autologous hematopoietic stem cell …...ii autologous hematopoietic stem cell transplantation for...
TRANSCRIPT
Autologous Hematopoietic Stem Cell Transplantation for Tolerance
Induction in a Mouse Model of Solid Organ Transplantation
By
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
© Copyright by Hassan Sadozai 2016
ii
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.
iii
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
iv
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.
v
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
vi
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
vii
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
viii
DISCUSSION ................................................................................................................................................ 98
CONCLUSIONS .......................................................................................................................................... 113
FUTURE DIRECTIONS ................................................................................................................................. 114
REFERENCES ............................................................................................................................................. 117
COPYRIGHT ACKNOWLEDGEMENTS ................................................................................................................... 163
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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
x
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
xi
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
xii
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 stem cell transplantation
IDO Indoleamine 2,3-dioxygenase
IFNγ Interferon gamma
IL Interleukin
IPEX Immune dysfunction, polyendocrinopathy, enteropathy X-linked syndrome
iTreg Induced (in vitro) regulatory T cells
LFA-1 Lymphocyte function associated antigen-1
LFA-3 Lymphocyte function associated antigen-3
LLPC Long-lived plasma cell
LSK Lin-Sca1+c-kit+ stem cells
mAb Monoclonal antibody
MLR Mixed lymphocyte reaction
MS Multiple sclerosis
mTOR Mammalian target of rapamycin
mTORi mTOR inhibitor
NFAT Nuclear factor of activated T cells
NF-κB Nuclear factor kappa B
xiii
NK Natural killer cells
NKT Natural killer T cells
PAMP Pathogen associated molecular pattern
pDC plasmacytoid DC
PE Phycoerythrin
PMN Polymorphonuclear cells (neutrophils)
pTreg Peripheral (induced) regulatory T cells
Rapa Rapamycin
SLE Systemic lupus erythematosus
SOT Solid organ transplantation
TCR T cell receptor
TEC Thymic epithelial cell
Teff Effector T cells
TGF-β Transforming growth factor beta
TNF Tissue necrosis factor
TolDC Tolerogenic DC
Treg T regulatory cells
tTreg Thymic derived (natural) regulatory T cells
TxHSCT HSCT group - BALB/C allograft in C57BL/6 recipient with HSCT and rapamycin
TxRapa Rapamycin group – BALB/C allograft in C57BL/6 recipients with rapamycin
TxRej Rejecting group – BALB/C allograft in C57BL/6 recipients with no treatment
TxSyn Syngeneic group – C57BL/6 isograft in C57BL/6 recipients with no treatment
WBC White blood cell
1
Introduction
1.1 Solid Organ Transplantation
1.1.1 Current Status of Solid Organ Transplantation
Currently, solid organ transplantation (SOT) is the most effective therapy for
patients with end-stage organ failure.1 The first successful solid organ transplant was a
kidney transplant from one identical twin to another by Joseph Murray in 1954.2 This
was followed shortly by the first liver transplant by Thomas Starzl in 1963 and the first
heart transplant in 1967.3,4 The first successful immunosuppressive drug regime
consisting of the anti-proliferative azathioprine, the corticosteroid prednisone and anti-
lymphocyte globulin (ALG) managed to improve 1 year graft survival to between 40 and
50% in the early 1960s.1,5 In the decades that followed there were significant advances
both in immunosuppressive drugs, as well as, surgical and organ preservation
techniques that allowed for improved graft survival. The modern era of
immunosuppression (IS) was ushered in by the discovery of the calcineurin inhibitor
(CNI), cyclosporine (CsA), which increased 1-year graft survival rates to between 70-
80%.6,7 Currently transplantation of kidney, liver, heart, lung and pancreas and small
bowel are routinely performed in clinical medicine.8,9 In past decade, pancreatic islet
cell transplantation has also being studied for treating a subset of Type-1 diabetes
mellitus patients.10 The primary success of SOT may be demonstrated by increased
survival benefit in terms of life-years over alternative therapies. In a recent study by
2
Rana et al., it was demonstrated that over 2 million life-years were saved by SOT over
a 25-year study period from 1987 to 2012.11 Furthermore, for certain end-stage organ
diseases, transplantation is far more cost-effective than existing alternative therapies. In
the context of end-stage renal disease, transplantation can result in $300,000 direct
savings per patient over 5 years compared with dialysis.12
In Canada, the number of transplants increased from 2,093 in 2009 to 2,356 in
2014.13 The majority (1,430) of these were kidney transplants followed by liver (537) but
over 4,514 patients were still on the waiting list at the end of 2014.13 While the gap
between donated organs and potential recipients continues to increase, the long-term
maintenance of graft function is also a fundamental obstacle in transplantation
medicine.14 In a US cohort of organ recipients studied from 1989 to 2008, it was shown
that while 1-year graft survival rates were above 80% for nearly all organ types, graft
survival falls below 60% for heart, lung, liver and intestine graft survival at the 10-year
mark.15 Notwithstanding, age-related differences in graft outcomes, much of the chronic
graft loss currently observed can be attributed to the adverse effects of long-term
immunosuppression (further discussed in 1.2.3). These include nephrotoxicity,
cardiovascular disease, de novo diabetes, opportunistic infections, recurrence of
original disease (e.g.hepatitis C virus) and cancer.15–19 Thus, the concept of
immunosuppression-free “immune tolerance” remains the holy grail of transplantation
medicine and an important area of research. Immune tolerance denotes an
immunological state whereby organ-specific unresponsiveness may be achieved
without the need for chronic immunosuppression while retaining a functional immune
system for protective immunity against pathogens.14,20 In the clinic, limited numbers of
3
patients have achieved “operational tolerance”, whereby the graft displays no signs of
immune rejection for at least 1 year in the absence of any immunosuppressive drugs.21
In order to understand how immune tolerance may be achieved, knowledge of the
complex immunobiology of graft rejection is necessary.
1.2 The Immune System
A detailed description of the immune system will not be presented as this is not
the main focus of this thesis. I refer readers to the following for a more detailed
description of advances of our understanding of the immune system.22–25 The immune
system is a heterogeneous network of organs, cells and molecules that functions to
distinguish self from non-self.22 This allows for recognition of foreign antigens while
retaining tolerance to self-tissue. The immune system is classically partitioned into two
separate components based primarily, on the pace and specificity of the response
(Table 1-1).24,26 The innate immune system comprises of physical and mechanical
barriers, such as the skin, as well as cells expressing a limited number of pattern-
recognition receptors (PRRs) that are encoded in the germ-line, and recognize
pathogen-associated molecular patterns (PAMPs). These PAMPs include viral nucleic
acids as well as, molecules found on bacterial and fungal cell walls.27–29 Thus, the
innate immune system furnishes the immediate host response to an invading pathogen.
There is continuous cross-talk between the adaptive and innate arms of the immune
response and research over the past two decades has demonstrated a vital role for the
innate immune system in the modulation of the adaptive immune response.30,31
4
The adaptive immune system is capable of a protracted response and eventual
clearance of a pathogen. It is able to do so by generating an immense variety of cell
surface receptors that can recognize and react to a plethora of antigens.32 The adaptive
immune system mediates its primary functions through two types of immune cells, T
and B lymphocytes.33 Both T and B cells have evolved the capacity to generate a
diverse repertoire of antigen receptor genes (> 108) through recombination of a limited
number of gene segments.34 While most self-reactive T and B cells are eliminated
through central and peripheral mechanisms reviewed in Chapter 1.3, some cells
manage to escape these mechanisms. Unchecked self-reactivity results in the wide
range of autoimmune pathologies observed in humans. Conversely, the large repertoire
of antigens recognized by the immune system poses a significant barrier to
transplantation of foreign tissue such as in allogeneic bone marrow or solid organ
transplantation.
5
Table 1-1 General Comparison of Innate and Adaptive Immunity
1.2.1 Immunology of graft rejection
1.2.1.1 Overview
A transplanted organ elicits a broad acting immune response involving
components of both the innate and adaptive immune system. Tissue damage as a
result of surgery and ischemia-reperfusion injury generate pro-inflammatory signals
such as damage-associated molecular patterns (DAMPs), that are recognized by the
INNATE IMMUNITY ADAPTIVE IMMUNITY
Encoding Receptors Germ-line Somatic
Receptor Recombination No Yes
Receptor Target Conserved (Invariable) Large variety of antigen specific receptors
Memory No Yes
Onset of Response Fast Slow
Cellular Components
Neutrophils Eosinophils Basophils Mast Cells
Macrophages Natural Killer Cells
T cells B cells
Soluble Components Complement
Cytokines Antibodies Cytokines
6
PRRs of innate immune cells as well as receptors of the complement system.35–37 The
innate immune response is non-specific but it serves to prime the adaptive arm of the
immune system which, if left unchecked, will ultimately result in graft loss. The antigen-
specific receptors of T and B cells can recognize various antigens in a transplanted
organ such as minor histocompatibility antigens, ABO blood group antigens and
endothelial cell antigens.36,38 However, the primary response is targeted to the major
histocompatibility complex (MHC) group of antigens.39,40
1.2.1.2 Major and Minor Histocompatibility Antigens
The MHC class I and class II antigens are structurally similar molecules that are
involved in antigen presentation to CD4+ (TH – helper T) and CD8+ (Tc – cytotoxic T )
cells.41,42 T cell activation is dependent upon recognition of antigen in the context of the
peptide:MHC (p:MHC) complex.41,43 MHC class I molecules are expressed in the
surface of all nucleated cells and present peptides of endogenous cytosolic origin
including viral and bacterial peptides in infected cells, to CD8+ T cells.44 MHC class II
molecules are expressed by cells of the thymic epithelium (TECs) and by professional
antigen presenting cells (APCs) such as dendritic cells (DCs), macrophages and B cells
and express peptides of extracellular origin which are degraded via the endocytic
pathway.45. Activated human T cells have also been shown to express MHC class II and
a majority of cell types can be induced to express MHC class II by interferon γ (IFNγ)
through the class II transactivator gene (CIITA).42,46
MHC antigens were discovered in mice by Peter Gorer and later it was George
Snell who described the multi-locus nature of MHC genes.47,48 Both MHC Class I and
7
Class II are encoded for by a set of closely related genes that are located on
chromosome 6 in humans and chromosome 17 in mice.49 The human MHC is termed
HLA (human leukocyte antigen) and is extremely polymorphic. HLA Class I molecules
are primarily encoded by the HLA-A, HLA-B and HLA-C loci. Additional loci HLA-E,
HLA-F, HLA-G have also been recently described and their exact functions are not well
described.50 The 3 primary HLA Class II loci are HLA-DQ, HLA-DP and HLA-DR.50 In
mice, the MHC genes are termed H-2 genes. In mice the three Class I loci are termed
K, D and L while Class II genes are located in the I region and are usually sub-classified
as I-A and I-E.48 In placental mammals, a third region of high gene-density termed MHC
Class III is present but it codes for molecules which are involved in the inflammatory
response and structurally different from the Class I and II molecules.51 HLA genes
display Mendelian inheritance and are expressed co-dominantly with the entire
complement of HLA genes derived from one parents termed the “haplotype”.52 In
laboratory strains of inbred mice, all mice are homozygous for the same haplotype
represented by superscript letters such as H-2b, H-2d etc.48 Finally, over 50 minor
histocompatibility (MiH) antigens have been described. These are T cell epitopes
derived from several different proteins encoded on various chromosomes and are
implicated in anti-donor immune responses in organ transplantation as well as HLA-
matched hematopoietic stem cell transplantation.53,54
1.2.1.3 Mechanisms of Allo-Recognition
Due to the central role of T cells in orchestrating graft rejection, this section will
focus on the mechanisms through which T cells recognize alloantigen. T cell allo-
8
recognition is proposed to occur through 2 well-described pathways. In the “direct”
pathway, recipient T cells are capable of directly binding to non self-peptide:MHC
complexes expressed on donor APCs that are transferred along with the allograft.55,56
As APCs express both MHC class I and II, both CD4+ and CD8+ can be activated by the
direct pathway. This observation is unique to transplantation and defies the canonical
understanding that T cells are restricted to recognition of only self-MHC during thymic
education.49 However, based on the principle of molecular mimicry, memory T cells
reactive for particular microbes may cross-react with allogeneic MHC directly, a concept
known as “heterologous immunity”.57 In a recent study by Macedo et al., it was shown
that virus-specific T cells contributed significantly to the alloreactive response.58 This
serves to expand the pool of potentially alloreactive T cells and in fact, studies have
shown that up to 10% of the T cell repertoire can is involved in the alloresponse to a
transplanted organ.59 In mice, the direct pathway of allo-recognition has been
extensively studied.60,61 In particular, the study by Pietra et al. demonstrated that even
in class II deficient, and recombination activating gene (RAG) deficient mice, injection of
wild-type CD4+ cells could mediate acute rejection in the absence of host MHC class
II.60 Hence, the direct allo-recognition pathway is a crucial mediator of the alloresponse,
and is believed to predominate early after transplantation prior to the depletion of donor
APCs by maintenance immunosuppression and recipient natural killer (NK) cells.62,63
The “indirect” pathway of allo-recognition occurs when alloantigen (mostly MHC
alloantigen) is processed and presented on recipient MHC complexes expressed host
APCs. This pathway alone was demonstrated to mediate rejection in preclinical studies
where using MHC class II deficient mice as donors resulted in graft rejection.64,65
9
However, graft rejection mediated through the indirect pathway is believed to be slower
and is deemed more relevant for chronic allograft rejection.66 The indirect pathway is
important for the production of alloantibody responses, in particular, the generation of
long-lived plasma cells (LLPCs) that reside in the bone marrow.62 Moreover, studies in
murine models of regulatory T cell (Treg)-mediated transplant tolerance have also
suggested that these Treg are activated through the indirect pathway.67,68 Finally, a third
pathway of “semi-direct” allo-recognition has also been proposed.69 This concept,
known as cross-dressing or cross-priming, may occur when recipient APC acquire and
present intact donor MHC and bound peptides on their surface , thereby activating both
CD4+ and CD8+ immunity.70 A recent study has demonstrated strong evidence for this
pathway in a murine model where recipient DCs were able to present intact donor MHC
class I to directly recruit CD8+ cells and mediate an acute rejection response.71
However, study is required to further delineate this model.
1.2.1.4 Types of Graft Rejection
Currently, histological analysis of the allograft biopsy serves as the best evidence
of rejection.72 The clinical stages of rejection are classified mostly on the
pathophysiological changes, and the pace of rejection. The three major classifications
are hyperacute, acute and chronic rejection.73 Various immune mechanisms are
involved in each stage of rejection.
10
1.2.1.4.1 Hyperacute rejection
Hyperacute rejection occurs within minutes to hours of the transplant and is a
consequence of pre-formed donor-specific alloantibodies (DSA) towards donor MHC,
graft endothelium or ABO blood groups as a result of a previously rejected graft,
pregnancy or blood transfusion.74 The DSA bind immediately to the graft and result in
activation and binding of complement subunits resulting in cell destruction via the
membrane attack complex.75 Additional complement byproducts such as C3a and C5a
are also potent chemokines.76 Antibody-binding can also result in injury by NK cells and
macrophages through antibody-dependent cell-mediated cytotoxicity (ADCC).77
Antibody deposition on endothelial cells also causes them to increase expression of
Von Willebrand factor (vWF) and the adhesion molecule p-selectin (CD62P). The
transplanted organ therefore suffers from endothelial necrosis, thrombosis and
increased local coagulation with some cases even resulting in hemorrhage as a result of
severely compromised vascular integrity.72,78 The organ ceases to function and nearly
all cases require excision of the organ.78
Due to the advent of HLA antibody flow crossmatch (FXM) platforms and
selection of ABO compatible donors, hyperacute rejection is a rare occurrence in the
clinic (<1%).73,74 Several treatments have been used in an attempt to reduce DSA in
pre-sensitized transplant patients. These include IVIG, plasmapheresis, the B-cell
depleting mAb rituximab, and the proteasome inhibitor Bortezomib but their success is
limited.78–81 In experimental models, hyperacute rejection is observed primarily in
xenograft models (transplants between different species) as all mammalian species
have pre-formed xenoreactive antibodies.82,83 Xenograft studies are of particular interest
11
as successful xenografting could provide a limitless supply of organs for clinical use.
Hence, research is being conducted on the use of genetically modified pigs lacking
epitopes (such as α-1,3-Gal) that are recognized by human xenoreactive antibodies.84
1.2.1.4.2 Acute Rejection
In the absence of predisposing factors for hyperacute rejection, the allograft is
still at risk of acute rejection which can occur anywhere in the range of 1 week to 1 year
post-transplantation and has an incidence of between 10 to 20% depending on organ
type and therapeutic regimens.74,85 Since acute allograft rejection is mediated by the
humoural and the cellular components of the immune system, each of these now
constitute a distinct sub-classification of acute rejection and warrant further discussion.
Acute humoural rejection
Acute humoural rejection-AHR (also known as acute vascular rejection), is
mediated through the emergence of de novo or pre-formed DSA.86 Occurring within
days to weeks post-transplantation, it contributes significantly to clinical episodes of
acute rejection.86 DSA are primarily formed towards HLA class I, and to a lesser extent
class II, but can also be directed towards autoantigens, endothelium, ABO antigens and
miH.86,87 In the context of transplantation, the interaction of alloantigen with the B cell
receptor (BCR) results in the activation of B cells and the production of IgM as the initial
anti-donor immunoglobulin (Ig).88 B cells can also receive activation signals from binding
of complement subunits C3b and C3d to co-receptors (CD21) on their surface.89 The
12
internalized alloantigen is then presented in the context of MHC class II of activated B
cells. In the secondary lymphoid organs, B cells undergo somatic hypermutation and
class-switch recombination which results in the production of higher affinity IgG
alloantibodies.88 As mentioned previously, CD4+ cells activated through the indirect
pathway of allo-recognition play an important role in activating and expanding the B cell
alloresponse through cognate TCR:BCR interactions and co-stimulation through T cell
bound CD40L (CD154) interacting with CD40 on B cells.90,91 In particular, T follicular
helper (TFH) cells are associated with B cells in the germinal center and play an
important role in the formation of long-lived plasma cells (LLPC) as described
previously.92 As a result of these interactions, AHR is often clinically associated with
acute cellular rejection. When AHR alone is implicated, the histopathological findings
are interstitial edema, thrombosis and neutrophil infiltration with absence of
mononuclear cell infiltrates.86,93 C4d, a byproduct of complement C4b, is also found
associated with acute humoural rejection, particularly in kidney transplantation.94,95
Treatment of AHR involves the previously described therapeutics (IVIG,
plasmapheresis) as well as B cell-targeted therapies (rituximab, bortezomib). Recently
eculizumab, a mAb against complement subunit C5, has been shown to reverse some
cases of AHR in kidney patients but further clinical study of its utility is required. In a few
patients who are refractory to other forms of treatment, splenectomy has been utilized
as a successful therapy although this renders them susceptible to life-long
immunodeficiency.96
13
Acute cellular rejection
Acute cellular rejection (ACR) results primarily from the T cell-mediated immune
response to alloantigen. In the early days of organ transplantation, ACR was the major
cause of graft loss.1,14 As previously described, during acute allograft rejection, both
CD4+ and CD8+ allorecognition occurs through either a semi-direct or direct pathway.
However, binding of TCR to MHC:peptide complexes is not sufficient for activation.
Currently, T cell activation is purported to occur through a 3 signal mechanism.97,98 The
first signal for activation is provided through binding of the TCR. The second signal is
relayed by interactions between molecules expressed on APCs and “positive” and
“negative” co-stimulatory molecules on the T cell surface.99 These are molecules that
belong to either the Ig family (e.g. CD28, CTLA-4, PD1), the TIM family (T cell Ig and
mucin domain), the TNF-TNFR family (e.g. CD40, OX40) or are cell surface adhesion
molecules (e.g. LFA-1).99 A schematic representation of the major co-stimulatory and
co-inhibitory receptor engagement is depicted in Figure 1-2. The best understood of
these pathways is the T-cell bound CD28, which is expressed on 50% of CD8+ and 95%
of CD4+ cells in humans (all T cell subsets in mice).100 CD28 comes into contact with
APCs (in a region known as the immunological synapse) and binds to B7-1 (CD80) and
B7-2 (CD86) to provide a co-stimulatory signal for T cell survival, proliferation and
cytokine production through its immunoreceptor tyrosine-based activation motif
(ITAM).101 If TCR binding occurs without CD28 co-stimulation, it results in the T cell
becoming anergic. Upon CD28 binding to B7-1/2, T cells also upregulate the expression
of CTLA-4, which shares homology with CD28 and can bind to B7-1/2 with greater
affinity than CD28 resulting in inhibition of Akt signalling and cell cycle arrest..102 Thus,
14
negative feedback loops such as CTLA-4 act as immune checkpoints to regulate T cell
activation (Fig 1-2).
Figure 1-1 Schematic of co-stimulatory and co-inhibitory receptors
This figure depicts the key co-stimulatory and co-inhibitory receptors involved in
T cell activation and inhibition. The first signal for T cell activation is provided by
TCR binding to a peptide:MHC complex. The second signal is mediated by
CD28 binding to CD80/86 on APCs. CTLA-4 which is upregulated upon T cell
activation can bind CD80/86 with higher affinity than CD28. T cells can also
license APCs by CD40-CD40L interactions. The PD-PD1L axis is important for
negatively regulating T cell activation and is crucial for self-tolerance.
15
The third signal for T cell activation is provided by cytokines, most importantly
interleukin (IL)-2.103 IL-2 binds to IL-2 receptor (IL-2R) to activate downstream
transcription through various signalling pathways such as the PI3K-Akt pathway which
lies upstream of the mammalian target of rapamycin (mTOR).104 mTOR signalling is
important for cellular metabolism, downstream DNA synthesis and clonal proliferation.73
Activation of T cells also leads to the formation of memory T cells which contribute to a
more vigorous secondary response upon re-exposure to alloantigen (second-set
rejection).105 Central memory (CD44+CD62Lhi) circulate in the blood and secondary
lymphoid organs but upon differentiation to effector memory cells and entering tissues,
these cells lose expression of the adhesion molecules CCR7 and CD62L (L-selectin)
preventing re-entry into peripheral lymph nodes.105
Once activated, CD4+ and CD8+ T cells mediate acute graft rejection through
various mechanisms. CD4+ cells are divided into functional subsets based on the types
of cytokines they secrete and their function in vivo (e.g TH1, TH2, TH17, TH9 etc).106 In
the context of organ transplantation, TH1 and TH2 are the best characterized subsets.
TH1 cells produce IL-2 and IFNγ causing activation of CD8+ cytotoxic T lymphocytes
(CTL) and NK cells, which also produce IFNγ once activated, thereby acting as a
positive feedback loop to TH1 cells.106 TH1 also prime B cells for alloantibody production
and recruit macrophages through the induction of delayed-typed hypersensitivity (DTH)
responses.106 Clinical studies in rejecting kidney patients have demonstrated a strong
role for TH1 cells expressing IFNγ.107 However, the observation that IFNγ expression
was required in a murine model for alloreactive Treg function, complicates its classical
perception as solely a rejection-associated biomarker.108 TH2 cells are marked by the
16
production of immunomodulatory cytokines such as IL-4 and IL-10 and are purported to
play an important role in B cell alloresponse. In mouse models, TH2 cells can cause
graft rejection in the absence of TH1 cells, possibly through the involvement of
eosinophils.109,110 Recent research has also outlined an important role for the TH17
subset of CD4+ cells through IL-17 mediated inflammation which depends on the
infiltration and function of neutrophils.106,111 As mentioned previously, TFH cells play an
important role in promoting the production of graft-specific alloantibodies.112 Finally,
CD4+ cells are believed to activate APCs through CD40-CD40L interacts, “licensing”
them to activate CD8+ CTLs.113,114 CTLs release cytotoxic granules containing perforin,
which perforates the target cell membrane and granzyme A and B, which induce
caspase-dependent apoptosis.36,57 CTLs can also upregulate the expression of Fas
ligand (FasL) which binds to Fas on target cells to induce apoptosis through the action
of caspases.115 In cardiac transplant rejection, Fas and FasL can be detected
histologically, with increased FasL expression in rejecting hearts.116 CTLs also release
cytokines such as IFNγ and tumour necrosis factor (TNF)-α to mediate graft damage.57
Finally, the roles of NK cells in acute cellular rejection are the being investigated. NK
cells can detect self/non-self by binding of their inhibitory receptors (KIR) to self-MHC
class I.36 NK cells also produce IFNγ and TNFα and possess the capacity for cell-
mediated cytotoxicity.57 A murine study blocking the NK cell NKG2D receptor, in CD28
deficient mice, led to graft acceptance.117 NK cells were also found to target donor DCs
in lymph nodes thereby promoting the indirect allorecognition pathway for T cells.63
Through the actions of these various cell types, ACR is characterized histologically by
the infiltration of macrophages and T cells in the allograft interstitium, widespread tissue
17
necrosis and edema.74 Due to the central role played by T cells in this process, most
immunosuppressive therapies for acute rejection target T cell proliferation and activation
(reviewed in Section 1.2.2). As a result of the success of these treatments, acute
cellular rejection rates are only observed in under 15% of all non-sensitized transplant
recipients.118
1.2.1.4.3 Chronic Rejection
Despite the tremendous advances in the prevention and treatment of hyperacute
and acute rejection, there has been very little improvement in the rates of chronic
rejection (CR) in the past 20 years.74,119 CR is a multifactorial, gradual process and
neither the etiology nor the pathophysiology of this late-term graft dysfunction are fully
understood. The time to onset for chronic rejection is highly variable ranging from
several weeks to several years post-transplant.74 In cardiac transplantation, cardiac
allograft vasculopathy (CAV) along with malignancy, are the most common causes of
patient mortality after 3 years post-transplantation.120 At 5 years post-transplant, CAV is
detectable in over 30% of heart transplant recipients.121 Similarly, chronic allograft
nephropathy is present in over 50% of kidney transplant recipients at 10 years post-
transplant.122 CR is mediated by both immune and non-immune mechanisms which
warrant further discussion.
As previously described, long-term immune damage of allografts is purported to
occur through a process involving the indirect pathway of T cell allorecognition and
involves the production of alloantibody.123 Clinically, the incidence of acute cellular
rejection is a known risk factor for later onset of CAV.124 However, the incidence of
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acute humoural or antibody-mediated rejection (AMR), is seen as a far more potent
prognostic factor for CAV. The risk of CR has been shown to increase incrementally
with each episode of AMR and overall, patients with AMR have a 9-fold higher
incidence of CAV versus patients with ACR alone.125 Even in the absence of AMR, the
presence of anti-donor HLA antibodies is a risk factor for CR of heart, kidney, liver and
lung transplants.126,127 Moreover, heart transplant patients can also develop CAV in the
absence of anti-HLA antibodies through autoimmune mechanisms that produce
antibodies against self-antigens such as myosin and vimentin.124,128,129 The heterotopic
cardiac transplantation model in mice, first described by Corry et al., has been used to
study the immune mechanisms involved in CR.130 Studies using this model have
demonstrated that chronic rejection in mice is strongly associated with the presence of
DSA even in mice treated with CD4+ and CD8+ depleting mAbs.131 In contrast, male
cardiac grafts are rejected in female recipients even in the absence of DSA and C4d
deposition, signaling the role for a T-cell mediated mechanism.132 These studies have
also signaled an important role for NK cells in CAV. NK cells have been demonstrated
to mediate CAV through an Fc-dependent mechanism involving the deposition of
alloantibody.133 NK cells were also implicated in causing CAV in a model of RAG
deficient mice infected with lymphocytic choriomeningitis virus (LCMV).134 These
findings can explain why infection with cytomegalovirus (CMV) in the clinical setting, is
associated with a higher incidence of CAV.135 A recent study demonstrated that in
antibody-deficient (AID/µS KO) mice, B cells can contribute to CR through antigen
presentation and supporting T cell infiltration.136 Finally, the non-immunological factors
19
associated with CAV are hyperlipidemia, hyperglycemia and pre-existing coronary
artery disease.124
Histologically, chronic rejection manifests as intimal thickening and fibrosis with
collagen deposition in the graft parenchyma and blood vessels.74 Further, endothelial
cell proliferation is observed resulting in the formation of neointima and vessel stenosis
as well as result in coronary atherosclerosis.105,124 Both the interstitium and
parenchyma may also be invaded by T cells and macrophages.72 The results from
studies conducted in both humans and mice using C4d to distinguish CR have been
inconsistent suggesting that C4d alone cannot be utilized as a histological marker of
CR.132,137 Despite, the continued emergence of novel immunosuppressive therapies,
there is currently no effective treatment for treatment of CR and most cases result in
graft loss. Furthermore, certain predisposing factors for chronic allograft dysfunction are
often the result of immunosuppression toxicity such as hyperlipidemia and recurrence of
CMV (discussed further in 1.2.2).124 Therefore, the ultimate goal of transplantation
medicine is to induce donor-specific immune tolerance without the need for long-term
immunosuppression.
1.2.2 Prevention of Rejection
1.2.2.1 Immunosuppression
The goal of current immunosuppressive therapy in the fields of autoimmune
disease and organ transplantation is to attenuate the immune response to self-antigen
or alloantigen respectively.138,139 Therefore, immunosuppressive agents used in
transplantation medicine are also used to treat autoimmunity. Treatment guidelines and
immunosuppressive drugs currently in use for organ transplantation and autoimmune
20
disease are reviewed elsewhere.140–143 Nevertheless, the major classes of
immunosuppressive drugs and their mechanisms of action warrant further discussion
here. In the context of organ transplantation, immunosuppressive drugs can be
classified as i) induction agents; which serve to broadly eliminate alloreactive
lymphocytes immediately post-transplant thus limiting the incidence of acute rejection
and ii) maintenance immunosuppression; monotherapy or drug cocktails that aim to
provide prophylaxis against rejection.144 Maintenance immunosuppressive drugs fall into
the following major classes, anti-metabolites, corticosteroids, calcineurin inhibitors,
mTOR inhibitors. Due to the use of rapamycin in our studies, mTOR inhibitors and their
mechanisms of action will be the primary focus of this section. Finally, due to their
relevance to transplantation, treatments for desensitization as well as emerging targeted
therapies will be discussed.
1.2.2.2 Induction Agents
ATG (Antithymocyte Globulin), is a cocktail of polyclonal antibodies, generated in
rabbits and horses against human thymocytes. Rabbit ATG has been demonstrated to
be superior to horse ATG in the prevention of acute rejection.145 ATG depletes T cells in
peripheral blood and lymphoid tissues as well as induces B cell apoptosis and interferes
with dendritic cell function.146 The most common side effect of polyclonal antibodies is
excessive cytokine release resulting in flulike symptoms of fever, malaise, nausea and
in rare cases, anaphylaxis.144
There has also been a drive towards development of mAbs to target T cells as
induction therapy. Orthoclone OKT3 is a mouse IgG2a antibody to CD3ε, that has been
21
used as an induction therapy in combination with other drugs for kidney and liver
transplantation, often to delay use of cyclosporine and its toxic side effects.147
Basiliximab, is a chimeric mouse/human mAb targeted to the α-chain of the IL-2
receptor (CD25). It was demonstrated to have similar efficacy to ATG and OKT3 in
reducing the incidence of acute rejection but fewer side effects in renal transplant
patients.148 Another humanized mAb, Alemtuzumab (Campath-1H) targets the
glycoprotein CD52, found on the surfaces of T and B cells as well as macrophages, NK
cells and granulocytes.149 In a recent trail, alemtuzumab was compared to ATG and
basiliximab and was found to be associated with a lower risk for acute rejection at 6 and
12-months post-transplantation.150 However, in the same trail, no differences were
observed between alemtuzumab and ATG for high-risk patients.
1.2.2.3 De-sensitization therapies
Sensitization to donor-antigen prior to transplantation is a major barrier to
successful transplantation, particularly in kidney transplantation.151 Current approaches
to de-sensitization of these patients involve plasmapheresis in addition to IVIG and B-
cell targeted therapeutics.152 IVIG is a polyclonal antibody blood product prepared from
the serum of about 1000 to 15000 patients and was developed as a therapy for patients
with antibody deficiencies and immune thrombocytopenic purpura (ITP).153 The major
mechanisms of IVIG are believed to be mediated through anti-idiotypic antibodies that
neutralize recipient antibodies.153 IVIG can also mediate its effects by inhibiting
complement and by binding to Fc receptors (such as the inhibitory FcγRIIB). IVIG has
been shown to inhibit T cell proliferation, induce apoptosis in B cells, reduce cytokine
22
synthesis and promote the expansion of Tregs.153,154 Clinically, IVIG alone has not been
proven to be very efficient at de-sensitization and requires additional therapies. 151
Rituximab is a mAb targeted to the B-cell surface antigen CD20, which plays a role in
regulating BCR-induced calcium influxes. CD20 is expressed on pre-B and mature B
lymphocytes but its expression is lost after differentiation to plasma cells.154 Although
originally approved for B-cell lymphomas, rituximab has been used in combination with
plasmapheresis and IVIG to desensitize kidney and heart transplant patients with high
DSA titres.155 Kohei et al. have shown that rituximab is capable of reducing DSA and
risk of chronic AMR episode in ABO incompatible kidney transplant recipients.156
Another study demonstrated the efficacy of IVIG in combination with rituximab versus
IVIG alone.157 Recently, novel therapies targeting plasma cells have been developed.
Bortezomib, is a first-in-class proteasome inhibitor, which binds reversibly to the 26S
proteasome in plasma cells resulting in apoptosis by counteracting survival signals.158
Tocilizumab is an antagonistic mAb to IL-6R and is designed to interfere with the IL-6
mediated progression of B cells to plasma cells.151 These novel therapies have
undergone clinical trials but results are mixed.151,159 Further research is warranted as
currently there is no FDA-approved therapy for the treatment of pre-sensitized
transplant patients.151
1.2.2.4 Anti-metabolites
Azathioprine, a prodrug of 6-mercaptopurine (6-MP) inhibits purine synthesis and
DNA replication thereby inhibiting cell proliferation.160 Several decades after its first
clinical use, research also demonstrated that azathioprine blocked Rac1 activation
23
downstream of the CD28 co-stimulatory signal, resulting in apoptosis.161 The
development of azathioprine and 6-MP allowed for the implementation of organ
transplantation as a viable therapeutic option for end-stage organ failure and resulted in
a Nobel Prize for the pharmaceutical pioneers who had developed these drugs,
Gertrude Elion and George Hitchings.162 From the 1950s onwards, 6-MP, and later,
azathioprine were incorporated for use in kidney and liver transplantation but were not
potent or specific enough to hinder acute allograft rejection.163,164 The major side
effects associated with azathioprine are myeloid suppression and hepatotoxicity.165,166
Mycophenolate Mofetil (MMF), a prodrug of the bioactive substance
mycophenolic acid (MPA), exerts its action through blocking inosine monophosphate
dehydrogenase which is required for guanosine synthesis.167 In 1995, the FDA
approved MMF after clinical trials had demonstrated its efficacy over azathioprine.168
MMF preferentially affects T and B lymphocytes, due to their lack of a purine salvage
pathway, and is therefore more selective than azathioprine. It also has been shown to
decrease nitric oxide (NO) production thereby counteracting the effects of classically
activated macrophages, which produce NO.169 The most common side effects of MMF
are symptoms of gastrointestinal distress, nausea and hematological cytopenias.170
Enteric-coated mycophenolate sodium (EC-MPS) has been developed to avoid the
gastrointestinal side effects associated with MMF, but its clinical utility in reducing these
side effects has not been demonstrated compared to MMF.144
24
1.2.2.5 Corticosteroids
The inclusion of the corticosteroid prednisone, as part of the first
immunosuppressive cocktail was pioneered by Thomas Starzl and significantly reduced
the incidence of acute rejection in renal transplantation.171 Prednisone belongs to the
glucocorticoid class (GC) of corticosteroids. Synthetic GCs such as prednisone,
prednisolone, methylprednisolone and hydrocortisone are the main corticosteroids used
in transplantation.73 GCs are currently not widely used for long-term maintenance
immunosuppression but pulse doses are still utilized as the first-line treatment for acute
rejection.155 Like other steroids, GCs mediate their effect through the GC receptor
(GCR), which upon activation, translocates to the nucleus and activate or repress gene
transcription. GCs are believed to counteract the pro-inflammatory transcriptional
activities of the molecules nuclear factor – kappa B (NF-κB) and activator protein 1 (AP-
1) as well as interfere with IL-2 signalling.144 GCs can also promote the transcription of
anti-inflammatory genes such as lipocortin-1.155 Thus GCs are highly potent
immunosuppressants and affect the immune system in many ways. These include
suppression of macrophage function, inhibition of T cell proliferation and expansion,
inhibiting the formation of cytokines such as IL-1, IL-8 and TNFα.73,172 There have also
been studies demonstrating the ability of GCs to reprogram DCs to a tolerogenic, IL-10
producing phenotype.173,174 As a result of their wide-ranging activity, there are several
side-effects associated with GC use in transplantation such as the risk for new-onset
diabetes mellitus (NODM), hyperlipidemia and osteoporosis.
25
1.2.2.6 Calcineurin Inhibitors (CNI)
Currently, the calcineurin inhibitors (CNIs) cyclosporine A (CsA) and Tacrolimus
(FK506) are the most widely used maintenance immunosuppression in solid and bone
marrow transplantation.155 CsA is a cyclic, 11-amino acid macrolide antibiotic isolated
from the fungus Tolypocladium inflatum and its application in the 1980s, significantly
improved short-term allograft survival.144 CsA functions by binding to the cytoplasmic
protein cyclophilin (an immunophilin), and together this heterodimer inactivates the
calcium-dependent serine phosphatase, calcineurin.175 Calcineurin is responsible for
dephosphorylating the cytoplasmic nuclear factor of activated T cells (NFAT), which
translocates to the nucleus and activates key cytokines required for T cell activation and
function such as IL-2, IL-4 and IFNγ.73,176 Tacrolimus, is a macrolide lactone isolated
from the bacterium Streptomyces tsukubaenis, and binds to the immunophilin FKB1A,
which then inhibits calcineurin with a higher potency than CsA.155 Both CsA and
tacrolimus are also observed to increase expression of transforming growth factor beta
(TGF-β).177 The major side effects associated with CsA are hirsutism, nephrotoxicity,
fibrosis and dyslipidemia. Comparatively, tacrolimus has a better safety profile but
significantly increases the risk for de novo diabetes mellitus.178 Although tacrolimus is
not believed to be less nephrotoxic than CsA, it is utilized more widely after trials that
showed that it resulted in a lower incidence of acute rejection compared to CsA.179
Alternately, patients with a high risk for diabetes, are still preferentially treated with
CsA.73
26
1.2.2.7 mTOR inhibitors (mTORi)
In 1999, the FDA approved sirolimus, or rapamycin for use in kidney
transplantation.180 Rapamycin, is a macrocylic lactone that was isolated in 1975 from
the bacterium Streptomyces hygroscopicus and initially investigated as an antibiotic.180
Further research showed that rapamycin bound to FKBP12 and inhibited the
serine/threonine protein kinase mTOR, also known as the mechanistic and in mammals,
mammalian target of rapamycin.181 mTOR is a crucial regulator of cell metabolism and
proliferation downstream of growth factor or cytokine signaling. Its role in the immune
system is the subject of much investigation.182–184 In T and B cells, mTOR lies
downstream of phosphoinositide 3-kinase (PI3K), which is induced by TCR/BCR
activation, co-stimulatory molecules (CD28) as well as cytokine (IL-2) signalling.183,185,186
In mammals, mTOR exists as two functionally and structurally distinct complexes known
as mTORC1 and mTORC2. These complexes are distinguished primarily by the
proteins associated with each compex.183 Whereas mTORC1 binds with the rapamycin-
sensitive, regulatory-protein of mTOR (RAPTOR), mTORC2 binds with the rapamycin-
insensitive companion of mTOR (RICTOR).181,187,188 Despite the evidence that only
mTORC1 is affected by rapamycin, recent studies have shown that rapamycin
treatment can also inhibit mTORC2 in certain cell lines and particularly, in naive T
cells.189,190 Downstream of mTORC1, S6 kinase 1 (S6K1) and eukaryotic initiation factor
4E-binding protein 1 (EIF4EBP1) regulate mRNA synthesis and translation. mTORC1
also negatively modulates autophagy and is involved in lipid metabolism.181,183 The
downstream effects of mTORC2 are not well-described but it is purported to rely on
downstream signaling by Akt, glucocorticoid regulated kinase 1 (SGK1) and protein
27
kinase C alpha (PKCα) to modulate cell shape and growth.191 Finally, rapamycin can
also inactivate ribosome synthesis and delay cell cycle progression by binding to p70S6
kinase.192 The multiple roles of mTORC1 and mTORC2 in cell biology have made them
an attractive target for cancer research.193
Early studies into the effects of rapamycin showed that it had potent in vitro
effects on inhibiting proliferation of human T cells, inducing anergy in TH1 cells and
blocking IL-2-dependent and independent proliferation of human B cells.194,195 Studies
have also shown its effects in reducing the production of harmful autoantibodies in
murine models of SLE (systemic lupus erythematosus).186 Studies have also shown that
rapamycin inhibits DC antigen uptake and maturation.196 The full role of mTOR inhibition
in innate immune function is not well described and is a highly researched area in
immunobiology.184 In contrast to its role as an immunosuppressant, research has also
demonstrated that rapamycin can improve CD8+ memory formation versus LCMV
infection in mice.197 Furthermore, studies performed in mice containing a CD4-specific
mTOR knockout (as full mTOR knockout is embryonic lethal), have demonstrated a
more nuanced role for the role of mTOR inhibition in T helper cells.198 It was
demonstrated that mTOR deficiency did not significantly alter T cell proliferation or IL-2
production but inhibited the differentiation of CD4+ cells into TH1, TH2 and TH17
subsets.198
Arguably, one of the most intriguing function of rapamycin is its ability to promote
the differentiation and expansion of CD4+CD25+ regulatory T cells (Tregs) that also
express the transcription factor Forkhead box P3 (FOXP3) .199–201 The mechanisms of
28
this induction and preferential expansion are not yet fully understood. FOXP3
expression is believed to be regulated in part by TGF-β signaling where TGF-β-induced
SMAD3 (mothers against decapentaplegic homologue 3) and NFAT act together to
increase FOXP3 transcription.196 Constitutively active mTOR counteracts this pathway
and thus rapamycin-mediated mTOR inhibition induces FOXP3 expression.196 As
expected, CD4+-specific mTOR deficiency results in preferential expansion of Treg cells
and constitutive SMAD3 phosphorylation.198 This increased SMAD3 signaling is
believed to be a result of increased sensitivity to TGF-β and not an increase in TGF-β
expression. In fact, high levels of TGF-β can induce FOXP3 expression even in the
presence of mTOR activity.202 Interestingly, TH17 could be converted to Tregs in the
presence of high TGF-β activity due to its ability to counteract the transcription factor
RORγt.202 The TGF-β-independent pathway of rapamycin-dependent Treg induction is
purported to be a result of PI3K-Akt-mTOR dependent chromosomal rearrangement
that favours FOXP3 expression when TCR and CD28 signalling is interrupted early (<18
hours).196,203 Using rapamycin in this setting greatly enhanced the formation of FOXP3+
Tregs and mAbs to TGF-β did not affect Treg promotion indicating a TGF-β-
independent pathway.203 Finally, it is also important to note that FOXP3+ Tregs have a
survival advantage over conventional T cells in the absence of mTOR activity due to
their ability to upregulate Pim-2 kinase, a serine/threonine kinase with characteristics
similar to Akt.204
Rapamycin, due to its multiple immunological effects, is therefore an ideal
candidate for an immunosuppressant in the context of organ transplantation. Several
studies, including previous work from our lab, have demonstrated the ability of
29
rapamycin to prolong cardiac allograft survival in murine models.205–208 Clinically,
sirolimus and its analogue, everolimus are used in CNI-sparing regimens to reduce the
risk of CNI-induced nephrotoxicity.180 Although they have not replaced CNIs as the
predominant drug for maintenance immunosuppression, several studies have shown
that using an mTOR inhibitor while minimizing tacrolimus preserved renal function in
heart, lung and liver transplant recipients while not increasing the risk of acute rejection
over standard therapy.209 In particular, everolimus has shown its efficacy as a CNI-
sparing regimen in liver transplant patients, who are at high risk for end-stage renal
disease.155 Recent data from the PROTECT trial has demonstrated that replacement of
CNIs with everolimus was just as efficacious as CNIs in terms of preventing acute
rejection, while preserving better renal function in these patients.210 Furthermore,
tacrolimus to sirolimus conversion in liver transplant recipients was also demonstrated
to lead to increased Tregs in peripheral blood mononuclear cells (PBMCs) and the
expression of immunoregulatory genes.211 Similarly, in cardiac transplantation,
everolimus with reduced-dose CNI has been demonstrated to be just as potent as MMF
in preserving graft survival and preventing acute rejection.212 Inhibiting mTOR is also
known to inhibit vascular endothelial growth factor (VEGF) signaling which is important
for neoangiogenesis and endothelial remodelling.183 Hence, data from recent trials using
both sirolimus and everolimus have demonstrated reduced incidence and severity of
CAV in patients treated with mTOR inhibitors compared with CNI and MMF treatment
respectively.213,214 Treatment with mTOR inhibitors is associated with reduced incidence
of CMV and BK virus recurrence as well as, reduced malignancy in kidney
transplantation.181 However, both sirolimus and everolimus have potent side effects
30
including hyperlipidemia, increased proteinuria, thrombocytopenia and reduced
spermatogenesis.181,209 The complete role and utility of mTOR inhibitors in organ
transplantation are still being investigated. Their ability to promote expansion of Tregs
warrants their inclusion in further studies designed to induce immune tolerance in the
clinic, which is the “holy grail” of transplantation immunology research.14
1.2.2.8 Novel therapies in the pipeline
As the scientific literature continues to further the understanding of mechanisms
of immune activation, novel therapies continue to emerge that are specifically targeted
to cell surface receptors or intracellular kinases in an attempt to selectively inhibit
alloreactive T and B cells.144 The importance of the role of co-stimulatory molecules
(signal 2) for T cell activation has generated interest in targeting them to suppress the
immune system (discussed further in 1.4.2). Belatacept (LEA29Y), is a fusion protein
comprised of the extracellular domain of the co-inhibitory molecule CTLA-4 and the Fc
portion of human IgG1.215 Belatacept was approved by the FDA in 2011, and early
results demonstrated that it had safer toxicity profiles compared to CNIs for kidney
transplantation but not improved efficacy.216 However, a recently published study by
Vincenti et al., demonstrated for the first time, the superiority of belatacept over CsA, in
terms of patient and graft survival for a follow-up period of 7 years.217 Both belatacept
(low-dosage and high-dosage) and CsA patients received MMF, basiliximab induction
and glucocorticoids. The belatacept (high and low-dosage) groups demonstrated
improved graft survival, lower DSA and fewer deaths associated with cardiovascular
31
complications versus the CsA group.217 Another targeted therapeutic that has been
recently studied is the oral Janus Kinase-3 (JAK3) inhibitor, Tofacitinib.218 JAK3 is
expressed predominantly in hematopoietic cells and is required for lymphocyte
activation.219 In a phase II clinical trial in renal transplant patients, high-intensity
tofacitinib exposure resulted in a lower incidence of acute rejection as well as lower
symptoms of chronic allograft nephropathy at 1-year post-transplant. However, this
treatment also significantly enhanced the risk for infections and PTLD.218,219 Another
related kinase inhibitor currently being investigated is Sotrastaurin.220 Sotrastaurin
targets protein-kinase C (PKC) which is required for early T cell activation and mediates
its effects by downstream NF-κB, NFAT and AP-1 signalling.220 However, phase II
studies in both kidney and liver transplant patients demonstrated that sotrastaurin had
either lower or equivalent efficacy and more serious adverse effects compared to
tacrolimus.221,222 The drive towards selective therapeutics in organ transplantation has
resulted in better targeted therapies and offers the potential to minimize and/or replace
current immunosuppressants. However, these novel therapies are also associated with
adverse effects and have not resulted in significant increases in long-term graft survival.
1.3 Immune Tolerance
1.3.1 Overview
A healthy immune system is defined by the ability to mount protective immune
responses against pathogens and foreign antigens while maintaining tolerance to self-
tissue. Immune tolerance therefore, can be defined as the lack of immune response to a
32
specific antigen in an otherwise functional immune system.14,223,224 Transplant tolerance
therefore denotes a situation where an organ is accepted without the need for long-term
immunosuppression thereby preserving protective immunity.14 The induction of
transplant tolerance remains the ultimate therapeutic goal of transplantation research.
As mentioned previously, limited numbers of patients, particularly liver transplant
recipients, achieve “operational tolerance” whereby they are able to retain graft function
in the absence of immunosuppressive drugs.20,21 In contrast to the clinic, there have
been several demonstrations of tolerance induction in experimental murine
models.225,226 In 1953, the concept of “acquired immune tolerance” was demonstrated
by Billingham, Brent and Medawar.227 This was performed by intra-uterine injections of
donor spleen cells thus exposing neonatal mice to donor antigen. These mice were then
capable of permanently accepting donor skin grafts.227 In 1956, Frank MacFarlane
Burnet proposed the theory of clonal selection as the mechanism through which self-
reactive lymphocytes are deleted to prevent immune recognition of self. In the decades
that followed, the process of intra-thymic clonal deletion was studied and classified as
central T cell tolerance (i.e. deletional tolerance).228 Similarly, the mechanisms for
controlling autoreactive mature T cells in the peripheral tissues i.e. peripheral tolerance,
have also been identified.229 In addition to T cells, B cells are subject to central and
peripheral mechanisms of tolerance.230,231 Due to their central role in transplantation,
this section will focus primarily on mechanisms underlying T cell tolerance.
33
1.3.2 B cell tolerance
Studies performed in transgenic mice have shed light on the mechanisms
underlying B cell self-tolerance, but the exact mechanisms in humans are not well
defined.232 In adult mammals, B cells arise from common lymphoid progenitors (CLPs)
in the bone marrow and undergo sequential developmental stages.231,233 Re-
arrangements of immunoglobulin heavy and light chains along with the expression of Ig-
α and Ig-β (which are critical for signal transduction), results in the formation of a fully
functional IgM BCR on immature B cells.233 These immature B cells begin to express
IgD as transitional B cells and migrate to the spleen to become fully functional mature B
cells.232 Early studies of both humans and mice demonstrated that a large proportion of
B cells (50-75%) expressed receptors reactive to self-antigens while in the bone
marrow. This proportion was significantly smaller (20%-40%) in B cell repertoires in the
spleen and peripheral blood.234,235 Using transgenic mouse models, three mechanisms
of self-education of B cells in the bone marrow have been identified. The dominant
mechanism is now known to be receptor editing, whereby self-reactive B cells undergo
persistent re-arrangement of their Ig light-chain genes replacing autoreactive BCRs with
non-autoreactive BCRs.236,237 It is estimated that nearly 35% of autoreactive immature
B cells undergo receptor editing while the remaining cells undergo clonal anergy or
deletion.232,238 Further clonal selection is posited to occur in the spleen between the
transition from transitional B cells to naive mature B cells. An important cytokine
modulating the emergence of mature B cells is B cell activating factor (BAFF).239 Self-
reactive B cells are demonstrated to express lower levels of BAFF receptor and as
such, BAFF preferentially selects for non-autoreactive B cells.238 Elevated BAFF is
34
associated with various autoimmune diseases in humans such Sjogrens syndrome, SLE
and rheumatoid arthritis.235 The mechanisms underlying B cell tolerance warrant further
study. Finally, recent studies have begun to unravel the roles of suppressive B cell
populations, i.e. B regulatory cells in maintaining peripheral immune tolerance
(discussed further in 1.3.5.2).240
1.3.3 Central T cell tolerance
The thymus is a rigorous environment for T cell development with only 1-2% of
total thymocytes emerging as mature T cells in mice.241 In the thymus, recognition of
self has contrasting effects on T cell development. It is essential for thymocyte survival
and maturation during the stage of positive selection. In the next stage, negative
selection results in “clonal deletion” of thymocytes that bind too strongly to self-MHC.242
Positive and negative selection occur primarily in different compartments of the thymus;
the cortex and the medulla respectively.24 In mice, multiple precursor cell types possess
the capacity for giving rise to thymocytes including common lymphoid progenitors
(CLPs), early thymic precursors (ETPs) and early lymphoid precursors.243,244 In the
initial stage of thymocyte maturation, they lack either CD4 or CD8 expression and are
termed double negative (DN). At this stage, VDJ recombination of the TCRβ chain in
combination with the CD3 and TCRα chain results in the formation of the pre-TCR.245
For cells that do not undergo TCRβ rearrangement, the re-arrangement of the γ chain
results in the formation of γδ T cells that are not self-MHC restricted and play an
important role in anti-microbial as well as anti-tumor immunity.246 Subsequently, the αβ
35
T cells upregulate both CD4 and CD8 expression to become double positive (DP)
thymocytes and their TCRα chain undergoes rearrangement to form a fully functional
TCR. The DP thymocytes then interact with MHC class I or II to become single positive
(SP) CD8+ or CD4+ cells respectively.243 Cells that do not bind to any MHC undergo cell
death through neglect.243 The differentiation of the CD4 lineage is dependent on the
transcription factors ThPOK and Gata3 whereas the Runx proteins Runx1 and Runx3
are involved in CD8 differentiation.247
SP thymocytes subsequently undergo negative selection (clonal deletion) of
autoreactive cells primarily in the thymic medulla.242 The medulla contains medullary
thymic epithelial cells (mTECs) that display tissue restricted antigens (TRA) to SP cells.
TRA (defined as antigens expressed in less than 5 body tissues) expression is
regulated by the transcription factor, autoimmune regulator (AIRE).242,248 In accordance
with the crucial role of AIRE, mutation in the AIRE gene in humans results in a multi-
organ disease called autoimmune polyendocrinopathy-candidiasis-ectodermal
dystrophy (APECED).249 For several years, AIRE was known to be the only regulator of
TRA expression even though TRA expression was observed to in AIRE-deficient
mice.250 In 2015, however, Takaba et al. identified Fezf2 as another transcription
involved in TRA expression.250 Fezf2 deficient mice also displayed signs of autoimmune
organ damage and production of autoantibody.250
The thymus is also populated by a small population of resident and migratory
DCs that play important roles in the presentation of thymic and peripheral antigens with
some evidence pointing to their role in protection from thymic viral infection.242,251 A
36
recent study in mice has also outlined the important role of circulating B cells that
migrate to the thymus and express AIRE to display endogenous self-antigens to CD4+
SP thymocytes.252 Thymocytes that bind self-MHC with high affinity are deleted through
apoptosis. Alternately, SP thymocytes with low affinity for self-MHC undergo maturation
to the next step.243 Co-stimulatory molecules are purported to play an important role in
this process. Buhlmann et al. also demonstrated using B7-1/2 double knock-out mice
that negative selection is predicated on co-stimulatory signaling through B7-1/2 and
CD28.253 Similarly, the study on aforementioned thymic B cells by Yamano et al.
showed that B cells required CD40-CD40L licensing to mediate their APC functions.252
Finally, the thymus also gives rise to Treg, NKT and CD8αα regulatory cells.254
Although the exact mechanisms are not fully understood, it is posited that this process
relies on TCR avidity for self-antigen where low avidity results in maturation, high avidity
in clonal deletion and intermediate avidity in the generation of Treg.255 In recent years,
the importance of this clonal diversion to Treg has been elucidated and is now
purported to be as important as clonal deletion in the maintenance of self-tolerance.229
1.3.4 Peripheral T cell tolerance
The thymus is stringent in its selection for functional and non-autoreactive T
cells. However, T cells with the potential for autoreactivity manage to escape to the
periphery where they necessitate control by the mechanisms of peripheral
tolerance.256,257 These suppressive mechanisms are either intrinsic such as ignorance,
37
anergy and activation-induced cell death (AICD), or due to the extrinsic modulation by
regulatory cell populations.229
1.3.4.1 Ignorance
Ignorance denotes a state whereby cells with self-reactive potential remain
inactivated and unable to respond to self.258 In 1991, Ohashi et al., demonstrated that
in the absence of LCMV infection, naïve transgenic (P14) CD8+ T cells specific for
LCMV glycoprotein (GP), did not attack islet cells that were expressing the LCMV-GP
under the control of a rat insulin promoter (RIP-GP mice).259 Infection of these mice with
LCMV resulted in induction of CD8+ islet cell destruction and diabetes indicating that the
T cells had become activated in presence of high levels of antigen.259 Thus, T cells
bearing TCRs with low avidity for self-antigens (TRA) may escape thymic deletion but
remain inactivated in the absence of high-antigen exposure. This is further achieved by
the physical separation of naive circulating lymphocytes that are restricted to the blood,
efferent lymph and secondary lymphoid organs.229,258 Conversely, antigen-experienced
T cells are able to enter peripheral tissues by downregulating CD62L and CCR7, where
they may be exposed to TRA.229 However, even in this setting, a stimulus may be
required to liberate self-antigens and cause inflammation. This was shown recently by
injecting OVA-specific transgenic OTI (CD8+) and OTII (CD4+) cells into transgenic mice
bearing the OVA protein in their skin. Even though antigen-experienced cells were
observed, a further inflammatory stimulus, in this case tape stripping, was required for T
cell homing to the skin and the induction of inflammatory skin disease.260
38
1.3.4.2 Anergy
T cell anergy is defined as a state of prolonged hyporesponsiveness towards an
antigen and is marked by cell cycle arrest as well as reduced TCR signaling and IL-2
expression.261 In CD4+ and CD8+ T cells, anergy also results in impaired production of
IFNγ and TNFα.262 There are multiple mechanisms that underlie the induction of anergy
in mature functional T cells.243,257,262 Several years ago, experimental studies
demonstrated that T cell activation by TCR binding in the absence of CD28 signaling
resulted in anergy.263,264 Subsequent studies have begun to unravel the molecular
pathways involved in the induction of anergy.262 Complete activation of T cells is
dependent on TCR and CD28 mediated recruitment of the transcription factors NFAT,
AP-1 and NF-κB for IL-2 expression. IL-2 expression and autocrine signaling through
the IL-2R, results in a complete activation of the PI3K-Akt-mTOR pathway which among
several other targets, degrades the cyclin-dependent kinase inhibitor p27kip1.265 In
contrast, TCR stimulation alone results in calcineurin/NFAT activation in the absence of
AP-1. This results in the formation of NFAT homodimers which induces a gene profile
characteristic of anergic T cells.261 These genes include E3 ubiquitin ligases such as
Cbl-b, GRAIL, Itch and Deltex-1 which target the molecular pathways downstream of
TCR and CD28 signaling.261,265 The anergic state also induces the activation of
transcription factors such as Erg1/2, cAMP response element modulator (CREM) and
Ikaros, which mediate direct repression of the IL-2 gene locus.265
39
The anergic state can also be induced by engaging co-inhibitory molecules on T
cells. CTLA-4 is an important inhibitory pathway as described previously. CTLA-4 is
induced after TCR and CD28 activation and binds with higher affinity to B7-1/2 on
APCs.266 The importance of CTLA-4 in maintaining peripheral tolerance is evident from
studies that showed that CTLA-4 deficient mice develop autoimmunity and CTLA-4
knockout CD4+ T cells are resistant to induction of anergy.267,268 Another crucial
inhibitory pathway is that of the immune checkpoint, programmed death-1 (PD-1)
(CD279), a receptor from the immunoglobulin family expressed on T cells, B cells and
myeloid cells.269 The ligands for PD-1 are PD-L1 (CD274) and PD-L2 (CD273), which
are expressed on T cells, B cells, APCs, endothelial cells and tumours.243 TCR
activation in addition to PD-1 ligation through its downstream immunoreceptor tyrosine-
switch motif (ITSM), results in activation of SHP-1 and SHP-2 phosphatases which
counteract the PI3K-Akt pathway.257 PD-1 and PD-L1 signaling is implicated to play a
role in negative selection in the thymus.270 In the periphery, T cell tolerance is
maintained by PD-1 by limiting early activation and expansion.270 Furthermore, PD-L1
expression on tolerogenic DCs was also shown to be necessary for the induction of
CD8+ T cell peripheral tolerance.271 Finally, PD-L1 signaling is also important for the
induction of peripheral CD4+FOXP3+ regulatory T cells.272 As expected, PD-1 deficient
mice display signs of lupus-like autoimmune disease.273
Finally anergy can also be induced in response to metabolic cues from the
environment.261 Studies using rapamycin have demonstrated induction of anergy in T
cells even in the presence of signal 1 and signal 2 (TCR and CD28) stimulation due to
the central role of mTOR in cell metabolism.195 This was further demonstrated by
40
utilizing leucine and glucose antagonists to induce anergy in the presence of both signal
1 and signal 2 of T cell activation.274 This sensing of hypoxia is believed to be the result
of adenosine monophosphate-activate protein kinase (AMPK), which is activated as a
result of low ATP levels in the cells and functions to counteract mTOR signaling.261
Clonal anergy in vitro can be reversed by the exogenous addition of IL-2.275 The
term “adaptive tolerance” has been coined to describe T cell anergy in vivo. This state,
which involves distinct signaling pathways, requires persistent exposure to antigen and
is refractory to exogenous IL-2 addition.276
1.3.4.3 Activation-induced cell death
Activation-induced cell death (AICD), is an important mechanism for T cell
homeostasis and for the prevention of autoimmunity in the peripheral immune
system.277,278 Upon primary antigen exposure, T cells undergo a primary expansion
phase and are resistant to AICD. During the contraction phase and upon re-stimulation,
T cells become sensitive to AICD.278 AICD is mediated in the target cell by the binding
of Fas (CD95) on the target cell to either soluble FasL (suicide) or a neighboring T cell
(fratricide).277 Fas belongs to the TNFR superfamily of transmembrane proteins. This
superfamily also contains other death receptors including TNFR1, DR3 (TNFRSF25),
DR4 (TRAILR1), DR5 (TRAILR2) and DR6 (TNFRSF21).279 Fas-induced apoptosis is
regulated primarily by the death-inducing signaling complex (DISC) comprised of the
adaptor FADD (Fas-associated death domain) protein and the proteases caspase-8 and
caspase-10.278 These activate downstream effector (executioner) caspase-3, caspase-6
41
and caspase-7 which mediate cell death by cleaving several critical proteins required for
cell function.279 Key cytokines such as IL-2, IL-4 and IFNγ increase sensitivity to
AICD.278 The role of Fas-induced AICD has an important role in prevention of
autoimmunity. In humans, autoimmune lymphoproliferative disorder (ALPS) is an
inherited disease based on mutations in the Fas, and in some cases the FasL and
caspase-10 genes.278,280 In mice, the MRL (Murphy Roths Large) strain develops multi-
organ autoimmunity, serum autoantibodies and lymphadenopathy which are the
hallmarks of SLE in humans.281 MRL/lpr mice were found to have a spontaneous
mutation in the lpr (fas) gene, and MRL/lpr mice have an accelerated autoimmune
pathogenesis compared to MRL mice.281 Finally, FasL is also expressed on astrocytes
and on cells in immunoprivileged sites, such as Sertoli cells, thyroid epithelium and
endothelial corneal cells.278
1.3.5 Suppression by immunomodulatory cells
In addition to cell-intrinsic mechanisms for maintaining peripheral tolerance,
extrinsic immunomodulation by regulatory cells is crucial for preventing excessive
immune activation and autoimmunity. Regulatory or tolerogenic cell subsets from both
the myeloid and lymphoid hematopoietic lineage have been identified and
described.282,283 As the focus of this section is on peripheral T cell tolerance, the cell
types that warrant further discussion are dendritic cells, regulatory B cells (Bregs) and
regulatory T cells (Tregs), all of which can suppress T cell function.282
42
1.3.5.1 Dendritic cells
As one of the three professional APCs, DCs provide a functional bridge between
innate and adaptive immunity.284 Upon sensing PAMPs and DAMPs through PRRs,
immature DCs become activated, upregulate their expression of co-stimulatory
molecules (CD80/86) and release inflammatory cytokines.285,286 DC subsets are highly
varied in phenotype and function and are found both in lymphoid and non-lymphoid
organs.284,287 DCs are broadly divided into two major subtypes based on their function
and ontogeny. Myeloid-derived classical, or conventional DCs (cDCs), express co-
stimulatory molecules and sample antigen for presentation to and activation of CD4+ T
cells.284 Plasmacytoid DCs (pDCs) arise from lymphoid progenitors and one of their
primary functions is to mediate anti-viral immune responses by production of large
quantities of type I interferons.286,287
Selective depletion of both pDCs and cDCs leads to fatal autoimmunity in mice
indicating a crucial role for DCs in maintaining immune tolerance.288 As discussed
previously, both resident and migratory DCs in the thymus, play an important role in
central T cell tolerance.242 On the other hand, DCs also important for maintenance of
peripheral T cell tolerance in the absence of inflammatory or pathogenic stimuli (i.e.
steady-state). The first evidence for the tolerogenic role of steady-state DCs was
described by Hawiger et al. in 2001.289 By producing a fusion protein comprised of DEC-
205 (a mAb towards a DC-specific endocytic receptor) and hen egg lysozyme (HEL),
HEL antigen was targeted directly to DCs. Despite production of IL-2 and an initial
proliferation of CD4+ T cells in these mice, they failed to produce IFNγ, IL-4 or IL-10.289
43
After 7 days, most activated T cells either underwent deletion or became anergic to re-
stimulation with HEL.289 Thus in the steady-state, DCs prevent T-helper subset
differentiation and mediate T cell anergy. It was demonstrated further that steady-state
presentation of antigen by DCs also induced CD8+ tolerance through co-inhibitory PD-1
and CTLA-4 signaling.271 Subsequent studies have delineated a wide range of DC
subsets with immunoregulatory roles.290 These include induction of FOXP3+ Tregs,
secretion of suppressive molecules like IL-10, TGF-β and indoleamine 2,3-dioxygenase
(IDO) and induction of apoptosis through FasL expression.285,290 Currently, tolerogenic
DCs (TolDCs) are believed to be immature or maturation-resistant DCs, with low
surface expression of co-stimulatory molecules, that modulate T cell function through
one or more of the aforementioned mechanisms.285,286 TolDCs from both mice and
humans can be generated in vitro using pharmacological treatments such as
dexamethasone, rapamycin, 1α,25-dihydroxyvitamin D3, sanglifehrin A or cytokine
treatment with IL-10, IDO or TGF-β and are being studied for their ability to promote
tolerance in autoimmunity and transplantation.285
1.3.5.2 Regulatory B cells
Since their discovery, B cells have been primarily conceived of as potent
activators of the immune system through antibody production and APC functions.
However, an increasing body of evidence in the literature points to the role of B cells as
negative regulators of the immune system.240,291,292 As early as the 1970s, the
suppressive role of B cells was observed in a guinea-pig model of delayed-type
44
hypersensitivity.293 In 1996, Janeway’s group generated evidence for a role of B cells in
mediating self-tolerance. B-cell deficient mice displayed an increased severity of and
inability to recover from experimental autoimmune encephalomyelitis (EAE), a murine
model of multiple sclerosis.294
Currently, B regulatory cells (Bregs) in both mice and humans lack a unique
phenotypic marker similar to FOXP3+ in CD4+CD25+ Tregs. Instead, all of these Breg
subsets are identified by their functional capacity to produce large amounts of IL-
10.291,292 In mice, the major mechanisms of Breg immunoregulation consist of inhibition
of TH1 differentiation, inhibition of DC maturation and induction of Tregs.295 Moreover,
Breg activation is believed to occur through CD40 or TLR stimulation.296,297 Two primary
Breg populations have been identified in mice. A transitional B cell population in the
marginal zone (T2-MZP-B cells) were identified by Evans et al. as suppressive cells in a
mouse model of collagen-induced arthritis.298 These cells are
CD19+CD21hiCD23+CD24hi and their suppressive capacity is IL-10 dependent.298 A
rare population of Bregs in the spleen (1-2%), termed B10 cells were identified by
Yanaba et al. in 2008.299 These cells were CD1dhiCD5+ and suppressed contact
hypersensitivity in mice in an IL-10 dependent manner.299 A subsequent study also
demonstrated that these cells could differentiate into Ab-producing plasmablasts after
transiently producing IL-10.300 It was posited that these Ab-production by these cells
could further suppress immune activation by reducing antigen load.
In humans, various Breg subsets have been identified and have been implicated
in protection from autoimmunity.291 The mechanisms of human Breg-mediated
45
immunosuppression involve TH1 differentiation and proliferation, but require further
characterization.291,295 Blair and colleagues, identified a subset of B cells that are
CD19+CD24hiCD38hi and produce IL-10 in response to CD40 stimulation in vitro.301 It
was demonstrated that this subset of Bregs was dysfunctional in patients suffering from
SLE.301 A reduced Breg frequency is also observed in other autoimmune pathologies
such as rheumatoid arthritis and multiple sclerosis.302,303 The multiple roles of Bregs in
maintaining and promoting immune tolerance have only recently been discovered.
Bregs have been implicated in promoting allograft tolerance in mouse models and a B
cell signature has been observed in operational tolerance in kidney transplantation.304–
306 Thus, Bregs are a potential candidate for adoptive cell therapy in tolerance induction
trials. However, further studies are necessary to characterize the phenotypes, functions
and isolation techniques of putative Breg populations in humans.
1.3.5.3 Regulatory T cells
Peripheral T cell tolerance is in part maintained by thymic selection and T cell-
intrinsic inactivation mechanisms.229 However, regulatory T cells (Tregs) are now
recognized as a major mechanisms of T cell suppression and prevention of
autoimmunity.307,308 The concept of “suppressor T cells” was first posited through the
work of Gershon and Kondo in 1970.309 Subsequent studies identified the IL-2Rα chain
CD25, as a marker for an immunosuppressive T cell population that was derived from
the thymus and was crucial for the prevention of autoimmunity.310,311 Finally, in 2003 the
X-linked transcription factor FOXP3, was identified as a master regulator of Tregs.312,313
46
Once foxp3 is expressed, it induces the transcription of signature Treg genes including
foxp3 itself.314 These cells are crucial for the maintenance of self-tolerance. FOXP3-
mutated Scurfy mice exhibit fatal autoimmune inflammation with increased TH1, TH2 and
TH17 activation.315 In humans, FOXP3 inactivating mutations result in immune
dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome.316
However, constitutive FOXP3 expression is not a requisite for the function of certain
regulatory T cells such as Type 1 regulatory (Tr1) cells.317 In addition to Tr1 cells other
T cells with immunoregulatory functions include Th3 cells, CD8+ Treg, CD8αα intra-
epithelial lymphocytes, double negative (DN) T cells and γδ T cells.246,308,318 However,
due to their central role in peripheral tolerance and relevance to this dissertation, this
section will focus on CD4+CD25+FOXP3+ cells as the major type of Treg.
1.3.5.3.1 CD4+CD25+FOXP3+ Tregs
In normal naive mice, Tregs comprise 5-10% of all CD4+ T cells.319 As previously
discussed, natural or thymic Tregs (tTregs), arise in the thymus through the
developmental processes of clonal diversion.320 Alternatively, limited numbers of
peripheral Tregs (pTregs) can be generated in the steady-state through the TGF-β
dependent mechanisms.321 This is posited to occur primarily in tolerogenic tissue
environments such as mucosal surfaces.307 However, pTregs can expand significantly
in the context of immune inflammation or due to pharmacological treatments.322
Moreover, in vitro TCR signaling in the presence of TGF-β and IL-2 can also give rise to
induced Tregs (iTregs) that express FOXP3.323 It is important to note that while these
47
different Treg populations exhibit FOXP3 expression, there are functional and genetic
differences between them. For instance, pTregs generated in vivo show potent in vitro
suppressive capacity whereas TGF-β induced iTregs are not as suppressive and do not
acquire the complete gene signature of pTregs.322 Feuerer et al. conducted an in-depth
microarray analysis of various Treg subtypes in mice and found differently expressed
genes between each subtype.324 Therefore, further study is required to find phenotypic
markers that could be used to isolate and study each subset of Tregs. Recently, Helios,
an Ikaros-family transcription factor, was studied as a candidate to distinguish tTregs
from pTregs.325 However, subsequent studies showed that Helios could be expressed in
activated T cells as well as iTregs.322 Another candidate marker, the cell surface
molecule Neuropilin-1 (Nrp-1) was observed to be expressed on tTreg.326 However,
pTregs can upregulate Nrp-1 during inflammatory settings.322 Thus, neither Helios nor
Nrp-1 are steady markers for natural thymic-derived Tregs in mice while their
expression in human Tregs require further investigation.322 Isolation of pure Tregs in
both mice and humans for functional studies is hindered by the lack of an exclusive cell
surface marker. Tregs express cell surface markers that are integral to their function
but are also expressed on other cell types such as CD25, CTLA-4, lymphocyte
activation gene-3 (LAG-3) and T cell immunoreceptor with Ig and ITIM domains
(TIGIT).327,328 A further complication observed in human T cells, FOXP3 in the absence
of suppressor Treg function.329 In human Tregs, the cell surface molecule CD127 (IL-
7Rα), was found to be inversely correlated to Treg suppressive function.330 Thus,
several studies have begun investigating Tregs that exhibit the
CD4+CD25+FOXP3+CD127low phenotype for human clinical studies.282 The critical role
48
for Tregs in maintaining peripheral tolerance is evident from studies in patients with
autoimmunity. Several studies in patients with SLE, MS, RA and type 1 diabetes (T1D)
demonstrate perturbations in Treg frequencies compared to healthy controls.308,322
However, the wider role for Tregs in several other contexts is also being recognized
including cancer and persistent viral infections.331,332 Finally, Tregs are an important
candidate for tolerance induction in transplantation.321
1.3.5.3.2 Mechanisms of Treg suppression
Tregs are considered major regulators of the immune system due to their broadly acting
suppressive functions. These functions can be grouped into four major mechanisms of
action; i) inhibition of APC maturation and function, ii) cytolysis of target cells, iii)
metabolic disruption of effector T cells (Teff), iv) secretion of immunosuppressive
factors. These mechanisms have been extensively reviewed in the literature by us and
other groups.318,328,333–335 One salient feature of Tregs that is important to note is that
unlike activated T effector cells, Tregs do not produce IL-2. Instead, they rely on
paracrine IL-2 signaling and genetic ablation of either IL-2 or IL-2R in mice results in a
50% reduction in the proportion of Treg thymocytes.320 The major mechanisms of Treg
immunosuppressive function are outlined in Figure 1-2 below. Moreover, the major cell
surface receptors and immunosuppressive molecules produced by Treg cells and their
effects are presented in Table 1-2.328
49
Figure 1-2 Treg-mediated mechanisms of immunosuppression
The major mechanisms of Treg function include APC modulation, direct cytolysis of target
cells, metabolic disruption of effector T (Teff) cells and secretion of a number of
immunosuppressive molecules. Copyright© Chruscinski et al. 2015.
Reprinted from Rambam Maimonides Med. J. 6, e0024, Role of Regulatory T Cells (Treg)
and the Treg Effector Molecule Fibrinogen-like Protein 2 in Alloimmunity and Autoimmunity,
Chruscinski, A. Sadozai, H et al., (2015).328
50
Table 1-2 Treg effector molecules
Cell Type Ligand/
Receptor
Target Cell Mechanism
CTLA-4 Treg B7 molecules
(CD80/CD86)
DC Inhibition of DC activation through the trans-endocytosis
and degradation of CD80 and CD86 molecules by Treg
Sterically hinders the association of naïve T cells with DC
through coordinated activity with LFA-1
Negative regulation of effector T cell survival by signaling
through Foxp3
IL-2 Activated
T cells
High affinity
IL-2 receptor
Treg IL-2 deprivation by Treg in low-affinity TCR and antigen:MHC
interactions induce T cell apoptosis
TIGIT Treg,
T cells
NK cells
CD155 (PVR)
CD112
(PVRL2)
DC Inhibition of IL-12 (p40) production by DC
Binds CD155 (PVR) and CD112 (PVRL2) on APCs
Increases IL-10 expression inducing tolerogenic DC which
suppress T cell proliferation and IFNγ production
LAG-3 Treg MHC-II DC Inhibits DC maturation
Inhibits co-stimulation of naïve T cells by DC
CD39
/CD73
Activated
Treg
Treg Activated
T cells
DC
CD39 converts ATP in the extracellular space into ADP and
AMP decreasing inflammation
CD39 increases suppressive activity of Treg
CD73 converts AMP to adenosine which inhibits DC function
and activated T cells
IL-10 Treg IL-10R T cells
DC
Inhibits T cell proliferation, decreases production of IL-2,
TNF-α and IL-5
Impairs Th1 responses by inhibiting DC activation and
secretion of IL-2
TGF-β Treg TGF-βR T cells Direct suppression of effector T cells
Inhibits cytokine production and cytotoxic function of T cells
IL-35 Treg IL-35R Naïve T cells,
DC
Direct inhibition of T cell proliferation
Induction of naïve T cells to become activated IL-35 Treg
Gzmb Treg Perforin-
independent
entry into
target cell
Activated
T cells
DC
Induction of apoptosis in target cells
FGL2 T cells,
Treg,
activated
Treg
FcγRIIB/ RIII DC Inhibition of DC maturation
Suppression of Th1 and Th17 effector T cell responses
Abbreviations: ADP, adenosine diphosphate; AMP, adenosine monophosphate; APC, antigen presenting cell; ATP,
adenosine triphosphate; CTLA-4, cytotoxic-T-lymphocyte-associated protein 4; DC, dendritic cell; FGL2, fibrinogen-
like protein 2; Foxp3, forkhead box p3; Gzmb, granzyme B; IL, interleukin; LAG-3, lymphocyte activation gene 3; LAT,
linker for activation of T cells; LFA-1, lymphocyte function-associated antigen 1; MHC, major histocompability
complex; PD-1, programmed cell death-1; PVR, poliovirus receptor; PVRL, poliovirus receptor ligand; TCR, T cell
receptor; TGF, Transforming Growth Factor; TIGIT, t cell immunoreceptor with Ig and ITIM domains.
51
1.4 Strategies to Induce Transplant Tolerance
1.4.1 Overview
The seminal work of Billingham, Brent and Medawar demonstrated that immune
tolerance in the absence of immunosuppression was theoretically possible.227 Their
approach was based on work performed by Ray Owen in the 1940s, who showed that
freemartin (dizygotic) twin cattle had shared placental circulation and chimeric immune
systems.336 Billingham and Medawar among others, demonstrated that these dizygotic
twins could accept skin grafts from each other.336 However, induction of tolerance in
their murine studies was considered a purely theoretical approach not deemed
applicable to clinical immune tolerance. However, research in the past two decades
have greatly furnished our understanding of human immune function, particularly in the
activation of T cells and the immunobiology of suppressor cell types.243,337 Moreover,
our understanding of hematopoiesis and immune system development, has allowed for
approaches based on Medawar’s initial concept of neonatal acquired tolerance.338
Currently, multiple strategies are being employed to induce long-term allograft specific
tolerance in the clinic. These strategies can be divided into three major approaches for
tolerance induction. These are i) co-stimulatory blockade ii) administering or inducing
Treg iii) hematopoietic stem cell transplantation.282,318,337,339,340 This section will discuss
all three major approaches with a primary focus on therapies that have gone or are
currently undergoing clinical trials for tolerance induction in solid organ transplantation.
52
1.4.2 Co-stimulatory blockade
Due to the central role of co-inhibitory and co-stimulatory receptors in T cell
activation, they are an attractive target for tolerance inducing regimens. As mentioned
previously, these molecules belong to either the Ig family (e.g. CD28, CTLA-4, PD1),
the TIM family (T cell Ig and mucin domain), the TNF-TNFR family (e.g. CD40, OX40) or
are cell surface adhesion molecules (e.g. LFA-1).99,337 The first well-characterized
regulatory axis was that observed between CD28, CTLA-4 and CD80/86.101 As
mentioned previously, Belatacept (CTLA-4Ig) has recently been demonstrated to have
efficacy over CsA but its use alone cannot induce tolerance..215,217 Furthermore, two
important considerations argue against the value of this approach in favour of a CD28
selective blockade. First, CTLA-4 signaling is also important for Treg function and
second, that CD80 was recently found to bind to PD-L1 on T cells resulting in
inhibition.320,341 Thus, the selective CD28 agonist TGN1412 was developed and tested
in a phase I safety trial in healthy volunteers. However, this resulted in a cytokine storm
in all patients marked by severe systemic inflammatory symptoms and all patients
required intensive care till the symptoms subsided.342 Subsequent studies are now
being conducted with engineered anti-CD28 antibodies that preserve CTLA-4 function
while selectively blocking CD28. These studies have shown to promote Tregs and
prolong allograft survival in murine and NHP preclinical models.337
There is also interest in targeting the CD40-CD154 pathway for tolerance
induction in transplantation due to its crucial role in promoting both cellular and humoral
immunity.343 Targeting CD154 was demonstrated to promote long-term graft survival in
53
both mice and NHP studies.344 Human trials with anti-CD154 were quickly halted as it
was not yet known that platelets express CD154 and that administration of anti-CD154
resulted in thromboembolic complications in these patients.345 Targeting CD40 with
novel non-depleting mAbs has showed efficacy in NHP models of renal, cardiac and
islet transplantation.346 Currently, an anti-CD40 mAb (ASKP1240) is undergoing phase
IIa clinical trials in kidney transplant recipients.346
Cell surface adhesion molecules are crucial for cell trafficking to sites of
inflammation. The adhesion molecule LFA-1 (lymphocyte function associated antigen-
1), is expressed on memory T cells. It is purported to have a role in T cell activation in
the immunological synapse and T cell migration into tissues by binding to ICAM
(intracellular adhesion molecule) on inflamed endothelium.347 Efalizumab (anti-LFA-1
mAb) was first approved for psoriasis and underwent clinicial trials in islet and renal
transplant recipients. Initial results demonstrated encouraging results including insulin-
free survival and an increased Treg signature when used in combination with
sirolimus.337 However, further development for transplantation and autoimmune disease
was halted after the development of progressive multifocal leukoencephalopathy (PML),
a fatal disorder associated with the JC polyomavirus.337 An additional focus has been to
disrupt the pathway between the T cell surface antigen CD2, expressed highly on
effector memory T cells with LFA-3 (lymphocyte functional associated antigen 3).
Alefacept, a LFA-3 fusion protein has been shown to prolong allograft survival in
synergy with anti-CD154 treatment in NHP kidney transplant recipients.348 However,
subsequent studies in NHP models in the absence of anti-CD154 treatment did not
demonstrate the same efficacy.337 Other pathways that have shown promise in
54
preclinical transplant tolerance studies are those associated with PD-1 and inducible T
cell co-stimulator protein (ICOS).337,346 ICOS is of particular interest as it has been
shown to play a critical role in TH17 differentiation, which is now being recognized as a
major subset involved in the alloresponse.106,349 To date, none of the co-stimulatory
blockade treatments tested clinically have resulted in organ-specific immune
tolerance.337 Further study is required to unravel the complex interplay between each of
these co-stimulatory and co-inhibitory pathways to generate rational and potentially,
combinatorial targeting of these receptors.
1.4.3 Induction or administration of Tregs
Research in the past two decades have unveiled the crucial role of Tregs in
promoting and maintaining allograft tolerance.282,321 The two approaches currently being
studied to utilize Tregs for transplant tolerance are induction of Tregs in vivo through
pharmacological modulation and infusion of ex vivo generated Tregs.350 Treatment with
mTOR inhibitors in humans have been shown to expand CD4+CD25+FOXP3+ Tregs in
diabetes patients and healthy volunteers.199,351 In the context of solid organ
transplantation, Levitsky et al. demonstrated that conversion from tacrolimus to
sirolimus in liver transplant recipients resulted in increased Tregs in peripheral blood
and bone marrow.211 Sirolimus treated patients also displayed an immunoregulatory
gene signature and diminished MLR responses.211 A similar expansion of Tregs was
recently reported in a renal transplant cohort converted from CNIs to everolimus, an
analog of rapamycin.352 Other candidate drugs for the expansion of Tregs include IL-2,
55
ATG and the aforementioned LFA-3 fusion protein, Afelacept. However, to date, none of
these have been tested for Treg expansion in clinical trials of solid organ
transplantation.350 Furthermore, the major focus of current clinical Treg therapy for
transplantation is on the transfer of ex vivo generated Tregs.282 In 1993, Qin et al. first
demonstrated that the capacity to adoptively transfer tolerance in murine allografts, i.e.
“infectious tolerance” involved a CD4+ T cell population.353 Recent studies have now
shown that transfer of Tregs can treat both acute and chronic allograft rejection, as well
as, prevent GVHD (graft-versus-host-disease) in murine models.318,321 Initial studies in
small human cohorts demonstrated that infusion of Tregs could prevent the induction of
GVHD in patients after allogeneic hematopoietic stem cell transplantation (HSCT).354
These patients received either ex vivo derived natural, or thymic Tregs (tTregs) or
umbilical cord blood purified Tregs. These studies were too preliminary to analyze
efficacy of treatment but they demonstrated the safety of performing Treg infusion in
human patients.355 The ONE study is a multi-center phase I/IIa trial that commenced in
2014, with the aim of examining autologous ex vivo expanded Tregs in renal transplant
patients (www.onestudy.org).356 The study will also test other regulatory cell types such
as Tr1 cells, TolDCs and regulatory macrophages. Participants will be followed for 60
weeks and biopsy-proven rejection has been established as the clinical endpoint for this
trial.356 This study will yield crucial information about the feasibility and efficacy of Treg-
based cell therapy for tolerance induction in transplantation. The primary concerns for
using ex vivo Tregs are the cost-effectiveness of GMP manufacture of the Treg product,
the shelf-life of manufactured products and finally, the stability of the Treg phenotype in
vivo.355
56
1.4.4 Hematopoietic stem cell transplantation
Both autologous and allogeneic hematopoietic stem cell transplantation (HSCT)
have been implemented clinically for the purposes of tolerance induction (Table 1-
3).338,357–359 Based on the concept of mixed chimerism, combined kidney and bone
marrow/HSCT has been examined as a tolerance inducing regimen in limited numbers
of patients.340,360 Alternatively, based on the preclinical observations of Van Bekkum et
al., autologous HSCT has been utilized for nearly two decades to abrogate various
types of autoimmune disease.361,362 Although some of the putative tolerogenic
mechanisms differ between allogeneic and autologous HSCT, both treatments
represent an attempt to induce tolerance by “re-education” of the recipient’s immune
system.339,340 As the use of autologous and allogeneic HSCT for malignant disease has
been reviewed extensively elsewhere, this section will focus on HSCT for tolerance
induction in human patients.363–365
57
Table 1-3 Overview of Autologous versus Allogeneic HSCT
1.4.4.1 Hematopoietic stem cells
The bombings of Hiroshima and Nagasaki, and the dawning of the atomic age
revealed the intense myeloablative effects of ionizing radiation.366 It was later observed
that allogeneic bone marrow transplantation could rescue lethally myelosuppressed
mice and humans.366 In a series of seminal studies starting in 1961, Till and McCulloch
demonstrated that the bone marrow consisted of a sub-population of cells with the
ability to give rise to multiple myeloerythroid cell types and to self-renew.367–369 These
AUTOLOGOUS HSCT ALLOGENEIC HSCT
INDICATIONS Myeloma
Non-Hodgkin Lymphoma
Autoimmune disease
Acute myeloid leukemia
Acute lymphoblastic leukemia
Fanconi Anaemia
Organ Transplantation
CONDITIONING
REGIMENS
Chemotherapy+/irradiation Chemotherapy+/irradiation
STEM CELL
SOURCES
Autologous Peripheral Blood
Stem Cells
Allogeneic Peripheral Blood Stem
Cells
Cord Blood Stem cells
COMPLICATIONS Treatment-related mortality and
Toxicity
(NO GVHD)
Disease Relapse
Infectious Disease
Treatment-related mortality and
Toxicitity
(GVHD)
Disease Relapse
Infectious Disease
58
cells were termed hematopoietic stem cells (HSCs) and are now defined by their
multipotent capacity to generate all of the hematopoietic lineages and to self-replicate
into daughter cells.370 Hematopoiesis is a highly regulated hierarchical process with
HSCs at the apex of that hierarchy as the multipotent progenitors of all hematopoietic
lineages (Figure 1-3).366,371 According to canonical understanding, these multipotent
cells follow a stepwise development into oligopotent and finally unipotent cell types that
give rise to all the lineages.366 However, in 2015, Notta et al., provided evidence against
the existence of the oligopotent common myeloid progenitor (CMP) stage in both mice
and human bone marrow which is believed to give rise to myeloid, erythroid and
megakaryocyte lineages.372 This study re-defined classical views of hematopoietic
development and further study is required to elucidate the potential heterogeneity within
each major progenitor cell type for a more accurate map of hematopoiesis.
Another major focus of research in immunology has been the identification of cell
surface markers for HSCs in humans and mice. Early studies identified a population of
cells with potent HSC capacity in vivo that were termed LSK and constituted about
0.05% of adult mouse bone marrow. These cells lacked expression of lineage-specific
markers (e.g. B220,CD3,Gr-1,Ter119) and displayed stem cell antigen 1 (Sca1) and the
trans-membrane tyrosine kinase c-kit (CD117), the receptor for stem cell factor also
known as steel factor. 373,374 LSK cells nevertheless, are a heterogeneous population of
long-term (LT-), intermediate-term (IT-) and short-term (ST-) HSCs in addition to
multipotent progenitors (MPPs) (Figure 1-3). These represent three distinct populations
classified by their repopulating activity and longevity in mice.371 The SLAM (signaling
lymphocytic activation molecule) family of glycoproteins have been used to identify LT-
59
HSCs.375,376 LT-HSCs comprise 10% of the LSK population and are
CD150(slamf1)+CD48-CD49blo.371 Whereas murine HSCs lack expression of CD34, in
humans, CD34 expression delineates cells with HSC and MPP activity.371,377 In 2011,
Notta et al. demonstrated that that single cells from the CD34+CD90+CD49f+
phenotypic subset could fully reconstitute humanized NOD-SCID-IL2RyKO (NSG)
recipient mice providing proof of a LT-HSC population in humans.378
In clinical medicine, bone marrow obtained from the iliac crest was the first
source of HSCs.379 However, due to the highly invasive nature of this procedure, there
was a shift towards using peripheral blood mobilized stem cells (PBSCs) in the
1990s.380 This technique involves treatment with chemotherapy along with G-CSF to
promote stem cell entry into peripheral circulation. Studies have shown that treatment
with AMD3100, an inhibitor of the chemokine receptor (CXCR4), which is involved in
HSC homing to the bone marrow, is more effective than G-CSF alone.381,382 PBSCs are
then isolated using magnetic-activated cell sorting (MACS) for CD34+ cells.383 Several
studies have demonstrated the safety and efficacy of this treatment.383 However, bone
marrow transplantation remains common in the pediatric population due to improved
outcomes versus PBSCs.357
60
Figure 1-3 Schematic of mouse and human hematopoeitic development
A comparative depiction of surface markers of major cell types in the hematopoietic hierarchies
in both mice and humans. Recent evidence generated by Notta et al. will require redefining
classically accepted hierarchies.372 This schematic nevertheless, remains useful for highlighting
the differences between murine and human hematopoietic lineages. CMP – common myeloid
progenitor, ETP – early thymic progenitor, GMP – granulocyte/macrophage progenitor,HSC-
hematopoietic stem cell, LMPP- lymphoid primed multipotent progenitor, MEP –
megakaryocyte/erythrocyte progenitor, MPP – multipotent progenitor, MLP – multi-lymphoid
progenitor. Reprinted from Cell Stem Cell, Volume 10. Doulatov et al., Hematopoiesis: A
Human Perspective, Pg. 124. (2012) with permission from Elsevier.371
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1.4.4.2 Allogeneic HSCT and mixed chimerism
Allogeneic bone marrow transplantation (BMT) was first used to treat primary
immune deficiencies and cancer.384 In the past thirty years, a few groups have also
attempted to utilize allogeneic HSCT to induce solid organ transplant tolerance.360 In
1984, Ildstad and Sachs demonstrated acceptance of allografts in mice treated with
total body irradiation and reconstituted with mixed bone marrow from syngeneic and
allogeneic strains of mice.385 GVHD was avoided through use of T-cell depleted bone
marrow. In 1989, Sharabi and Sachs used a non-lethal conditioning regimen using T
cell depleting mAbs along with local thymic irradiation, to induce long-term mixed
chimerism and graft acceptance.386 Studies in mice utilizing the mixed chimerism
approach have shown an indispensable role for intra-thymic deletion of donor-reactive T
cells.340 Further tolerization is posited to occur through the various peripheral self-
education mechanisms.340 Studies in NHP models demonstrated that allograft
tolerance could be established but mixed chimerism was transient potentially due to the
large repertoire of memory T cells in NHPs versus in-bred laboratory mice.387 This
indicates a potential role for regulatory mechanisms in maintaining tolerance in NHP
models after mixed chimerism is lost.360 Studies in NHP models also presented
evidence for the utility of co-stimulatory blockade in enhancing but not establishing long-
term mixed chimerism.388
Currently, three groups have examined the potential of mixed chimerism for
tolerance induction in clinical transplantation utilizing three different protocols. At
62
Stanford, total-lymphoid irradiation (TLI) and ATG was used to induce mixed chimerism
in HLA-matched kidney transplant recipients. Of the 16 patients receiving this treatment,
15 developed long-term mixed chimerism (>6 months) and 8 patients were successfully
withdrawn from all IS between 6-12 months post-HSCT.389 In the most recent report by
this group, it was shown that out of 22 patients that received the protocol, 18 patients
who established persistent mixed chimerism (>12 months), were successfully withdrawn
from IS and no incidence of GVHD or rejection was observed for up to a 7 year post-
withdrawal observation period.390 The same protocol was tested in HLA-mismatched
kidney transplant recipients but did not result in tolerance rendering this approach
currently unsuitable for non-HLA identical organ transplants.360,391 A second protocol
was established at the Massachusetts General Hospital (MGH) based on a non-
myeloablative conditioning regimen involving cyclophosphamide and thymic
irradiation.392 This trial was performed in 10 HLA-mismatched kidney transplant
recipients with varying underlying indications for transplant. 7 out of 10 patients were
successfully withdrawn from IS after 8-14 months. Mixed chimerism was transient and
non-detectable after 21 days of HSCT with only 4 patients showing longer (<105 days)
lasting mixed chimerism. Nevertheless, treated patients demonstrated reduced donor-
specific MLR and CTL activity in vitro with elevated mRNA levels of FOXP3 in the
graft.392 The long-term follow-up to this patient cohort shows mixed results for tolerance
induction. As of the last report in 2014, only 4 out of 10 patients remained IS-free
without any complications, with one patient IS-free for a period of 10 years.393 While
there was no serious opportunistic infections, 3 patients were required to re-start IS
after 6-8 years as they started to show histological signs of rejection and 3 patients
63
experienced graft loss due to rejection or thrombotic microangiopathy.393 All patients
suffered from HSCT-associated engraftment syndrome that causes fever, skin rash and
diarrhea.394 A third clinical protocol was developed at Northwestern University based on
a high-intensity conditioning regimen using cyclophosphamide, fludarabine and total
body irradiation (TBI- 200 cGy).395 Of note, this trial involved manipulation of the PBSC
grafts by the company Regenerex LLC, to enrich for HSCs and a population of CD8+
facilitating cells (FCs). The full phenotype of these proprietary FCs are hitherto
undisclosed. As of 2015, this group has reported that 12 patients out of a cohort of 19,
had been withdrawn from IS for between 8 months to 48 months without incidence of
acute rejection.396 The high-intensity nature of this regimen resulted in stable donor
chimerism in the recipients with only one patient developing stable mixed chimerism.
Although only one patient reportedly developed GVHD, this treatment was associated
with severe neutropenia and a high incidence of opportunistic bacterial or fungal
infections.396 The 3 clinical approaches for tolerance induction using mixed chimerism
have demonstrated its utility for prolonging graft survival in limited numbers of patients.
However, the results from all 3 studies are mixed with some patients undergoing
rejection episodes or requiring IS after withdrawal.397 The primary successes of this
technique appear to be associated with the length of the mixed chimeric state. After
mixed chimerism is lost, there is a potential for the activation of Treg-mediated
tolerogenic mechanisms as demonstrated by the elevated FOXP3 expression in the
graft in the MGH cohort.392 Although, there was not significant incidence of GVHD
associated with any of the 3 protocols, stable allogeneic chimerism always poses the
risk for GVHD.397 Finally, finding HLA-matched allogeneic donors is an additional
64
challenge associated with the mixed chimerism approach. These considerations warrant
that alternative mechanisms for immune re-education be investigated as well.
1.4.4.3 Autologous HSCT
In 1989, Van Bekkum et al. studied autologous BMT as a treatment for adjuvant-
induced arthritis in rats.361 Due to the technical complications of autologous HSCT in
rodent models, bone marrow from syngeneic or congenic (CD45.1 vs. CD45.2) mice is
utilized and deemed to be an autologous HSCT.398 As the authors noted in the rat
model, treatment with TBI and syngeneic bone marrow transfer resulted in significant
reduction in arthritis severity. As a control for TBI, a group of rats was also treated with
CsA but did not cause significant recovery from arthritis versus the autologous BMT
group.361
As a result of work with rodent models, autologous HSCT was quickly adopted as
a treatment for patients with treatment-refractory autoimmune disease (AID). It is
estimated that as of 2011, nearly 3000 patients worldwide had been treated with
autologous HSCT for AID.399 Currently, conditioning regimens for autologous HSCT are
highly varied and are generally classified into the following categories, i) “high intensity”
regimens involving the use of high doses of busulphan or TBI, ii) “low-intensity”
regimens involve fludarabine-based, or cyclophosphamide-alone regimens,
iii)“intermediate-intensity” chemotherapeutic regimens such as BEAM (carmustine,
etoposide, cytarabine and melphalan) or cyclophosphamide in combination with ATG
primarily for rheumatological conditions (e.g.SSc, SLE).400 Clinical trials of autologous
65
HSCT have been performed in a wide range of autoimmune pathologies including
multiple sclerosis, systemic sclerosis (SSc), SLE, Crohn’s disease, juvenile arthritis and
T1D.400,401 These trials have shown treatment safety and efficacy with an increased
patient quality of life and stabilization of disease.400,402 Recent clinical trials have shown
significant abrogation of autoimmune disease and have shown that autologous HSCT is
superior to current standard of care for those diseases. In 2011 Burt et al., reported the
efficacy of non-myeloablative autologous HSCT in systemic sclerosis.403 Ten out of
nineteen patients treated with HSCT had improved modified Rodnan skin scores
(mRSS) and pulmonary function as measured by forced vital capacity, compared to the
9 cyclophosphamide-alone treated control arm patients. Furthermore, 8 out of 9 control
arm patients had disease progression compared to none in the HSCT-treated arm.403
Another seminal clinical trial was recently performed in MS patients by our collaborator
Dr. Harold Atkins at the Ottawa Hospital Research Institute.404 This phase II, single-arm
trial included 24 patients (aged 18-50) who had progressive disease and poor
prognosis. Patients were treated with busulphan, cyclophosphamide and ATG followed
by autologous HSCT and monitored for a median period of 6.7 years post-HSCT. Prior
to HSCT, the cohort of 24 patients had 167 clinical relapses over 140 patient-years and
188 gadolinium-enhancing lesions over 48 MRI (magnetic resonance imaging) scans.404
Post-HSCT, there were no new T2-weighted or Gd lesions over 188 MRI scans and no
clinical relapses for a follow up period of up to 12 years. The Expanded Disability Status
Scale (EDSS) score was improved in 35% of patients. HSCT-associated mortality was
low with 1 out of 24 patients succumbing to treatment-related toxicity.362 This study was
the first evidence of long-term disease abrogation in MS due to autologous HSCT.
66
Although this clinical study did not report the immunological changes associated with
HSCT, other groups have studied potential immune potential mechanisms of immune
re-education associated with autologous HSCT. One of the primary mechanisms is the
re-setting of the immune system with a shift from memory T and B cells to newly
emerging naive cells.400,405 Recently, Muraro et al. performed high-throughput
sequencing of the TCRβ chain before and after HSCT in patients being treated for
MS.359 This study showed that while CD4+ repertoires were almost entirely renewed
post-HSCT, CD8+ were not effectively depleted and reconstituted the CD8+
compartment.359 In 2016, Delemarre et al. also provided evidence of a crucial effect of
autologous HSCT on the Treg compartment. By comparing Treg TCR diversity pre- and
post-HSCT, the authors demonstrate that Treg TCR diversity was significantly
increased after HSCT.406 The primary limitations of this study were that it was
performed in a pediatric AID patient population for patients suffering from juvenile
arthritis and dermatomyositis, and the patient cohort was small (n=4). Further studies in
adults and various time points post-HSCT will be required to uncover the role of Treg
diversity in abrogating autoimmunity post-HSCT. Nevertheless, this study provides
additional proof of the potent immune re-education capacity of autologous HSCT.
Another important phenotypic observation in AID patients receiving autologous HSCT is
a significant increase of CD4+CD25+FOXP3+ Tregs in patients compared to pre-
transplant levels.407,408 In 2013, Abrahamsson et al. performed multiparameter
immunophenotyping in MS patients receiving non-myeloablative HSCT.408 In addition to
a transient but significant increase in CD4+FOXP3+ Tregs, the study also unveiled
perturbations in other cell populations that may be important for promoting tolerance.
67
These involved a significant increase in CD56high NK cells that have putative
immunoregulatory roles and a pronounced depletion of CD8+CD161high.408
CD8+CD161high cells represent a population termed mucosal-associated invariant T cells
that are found to infiltrate the brain in MS patients, and produce high levels of the pro-
inflammatory cytokines IFNγ and IL-17.408,409 These results indicate that tolerance
induction and disease-abrogation post-HSCT in these patients is likely a multifactorial
process that results from a system-wide resetting of the immune system. In 2014, a
clinical report also described that HSCT in SSc patients also resulted in the emergence
of a putative Breg population designated as CD19+CD24highCD38high at 6 and 12 months
post-transplant.410 This was also associated with a significant reduction in B memory
cells (CD19+CD27+IgD-) coinciding with an increase in naive (CD19+CD27+IgD+) B cells
.411 Further studies from this group are yet to be reported. In particular, the
immunological functions of this Breg population will need to be elucidated in the context
of AID. The evidence from clinical studies of autologous HSCT indicate several
phenotypic and functional alterations that can play a role in tolerance induction. These
studies provide a rationale for expanding its use in AID and potentially in organ
transplantation. The primary complications of autologous HSCT are treatment-related
mortality, which can be potentially as high as 15%, as well as treatment-related
morbidity including opportunistic infections, infertility and risk of malignant diseaase.358
The major advantages of this approach however are that there is no risk of GVHD and
that autologous PBSCs are readily available and easily stored.
At the Multi-Organ Transplant Program of the University Health Network, we
observed a clinical case of a patient who underwent autologous HSCT for underlying
68
malignant disease and subsequently maintained stable graft function after IS withdrawal
(Levy, unpublished data). This resulted in the establishment the ASCOTT study, a
phase I clinical trial in liver transplant patients who will undergo autologous HSCT for
tolerance induction (NCT02549586). In parallel to the clinical study, there is a need for a
preclinical murine model to provide mechanistic proof-of-concept of the utility of this
technique in SOT. The establishment and investigation of the mouse model is the focus
of this dissertation.
69
Hypotheses and Aims
The overall aim of this thesis project was to establish a murine model of autologous
HSCT and examine its potential for tolerance induction in a murine allograft model.
Previously, in our lab we established the heterotopic cardiac allograft model first
described by Corry et al.130,206 The specific hypothesis that we tested in this thesis was
as follows; Tolerance will develop to MHC-mismatched cardiac allografts in mice
following autologous HSCT.
The aims of this study were to:
1. Establish a mouse model of autologous HSCT which reconstitutes all hematopoietic
compartments in the recipient.
2. Examine the potential of HSCT to induce tolerance in a murine model of heterotopic
cardiac transplantation.
3. Elucidate immunological mechanisms post-HSCT in allograft recipient mice.
70
Materials and Methods
3.1 Mice
C57BL/6J (H-2b), B6.CD45.1 (Ptprca, H-2b), BALB/cJ (H-2d) and C3H/HeJ (H-2k) were
purchased from the Jackson Laboratory (Bar Harbor, ME) and were kept in specific-
pathogen free housing at the University Health Network Animal Resource Center.
Animals were treated in accordance with guidelines set by the Canadian Council for
Animal Care and the University Health Network. All mice used were female and
between 6 to 12 weeks of age. CD45.1 and CD45.2 (BL/6) congenic mice were used as
HSC donor and recipient respectively so that post-HSCT hematopoietic lineages could
be tracked and to determine degree of chimerism . This allows for analysis of pre- and
post-HSCT hematopoietic populations.
3.2 Heterotopic cardiac transplantation
Intra-abdominal heterotopic cardiac transplantation was performed as previously
described by Corry et al.130 All surgeries were performed by microsurgeon Dr. William
(Wei) He from the Levy lab. Briefly, six week old donor BALB/cJ mice were injected i.p.
with pentobarbital and placed under an operating microscope (25x). 1ml of 300U
heparin was then injected into the inferior vena cava. Donor hearts were harvested by
ligating the inferior vena cava (IVC), the superior vena cava and the azygous vein with
6-0 silk sutures and dividing them superior to the ligatures. The ascending aorta and
pulmonary artery were then separated and the pulmonary artery was transected at the
71
point of bifurcation. The heart was then excised and placed in a 4°C cold saline solution
(Baxter Healthcare, IL).
Recipient 12-week-old recipient C57BL/6J mice were anesthetized with an i.p. injection
of pentobarbital and placed under the operating microscope. A long midline abdominal
incision was made and the contents of the abdomen were moved aside and covered
with gauze, exposing the abdominal aorta and IVC. The abdominal aorta and IVC were
mobilized and clamped off at the point of bifurcations of the renal and iliac vessels. The
lumbar vessels were ligated with 10-0 sutures. The donor’s aorta was anastomosed
with the recipient’s abdominal aorta. In similar fashion, the donor’s pulmonary artery
was sutured to the recipient’s IVC. Afterwards the clamps on the recipient’s abdominal
aorta and IVC were removed. This allows for retrograde blood flow from the recipient
aorta to the donor coronary arteries draining into the donor right atrium where it is
pumped into the right ventricle and eventually via the donor pulmonary artery to the
recipient IVC.
Graft function was assessed daily via manual palpation and scored for beating rate and
strength on a scale from 0-3 with 0 being a total cessation of graft function. Grafts that
continued to beat ≥ 70 days post-transplant were considered to be accepted. After 70
days, or at cessation of graft beating, mice were sacrificed and graft function was
verified by direct visual examination. Grafts were subsequently harvested for
histological studies. Prior to sacrifice, saphenous vein blood was collected in EDTA
coated capillary tubes and submitted for complete blood counts using the Hemavet
950FS (Drew Scientific, Miami Lakes, FL).
72
3.3 Flow cytometry
Flow Reagents. The following anti-mouse mAbs were utilized for FACS analyses of
reconstitution studies. All mAbs were purchased from Biolegend (San Diego, CA):
Brilliant Violet™ (BV) 650 anti-CD45.1 (clone-A20), BV785 anti-CD45.2 (clone-104),
BV510 anti-CD3ε (clone-145-2C11), BV605 anti-CD19 (clone-6D5), PerCP/Cy5.5 anti-
CD4 (clone-GK1.5), APC/Cy7 anti-mouse CD8α (clone-53-6.7). For Treg staining, the
following anti-mouse mAbs were used: FITC anti-CD4 (clone-GK1.5), APC anti-CD25
(clone-PC61), PE anti-Foxp3 (clone-150D). In all flow cytometric studies, Fc blocking
was performed using a mAb to mouse CD16/32 (TruStain FcX™ clone 93) from
Biolegend. For all studies viability was assessed by staining with fixable viability dye
eFluor®450 (eBioscience, San Diego, CA) also referred to as pacific blue.
Cell Suspensions. Single cell suspensions of spleen and bone marrow cells were
prepared as follows. Spleens were mechanically disassociated on a 40 µm nylon filter.
Bone marrow was obtained from crushed femurs and tibias. Subsequently, red blood
cell lysis was performed using RBC lysis buffer (eBioscience) followed by a final
filtration step through a 40 µm filter. For surface staining, 1x106 spleen or bone marrow
cells were re-suspended in 100 µL of FACS buffer (1XPBS supplemented with 1%FBS
and 5 mM EDTA) and incubated with flow mAbs at 4°C for 30 minutes (min). After
surface staining, cells were washed twice and incubated with pacific blue viability dye
for 30 min at 4°C. Finally, cells were fixed using 2% paraformaldehyde (PFA), washed 2
times and re-suspended in 400 µl of FACS buffer for analysis. For staining of peripheral
blood, 100 µl of saphenous vein blood was treated initially with RBC lysis buffer
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(eBioscience). After stopping the reaction, cells were surface stained and followed by
staining with pacific blue viability dye. Finally, the cells were placed in 1X Fix/Lyse buffer
(eBioscience) to further lyse red blood cells and fix cells for further analysis. Cells were
washed 2 times and re-suspended in 400 µl of FACS buffer for analysis.
Analysis. Single cell suspensions were analyzed on a BD LSRII flow cytometer (BD
Bioscience, Franklin Lakes, NJ). Data were analyzed using the program FlowJo version
9.6 (Treestar Inc. Ashland, OR). For all analyses, cells were gated on singlets, live cells.
Lymphocytes were gated on using forward and side scatter.
3.4 Purification of LSK cells
LSK isolation protocols were adapted from earlier studies.412,413 Femurs and tibias were
harvested from four 12 week old donor CD45.1 female mice. Bone marrow was
obtained by crushing femurs in 10cm tissue culture dishes (Sarstedt AG&Co,
Numbrecht, Germany) containing 10ml PBS using a glass mortar. Crushed femurs were
washed by centrifuging 1200RPM for 5 min in α-modified Eagle’s Medium (Invitrogen,
Carlsbad, CA) with 10% fetal bovine serum (ThermoFisher, Waltham, MA) and 0.5 µM
2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). The pellet was filtered through a 40-
µm nylon filter and centrifuged as above. Subsequently the cell pellet was re-suspended
in 4 mL of RBC Lysis Buffer (eBioscience, San Diego, CA) and incubated at room
temperature (RT). After 5 min, the reaction was stopped and cells were counted prior to
lineage depletion.
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Lineage negative (Lin-) cells were enriched by labelling bone marrow cells with the
mouse lineage cell depletion kit (Miltenyi Biotec, Bergisch Gladbach, Germany) followed
by magnetic separation using the autoMACS® Pro Separator (Miltenyi Biotec). Lin- cells
were counted and stained with APC anti-mouse CD117 (c-kit) antibody (clone 2B8-
Biolegend, San Diego, CA) and PE anti-mouse Ly-6A/E (Sca1) antibody (clone D7-
Biolegend). As the lineage cell depletion kit contains biotinylated mAbs, FITC
Streptavidin was utilized to assess enrichment pre- and post-lineage depletion. A 10-
fold enrichment in Lin- cells was observed post-lineage depletion (Fig 4-1). After 30 min
incubation at 4°C, cells were washed twice and re-suspended in 200 μL of FACS buffer.
5 µl of the viability dye Propidium Iodide (PI) was added to the cells, and the sample
was submitted the Sickkids-UHN Flow Cytometry Facility for LSK sorting using a BD
FACSAriaII cell sorter (BD Biosciences, San Jose, CA).
3.5 Hematopoietic stem cell transplantation
Sorted LSK cells were washed 2 times with PBS and counted using a hemocytometer.
CD45.2 B6 recipient mice were lethally irradiated at a dose of 13 Grays (Gy) using a
GAMMACELL® 40 cesium-137 irradiator (Best Theratronics, Ottawa, ON). The dose
was fractionated into two doses of 6.5 Gy. Within 2h of the final irradiation recipient
mice received intravenous (i.v.) injections of 200 µl of PBS containing 5000 viable
CD45.1 LSK cells. HSCT-treated mice were weighed weekly and were monitored daily
for morbidity and mortality.
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3.6 Treatment groups
The HSCT treatment protocol was adapted from earlier studies for use in our murine
heterotopic heart transplant model.414 Recipient CD45.2 BL/6 female mice (12 weeks of
age) were allografted as described above. To prevent acute allograft rejection, allograft
recipients were treated with rapamycin (Pfizer, New York City, NY) at a dosage of 2
mg/kg per day for a total of 14 days starting on day -1 to day 13 post-heart
transplantation. Six days post-transplant, the mice were treated with HSCT as described
above. This group, designated as TxHSCT, and all control groups used in this study are
tabulated in Table 3-1. The control groups included were as follows: TxRej-an
allogeneic transplant group that did not receive any treatment (BALB/c > C57BL/6);
TxRapa-an allogeneic transplant group treated with 14 days of rapamycin (BALB/c >
C57BL/6); TxSyn-a syngeneic transplant group that received no treatment and an
untreated non-transplanted C57BL/6 (CD45.2) control group (Non-Tx).
Table 3-1 Heterotopic heart transplantation treatment groups
Treatment Group Graft Rapamycin (1mg/kg) HSCT
1. TxRej ALLOGRAFT NO NO
2. TxRapa ALLOGRAFT YES- 14 days NO
3. TxHSCT ALLOGRAFT YES-14 days YES – POD7
4.TxSyn SYNGENEIC NO NO
5. Non-Tx Controls C57BL/6
NO NO NO
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Figure 3-1 Schematic of HSCT treatment for tolerance induction in cardiac
allotransplant model
HSCT treatment were examined in a previously established murine model of heterotopic
heart transplantation. A short course of rapamycin treatment was used to prevent acute
rejection and expand Tregs. On post-operative day 7 (POD7), mice underwent TBI
(13Gy) and were reconstituted with 5000 LSKs from congenic CD45.1 donor mice.
Rapamycin will be withdrawn at Day 13. Allograft survival was monitored via manual
palpation and grafts surviving > 70 days was deemed accepted. On POD45, mice from
each treatment group (Table 3-1) were sacrificed for immunological studies.
77
3.7 Histology and immunohistochemistry
Transplanted and native BALB/c hearts were extracted and dissected along the
transverse (horizontal) axis into 2 nearly equivalent sections. The apex of the graft
proximal to the anastomoses with the recipient’s vessels was discarded and not utilized.
The two sections from each heart were placed into 10% buffered formalin and
embedded in OCT (Sakura Fintek, Torrence, CA) respectively.
The tissue fixed in 10% buffered formalin was submitted for sectioning and staining to
the Pathology Research Program Core Facility (PRP) at the Toronto General Hospital.
The tissue was subsequently embedded in paraffin, cut into 5 µm thick sections and
stained with hematoxylin and eosin (H&E) and Masson’s Trichrome (MT) dyes.
Immunoperoxidase staining was performed using anti-rat/mouse Foxp3 (clone FJK-16s,
eBioscience, San Diego, CA), anti-mouse CD3 (clone 17A2, eBioscience) and anti-
mouse B220 (BD Pharmingen-RA3-6B2) antibodies. Representative images of the
histology were taken by a trained pathologist and selected slides were scanned for
morphometric analysis at the STTARR research facility at the University Health Network
using an Aperio ScanScope XT whole slide scanner (Leica Biosystems, Wetzlar,
Germany). Positively stained cells were quantitated using TissueStudio® (Definiens,
Carlsbad, CA).
3.8 Mixed lymphocyte reaction
Mixed lymphocyte reactions were performed as per the Current Protocols in
Immunology.415 Spleens were extracted from donor (BALB/c), recipient (C57BL/6) and
78
third-party (C3H/HeJ) mice, mechanically disassociated and RBCs were lysed. The
splenocytes were then layered onto Lympholyte-M (Cedarlane, Burlington, ON) and
spun at 1250xg at RT for 20 min. The interphase was collected and washed twice with
complete αMEM (prepared as described above). Allogeneic donor (BALB/c), third-party
(C3H) and syngeneic (C57BL/6) splenocytes were irradiated with 20 Gy using a
GAMMACELL® 40 cesium-137 irradiator (Best Theratronics, Ottawa, ON). In a U-
bottom 96-well suspension cell plate (Sarstedt AG&Co, Numbrecht, Germany), 2x105
responder splenocytes from C57BL/6 mice were co-cultured in triplicate with irradiated
4x105 syngeneic (C57BL/6), donor (BALB/c) or third-party (C3H) stimulator cells. Mixed
lymphocyte co-cultures were incubated at 37°C for 48 hours. After 48 hours, 1 µCi of
3H-thymidine (PerkinElmer, Boston, MA) was added to each well and cells were
incubated for an additional 18 hours. Plates were harvested using a UNIFILTER-96
Filtermate Harvester (PerkinElmer) and counts per minute (CPM) were recorded on a
TopCount Microplate scintillation counter (PerkinElmer). The proliferation data are
presented as fold change by calculating a “Stimulation Index” (SI) as per the Current
Protocols in Immunology .415 This is calculated by dividing the arithmetic mean of CPM
from triplicate experimental cultures by CPM from triplicate control (syngeneic) cultures.
3.9 Flow cytometry for donor-specific antibodies (DSA)
A flow cytometry-based cross match assay was adapted from earlier studies.206,416 Sera
was obtained from saphenous vein peripheral blood of C57BL/6 graft recipients and
non-transplanted controls. 1x106 donor BALB/c splenocytes were treated with Fc block
and incubated in duplicate with 2.5 µl of serum from recipient or non-transplanted mice
79
for 30 min at 4°C. Afterwards, cells were washed and incubated with polyclonal FITC-
conjugated anti-mouse IgG (Immunology Consultants Laboratory, Portland, OR)
followed by APC anti-mouse CD3ε (Biolegend-clone:145-2C11). Cells were finally
stained with pacific blue viability dye and analyzed using a BD LSRII flow cytometer (BD
Bioscience, Franklin Lakes, NJ). Data were analyzed using the program FlowJo version
9.6 (Treestar Inc. Ashland, OR). Levels of anti-donor MHC Class I antibodies were
determined by gating on live CD3+ cells and reported as median fluorescence
intensities (MFI).
3.10 Statistics
Data are shown as mean ± SEM unless otherwise stated. Statistical significance was
determined using Students t-tests or one-way ANOVA followed by Tukey’s post-hoc test
on Prism version 5 (Graphpad Software, La Jolla, CA). Survival data was plotted on
Kaplan-Meier curves using log-rank tests to assess for statistical significance. Statistical
results with P≤0.05 were considered significant.
80
Results
4.1 Purification of LSK cells and dose selection
To establish our autologous HSCT model, we aimed to isolate and purify the Lin-
Sca1+c-kit+ (LSK) population of murine HSCs from donor CD45.1 mice.413 As described
in the methods (section 3.4), we implemented a MACS-based lineage depletion protocol
followed by FACS for the stem cell markers Sca1 and c-kit (Figure 4-1). LSK were gated
on propidium iodide (PI)-negative populations to exclude dead cells. LSK cells were re-
suspended in PBS and injected intravenously to recipient CD45.2 mice. Subsequently,
we performed a dose-escalation study to investigate which dose resulted in complete
CD45.1 chimerism in the long-term. 1500, 3000 and 5000 LSKs were injected into mice
conditioned with 13Gy of TBI. At Day 100 post-HSCT, mice were sacrificed and level of
CD45.1 chimerism was determined using flow cytometry (Figure 4-2). All treatment
groups achieved > 97% CD45.1 chimerism in peripheral blood mononuclear cells with
no statistically significant differences between treatment groups. However, best results
were observed in the 5000 LSK cell group which tended to display higher (>99%)
CD45.1 chimerism.
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Figure 4-1 Isolation of LSK cells
This figure shows gating strategy for LSK cells pre- and post- lineage depletion to outline the
LSK purification process. Bone marrow was isolated by crushing femurs from CD45.1 donor
mice. RBC lysis was performed and cells were stained with the murine lineage depletion kit
antibody cocktail (Miltenyi Biotec). Lineage depletion was performed using autoMACS robotic
separation. Lin- cells were enriched 10-fold after magnetic sorting. Cells were stained with
mAbs to c-kit (APC) and Sca1 (PE) and submitted for LSK sorting on the Sca1 and c-kit
double positive population. As the lineage depletion cocktail was biotinylated, FITC-
streptavidin was used to distinguish between lineage positive and negative cells. Propidium
iodide was used as a viability dye for the exclusion of dead cells. Data are representative of 3
independent isolations using pooled bone marrow from n=4 mice at each time point.
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4.2 HSCT with LSK cells results in full hematopoietic reconstitution
and is not impaired by rapamycin treatment
We selected 5000 LSK cells as our optimal stem cell dosage. Subsequently, we
examined the ability of LSK cells to fully reconstitute all the hematopoietic lineages post-
HSCT. We were also concerned about performing LSK engraftment under the cover of
rapamycin, a potent immunosuppressant. As per Figure 3-1, our experimental plan for
allograft tolerance induction involves using a short course of rapamycin treatment pre-
Figure 4-2 LSK cell dose selection
Flow cytometry plots of CD45.1 versus CD45.2 populations in peripheral blood
mononuclear cells from mice treated with 5000, 3000 and 1500 LSK cells
respectively. There were no statistically significant differences in long-term CD45.1
chimerism in any of the treatment groups as determined by ANOVA. Gating was
performed on pacific blue negative (viable) cells. Data are representative of n=3
mice per group.
83
and post-HSCT to prevent acute rejection and expand Tregs. Hence, we investigated
the effects of rapamycin treatment on long-term hematopoietic reconstitution after LSK
cell transfer. In the absence of an allograft, hematopoietic reconstitution was assessed
in a treatment group of mice receiving LSK cells alone compared to a treatment group
receiving LSK cells plus rapamycin as per our dosing regimen outlined in Figure 3-1.
At Day 100, mice from both the LSK and LSK+Rapa groups were sacrificed and
peripheral blood, spleen and bone marrow cells were stained for CD45.1, CD45.2, CD4,
CD8 and CD19 (Figure 4-3). There were no statistically significant differences between
the two groups in terms of CD45.1 (donor) chimerism in PBMC (Figure 4-3A). Both
groups had over 99% donor chimerism indicating successful engraftment of LSK cells in
the absence and presence of rapamycin treatment. Furthermore, flow cytometric
analyses demonstrated that frequencies of CD4+, CD8+ and CD19+ lymphocytes were
not significant different between groups in PBMC (Figure 4-3B), splenic lymphocytes
(Figure 4-3C) and bone marrow lymphocytes (Figure 4-3D). We also performed
complete blood count analysis (CBC) to examine full hematopoietic reconstitution in
both treatment groups (Table 4-1). Both treatment groups were within the normal range
for white blood cells, neutrophils (PMNs), platelets and hematocrit indicating
multilineage long-term reconstitution. At Day 100 post-LSK cell transfer, there were no
significant differences between the two groups for any of the aforementioned
parameters.
84
Figure 4-3 Rapamycin does not impair long-term hematopoietic reconstitution
after HSCT with LSK cells
To study the effects of rapamycin on long-term LSK cell reconstitution, mice were
irradiated and treated with either 5000 LSK cells or 5000 LSK cells and rapamycin.
Hematopoietic reconstitution was assessed by flow cytometry. There were no
statistically significant differences between groups in A) CD45.1 chimerism in PBMC or
the frequencies of CD4+, CD8+ and CD19+ lymphocytes in B) peripheral blood C) spleen
or D) bone marrow. Differences between groups means were analyzed using ANOVA
followed by Tukey’s post-hoc test. Data are represented as mean ± SEM (n=4 mice per
group).
85
Table 4-1 Complete blood counts at Day 100 post-HSCT (data are shown as mean
±SEM)
Treatment
Group
WBCs
(1x103/uL)
PMNs
(1x103/uL)
Lymphocytes
(1x103/uL)
Hct
(%)
Platelets
(1x103/uL)
Normal Ranges 1.8-10.7 0.1-2.4 0.9-9.3 35.1-45.4 592-3000
LSK
(n=4)
6.5±1.4 1.2±0.2 4.2±0.8 38.7
±0.5
798.8
±110.7
LSK + Rapa
(n=4)
6.4±1.3 1.1±0.1 4.5±0.9 36.3
±0.6
783.7
±55.5
WBC- White blood cells PMN – polymorphonuclear cells (neutrophils) Hct - Hematocrit
86
4.3 HSCT promotes long-term cardiac allograft survival
HSCT was observed to significantly prolong allograft survival compared to
untreated and rapamycin-alone treated allograft recipients (Figure 4-4). TxHSCT mice
had a median survival time (MST) of 55 days with one recipient surviving long term till
Day 70. As expected, the untreated recipients (TxRej) suffered from acute rejection
culminating in 100% graft loss by Day 10 with a MST of 8 days. TxRapa recipients had
a MST of 31 days indicating that rapamycin alone cannot induce long-term graft survival
in our MHC-mismatch model. As expected, isograft recipients (TxSyn) had 100% long
term survival and were visually examined to be beating at the Day 70 endpoint (Figure
3-1). A small group (n=3) of isograft recipients were also treated with 5000 LSKs and
13Gy of TBI to examine whether radiation-induced damage could trigger graft loss in
the absence of immunological rejection. At Day 70, 100% of TxSynHSCT allografts
were surviving despite a reduced cardiac function score as assessed by manual
palpation (data not shown). These results indicate that while TBI is not sufficient to
induce graft loss in the context of an isograft it may result in graft damage.
Histological studies were performed on grafts sacrificed at peak of rejection for
each group. Peak of rejection for each group was based on median graft survival time
and reduction graft function as defined by manual palpation score. Thus, peak of
rejection was deemed to be POD 5 for the non-treated rejecting (TxRej), POD 20 for the
TxRapa group and POD 30 for the TxHSCT. Syngeneic grafts from the TxSyn group
were sacrificed at Day 30 and used as histological controls. Upon visual examination of
H&E histology at 100X magnification (Figure 4-5A), it was observed that both the TxRej
and TxRapa groups exhibited hallmarks of acute cellular rejection marked by dense
87
mononuclear infiltrates (depicted by asterisks on image) and myocyte necrosis. In
contrast, allografts from the TxHSCT group had markedly reduced cellular infiltration
and tissue morphology comparable to that of syngeneic isografts which survive
indefinitely. A higher magnification (400X) array of H&E stains is also presented for
each group (Figure 4-5B). Visual inspection of these representative images revealed
extensive infiltration of mononuclear cells in the endothelium (vasculitis- depicted by
black arrows on image) and in cardiac tissue in the non-treated rejecting (TxRej) and
the TxRapa (rapamycin-only) allografts. In contrast, blood vessels from TxHSCT
allografts have very limited vasculitis and markedly reduced cellular infiltration. Blood
vessels from the TxSyn group have near normal appearance and are patent.
Representative images of immunoperoxidase staining for CD3+ cells are depicted for
cardiac grafts harvested from each treatment group at the same time point as the H&E
stained grafts (Figure 4-5C). Visual inspection of these images reveals that non-treated
rejecting (TxRej) and TxRapa allografts have significantly high numbers of graft-
infiltrating CD3+ cells. In contrast, TxHSCT allografts exhibit moderate to low numbers
of intra-graft CD3+ cells while syngeneic grafts (TxSyn) exhibit very low CD3+ cell
infiltration.
Immunostaining was also performed to investigate graft-infiltrating B, Tconv and
Treg cells in each treatment group. A single time point was selected for comparative
morphometric analyses. On POD 45, grafts were harvested and stained for infiltrating
CD3, B220 and FOXP3 positive cells (Figure 4-6). Grafts from the TxRej group which
reject entirely by POD 11, were not included for analysis due to extensive necrotic
damage and poor morphology at POD 45. Visual analyses of these representative
88
images reveal that TxHSCT allografts have significantly fewer CD3+ cells (Figure 4-6A),
markedly higher FOXP3+ infiltrates and considerably lower B220+ cells (Figure 4-6C)
than TxRapa allografts. Syngeneic grafts from the TxSyn group exhibit low levels of all
infiltrating cell types (B cell, Tconv and Treg).
These aforementioned images were analyzed for morphometry of the
immunoperoxidase staining using TissueStudio® (Definiens, Carlsbad,CA).
Morphometry readouts for each marker (CD3, FOXP3 and B220) are depicted in Figure
4-7. Allografts from the TxRapa group had significantly higher infiltration of CD3+ cells
per mm2 (3344 ± 636) versus TxHSCT (1217 ± 266) and TxSyn (1141 ± 273) treatment
groups (Figure 4-7A). Mice treated with HSCT and rapamycin also displayed drastically
diminished levels of infiltrating B cells (B220+) versus TxRapa (Figure 4-7B). The
numbers of B220+ cells per mm2 in the TxRapa grafts (228 ± 7.3) were over 10-fold
higher than the numbers in the TxHSCT (17 ± 4.1) group, which had nearly the same
numbers of B cells as the TxSyn control group (20 ± 4.0). We also wished to examine
graft-infiltrating Tregs. Treatment with HSCT and rapamycin resulted in statistically
higher numbers of FOXP3+ infiltrating cells per unit area, compared to rapamycin alone
or in syngeneic control grafts (Figure 4-7C). FOXP3+ cells per mm2 in the TxHSCT
allografts (350 ± 25) were over 3-fold higher than in TxRapa (98 ± 18) and over 10-fold
higher than in control TxSyn (28 ± 14) grafts. Treg to T cell ratios were examined in the
grafts by expressing the immunostaining data as a ratio of FOXP3+ to CD3+ cells
normalized for cells per unit area (Figure 4-7D). Graft prolongation in the TxHSCT group
correlated with elevated Treg to T cell ratios. TxHSCT (20.8 ± 5.6%) grafts were
observed to have statistically higher Treg to Tconv ratios versus the TxRapa (5.1 ±
89
0.9%) and the control TxSyn (2.7 ± 1.3%) grafts. Collectively, these data suggest that
graft prolongation in the TxHSCT group are associated with significantly reduced Tconv
and B lymphocytes and increased levels of Tregs. TxHSCT allografts closely resembled
sygeneic grafts in morphology and lack of cellular infiltration, except for graft-infiltrating
FOXP3+ Tregs. These results indicate that HSCT and rapamycin treatment protects
against cell-mediated rejection versus rapamycin alone. Thus, graft loss in the TxHSCT
group may be due to the toxicity associated with the TBI conditioning regimen.
90
Figure 4-4 HSCT prolongs cardiac allograft survival
Heterotopic heart transplant survival in HSCT-treated and control groups of mice. Upon
cessation of beating, mice were sacrificed and graft loss was confirmed by visual
examination. Allografts in HSCT-treated recipients survived significantly longer (▲:
median survival time = 55 days) than untreated recipients (●: median survival time = 8
days) or rapamycin-alone treated recipients (■: median survival time = 31 days) with 1
graft surviving indefinitely. Control syngeneic graft recipients survived indefinitely as
expected (▼: survival > 70 days). Syngeneic graft recipients were treated with
autologous HSCT to assess the effects of the conditioning regimen (TBI) on graft
survival. Syngeneic recipients treated with HSCT also had indefinite survival (♦: survival
> 70 days). *** P<0.001 versus rapamycin only and untreated controls as determined by
log-rank test.
91
Figure 4-5 HSCT treatment preserves cardiac allograft morphology
Cardiac grafts were sectioned and stained with H&E and anti-CD3 at various timepoints
coinciding with peak of rejection. Syngeneic grafts which survive indefinitely are used as
histological controls. A) Grafts from all treatment groups stained with H&E at 100X magnification.
TxRej and TxRapa grafts exhibit myocyte damage, dense mononuclear infiltrates and vasculitis.
TxHSCT and syngeneic (TxSyn) grafts have preserved morphology with the absence of cellular
infiltrates. Asterisks(*) indicate foci of dense mononuclear cellular infiltration. B) Grafts from all
treatment groups stained with H&E at 400X magnification. High magnification reveals that TxRej
and TxRapa grafts have vasculitis. TxHSCT and TxSyn have patent vessels with normal
appearance and absence of vasculitis. Arrows point to vessels. C) Representative CD3+
immunoperoxidase staining of all treatment groups. TxRapa and TxRej grafts exhibit foci of high
CD3+ cell infiltration. In contrast, visual examination reveals significantly reduced CD3+ cells in
HSCT treated grafts (TxHSCT) and sygeneic grafts. Representative graft histology (n=3-4 mice
per group) are presented.
92
Figure 4-6 Representative immunoperoxidase staining of graft infiltrating cells
Cardiac grafts from POD 45 were harvested, sectioned and stained for anti-CD3,
FOXP3 and B220 cells. Untreated rejecting (TxRej) grafts were not included due to
extensive necrosis and poor tissue morphology. Representative images from each
group are presented at 200X magnification (n=3/4 mice per group). A) Representative
staining for graft infiltrating CD3+ cells. TxRapa grafts exhibit extensive CD3+ cell
infiltration. In contrast TxHSCT allografts and TxSyn isografts display markedly reduced
CD3+ cell infiltration. B) Representative staining for graft infiltrating FOXP3+ cells.
Examination of TxHSCT allografts reveals high numbers of intra-graft FOXP3+ cells in
contrast to TxRapa allografts or syngeneic (TxSyn) grafts. C) Representative
immunostaining for B220+ cells. Upon visual examination, TxRapa allografts exhibit
markedly increased B220+ (B cell) infiltration in the graft compared to HSCT treated
(TxHSCT) and isografts (TxSyn).
93
Figure 4-7 Morphometric analyses of immunoperoxidase staining
Graphs of morphometric analysis performed on immunohistochemistry stains performed
on post-operative day 45 (POD45). Representative images are provided in Figure 4-6.
Data are presented as means ± SEM for each group A) Absolute number of CD3+ cells
per unit area, B) absolute number of B220+ cells per unit area, C) absolute number of
FOXP3+ cells per unit area, D) ratio of FOXP3+ to CD3+ cells in the grafts. Differences in
group means were analyzed by ANOVA followed by Tukey’s post-hoc test
****P<0.0001, ***P<0.001,**P<0.01,*P<0.05.
94
4.4 HSCT-treated mice maintain primary immune response in
vitro
At POD 45 we performed a one-way MLR to assess T cell proliferation in
response to donor (BALB/C) and third-party (C3H) stimulators (Figure 4-5A and 4-5B
respectively). There were statistically significant differences in MLR responses to
BALB/C between TxRej mice and all other treatment groups. As expected, untreated
allograft recipients (TxRej) that had rejected their grafts by Day 10 demonstrated a
secondary immune response in vitro (SI =15.0 ± 1.3). In contrast, allografted mice
receiving HSCT and rapamycin (TxHSCT) maintained a primary immune response to
donor (SI = 9.8 ± 0.9). Despite rejecting their grafts by Day 32, allograft recipients given
rapamycin alone (TxRapa) also maintained a primary MLR response to BALB/C
stimulators (SI = 7.6 ± 1.2). Both syngeneic graft recipients (TxSyn) and non-
transplanted controls (Non-Tx) which had not been previously exposed to BALB/C
antigen demonstrated a primary immune response as expected (SI = 5.8 ± 0.4 and SI =
6.1 ± 1.0 respectively). There were no statistically significant differences in SI between
the TxHSCT, TxRapa, TxSyn or Non-Tx control groups. When co-cultured with third-
party (C3H) stimulators, all treatment groups had a primary MLR response (SI < 5 for all
groups).
95
Figure 4-8 Lymphocytes from HSCT treated mice maintain primary immune
response in vitro
A mixed lymphocyte reaction (MLR) was performed using responder splenocytes from
each treatment group. MLR proliferation was assessed by incorporation of 3H-thymidine
and expressed as stimulation indices (SI) as per the Current Protocols in Immunology.
Background counts were assessed by co-culturing with syngeneic stimulator
splenocytes and were <500 CPM. Experimental counts were assessed by co-culturing
with allogeneic stimulator splenocytes from A) donor (BALB/C) or B) third-party (C3H)
mice and were > 1500 CPM. Data are presented as mean ± SEM. Differences between
group means were analyzed by ANOVA followed by Tukey’s post-hoc
test.***P<0.001,**P<0.01,*P<0.05.
96
4.5 HSCT treatment markedly diminishes DSA and expands
Tregs
At POD 45, we also performed quantitation of Tregs in the spleens of mice from all
treatment groups and donor-specific antibodies in recipient mice sera (Figure 4-6). We
investigated the frequencies of CD25+FOXP3+ cells in the CD4+ compartment of splenic
lymphocytes. As a proportion of CD4+ cells, Tregs were significantly higher in the
TxHSCT group (7.2 ± 0.7%) compared to all other treatment groups (Figure 4-6A).
Although Tregs tended to be higher in the TxRapa group (4.2 ± 0.7%) compared to
TxSyn (2.9 ± 0.3%), TxRej (3.7 ± 0.5%) and Non-Tx (3.0 ± 0.6%) control mice, these
differences were not statistically significant. On POD 45, sera were also isolated from
mice in all treatment groups and non-transplanted controls. As described in the methods
section (section 3.9), we performed a flow cross-match assay to assess levels of DSA.
As expected, in isografted TxSyn and non-transplanted control mice which had not
previously been exposed to BALB/C antigen, DSA was deemed non-detectable (Figure
4-6B). Even though these samples had background fluorescence (MFI =128.5 ± 13.3
and 90.5 ± 13.1 for Non-Tx and TxSyn respectively), it was not significantly different to
control wells in which no serum was added (80.2 ± 11.5%). Thus, these background
levels were deemed to be below the limit of detection. We observed that the TxHSCT
group had markedly diminished levels of DSA (MFI = 2854 ± 143). These levels were
15-fold lower than the DSA levels (MFI = 45049 ± 3498) in untreated recipients (TxRej) .
We also noted that despite treatment with a potent immunosuppressant, mice given
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rapamycin alone (MFI = 21476 ± 1799) continued to exhibit high levels of DSA (8-fold
greater than TxHSCT) albeit lower than DSA in the TxRej group.
Figure 4-9 Splenic Treg and DSA quantitation
HSCT treatment markedly increases proportions of Tregs and reduces DSA levels. A)
Quantification of flow cytometric profiles. CD25+FOXP3+ Treg are shown as a proportion
of CD4+ cells in the spleen. B) Sera from mice in each transplanted group and non-
transplanted controls were analyzed for DSA using a flow cross-match assay.
Alloantibody levels are depicted by median fluorescence intensity (MFI). The
segmented axis is used to compare results that show several fold-differences.
Syngeneic graft recipients and non-transplanted controls had MFI levels comparable to
non-serum controls and were deemed below limit of detection. Data in both A and B are
expressed as mean ± SEM. Differences between group means were analyzed by
ANOVA followed by Tukey’s post-hoc test.****P<0.0001,**P<0.01,*P<0.05.
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Discussion
Although solid organ transplantation is a highly successful therapy for patients
with end stage organ failure, the need for long term immunosuppression limits long term
survival and quality of life. One solution to this problem is a therapy that can induce
organ-specific immune tolerance in the clinic.
A number of approaches are being studied both pre clinically and clinically
including allogeneic stem cell transplantation to produce mixed chimerism, infusion of
Treg cells and the identification of biomarkers that will identify patients who have
developed tolerance.354,360,417 Each of these treatments has distinct advantages and
disadvantages that warrant further discussion. The success of the mixed chimerism
approach is predicated on the successful establishment of multi-lineage hematopoietic
chimerism. Studies performed in pre-clinical models demonstrated the capacity of multi-
lineage chimerism to induce tolerance by both central (deletional) and peripheral
mechanisms of tolerance.360 The trafficking of donor APC to the host thymus in these
murine models results in intra-thymic chimerism and deletion (negative selection) of
alloreactive clones.418,419 Evidence for clonal deletion in human patients was provided
by high-throughput sequencing of the TCRβ chain in three tolerant patients who had
been treated with the MGH combined kidney and bone marrow (CKBM) transplant
protocol.420 In contrast, non-tolerant recipients of the MGH protocol or conventional
kidney transplant recipients did not display similar reductions of donor-reactive
clones.420 Allogeneic HSCT resulting in mixed or complete chimerism also involves
mechanisms of peripheral tolerance.397 An early increase of Treg proportions has been
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reported in post-allogeneic HSCT in patients from all three clinical cohorts (MGH, SU
and NW).395,421,422 Patients treated with the Stanford protocol, which involves total
lymphoid irradiation and ATG, also displayed increased ratios of NKT to T cells. These
observations are functionally validated by the donor-specific hyporesponsiveness
observed in vitro using lymphocytes from patients who were successfully weaned off IS
using the allogeneic HSCT approach.395,422 Despite these promising results, it is now
clear that the allogeneic HSCT approach has severe limitations. The use of allogeneic
bone marrow poses a risk for GVHD. At MGH, the HLA-matched CKBM has also been
utilized in patients with multiple myeloma and end-stage renal disease. In this cohort, 2
out of 7 patients developed chronic GVHD while one patient suffered from acute
GVHD.423 However, GVHD was largely absent in all of the three recent clinical cohorts
of patients treated with allogeneic HSCT.397,424 GVHD was reportedly avoided by using
TLI and ATG in the SU group while the NW group posited that this avoidance of GVHD
was a result of using proprietary facilitating cells (FCx).397 However, long-term follow up
has revealed that GVHD does occur in a subset of patients over time. In a recent clinical
abstract, Leventhal et al. reported that two patients from the NW cohort developed
GVHD.425 Finally, it is important to note that the success rate of tolerance induction in
these trials is low. In the MGH trial of CKBM transplant in HLA-mismatched patients,
only 7 out of 10 treated patients were successfully weaned off IS. Moreover, three out of
these 7 patients were subsequently returned to systemic IS after experiencing rejection
episodes within 5 years of IS withdrawal.393,426 Only a single patient has achieved long-
term (~11 years) IS-free graft survival. Similarly, in the SU cohort, long-term follow up
data is only available for HLA-matched patients. In 22 HLA-matched patients, only 16
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could be successfully withdrawn from IS and currently the longest time off IS for a
patient is under 6 years.390,427 A total of 6 HLA-mismatched patients have also been
treated with the SU protocol recently but tolerance induction results from this cohort are
not yet available.390 Finally, in the NW cohort of patients, only 12 HLA-mismatched
patients are currently off IS for a maximum period of 6 years. Furthermore, this cohort
has also experienced a high rate of opportunistic infections with 11 out of 19 treated
patients developing bacterial or fungal infections that required treatment.396 In their most
recent report, Leventhal et al. also describe the loss of two renal transplants due to
infection.425 Collectively, these results indicate that the allogeneic HSCT approach is not
the most effective therapy for tolerance induction in clinical transplant recipients. A
crucial observation in all three clinical cohorts was that a large subset of patients had
only transient (<100 days) mixed chimerism. Patients weaned off IS in the MGH cohort
had transient chimerism persisting for only 2-3 weeks.424 Patients treated with the SU
protocol developed a stable mixed chimerism (2-4 years) but long-term data on the
persistence of chimerism in these patients are not yet available.397,424 Finally, 11 out of
12 IS-free patients in the NW cohort developed full donor chimerism, which significantly
increases the risk for GVHD.396 A further consideration is that out of the 41 patients from
all three cohorts that are currently reported to be off IS, only 16 are HLA-mismatched.397
In the absence of the tolerizing effects of the mixed chimerism state, the risk for GVHD
and/or graft rejection for HLA-mismatched patients will significantly increase in the long-
term. The mixed chimerism approach is hypothesized to induce lifelong tolerance on the
indispensable condition of lifelong multilineage hematopoietic chimerism. Whereas mice
achieve lifelong mixed chimerism, studies have shown that mixed chimerism was only
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transient in NHP models of renal transplantation.360,388 Studies demonstrated that while
tolerance was induced in these NHP models even with transient mixed chimerism, the
transplants could only be performed while peripheral mixed chimerism lasted.360 Thus, a
loss of mixed chimerism or the development of full donor chimerism, the risk of GVHD
and development of opportunisitic infections is a serious limitation to the use of this
approach to induce tolerance in these patients.
The approach of using ex vivo expanded autologous Tregs for the purposes of
tolerance induction in transplantation has also recently attracted interest.428,429 The
concept of infusing autologous Tregs is promising due to their central role in maintaining
peripheral tolerance. Studies in murine models have shown that adoptive transfers of
Tregs can induce allograft tolerance.430 However, no such study has been reported to
date in human patients. As discussed previously, the ONE study is a multi-center phase
I/IIa trial that will examine the potential of ex vivo expanded Tregs to induce tolerance in
renal transplantation (www.onestudy.org).356 Thus far, Treg infusions in humans have
been used successfully to prevent GVHD in limited numbers of allogeneic bone marrow
recipients.431 Two studies have also recently shown safety of Treg infusion in Type 1
diabetes patients but long-term follow up of these patients is required to determine if
self-tolerance can be established.431 Despite the encouraging safety profiles, there are
several concerns regarding Treg infusions as a therapy for organ transplantation. First,
current trials have utilized polyclonal freshly isolated and expanded Tregs. However,
studies have shown that alloantigen-specific Tregs can be generated in vitro and have
superior suppressive capacity compared to polyclonal Tregs.432,433 The type of Treg
used and the methods for ex vivo generation of these cells will necessitate further
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optimization in the context of organ transplantation. Furthermore, it will be important to
ensure that infused Tregs stably continue to express FOXP3. The demethylation of key
regions of the FOXP3 gene are important for maintaining stability.434 Thus, the
demethylating agent azacytidine has been tested in a phase I trial to promote Treg
stability and prevent GVHD in patients receiving allo-HSCT for acute myeloid leukemia.
It was demonstrated that azacytidine increased post-transplant numbers of Tregs and
prevented GVHD while promoting a favourable CD8+ graft versus leukemia effect.435
Thus, attempts to induce long-term FOXP3 stability might also activate graft-specific
CD8+ T cells. The longevity of these infused Tregs is also a key concern. It has been
posited that continued treatment with IL-2 may allow for long-term survival of infused
Tregs but as IL-2 is also required for effector T cells and is used regularly for cancer
treatment, this approach may counteract the tolerogenic effects of the Treg transfer.431
A final consideration is that Treg transfer is a highly personalized therapy requiring ex
vivo manipulation of cells in a GMP grade facility. The cost of a single injection of Tregs
in a UK-based GMP facility was reported to be £20,000 (CAD 35,000) and multiple
injections may be required to successfully induce tolerance thereby significantly
increasing the cost per patient for this treatment.431 As a whole, these considerations
raise concerns about the practicality of Treg infusions as a treatment and may be
ultimately non-viable for the larger population of organ transplant recipients.
In the quest to achieve immunosuppression-free graft survival in organ
transplantation, several groups have also focused on the discovery of biomarkers that
can identify patients that are “operationally tolerant” and can be safely weaned off
immunosuppression.20,306,436 As discussed previously, operational tolerance is defined
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as a state of rejection-free graft survival in the absence of immunosuppression for at
least one year.21 The identification of reliable biomarkers would improve the field of
organ transplantation in several ways.437 First, it would significantly ameliorate long-term
health and quality of life for patients who are successfully weaned off
immunosuppression. Second, it would provide a technique to gauge the establishment
of tolerance and define end-points for the various clinical trials attempting to induce
clinical transplant tolerance. Finally, identification of genomic or proteomic biomarkers
for tolerance would provide mechanistic insight into tolerance induction in humans,
which is currently not well understood.437 Currently, reliable biomarkers for operational
tolerance have only been identified in cohorts of liver and renal transplant patients.20 It
has been observed that due to the immunoprivileged status of the liver, nearly 20% of
liver transplant recipients are operationally tolerant to their graft.437 The number of
operationally tolerant kidney recipients is far less with only 100 cases of reliable
operational tolerance described since the 1970s.438 A further complication is that the
biomarkers identified are not unique to all organ types. There are also differences
between intra-graft and peripheral expression profiles in operationally tolerant
patients.437 In operationally tolerant liver transplant patients, studies have identified
transcripts associated with NK and γδ cells as potential biomarkers in peripheral
blood.439 Conversely, in kidney transplant recipients, operational tolerance appears to
be related to B cell associated gene expression signatures.305,306 Thus, there is no
universal biomarker for tolerance that can reliably predict or assess operational
tolerance in organ transplantation. While the search for biomarkers of tolerance is an
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important endeavour within the field of transplantation immunology, there is an unmet
need for therapies that can actively induce tolerance in the clinic.
Autologous HSCT involves the re-setting of the immune system by reconstituting
a myeloablated host with previously isolated autologous bone marrow or peripheral
blood stem cells.440 Recently, patients treated with autologous HSCT for AID have been
shown to halt progression of disease and ameliorate quality of life. Based on these
reports, we have established a murine model of autologous HSCT following SOT. In this
thesis we have established HSCT as a potential mechanism of re-educating the
immune response in the setting of allotransplantation. A comparison of allogeneic
versus autologous HSCT is provided in Table 1-3.
For the first aim of this thesis, we established a murine model of autologous
HSCT that was capable of long-term multilineage hematopoietic reconstitution.
Currently, the majority of HSCT in mouse models is performed with total or T cell-
depleted bone marrow.441 However, the clinical relevance of using bone marrow can be
called into question given that currently, autologous HSCT in humans predominantly
utilizes mobilized and magnetically sorted CD34+ PBSCs.380 Another concern is that
bone marrow functions as a reservoir for memory T and B cells, as well as, long-lived
plasma cells.442–444 Thus, using highly purified LSK cells can prevent carryover of
mature, potentially alloreactive memory cells. Due to the indispensable use of
rapamycin in our murine allograft model, we also sought to test whether rapamycin
would interfere with LSK cell engraftment and long-term hematopoietic reconstitution.
Our studies demonstrated that performing LSK cell transfers under rapamycin treatment
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does not impair long-term hematopoietic reconstitution. Furthermore, we utilized a
peripheral blood CBC analyzer to demonstrate full reconstitution of WBC, platelets,
PMNs and RBCs (hematocrit). Thus, we propose that LSK cells are an ideal population
of readily purified murine HSCs for future studies of autologous HSCT in mice.
We also demonstrated as part of aim 2 of this thesis, that autologous HSCT
significantly prolonged cardiac allograft survival and in some cases produced long term
survival in recipients of fully MHC-mismatched heart allografts. As expected, at the
histological peak of rejection, grafts from both the TxRej and TxRapa groups
demonstrated classical histological signs of acute graft rejection consisting of dense
mononuclear infiltrates, myocyte necrosis and vasculitis. In contrast, grafts from the
TxHSCT group did not show strong evidence of cell mediated rejection but ultimately,
most grafts were most. We also performed immunostaining for CD3 on grafts harvested
from the treatment groups at earlier time points. Our immunostaining revealed a far
higher intensity of immunoperoxidase CD3 staining in the TxRej and TxRapa groups
versus the TxHSCT and the TxSyn groups. Morphometric analyses revealed that grafts
from TxHSCT mice had significantly reduced numbers of CD3+ infiltrating cells. In fact,
these numbers were not significantly higher than those found in syngeneic grafts which
display inflammation associated immune infiltration but do not undergo cell-mediated
rejection. Allografts treated with HSCT also demonstrated a higher absolute number of
FOXP3+ infiltrating cells and a significantly higher ratio of FOXP3+ to CD3+ cells, versus
rapamycin-only (TxRapa) or syngeneic grafts. These results are in accordance with
previous studies from our lab, performed in murine models of cardiac
allotransplantation.206,445 In both studies, long-term allograft survival was associated
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with an increased ratio of FOXP3+ (Tregs) to CD3+ (T cells). Using a rapamycin-induced
model of cardiac tolerance with BALB/C (H2d) donors and C3H/HeJ (H2k) recipients, we
showed that tolerance was dependent on graft-infiltrating Treg cells.206 Furthermore,
depletion of Tregs using anti-CD25 mAbs resulted in abrogation of tolerance. These
observations have also been reported in other studies. Lee et al. in 2005, demonstrated
an indispensable role for FOXP3+ cells in a murine model of cardiac allograft tolerance
using CD154 co-stimulation blockade and donor-specific transfusion (DST).446
Thymectomy or anti-CD25 mAbs prevented establishment of tolerance. Moreover, this
study showed that trafficking of Tregs to the graft was dependent on the expression of
the chemokine receptor CCR4 as CCR4 KO mice were also incapable of developing
tolerance using CD154 and DST.446 Studies in clinical transplantation have also showed
an important role for FOXP3 infiltration as a marker for tolerance. Bestard et al. have
shown that the FOXP3 to CD3 ratio is an accurate marker for predicting rejection in
kidney transplant patients with rejection occurring in patient who have low FOXP3/CD3
ratios.447 In the cohort of patients developing tolerance post allo-HSCT at MGH, Kawai
et al. reported an increased intragraft gene expression of FOXP3.392 As previously
described, Levitsky et al. reported markedly improved immunoregulatory profiles in liver
transplant patients converted from tacrolimus (CNI) to sirolimus (rapamycin).211 In
particular, this study noted that increased numbers of Tregs in the peripheral blood were
strongly correlated with increased FOXP3 to CD3 ratios in the graft of sirolimus-
converted patients.211 These observations are also in accordance with the data
generated in this thesis demonstrating that increased splenic Treg proportions in the
TxHSCT group are correlated with increased intra-graft FOXP3+ to CD3+ ratios. It is
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also important to note that FOXP3+ cells may infiltrate the graft prior to rejection. In a
recent study, Boer et al. assessed infiltration of natural (thymic) Tregs in
endomyocardial biopsies of heart transplant recipients who had suffered from acute
rejection or were rejection-free.448 Whereas, natural Tregs were increased in patients
undergoing acute rejection, total Treg numbers were higher in non-rejecting patient
biopsies, or biopsies taken from rejecting patients prior to onset of acute rejection.448
These results implicated an important role for Tregs in preventing rejection. In our
study, the preferential recruitment of FOXP3+ cells to the HSCT-treated grafts versus
rapamycin-alone treated grafts, suggests that HSCT treatment expands Tregs with far
greater efficacy than rapamycin alone. Our immunostaining data for graft-infiltrating
B220+ (B cells) showed that B cells were also significantly lower (>10 fold) in TxHSCT
grafts versus TxRapa grafts. Flow cytometric analyses of the splenic compartment in
the TxRej, TxRapa, TxHSCT and TxSyn groups revealed no significant differences in
frequencies of B cells (data not shown). These observations indicate either the
depletion of alloreactive B cell clones or a B cell targeted suppressive mechanism.
Thus, further studies are required to uncover the exact mechanism of HSCT on B cells
re-education in our model. Recent studies have also uncovered the importance of the
antigen presenting role of graft-infiltrating B cells. In 2014, Zeng et al. showed that
chronic rejection of murine cardiac allografts was predicated on the antigen-presenting,
as opposed to the antibody producing roles of B cells.136 These studies require further
validation in human patients to examine if B cells play analogous roles in chronic
rejection of human cardiac transplants. Nevertheless, these observations warrant
further study as autologous HSCT potentially offers a mechanism for reducing the risk
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for chronic allograft rejection, the most common cause of late-term graft loss in human
heart transplant patients.449
A potential cause of graft loss in the HSCT-treated group may be attributed to the
effects of TBI on cardiac tissue. Radiation-induced heart disease (RIHD) in patients
undergoing radiotherapy is now a well described clinical occurrence.450,451 RIHD
includes a wide spectrum of cardiac pathologies including cardiomyopathy, myocardial
fibrosis, pericarditis, valvular disease, coronary artery disease and arrhythmias.451
Cardiovascular disease is a leading cause of death in Hodgkin’s lymphoma patients
who often receive radiotherapy.452 Similarly, meta-analysis studies have shown a 62%
increase in cardiac deaths in breast cancer patients undergoing clinical trials involving
radiotherapy.453 The mechanisms of radiation-induced cardiac damage are not fully
understood but are known to involve endothelial damage and recruitment of
inflammatory cells all of which culminate in tissue fibrosis.451 In the acute phase post
exposure to ionizing radiation, damaged endothelial cells express adhesion molecules
and recruit inflammatory cells that express TNF, IL-1, IL-6 and IL-8.451 Classical pro-
fibrotic cytokines such as platelet-derived growth factor (PDGF), basic fibroblast growth
factor (bFGF) and TGF-β, are also released in the acute phase. This sets the stage for
a chronic fibrosis phase resulting in collagen deposition that affects the function of
myocytes, vascular endothelium and the pericardium.451 It has also been proposed that
upregulated NF-κB and chronic oxidative stress can mediate vascular damage post
radiation exposure.450 These widespread effects of radiation-induced cardiac damage
may explain the graft loss in the TxHSCT group even in the absence of high numbers of
mononuclear cell infiltration and vasculitis. This is also supported by Masson’s trichrome
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staining of TxHSCT grafts harvested at POD 45, which display extensive staining for
collagen deposition (data not shown). Therefore, we propose that future studies using
our model could adopt a lower dose of TBI or switch to chemotherapy-based
myeloablative regimens.
The immunological studies in our model provide evidence for a “re-education” of
the immune system in the setting of allotransplantation. Data generated here strongly
suggests the role for active immune regulation mechanisms as opposed to clonal
deletion. This assertion is supported by the observation of significantly higher
proportions of splenic and intra-graft Tregs in the TxHSCT group compared to all other
groups on POD 45. This observation is in accordance with studies in human patients
undergoing autologous HSCT for AID.408 Several mechanisms may be involved in the
elevated proportions of splenic Treg in TxHSCT mice. First, rapamycin treatment in the
presence of a newly emerging T cell repertoire could significantly skew the T cell
populations towards a high ratio of Tregs to effector T cells. Battaglia et al. showed that
rapamycin can cause preferential expansion and increase of Treg proportions in vitro.199
In 2007, Noris et al. also observed Treg expansion in patients who received sirolimus
after cytoreductive therapy.454 Thus, rapamycin treatment should have also resulted in
preferential expansion of Tregs in the TxRapa group but splenic Treg proportions in
TxRapa were not significantly higher than Non-Tx control, TxRej and TxSyn mice on
POD 45. Thus, alternative mechanisms of Treg expansion may also be contributing to
the high proportions of splenic Treg observed in the TxHSCT group. Delemarre et al.
recently showed that autologous HSCT in human patients, results in a renewal and
diversification of the Treg TCR repertoire.406 The authors also performed murine
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congenic (CD90.1/90.2) bone marrow transplants using 7.5Gy of TBI mice to analyze
Treg repopulation. It was observed that Treg populations from donor and host are
maintained in host mice at 3 weeks post-BMT while at 7 weeks, the entire Treg
compartment is donor derived.406 These thymic derived congenic donor Treg also
displayed proliferative capacity and an increased IL-10 mRNA expression compared to
host Treg. This increased suppressive function was purported to be a result of the
increased Treg TCR diversity that was observed in human patients post-HSCT.406 As
discussed above, HSCT-treatment in our model may also have significantly expanded
Tregs that persisted both intra-graft and in the periphery at higher levels compared to all
other treatment groups. Further work is warranted in the setting of both murine and
human autologous HSCT in organ transplantation to examine the phenotype and
functionality of pre- and post-HSCT Tregs.
Another line of evidence for a peripheral tolerogenic mechanism in our model, is
the data from in vitro MLR assays. Mice in the TxHSCT group maintained a primary
immune response to BALB/C stimulators indicating a peripheral suppressive versus a
central (deletional) mechanism of tolerance to donor antigen in this setting. An elevated
proportion of Tregs in these may be the major contributors towards suppressing donor
specific T cell responses in vitro and in vivo. As demonstrated by Muraro et al., HSCT in
human patients results in a nearly complete renewal of the CD4+ but not the CD8+ T cell
repertoire. Based on our results so far, we cannot rule out the contribution of CD8+ T
cells as well as innate immune cells such as NK cells, towards graft loss post-HSCT in
our model. Of note, we also observed that while all rapamycin only (TxRapa) treated
mice rejected by POD 32 and did not have increased Treg levels on POD 45, these
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mice displayed a primary MLR response in vitro. These results can be explained by a
number of reasons. First, rapamycin is known to significantly reduce DC maturation and
function.455 In 2007, Turnquist et al. demonstrated that ex-vivo rapamycin-conditioned
murine DCs were potent suppressors of allogeneic MLRs and could promote indefinite
cardiac allograft survival.456 Thus, rapamycin conditioning in these mice may have led to
anergy induction and deletion of potentially alloreactive CD4+ T cell clones as a result of
binding to immature DCs. Furthermore, rapamycin has been observed to play a
contradictory role in promoting both CD8+ memory as well as CD4+ Tregs due to similar
metabolic demands.457 Araki et al. showed that rapamycin treatment significantly
improved LCMV-specific CD8+ memory cells signaling a potential immune activating
role for rapamycin.197 However, in 2010, Ferrer et al. used a transgenic mouse model to
show rapamycin could improve CD8+ cell responses towards antigen when presented in
the context of a bacterial pathogen but not towards antigen presented in the context of
an allograft.458 Furthermore, there is also some evidence for the role of mTOR inhibition
in promoting apoptosis of memory CD4+ T cells.459 These data show that rapamycin can
significantly diminish alloreactive memory T cell responses thereby maintaining a
primary immune response to alloantigen in vitro. Therefore, the rejection of allografts in
TxRapa mice may be attributed to the role of B cells, NK cells and other innate cell
types. In fact, it was shown that rapamycin-only treated mice had high levels of DSA.
Our study also showed a significant re-education of the B cell repertoire post-
HSCT. As discussed above, there was a marked reduction in graft-infiltrating B cells in
TxHSCT mice. Our data also showed an important role for re-education of B cells in the
periphery. DSA levels in TxHSCT mice were 16-fold and 8-fold lower than in untreated
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rejecting and rapamycin only treated mice respectively. Studies performed in human
AID patients treated with autologous HSCT have demonstrated a potent re-education of
the B cell repertoire.411,460 In 2009, Alexander et al. reported the effects of autologous
HSCT in a small cohort of 7 SLE patients.460 HSCT in these patients re-set the B cell
repertoire towards a predominantly naive B cell repertoire and significant lowered (9-
fold) the frequencies of CD19+CD27+IgD- memory B cells in peripheral blood.
Furthermore, autoantibodies were also significantly abolished in these patients. In 6 out
of 7 patients, anti-dsDNA antibodies dropped to below limit of detection, while in 4
patients, antinuclear antibodies (ANA) fell to below clinically significant values.460 The
mechanisms underlying this abrogation of autoantibody production are not well
understood. However, it is known that long-lived plasma cells (LLPCs) in the bone
marrow a major source of antibody production.461 Thus, autologous HSCT may alter this
repertoire through newly emerging B cells outcompeting these LLPCs or directly
inhibiting their survival through an unknown suppressor mechanism. Either of these
mechanisms may be underlying the significant reduction of DSA observed post-HSCT in
our mouse model and warrant further study.
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Conclusions
In conclusion, this thesis has established HSCT as a potential means to re-
educate the immune response in the setting of allotransplantation The re-education
affected both T and B cell responses. These results are consistent with the data
generated by Atkins et al showing that autologous HSCT can abrogate MS progression
in human patients.404 Thus, our data provide a rationale for the clinical study to examine
the use of autologous HSCT in the setting of liver transplantation to induce tolerance
(ASCOTT- NCT02549586).
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Future Directions
The studies performed and the results generated by this dissertation provide
many avenues for further research. The two major approaches for future research in
this area are to further elucidate the mechanisms associated with autologous HSCT in
murine models and to perform research using clinical samples from the ASCOTT trial
for investigating re-education in the human SOT recipients. The autologous HSCT and
cardiac allograft model established in this thesis can be further studied to gain
mechanistic insights into the tolerogenic potential of autologous HSCT in SOT.
However, this approach can also be expanded to other murine allograft models such as
kidney and lung.226 Our studies demonstrated that Tregs were significantly increased in
the TxHSCT group implicating a role of Tregs in prolonging cardiac allograft survival in
this model. Thus, future experiments could administer anti-CD25 mAbs to HSCT-treated
mice and monitor for accelerated rejection as performed in a previous study by our
group.206 As discussed in the previous section, Delemarre et al. also demonstrated that
donor derived Tregs post-BMT from congenic donors, had high proliferative capacity
and increased IL-10 expression.406 Thus, future functional studies in our mouse model
should examine the mRNA levels of key suppressive cytokines (TGF-β, IL-10 and IL-34)
in Tregs from TxHSCT, TxRapa and TxRej groups. Another key observation was the
marked reduction of DSA in the mice treated with HSCT. Future studies can be
performed to uncover the mechanisms through which this reduction may occur. LLPCs,
as described earlier, are known reside in the bone marrow and function as long-term
producers of alloantibodies.88 Hence, multiparameter flow cytometric profiling of B cell
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subsets including LLPCs, should yield evidence for perturbations in the B cell repertoire
as observed in human patients undergoing autologous HSCT for AID.411 Multiplex
ELISA can also be carried out using available bead-arrays such as Legendplex™ from
Biolegend (San Diego, CA) for a wide range of pro- and anti-inflammatory cytokines
such as IL-10, TGF-β, IL-4, IFNγ and IL-1. We deem radiation-induced cardiac damage
to be a major cause of graft loss in our studies. Therefore, future studies could
incorporate our LSK-based HSCT model with chemotherapy-based myeloablative
regimens. In 2014, Gorczynski et al. showed that T cell-depleted BMT using busulphan
and cyclophosphamide as a conditioning regimen resulted in significantly prolonged
skin allograft survival and reduced in vitro responsiveness to donor antigen.462 Thus,
LSK studies can be performed in this setting to evade the potential damage associated
with TBI. Finally, future work can also focus on examining intra-graft mRNA expression.
In our previous work, we demonstrated that tolerant murine cardiac allografts
demonstrated a 6-gene profile associated with tolerance.206 Future experiments could
involve studying the expression of these 6 genes in HSCT-treated allografts in our
model.
In parallel to the murine work, the establishment of the ASCOTT trial
(NCT02549586), offers an exciting opportunity to study the mechanisms of HSCT-
mediated immune re-education in SOT patients. As demonstrated by the TCR
sequencing studies of Muraro et al. and Delemarre et al. an in-depth view of renewal
the Tconv/Treg repertoire can be generated using this approach.359,406 Similar studies in
SOT patients can be performed to examine alloreactive T cell/Treg repertoires. Due to
the large proportion of T cells that can respond to alloantigen as a result of heterologous
116
immunity (5-10%), it will be essential to examine the ability of autologous HSCT to reset
this alloreactive repertoire.56 For HSCT-treated patients who have a living-donor
transplant, future studies will have the advantage of opportunity to perform in vitro MLR
and CTL assays to assess donor-specific responsiveness. Finally, as the
immunobiology of graft rejection is far more complex in NHPs and humans compared to
mice, future studies will require detailed immunophenotyping studies in SOT patients
who undergo HSCT. These studies can follow a three-pronged approach. First, as the
studies in AID patients demonstrate, several cell types are perturbed post-HSCT.408
Thus immunophenotyping of PBMCs in these patients will require a high-throughput
approach to examine several cell subsets. Currently, flow cytometry is limited at 16-17
markers due to issues of spectral overlap. However, with the advent of mass cytometry
(CyTOF), currently around 45-50 cell markers can be examined in a single sample.463
Thus future studies in ASCOTT patients can utilize CyTOF as a high-throughput
phenotyping platform in conjunction with multiplex ELISA for proteomic and multiplex
qPCR for genomic assessments in these patients. This three-pronged approach has the
potential to not only characterize the tolerant patients in this trial but also discover
potentially novel mechanisms of tolerance induction in SOT.
117
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Copyright Acknowledgements
This thesis contains one figure (Figure 1-2) that was reproduced under the Creative
Commons License from an open-access publication for which I am a co-author. A
second figure (Figure 1-3) from Doulatov et al.371 was reproduced with permission from
Elsevier and the license is provided on the next page.
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