THE ROLE OF HUMAN LEUKOCYTE ANTIGEN-G IN

HEART TRANSPLANTATION

by

Rohit Sheshgiri, BSc (Hons)

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 Rohit Sheshgiri (2008)

ii

ABSTRACT

The Role of Human Leukocyte Antigen-G in Heart Transplantation MSc Thesis, 2008 Rohit Sheshgiri, BSc (Hons) The Institute of Medical Science, University of Toronto

Human leukocyte antigen-G (HLA-G), a protein expressed primarily by fetal trophoblasts,

plays an essential role in maintaining fetal immune tolerance and has previously been

detected following heart transplantation. We sought to establish the value of HLA-G in

identifying freedom from moderate or severe rejection post-heart transplant, and the

capability of its expression in vitro. After assessing myocardial HLA-G expression through

immunohistochemistry, we demonstrated that it was significantly more prevalent in non-

rejecting than rejecting heart transplant recipients. Utilizing vascular endothelial and smooth

muscle cell culture models, we also determined that while HLA-G expression remains tightly

regulated, its expression in vitro can be induced following progesterone treatment in a dose-

dependent manner. Hence, HLA-G may reliably identify patients with a low immunological

risk of developing subsequent clinically significant rejection post-heart transplant.

Furthermore, HLA-G expression can be induced in cultured endothelial and smooth muscle

cells, which might represent a strategy to protect against allograft rejection and vasculopathy.

iii

ACKNOWLEDGEMENT

I will forever remain grateful to my supervisors, colleagues, collaborators and friends who

have been instrumental to the completion of this thesis. Without their help, this body of work

would not have been possible.

I am especially appreciative of my supervisors, Dr. Vivek Rao and Dr. Diego

Delgado, for their mentorship, encouragement and guidance throughout my training program.

Both have been responsible for designing and overseeing this thesis, and have provided me

with the freedom to expand it into a variety of other studies. Most importantly, I was given

the opportunity to explore my own academic interests while undertaking this project. For

these reasons and more, I will never forget the impact they have had on my academic career.

I would like to express my gratitude to Dr. Heather Ross and Dr. Jagdish Butany for

providing me with invaluable advice and expertise during my time with the Heart Transplant

Program. I am also indebted to Laura Tumiati, Dr. Edgardo Carosella, Dr. Nathalie Rouas-

Freiss, Dr. Clifford Librach and Rong Xiao for offering much insight and technical help

along the way.

I am thankful to all my colleagues, especially Danny, Jessica, Mitesh and Elissa for

making my experience a pleasant and memorable one, and to my family for their constant

support and encouragement through the good times and bad. Finally, I thank the Heart and

Stroke/Richard Lewar Centre of Excellence in Cardiovascular Research, Astellas Pharma

Canada and the Institute of Medical Science for supporting our investigations.

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TABLE OF CONTENTS

Title Page …i

Abstract …ii

Acknowledgement …iii

Table of Contents …iv

List of Abbreviations …vii

List of Figures and Tables …ix

Chapter 1 KNOWLEDGE TO DATE …1

1.1 Introduction …2

1.2 Heart Transplantation and Immunology …21.2.1 Heart Transplantation …21.2.2 Innate and Adaptive Immunity …5 1.2.3 The Major Histocompatibility Complex …6

1.2.4. Allorecognition Pathways …8

1.3 Human Leukocyte Antigen-G …10 1.3.1 Structure …10 1.3.2 Receptors …11 1.3.3 Inhibition of Natural Killer Cell Function …121.3.4 Modulation of T Cell Function …141.3.5 Inhibition of Antigen-Presenting Cell Function …161.3.6 Pregnancy …181.3.7 Solid Organ Transplantation …191.3.8 Cancer …251.3.9 Inflammation …29

Chapter 2 PROPOSED INVESTIGATIONS …32

2.1 Rationale …33

2.2 Hypotheses …34

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Chapter 3 MYOCARDIAL HLA-G RELIABLY INDICATES A LOW RISK OF ACUTE CELLULAR REJECTION FOLLOWING HEART TRANSPLANTATION …35

3.1 Methods …363.1.1 Patients …363.1.2 Biopsy Scoring …363.1.3 Tissue Samples …383.1.4 Immunohistochemistry …383.1.5 Statistical Analysis …40

3.2 Results …413.2.1 Baseline Characteristics …413.2.2 HLA-G Expression …42

Chapter 4 PROGESTERONE INDUCES EXPRESSION OF HLA-G IN VASCULAR ENDOTHELIAL AND SMOOTH MUSCLE CELLS IN VITRO …44

4.1 Methods …454.1.1 Cell Cultures …454.1.2 Treatments and Interventions …464.1.3 Protein Extraction …474.1.4 Enzyme-Linked Immunosorbent Assays …484.1.5 Protein Determination …494.1.6 Flow Cytometry …504.1.7 Viability Assays …514.1.8 Statistical Analysis …52

4.2 Results …524.2.1 Induction Experiments …524.2.2 The Effect of Progesterone …534.2.3 Inhibition Experiments …534.2.4 Viability Studies …53

Chapter 5 FIGURES AND TABLES …55

Chapter 6 DISCUSSION …70

6.1 Myocardial HLA-G Reliably Indicates a Low Risk of Acute Cellular Rejection Following Heart Transplantation …71

vi

6.2 Progesterone Induces Expression of HLA-G in Vascular Endothelial and Smooth Muscle Cells in vitro …75

6.3 Future Perspectives …80

Chapter 7 REFERENCES …85

vii

LIST OF ABBREVIATIONS

AEC 3-amino-9-ethylcarbazole ANOVA Analysis of variance APC Antigen-presenting cell β2M Beta-2-microglobulin BEC Biliary epithelial cell BNP B-type natriuretic peptide BSA Bovine serum albumin CAV Cardiac allograft vasculopathy CD Cluster of differentiation CRP C-reactive protein CSF Cerebrospinal fluid DMEM Dulbecco's modified Eagle medium DMSO Dimethyl sulfoxide E- Exon EBM-2 Endothelial cell basal medium-2 EDTA Ethylene diamine tetra-acetic acid EGM-2 Endothelial cell growth medium-2 EGM-2 MV Microvascular endothelial cell growth medium-2 ELISA Enzyme-linked immunosorbent assay FasL Fas ligand FBS Fetal bovine serum H/R Hypoxia followed by reperfusion HAEC Human aortic endothelial cell HCAEC Human coronary artery endothelial cell HCASMC Human coronary artery smooth muscle cell HCl Hydrochloric acid HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HLA- Human leukocyte antigen HLA-G Human leukocyte antigen-G HLA-G- Human leukocyte antigen-G-negative HLA-G+ Human leukocyte antigen-G-positive HRP Horseradish peroxidase I- Intron IFN-γ Interferon-γIL Infiltrating leukocyte IL-10 Interleukin-10 ILT- Immunoglobulin-like transcript ISHLT International Society for Heart and Lung Transplantation IVF In vitro fertilization KIR2DL4 Killer cell immunoglobulin-like receptor 2DL4 mAb Monoclonal antibody MC Myocardial cell MHC Major histocompatibility complex

viii

MHC-I Major histocompatibility complex class I MHC-I b Major histocompatibility complex class Ib MHC-II Major histocompatibility complex class II MICA Major histocompatibility complex class I-related chain A MLR Mixed lymphocyte reaction MS Multiple sclerosis N/A Not applicable NK Natural Killer PBMC Peripheral blood mononuclear cell PBS Phosphate buffered saline PBSG/BSA Phosphate buffered saline with glucose and bovine serum albumin PE Preeclampsia PIR-B Paired immunoglobulin-like inhibitory receptor-B PRA Panel reactive antibodies PRE Progesterone response element RCC Renal cell carcinoma RIPA Radio-immunoprecipitation assay sHLA-G Soluble human leukocyte antigen-G SMA Smooth muscle actin SmBM-2 Smooth muscle basal medium-2 SmGM-2 Smooth muscle growth medium-2 Treg Regulatory T VCAM-1 Vascular cell adhesion molecular-1 XTT Sodium 3’-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-

6-nitro)-benzene sulfonic acid hydrate

ix

LIST OF FIGURES AND TABLES

Figure 1. Protein structures of the HLA-G isoforms …56

Figure 2. Structures of HLA-G mRNA transcripts …57

Figure 3. Immunohistochemical staining of MHC-I proteins in myocardial biopsies of heart transplant recipients …58

Figure 4. HLA-G expression profile in non-rejecting and rejecting patients …59

Figure 5. Characterization of cultured endothelial and smooth muscle cells through flow cytometric analysis …60

Figure 6. Assessment of HLA-G expression in endothelial and smooth muscle cell cultures following progesterone treatment …61

Figure 7. Flow cytometric analysis of HLA-G expression in cultured endothelial and smooth muscle cells following progesterone treatment …62

Figure 8. Time course experiments assessing progesterone treatment on HLA-G expression …63

Figure 9. The effect of mifepristone, a progesterone receptor antagonist, on progesterone-induced HLA-G expression in cultured endothelial and smooth muscle cells …64

Table 1. Baseline characteristics of non-rejecting and rejecting heart transplant patients …65

Table 2. Expression of HLA-G in non-rejecting and rejecting patients …66

Table 3. Expression of HLA-G in cultured endothelial and smooth muscle cells following exposure to interventions of interest …67

Table 4. Viability and proliferation ability of treated endothelial and smooth muscle cell cultures …68

Table 5. Viability and proliferation ability of treated endothelial and smooth muscle cell cultures following H/R stress …69

1

CHAPTER 1:

KNOWLEDGE TO DATE

2

1.1 INTRODUCTION

In 1986, Ellis et al. described a novel major histocompatibility complex (MHC) molecule

isolated from cytotrophoblast cell membranes (1), which was subsequently cloned the

following year as reported by Geraghty et al. (2). This protein, termed human leukocyte

antigen-G (HLA-G), was mapped to the short arm of chromosome 6 (3) and was initially

believed to be restricted to cytotrophoblast cells of the fetus during early gestation, based on

the results of pioneering studies (4-6). However, in recent years HLA-G has been shown to

be expressed during a number of physiological conditions, both pathological and non-

pathological. Considered a non-classical MHC molecule because of its role in immune

suppression rather than immune activation, its functions are now known to be much more

complex than initially believed and have been extended beyond the scope of pregnancy into a

variety of other milieus including transplantation, cancer and inflammation. This thesis

examines the role of HLA-G in the context of heart transplantation.

1.2 HEART TRANSPLANTATION AND IMMUNOLOGY

Heart Transplantation

Heart failure, a condition characterized by impaired cardiac function leading to insufficient

blood supply to the tissues, is increasing in prevalence worldwide and results in high rates of

morbidity and mortality while commanding tremendous human and economic resources (7-

9). Presently, heart transplantation remains the definitive treatment of choice for patients

suffering from end-stage congestive heart failure failing maximal medical therapy (10). Since

the first heart transplant operation performed by Christiaan Barnard in 1967 (11), over

3

76,000 procedures reported to the registry of the International Society for Heart and Lung

Transplantation (ISHLT) and tens of thousands of unreported cases have been performed

worldwide (12).

The vast majority of heart transplant operations have been undertaken due to existing

coronary artery disease or cardiomyopathy (12). While transplantation provides a reliable

therapeutic option for these conditions, advances in the cardiovascular field have introduced

a new set of problems. Improvements in patient outcomes have continued to expand the pool

of eligible recipients, resulting in longer waiting times and pre-operative mortalities (13) due

to the limited availability of suitable donor organs (14). To help combat this problem,

suboptimal or marginal hearts may be used, albeit at the expense of long-term cardiac

function and patient survival (15,16). Improvements in early post-transplant graft and patient

survival have also introduced a new set of late-onset complications (17).

Survival following heart transplantation is limited by several factors. While graft

failure and infection are primarily responsible for early death, malignancy and cardiac

allograft vasculopathy (CAV) are the leading causes of late morbidity and mortality (12). The

effects of lifelong immunosuppressive therapy in conjunction with the aging patient

population are widely regarded to be responsible for the increased risk of neoplastic

transformation post-cardiac transplant (18-21). It is also known that patients with a prior

history of cancer have an increased risk of developing post-transplant malignancies (20). Yet,

it is still unclear which components of an immunosuppressive regimen increase the risk of

developing the solid and hematologic malignancies seen following cardiac transplantation.

Allograft vasculopathy, characterized by a progressive, diffuse and concentric

thickening of the arterial intima, is another major post-transplant complication that affects

4

long-term graft survival (22-24). CAV is a multifactorial process that is the consequence of

immunologic factors such as acute rejection episodes, HLA-mismatches and anti-HLA

antibodies, as well as non-immunologic risk factors including donor and recipient-related

characteristics, ischemia-reperfusion injury, viral infections, immunosuppressive protocols,

smoking, obesity, diabetes, dyslipidemia and hypertension (25). Ultimately, endothelial

injury is the key event which triggers the proliferative and fibrotic processes that drive the

pathophysiology of CAV (26). Therefore, current research aimed at preventing CAV is

mostly focused on inhibiting intimal proliferation and the known mechanisms that damage

the endothelium.

The risk of post-transplant death is at its greatest in the early post-operative period,

with a survival rate of approximately 85% in the first year post-heart transplant (12). While

the mortality rate is steepest within the first six months, patient survival thereafter essentially

decreases at a steady linear rate with a median survival time of close to 11 years or, for those

who survive the first post-operative year, 13 years (12). In adult heart transplant recipients,

although the median patient survival time has been improving in each successive era, the

major gains are mostly noticed within the first year, with little change in the long-term

mortality rate (12). Successful basic science and clinical research have helped improve early

survival, but have not solved the problem of late-stage morbidities.

Therefore, it is worthwhile to direct current and future research endeavours towards

combating the late-stage complications which arise post-heart transplant. One such area of

research is the generation of allograft tolerance, widely regarded as the ultimate endpoint in

organ transplantation. Full graft tolerance will ultimately abrogate the need for lifelong

immunosuppression decreasing the risk of developing malignancies, and prevent immune

5

attack against the allograft resulting in protection from CAV. Accordingly, the two major

long-term challenges post-transplant can be overcome by tolerance through inhibition of

immune responses aimed specifically at the allograft.

Innate and Adaptive Immunity

The immune system is a functional network of organs, glands, tissues, cells, proteins and

secreted factors which collectively protect the host from foreign pathogens. Comprising a

series of complex defence mechanisms and responses, it is a fundamental body system

crucial for the maintenance of proper physiological function. It is traditionally divided into

two major areas consisting of the innate and adaptive immune systems which work

concurrently to prevent and combat infection.

Innate immunity is a multi-faceted system which serves as the body’s first line of

defence. It includes anatomical barriers which help prevent pathogens from entering the host,

and circulating factors which play a role in initiating inflammatory responses and leukocyte

recruitment (27). Cells involved in innate immunity exert their function in a variety of ways.

Macrophages, neutrophils and dendritic cells are phagocytic, while natural killer (NK) cells

exhibit direct cytotoxic effects (27). Other cells such as basophils, eosinophils and mast cells

release soluble factors to combat pathogens (27). Cells of the innate immune response are not

antigen-specific and rely on germline-encoded receptors to recognize structures common to

many pathogens (28). Innate immunity does not result in immunologic memory and,

consequently, generates a rapid but non-specific response when activated (28).

The adaptive immune system, conversely, is highly specialized, antigen-specific and

becomes activated once the innate defences are breached. Lymphocytes play a major role in

the adaptive immune response and can detect antigens which overcome the innate immune

6

response. During lymphocyte development, each cell expresses receptors with a particular

antigenic specificity (29). Due to the millions of different variants of genes encoding the

receptor proteins, the entire lymphocyte receptor repertoire is able to recognize an almost

infinite variety of antigens (29). Only when a lymphocyte with particular specificity

encounters its antigen will it become activated and clonally expand into an effector cell

population (29). Following pathogen clearance, a small number of cells persist, resulting in

immunologic memory and, thus, ensuring a stronger and quicker response upon subsequent

encounter with the antigen (27).

The innate immune system plays a crucial role in host protection during the early

phase of infection, since there is a lag time between antigen exposure and maximal response

of the adaptive immune system (27). The innate immune system also initiates and directs the

subsequent adaptive response, and participates in the removal of pathogens targeted by this

response (30). Yet, while innate immunity is essential for the control of common infections,

pathogens can evade or overwhelm it. Consequently, the adaptive system has evolved in

conjunction with the innate system to more efficiently protect against pathogens and prevent

reinfection (30). Therefore both systems of defence are vital in combating infections and

maintaining normal physiological function.

The Major Histocompatibility Complex

Human leukocyte antigens (HLA) are crucial for the functioning of the immune system.

Classical HLA are membrane glycoproteins encoded by the MHC, a genomic region located

on chromosome 6 (31). These proteins serve to bind antigenic peptides derived from

pathogens and display them on the cell surface for recognition. The MHC is polygeneic,

containing numerous MHC class I (MHC-I) and MHC class II (MHC-II) genes, which

7

enables individuals to possess MHC molecules with a wide range of peptide-binding

specificities (27). The MHC is also highly polymorphic, with multiple variants of each gene

existing within the population (27). In humans, there are three classical MHC-I (HLA-A, -B

and -C) and MHC-II (HLA-DR, -DP and -DQ) structures, all of which are able to bind a

different range of peptides (31).

The receptors of circulating T cells are able to recognize particular antigenic peptides

presented at the cell surface in the presence of MHC proteins. Immature lymphocytes with

receptors specific for self antigens are deleted from the lymphocytic pool to prevent

autoimmunity (27). Classical MHC-I molecules bound to foreign peptides are recognized by

CD8+ T cells, while MHC-II/peptide complexes are bound by receptors of CD4+ T cells (32).

Naive T cells which are not self-reactive initially become activated by MHC/antigen

complexes presented by antigen-presenting cells (APC) such as dendritic cells and

macrophages (32). Once activated, naive T cells differentiate into effector cells. CD8+ T cells

eliminate infected cells through direct cytotoxicity (27). CD4+ T cells help activate CD8+ T

cells of the same antigen specificity, activate macrophages to destroy intracellular pathogens,

and activate B cells with the same receptor specificity to produce and secrete antibodies (27).

Consequently, effector T cells and activated B cells are fundamental to the adaptive immune

response.

Unlike T cells, NK cells play a major role in innate immunity and are inhibited by

MHC-I proteins (33). Although they are directly cytotoxic to their targets, they possess killer

cell immunoglobulin-like inhibitory receptors which recognize MHC-I proteins to ensure

tolerance to the host (33). Similarly, the adaptive immune system also requires some form of

regulation. Indeed, there exists a population of naturally occurring CD4+CD25+Foxp3+

8

regulatory T (Treg) cells which help control adaptive immune responses by inhibiting

proliferation of CD4+ and CD8+ T cells (34). Even though the innate and adaptive immune

systems have evolved to combat pathogens, they are ultimately subject to regulation in order

to prevent overactive responses.

In addition to the classical MHC-I and MHC-II genes, there exists another MHC

subset which exhibits limited polymorphism (35). These genes have been termed MHC Class

Ib (MHC-Ib) and encode non-classical MHC-I molecules which are, in most instances,

expressed at much lower levels on the cell surface (35). While the precise roles of these

MHC-Ib molecules have not been well defined, one such protein, HLA-G, has recently been

studied in the context of maintaining immune tolerance in a variety of physiological

situations, including organ transplantation, where allograft rejection directly and indirectly

affects long-term graft function and patient survival.

Allorecognition Pathways

Foreign proteins, such as those within an allograft, can induce strong immune responses

leading to graft rejection in a transplant recipient. In fact, without the aid of lifelong

immunosuppressive therapy, transplanted organs are unable to survive as a result of immune-

mediated attack. Due to their polymorphic nature and high expression levels, donor MHC

proteins have been heavily implicated in alloresponses. Three pathways of allorecognition

leading to graft rejection have been described so far. The direct pathway refers to host CD4+

and CD8+ T cells recognizing intact donor MHC-I and -II proteins expressed by foreign cells

of the allograft, following activation by donor “passenger” APC brought in with the graft

(36). Early studies have demonstrated that donor APC indeed play an important role in the

development of rejection (37).

9

However, the direct pathway appears to become less important in mediating long-

term alloresponses following transplantation, possibly because passenger APC are depleted

(38). Clinical studies have demonstrated that direct alloresponses diminish over time equally

in heart transplant patients with stable function and those with chronic rejection, suggesting

that alternate mechanisms play a role in maintaining rejection (39). Therefore, much research

has focused on the indirect pathway, which is believed to contribute to the late, more chronic

form of allograft rejection because of its persistent nature (40). Indirect allorecognition

involves presentation by recipient APC of donor-derived MHC peptides in the presence of

recipient MHC-II molecules to CD4+ T cells (40). Host APC constantly circulating through

the graft acquire, process and present donor MHC peptides to CD4+ T cells, which in turn

activate other arms of the adaptive immune response. In fact, the ability of the indirect

pathway to mediate rejection has been demonstrated by numerous animal studies (41,42).

A third, semi-direct pathway linking the direct and indirect allorecognition pathways

has also been recently proposed. This pathway holds that dendritic cells can acquire intact

MHC molecules from other cells and present them directly to T cells (43). Therefore,

following transplantation, recipient dendritic cells acquire intact donor MHC proteins from

allograft cells and directly initiate anti-donor immune responses (43). The semi-direct and

indirect pathways might be responsible for persistent, late-stage rejection because, unlike the

direct pathway, they can remain constantly active without the requirement for donor APC

(43). While the precise contribution of each pathway to allograft rejection is uncertain, it is

widely accepted that immune-mediated rejection ultimately leads to the development of long-

term complications following solid organ transplantation. Hence, strategies aimed at

preventing and detecting rejection are of considerable importance.

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1.3 HUMAN LEUKOCYTE ANTIGEN-G

Structure

While the genetic and protein structures of HLA-G are similar to those of other MHC-I

antigens (27,44), seven different HLA-G isoforms have been described to date, including

four membrane-bound (HLA-G1, -G2, -G3 and -G4) and three soluble (HLA-G5, -G6 and -

G7) proteins (Figure 1), rendering HLA-G unique among the MHC-I molecules (45,46).

Alternate splicing of the primary HLA-G mRNA transcript (Figure 2) is an important

property, because it can result in expression of truncated and soluble isoforms which may be

differentially expressed in different cell types or physiological situations (47).

The major cell-surface isoform, HLA-G1 and its circulating equivalent HLA-G5 have

been studied the most extensively because they possess the complete HLA-G structure. As

revealed by crystallographic studies, the structure of HLA-G1 is typical of MHC-I proteins,

consisting of cytoplasmic and transmembrane segments, and a heavy chain made up of three

globular domains, namely α1, α2 and α3, non-covalently bound to a beta-2-microglobulin

(β2M) structure (44,48). The cytoplasmic tails of HLA-G1 and the other membrane-bound

isoforms are shortened due to a stop codon in exon 6, which is a feature unique to HLA-G

compared to the classical MHC-I proteins (2). The α1 and α2 domains of the heavy chain

form an antigen-binding cleft, which binds a nonameric peptide, while the α3 domain is

involved in coreceptor binding (44,49). Classical MHC-I proteins show considerable

polymorphism, particularly around the antigen binding site, rendering them ideal for

presentation of antigenic peptides to the cell surface (44,49). Conversely, HLA-G and other

non-classical MHC-I antigens show limited polymorphism (35,50). The repertoire of

11

peptides to which HLA-G binds is limited (51), indicating that HLA-G is not well suited for

antigen presentation.

The other HLA-G isoforms are truncated versions of the complete protein. These are

smaller, less complex and do not associate with β2M or peptides for antigen presentation

(47). The structures of HLA-G2, -G3 and -G4 are similar to HLA-G1, except they lack the

α2, α2 and α3, or α3 domains, respectively (52,53). HLA-G5, the soluble counterpart to

HLA-G1, possesses the heavy chain associated with β2M, but lacks the cytoplasmic and

transmembrane segments (47). Likewise, HLA-G6 and -G7 are the soluble counterparts to

membrane bound -G2 and -G3, respectively (47). HLA-G6 lacks the α2 domain, while the α2

and α3 domains are missing in -G7 proteins (47). The open reading frame of soluble HLA-

G5 and G6 mRNA continues into intron 4 before terminating, resulting in a 21 amino acid-

long tail after the α3 domain (54,55). On the other hand, the HLA-G7 isoform consists of the

α1 domain linked to only two amino acids because the open reading frame of the -G7 mRNA

terminates shortly into intron 2 (47), resulting in a severely truncated protein.

Receptors

Unlike classical MHC-I proteins, which are primarily responsible for antigen presentation,

HLA-G does not appear to play a role in activating the immune response. HLA-G binds to

several different inhibitory receptors expressed by T cells, B cells, NK cells and APC and,

contrary to traditional MHC proteins, exerts immunosuppressive effects. Three different

inhibitory HLA-G receptors have been identified thus far: the immunoglobulin-like transcript

2 (ILT2) receptor, which has been detected in monocytes, macrophages, dendritic cells, B

cells as well as subsets of T cells and NK cells (56-58); the immunoglobulin-like transcript 4

(ILT4) receptor, expressed by APC, namely monocytes, macrophages and dendritic cells (58-

12

60); and the killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), which is mainly

expressed by NK cells (61-63). According to the revised nomenclature, these three receptors

have been recently renamed CD85j, CD85d and CD158d, respectively (64). In addition,

HLA-G has been shown to ligate the CD8 coreceptor expressed by certain T cells and NK

cells (65,66).

The ITL2 and -4 receptors, have broad specificities and bind classical MHC-I

molecules in addition to HLA-G, suggesting that they are important regulators of the immune

response (56,59). However, both receptors bind to HLA-G with a higher affinity than that

with which they bind classical MHC-I proteins, indicating that ILT/HLA-G interactions play

a major role in controlling NK cell, T cell and APC activity (67). Both receptors also have

higher affinity for HLA-G dimers, which are linked by an intermolecular disulfide bond (68).

An important difference between the ILT2 and -4 receptors is that HLA-G must associate

with β2M to bind to the former (69). Nonetheless, both possess inhibitory properties and

modulate the immune response accordingly. The KIR2DL4 receptor, unlike ILT2 and -4,

binds exclusively to HLA-G and not to classical MHC-I molecules (61). It has been shown to

possess both inhibitory (61,62) and stimulatory (62,63) properties. As a result of the

somewhat controversial nature of KIR2DL4, the immunosuppressive effects of HLA-G have

mostly been described through mechanisms involving the ILT2 and -4 receptors.

Inhibition of Natural Killer Cell Function

The immunosuppressive role of HLA-G is most evident by its ability to inhibit NK cell

function, as revealed by numerous investigations. Rouas-Freiss et al. have demonstrated that

HLA-G1 expression by ex vivo cultured cytotrophoblasts inhibits maternal uterine NK cell-

mediated lysis (70). This inhibition was noticed in semi-allogeneic combinations of maternal

13

uterine NK cells and their own trophoblast cells, in addition to allogeneic combinations with

trophoblast cells from different mothers, providing direct evidence that HLA-G1 expression

at the maternal-fetal interface inhibits NK cell function. Moreover, cultured HLA-G-negative

(HLA-G-) cells transfected with HLA-G1 or HLA-G2 are able to evade cell lysis mediated by

peripheral blood NK cells, illustrating inhibition of NK cells from various sources (70,71). In

fact, Riteau et al. have shown that HLA-G- cell lines transfected with HLA-G1 are able to

inhibit NK cells obtained from multiple donors, highlighting its ability to inhibit NK cells of

different phenotypes and functionalities (72).

Recently, it has been demonstrated by Caumartin et al. that NK cells in vitro, after

coming into contact with HLA-G-positive (HLA-G+) cells, can acquire HLA-G through

intercellular membrane exchange (73). Interestingly, acquisition of membrane-bound HLA-G

blocks NK cell proliferation and inhibits their cytolytic function toward target cells.

Furthermore, these HLA-G+ NK cells are capable of inhibiting the cytolytic functions of

other, proximal NK cells via HLA-G/ILT2 interactions. Acquired HLA-G expression is

temporary because NK cells do not constitutively express HLA-G and, due to membrane

dynamics, shedding and/or recycling, lose this expression (73). Nonetheless, taken together,

HLA-G+ cells can inhibit effector NK cells and exchange membrane patches to yield non-

proliferating HLA-G+ NK cells, which in turn inhibit other functional NK cells. This study

illustrates a potential mechanism of how a small number of HLA-G+ cells among a

population of HLA-G- cells can extend their immunosuppressive influence throughout the

local microenvironment.

In the xenogeneic milieu, membrane-bound HLA-G1 when transfected into porcine

endothelial cells, inhibits transendothelial migration of human NK cells (74,75). Contact

14

between HLA-G and ILT2 receptors on NK cells is required to inhibit this migration (75).

Such an interaction might be useful to limit intragraft migration of NK cells in the context of

xenotransplantation, where NK cells play a major role (76). This further demonstrates the

immunomodulatory capability and potential clinical applicability of HLA-G in the realm of

transplantation.

It is noteworthy to mention the strength of the inhibitory signal HLA-G delivers to

NK cells. Although other MHC-I proteins inhibit NK cell function (33), transfection of HLA-

G1 into cells expressing HLA-A, -B, -C and -E boosts this inhibitory response, illustrating its

role as the major NK inhibitory ligand (72). HLA-G1 has also been shown to inhibit NK cell

activity even when co-expressed with MHC class I-related chain A (MICA) antigens (77),

which give strong activating signals to NK cells (78). The fact that HLA-G overrides the NK-

activating signal mediated by MICA illustrates the strength of its inhibitory signal. Soluble

HLA-G (sHLA-G) has also been shown to inhibit NK cell cytotoxicity demonstrating that, in

addition to membrane-bound forms, sHLA-G proteins possess immunosuppressive functions

and may potentially be used therapeutically in the clinical setting (79). In fact, inhibition of

NK cell lysis by the recombinant sHLA-G heavy chain (80) and by sHLA-G5 and sHLA-G1

released by proteolytic shedding (81) has also been reported.

Modulation of T Cell Function

While NK cells are mainly involved with innate immunity, the immunosuppressive

properties of HLA-G can also be directed against adaptive immune responses. Impairment of

CD4+ and CD8+ T cell function has indeed been well documented. Direct evidence is

illustrated by the fact that HLA-G1, when transfected into target cells, blocks cytotoxic

responses of CD8+ T cells specific for antigens expressed by these target cells (82). Soluble

15

HLA-G has also been shown to induce apoptosis in CD8+ T cells by interacting with CD8,

leading to Fas ligand (FasL) upregulation, FasL secretion, Fas/FasL interaction and,

ultimately, apoptotic signaling (65,66,83). Furthermore, in vitro studies have demonstrated

that soluble HLA-G5 inhibits CD4+ and CD8+ T Cell proliferation following an allogeneic

response induced by T cell receptor activation, by binding to ILT2 receptors and arresting

cell cycle progression (84). The effects of HLA-G may also be enhanced by upregulation of

inhibitory receptors in CD4+ cells via interactions with these inhibitory receptors and

activation of a positive feedback loop (85).

The effects of HLA-G on T cell function have been extensively studied through

mixed lymphocyte reactions (MLR). Addition of sHLA-G proteins to the medium of MLR

significantly inhibits T cell alloproliferative responses through interactions with inhibitory

ILT4 receptors of APC and ILT2 receptors of T cells (86,87). Moreover, T cells primed with

HLA-G1-expressing APC show significantly reduced alloproliferation and display decreased

expression of the CD4 and CD8 coreceptors compared to T cells primed with HLA-G- APC

(88). These CD4low and CD8low T cells induced by HLA-G+ APC were shown to significantly

inhibit alloproliferation during MLR when used as third party cells compared to the CD4high

and CD8high T cells induced by HLA-G- APC, suggesting a regulatory role (88). Several

other investigations have also illustrated the ability of HLA-G to induce differentiation into

suppressive T cells that are different from traditional CD4+CD25+Foxp3+ Treg cells. HLA-G+

APC inhibit the proliferation of CD4+ T cells, cause long-term unresponsiveness and induce

them into differentiating into Treg cells, which in turn can suppress allogeneic CD4+ T cells in

MLR (89). Similarly, T cells pre-treated with HLA-G5 function as Treg cells by inhibiting the

alloresponse of responder T cells in MLR, when added as third party cells (87).

16

Recently, a small fraction of CD4+ and CD8+ cells from peripheral blood have been

found to stably express HLA-G, and show less proliferation to allogeneic stimuli compared

to their HLA-G- counterparts (90). These CD4+HLA-G+ and CD8+HLA-G+ cells represent

novel Treg cell subsets, distinct from the traditional CD4+CD25+Foxp3+ population, and are

independently able to suppress lymphocytic proliferation of both, CD4+HLA-G- and

CD8+HLA-G- populations (90). Resting and activated CD4+ T cells also have the ability to

rapidly acquire HLA-G1 through membrane exchange with HLA-G+ APC (91). Following

acquisition of membrane-bound HLA-G, these effectors become Treg cells which have the

ability to inhibit alloproliferative responses. While these cells are distinct from

CD4+CD25+Foxp3+ Treg cells, they temporarily function as such through the HLA-G1 they

acquire but do not constitutively express. Acquisition of HLA-G1 by CD4+ T cells might

explain how a few HLA-G+ cells can protect against immune aggression towards HLA-G-

target cells in the local milieu. There is, thus, considerable evidence demonstrating that in

addition to directly inhibiting effector CD4+ and CD8+ T cells, HLA-G can generate

populations of Treg cells to suppress these effectors.

Inhibition of Antigen-Presenting Cell Function

In addition to protecting against NK cells and T cells, HLA-G has been shown to inhibit APC

function, highlighting another mechanism by which it mediates evasion of the adaptive

immune response. Interactions between recombinant HLA-G complexes and the ILT4

receptor on human dendritic cells, in vitro, results in impaired dendritic cell maturation

characterized by reduced cell surface expression of MHC-II and co-stimulatory molecules

typically induced by the maturation stimulus (92). The HLA-G/ILT4 interaction has also

17

been shown to diminish the ability of dendritic cells to subsequently induce allogeneic T cell

proliferation (92).

Such findings have been applied and expanded upon in murine models. In studies

conducted by Horuzsko et al., dendritic cell maturation was found to be impaired in HLA-G

transgenic mice, compared to their non-transgenic counterparts (93). Moreover, skin

allografts from non-transgenic mice applied to these HLA-G transgenic mice survived longer

than controls and resulted in less potent T cell responses. It has also been reported that

allogeneic skin grafts among non-transgenic mice survive longer if recipients are previously

injected with recombinant HLA-G complexes (94). In such models, inhibition of dendritic

cell function occurs through HLA-G interactions with murine paired immunoglobulin-like

inhibitory receptor-B (PIR-B), a close homologue of the ILT4 receptor expressed by human

APC (94). Intriguingly, prior injection of dendritic cells whose PIR-B receptors were

stimulated with HLA-G results in longer skin allograft survival in mice (92). These mice also

contain markedly increased numbers of Treg cells (92), representing another possible

mechanism of how dendritic cells stimulated with HLA-G can prolong allograft survival.

Increased skin allograft survival has also been noted following injection of

recombinant HLA-G complexes in transgenic mice expressing human ILT4 receptors

exclusively on dendritic cells, via HLA-G/ILT4 binding (95,96). Recombinant HLA-G

complexes have also been shown to impair dendritic cell maturation, induce T cell anergy,

diminish CD4+ and CD8+ T cells responses and generate Treg cells in ILT4 transgenic mice

compared to their non-transgenic counterparts (95,96). Thus, transgenic animal models have

clearly demonstrated how HLA-G can impair APC maturation and, consequently, diminish

their ability to activate T cells.

18

Pregnancy

The role of HLA-G has been studied extensively in a variety of physiological situations

because of its ability to modulate the immune response. After being first identified in

cytotrophoblasts (1,4,6), HLA-G was initially believed to be solely expressed during

pregnancy. Cytotrophoblasts are cells derived from the trophoderm, which comprises the

outer layer of the blastocyst (97,98), and are thus semi-allogeneic to the mother. During

embryo implantation, cytotrophoblast cells invade the maternal uterine wall and come into

direct contact with the maternal blood to allow for uteroplacental circulation (98). The

paradox of pregnancy is based on this interface because, while the immune system has been

designed to attack non-self molecules that invade the host, the semi-allogeneic fetus

expresses non-self paternal antigens. Yet, it remains an immune privileged site, protected

from rejection.

Due to its immunosuppressive properties, HLA-G expression by cytotrophoblasts is

now widely accepted to maintain immune tolerance during pregnancy. Serum levels of

sHLA-G are significantly greater in pregnant than non-pregnant females, supporting this

notion (99). In fact, Fuzzi et al. provide strong indications that HLA-G expression is a

fundamental prerequisite for a successful pregnancy (100). In this investigation, 18/75

(24.0%) patients undergoing in vitro fertilization (IVF) with HLA-G+ embryo culture media

had clinical pregnancies compared to 0/26 (0%) patients with HLA-G- media, demonstrating

that sHLA-G expression in embryo culture media following IVF is required for a clinical

pregnancy. Moreover, in women with intact pregnancies after IVF, pre-ovulatory sHLA-G

levels were significantly greater than in women experiencing early abortions and in healthy

controls, without differences in total sHLA class I proteins, implying that maternal sHLA-G

19

proteins support early implantation (101). The same differences were seen for 9 gestational

weeks, indicating that HLA-G consistently plays an important role and may be used to

monitor the status of pregnancies.

Low HLA-G expression has also been associated with the development of

preeclampsia (PE). Yie et al. have shown that serum and placental levels of HLA-G are

significantly lower in PE patients than in healthy pregnant women (102). Reduced HLA-G

expression may contribute to the development of PE because the maternal immune system

becomes activated in patients with this condition (103). Taken together, there is strong

evidence illustrating that HLA-G plays an important role during gestation.

Solid Organ Transplantation

Because of its ability to suppress immune responses HLA-G has been implicated in a variety

of other situations extending beyond the scope of pregnancy. Evidence supporting the

protective role of HLA-G in solid organ transplantation was first presented by Lila et al. in a

pilot study encompassing 31 heart transplant patients (104). It was found that 5 (16.1%)

patients displayed HLA-G expression in myocardial biopsies and serum samples, and that

this expression was inversely correlated with acute and chronic allograft rejection. In a

follow-up single-centre study (105) encompassing a larger number of patients, post-

transplant myocardial biopsies from 9/51 (17.6%) contained detectable HLA-G proteins. In

addition, all these HLA-G+ patients displayed soluble HLA-G5 or -G6 protein expression in

sera. Interestingly, the mean acute rejection score in the first year post-operative year was

significantly lower in HLA-G+ (1.2±1.1) than HLA-G- (4.5±2.8) patients. Furthermore, none

of the HLA-G+ patients suffered from chronic rejection as assessed by annual coronary

angiography, compared to 15/42 (35.7%) HLA-G- patients, indicating that HLA-G is

20

associated with protection from early rejection episodes and graft vasculopathy. A one year

follow-up of the study patients revealed that HLA-G status remained unchanged. Patients

who were HLA-G+ did not lose this expression, while HLA-G- patients did not present HLA-

G+ biopsies during the follow-up visit, suggesting that HLA-G expression appears to remain

constant.

Several investigations have also examined the role of sHLA-G following heart

transplantation and have revealed that, while there is considerable intra- and inter-patient

variability, high serum HLA-G concentrations are generally associated with fewer rejection

episodes, suggesting inhibition of the immune response directed against the allograft (106-

108). Luque et al. have shown that patients with sHLA-G levels < 30 ng/mL one week post-

transplant appeared to have a higher incidence of recurrent severe rejection in the first year

versus those with higher concentrations (108). More recently, Lila et al. have shown that

serum HLA-G levels from 1 to 18 years post transplant are not only associated with fewer

acute rejection episodes, but also chronic rejection as measured by angiogram and stress

echocardiography (106). In this study encompassing 75 patients, all 41 with HLA-G ≤ 6

ng/mL experienced chronic rejection, while the 34 patients with sHLA-G > 6 ng/mL did not.

Furthermore, no acute rejection episodes were detected in patients with sHLA-G levels > 200

ng/mL. Among patients with sHLA-G concentrations ≤ 200 ng/mL, such episodes were more

prevalent. It has also been suggested that higher pre-transplant sHLA-G levels might be

inversely associated with recurrent acute rejection post-transplant (107). However, more

studies with larger sample sizes need to be conducted to make definitive conclusions.

While differential HLA-G expression is likely the result of several factors, including

genetics (109), immunosuppressive therapy has been suggested to modulate sHLA-G

21

concentrations post-heart transplant, since these levels have been shown to increase within 4

hours of drug administration in some patients (107,108). A recent study has illustrated two

patterns of serum HLA-G expression in heart transplant patients over a period of 12 hours

following administration of immunosuppressive therapy consisting of mycophenolate mofetil

or everolimus, methylprednisolone, and cyclosporine or tacrolimus during the first month

post-transplant (107). In this investigation, 7/12 patients (58.3%) displayed consistently low

levels following drug administration, while 5/12 (41.7%) patients presented a greater than

two-fold increase in sHLA-G levels within 4 hours followed by a return to basal levels within

10 hours post-dose, suggesting that immunosuppressive agents may influence HLA-G

expression.

Following the discovery of HLA-G expression in heart transplant patients, several

centres began assessing the role of HLA-G in other solid organ transplants. The protective

effect of HLA-G expression following kidney transplantation was recently reported by Qui et

al. who showed that sHLA-G is inversely related to both allograft failure from chronic

rejection and HLA antibody production (110). In this single-centre investigation, 13/26

(50.0%) patients with functioning kidneys compared to only 8/39 (20.5%) patients with

rejecting kidneys had detectable HLA-G levels in sera, suggesting a correlation between

sHLA-G and allograft function. Furthermore, 83/224 (35.5%) serum samples without HLA

IgG titres versus 19/96 (19.8%) samples with IgG titres were HLA-G+, indicating a negative

association between serum HLA-G and HLA IgG levels. Since antibody production is known

to be associated with acute and chronic allograft rejection following organ transplantation

(111), sHLA-G expression in these patients might inhibit the immune response and protect

the allograft from dysfunction. However, no significant association was noted between

22

sHLA-G and MICA antibodies, which are also associated with non-functioning kidney

allografts (112). It is important to note that some patients who were HLA-G+ lost this

expression, while some HLA-G- patients became HLA-G+, implying that sHLA-G status

might change following kidney transplantation.

Expression of HLA-G in kidney biopsy specimens post-transplant also appears to

confer protection against allograft rejection in renal transplant patients, as illustrated by a

study conducted by Crispim et al. (113). In this single-centre experience, 40/73 (54.8%)

study patients displayed HLA-G expression in tubule epithelial, glomerular or infiltrating

cells, while non-transplanted, healthy kidney tissue displayed no HLA-G expression. Among

the 23 patients with acute rejection or chronic allograft nephropathy, only 4 (17.4%) were

HLA-G+. Significantly more non-rejecting patients (36/50, 72.0%) presented biopsies with

HLA-G staining, indicating a strong relationship between HLA-G expression and freedom

from acute and chronic rejection. HLA-G expression also appeared to be associated with

tacrolimus therapy since 31/48 (64.6%) patients on tacrolimus were HLA-G+, compared to

9/25 (36.0%) patients not on tacrolimus. It should be noted that this study does not accurately

reflect the true proportion of HLA-G+ patients post-renal transplant because kidney biopsies

were not routinely performed in these patients. Biopsies used in this investigation were only

obtained from patients presenting clinical and/or laboratory evidence of acute or chronic

renal dysfunction. Therefore, the actual frequency of HLA-G expression in kidney transplant

recipients is not known. Furthermore, only one biopsy was collected from each patient with

no follow-up, so it is unknown if this expression is maintained over time in kidney transplant

patients.

23

Combined kidney-liver transplant patients have also been assessed for HLA-G

expression as described by Creput et al. (114). Out of 40 combined kidney-liver transplant

study patients, HLA-G was expressed by biliary epithelial cells (BEC) in 9 liver graft

biopsies and infiltrating mononuclear cells in 5 biopsies, while remaining undetected in

specimens of non-transplanted liver tissue from healthy individuals. HLA-G proteins were

also detected in a small number of renal biopsies from the same patient cohort. While the

sample size was too small to establish a correlation between renal HLA-G expression and

rejection, HLA-G expression in liver biopsies appeared to confer protection to the allograft.

No patient displaying BEC expression of HLA-G compared to 11/31 (35.5%) patients

without BEC HLA-G expression suffered from acute liver rejection. Since BEC are

susceptible targets for rejection (115,116), HLA-G may suppress the immune response

directed against the allograft. Interestingly, none of the BEC HLA-G+ patients suffered from

acute renal rejection, suggesting that HLA-G expression by the liver might confer protection

to the kidney allograft. Moreover, serum HLA-G levels following combined kidney-liver

transplantation have been reported to be significantly elevated, and may potentially be useful

in monitoring graft function post-transplant (117).

A subsequent study described by Le Rond et al. has demonstrated that high sHLA-G

levels from kidney-liver transplant recipients are also associated with freedom from allograft

rejection (87). Furthermore, follow-up measurements 2 years later reveal that these high

levels are maintained. Interestingly, addition of sera from HLA-G+ patients to the medium of

MLR between healthy donors significantly inhibited T cell alloproliferative responses, which

was not noticed following addition of HLA-G- sera to MLR. Furthermore, peripheral blood

mononuclear cells (PBMC) obtained from HLA-G+ patients were unresponsive to allogeneic

24

stimulation in vitro compared to healthy controls suggesting, that sHLA-G inhibits PBMC

alloproliferation in vivo. Moreover, these PBMC from the HLA-G+ kidney-liver transplant

recipients inhibited alloproliferative responses of MLR compared to healthy control donor

PBMC when used as third party cells, indicating sHLA-G is associated with higher Treg cell

levels in vivo.

In an another investigation, Le Rond et al. have shown that circulating CD4+ and

CD8+ T cells obtained from two kidney-liver transplant patients, who had high sHLA-G

levels in the sera and didn’t suffer from allograft rejection, expressed membrane-bound

HLA-G (118). In fact, in vitro studies have revealed that some CD4+ and CD8+ T cells

express HLA-G following allogeneic stimulation (86,118). While shedding of HLA-G by

allograft cells might contribute to sHLA-G levels, HLA-G+ T cells following allogeneic

stimulation might represent another source of the HLA-G detected after transplantation, and

may possess clinical relevance due to their immunosuppressive properties.

As reported by Naji et al., in addition to sHLA-G, circulating levels of the

suppressive CD4low and CD8low T cells are found to be elevated in patients following liver

transplantation and combined kidney-liver transplantation (88). Moreover, plasma CD4 and

CD8 levels are significantly greater in these patients, indicating increased shedding of these

coreceptors. These findings are supported by in vitro investigations illustrating that CD4low

and CD8low T cell populations are induced by HLA-G and possess immunosuppressive

properties (88), thus highlighting the clinical relevance of HLA-G post-transplant in

contributing to immune tolerance.

Although HLA-G appears to inhibit the alloresponse in combined kidney-liver

transplant patients, such an effect is not noticed following liver transplantation as described

25

by Creput et al. (114). While 6/58 (10.3%) of liver transplant patients in this single-centre

investigation presented with BEC HLA-G expression, there was no significant correlation

between HLA-G expression and acute or chronic liver rejection, suggesting that HLA-G is

more frequently expressed and more associative with freedom from rejection in combined

kidney-liver transplant patients. Nonetheless, circulating HLA-G levels have been linked

with improved allograft function post-liver transplant as reported by Basturk et al. (119).

This study showed that a serum HLA-G concentration < 30 ng/mL was associated with

increased levels of markers of liver dysfunction including aspartate aminotransferase, direct

bilirubin, total bilirubin and alkaline phosphatase. This finding implies that low sHLA-G

levels in liver transplant patients are associated with liver function test results falling above

the normal range, suggesting graft dysfunction or rejection. Interestingly, this study also

suggested that in Treg cells obtained from liver transplant recipients, prednisolone treatment

increased expression of membrane-bound HLA-G1, while cyclosporine treatment resulted in

sHLA-G expression, providing more evidence that HLA-G expression might be modified by

immunosuppressive therapy.

Cancer

In addition to defending the host from pathogen invasion, the immune system is widely

believed to play a major role in cancer immunosurveillance, which refers to its ability to

identify cancerous cells based on expression of tumour-specific molecules, and eliminate or

control them before they can harm the host (120,121). The fact that immunosuppressed

transplant patients (18,21,122) and individuals with primary immunodeficiencies (123,124)

are at greater risk for developing malignancies provides further evidence that the immune

system helps protect the host by inhibiting tumour formation and participating in tumour

26

elimination. More recently, cancer immunosurveillance has been incorporated into a broader

concept known as immunoediting, which consists of three phases: immune-mediated

elimination of cancerous or precancerous cells; equilibrium between host and tumours that

are not eliminated; and tumour escape from the immune system, leading to progressively

growing malignancies (120,121). Therefore, this concept holds that cancerous cells that are

not eliminated have been shaped by the immune system into achieving a phenotype that

allows for escape from immune attack, survival and the potential to develop into

malignancies.

Due to its immunosuppressive nature, HLA-G expression by tumours during all

phases of cancer immunoediting is widely believed to represent a survival strategy (125).

When cancerous cells are under attack by the immune system during the initial elimination

phase, HLA-G expression might be advantageous since it can protect against both, the innate

and adaptive immune responses. During the equilibrium phase, when tumours that ultimately

survive are sculpted by the immune system into becoming less immunogenic, HLA-G

expression might also prove beneficial. Alterations in DNA methylation are believed to play

a major role in the complex, multifaceted process of carcinogenesis by influencing gene

expression and cell proliferation (126). For instance, activation of genes of the MAGE

family, which are involved in tumour-specific antigen expression, occurs via demethylation

(127). Interestingly, DNA demethylation reverses HLA-G gene repression, thereby resulting

in a less immunogenic phenotype leading to positive selection during the equilibrium phase

of cancer immunoediting.

Because the immune system plays an important role in cancer elimination, HLA-G

has been extensively researched in this field (125,128,129). Early studies by Amiot et al.

27

showed the presence of HLA-G mRNA transcripts in malignant circulating cells obtained

from patients presenting with various hematological cancers (130,131). Shortly thereafter, in

a pioneering investigation conducted by Paul et al., high levels of HLA-G mRNA with

varying patterns of HLA-G protein expression were detected in melanoma cell lines, some of

which had the ability to inhibit NK cell lysis (132). Moreover, to illustrate the in vivo

relevance of these finding, higher levels of HLA-G transcripts were observed in a skin

melanoma biopsy than healthy skin from the same patient (132). Subsequent studies have

confirmed high levels of HLA-G mRNA and protein expression in melanoma biopsies

(133,134), and have shown increased HLA-G expression by melanocytic and inflammatory

infiltrating cells in metastatic melanoma lesions compared to benign lesions, indicating an

association with malignant transformation (135).

These early discoveries have led to a plethora of studies assessing HLA-G expression

in the context of cancer. While absent in healthy tissue, HLA-G has been detected in some

renal cell carcinoma (RCC) lesions, despite a high frequency of infiltrating tumour-specific T

cells, indicating protection from immune attack (136,137). This expression is also noticed in

malignant and premalignant lesions in renal transplant recipients, whereas healthy renal

tissue from individuals with RCC displays no HLA-G expression (138). Constitutive and

IFN-γ-mediated HLA-G mRNA and protein expression have been observed in certain RCC

cell lines in vitro, but not in normal cultured kidney cells (136). Functional studies have

revealed that compared to HLA-G- renal cell lines, these HLA-G+ cultures can inhibit

allogeneic NK cells and antigen-specific T lymphocytes (136), suggesting a potential

mechanism of tumour survival. In patients with B cell chronic lymphocytic leukemia, HLA-

G expression in malignant cells is associated with a significantly shorter progression-free

28

survival time, decreased humoral and cellular responses, and was found to be an independent

predictive factor of disease progression (139). In breast cancer tissue specimens, but not

healthy mammary tissue, epithelial cells as well as infiltrating macrophages and CD8+ T cells

have been found to express HLA-G, suggesting that it might be activated by host anti-tumour

responses (140). Similar investigations have shown HLA-G mRNA and protein expression in

ovarian and breast ductal carcinomas, while being absent in healthy tissue and benign

tumours from these organs (141). In lung cancer patients, HLA-G proteins have been

detected in lung carcinoma specimens (142) and in activated macrophages and dendritic cells

infiltrating the tumours (143). Investigations have also revealed that HLA-G is expressed in

glioma cell lines and brain tumour tissue samples (144). In vitro studies showed that a few

HLA-G+ glioma cells can protect HLA-G- glioma cells by inhibiting direct alloreactive lysis,

alloproliferative responses and efficient priming of cytotoxic T cells (144).

Soluble HLA-G proteins, which likely originate from tumours or inflammatory cells,

have also been detected in cancer patients. Compared to healthy controls, concentrations of

sHLA-G are significantly increased in malignant melanoma, breast cancer, ovarian cancer

and glioblastoma patients (145) as well as those suffering from lymphoproliferative disorders

(146,147). These levels have also been reported to be significantly higher in malignant versus

benign ascitic fluid (141). In patients with such malignancies, shedding and secretion of

HLA-G proteins by tumour cells might contribute to the increased sHLA-G concentrations in

the circulation. Alternatively, cells involved in the immune response might represent another

source of HLA-G. For example, increased expression of membrane-bound HLA-G in

circulating monocytes is associated with the high serum HLA-G levels in melanoma patients

(148). Accordingly, sHLA-G likely contributes to tumour survival by inhibiting anti-tumour

29

responses at the local tumour site and in the systemic circulation, and might function as a

marker of disease progression.

Some forms of cancer such as uveal melanoma and laryngeal carcinoma are not

associated with HLA-G expression (128,149), implying that HLA-G expression is dependent

on the nature of the malignancy. Differential HLA-G mRNA and protein expression in

various cancers might be the result of differences in tumour biology, the methods used to

detect HLA-G and/or the patient populations. Alternatively, due to the tight HLA-G

regulatory mechanisms, the tumour microenvironment and the pathogenesis underlying

tumour formation might affect HLA-G expression. Nevertheless, there has been considerable

interest in assessing the role of HLA-G in tumour survival, with more than one thousand

cancerous tissue specimens originating from various types of malignancies having been

assessed for HLA-G expression to help address this issue (128). Cumulatively, these studies

reveal a strong association between HLA-G expression and tumour survival, emphasizing the

role of this protein as a potential suppressor of immune-mediated attack. Hence, HLA-G may

contribute to impairment of immune responses against cancerous cells and may favour

tumour progression.

Inflammation

Recent evidence has shown that the multi-faceted role of HLA-G can be extended beyond the

scope of pregnancy and into inflammatory situations, which is often the result of overactive

immune responses (150). Expression of HLA-G in lesions arising from inflammation might

represent a protective, inhibitory response to immune-mediated attack. Considerable

expression of HLA-G and its receptor, ILT2, have been noted in acute and chronic active

plaques, perilesional areas and normal appearing white matter in brain specimens obtained

30

from multiple sclerosis (MS) patients (151). In addition, HLA-G levels in cerebrospinal fluid

(CSF) samples from MS patients are significantly greater than levels found in healthy

individuals (151). While the mechanisms of HLA-G expression are vague, it might represent

an inhibitory response aimed at diminishing the harmful effects of T cell infiltration in

neuroinflammation.

HLA-G has been detected in a variety of other inflammatory diseases. Biopsies of

lesions from patients with chronic atopic dermatitis exhibit HLA-G expression (152),

suggesting a possible inhibitory response to infiltrating T cells. HLA-G proteins and ILT2

receptors have also been detected in psoriatic plaque lesions (153), indicating that infiltrating

T cells might be subjected to regulatory mechanisms aimed at downregulating their

deleterious effects. In another investigation conducted by Wiendl et al., muscle fibres and

infiltrating cells of patients with various inflammatory myopathies, have been shown to

express HLA-G, unlike healthy individuals or patients with degenerative muscular diseases

(154). This expression was also noted in cultured myoblasts stimulated with the

inflammatory cytokine IFN-γ (154). Other studies have show that gene transfer of HLA-G1

and -G5 into cultured myoblasts rendered theses cells resistant to immune-mediated lysis

through inhibition of NK cells and CD4+ and CD8+ T cells (155), suggesting a role for HLA-

G as a protective mechanism from strong immune responses.

Moreover, HLA-G+ suppressive T cells have been noted in sites of inflammation.

While absent in biopsies of healthy muscle, CD8+HLA-G+ Treg cells have been detected in

muscle biopsies of patients with various inflammatory myopathies (90), which are often the

result of attack from cytotoxic T cells (156). CD4+HLA-G+ cells are also elevated in the CSF

of MS patients with acute relapse (90). The increased numbers of suppressive HLA-G+ T

31

cells during inflammation suggests an immunoregulatory role in modulating inflammatory

responses. While T cell infiltration promotes the pathogenesis of inflammatory diseases,

regulatory pathways likely control such responses. The fact that HLA-G expression has been

found to be expressed in lesions arising from various inflammatory conditions, while being

undetected in healthy tissue, suggests that it likely plays a protective role in controlling

inflammation.

32

CHAPTER 2:

PROPOSED INVESTIGATIONS

33

2.1 RATIONALE

While HLA-G expression has been detected post-heart transplant, it remains unclear whether

this protein can reliably predict freedom from clinically significant rejection in such patients.

Although other clinical markers of negative outcomes post-heart transplant such as B-type

natriuretic peptide (BNP), C-reactive protein (CRP) and cardiac troponins have been

established (157-160), all are indicators of cardiac dysfunction rather than rejection.

Conversely, HLA-G possesses immunosuppressive properties and might have the potential to

reliably identify freedom from clinically significant rejection in heart transplant patients.

Additionally, is it still unknown how HLA-G expression is induced following heart

transplantation. Due to the ability of HLA-G to directly inhibit immune responses, induction

of this expression might represent a valuable strategy to protect against rejection. Expression

of HLA-G in vascular endothelial and smooth muscle cells is of particular interest because

theses cells are frequent targets of the immune response post-transplant (161). Furthermore,

inflammatory cells are present in close proximity to the luminal endothelium of allograft

vessels and likely contribute to the progression of transplant vasculopathy (162). Yet, it is

still vague whether mature vessels are capable of HLA-G expression.

Our studies were therefore designed with the purpose of gaining a better

understanding of HLA-G expression and its clinical relevance in the context of heart

transplantation. To achieve this, we proposed two investigations to:

1) Determine the usefulness of HLA-G in identifying freedom from clinically

significant rejection using a retrospective case-control design

2) Assess the ability of various interventions to induce HLA-G expression in

cultured vascular endothelial and smooth muscle cells

34

2.2 HYPOTHESES

We hypothesize that:

1) There are significant differences in the HLA-G expression profiles of rejecting

and non-rejecting patients

2) Expression of HLA-G can be induced in our cell culture models in vitro

The results of our studies will help us better understand the clinical validity of the HLA-G

protein in heart transplantation and if in vitro expression of HLA-G expression can be

induced in target cells. Our results may form the basis for subsequent experiments and may

also justify future in vivo clinical studies. Ultimately, such investigations might result in the

clinical application of HLA-G as either a biological prognostic indicator or a potential

therapeutic target. The role of HLA-G as biological marker may enable better monitoring of

heart transplant recipients, while induction of HLA-G expression might help protect against

rejection and, subsequently, allograft vasculopathy in these patients.

35

CHAPTER 3:

MYOCARDIAL HLA-G RELIABLY INDICATES A LOW RISK OF A CUTE CELLULAR REJECTION FOLLOWING HEART TRANSPLANTATION

36

3.1 METHODS

Patients

This study, approved by the local research ethics board, included 67 heart transplant

recipients selected on the basis of retrospective myocardial biopsy rejection scores, who

underwent heart transplantation at Toronto General Hospital from May 1995 to October

2006. All rejection scores were graded according to the revised version (163) of the 1990

working formulation for standardized cardiac allograft rejection grading, set forth by the

ISHLT in 2004 (164). Post-transplant serial myocardial biopsies were performed weekly

during the first month, biweekly until month 3, monthly until month 6 and every three

months until one year post-transplant. Additional biopsies were taken if clinically indicated.

For immunosuppression, all patients received triple therapy consisting of cyclosporine or

tacrolimus, azathioprine and prednisone. However, mycophenolate mofetil was introduced

routinely in preference to azathioprine in 1998, resulting in a switch to mycophenolate

mofetil from azathioprine in many patients. For induction therapy, rabbit antithymocyte

serum was used in all patients until 1999, when it was substituted for basiliximab. Since

2003, all patients received rabbit anti-thymocyte globulin for induction therapy in place of

basiliximab.

Biopsy Scoring

Biopsies were scored using the revised ISHLT scale: Grade 0R, no rejection; Grade 1R, mild

rejection involving interstitial and/or perivascular inflammatory cell infiltration with up to

one focus of myocyte damage; Grade 2R, moderate rejection involving two or more foci of

cellular infiltration with associated myocyte damage; Grade 3R, severe rejection involving

37

diffuse cellular infiltration with multifocal myocyte damage with or without edema,

hemorrhage and/or vasculitis (163). Since all rejection scores were determined by one

cardiac pathologist at this institution, there was no inter-observer variability. Biopsies were

scored using the 2004 revised ISHLT classification because it is more accurate, reproducible

and clinically relevant when used for clinical diagnosis or investigation (165). Rejection

scores of tissue specimens obtained before the implementation of the new classification were

converted to scores based on the revised heart biopsy grading scale. Patients were divided

into two groups based on their cardiac allograft rejection profiles. Group A consisted of 29

patients who did not suffer from any moderate or severe rejection episodes following heart

transplantation (all biopsies were of Grade ≤ 1R) and were considered to be non-rejectors.

All 38 patients comprising Group B suffered from sustained (at least 2) moderate or severe

acute rejection episodes (all biopsies were of Grade ≥ 2R) within a one-year period and were

classified as rejectors. Out of the 212 heart transplant recipients screened, those who did not

fit the criteria for Groups A and B were excluded from this study. An additional 20 eligible

patients were also excluded due to poor tissue quality. As a result, this investigation is not

representative of the entire transplant population at this institution. Biopsies were assessed

for rejection independently of quilty lesions, which are nodular endocardial infiltrates

capable of causing variability in the diagnosis of allograft rejection (166). Since our centre

routinely ensures that there are no instances of quilty lesions masquerading as rejection, all

biopsies were appropriately graded. Because quilty lesions are not regarded as clinically

significant, we did not take them into consideration for the purposes of this study. Screening

for antibody-mediated rejection assessed by C4d staining was only performed when

clinically indicated.

38

Tissue Samples

Myocardial biopsies obtained from the right interventricular septum of each patient were

used for the assessment of HLA-G expression. Due to the retrospective nature of this study,

the patients’ HLA-G status was unknown to the cardiac pathologist at the time of biopsy

scoring. In Group A, biopsies were taken at 1, 6 and 12 months post-transplant. In Group B,

three biopsies were also analyzed: one during an episode of severe rejection (Grade ≥ 2R),

one approximately 6 months before (Grade 0) and one approximately 6 months after (Grade

0) the episode of rejection. Among these patients, 26 had their biopsy samples analyzed

within the first three years post transplant, while specimens from the remaining 12 were

taken thereafter. Although biopsies from the two groups of patients were obtained at different

times post-transplant, endomyocardial HLA-G expression has been shown to be unaffected

by time after heart transplantation (104,105). All specimens had been previously fixed in

10% neutral buffered formalin (Fisher Scientific, Waltham, MA, USA), embedded in

paraffin blocks and mounted onto slides following routine endomyocardial biopsy procedures

to survey cardiac allograft rejection. All samples were assessed for HLA-G through

immunohistochemical analysis. To confirm tissue integrity and to avoid the risk of false

negative staining, all biopsies were stained for the classical MHC-I proteins HLA-B and/or

HLA-C, which are highly expressed in the heart, through immunohistochemical analysis.

Immunohistochemistry

The following monoclonal antibodies (mAb) were used for immunohistochemistry: HC10,

IgG2a, specific for a determinant expressed on β2M-free HLA-B and HLA-C heavy chains

(167), kindly provided by D. Schutz and H. Ploegh (Harvard University, Cambridge, MA,

USA); 4H84, IgG1, specific for the α1 domain common to all HLA-G isoforms (168), kindly

39

provided by M. McMaster (University of California, San Francisco, CA, USA); and MEM-

G/2 (Exbio Praha, a.s., Vestec, Czech Republic), IgG1, specific for the heavy chain of all

HLA-G isoforms (169).

Tissue sections were deparaffinized with toluene (Sigma-Aldrich Corporation, St.

Louis, MO, USA) for 15 min and rehydrated with ethanol for 10 min and water for 5 min.

Deparaffinized sections were submitted to epitope retrieval by high temperature in 10 mM

sodium citrate buffer (pH 6.0) for 15 min using a commercial microwave oven to optimize

immunoreactivity. After cooling, slides were rehydrated for 5 min with a wash solution of

0.1% saponin and 10 mM HEPES in phosphate buffered saline (PBS). Endogenous

peroxidase activity was quenched by treating sections for 5 min at room temperature with 3%

hydrogen peroxide in water. Slides were then treated with wash buffer for 2 min followed by

a solution of 20% human serum and 5% BSA (bovine serum albumin, Sigma) in water for 20

min to prevent non-specific binding. They were subsequently washed, treated with the

primary mAb at a concentration of 2 µg/mL in PBS for 30 min at room temperature, and

washed three times. An isotype-matched antibody was used under similar conditions to

control non-specific staining. Using the UltraTech-Peroxidase Detection Kit (Beckman

Coulter, Inc., Fullerton, CA, USA), slides were treated with a biotinylated secondary

antibody for 10 min, washed three times, and treated with streptavidin-horseradish

peroxidase (HRP) for 10 min. After three more washing steps, expression of all HLA

proteins in post-transplant cardiac biopsy tissue was detected by covering them with the AEC

substrate-chromogen (Dako Denmark A/S, Glostrup, Denmark) for 15 min. Slides were then

washed and counterstained with hematoxylin (Sigma) for 5-10 min. Finally, tissue sections

40

were washed and mounted with hot Glycergel mounting medium (Dako), Trophoblast tissue

sections were used as positive controls for HLA-G expression.

We assessed HLA-G staining in three endomyocardial biopsies of all patients in both

groups. Only tissue specimens which stained positive for HLA-B and/or HLA-C were used in

this study. A specimen was considered positively labeled if > 20% of either allograft

myocardial cells or infiltrating leukocytes in the paraffin-embedded tissue section contained

visually detectable red chromogen in the membrane and/or cytoplasm. Patients were

considered HLA-G+ if at least one tissue sample was stained with the either the 4H84 or

MEM-G/2 mAb, both of which recognize all HLA-G isoforms. The remaining patients were

considered HLA-G-. All patients were assigned a random study number ensuring that at the

time of HLA-G staining the patients’ clinical information and rejection scores were

unknown. Immunohistochemical analyses were performed in the laboratory of N. Rouas-

Freiss (Saint-Louis Hospital, Paris, France).

Statistical Analysis

Unless specified, values are expressed as mean ± standard deviation. To determine the

statistical significance of differences between proportions and means, we performed chi-

square tests and unpaired t tests, respectively. We considered p < 0.05 to be statistically

significant; however, exact p values are provided to enable the reader to determine clinical

significance.

41

3.2 RESULTS

Baseline Characteristics

Among the 67 study patients (55 male, 12 female), who had a mean operative age of 49±13

years (range 18-69), cardiac transplantation had been performed due to idiopathic

cardiomyopathy (49% of cases), ischemic cardiomyopathy (34%), hypertrophic obstructive

cardiomyopathy (6%), myocarditis (5%) and other pre-transplant heart diseases (6%),

including amyloidosis, rheumatic disease and congenital heart disease. Group A (non-

rejecting) consisted of 29 patients (24 male, 5 female) with a mean age of 51±12 years (range

25-68), while 38 patients (31 male, 7 female) with a mean age of 47±13 years at the time of

heart transplantation (range 18-69) were included in Group B (rejecting). At the time of chart

review, 12 Group A patients were one year post-transplant, 7 between 1-3 years and 10

between 3-9 years. Among Group B patients, 14 developed sustained rejection within the

first year post-transplant, 12 between years 1-3, and 12 between 3-9 years following

transplantation.

There were no significant differences in mean age, gender, indication for heart

transplantation, donor characteristics or pre-transplant panel reactive antibodies between the

two groups (Table 1). Not surprisingly, significantly more patients in Group B (39%

compared to 10% in Group A) received sirolimus (p=0.01). In Group A, 2 patients were on

sirolimus due to renal dysfunction and 1 due to graft vasculopathy. In Group B, the

indications to use this agent were renal dysfunction in 7, graft vasculopathy in 4, recurrent

rejection in 3 and cancer in 1 patient. The incidence of renal dysfunction was 7% in Group A

versus 18% in Group B (p=0.17). There were no significant differences in other

immunosuppressive medications (Table 1). None of the patients had hemodynamic

42

decompensated cellular rejection. None of the patients developed left ventricular dysfunction

measured by echocardiogram following transplantation. As there were no clinical indications,

no patient required screening for antibody-mediated rejection.

HLA-G Expression

HLA-G status of biopsy specimens was determined by immunohistochemical staining with

either the MEM-G/2 or 4H84 mAb, while tissue integrity was confirmed by staining of the

classical MHC-I proteins, HLA-B and/or HLA-C (Figure 3). Cells that stained positive for

HLA-G included cardiomyocytes and infiltrating inflammatory cells. While the overall rate

of HLA-G expression in the study patient cohort was 29/67 (43.3%), it is important to note

that this proportion only takes into account the rejecting and non-rejecting patients with good

tissue quality, not the entire heart transplant population at our centre. We compared

differences in HLA-G status between the two patient groups (Table 2), and observed that the

proportion of non-rejecting patients who had at least one HLA-G+ endomyocardial biopsy

(86%) was significantly greater (p < 0.001) than that of the rejecting patients (11%). The

sensitivity and specificity of myocardial HLA-G expression as a test to diagnose the absence

of moderate or severe acute cellular rejection at any time post transplant was 86% and 89%,

respectively, while the positive and negative predictive values were 86% and 89%,

respectively. Furthermore, the proportion of HLA-G+ tissue samples obtained from the non-

rejecting patients in our study population (53%) was significantly higher (p < 0.001) when

compared to 4% of biopsies with detectable HLA-G expression from rejecting patients

(Table 2).

As illustrated by the rejection profile of study patients (Figure 4), age, gender, time

after transplantation and rejection score did not have an effect on HLA-G expression. Among

43

Group B patients, one expressed HLA-G before, one during, and two after the Grade ≥ 2R

rejection episode. These patients did not display increased HLA-G expression during

episodes of no rejection versus episodes of severe acute cellular rejection, indicating that

there was no correlation between HLA-G status and ISHLT rejection severity at the time of

biopsy.

It is important to note that although 4 patients in Group B were considered HLA-G+,

all had only one HLA-G+ biopsy. Expression of HLA-G was not detected in the other biopsy

specimens from rejecting patients. In contrast, 15 out of 25 HLA-G+ patients (60%) from

Group A had more than one HLA-G+ biopsy (p=0.03), suggesting that HLA-G expression in

Group A HLA-G+ patients was more prominent and sustained than in Group B HLA-G+

patients. Interestingly, none of the patients in Group A experienced any moderate or severe

rejection episodes up to 9 years post transplant.

44

CHAPTER 4:

PROGESTERONE INDUCES EXPRESSION OF HLA-G IN VASCULAR ENDOTHELIAL AND SMOOTH MUSCLE CELLS IN VITRO

45

4.1 METHODS

Cell Cultures

Commercially available human coronary artery endothelial cell (HCAEC), aortic endothelial

cell (HAEC) and coronary artery smooth muscle cell (HCASMC) cultures (Lonza

Walkersville, Inc., Walkersville, MD, USA) were grown to full confluence in a sterile,

humidified tissue incubator (Model MCO-18AIC, Sanyo Electric Co., Ltd, Moriguchi, Japan)

at 37 °C and 5% CO2 for all experiments, unless otherwise indicated. HCAEC and HCASMC

were procured from the left and right coronary arteries and HAEC from the ascending and

descending aortas of cadaveric donors. HCAEC and HAEC were cultured in Endothelial Cell

Basal Medium-2 (EBM-2) supplemented with Microvascular Endothelial Cell Growth

Medium-2 (EGM-2 MV) and Endothelial Cell Growth Medium-2 (EGM-2) bullet kits,

respectively, while HCASMC were grown in Smooth Muscle Basal Medium-2 (SmBM-2)

medium supplemented with Smooth Muscle Growth Medium-2 (SmGM-2, Lonza). These

supplements provided HCAEC and HCASMC cultures with 5% fetal bovine serum (FBS),

and HAEC cultures with 2% FBS. All culture media were further treated with 100 U/mL

streptomycin and 100 µg/ml penicillin (Invitrogen Corp., Carlsbad, CA, USA), to protect

against contamination. From the primary culture, cells were passaged once and

cryopreserved in solution consisting of culture medium, FBS (Invitrogen) and dimethyl

sulfoxide (DMSO, Sigma) in a 5:4:1 ratio, respectively, until further use. Cells had been

cryopreserved for no longer than 6 months and were subsequently passaged no more than

three times when being used for experimental purposes. Cells were grown in 6 cm

polystyrene culture dishes for flow cytometric experiments and 6-well plates (Corning

Incorporated, Corning, NY, USA) for protein expression by enzyme-linked immunosorbent

46

assay (ELISA) in 4 mL and 2 mL medium, respectively. For viability studies, cells were

cultured with 1 mL medium in 24-well plates (Corning). All cell cultures were initially

grown in 10 cm polystyrene dishes with 10 mL medium (Corning) for expansion, before

being transferred to the smaller culture dishes or plates for experimentation. To passage cells,

culture media from the 10 cm dishes were aspirated and cells washed with 10 mL PBS. Cells

were detached by adding 2 mL 0.25% trypsin solution with ethylene diamine tetra-acetic acid

(EDTA, Invitrogen) and incubating for 2 min. At least 4 mL medium was then added to

culture dishes to block the enzymatic activity of trypsin. Cells were separated by repeated

pipetting, suspended in the supernatant and transferred into new culture dishes or plates.

Medium was then added, if necessary, to achieve the full desired volume. All culture media

were replaced within 48 h to allow the cells being used for expansion or experimentation to

grow to full confluence and to prevent contamination.

Treatments and Interventions

To determine if vascular endothelial and smooth muscle cells grown in culture could be

induced into expressing HLA-G, they were treated with interventions that have been shown

to modulate HLA-G levels in vitro and in vivo. Cells were subjected to cytokines:

recombinant IFN-γ and IL-10 (Sigma) at 0.1-100 ng/mL for 2-24 h; hypoxia (pO2 < 0.1%) for

2-12 h followed by reoxygenation (pO2=21%) for 2 hr (H/R), which mimics the relevant

ischemia and reperfusion time periods observed in clinical transplantation;

immunosuppressive agents (LC Laboratories, Woburn, MA, USA): cyclosporine (100-1000

ng/mL), sirolimus and tacrolimus (0.1-100 ng/mL) for 2-24 h, which encompass clinically

relevant concentrations; and progesterone (100-10,000 ng/mL) for 2-24 h, with or without

mifepristone (Sigma), its receptor antagonist (1000 ng/mL). The vehicles for our treatments

47

were: PBS (pH 8.0) for IFN-γ; 5mM sodium phosphate buffer (pH 7.2) for IL-10; DMSO for

the immunosuppressive agents; and anhydrous ethyl alcohol for progesterone and

mifepristone. Cells used as controls were treaded with vehicle alone. For H/R experiments,

cells were subjected to an initial stabilization period by replacing the culture media and

incubating them under normoxic conditions for 30 min. Following this period, the

supernatant was aspirated, cells were washed and 0.7 mL, 0.5 mL or 0.3 mL fresh media

were added to the 6 cm dishes, 6-well plates or 24-well plates, respectively. These were then

placed in a sterile, humidified hypoxia chamber (BioSpherix, Ltd, Lacona, NY, USA) at 37

°C, 5% CO2 and 0.1% O2 for the required hypoxic time period. Gas concentrations were

maintained with the ProOX (Model 110) O2 controller and ProCO2 (Model 120) CO2

controller (BioSpherix). After the period of hypoxia, cultures were subjected to

reoxygenation by replenishing the media and incubating the cells under normoxic conditions

for the required time period. Cells kept under normoxia for the entire H/R duration were used

as controls. Following all treatments and interventions, cultures were assessed for cell

proliferation ability and viability or HLA-G protein expression by ELISA and flow

cytometry.

Protein Extraction

Following the treatment period, cells in 6-well plates were washed twice with PBS to remove

residual medium, non-adherent cells and other minor contaminants. After aspiration of the

final wash solution, cells were incubated at 4 °C for 10 min with 250 µL per well of cold

radio-immunoprecipitation assay (RIPA) extraction buffer (Sigma) with protease inhibitors.

The same extraction buffer batch was used when preparing protein standards for subsequent

determination of total protein content. Cells were then harvested by scraping the wells with

48

cell lifters (Fisher) and immediately transferring the lysates to 1.5 mL microcentrifuge tubes

(Fisher). Lysates were clarified by centrifugation at 8000 x g for 10 min at 4 °C to pellet the

cell debris. Supernatants containing soluble proteins were aliquoted, flash frozen in liquid

nitrogen and stored at -80 °C for determination of HLA-G and total protein content.

Enzyme-linked Immunosorbent Assays

Antibodies and purified HLA-G protein standard containing all isoforms were kindly

provided by C. Librach (University of Toronto, Toronto, ON, Canada). For each ELISA,

wells of a 96-well polystyrene plate (Corning) were filled with 100 µL of the 2C/C8 mAb,

which has an epitope on the HLA-G heavy chain, at a concentration of 10µg/mL in PBS, and

kept at 4 °C overnight. Wells were washed three times with a PBS washing solution

containing 0.05% Tween-20 (Fisher) and blocked with 5% milk in PBS for 4 h at room

temperature. Duplicate cell lysate samples, extracted with RIPA extraction buffer (Sigma)

following treatment or intervention, were added to a final volume of 100 µL in each well and

incubated at 4 °C overnight. Wells were washed three times with washing solution, before

100 µL of the biotinylated 3C/G4 mAb (0.4 mg/mL in PBS), which also binds to a distinct

site on the HLA-G heavy chain, was added. The plates were then incubated for 2 h at room

temperature. All wells were washed four times before adding 100 µL of a 1:2000 dilution of

streptavidin-HRP (Sigma) in PBS containing 1% BSA, and then incubated for 1 h at room

temperature. Wells were subsequently washed four times before adding 100 µl of

tetramethylbenzidine substrate solution (Sigma). After 10-15 min incubation at room

temperature the reactions were stopped by the addition of 50 µL of 1M HCl (Sigma) to each

well. Each plate was read spectrophotometrically at a wavelength of 450 nm on an automated

ELISA plate reader (Molecular Devices, Sunnyvale, CA, USA). Absorbance readings of two

49

wells without the addition of samples or standards, but otherwise treated in the same manner,

were used as blanks and were subtracted from all sample readings. HLA-G levels were

derived from an HLA-G protein standard curve based on absorbance readings of purified

HLA-G protein at concentrations of 2, 1, 0.2, 0.04 and 0.02 and 0 µg/mL in PBS. The lower

detection limit of this ELISA was 10 ng/mL. All HLA-G concentrations in each cell lysate

sample were normalized using the total cellular protein concentrations. This assay was

specific for all HLA-G isoforms. Measurement of HLA-G in cell lysates ensured that all

membrane-bound and soluble isoforms could be detected. We did not measure levels of

soluble and shed HLA-G in culture supernatants as they fell below the lower detection limit

of the ELISA.

Protein Determination

Total protein concentrations in each cell lysate sample were determined with the Bio-Rad DC

protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Working Reagent A was

prepared by combining 2% Reagent S with Reagent A. Protein standards of 0, 0.05, 0.1, 0.25,

0.5 and 0.77 mg/mL of BSA in RIPA extraction buffer containing the protease inhibitors

were also made. Once prepared, 10 µL of each sample and standard was combined with 50

µL of Working Reagent A and 400 µL of Reagent B. Following this, 200 µL of the samples

and standards were transferred to a 96-well polystyrene plate. After being incubated for

approximately 15 min at room temperature, the plate was read by a spectrophotometer

(µQuant Universal Microplate Spectrophotometer, BioTek Instruments, Inc., Winooski, VT,

USA) at an absorbance wavelength of 750 nm. Absorbance readings from blank wells were

subtracted from the values of the samples and standards. Total protein concentrations of

50

samples were calculated by plotting the corrected absorbances on the linear protein standard

curve.

Flow Cytometry

To assess cell surface protein expression, cells cultured in 6 cm dishes were washed with

PBS and detached by treating with 1 mL Accutase cell detachment medium (eBioscience,

Inc., San Diego, CA, USA) at 37 °C for 5 min. An equal number of cells from each 6 cm dish

were transferred into 2 polypropylene round-bottom tubes (BD Biosciences, San Jose, CA,

USA), to be assessed for protein expression or used as isotype controls. Non-specific binding

was controlled by decanting the supernatant, adding 50 µL of PBS with 20mMol glucose

(Fisher) and 5% BSA (PBSG/BSA) with 40% mouse serum (Sigma) and incubating at 4 °C

for 10 min. For determination of protein expression, 50 µL PBSG/BSA with a primary

antibody was added to the cell suspension and incubated for 45 minutes in the dark at 4 °C. If

necessary, cells were washed once with PBSG/BSA and incubated in the dark for 30 minutes

at 4 °C in 100 µL PBSG/BSA containing a secondary antibody. After two washing steps,

cells were resuspended in 200 µL PBSG/BSA and cell-surface HLA-G expression was

assessed using a Coulter Epics XL-MCL flow cytometer (Beckman Coulter). IsoFlow sheath

fluid (Beckman Coulter) was used to top up sample volumes in the round-bottom tubes. To

assess HLA-G expression we used a primary mouse anti-human HLA-G antibody, MEM-G/9

(AbD Serotec, Planegg, Germany), at 10 µg/mL. Cells serving as negative controls were

incubated with isotype-matched mouse IgG (AbD Serotec) at the same antibody

concentration in similar fashion. A goat anti-mouse IgG conjugated to Alexa Fluor

(Invitrogen) at 2.5 µg/mL was used as the secondary antibody. For phenotypic

characterization of HCAEC and HAEC cultures, cells were incubated with the primary

51

mouse anti-human CD31 antibody conjugated to phycoerythrin and compared to a

phycoerythrin-conjugated isotype matched mouse IgG (Beckman Coulter) at the same

concentration, without the need for a secondary antibody. For smooth muscle cell

characterization, cells were assessed for intracellular α-smooth muscle actin (SMA)

expression with a cyanine 3-primary conjugated mouse anti-SMA (Sigma) antibody. To

enable intracellular staining, cells were first simultaneously fixed and permeabilized by

resuspending in 100 µL Cytofix/Cytoperm solution (BD Biosciences) and incubating at 4 °C

for 20 min. Cells were washed and stained as previously described. For characterization of

smooth muscle cells, the Perm/Wash buffer (BD Biosciences) was used for all washing and

incubation steps to keep the HCASMC permeabilized.

Viability Assays

The XTT (sodium 3’-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)-

benzene sulfonic acid hydrate) based Cell Proliferation Kit II (Roche Diagnostics

Corporation, Basel, Switzerland) was used for the quantification of cell proliferation and

viability. XTT labeling mixture was first prepared by mixing 2% electron coupling reagent in

XTT labeling reagent. Cells were then incubated for 4 h in 750 µL phenol red-free

Dulbecco's Modified Eagle Medium (DMEM, Invitrogen)supplemented with 5% FBS and

20% XTT labeling mixture per well. Cell viability was determined by the ability of

metabolically active cells to convert the yellow XTT salt to an orange formazan dye. Sample

absorbance was measured by an ELISA plate reader at 450 nm with a reference wavelength

of 650 nm. Cell viability was calculated as a percentage of the absorbance of control wells.

52

Statistical Analysis

ELISA, protein determination and viability experiments were all performed in duplicate. The

mean of the two readings was used as the absorbance of a sample or protein standard. Data

are represented as mean ± standard error. Comparisons between multiple groups were

performed by two-factor ANOVA. We used unpaired t tests for inhibition experiments

involving comparisons between two groups. We considered p < 0.05 to represent statistical

significance.

4.2 RESULTS

Induction Experiments

Phenotypic characterization of endothelial and smooth muscle cells was confirmed through

flow cytometric analysis. HCAEC and HAEC showed cell-surface expression of CD31,

while HCASMC cultures stained strongly for intracellular α-SMA (Figure 5). To determine

HLA-G expression in HCAEC, HAEC and HCASMC cultures, we subjected them to our

interventions of interest and assessed protein expression via ELISA and flow cytometry

(Table 3). Dose response and time course experiments were performed in all cell types for all

treatments. HCAEC, HAEC and HCASMC cultures did not express HLA-G at baseline.

Expression of HLA-G was not detected following treatment with cytokines (IFN-γ and IL-

10) and immunosuppressive agents (cyclosporine, sirolimus and tacrolimus), at all doses and

time points. We also did not detect HLA-G expression following exposure to different

periods of H/R. However, HLA-G expression was induced in progesterone-treated HCAEC,

HAEC and HCASMC cultures.

53

The Effect of Progesterone

Because HLA-G expression was only induced after incubation with progesterone, cells were

treated with varying progesterone doses for 24 h. Total HLA-G protein expression increased

in a dose-dependent manner (p < 0.001) as detected by ELISA (Figure 6), while cell surface

expression was confirmed by flow cytometry (Figure 7) in HCAEC, HAEC and HCASMC

cultures. Time course experiments showed maximal HLA-G expression within 12 h in all cell

cultures (p < 0.001) (Figure 8).

Inhibition Experiments

To determine if progesterone-induced HLA-G expression could be inhibited by the

progesterone receptor antagonist, mifepristone, cultured cells were incubated with

progesterone (10,000 ng/mL) in the presence or absence of mifepristone (1000 ng/mL) for 24

h. As measured by ELISA (Figure 9), HLA-G expression in HCAEC, HAEC and HCASMC

cultures was partially blocked by mifepristone (p < 0.05), indicating that the mechanism of

progesterone-induced HLA-G expression was through receptor binding.

Viability Studies

Cells were assessed for proliferation ability and viability following 24 h exposure to

progesterone with or without mifepristone to ensure that differences in HLA-G expression

were not the result of cellular injury. There were no significant differences in HCAEC,

HAEC or HCASMC viability following incubation with our interventions compared to

vehicle, indicating that these cultures were not injured by progesterone or mifepristone

(Table 4). In order to assess whether HLA-G expression might confer protection against H/R

injury, cells were subjected to 12 h hypoxia and 2 h reperfusion following 24 h progesterone

54

treatment. Viability studies demonstrated that HCAEC, HAEC and HCASMC cultures were

not protected against H/R injury following progesterone-induced HLA-G expression (Table

5). Relative to vehicle-treated cells under normoxia, there were no significant differences in

proliferation ability and viability following H/R stress in progesterone-treated cells compared

to vehicle-treated cells.

55

CHAPTER 5:

FIGURES AND TABLES

56

Figure 1. Protein structures of the HLA-G isoforms. To date, seven HLA-G variants have been reported. The transmembrane domain common to HLA-G1 to -G4 enable membrane anchoring. The HLA-G5 to -G7 structures are the soluble counterparts of HLA-G1 to -G3, respectively. HLA-G1 and -G5 possess all globular domains and non-covalently associate with β2M. Adapted from Carosella et al. (46).

57

Figure 2. Structures of HLA-G mRNA transcripts. HLA-G protein variants are the result of alternate splicing of the primary mRNA transcript. Exon 1 (E1) encodes the leader peptide, while exons 2, 3 and 4 encode the α1, α2 and α3 globular domains, respectively, of the heavy chain. Unlike the α1 domain which is common to all isoforms, α2 and α3 are absent in some HLA-G proteins. Exon 5 encodes the transmembrane region for the cell surface isoforms, HLA-G1 to -G4. Translation of these proteins terminates in exon 6 which encodes a shortened cytoplasmic domain. Intron 4 (I4) is retained in the mature mRNA transcripts of HLA-G5 and -G6, giving rise to a tail 21 amino acids in length. Similarly, the -G7 isoform has a short tail 2 amino acids long due to the presence of intron 2 in the mature transcript. The HLA-G5 to -G7 isoforms lack the transmembrane domain because of stop codons located within introns 2 and 4 and, thus, do not associate with the cell membrane. Adapted from Carosella et al. (46).

58

Figure 3. Immunohistochemical staining of MHC-I proteins in myocardial biopsies of heart transplant recipients. Expression of HLA-B and/or HLA-C in allograft myocardial cells (MC) and infiltrating leukocytes (IL) during Grade 0 rejection (a) or Grade ≥ 2R rejection (b) was revealed by the HC10 mAb to ensure tissue integrity. The MEM-G/2 mAb enabled detection of HLA-G+ (c) and HLA-G- (d) specimens, respectively, based on the presence or absence of red chromogen. HLA-G+ and HLA-G- biopsies were also identified by the staining (e) or absence of staining (f) with the 4H84 mAb, respectively.

59

Figure 4. HLA-G expression profile in non-rejecting and rejecting patients. Group A: patients without clinically significant rejection. Group B: patients with sustained moderate to severe rejection. The second biopsies of Group B patients were obtained during an episode of Grade ≥ 2R rejection. All other biopsy scores in both groups of patients were of rejection Grade ≤ 1R.

60

Figure 5. Characterization of cultured endothelial and smooth muscle cells through flow cytometric analysis. Cell surface protein expression of CD31 by HCAEC (a) and HAEC (b) confirmed endothelial phenotype. Intracellular α-smooth muscle actin (α-SMA) staining by HCASMC (c) confirmed smooth muscle lineage. More than 90% of cells stained positive for their respective markers.

61

Figure 6. Assessment of HLA-G expression in endothelial and smooth muscle cell cultures following progesterone treatment. HCAEC, HAEC and HCASMC were incubated for 24 h at 100-10,000 ng/ml. Treatment with vehicle alone failed to induce HLA-G expression in all cell types. HLA-G protein expression, determined by ELISA, increased with incremental treatment doses of progesterone in all cell types (p < 0.001). HLA-G concentrations were normalized for total cellular protein content.

62

Figure 7. Flow cytometric analysis of HLA-G expression in cultured endothelial and smooth muscle cells following progesterone treatment. HCAEC (a), HAEC (b) and HCASMC (c) were incubated with low (100 ng/mL) or high (10,000 ng/mL) doses for 24 h. Cell surface expression of HLA-G was not detected in vehicle-treated cells, but was present in all cultures following progesterone treatment.

63

Figure 8. Time course experiments assessing progesterone treatment on HLA-G expression. HCAEC (a), HAEC (b) and HCASMC cultures (c) showed maximal HLA-G expression within 6-12 hours of exposure following 10,000 ng/mL (p < 0.001). HLA-G levels were measured by ELISA and normalized for total cellular protein.

64

Figure 9. The effect of mifepristone, a progesterone receptor antagonist, on progesterone-induced HLA-G expression in cultured endothelial and smooth muscle cells. Compared to cells treated with 10,000 ng/mL progesterone alone (controls), HCAEC, HAEC and HCASMC incubated with 1000 ng/mL mifepristone in combination with 10,000 ng/mL progesterone had significantly reduced HLA-G expression. HLA-G levels were determined by ELISA and normalized for total protein content. All cultures were incubated for 24 h with treatment. *p < 0.05 compared to control.

65

Table 1. Baseline characteristics of non-rejecting and rejecting heart transplant patients.

Group A (n = 29)

Group B (n = 38) P Value

Demographics

Gender (male/female) 24/5 31/7 0.90

Mean Age (range) 51±12 (25-68) 47±13 (18-69) 0.28

Reason For Transplantation

Idiopathic Cardiomyopathy 13 (45) 20 (53) 0.53

Ischemic Cardiomyopathy 11 (38) 12 (32) 0.59

Other 5 (17) 6 (16) 0.87

Immunosuppressive Therapy

Cyclosporine 23 (79) 29 (76) 0.77

Tacrolimus 8 (28) 14 (37) 0.42

Mycophenolate Mofetil 25 (86) 36 (95) 0.23

Sirolimus 3 (10) 15 (39) 0.01

Prednisone 28 (97) 34 (89) 0.27

Donor Characteristics

Mean Age (range) 38±16 (15-67) 35±12 (15-56) 0.35

Gender (male/female) 20/9 26/12 0.96

Gender Mismatches 10 (34) 11 (29) 0.63

Pre-transplant PRA

0% 20 (69) 30 (79) 0.35

1-19% 6 (21) 4 (11) 0.25

20-100% 3 (10) 4 (11) 0.98

Unless specified, values in parentheses denote percentages. Non-rejecting and rejecting patients were separated into Groups A and B, respectively. PRA: Panel Reactive Antibodies.

66

Table 2. Expression of HLA-G in non-rejecting and rejecting patients.

Group A Group B P Value

HLA-G+ 25 (86) 4 (11) Patients

HLA-G- 4 (14) 34 (89) < 0.001

HLA-G+ 46 (53) 4 (4) Biopsies

HLA-G- 41 (47) 110 (96) < 0.001

Values in parentheses denote percentages. Non-rejecting and rejecting patients were separated into Groups A and B, respectively.

67

Table 3. Expression of HLA-G in cultured endothelial and smooth muscle cells following exposure to interventions of interest.

Intervention Dose Time HLA-G

IFN-γ 0.1-100 ng/mL 2-24 hr -

IL-10 0.1-100 ng/mL 2-24 hr -

H/R N/A 2-12 hr/2 hr -

Cyclosporine 100-1000 ng/mL 2-24 h -

Sirolimus 0.1-100 ng/mL 2-24 h -

Tacrolimus 0.1-100 ng/mL 2-24 h -

Progesterone 100-10,000 ng/mL 2-24 hr +

Plus signs indicate the presence of HLA-G following treatment in HCAEC, HAEC and HCASMC cultures. Minus signs indicate undetectable HLA-G expression that fell below the lower detection limit of the ELISA. N/A: not applicable.

68

Table 4. Viability and proliferation ability of treated endothelial and smooth muscle cell cultures.

Progesterone/Mifepristone Dose (ng/mL)

0/0 100/0 1000/0 10,000/0 10,000/1000

HCAEC 100 ± 5 102 ± 6 94 ± 9 98 ± 4 93 ± 8

HAEC 100 ± 6 100 ± 10 98 ± 7 96 ± 10 93 ± 9

HCASMC 100 ± 6 97 ± 5 104 ± 5 93 ± 5 96 ± 5

Data are represented as percentages of vehicle-treated cells.

69

Table 5. Viability and proliferation ability of treated endothelial and smooth muscle cell cultures following H/R stress.

Progesterone/Mifepristone Dose (ng/mL)

0/0 100/0 1000/0 10,000/0 10,000/1000

HCAEC 70 ± 3 73 ± 7 74 ± 7 74 ± 4 74 ± 5

HAEC 65 ± 7 70 ± 5 67 ± 6 67 ± 5 68 ± 5

HCASMC 82 ± 5 82 ± 4 84 ± 6 84 ± 4 87 ± 4

Data are represented as percentages of vehicle-treated cells under normoxic conditions.

70

CHAPTER 6:

DISCUSSION

71

6.1 MYOCARDIAL HLA-G RELIABLY INDICATES A LOW RISK OF ACUTE CELLULAR REJECTION FOLLOWING HEART TRANSPLANTATION

Currently, allograft rejection post-heart transplant is best monitored through myocardial

biopsies (170). However, assessment of rejection by this procedure is expensive, invasive

and subjective (171). For these reasons, it is important to develop non-invasive techniques to

monitor allograft status. Although molecules such as BNP, CRP and cardiac troponins have

been described as potential biological markers post-transplant, they indicate cardiac damage

rather than rejection. Therefore, our retrospective investigation was undertaken to assess the

value of HLA-G expression in determining freedom from clinically important rejection in

heart transplant patients. This study demonstrates that myocardial HLA-G expression

following heart transplantation is more prevalent in patients who exhibit lower rejection

scores. Both, the frequency of HLA-G expression and the proportion of HLA-G+ patients

were significantly greater in Group A (non-rejecting) versus Group B (rejecting), indicating

that HLA-G expression portrays a low risk of cardiac allograft rejection. Thus, myocardial

HLA-G expression post-transplant might reliably aid in identifying patients with a low

immunological risk of developing subsequent clinically relevant moderate or severe acute

cellular rejection episodes.

Our results are comparable to observations from other studies which demonstrate that

HLA-G expression is associated with fewer acute rejection episodes in the first post-

operative year, and absence of chronic rejection as assessed by coronary angiography in heart

transplant patients (104,105). However, using a case-control design we have illustrated for

the first time differential expression of HLA-G in two distinct populations of heart transplant

recipients, whereby non-rejecting patients were found to have significantly increased

72

myocardial HLA-G expression compared to rejecting patients post-transplant. Additionally,

we show for the first time that HLA-G expression is inversely associated with moderate and

severe rejection episodes after the first post-operative year, as Grade ≥ 2R rejection was

undetected in Group A patients up to 9 years post-transplant. Interestingly, unlike previous

reports of unvarying HLA-G status (104,105), HLA-G appears to have a dynamic pattern of

expression in our study population, a finding which is consistent with fluctuations in post-

transplant soluble HLA-G levels observed in other studies (107,108). While there was no

direct association between HLA-G status and rejection score at the time of a myocardial

biopsy, detection of an HLA-G+ biopsy appears to reliably indicate low risk of developing

moderate or severe graft rejection post-transplant, since 86% of recipients with no Grade ≥

2R rejection were HLA-G+. Nonetheless, prospective investigations involving larger patient

cohorts are required to investigate the utility and variability of HLA-G expression post-heart

transplant.

In addition to determining HLA-G status of heart transplant patients through

immunohistochemical staining of biopsies obtained from allografts, measuring sHLA-G in

these recipients might represent another potential strategy to monitor the degree of allograft

rejection. High sHLA-G concentrations in heart transplant patients appear to be associated

with reduced incidences of acute and chronic rejection (106-108). Similar phenomena have

been reported across the solid organ transplantation milieu. Soluble HLA-G levels in liver

and/or kidney transplant patients have a negative relationship with rejection and graft failure

(110,117,119). Moreover, post-transplant sHLA-G is inversely correlated with anti-HLA IgG

antibody production (110), possibly suggesting protection from humoral rejection. Since

HLA antibodies are linked to poor patient outcomes and rejection episodes post-cardiac

73

transplant (172), sHLA-G might possess further clinical relevance. Hence, quantifying

sHLA-G levels in conjunction with myocardial HLA-G staining might improve stratification

of patients according to immunological risk.

The use of ISHLT rejection scores represents a major limitation of this study. As all

biopsies were graded by one cardiac pathologist, rejection scores are subjective and may not

have been graded similarly by other pathologists. Furthermore, because the biopsies were not

graded concurrently, rejection scores are susceptible to intra-observer variability. Patient

clinical information may have also influenced the rejection grading. Another limitation of

this investigation is the use of retrospective biopsy samples. Incorporating such a procedure

into clinical practice might be challenging due to tissue degradation and different biopsy

storage approaches across heart transplant centers. Several patients were excluded from our

study as a result of poor tissue quality. Furthermore, tissue specimens show diminished

immunoreactivity over time. Even with the reassurance that the biopsies stained positively

for HLA-B and/or HLA-C, HLA-G might degrade differently and harbour false negatives.

The retrospective nature of this study also did not allow for quantification of soluble HLA-G;

however, previous studies have shown a significant correlation between myocardial and

soluble HLA-G expression (104,105). Nevertheless, to effectively utilize the HLA-G status

of patients in the clinical setting as a prognostic indicator of immunological risk, tissue

samples used for HLA-G measurement should be obtained prospectively during routine

myocardial biopsy procedures and employed with soluble HLA-G levels to help predict the

rejection profile of heart transplant recipients. It is important to note that HLA-G testing

should be performed repeatedly due to the risk of false negative staining.

74

Although our investigation in conjunction with other studies have demonstrated the

beneficial nature of HLA-G expression as it applies to protection from allograft rejection, it

remains unknown whether myocardial HLA-G expression following heart transplantation is

donor- or recipient-specific, why this expression is only presented by certain patients and

how it is induced. Nevertheless, both genetic and non-genetic factors are believed to be

responsible for this phenomenon. Distinct HLA-G alleles have been associated with

variations in HLA-G splicing patterns and mRNA levels (173). Additionally, different HLA-

G alleles have been shown to differentially influence soluble HLA-G concentrations (109),

which raises the possibility for differential HLA-G expression post-transplant based on

genotype. Immunosuppressive therapy has also been suggested to increase sHLA-G levels in

some heart transplant patients soon after administration (107,108), which might partially

explain the presence or absence of HLA-G expression in certain patients at different stages

post-transplant. With the exception of sirolimus, however, we did not detect significant

differences in immunosuppressive regimens between the two patient cohorts. It is unclear

whether sirolimus influenced our results because of the limited sample size. Given that HLA-

G has not been detected in healthy cardiac tissue and that sHLA-G levels are generally higher

in heart transplant patients, stressors in the peri- and/or post-operative period may represent

another likely explanation for HLA-G expression. This situation is not unexpected

considering mechanical and pathological stress may induce re-expression of cardiac fetal

genes (174-180). Ultimately, HLA-G expression in certain heart transplant patients is likely

the result of numerous non-genetic peri- and post-transplant factors affecting those who are

genetically-susceptible.

75

Is summary, our retrospective study illustrates an applicable inverse relationship

between myocardial HLA-G expression and clinically relevant cardiac allograft rejection,

suggesting that myocardial HLA-G staining indicates an improved tolerance profile post-

transplant. Yet, while HLA-G status may prove clinically useful as a prognostic indicator of

immunological risk, the ultimate goal of research in the field of organ transplantation is to

inhibit the initiation and progression of allograft rejection in transplant patients, without the

administration of lifelong immunosuppression to thereby uphold viable immune responses

against other pathogens. Therefore, it is important to scrutinize the potential strategies that

can hinder the rejection process, such as elucidating the mechanisms that contribute to HLA-

G expression following organ recovery, storage or transplantation. In heart transplant

patients, CAV (22-24) due in part to immune-mediated factors, and malignancy (18-21) due

to constant immunosuppression, represent the most prevalent long-term complications.

Induction of HLA-G expression in transplant recipients could represent a possible therapeutic

strategy to limit allograft rejection and reduce immunosuppressive medications along with

their associated negative side effects, and thus potentially improve long-term outcomes by

protecting against CAV and malignancy.

6.2 PROGESTERONE INDUCES EXPRESSION OF HLA-G IN VASCULAR ENDOTHELIAL AND SMOOTH MUSCLE CELLS IN VITRO

Following the results of our retrospective investigation which suggested that HLA-G

expression might reliably indicate freedom from clinically relevant rejection post-heart

transplant, we assessed whether vascular endothelial and smooth muscle cultures were

capable of in vitro HLA-G expression, as these could represent potential targets for future

76

therapies aimed at protecting against allograft vasculopathy. While HLA-G has been detected

in endothelial cells of chorionic fetal vessels during embryonic development, this expression

is lost in endothelial cells lining mature vessels (181,182). Our experiments, for the first time,

illustrate in vitro HLA-G expression in adult HCAEC, HAEC and HCASMC cultures.

Interestingly, increasing HLA-G levels were detected following treatment with incremental

progesterone doses in all cell types, without any changes in cellular proliferation ability or

viability; yet no expression was found after exposure to our other interventions including

cytokines, H/R stress or immunosuppressive agents, which may be present in the peri- or

post-transplant milieu, and have been shown experimentally to induce or upregulate HLA-G

mRNA and/or protein expression.

Treatment of normal blood monocytes, macrophage cell lines and constitutive HLA-

G-expressing cell lines (183-186) with IFN-γ has been shown to raise HLA-G mRNA levels,

leading to increased intracellular and cell-surface protein expression in vitro. This effect is

not unexpected considering IFN-γ and IFN-γ receptors are synthesized in first trimester

human trophoblasts (187,188), which coincide with HLA-G expression. Additionally, IL-10

has been shown to induce HLA-G mRNA and protein expression in peripheral blood

monocytes, trophoblasts and cells lines which constitutively express HLA-G (189,190).

Recent evidence also suggests that HLA-G expression in PBMC may be the result of an IL-

10 autocrine feedback loop (191), consistent with the fact that human cytotrophoblasts

produce and secrete IL-10 (192). However, treatment with IFN-γ or IL-10 did not induce

HLA-G expression in our studies, indicating that HLA-G expression appears to be tightly

regulated and specific for cell type.

77

We also examined the effect of H/R on HLA-G expression in our cell culture models,

because allografts are subjected to ischemia-reperfusion injury in the peri-operative period. It

has been shown that soluble and membrane-bound HLA-G mRNA expression is inversely

related to oxygen concentrations in primary cultures of extravillous cytotrophoblasts (193).

Furthermore, hypoxia has been demonstrated to differentially affect HLA-G expression in

various tumor cell lines by inducing HLA-G gene transcription in some HLA-G- lines and

decreasing constitutive expression in certain HLA-G+ lines, indicating hypoxic injury is

capable of modulating HLA-G expression in different cell types (194). Interestingly, the

HLA-G promoter contains a heat shock element which binds to heat shock factor 1 (195), a

transcriptional factor activated during conditions of environmental stress (196), thus

providing more evidence that HLA-G expression might be stress-inducible. For this reason,

we mimicked the ischemic and reperfusion times commonly observed in clinical

transplantation by exposing our vascular and smooth muscle cell cultures to H/R injury, but

were unable to detect any expression following this intervention.

Differential patterns of HLA-G expression following heart transplantation have been

noted in patients from our retrospective study and other investigations (104,105). Yet it

remains unknown why HLA-G is detected in only some patients post-transplant. While

genetic predisposition of the donor allograft and/or recipient represents a likely explanation

(109), immunosuppressive therapy might also play a role since different regimes are tailored

for different patients. We therefore assessed HLA-G expression after treatment with

cyclosporine, sirolimus or tacrolimus, as recent studies have implicated immunosuppressive

agents with increased serum HLA-G concentrations following heart transplantation

(107,108). After treating cell cultures with clinically relevant doses of these interventions, we

78

were unable to detect HLA-G protein expression, leading us to believe that HLA-G

expression in response to immunosuppressive therapy might be specific for cell type.

Finally, we determined the effects of progesterone and its receptor antagonist,

mifepristone, on HLA-G expression. Progesterone has been recently demonstrated to

enhance HLA-G expression in the JEG-3 choriocarcinoma cell line and isolated first

trimester cytotrophoblasts (197) through receptor activation followed by binding to a

progesterone response element (PRE), which shares 60% homology with the wild-type

mouse mammary tumor virus PRE sequence (198). Furthermore, progesterone receptors are

present in human heart as well as vascular endothelial and smooth muscle cells (199,200),

indicating that these tissues are possible targets for progesterone-induced HLA-G expression.

Intriguingly, our results show induction of this expression following treatment with

progesterone, and partial inhibition after co-incubation with mifepristone in HCAEC, HAEC

and HCASMC cultures, suggesting that the effect of progesterone was the result of

progesterone receptor activation. Complete inhibition was not noticed, however, since

mifepristone is a competitive inhibitor of progesterone. Following this finding, we assessed

the effect of HLA-G expression on protection from H/R injury. However, while HLA-G

upregulation might represent a protective response against hypoxic injury in tumor cells

(194), we found that progesterone-induced HLA-G expression did not protect against H/R

injury in our cell cultures.

Induction of HLA-G expression in endothelial cells is of particular interest as they are

primary targets of circulating T cells post-transplant, due to expression of classical MHC-I

and II antigens (161). After binding to classical MHC proteins, T cells secrete a host of

cytokines, leading to recruitment of inflammatory cells and proliferation of smooth muscle

79

cells, which can eventually progress into CAV (201,202). Endothelial HLA-G expression

following progesterone treatment could represent a strategy to inhibit adjacent T cells and

prevent the progression of CAV. Intriguingly, progesterone appears to have numerous other

beneficial effects on the vasculature such as suppressing endothelial VCAM-1 mRNA and

protein expression (203), which has been implicated in leukocyte adhesion (204), and

inhibiting proliferation of endothelial (205) and smooth muscle cells (206), all of which

contribute to atherosclerosis (206,207) and CAV (201,202).

Vasculopathy remains a primary complication post-heart transplant and represents a

major cause of late morbidity and mortality (22-24). Although it is the result of immune and

non-immune factors (25), the fact that transplant patients require lifelong

immunosuppression indicates that the immune system constantly directs responses to the

allograft likely through the indirect (40) and semi-direct (43) allorecognition pathways and,

over time, contributes heavily to CAV development. Hence, induction of HLA-G expression

in these patients might prove clinically relevant due to its inhibitory effects on NK cells (70-

75,77,79-81), T cells (65,66,82-89) and APC (92-96), and its ability to protect against CAV

(104-106). Expression of HLA-G post-transplant might also reduce the requirement for

immunosuppressive agents and thus lessen their associated negative side effects such as

malignancy, the leading complication post-heart transplant (18-21).

The beneficial effects of post-transplant soluble and membrane-bound HLA-G

expression are evident throughout the solid organ transplant milieu, including heart (104-

108), kidney (110,113), liver (88,119) and combined kidney-liver (87,88,114,117).

Investigations involving murine models also demonstrate longer skin allograft survival in the

presence of transgenic or recombinant HLA-G (92-96). Yet, the mechanisms of post-

80

transplant HLA-G expression remain vague. Peri- and post-transplant interventions are likely

to be responsible for this phenomenon because HLA-G is not detected in healthy organ

tissue. Therefore, our investigations were designed to assess whether factors such as

cytokines, H/R stress or immunosuppressive agents resulted in HLA-G expression in vitro.

While we did not detect HLA-G expression in response to these interventions, HLA-G was

detected after progesterone treatment. This result is consistent with the fact that progesterone

levels increase during pregnancy, a physiological situation resulting in fetal HLA-G

expression. Nevertheless, it still remains important to elucidate the precise mechanisms that

contribute to HLA-G expression following heart transplantation. Our experiments

demonstrate that although HLA-G is tightly regulated in human tissue, vascular endothelial

and smooth muscle cells are capable of HLA-G expression in vitro. Induction of this

expression in vivo might represent a novel therapeutic strategy to protect against acute

rejection episodes and CAV.

6.3 FUTURE PERSPECTIVES

Transplantation remains the definitive option for patients with advanced heart disease (10).

Successful clinical and basic science research has translated into improved short-term

outcomes following heart transplantation. Additionally, medical advances have resulted in

higher risk patients being eligible for transplantation, and the use of donor hearts that

previously may have not been suitable. For these reasons, improvements in patient survival

may actually be more meaningful than what they appear (12). However, despite such

advancements in early patient survival, the mortality rate in patients surviving past the first

post-operative year has remained unchanged (12). Therefore it is worthwhile to aim current

81

and future strategies towards solving the long-term problems which arise post-transplant. In

the context of heart transplantation, HLA-G is negatively associated with long-term rejection

as demonstrated by this investigation and those conducted by others (104-106). It is therefore

an attractive protein in this context because of its potential clinical implications.

In order to minimize inter-assay variability, standardized HLA-G assays need to be

developed if sHLA-G concentrations are to be used for future investigation or clinical

assessment. This will ensure that levels are measured accurately thus enabling results to be

reproducible and applicable across different transplant centres. HLA-G detection protocols

across centres must also remain consistent to maintain reproducibility. All subsequent assays

must be conducted with plasma rather than serum samples, since sHLA-G levels in plasma

are often greater than in serum (208). Therefore, serum HLA-G measurements, as reported in

several earlier clinical investigations, may not accurately true sHLA-G levels.

If HLA-G is to be utilized therapeutically in heart transplant recipients, either through

administration of recombinant protein or by induction of its expression via pharmacologic

mechanisms or gene therapy, the side-effects of such strategies need to be assessed. An

important consideration is the risk of malignancy which is a major complication post-heart

transplant (12). The relationship between HLA-G expression and cancers has been well

documented, suggesting that this expression might enable tumor survival (125,128,129).

However, it is important to emphasize that HLA-G has not been shown to induce malignant

transformation. Its association with cancer is likely the consequence of HLA-G upregulation

by malignant cells as a survival strategy. There is no evidence suggesting that pre-existing

HLA-G leads to the generation of new tumours. Furthermore, the onset of carcinogenesis

following heart transplantation is likely caused by immunosuppressive agents affecting the

82

cancer immunosurveillance capability of the recipient (18-21). If HLA-G decreases or

ultimately abolishes the need for lifelong immunosuppression, the risk of post-transplant

malignancies may decrease.

There remain many unanswered questions with respect to HLA-G in the realm of

heart transplantation. There is a poor understanding of the relationship between HLA-G on

antibody-mediated rejection, which is clinically relevant in heart transplant patients (172).

Given that HLA-G inhibits CD4+ cells, it is likely to modulate the humoral response to some

degree. It should also be assessed whether pre-transplant sHLA-G levels have any clinical

value. These levels might be associated with freedom from rejection and vasculopathy post-

transplant. Moreover, it remains unknown why HLA-G is detected in certain patients only.

Genetic factors likely play a role since as different HLA-G alleles have been shown to result

in differential protein expression (109). Yet, the fact that HLA-G has not been detected in

healthy cardiac tissue but in biopsies of post-transplant allografts indicates that there are

factors present in the peri- or post-transplant stages that induce expression of this gene. Our

experiments revealed that stressors such as cytokines, H/R or immunosuppressive agents

were unable to induce this expression in vitro. Nonetheless, genotype might predispose

certain individuals to express HLA-G post-transplant. Future investigations might assess the

HLA-G genotypes of organ donors and recipients to determine whether this has an affect on

post-transplant expression. Such issues may be better clarified through prospective

investigations.

Our in vitro investigations illustrate induction of HLA-G expression in vascular

endothelial and smooth muscle cells following progesterone treatment. Yet, age and gender

did not appear to have a relationship with HLA-G expression among patients in our clinical

83

investigation. Future experiments may examine the role of progesterone on HLA-G

expression and transplant outcomes, to determine if it is indeed protective in vivo. These

investigations might also be expanded to co-culture models to assess whether progesterone

treated cells are protected from T cell and NK cell responses. Additionally, it may be

worthwhile to examine the role of circulating progesterone levels on HLA-G expression in

vivo. For instance, fluctuations in progesterone levels during the menstrual cycle may

influence sHLA-G levels.

Transplanted organs, undoubtedly, do not survive as long or perform as well as

healthy organs due to a multitude of factors. Research in the realm of transplantation is

directed towards advancements in several areas including surgical technique, donor organ

preservation, immunosuppressive therapy, tolerance induction and xenotransplantation.

Improvements in these areas can improve patient survival, reduce negative side effects and

potentially solve the organ shortage problem. The generation of tolerance induction,

however, is considered the ultimate goal of transplantation research. Since the fetus can be

considered a semi-allograft, it might be worthwhile to study the mechanisms of fetal

protection during pregnancy and to determine whether these mechanisms can be applied to

the realm of transplantation. Expression of HLA-G is one such strategy by which an

immunosuppressive protein expressed primarily during pregnancy is applicable to the post-

transplant setting. Other immunosuppressive molecules that participate in fetal tolerance,

such as galectin-1 (209), might represent useful candidate proteins for gene therapy

following transplantation to combat rejection and vasculopathy. Applying concepts from

pregnancy to transplantation might ultimately represent an effective strategy to induce

allograft tolerance.

84

The negative relationship between allograft and/or soluble HLA-G expression and

graft dysfunction secondary to rejection has been well documented following solid organ

transplantation. The clinical relevance of HLA-G in the transplant milieu can be attested to

the fact that such findings are based on studies encompassing over one thousand heart, liver

and kidney transplant patients (46). Allograft HLA-G expression inhibits NK cells, T cells

and APC in the local milieu and, unlike medical therapy, does not systemically

immunosuppress the host, making it particularly attractive. Our studies have shown that

HLA-G expression reliably indicates freedom from clinically significant rejection and that

this expression can be induced in vitro. Therefore, there is considerable potential for HLA-G

expression as a prognostic indicator or as a therapeutic endpoint to protect against allograft

rejection and vasculopathy in heart transplant recipients.

85

CHAPTER 7:

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