Autologous Hematopoietic Stem Cell Transplantation for Tolerance

Induction in a Mouse Model of Solid Organ Transplantation

Hassan Sadozai

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of the Institute of Medical Science

University of Toronto

Autologous Hematopoietic Stem Cell Transplantation for Tolerance Induction in a

Mouse Model of Solid Organ Transplantation

Hassan Sadozai

Masters of Science, 2016

Institute of Medical Science

University of Toronto

ABSTRACT

The need for long term immunosuppression negatively impacts long term survival and quality of life for solid organ transplant patients. One solution for this would be establishment of immune tolerance. Autologous HSCT has been used clinically for the restoration of self-tolerance in autoimmune disease. We tested whether autologous

HSCT and a short treatment with rapamycin could result in tolerance to fully MHC- mismatched allografts. Mice that received HSCT and a short course of the mTORi rapamycin demonstrated significantly prolonged allograft survival compared to untreated and rapamycin-only treated controls. Immunologic studies showed that

HSCT-treated mice displayed active immune regulation as demonstrated by a primary

MLR response, markedly diminished donor specific antibody levels and significantly higher frequencies of CD4 +CD25 +FOXP3 + Tregs. These data provide a rationale for human clinical trials to examine the ability of autologous stem cell transplantation to induce tolerance in liver transplant patients (ASCOTT) which are now ongoing.

Acknowledgments

I would like to express sincere gratitude for everyone who has helped me throughout my Master’s degree. Their invaluable contributions have made this work possible and enriched my graduate experience.

I would like to humbly thank my supervisor Dr. Gary Levy for his support and guidance. His input has greatly honed my ability to think critically and design well- thought out experiments. My experience in his lab has also imbued me with a love for immunology, a subject I hope to pursue in my future academic career. I would also like to thank members of my advisory committee, Drs. Reginald Gorczynski, Li Zhang and

Mark Minden. Their support and scientific input has been indispensable to the progress and completion of this work. In particular, I would like to thank Reg Gorczynski for his rapier wit, invaluable assistance and profound insights into not only my work but also the entire field of immunology. I would also like to thank our collaborators for providing their expertise and assistance: Dr. Harold Atkins (Ottawa Hospital Research Institute),

Dr. Oyedele Adeyi and Dr. Clinton Robbins. Dr. Adeyi’s input and direction has tremendously enriched this work.

I wish to express my sincere gratitude to all members of the Levy Lab. I am very thankful to Dr. Andrzej Chruscinski, whose tireless efforts and excellent scientific guidance have significantly improved this thesis. I am also sincerely indebted to Dr. Wei

He, whose helpful demeanour and superb surgical expertise, made this work possible. I am also very thankful to Jianhua Zhang for her technical assistance and cheerful disposition. I am very grateful to my peers and cherished friends, Vanessa Rojas iii

Luengas, Kaveh Farrokhi, Mani Mian and Angela Li for their encouragement, support and technical assistance without which, this work would not have been possible. I am also thankful to all current and past members of the Levy Lab including Dr. Nazia

Selzner, Dr. Peter Urbanellis, Dr. Agata Bartczak, Anna Cocco, Kai Yu, Charmaine

Beal, Olga Luft, Albert Nguyen and Justin Manuel. I am very thankful to Andre Siegel for his assistance and his advice. I also wish to express gratitude to members of the

Gorczynski Lab, Dr. Ismat Khatri, Camila Balgobin, Fang Zhu and Anna Curry, whose help and kindness significantly enriched my graduate experience. I am also indebted to all the talented summer students I have had the opportunity to train and befriend; Celine

Yoo, Dario Ferri, Conan Chua and Nancy Qin. I would also like to thank my friends Alya

Bhimji, Sherine Ensan, Natalie Simard and Arian Khandani for their scientific insights, encouragement and invaluable support. I am very thankful to my friend Novina Wong for her continual support and camaraderie. Finally, my sincerest gratitude goes out to the

IMS department for ensuring that this graduate degree will be a most memorable one.

In particular, I would like to thank Dr. Howard Mount for his wise counsel and helpful advice over the years.

Finally, I would like to thank my friends and extended family for their inveterate support of my career aspirations and academic pursuits. I would like to thank my parents and my sister who continue to inspire me with excellence in their respective fields. I am also thankful to other members of my family who have encouraged me in my career. In particular, I am thankful to my bosom friend Dorsa Saeidi, whose loving support and profound insights push me to work diligently and be a better person. Merci

à tous.

Contributions

I would like to thank the following individuals for their contributions towards this thesis

Dr. Andrzej Chruscinski – Experiment design and data collection

Dr. Reginald Gorczynski – Experiment design and scientific input

Dr. Oyedele Adeyi – Selection of pictures for histology, pathology scores and morphometric analyses

Dr. William (Wei) He – Murine heterotopic heart transplants and data collection

UHN Flow Cytometry Facility – Sorting of labelled LSK cells

UHN STTARR Facility – Morphometric analyses and slide scanning

UHN Animal Resource Center – Tail vein injections, murine handling and housing, animal ordering

Table of Contents

ACKNOWLEDGMENTS ...... III

CONTRIBUTIONS ...... V

TABLE OF CONTENTS ...... VI

LIST OF TABLES ...... IX

LIST OF FIGURES ...... X

ABBREVIATIONS ...... XI

INTRODUCTION ...... 1

1.1 SOLID ORGAN TRANSPLANTATION ...... 1 1.1.1 Current Status of Solid Organ Transplantation ...... 1

1.2 THE IMMUNE SYSTEM ...... 3 1.2.1 Immunology of graft rejection ...... 5 1.2.1.1 Overview ...... 5 1.2.1.2 Major and Minor Histocompatibility Antigens ...... 6 1.2.1.3 Mechanisms of Allo-Recognition ...... 7 1.2.1.4 Types of Graft Rejection ...... 9 1.2.1.4.1 Hyperacute rejection ...... 10 1.2.1.4.2 Acute Rejection ...... 11 1.2.1.4.3 Chronic Rejection ...... 17 1.2.2 Prevention of Rejection ...... 19 1.2.2.1 Immunosuppression ...... 19 1.2.2.2 Induction Agents ...... 20 1.2.2.3 De-sensitization therapies ...... 21 1.2.2.4 Anti-metabolites ...... 22 1.2.2.5 Corticosteroids ...... 24 1.2.2.6 Calcineurin Inhibitors (CNI) ...... 25 1.2.2.7 mTOR inhibitors (mTORi) ...... 26 1.2.2.8 Novel therapies in the pipeline ...... 30

1.3 IMMUNE TOLERANCE ...... 31 1.3.1 Overview ...... 31 1.3.2 B cell tolerance ...... 33 1.3.3 Central T cell tolerance ...... 34 1.3.4 Peripheral T cell tolerance ...... 36

1.3.4.1 Ignorance ...... 37 1.3.4.2 Anergy ...... 38 1.3.4.3 Activation-induced cell death ...... 40 1.3.5 Suppression by immunomodulatory cells ...... 41 1.3.5.1 Dendritic cells ...... 42 1.3.5.2 Regulatory B cells ...... 43 1.3.5.3 Regulatory T cells ...... 45 1.3.5.3.1 CD4 +CD25 +FOXP3 + Tregs ...... 46 1.3.5.3.2 Mechanisms of Treg suppression ...... 48

1.4 STRATEGIES TO INDUCE TRANSPLANT TOLERANCE ...... 51 1.4.1 Overview ...... 51 1.4.2 Co-stimulatory blockade ...... 52 1.4.3 Induction or administration of Tregs ...... 54 1.4.4 Hematopoietic stem cell transplantation ...... 56 1.4.4.1 Hematopoietic stem cells ...... 57 1.4.4.2 Allogeneic HSCT and mixed chimerism ...... 61 1.4.4.3 Autologous HSCT ...... 64

HYPOTHESES AND AIMS ...... 69

MATERIALS AND METHODS ...... 70

3.1 MICE ...... 70

3.2 HETEROTOPIC CARDIAC TRANSPLANTATION ...... 70

3.3 FLOW CYTOMETRY ...... 72

3.4 PURIFICATION OF LSK CELLS ...... 73

3.5 HEMATOPOIETIC STEM CELL TRANSPLANTATION ...... 74

3.6 TREATMENT GROUPS ...... 75

3.7 HISTOLOGY AND IMMUNOHISTOCHEMISTRY ...... 77

3.8 MIXED LYMPHOCYTE REACTION ...... 77

3.9 FLOW CYTOMETRY FOR DONOR -SPECIFIC ANTIBODIES (DSA) ...... 78

3.10 STATISTICS ...... 79

RESULTS ...... 80

4.1 PURIFICATION OF LSK CELLS AND DOSE SELECTION ...... 80

4.2 HSCT WITH LSK CELLS RESULTS IN FULL HEMATOPOIETIC RECONSTITUTION AND IS NOT IMPAIRED BY RAPAMYCIN TREATMENT . 82

4.3 HSCT PROMOTES LONG -TERM CARDIAC ALLOGRAFT SURVIVAL ...... 86

4.4 HSCT-TREATED MICE MAINTAIN PRIMARY IMMUNE RESPONSE IN VITRO ...... 94

4.5 HSCT TREATMENT MARKEDLY DIMINISHES DSA AND EXPANDS TREGS ...... 96 vii

DISCUSSION ...... 98

CONCLUSIONS ...... 113

FUTURE DIRECTIONS ...... 114

REFERENCES ...... 117

viii

List of Tables

Table 1-1 General Comparison of Innate and Adaptive Immunity ...... 5

Table 1-2 Treg effector molecules ...... 50

Table 1-3 Overview of Autologous versus Allogeneic HSCT ...... 57

Table 3-1 Heterotopic heart transplantation treatment groups ...... 75

Table 4-1 Complete blood counts at Day 100 post-HSCT (data are shown as mean ±SEM) ...... 85

List of Figures

Figure 1-1 Schematic of co-stimulatory and co-inhibitory receptors ...... 14

Figure 1-2 Treg-mediated mechanisms of immunosuppression ...... 49

Figure 1-3 Schematic of mouse and human hematopoeitic development ...... 60

Figure 3-1 Schematic of HSCT treatment for tolerance induction in cardiac allotransplant model ...... 76

Figure 4-1 Isolation of LSK cells ...... 81

Figure 4-2 LSK cell dose selection ...... 82

Figure 4-3 Rapamycin does not impair long-term hematopoietic reconstitution after HSCT with LSK cells ...... 84

Figure 4-4 HSCT prolongs cardiac allograft survival ...... 90

Figure 4-5 HSCT treatment preserves cardiac allograft morphology ...... 91

Figure 4-6 Representative immunoperoxidase staining of graft infiltrating cells ...... 92

Figure 4-7 Morphometric analyses of immunoperoxidase staining ...... 93

Figure 4-8 Lymphocytes from HSCT treated mice maintain primary immune response in vitro ...... 95

Figure 4-9 Splenic Treg and DSA quantitation ...... 97

Abbreviations

Ab Antibody

ACR Acute cellular rejection

Ag Antigen

AHR Acute humoural rejection

AICD Activation induced cell death

ALPS Autoimmune lymphoproliferative syndrome

AP-1 Activator protein 1

APC Allophycocyanin

APC Antigen presenting cell

APECED Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy

BCR B cell receptor

CAV Chronic allograft vasculopathy

CD Cluster of differentiation cDC conventional DC

CNI Calcineurin inhibitor

CR Chronic rejection

CsA Cyclosporine A

CTL Cytotoxic T lymphocyte

CTLA-4 Cytotoxic T lymphocyte antigen-4

DAMP Damage associated molecular pattern

DC Dendritic cell

DN Double negative

DP Double positive

DSA Donor specific antibody xi

Fc Constant region fragment

FcR Fc receptor

FITC Fluoroscein isothiocyanate

FKBP12 FK-506 binding protein 1A, 12 kDa

FOXP3 Forkhead box P3

H&E Hematoxylin and eosin

HAR Hyperacute rejection

Hct Hematocrit

HSCT Hematopoietic s