . Effects of different drugs on aGVHD......................................................101
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List of Tables
Table 1 . Trivalent arsenic induces immune cell apoptosis: sensitivity and mechanisms of action................................................................................................................13
Table 2 . Use of trivalent arsenic in mouse models of immune-mediated diseases..... 24 Table 3 . Trivalent arsenic facilitates tumor immunotherapy...................................... 27 Table 4 . Revised Glucksberg aGVHD grading system (Harris et al., Biol Blood
Marrow Transplant, 2016)....................................................................................50 Table 5 . Overview of mouse models for aGVHD based on TBI (Boieri et al., Front
Immunol, 2016).................................................................................................... 58 Table 6 . Mouse GVHD scoring (modified from Cooke et al., Blood, 1996)............104
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Table of Contents
Acknowledgments.......................................................................................................... 2 List of abbreviations.......................................................................................................4 List of Figures................................................................................................................ 6 List of Tables..................................................................................................................7 1. Introduction................................................................................................................ 9
1.1. Immunomodulatory properties of trivalent arsenic....................................... 10 1.1.1. Pharmaceutical mechanisms of action............................................... 10 1.1.2. The multifaceted effects of trivalent arsenic on immune cells...........15 1.1.3. Trivalent arsenic in mouse models of immune-mediate diseases...... 22 1.1.4. Trivalent arsenic and tumor immunotherapy..................................... 26
1.2. Plasmacytoid dendritic cells in autoimmunity/alloreactivity........................ 29 1.2.1. Definition of pDCs............................................................................. 29 1.2.2. Development of pDCs........................................................................ 31 1.2.3. Functions of pDCs..............................................................................35 1.2.4. pDCs in autoimmunity....................................................................... 43 1.2.5. pDCs in alloreactivity.........................................................................45
1.3. Mouse models of acute GVHD..................................................................... 48 1.3.1. Acute GVHD (aGVHD).....................................................................48 1.3.2. Challenges in aGVHD prophylaxis/treatment....................................54 1.3.3. Translational value of existing aGVHD mouse models.....................57
2. Results...................................................................................................................... 62 2.1. Part 1. Immunomodulatory effects of arsenic trioxide on plasmacytoid dendritic cells and study of mechanism............................................................... 62 2.1.1. Article 1: Arsenic trioxide induces regulatory functions of plasmacytoid dendritic cells through interferon-alpha inhibition................ 63 2.2. Part 2. Effects of arsenic trioxide and other immunomodulatory drugs in a novel mouse models of acute graft-versus-host disease.......................................96 2.2.1. Results................................................................................................ 97 2.2.2. Materials and Methods..................................................................... 102
3. Discussions.............................................................................................................105 3.1. As2O3, a promising drug for diseases with IFN-I signature........................ 105 3.2. A novel aGVHD model with chemotherapy-based conditioning and G-CSF mobilized graft: advances and limitations..........................................................108
4. Conclusion..............................................................................................................111 5. References.............................................................................................................. 112 6. Annex..................................................................................................................... 149
Article 2..............................................................................................................149 Article 3..............................................................................................................162
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1.Introduction
Inorganic arsenic compounds (iAs) have been used in traditional Chinese and Western medicine for over 2400 years (Emadi et al., 2010). Arsenic trioxide (As2O3) was rediscovered in the 1970s in the treatment of acute promyelocytic leukemia (APL) with striking efficacy and good safety profile (Wang et al., 2008; Shen et al., 1997). So far, As2O3, together with all-trans retinoic acid (ATRA), has revolutionized the treatment of APL (Lo-Coco et al., 2013; Cicconi et al., 2016). In the recent decades, the successful treatment of As2O3 in mouse models of several autoimmune and inflammatory diseases has shed light on this old drug as a ‘novel’ immunomodulator (Bobe et al., 2006; Kavian et al., 2012a; 2012b). Consistently, As2O3 has also shown efficacy in a mouse model of graft-versus-host disease (GVHD), which is a major inflammatory complication after allogeneic hematopoietic cell transplantation (HCT). Alloreactivity is the cause of GVHD, which happens when immunocompetent T cells in the donated tissue (the graft) recognize the recipient (the host) as foreign, and attack the target organs. However, despite these exciting findings, the mechanisms underneath are largely unknown. Herein, we focused on plasmacytoid dendritic cells (pDCs), which is a unique subset of dendritic cells specialized in secreting high levels of type-I interferons (IFN-I), and have been reported to be implicated in the pathogenesis of autoimmune diseases such as systemic sclerosis (SSc) (Ah Kioon et al., 2018). Moreover, pDCs are also demonstrated to play a important role in GVHD pathophysiology (Bossard et al., 2012; Malard et al., 2013; Waller et al., 2014). Given the therapeutic potential of As2O3, and pathogenetic role of pDCs in both autoimmunity and alloreactivity, respectively, the first part of this thesis aimed to explore the in vitro effects of As2O3 on pDC from both healthy donors and from SSc. Following the in vitro discoveries, in the second part we constructed a novel clinical-relevant mouse model of acute GVHD (aGVHD), and tested efficacy of As2O3 and other potential anti-aGVHD drugs in this model.
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