Materials and Methods

ΠΑΝΕΠΙΣΤΗΜΙΟ ΠΑΤΡΩΝ
ΣΧΟΛΗ ΕΠΙΣΤΗΜΩΝ ΥΓΕΙΑΣ
ΤΜΗΜΑ ΙΑΤΡΙΚΗΣ
ΜΕΤΑΠΤΥΧΙΑΚΟ ΠΡΟΓΡΑΜΜΑ ΣΠΟΥΔΩΝ
«ΒΑΣΙΚΕΣ ΙΑΤΡΙΚΕΣ ΕΠΙΣΤΗΜΕΣ»
ΚΑΤΕΥΘΥΝΣΗ: ΠΑΘΟΒΙΟΧΗΜΕΙΑ
ΑΙΜΑΤΟΛΟΓΙΚΟ ΤΜΗΜΑ ΠΑΘΟΛΟΓΙΚΗΣ ΚΛΙΝΙΚΗΣ
ΕΡΓΑΣΤΗΡΙΟ ΜΕΤΑΜΟΣΧΕΥΣΗΣ ΜΥΕΛΟΥ ΤΩΝ ΟΣΤΩΝ
«Eπαγωγή της έκφρασης του μορίου HLA- G in vitro σε
λεμφοκύτταρα περιφερικού αίματος υγιών ατόμων και
λειτουργικός χαρακτηρισμός αυτών»
Ζούδιαρη Αναστασία
Βιολόγος
2013
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Στους γονείς μου…
«Θέλησα τον κόσμο να αγγίξω ξανά..»
Ν. Ζούδιαρης
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COMMITTEE
Alexandros Spyridonidis
Associate Professor, Medical School, Patras University
Athanasia Mouzaki
Professor, Medical School, Patras University
Solomou – Liosi Elena
Lecturer, Medical School, Patras University
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Acknowledgements
This work was carried out in the Laboratory of Haematology,
Haematology Division, Department of Internal Medicine between the
years 2009-2012.
I would like to thank my supervisor Prof. A. Spyridonidis for giving me
the opportunity to enter the research world and to find out my personal
expectations.
I would like to specially thank Prof. A. Mouzaki for her discrete presence
and support in general academic pursuits that I had during my master
studies.
I would also like to thank Lect. E. Solomou-Liosi for agreeing to be on
my thesis committee.
I would like to express my gratitude to my former colleagues and
intimate friends Georgia Oikonomopoulou and Ioanna Lazana for their
major support all these years and for their attempt to color my life.
Georgia was my guidance in the lab and helped me a lot in different
aspects of my master studies. Ioanna was the most important being
without her guidance, support and good nature I would never have been
able to finish this master thesis.
I would also like to thank my friend Dimitra Kokkinou for her constant
presence, our daily conversations and the patience that she exhibited
when things went wrong. Dimitra helped me a lot performing FACS
sorting and discussing part of this project.
Special thanks to my family and friends outside the lab. I am so happy
that you belong in my life.
I would like to thank all the members of the lab (Elena Kalyvioti, Fotis
Karagiannis, Stella Karambina, Eustathia Voulgari, Viktoria Katsarou,
Christos Sotiropoulos) for the friendly atmosphere.
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Table of contents
Summary ................................................................................................................................. 11
I. Introduction .......................................................................................................................... 13
1.1. Haematopoietic Stem Cell Transplantation ...................................................................... 14
1.1.1
Stem Cell Sources................................................................................................ 14
1.1.2
Types of haematopoietic stem cell transplantation .............................................. 15
1.2. Graft-versus-host disease (GvHD) ................................................................................... 15
1.2.1 Future Strategies – Cellular Therapies ....................................................................... 17
1.2
Mesenchymal stem cells (MSCs) for treating GvHD .................................................. 18
1.4. Naturally occurring T regulatory (nTregs) cells for treating GvHD ................................ 19
1.4.1 Natural occurring T regulatory cells – General .......................................................... 19
1.4.2 Inducible T regulatory cells ........................................................................................ 21
1.5 The non-classic HLA class I molecule, human leukocyte ................................................. 26
antigen (HLA)-G ..................................................................................................................... 26
1.5.1 HLA-G: Structure and Polymorphism........................................................................ 26
1.5.2 The functional properties of HLA-G molecule .......................................................... 28
1.5.3 Regulation of HLA-G expression ............................................................................... 31
1.5.3.1 Epigenetic regulation ........................................................................................... 32
1.5.4 HLA-G expression in healthy individuals .................................................................. 32
1.6 Epigenetics ........................................................................................................................ 33
1.6.1 General ....................................................................................................................... 33
1.7 Epigenetics and Demethylating Agents............................................................................. 34
1.7.1 General ....................................................................................................................... 34
1.7.2 Chemical Structure ..................................................................................................... 35
1.7.3 Mode of action............................................................................................................ 36
1.7.4 Future perspectives ..................................................................................................... 37
1.8 Epigenetics and Histone modifications ............................................................................. 38
Aim of the Study ..................................................................................................................... 40
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II. Materials and Methods........................................................................................................ 42
2.1 Cell Isolation ..................................................................................................................... 43
2.1.1 Cell Separation over Ficoll ......................................................................................... 43
2.2 Cell treatments and culture ................................................................................................ 44
2.2.1 Demethylating treatment ............................................................................................ 44
2.2.2 Histone deacetylase (HDAC) inhibitory treatment..................................................... 44
2.2.3 Cell cultures ................................................................................................................ 44
2.3 Flow Cytometry ................................................................................................................. 44
2.4 Fluorescence Activated Cell Sorting (FACS) ................................................................... 46
2.5 Suppression assays ............................................................................................................ 47
2.5.1 Transwell System ....................................................................................................... 49
2.5.2 Blocking Experiments ................................................................................................ 50
2.6 T cell stimulation assays .................................................................................................... 50
III. Results ............................................................................................................................... 53
3.1 The effect of the hypomethylating agent 5-aza-dC on HLA-G expression and cell viability
in human peripheral blood T cells in vitro .............................................................................. 54
3.2 Confirmation of HLA-G induction on CD3+ T cells after treatment with 5-aza-dC ......... 60
3.3 Aza-treated HLA-Gpos T cells are suppressive and this suppression is partially reduced
after neutralization of HLA-G and is not dependent on cell-to-cell contact ........................... 62
3.4 Aza-treated HLA-Gpos T cells show a reduced proliferation to allogeneic stimuli ......... 67
IV. Discussion ......................................................................................................................... 70
4. Discussion and perspectives ................................................................................................ 71
V. References .......................................................................................................................... 74
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Summary
There is an urgent need for novel preventive and therapeutic strategies for graft versus
host disease (GvHD) occurring after allogeneic hematopoietic cell transplantation
(allo-HCT). T-cell-based immunotherapies have been developed, however there are
still some hurdles for the use of currently availably regulatory T-cells in clinical
practice (naturally occurring FOXP3+ nTregs and inducible regulatory T cells),
mainly owing to the lack of specific cell surface markers. The hypomethylating agent
azacytidine (5-aza-dC) has been shown to generate immunoregulatory T-cells ex vivo.
Interestingly, it has been shown that genes other than FOXP3 are responsible for the
suppressor function of 5-aza-dC induced T-regs. HLA-G is a surface molecule with
potent immunoregulatory functions which is normally expressed during pregnancy
protecting the “semi-allogeneic” fetus from maternal immune attack and then is
epigenetically repressed. The aim of this study was the induction of HLA-G
expression in T-lymphocytes with the use of the demethylating agent 5-Aza-dC and
investigation of their possible immunoregulatory properties. Our results showed that
short in vitro treatment of peripheral blood T-cells with 5-aza-dC induces HLA-G
expression and, more importantly, these induced HLA-G+ T-cells could suppress
lymphoproliferation when added as third party cells in mixed lymphocyte cultures.
This suppression seems to be reduced after HLA-G neutralization and cell-to-cell
contact independent. Furthermore, these induced HLA-G+ T-cells show a reduced
proliferation to allogeneic stimuli. Taken together, our results indicate the ex vivo
production of HLA-Gpos T-lymphocytes with immunoregulatory properties. Our long
term goal is the use of this population as adoptive cellular therapy for GvHD and
other T-cell mediated diseases.
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I. Introduction
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Introduction
1.1. Haematopoietic Stem Cell Transplantation
Haematopoietic stem cell transplantation (HSCT) is the procedure of choice for the
cure of many malignant and non diseases. It refers to the provision of haematopoietic
stem cells to repopulate the bone marrow and generate a new immune system, after
their exposure to high-doses chemotherapy and/or total body irradiation, leading to
their elimination.
1.1.1 Stem Cell Sources
Haematopoietic stem cells (HSCs) reside in the medulla of bone marrow and give rise
to all the blood cell types: T, B and NK cells, lymphocytes, erythrocytes,
megakaryocytes / platelets, granulocytes (neutrophils, baseophils, eosinophils),
monocytes, macrophages and dendritic cells. They are a rare and heterogeneous
population, constituting 1:10,000 of bone marrow cells (Morrison and Weissman
1994; Bonnet 2002). Typically, HSC is a cell owing the following properties: (i).
longterm self renewal, (ii). ability of differentiating to a variety of specialized cells
(multipotency) and (iii). repopulating capacity .
Today, several alternatives are available for the isolation of stem cell transplant and it
can be performed as follows:

Bone Marrow: The classic source of haematopoietic stem cells is bone
marrow (Thomas and Storb 1970) having a 18-fold increase in the percentage
of CD34 as opposed to the respective percentage among circulating total
nucleated cells at steady state in healthy individuals. Over the last 40
years, bone marrow harvesting has become a routine procedure for HSC
transplantation. It refers to the isolation of haematopoietic stem cells by
repeated aspirations of the posterior iliac crests of the donor, using a special
needle and syringe, under general or local anesthesia in hospital.

Peripheral Blood: Stem cells mobilization from bone marrow under the
effect of the granulocyte colony-stimulating factor (G-CSF) has led to the
conception and to further implementation of collecting stem cells from
peripheral blood via leukocytopheresis (Schroeder, Kamperis et al. 2006).
For allogeneic transplantation, it is now very often used as an alternative to
bone marrow.

Cord Blood: Umbilical cord blood and placenta are highly enriched in
haematopoietic stem cells, making them an attractive candidate for stem cell
14
Introduction
collection (Schroeder, Kamperis et al. 2006). Haematopoietic stem cells have
been harvestd routinely from the umbilical cord blood and cryopreserved at
birth for their later use as child/adult stem cell transplants, now accounting for
over 20% of all stem cell transplants (Gluckman and Rocha 2005).
1.1.2 Types of haematopoietic stem cell transplantation
There are two types of transplants:

Autologous: An autologous transplant refers to the administration of highdose chemotherapy followed by infusion of haematopoietic stem cells that
have been previously collected from the patient himself. The following
administration of haematopoietic stem cells aims to replace the bone marrow
that has been previously destroyed, in order to generate a new haematopoietic
system.

Allogeneic: An allogeneic transplant refers to the administration of highdose chemotherapy, in order to destroy the cancer cells, followed by
infusion of haematopoietic cells that have been previously collected from a
donor, to replace the destroyed bone marrow. Allogeneic transplantation is
commonly preferred for diseases such as leukemia, aplastic anemia,
myelodysplasia, myelofibrosis etc, affecting the bone marrow. This type of
transplant is associated with lower relapse rates due to the graft versus tumor
(GvT) effect. GvT effect is mediated through the donor T cells which
recognize attack and eradicate the antigenically foreign cancer cells. However,
besides this beneficial effect, the donor T cells may simultaneously cause
the life-threatening graft versus host disease (GvHD).
1.2. Graft-versus-host disease (GvHD)
Graft-versus-host disease is the major complication of allogeneic HSCT. GvHD is
initiated by donor T cells that respond to alloantigen presented on host antigen
presenting cells. GvHD is more frequent and severe when HLA-mismatched grafts are
used (major mismatch), but also occurs with a frequency of up to 60% when the donor
and the recipient are fully HLA matched (minor mismatch). GvHD is a principle
contributor to transplant-related non-relapse morbidity and mortality following
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Introduction
allogeneic HCT and represents the major non-relapse barrier to the success of this
otherwise potentially curative treatment approach (Passweg et al, 2012).
GvHD occurs as an acute (aGvHD) or chronic (cGvHD) clinical syndrome, somewhat
arbitrarily defined as occurring prior to or after 100 days post transplant, respectively.
Specifically, aGvHD manifest most commonly as an acute inflammatory process
principally involving the integument, intestinal tract and liver and frequently presents
as a maculopapular rash, nausea, vomiting and diarrhea and hepatic cholestasis,
respectively. In contrast, cGvHD is a chronic inflammatory process leading to fibrosis
of involved organs and frequently presents clinically with SICCA syndrome-like
features, scleradermatous-like skin changes, chronic fibrosing pulmonary, hepatic and
intestinal manifestations and cytopenias. The severity of aGvHD is determined by a
staging/grading system grade I-IV, with higher grades related to a worsening
prognosis and likelihood of response to any therapy. Chronic GvHD historically has
been determined as limited or extensive and represents the leading cause of late
treatment related deaths among recipients of allogeneic HCT (Mc Guirk; Weiss,
2011).
The pathophysiology of GVHD involves cellular as well as cytokine-mediated
mechanisms. Tissue damage and cytokine dysregulation caused by the conditioning of
the patient provide a pro-inflammatory environment even before allogeneic T cells
enter the body. Host reactive donor T cells then respond to major or minor
histocompatibility antigens consisting of mismatched HLA/peptide complexes or
matched HLA molecules loaded with host-specific peptides derived from
polymorphic proteins, respectively. Residual host antigen-presenting cells (APC) play
a crucial role for the early activation of donor T cells, since they present the relevant
alloantigens and provide potent co-stimulation for the initiation of the alloresponse.
Depending on the degree of HLA disparity and the (yet unknown) number of protein
polymorphisms between donor and recipient, the precursor frequency of alloreactive
T cells regularly exceeds that of T cells reacting to environmental antigens. The
combination of high precursor frequency, high T cell receptor (TCR) avidity, strong
costimulation and a pro-inflammatory cytokine milieu results in the early activation
and expansion of alloreactive T cells that further perpetuates the inflammatory
process. Responding T cells differentiate into effector cells and either cause target
tissue destruction directly or, by modulating the activity of other cell populations of
the innate and adaptive immune system, indirectly (Ferrara et al. 1999).
Several well defined risk factors associated with the development and severity of
GvHD include human leukocyte antigen (HLA) mismatching between donor and
recipient, sex mismatching, advanced recipient and/or donor age, stem cell source,
and methodology of GvHD prophylaxis.
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Introduction
Two principal approaches to the management of GvHD include prevention and
treatment. The most commonly employed strategies to prevent GvHD include optimal
HLA matching at MHC class I and II loci between donor and recipient, and blocking
T-cell antigen recognition and resultant proliferation during the early initiating phases
of GvHD through pharmacologic prophylaxis. Less common, but increasingly utilized
approaches include graft manipulation through in vivo or ex vivo T-cell depletion
strategies and limiting tissue damage caused by the preparative regimen. Once an
inflammatory cascade is triggered and donor’s T-cells begin destroying host tissues,
the treatment regimens for GvHD ensue. GvHD treatment involves various
immunosuppressive therapies. The standard initial treatment is steroid therapy (Mc
Guirk; Weiss, 2011).
1.2.1 Future Strategies – Cellular Therapies
Despite the aforementioned therapeutic measures, aGvHD will eventually
progress, resulting in high rates of morbidity and mortality. Notably, only 30%40% of patients will respond to steroid therapy, with overall survival ranging
between 50%-60%, while for the case of steroid-refractory disease, the percentage of
long-term survival shrinks to 5-30%. Furthermore, up to 70% of patients will develop
chronic GvHD. All the above underline the pressing need for developing new
treatment approaches for aGvHD.
Peripheral tolerance mechanisms that prevent autoimmunity or contribute to tolerance
induction after solid organ transplantation are also operational in allogeneic SCT and
eventually confine such alloaggression These mechanisms include activation-induced
cell death, clonal exhaustion, T cell anergy, or ignorance and peripheral deletion. In
addition, active suppression of alloreactive cells has increasingly been recognized as a
major means for the generation and maintenance of alloantigen-specific
unresponsiveness, as studied extensively in a number of trans-plantation models. In
experimental hematopoietic SCT, several cell populations have been implicated in
these suppressive effects, such as NKT cells, CD4–CD8– (double negative) T cells,
cells containing “veto” activity, as well as regulatory T cell populations (Treg)
generated in vitro or in vivo by various manipulations. More recently the immune
regulatory potential of mesenchymal stem cells (MSCs) represents a novel treatment
for GvHD. In addition, adoptive Treg cellular therapies have been found to be
efficacious in controlling alloimmune responses in the settings of GvHD in animal
models (Hoffmann et al, 2005).
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Introduction
1.2 Mesenchymal stem cells (MSCs) for treating GvHD
One promising treatment for GvHD involves the infusion of third party, HLAdisparate; unrelated mesenchymal stromal cells (MSCs) which are non-hemopoietic
cells with the capacity to self-renew and differentiate into various cell lineages of
mesenchymal origin and were originally identified by Friedenstein et al. These cells
which comprise a very small population (<0.1%) of adult bone marrow cells, provide
the supportive niche for hematopoietic stem cells (HSCs); however, recent studies
favor osteoblasts, a progeny of MSCs, as the main cell involved in HSC niche
(Battiwalla M, Hematti P, 2005).
These cells can be obtained from bone marrow, adipose tissues, fetal liver and
umbilical cord blood. The in vivo and in vitro properties of MSC suggest their
potential use in a broad range of inflammatory and immune-mediated conditions, such
as GvHD. MSC are a population of undifferentiated multipotent mesenchymal
stromal cells which express HLA class I and do not express HLA class II or co
stimulatory molecules CD40, CD80 or CD86 and have been demonstrated to
modulate immune and inflammatory response in animal models of inflammatory
disease including GvHD, and to facilitate repair of connective tissues (Battiwalla M,
Hematti P, 2005).
MSCs inhibit the activation of and proliferation of activated T-cells that have been
induced by a variety of stimuli and down-regulate inflammatory cytokine expression
such as tumor necrosis factor (TNF)-α, IL2R-α, , and interferon- γ (IFN-γ). On this
basis, it was conceived the idea of using MSCs as therapeutic cells for treatment of
aGvHD. Le Blanc et al. was the first who used MSCs for steroid-resistant
aGvHD, reporting responses in 55% of patients (Le Blanc, Frassoni et al. 2008).
However, the following study of Von Bonin et al. demonstrated that despite the
use of MSCs as third party cells, patients eventually developed aGvHD (von
Bonin, Kiani et al. 2009). Important for the availability of off-the-shelf cell therapy,
MSCs from freshly expanded samples or from cryogenically stored/thawed cell
preparations have been used with no apparent differences in response. MSCs have
been shown to be safe: no ectopic tissue formation has been derived from infused
MSCs. Finally, MSCs did no harm: no clearly defined increased incidence of
opportunistic infections or relapse of malignancy was reported. In summary, the data
support the concept of MSCs as a safe, well-tolerated and variably effective treatment
for GvHD. Importantly; MSCs can be cryogenically banked, thawed and given
without the need for donor-recipient matching.
MSCs have specific problems that limit their usefulness. For example, MSC isolation
requires aspiration from the marrow cavity which is a painful, invasive procedure,
18
Introduction
with certain risks. Several studies indicate that adult-derived MSCs have limited
expansion potential or slower expansion in vitro compared to fetal-derived MSCs.
Adult MSCs may be less responsive than fetal or neonatal MSCs in certain
applications because they may not expand to clinically relevant number and may be
unsuitable for therapy. It is well understood that MSCs have limitations that may
affect their clinical potency and impact (Yoo KH et al, 2009).
In conclusion, the clinical experience with MSC for the treatment of GvHD is
encouraging, but incomplete. Significant questions remain regarding the optimal
culture conditions, biodistribution, and persistence of MSC, as well as potential longterm toxicities and risk for infection. Future clinical trials should be designed to
determine the optimal dose and schedule of MSC administration, so that MSC are
used most effectively (Horwitz et al, 2011).
1.4. Naturally occurring T regulatory (nTregs) cells for
treating GvHD
1.4.1 Natural occurring T regulatory cells – General
Regulatory T cells are crucial for maintaining tolerance and fundamental in
controlling various immune responses by downregulating undesired immune
responses to self and non-self antigens. Several different regulatory T-cell subsets
have been described.
The best-described population of Tregs is the so-called natural Tregs. These are CD4+
T cells arising during T-cell development in the thymus (thymus-derived) and
constitutively expressing high levels of the α chain of the interleukin (IL)-2 receptor
(CD25). They represent about 5–10% of total CD4+ T cells in the periphery In vivo,
CD4+CD25+ Treg cells contribute to the maintenance of self-tolerance and thereby
protect from a variety of autoimmune diseases (Sakaguchi S et al, 2006). They control
the size of the peripheral T cell pool and modulate immune responses to infections, to
tumors and to allogeneic organ grafts. Naturally occurring CD4+ Tregs constitutively
express a variety of cell surface molecules commonly associated with
activated/memory cell phenotype. These include CD25, CD45RBlow CD62L,
cytotoxic T-lymphocyte anti-gen-4 (CTLA-4, or CD152) and glucocorticoid-induced
tumor necrosis factor receptor (GITR) family-related gene. Although none of these
surface markers is uniquely expressed by natural Tregs, their level of expression and
constitutive nature have made them useful as functional descriptors and enabled the
consistent isolation and investigation of CD4+ T cells with suppressive capabilities.
Work in recent years has shown that the forkhead family transcription factor (FoxP3)
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Introduction
is critically important for the development and function of Tregs. Genetic mutations
in the gene encoding FoxP3 have been identified in both humans and mice, and result
in fatal autoimmune diseases. Humans with mutations in FoxP3 suffer from a severe
and rapidly fatal autoimmune disorder known as the immune dysregulation,
polyendocrinopathy, enteropathy X-linked (IPEX) syndrome. An analogous disease
also occurs in mice, known as ‘scurfy’, owing to a spontaneous mutation in FoxP3.
Furthermore, ectopic expression of FoxP3 can phenotypically and functionally
convert effector T cells (Teffs) to Tregs. It appears that in mice, FoxP3 expression is
both necessary and sufficient for Treg development. However, there is accumulating
evidence demonstrating that, in contrast to mice, human FoxP3 is transiently
expressed in activated T cells where it could be detected within 24h and peaked at
72h. The observation that abundant FoxP3 messenger RNA (mRNA) was detected in
the recently activated CD4+ CD25+ cells lacking regulatory function strongly suggests
that in humans, FoxP3 expression alone is not sufficient to indicate regulatory activity
of CD4+ CD25+ cells. The quest to identify molecules, especially cell surface markers
that uniquely define Tregs, has led to a recent identification of an additional potential
marker, the IL-7 receptor a chain (CD127) where its expression it remains low or
even undetectable (NT Le and N Chao, 2007).
Table 1.1: The table summarizes the different types of Treg subsets in the immune system
(NT Le and N Chao, 2007)
Moreover they are characterized by their inability to produce interleukin-2 (IL-2) and
to proliferate in vitro. Their most characteristic feature, however, is their functional
behavior, i.e., their anergic state and suppressive activity. Anergy, i.e. the impaired
proliferative response of Treg cells to standard T cell stimuli in vitro does not,
however, indicate that these cells are generally insusceptible to activation. On the
contrary, to gain suppressive function, CD4+CD25+ Treg cells require antigenspecific activation via their TCR. Furthermore, their inability to proliferate upon
20
Introduction
TCR/CD28 engagement is a result of the failure of these cells to actively transcribe
and secrete IL-2 (Thornton and Shevach 2000).
Once activated, they suppress the proliferation and cytokine secretion of co-cultured
conventional CD4+ and CD8+ T cells antigen-nonspecifically by inhibiting IL2
transcription. Treg cells can also suppress other cell types, such as antigen-presenting
cells. Most in vitro studies indicate that Treg cells mediate suppression by an
undefined cell-contact-dependent mechanism. However, in vivo, suppression may be
mediated by suppressive cytokines. Treg cells may facilitate the production of IL-10
and transforming growth factor-β (TGF-β) by other T cells through “infectious
tolerance” (recognize antigens derived from pathogens - essential step in their
regulatory function). Alternatively, under certain conditions, Treg cells might acquire
the ability to produce TGF-β and/or IL-10. The relationship between nTreg cell
anergy and suppressive activity is unknown. Initial studies showed that abrogation of
the anergic state by in vitro TCR stimulation in the presence of a high dose of IL-2 or
CD28 ligation results in simultaneous loss of suppressive activity. Moreover, these
studies showed that this anergic state is a default state, as CD4+CD25+nTreg cells
spontaneously revert to the original anergic state and regain the suppressive activity
once IL-2 or anti-CD28 costimulation is removed. IL-2 is crucial for the generation,
expansion, survival and effector function of Treg cells. As Treg cells do not produce
IL-2 themselves, their capacity to use IL-2 secreted by target T cells appears to be
essential for their suppressive activity (Sakaguchi S at al, 2008).
Table 1.2: Mechanism of suppression: natural Tregs versus inducible Tregs.
1.4.2 Inducible T regulatory cells
In contrast to the intrathymic generating of natural Tregs, other types of Tregs can be
induced (inducible Tregs, iTregs) are generated in the periphery, presumably to help
terminate the response when the pathogen is eliminated and to prevent secondary
autoimmunity during the course of a normal immune response. Their generation is
dependent on the peripheral factors such as the maturation and type of the stimulating
21
Introduction
antigen-presenting cells (APC), availability of immunosuppressive cytokines such as
TGF-b and the presence of low-dose antigenic stimulation (NT Le and N Chao,2007).
Many different subsets of inducible regulatory T cells have been reported. Among
them are the T-cell subset (known as T regulatory type 1 (TR1) cells) that produces
high levels of interleukin-10 (IL-10). In addition, inducible FOXP3+CD4+CD25+
human regulatory T cells can be generated in vitro from CD4+CD25– T cells in the
presence of transforming growth factor-β (TGFβ) (Th3 cells). Th3 cells were first
identified because of their role in oral tolerance, through the secretion of transforming
growth factor (TGF)-b. Tr1 cells are very similar to Th3 cells but secrete large
amount of IL-10 and were characterized on the basis of their role in preventing
autoimmune colitis. (Roncarolo MG and Battaglia M 2007)
The most characterized category of inducible Tregs are Tr1 cells with a unique
cytokine production profile (IL-2low/–IL-4–IL-5+IL-10+TGFβ+) which distinguishes
them from T helper 0 (Th0), Th1 and Th2 cells. To date, no specific cell-surface
marker for Tr1 cells has been identified. TR1 cells have a very low proliferative
capacity following activation in vitro through the T-cell receptor (TCR), in part due to
autocrine production of IL-10. However, their proliferative activity in vivo is
unknown. Tr1 cells regulate immune responses through the secretion of the
immunosuppressive cytokines IL-10 and transforming growth factor-β (TGFβ), and
they suppress both naive and memory T-cell responses and downregulate the
expression of co-stimulatory molecules and pro-inflammatory cytokines by antigenpresenting cells (APCs). Tr1 cells are inducible, antigen specific and need to be
activated through their TCR to exert their suppressive functions. However, once
activated, they mediate suppression in an antigen non-specific manner. (Roncarolo
MG and Battaglia M 2007)
Although the inducible Tregs are largely contained within the CD4 T-cell
compartment, they are distinct from the natural Tregs in various aspects based on their
potential to produce certain signature cytokines after antigen priming. Other T-cell
populations with demonstrable immunosuppressive function, such as CD8 Tregs and
natural killer Tregs, have also been reported in different models of autoimmune
diseases and transplantation tolerance. There are also some other categories of
induced Treg cells which include counter-regulatory IFN-γ-producing Th1 cells and
IL-4-producing Th2 cells. Although CD4+CD25+ nTreg cells preferentially express
the cell surface or intracellular molecules described above, these markers are not
reliable indicators of iTregs, and there is no unique cell surface marker to distinguish
them from other T-cell subsets. So inducible Tregs do not have the classical CD4+
CD25+ T-cell phenotype and are rather defined on the basis of their cytokine profile.
Although FoxP3 is expressed by natural Tregs, at present it is not clear whether
FoxP3 also regulates the development of either Th3 or Tr1 cells. Indeed, Foxp3 + and
22
Introduction
Foxp3– iTreg cells can be generated in vitro or in vivo under various stimulation
regimes and generally function in a cytokine-dependent manner in vitro and in vivo.
(NT Le and N Chao 2007)
In contrast to natural Tregs, the inducible Tregs, including both Th3 and Tr1 cells,
appear to function independently of cell–cell interaction and suppress immune
responses through the secretion of cytokines such as IL-10 and TGF-b. Perhaps, the
model of ‘linked suppression’ (inducing the inducible Tregs - a potentially
alloreactive T cell comes under the influence of a Treg as both cells recognize their
respective alloantigens presented by the same APC) which involves both direct cell
contact and soluble cytokines could reconcile some of the apparent disparities
regarding themechanisms of suppression by Tregs.(Davies et al, 1996)
Figure 1.1: nTreg and iTreg CD4 cells control T cell responses. nTreg and iTreg CD4
populations potentially regulate the function of activated effector T cells in many different
immunological contexts, and the mechanisms to achieve this regulation are diverse. Although
CD4 nTreg cells emerge from the thymus, CD4 iTreg cells originate from the activation
and differentiation of conventional CD4 T cells in the periphery. CD4 iTreg cells operate
via the secretion of immunosuppressive cytokines including IL-10 and TGF- 1, and nTreg
cells might opt for cytokine-dependent (A), cell contact-dependent (B), or cytokine/cellular
23
Introduction
contact-dependent (A B) modes of action to control similar T cell responses. The
relative contribution of each subset in the overall regulation of immune responses is
unclear, but both can conceivably synergize to achieve this outcome. (NT Le and N
Chao 2007)
Little is known as to how the immunosuppressive activity of Tregs is downregulated
and/or terminated once it is no longer beneficial to the host. As Tregs are intrinsically
hypoproliferative and hyporesponsive, it is reasonable to propose that once the
inflammatory ‘cytokine storm’ subsides and in the absence of antigenic stimulation,
Tregs will resume to their original anergic state with diminishing immunosuppressive
activity. Further studies are certainly needed to address this important issue (NT Le
and N Chao et al, 2007)
The possibility that regulatory T cells might be used for the treatment of T-cellmediated diseases has recently gained increasing momentum. The transfer of freshly
isolated Treg cells together with the bone-marrow allograft has been shown to
ameliorate GvHD and facilitate engraftment in mouse models of HST. Further studies
revealed that transfer of Tregs in a ratio of 1:1 with effector cells protected from
aGvHD and significantly decreased the GvHD-related mortality (Cohen, Trenado et
al. 2002), (Hoffmann, Ermann et al. 2002). GvHD was also the first model in which it
was shown that the adoptive transfer of donor Treg cells that were polyclonally
expanded ex vivo is as efficient as the transfer of freshly isolated Treg cells in curing
disease that results from transplantation in mice models. Thus, the data accumulated
from the animal studies clearly indicate that CD4+CD25+Tregs play a vital role in
down regulating GvHD. Thanks to these promising results generated in the animal
models and to the lack of antigen-specific requirements for the transferred Treg cells,
haematopoietic stem cell transplantation is the setting for the first human clinical trial
with Treg cells generated ex vivo. Preliminary results from clinical trials with freshly
ex vivo isolated Treg cells and ex vivo generated Tr1 cells in patients undergoing
stem cell transplantation show feasibility and suggest a good safety profile. Efficacy
data will hopefully be generated in the coming years.(Hippen et al, 2011)
The advantages of the adoptive transfer of regulatory T cells over conventional
treatments are numerous. Some of these benefits include: the potential for antigen
specificity with the lack of general immunosuppression, the possibility of inducing
‘physiological’ long-lasting regulation in vivo, and the fact that regulatory T-cellbased immunotherapy could be a custom-made product, designed ad hoc for each
patient, with very limited or absent side effects.
The obstacles that limit regulatory T-cell-based immunotherapy at present are mainly
technical and relate to cell manipulation. The major limitation for the clinical
application of Treg -based therapy to prevent GvHD is obtaining sufficient n umbers
24
Introduction
of antigen-specific Treg and maintaining their regulatory properties after infusion.
Circulating Treg cells can be directly isolated from the circulating pool of CD4+ T
cells but because of their limited number they need to be further expanded in vitro.
Moreover lacking of specific cell surface markers and losing of their their suppressive
function is a major obstacle for their efficient purification after ex vivo expansion.
Several other points need to be considered for the development of regulatory T cells
as a medicinal product and many different quality controls are needed. Cell isolation
and manipulation are therefore prerequisite for regulatory T-cell-based
immunotherapy So, adoptive cell therapy remains a great challenge for many groups
worldwide. Safety of the infused ex vivo manipulated product is clearly a high
priority. The risk of uncontrolled cell proliferation, pan immunosuppression and
consequent tumour development should be carefully monitored. Furthermore, superior
efficacy of regulatory T-cell-based immunotherapy over conventional therapy should
be clearly demonstrated.
The aforementioned obstacles of T cell therapy make the investigation of new
therapeutic and/or preventive strategies of paramount importance.
Table 1.3: Regulatory –cell based immunotherapy in humans (Roncarolo MG and Battaglia
M, 2007)
25
Introduction
1.5 The non-classic HLA class I molecule, human leukocyte
antigen (HLA)-G
Since its first description in pregnancy, approximately 25 years ago, human leukocyte
antigen (HLA)-G has been extensively studied by an increasing number of
groups worldwide. HLA-G was initially detected as an unusual form of HLA class I
molecule, expressed by extravillous trophoblast from normal human placenta (Ellis,
Sargent et al. 1986). In 1990, Kovats et al. described the novel HLA class I
antigen, HLA-G, to be non-polymorphic and prominent in the first trimester
extravillous cytotrophoblasts, suggesting that this cell type specific molecule may be
essential to maintenance of the trophoblast and thus the fetus (Kovats, Main et
al. 1990).Human leukocyte antigen (HLA)–G is a nonclassic HLA class I molecule
that is characterized by a low polymophism, a highly restricted constitutive tissue
expression, and a tolerogenic function.
1.5.1 HLA-G: Structure and Polymorphism
The human major histocompatibility complex (MHC), namely human leukocyte
antigen (HLA), is located on the short arm of chromosome 6 (6p) and divided into the
following three subgroups: (a). class I: encoding the classic HLA-A, -B and -C
antigens, (b). class II encoding the classic HLA-DP, -DQ nad -DR antigens and (c).
class III. The gene structure of HLA-G is similar to that of the classic HLA class I
genes and it consists of 8 exons and seven introns. Exon 1 encodes the signal peptide,
exons 2, 3 and 4 encode the α1, α2 and α3 extracellular domains, respectively and
exon 5 the transmembrane domain. Exon 6 encodes the cytoplasmic domain of
heavy chain, which is shorter compared to the classical HLA class I genes, due
to a premature stop codon existing in its second codon. Exon 7 is always
absent from the mature mRNA and, due to the stop codon in exon 6, exon 8
is not translated (Carosella, Moreau et al. 2003). It has been previously documented
that in contrast to the highly polymorphic classical HLA class I molecules, HLA-G
gene has a low level of allelic polymorphism , having only 44 HLA-G alleles.
The HLA-G primary transcript may generate seven different mRNAs, by
alternative splicing, encoding seven protein isoforms; four membrane-bound
(HLA-G1, -G2, -G3, -G4) and three soluble (HLA-G5, -G6 and -G7). The
‘complete’ HLA-G1 isoform exhibits a structure similar to that of the membranebound classical HLA molecule: a heavy chain of three globular domains
noncovalently bound to β2m. The HLA-G2 isoform lacks the α2 domain, encoded by
exon 3, the HLA-G3 has no α2 and α3 domains, encoded by exons 3 and 4 and the
HLA-G4 has no α3 domain, encoded by exon 4 (Carosella, Moreau et al. 2003).
26
Introduction
Translation of intron 4 yields the HLA-G5 and HLA-G6 soluble forms, lacking
the transmembrane domain and the cytoplasmic tail, which are substituted by a
specific C-terminus sequence. The soluble HLA-G7 has only the α1 domain
linked to two amino acids encoded by intron 2, which is retained in the
corresponding transcript. All alternative transcripts lack exon 7. Beyond the
alternative spicing, soluble HLA-G isoforms may also be generated by cleavage
of the membrane-bound form from the cell surface by metalloproteinases .
Studies have converged that the ‘complete’ HLA-G1 isoform and its soluble
counterpart HLA-G5 are the most abundant expressed, while the ‘truncated’ isoforms
are expressed only in low levels. (Carosella, Favier et al, 2008)
A classic HLA class I molecule consists of three segments: (i). the
extracellular portion consisting of the β2-microglobulin (β2m) and the α1, α2
and α3 domains of the heavy chain, (ii). the transmembrane region of the
heavy chain and (iii). the intracellular segment of the heavy chain). Despite the
high similarity in the structure of the complete HLA-G1 and -G5 molecules with the
respective of the classic HLA class I molecules, there are some major differences.
These include the followings: (i). the α3 domain of HLA-G1/ -G5 is more
hydrophobic leading to a higher affinity for ILT2, than HLA class I molecules,
(ii). the HLA-G1 and -G5 molecules can also be found as β2m-free heavy chains
and (iii). the HLA-G molecules can be found as disulphide-bonded
homodimers.
27
Introduction
Figure 1.2: The generation of multiple isoforms of HLA-G by alternative mRNA splicing.
HLA-G heavy-chain proteins are encoded by up to six exons, each of which corresponds to a
discrete domain of the mature, full-length HLA-G1 molecule. (HLA-G mRNAs also contain
transcripts of two further exons 7 and 8, but these are always non-coding). During processing,
nascent HLA-G mRNA is spliced into one of at least six different forms, some of which lack
exons 3 and/or 4 and some of which retain intron 4.These mRNA species might be translated
subsequently into six protein forms, including truncated forms. Because of the retention of
intron 4, which contains a premature stop codon (marked by a red asterisk), two soluble forms
might be generated, which do not include the transmembrane region. Both HLA-G1 and
soluble HLA-G1 (sHLA-G1) associate with β2-microglobulin (Bainbridge D et al, 2001).
1.5.2 The functional properties of HLA-G molecule
Since its first description in pregnancy, HLA-G has been considered an
important mediator of immunotolerance, serving as an inhibitory ligand for immune
cells .The data accumulated on the immunological interplay between HLA-G
and immunocompetent cells are now compelling. Due to their abundant
expression, the HLA-G1 and HLA-G5 molecules have been studied the most,
while little is known about the function of the least expressed truncated isoforms.
-HLA-G1 functions (Carosella, Favier et al, 2008):
Concerning the function of HLA-G1 molecule on immune cells, experiments using
HLA-G1 transfected cell lines have previously documented that it may:
28
Introduction
(i). inhibit the CD4+ T cell proliferative response in vitro in allogeneic mixed
lymphocyte reactions
(ii).disrupt the cytolytic function of both uterine and peripheral blood NK cells,
through binding to ILT2 receptor
(iii). impair the antigen-specific cytolytic function of cytotoxic T lymphocytes in
a dose-dependent way
(iv). inhibit the proliferation of T cells and peripheral blood NK cells through HLA-G:
ILT2 interaction which results in induction of cell cycle arrest at G1 phase
(v). hamper the maturation and function of dendritic cells both in vitro and in
vivo
(vi). induce the apoptosis of CD8+cells after ligation to CD8 and via a FasFasL-dependant mechanism
Alongside its aforementioned direct inhibitory functions, the HLA-G1 molecule
has been described to exert further indirect tolerogeneic functions, which
include the followings:
(i). induction of longterm CD4+ T cell unresponsiveness
(ii). differentiation of CD4+ T cells into regulatory / suppressor cells able to
inhibit allogeneic responses
(iii). induction of tolerogeneic APCs
(iv). up-regulation of inhibitory receptor expression and expression of HLA-G
itself
In general, HLA-G is able to create a tolerogeneic environment by altering the
functions of immunocompetent cells. HLA-G1 or soluble HLA-G5 have similar
functions; however the other HLA-G isoforms have been less studied and little is
known about their function except that membrane bound HLA-G2, G3, and G4 can
inhibit NK and T cells cytolysis in vitro. (Carosella, Favier et al, 2008)
29
Introduction
Figure 1.3: HLA-G functions. HLA-G is able to create a tolerogeneic environment by
altering the functions of immunocompetent cells. HLA-G has proven to inhibit the
proliferation of T cells as well as their alloreactivity through binding to ILT2
receptor. Furthermore, it has been shown to inhibit proliferation of CD8+ T cells along with
their cytotoxic function by interacting with ILT2 receptor, while binding of CD8 coreceptor promotes CD8 apoptosis. HLA-G affects NK functions by inhibiting their
proliferation and cytolytic functions (through ILT2), while increased secretion of IFNγ
and proangiogenic factors has been also reported and been attributed to HLAG:KIR2DL4 interactions. NK apoptosis after HLA-G binding to CD8 co-receptor has
been also documented. Decreased production of HLA antibodies has been also reported.
The immunological functions of HLA-G on myeloid cells include the inhibition of DC
maturation and antigen presentation, generation of tolerogeneic APCs and induction of
suppressor T cells.(I.Lazana Doctoral Thesis)
HLA-G1 does not induce immune responses through TCR interactions but
rather through binding to inhibitory receptors. To date, 3 HLA-G inhibitory
receptors have been described: (i). ILT2/CD85j/LILRB1 (ILT2), which is
expressed by B cells, some T cells, some NK cells and all monocytes/
dendritic cells, (ii). ILT4/CD85d/LILRB2 (ILT4), which is myeloid-specific, being
expressed only by monocytes/ DCs and (iii). KIR2DL4/CD158d
(KIR2DL4),
which is restricted to NK cells (Contini P. et al,2000).
30
Introduction
Figure 1.4: HLA-G is well known to act through binding of inhibitory receptors, such as
immunoglobulin-like transcript 2 (ILT2), ILT4, and killer-cell immunoglobulin-like
receptor,2 domains, long cytoplasmic tail, 4 (KIR2DL4), that are differentially expressed by
immune cells, but binding to CD8 has also been reported (Contini P. et al,2000)
Beyond the inhibitory functions of HLA-G1 molecule on all cell actors of an immune
response (i.e. NK cells, T cells, APCs), HLA-G1 has been also shown to act through
modulation of cytokines release. It has been previously demonstrated that the
recognition of membrane-bound HLA-G1 by target cells shifts the balance of Th1 and
Th2 cytokines secreted by PBMCs or decidual tissues to Th2 polarization.
1.5.3 Regulation of HLA-G expression
Beyond the maternal-fetal interface and embryonic tissues, HLA-G expression
has been also described in adults’ tissues and particularly in immunoprivileged
sites. Specifically, HLA-G constitutive expression has been documented in adult
thymic medulla , cornea, pancreatic islands and erythroid and endothelial-cell
precursors and mesenchymal stem cells (MSCs). The reason why HLA-G
expression is detected only in specific tissues remains elusive.
However, upregulation of HLA-G expression has been described in various
pathologic conditions such as those involving cancers, transplantation, inflammatory
and autoimmune diseases , and viral infections .
The identification of HLA-G expression in conditions such as pregnancy,
transplantation and cancer, highly suggests that microenvironmental factors influence
the HLA-G gene expression. Indeed, there are compeling evidence indicating the role
31
Introduction
of several cytokines in regulation of HLA-G expression Notably, both proinflammatory (i.e. TNF-α, IL-1β, IL-2) and anti-inflammatory (i.e. IL-10, IL-4, IL-5,
IL-6) cytokines have been associated with a positive modulation of HLA-G
gene expression . Transforming or growth factors, such as TGF-β, GM-CSF, GCSF, CSF-1, LIF and EGF have been also found to upregulate HLA-G
(Moreau, Mouillot et al. 2003).
1.5.3.1 Epigenetic regulation
Despite the importance and high contribution of several microenvironmental
factors in maintenance or up- / down- regulation of HLA-G expression, they
seem to be incapable of inducing it. Indeed, the aforementioned factors seem to have
no effect on HLA-G gene transcription in cells in which the HLA-G gene is
repressed (Frumento, Franchello et al. 2000). This observation strongly supports the
hypothesis that distinct epigenetic mechanisms exist capable of controlling the
HLA-G gene. Both DNA methylation and histone deacetylation have been
recognized as major determinants in epigenetic gene silencing, playing a key role in
transcriptional control. A recent study of P. Moreau et al. proposed that HLA-G
expression is regulated by epigenetic mechanisms, demonstrating that DNA
methylation and histone deacetylation are involved in HLA-G expression
blockade (Moreau, Mouillot et al. 2003). On this basis, Polakova et al.,
exposing several cell lines to the demethylating agent 5-aza-2’-deoxycytidine (5-azadC) and to a histone deacetylase inhibitor revealed a significant activation of
HLA-G transcription (Polakova, Bandzuchova et al. 2007). Further studies with
primary malignant cells isolated from leukemia patients confirmed the potency of 5aza-2’-deoxycytidine (5-aza-dC) to reverse HLA-G gene silencing (Polakova,
Bandzuchova et al. 2009). Moreover, C.C. Chang et al. proposed the epigenetic
alterations occurring during malignant transformation to activate the HLA-G gene,
as a model of HLA-G regulation in melanoma cells in vivo (Chang, Murphy et al.
2003).
1.5.4 HLA-G expression in healthy individuals
Beyond the maternal-fetal interface and embryonic tissues, HLA-G expression
has been also described in adults’ tissues and particularly in immunoprivileged
sites. Specifically, HLA-G constitutive expression has been documented in adult
thymic medulla (Mallet, Blaschitz et al. 1999), cornea (Le Discorde, Moreau et
al. 2003), pancreatic islands (Cirulli, Zalatan et al. 2006) and erythroid and
endothelial-cell precursors (Menier, Rabreau et al. 2004).
32
Introduction
On the basis of this broader HLA-G expression, human blood cells have been
investigated for HLA-G transcription and translation. Basic HLA-G
transcriptional activity has been documented in both lymphocytes and monocytes
(Amiot, Onno et al. 1998), (Amiot, Onno et al. 1996), (Moreau, Adrian-Cabestre et
al. 1999), while the HLA-G gene has been found to be repressed in stem cells
and in committed progenitor cells expressing CD34 cell surface antigen (Amiot,
Onno et al. 1996).
Despite the initial controversial data concerning the HLA-G protein expression
by human lymphocytes, Feger et al. (Feger, Tolosa et al. 2007) has recently
reported the identification of a novel subset of naturally occurring regulatory
cells expressing HLA-G. These cells were shown to be hyporesponsive to
secondary stimuli and immunosuppressive and to be of thymic origin. They are
negative for CD25 and FoxP3 expression and exhibit potent suppressive properties
that are mediated partially by HLA-G. Further, HLA-Gpos Treg -mediated
suppression critically depends on the secretion ofIL-10 but not TGF-β (Huang YuHwa et al, 2009).
Interestingly, mesenchymal stem cells (MSCs) have been described to express both
membrane-bound and soluble HLA-G and this expression has been shown to
be critical to their suppressive functions (Selmani, Naji et al. 2008). The
existence of HLA-G–dependent suppressor (regulatory) cells highlights a role for
HLA-G in long-term immunomodulatory mechanisms and increases its in vivo
relevance. Identifying such cells in situ may serve as a marker of poor (e.g., as in
cancer) or favorable (e.g., as in transplantation) prognosis, and manipulating these
regulatory cells for the purpose of immunotherapy in cancer or in transplantation may
prove to be therapeutically useful.
1.6 Epigenetics
1.6.1 General
Epigenetics refers to “mechanisms that initiate and maintain patterns of gene
expression and gene function in a heritable manner without changing the sequence of
the genome”. Epigenetics impact gene regulation though DNA methylation, histone
protein modifications and chromatin organization. There are specific areas in the
genome that hold a greater concentration of CpG dinucleotides than expected, based
on ‘background’ incidence. These regions are termed ‘CpG islands’ and correlate with
promoter sequences of housekeeping genes and approximately 40% of tissue-specific
genes. CpG islands (regions longer than 500bp) localized in promoter regions are
usually unmethylated in normal tissues, regardless of the transcriptional activity of the
33
Introduction
gene. Overall, DNA methylation of CpG islands is associated with histone
hypoacetylation that collectively repress transcription. DNA methyltransferases
methylate cytidines at CpG dinucleotide sites. This modification can interfere with the
recognition and attachment of DNA binding proteins to genomic cis regulatory
elements. Additionally, methyl-CpG binding proteins are recruited to methylated
CpGs (mCpG) and, in turn, recruit histone deacetylases (HDACs), histone
methyltransferases (HMTs) and chromatin-remodeling factors to methylated regions.
These modifications condense chromatin structure, sequestering potential binding sites from
transcriptional activators and restricting occupancy (Christian Judith K, 2002).
DNA methylation is a post-replication modification catalyzed by DNA
methyltransferases (Dnmts). In mammals, the Dnmts catalyze the transfer of a methyl
group from S-adenosyl-methionine to the carbon 5 of cytosine in the CpG
dinucleotides. Cytosine methylation in CHG and CHH sites (where H= A, T or C) is
found in embryonic stem cells and disappear after differentiation. Other forms of
DNA methylation found in humans are methylcytosines and hydroxymethylcytosines,
but their significance still remains unknown. To date, five Dnmts have been
identified: Dnmt1, Dnmt2, Dnmt3A, Dnmt3B and Dnmt3L. However, only Dnmt1,
3A and 3B have methyltransferase activity in vivo. Dnmt2 has very little
methyltransferase activity in vitro. Dnmt1 primarily functions to maintain the
genomic methylation status following replication. DNA methylation can repress
transcription through transcription factor binding exclusion (Christian Judith K, 2002).
1.7 Epigenetics and Demethylating Agents
1.7.1 General
More than 40 years ago, the azanucleosides 5-azacytidine (Azacytidine, Vidaza;
Pharmion Corporation) and its deoxy derivative 5-Aza-2’-Deoxycitidine (Decitabine)
were developed as classical cytostatic agents. Several years later, it was shown that
these compounds inhibit DNA methylation in human cell lines, which provided a
mechanistic explanation for their differentiation-modulating activity. In addition, this
observation also initiated the development of azanucleosides as epigenetic drugs.
They are pyrimidine nucleoside analogs that were chemically synthesized and
characterized in Czechoslovakia by František Šorm and his fellow investigators in the
1960s (Sorm et al. 1964). Shortly after, 5-Azacytidine was also microbiologically
isolated from the fermentation beer of Streptoverticillium ladakanus (Hanka et al.
1966). The new agent was shown to possess a wide range of biological effects,
including antimicrobial, abortive, mutagenic, leukopenic, immunosuppressive,
cytotoxic, and antineoplastic activity (von Hoff et al. 1976). Particular interest was
34
Introduction
evoked when the antitumor activity in leukemia cell lines was established (Li et al.
1970; Sorm and Vesely 1964), and in vivo studies confirmed the cytotoxicity by
demonstrating a prolonged survival of mice with L1210 leukemias after
administration of 5-Azacytidine (Presant et al. 1975).
In the 1970s, the clinical efficacy of 5-Azacytidine was tested in a wide range of
solid tumors and leukemias. While treatment results in solid tumors were generally
discouraging, consistent antitumor activity was observed in patients with acute
myeloid leukemia (AML) and myelodysplastic syndromes (MDS) (von Hoff et al.
1976). In 1980, Jones and Taylor discovered that 5-Azacytidine could inhibit DNA
methyltransferase activity (Jones and Taylor 1980). Accordingly, when it was
recognized that aberrant DNA methylation is critically involved in the development of
many neoplasias, including MDS, the demethylating agents attracted new attention.
Since there was no satisfactory treatment option for the majority of MDS patients, and
early studies had shown responses to 5-Azacytidine, MDS offered an appropriate
disease entity to study the effects of the drug on DNA methylation, gene transcription,
and cell differentiation. In the mid1980s, trials exploring the usefulness of 5Azacytidine in MDS were initiated, and confirmed the clinical efficacy, safety, a
reduced risk for trans-formation into AML, and a beneficial impact on quality of life
over best supportive care (Kornblith et al. 2002; Silverman et al. 2002). Thus, in
May 2004 5-Azacytidine was approved by the US Food and Drug Administration
(FDA), and it has been postulated that it should be considered as the first-line therapy
for MDS (Kaminskas et al. 2005).
1.7.2 Chemical Structure
5-Azacytidine (4-amino-1-b-d-ribofuranosyl 1-1,3,5 triazine-2-one or 1-b -dribofuranosyl-5-azacy-tosine; C8H12N4O5; molecular weight 244) is a ring analog of
the naturally occurring pyrimidine nucleoside cytidine, from which it differs only by a
nitrogen in place of the fifth carbon . 5-Azacytidine is a white to off-white solid that is
stable at 25°C, not light sensitive, sparingly sol-uble in water, and unstable when
reconstituted in aqueous solution. Hydrolytic degradation results in a 21–36% loss
over 8 h at 25–30°C, and a 2–3% loss at 5°C (Kaminskas et al. 2005).
35
Introduction
Figure 1.5: Chemical structure of 5-Aza-2’-Deoxycitidine
1.7.3 Mode of action
Two main mechanisms of antineoplastic action have been identified for 5Azacytidine, namely the capacity to [a] incorporate directly into RNA with
subsequent disruption of RNA metabolism, and [b] to inhibit DNA methylation
Upon uptake into cells, 5-Azacytidine is phosphorylated by several kinases (uridine
cytidine-, pyrimidine monophosphate-, and diphosphate-kinases) to 5-aza2’deoxycytidine di-, and subse-quently triphosphate. The ribose structure needs to be
metabolized by ribonucleotide reductase (RNR) first to be integrated into DNA.
Incorporation of 5-Azacytidine triphosphate into RNA occurs directly, and causes a
disruption of nuclear and cytoplasmic RNA metabolism with subsequent inhibition of
protein synthesis (Li et al. 1970). As 5-azacitidine is a ribonucleoside, it incoporates
into RNA to a larger extent than into DNA. The incorporation into RNA leads to the
dissembly of polyribosomes, defective methylation and acceptor function of transfer
RNA, and inhibition of the production of protein.
The second mechanism of action is the inhibition of DNA methylation by trapping
DNA methyltransferases .It inhibits the enzyme in its progression along the DNA
duplex and functionally depletes it from the cell. In general, DNA methylation refers
to the addition of a methyl group to the cytosine residue of a CpG site. So called CpG
islands are genomic regions with a high frequency of CG dinucleotides (the “p” in
CpG notation refers to the phosphodiester bond between the cytosine and the
guanine), that are typically located in proximity to promoters. The degree of
methylation of CpG islands plays a role in the control of gene transcription. Usually,
fully methylated sites are associated with suppression of gene expression, while
hypomethylated or unmethylated CpG islands are linked to active transcription.
Forming a tight-binding complex 5-Azacytidine irreversibly binds to
methyltransferase. It inhibits the enzyme in its progression along the DNA duplex and
36
Introduction
functionally depletes it from the cell. Consequently, unmethylated DNA can lead to
the transcription of previously quiescent genes (Jones and Taylor 1981; Taylor and
Jones 1982). Already minor substitution of cytosine residues (~0.3%) suffices to
inactivate more than 95% of methyltransferase activity in the cell (Creusot et al.
1982).
At lower doses, 5‐azadC sequesters and promotes degradation of DNA methyltransfer
ase (DNMT), inducing DNA hypomethylation, thereby causing re‐expression of
genes, leading to differentiation and/or apoptosis of the myeloid leukaemic cells.
The alternate mechanism of action includes induction of DNA damage , inhibition
of NFkB synthesis and enhancement of anti‐tumour immune responses.
1.7.4 Future perspectives
DNA (hyper-) methylation is believed to contribute to cancer initiation and
progression by silencing tumor suppressor genes and other genes critical for
regulation of the cell cycle, cell growth, differentiation, and apoptosis (Bird 1996). In
this setting, 5-Azacytidine can restore the expression of potentially important genes
by demethylating such pathologically hypermethylated regions (Silverman 2001). In
addition to these modes of action, 5-Azacytidine has been reported to inhibit DNA
histone acetylation, another regulatory mechanism in gene silencing (Chiurazzi et al.
1999). Moreover, 5-aza-dC is able to induce Dnmt1 (DNA methylatrasferase 1)
degradation by the proteosomal pathway. By this way DNA methylation can be
controlled in a replication-independent manner since it binds chromatin throughout
G2 and M cell cycle phases. As mentioned before, 5-aza-dC can be incorporated into
RNA, resulting in the inhibition of RNA and protein synthesis independently of any
effect on the genome. Therefore, 5-aza-dC and its analogs can influence cellular
systems through their ability to decrease global methylation in replication-dependent
or –independent manners, through DNA damage response, or through inhibition of
mRNA and protein synthesis.
Next to pathological hypermethylation, also physiologically methylated CpG sites
may be targets for methyltransferase inhibitors. Recent data imply that a stable and
permanent expression of the human transcription factor forkhead box P3 (FOXP3) in
regulatory T cells might be crucial in the prevention of autoimmunity, allergy, and
graft-vs.-host disease after allogeneic hematopoietic cell transplantation. Apparently,
DNA methylation patterns in the FOXP3 locus can serve to discriminate FOXP3+
regulatory T cells with suppressive capacity (demethylated promotor region) from
activated FOXP3+ conventional T cells that lack this protective function (methylated
CpG sites) (Polansky et al. 2008). Experimental data support the hypothesis that
inhibition of methyltransferases stabilizes transcription of FOXP3, which could result
in an increase of suppressive FOXP3+ regulatory T cells. It is conceivable that in the
37
Introduction
future demethylating agents might be used as a therapeutic tool for immune
modulation(Nagaretal.2008).It has been shown that5‐azaC treatment in the post bone
marrow transplant setting, leads to expansion of Tregs which may alleviate graft versu
s host disease (GvHD). Two elegant studies ( explored the possibility that aza may be
used to generate immunoregulatory T cells ex vivo. In vitro treatment of
conventional T-cells with 5-aza-dC inhibited their activation, proliferation and
secretion of proinflammatory cytokines and after longer exposure converted
them to immunosuppressive T-cells. Interestingly, although these aza-treated T-cells
expressed FOXP3+, the authors showed very elegant that their suppressor function
was independent on FOXP3 expression. Thus, genes other than FOXP3 are
responsible for the suppressor function of aza-induced T-regs. In an effort to
identify these target genes, the authors found 48 candidate genes that are de novo
expressed in the 5-aza-dC induced T-regs, however without including in their
analysis the HLA-G gene (Sanchez-Abarca et al,2010; Choi et al, 2010).
1.8 Epigenetics and Histone modifications
Histones are nuclear proteins involved in DNA packaging through chromatin
formation. A nucleosome is a chromatin unit formed by a core of histones (two
H2AH2B dimmers and a H3-H4 tetramer) and 147 base pairs of DNA wrapped
around an octamer of histones. Modification of the histone aminotails, in conjunction
with other nuclear proteins, link histones and DNA methylation play a role regulating
chromatin structure and gene accessibility. Histone acetyltransferases (HATs) are
responsible for the acetylation of specific lysine residues in the histone amino-tails.
On the other hand, histone hypoacetylation correlates with gene repression. It is
apparent that histone modifications, in conjunction with the transcriptional machinery
comprised of RNA polymerase and associated transcription factors, collectively
delineate the transcriptional state of the genome.
38
39
Aim of the Study
Allogeneic Haematopoietic Stem Cell Transplantation (allo-HSCT) is a
potentially curative treatment for many malignant and nonmalignant conditions.
However, its success is hampered by the occurrence of graft-versus-host disease
(GVHD), caused by donor T cells attacking the antigenically foreign tissues of
the recipient. GvHD is a major cause of high morbidity, impaired quality of life
and ultimately an increased risk for mortality. Current methods to control GvHD
include immunosuppressive drugs that are only partially effective, increasing the
relative risk of infection and T-cell depletion, associating with increased incidences of
graft failure and increased relapse rates of original malignancies. Consequently,
finding ways to control GvHD after allo-HSCT is of paramount importance. To this
end, natural suppressors of immune cell function have been explored for their
capacity to control GvHD. Natural Regulatory T cells (nTreg) despite their initial
promising results have been used with very limited success. As such, there is a
critical need to identify further immune regulatory cell types that could be used to
control GvHD. Of note, mesenchymal stem cells which are used for prevention and
treatment of GvHD in humans have been shown to act immunosuppressive
through HLA-G.
HLA-G is a non-classical HLA class Ib molecule characterized by a tissue distribution
restricted to immunoprivileged sites and unique regulatory properties promoting
immune tolerance. Importantly, HLA-G expression has been found to be up-regulated
in various pathological conditions, in order to create a tolerogeneic environment, with
either beneficial (transplantation) or deleterious (cancer) results. Moreover, HLA-G
gene is regulated at the post-transcriptional level by DNA methylation or histone
acetylation.
Our hypothesis is that anti-methylating agents such as 5-aza-dC could be used
to induce the expression of HLA-G in cells that do not express HLA-G, such
as T cells, and convert these T-cells into T regulatory cells (Tregs).
On this basis we aimed to investigate:
(i). To investigate the possibility of hypomethylating agents to induce HLA-G
transcription in T-lymphocytes of healthy individuals
(ii). To functionally characterize inducible HLA-G+ T-cells
Our ultimate goal was to identify a novel regulatory population with the perspective
to be used as an adoptive cellular therapy to prevent and/or treat GvHD after
allo-HSCT
40
41
II. Materials and Methods
Materials and Methods
2.1 Cell Isolation
2.1.1 Cell Separation over Ficoll
Peripheral blood mononuclear cells (PBMC) from healthy donors were isolated by
density-gradient centrifugation over Ficoll-Histopaque PLUS (GE Healthcare, BioSciences, Uppsala, Sweden). Isolation of the light density cells (mononuclear cells)
typically removes red cells and granulocytes which have a higher density. In brief,
the procedure was as follows:
i. Sterile Ficoll-Histopaque (previously left at room temperature) was
pipetted in 15ml sterile conical tubes.
ii. Heparinized PB was carefully layered on top of Ficoll in ratio 1:1 (Ficoll and PB
should not mixed)
iii. Tubes were centrifuged at 1750 rpm for 30 min at room temperature
with the brake off. Resultant layers are from top to bottom: plasma - platelets PBMC - Ficoll - red blood cells (with granulocytes)
iv. The buffy coat with PBMCs was carefully aspirated and transferred to one new
15 ml sterile tube (including some plasma is no problem, but aspirating Ficoll should
be minimized, as it is toxic to cells).
vi. Cells were washed twice in culture medium (Culture Medium (CM): RPMI, 10%
FBS, 1% P/S) at 1500 rpm for 5 min, resuspended in CM and counted using a
haemocytometer (Neubauer).
v. Cells were diluted at a concentration of 106 cells/ ml CM for further use.
Figure 2.1: Schematic representation of blood separation layer. Before centrifugation
(A), after centrifugation (B)
43
Materials and Methods
2.2 Cell treatments and culture
2.2.1 Demethylating treatment
Freshly isolated peripheral blood mononuclear cells (PBMC) were resuspended and
cultured in RPMI 1640 medium supplemented with 1% penicillin/streptomycin (P/S)
and 10% heat-inactivated Fetal Bovine Serum (FBS). Demethylating treatment of
cells was carried out with 5-aza-2-deoxycytidine (5-Aza-dC, 44 mM in DMSO,
Sigma) at different final concentrations of 0,5μΜ, 1μΜ, 2,5μΜ, 5μΜ and 10μΜ for 3
days. A freshly prepared working solution of 5-Aza-dC (100μΜ) was made with
RPMI 1640 culture medium in each experiment.Untreated PBMCs were used as
negative controls. In a series of experiments demethylating treatment was performed
to isolated by Fluorescence Activated Cell Sorting (see below) CD3+/HLA-G neg
cells from PBMC of healthy individuals.
2.2.2 Histone deacetylase (HDAC) inhibitory treatment
Freshly isolated peripheral blood mononuclear cells (PBMC) were resuspended and
cultured in RPMI 1640 medium supplemented with 1% penicillin/streptomycin (P/S)
and 10% heat-inactivated Fetal Bovine Serum (FBS). Histone deacetylase (HDAC)
inhibitory treatment was carried out with for 24 hours with trichostatin A (TSA)
(1mM in DMSO, Calbiochem) at final concentrations of 0.1, 1, and 10 μM. A freshly
prepared working solution of TSA (330μM) was made with RPMI 1640 culture
medium in each experiment. Untreated PBMCs were used as negative controls.
2.2.3 Cell cultures
All manipulations were performed in aseptic conditions under a laminar air flow hood
workplace. Cultures were maintained in a humidified incubator at 37°C and 5% CO2.
Cells were counted using a haemocytometer (Neubauer).
2.3 Flow Cytometry
Flow cytometry is a technology that simultaneously measures and then
analyzes multiple physical characteristics of single particles, usually cells,
as they flow in a fluid stream through a beam of light. A beam of light (usually
laser light) of a single wavelength is directed onto a hydrodynamically-focused stream
of fluid. A number of detectors are aimed at the point where the stream passes through
the light beam: one in line with the light beam (Forward Scatter or FSC) and several
perpendicular to it (Side Scatter (SSC) and one or more fluorescent detectors). Each
suspended particle from 0.2 to 150 micrometers passing through the beam scatters the
oway, and fluorescent chemicals found in the particle or attached to the particle may
be excited into emitting light at a longer wavelength than the light source. This
44
Materials and Methods
combination of scattered and fluorescent light is picked up by the detectors, and, by
analysing fluctuations in brightness at each detector (one for each fluorescent
emission peak), it is then possible to derive various types of information about the
physical and chemical structure of each individual particle. FSC correlates with the
cell volume and SSC depends on the inner complexity of the particle (i.e., shape of
the nucleus, the amount and type of cytoplasmic granules or the membrane
roughness). So, the properties measured include a particle’s relative size, relative
granularity or internal complexity, and relative fluorescence intensity. These
characteristics are determined using an optical-to-electronic coupling system that
records how the cell or particle scatters incident laser light and emits
fluorescence.
Figure 2.2: Flow Cytometry diagram (www.abcam.com)
Two-color flow cytometry was performed using the following monoclonal antibodies
(mAbs): anti-HLA-G FITC or PE (clone MEM-G/9), IgG2a specific for native HLAG1 and -G5 (Abcam, Cambridge, UK); anti-CD3
PC5 or FITC, anti-CD14 PC5
or FITC (Beckman Coulter, CA, USA).Propidium iodide was used for cell viability
measurement. First, PBMCs were incubated with the indicative mAbs (anti-CD3 or
anti-CD14) diluted 1/500 in Phosphate Buffer Saline (PBS, GIBCO) for 30min at 4 C,
then washed once with PBS. Then cells were incubated with the specific anti-HLA-G
45
Materials and Methods
diluted 3μl in 100μl PBS for 20min at 4°C and finally detected on EPICS XL-MCL
flow cytometer
(Coulter, Fullerton, CA, USA). At least 5x104 cells were
acquired and analyzed using the WinMDI 2.9 software. The percentage of HLA-Gpos
cells as well as their HLA-G expression intensity (defined as specific fluorescence
index) was measured. The Specific Fluorescence Index (SFI) was calculated using the
formula: SFI= mean fluorescence obtained with specific Ab/ mean fluorescence
obtained with control Ab. Matched isotype controls were systematically used to
subtract non-specific signals. In one series of experiments we aimed to measure cell
viability with propidium iodide (PI) performing three-color flow cytometry.
2.4 Fluorescence Activated Cell Sorting (FACS)
Fluorescence Activated Cell Sorting is a special category of flow cytometry, offering
the significant advantage of isolating one or two cell populations, based upon the cell
type (expressed as size and granularity) and expression of specific markers
(expressed as fluorescent characteristics). Before initiating the cell sorting, the
cells are labeled with one or more specific fluorescent mAbs, which recognize
antigens that are specifically expressed by the desired population. At the
beginning of cell sorting the cell suspension enters a flowing thin stream where
upon the effect of a specific fuid (sheath) it is separated into a single cell
flow. The source of such stream vibrates in extremely high frequency
(approximately 27,000 cycle per second) in order to break the stream into small
droplets (approximately 27,000). To this end, some droplets may contain a
single cell and other not. Just before its separation, the stream passes through a
laser beam, creating signals which are collected by different detectors. Notably, the
resulted forward scatter corresponds to the size of each cell and the side scatter to its
granularity. Furtermore, if the cell that passes through the beam is labeled with a
fluorochrome, additional fluorescence will be emmited which in turn will be detected
by specific detectors. The cell that meets the criteria that have been set for the
sorting (size and fluorescence) will be positively/negatively chaged. Next the stream,
which has been separated into droplets, passes through two opposed magnetic
plates (deflection plates); during this passage, the positively charged drops are
attracted by the negatively charged plate and the negatively charged drops by
the positive. At the end of the passage the drops end to two collection tubes where the
desired populations are collected. The cells that had not been charged, as they did not
meet any of the criteria set, are discarded .
46
Materials and Methods
Figure 2.3: Schematic presentation of fluorescence activated cell sorting
(http://www.mpi-bremen.de/en/Flow_Cytometry.html)
For isolation of aza-treated CD3+/HLA-Gpos and aza-treated CD3+/HLA-Gneg as
well as for CD3+/HLA-Gneg (not treated) cell populations, the following mAbs were
respectively used: CD3-PC5 and HLA-G-PE. A gate was set on all non-aggregated
cells followed by sorting of the desirable populations. Cells were collected in
tubes containing 1ml RPMI, 10% FBS. At the end of the sorting, the isolated
cells were immediately washed, resuspended in culture medium and used for various
purposes. Cell purity was assessed by flow cytometry and was ranged between 90 95%. The fluorescence activated cell sorter FACSVantage (Becton-Dickinson) was
used.
2.5 Suppression assays
To assess the suppressive nature of aza-treated HLA-Gpos or HLA-G neg T cells oneway mixed lymphocyte cultures (MLCs) were performed. In the MLC, suspensions of
responder PBMCS are cultured with allogeneic stimulator PBMCs. Briefly the
following procedure was performed:
- Unseperated PBMCs from a donor B were labeled with CFSE and used as
responder cells
47
Materials and Methods
- Irradiated PBMCs from a donor C were used as stimulator cells at a ratio
1:1 responder: stimulator
- FACS-sorted aza-treated HLA-Gpos or aza-treated HLA-Gneg T cells from a
donor A, were irradiated and used as third party cells in MLRs at various responder
to suppressor ratios (1:0.5, 1:1, 1:2, 1:3, 1:5). MLCs without third party cells were
used as controls.
- T cell proliferation was assessed by cytofluorimetric analysis for CFSE
staining intensity on day 6.
- T cell proliferation was determined using the CFSE Cell proliferation assay. CFSE
or CFDA-SE (carboxylfluorescein diacetate, succinimidyl ester) is colorless and nonfluorescent. CFSE passively diffuses into cells where the cellular esterases
cleave the acetate groups rendering the molecule fluorescent and cell
impermeable. The succinimidyl ester binds to intracellular free amines resulting in
long lived fluorescent adducts which are retained by the cells during throughout
development and meiosis. CFSE is partitioned equally among daughter cells with
each division and is not transferred to adjacent cells in a population .
Figure 2.4: CFSE cell proliferation assay
CFSE Cell Proliferation assay was performed
instructions, as follows:
according
to
manufacturer’s
 Resuspend cells of interest in prewarmed PBS/0.1% BSA at a final
concentration of 106 cells/ml
48
Materials and Methods
 Add 2μl of 5mM stock CFSE solution per ml of cells for a final
working concentration of 10μM
 Incubate dye at 37oC for 10min
 Quench the staining by the addition of 5 volumes of ice-cold culture media to
the cells
 Incubate 5min on ice
 Pellet cells by centrifugation
 Wash the cells by resuspending the pellet in fresh media (total of three
washes)
 Set up in vitro cell cultures under appropriate conditions
2.5.1 Transwell System
When indicated, suppression assays were performed in transwell systems. In these
experiments, 96-well plates with a chamber fitted with a filter membrane
containing pores of 0.4μm were used (Greiner bio-one, San Diego, CA). The MLR
was set up as mentioned above and was placed in the lower chamber of
transwell; the third party cells (FACS-sorted aza-treated HLA-Gpos or HLA-Gneg
T cells) were irradiated (25Gy) and placed in the upper chamber. The T cell
proliferative responses were measured by flow cytometry on day 6 using the CFSE
cell proliferation assay (see above).
Figure 2.5: Schematic representation of the Transwell System
49
Materials and Methods
Figure 2.6: Suppression assay (performed either in a transwell or not)
2.5.2 Blocking Experiments
When indicated, suppression assays were performed in the presence or in the absence
of an anti–HLA-G blocking antibody (mAb87G) blocking antibodies. mAb87G
(Exbio) recognizes transmembraneous HLA-G1 as well as soluble HLA-G5, which
represent the 2 major HLA-G isoforms. In these experiments the following
neutralized antibodies were used: a. A neutralized mAb directed to HLA-G (clone
87G; Exbio) was added in suppression assays at a concentration of 20μg/ml on
days 0 and 3. An irrelevant IgG2a isotype Ab was used as a control at the same
concentration.
2.6 T cell stimulation assays
T cell stimulation assays were pretreated as MLC as described above. For this reason
PBMC were obtained were obtained from healthy volunteers and aza treatment was
performed as mentioned above. After treatment we isolated aza-treated CD3+/HLAGpos and CD3+/HLA-Gneg populations by FACS sorting (purity >98%) as described
50
Materials and Methods
above. For allogeneic stimulation, these cells were labeled with CFSE and used as
responder cells in MLCs. Different ratios of irradiated allogeneic PBMCs obtained by
different donors were used as stimulator cells (responder/stimulator ratio: 1/0, 1/1,
1/2). The final cell concentration was up to 106 cells/ml of cultured medium and
placed in U-bottom 96 well-plates. T cell proliferation was measured after 6 days in
culture according to CFSE intensity by flow cytometry .
Figure 2.6: T cell stimulation assay
51
III. Results
53
Results
3.1 The effect of the hypomethylating agent 5-aza-dC on
HLA-G expression and cell viability in human peripheral
blood T cells in vitro
Our initial aim was to study the possible efficacy of the demethylation agent 5-aza-dC
and the histone deacetylase inhibitor trichostatin A (TSA) on the protein activation of
HLA-G molecule in whole PBMC and particularly in CD3+ T cells from healthy
donors. For this purpose, peripheral blood mononuclear cells (PBMCs) were isolated
from healthy donors and then 5-aza-dC and TSA was administered at different doses
(5-aza-dC: 500nM, 1μΜ, 2.5μΜ, 5.0 μΜ, 10μΜ for 3 days and TSA: 100nM, 1μΜ,
10μΜ for 1 day) in order to specify the optimal concentration in which could both
show:
a) the lowest toxic effects (viability kinetics)
b) maximum induced HLA-G protein expression
Viability kinetics was done by propidium iodide staining. The percentage of HLAGpos cells in total PBMCs, CD3 gated and CD14 gated cells (gating strategy is shown
at figure 3.1) as well as their HLA-G expression intensity (defined as Specific
Fluorescence Index –SFI) were measured by flow cytometry.
54
Results
Figure 3.1: In this figure we show a representative figure of gating strategy. We
exhibit the way gating was done and the way we use the isotype and specific antibody
in order to define positive population.
HLA-G expression (percentage of positive cells and SFI) was increased after
treatment with all the concentrations that we used in PBMCs and especially in CD3+
T cells (figure 3.2). Of note maximun HLA-G expression in PBMCs and CD3+ gated
cells was achieved after exposure to 10μΜ aza (figure 3.3, 3.4). In addition, 10μΜ of
aza could be used in a safe manner since viability of 10μΜ aza-treated cells was
similar to untreated cells (35,41 +/- 26,10 vs 34,75 +/- 19,80 respectively, figure 3.5).
Figure 3.2: A representative experiment is shown. HLA-G cell surface expression as
the percentage of HLA-Gpos cells and SFI expression is used. From top to bottom,
HLA-G expression on PBMC and CD3+ cells gated according already showed gating
strategy.
55
Results
Figure 3.3: The graph depicts the percentage of HLA-Gpos cells in PBMC (black
columns) and in gated CD3pos T cells (grey columns) before and after treatment with
aza on day 3. A statistically significant difference (p= 0.0192) of HLA-Gpos in CD3
population was observed after treatment with 10μΜ aza. Specifically a mean
percentage of 24,84 was found to express HLA-G after 10μΜ aza in comparison to a
mean percentage of 4,84 of control (no treatment).Moreover a mean percentage of
9,27 of HLA-G pos cells were also increased in PBMC in contrast to 5,28 of control.
Figure 3.4: The graph shows HLA-G expression as SFI in PBMC (black columns)
and in CD3pos (grey columns) with or without aza treatment on day 3. In PBMC SFI
was not changed. In gated CD3pos T lymphocytes HLA-G SFI is shown a variation.
56
Results
100
% PIneg cells
80
60
40
20
a
uM
+
10
M
5u
+
az
az
a
az
a
M
az
a
M
1u
+
2.
5u
+
+
0.
5
uM
no
az
az
a
a
0
Figure 3.5: The graph summarizes the results of PBMC PIneg cells after aza
treatment. PI staining was performed on day 3 of culture. Specifically, a mean
percentage of 35,41% of cells were viable (PIneg) after treatment with 10μΜ aza as
compared to the control (no aza) where a mean percentage of 34,75% of PBMC cells
were viable. This demonstrates that culture conditions as long as aza treatment could
affect PBMC viability.
We also observed that the percentage of CD3pos cells was not affected by aza
treatment (figure 3.6). Moreover, as far as CD14+ cells, we observed an increase in
their percentage of HLA-Gpos cells and their SFI expression (figure 3.7). Also, there
was a small decrease in their percentage in culture which probably caused due to
culture conditions. Interestingly, after TSA treatment with high dose (10μΜ), cell
viability decreased and no induction of HLA-G expression was observed (figure 3.8
and 3.9).
In order to obtain a sizeable population of aza-induced HLA-G positive T
lymphocytes, 10μΜ aza was used for the following experiments of functionally
characterization of those cells.
57
Results
% CD3 pos cells
60
40
20
az
2.
a
5u
M
az
+
a
5u
M
az
+
a
10
uM
az
a
M
+
1u
+
+0
.5
no
nM
az
a
az
a
0
Figure 3.6: The graph depicts the percentage of CD3pos before and after aza
treatment after staining with the specific antibody. There was not observed a
statistically significant decrease on their percentage. This was a rough proof of not
positive selecting of HLA-gpos T cells.
Figure 3.7: The graphic shows the % of HLA-Gpos cells (left Y axis) and the SFI
expression of HLA-Gpos cells (right Y axis) after gating on CD14+ cells before and
after treatment with aza. It is demonstrated an increase after treatment both in the
percentage of HLA-Gpos cells and SFI expression after exposure to 10μΜ of aza in
comparison to control experiments (82,84% vs 50,67% and 17,42 vs 4,23
respectively).
58
Results
Figure 3.8: A representative figure analysis of one specific experiment. HLA-G
expression is shown both in PBMC and CD3 gated cells. There was no induction of
HLA-G expression.
59
Results
Figure 3.9: Results of viable cells in PBMC before and after TSA treatment showing
a statistical significant decrease in PIneg cells after treatment with 10μΜ TSA as
compared to the control (mean: 9,16% vs 63,40% respectively, p=0,0232).
3.2 Confirmation of HLA-G induction on CD3+ T cells after
treatment with 5-aza-dC
We have already examined the activity of the demethylating agent aza on HLA-G
expression in lymphocytic population and we observed an induction of HLA-G
surface expression after treatment with 10μΜ of aza in those cells. In order to further
prove this induction and to exclude the possibility of any positive selection of
naturally occurring HLA-G positive T cells [Feger et al, 2007], we performed the
same kinetic experiments of aza in FACS-sorted HLA-G negative T lymphocytes
obtained by healthy individuals.
HLA-G expression as percentage of positive cells and SFI were increased after
treatment with the hypomethylating agent 5-aza-dC (aza) (figure 3.10 and 3.11).
60
Results
Figure 3.10: A representative experiment is shown. From left to right, FS/SS and
CD3 gating as well as HLA-G expression according to specific antibody is shown.
61
Results
Figure 3.11: The graph summarizes the results of the percentage of HLA-Gpos cells
(black columns) and SFI expression (grey columns) after treating isolated
CD3+/HLA-Gneg cells with various concentrations of aza.
3.3 Aza-treated HLA-Gpos T cells are suppressive and this
suppression is partially reduced after neutralization of HLAG and is not dependent on cell-to-cell contact

Aza-treated HLA-Gpos T cells are suppressive
Given the inhibitory role of HLA-G, we wondered if these cells might have a
suppressive effect on lymphocyte proliferation. To investigate this possibility, we
used MLCs where proliferation of CFSE-labeled allogeneic PBMC was assessed by
flow cytometry in the presence or absence of different ratios of allogeneic aza-treated
CD3+/HLA-Gpos or HLA-Gneg T cells (third-party population).
62
Results
Our results indicate that addition of isolated aza-treated CD3+/HLA-Gpos cells led to
significant reduction of CFSE-labeled allogeneic PBMC (Figure 3.12 and 3.13).
Specifically, maximum suppression was detected at responder to third party cells ratio
1:5 as compared to control MLC without third party cells (82,90%, +/- 10,36,
p=0,0010) as well as to MLCs containing their paired HLA-Gneg cells (p=0,0423)
(Figure 3.14). Concomitantly, addition of their negative counterparts (isolated azatreated CD3+/HLA-Gneg cells) increased cell proliferation at all responder to
suppressor ratios (Figure 3.14). These data suggest that HLA-Gpos induced T cells
after treatment with the hypomethylating agent aza were able to function as
suppressor cells.
Figure 3.12:
analysis.
A representative figure of gating strategy for suppression assays
63
Results
Figure 3.13: In this figure a representative experiment of suppression assay is
presented. Cells were gated according to gating strategy showed at figure 3.12. The
histograms depict proliferation of cells.
64
Results
Figure 3.14: The graph summarizes the results of the suppression assays. Results are
shown as mean+/-SEM of proliferation as compared to MLC without third
party cells. The horizontal axis indicate increasing ratios of responder : third party
cells. Asterisks indicate statistically significant inhibition of proliferation as
compared to control MLC (1:0).

Suppression by aza-treated HLA-Gpos T cells seems to be
reduced after HLA-G neutralization
To test the hypothesis that HLA-G is not just the phenotypic marker of a regulatory
population but also mediates suppression, we first tried to rescue proliferation of the
allogeneic PBMC used as responder cells in MLRs by neutralizing HLA-G with an
anti–HLA-G blocking antibody (mAb87G). mAb87G recognizes transmembraneous
HLA-G1 as well as soluble HLA-G5, which represent the 2 major HLA-G isoforms.
In our preliminary results HLA-G neutralization partially antagonized effects of azatreated CD3+/HLA-Gpos T cells in comparison with MLC with the same cells used as
third party. Of note when an irrelevant isotype antibody was added cell proliferation
partially declined (figure 3.15). Our data therefore possibly suggest that HLA-G
molecule contributes to the suppressive effect of our cells, but it seems not to be the
only molecule responsible for their action.
65
Results
Figure 3.15: This graph summarizes the results of suppression assays where
neutralized antibody to HLA-G (87G) and an irrelevant isotype were used. Circles
indicate the number of experiments at each specific culture condition. The ratio is
referred to responder to third party cells and their proliferation were measured in
comparison to MLC (1:0).

Suppression by aza-treated HLA-Gpos T cells seems not to be
dependent on cell-to-cell contact
To assess whether direct cell to cell contact is essential for the suppressive function of
the aza-treated HLA-Gpos T cells, we performed the suppression assays in a transwell
system, where responder and suppressor cells were not in direct contact.
When suppression assays were performed in a transwell system, in which aza-treated
CD3+/HLA-Gpos cells were separated from the responder cells with a membrane, we
found that aza-treated CD3+/HLA-Gpos T cells maintained their suppressive effect
when comparing proliferation with or without cell-cell contact.(figure 3.16).
66
Results
Figure 3.16: The graph shows the results of suppression assays performed in
transwell system. Different responder to third party cells ratios were used. Cell
proliferation was measured in comparison to proliferation of MLC without third party
cells.
3.4 Aza-treated HLA-Gpos T cells show a reduced
proliferation to allogeneic stimuli
Recent studies have shown that natural occurring HLA-Gpos T cells show a reduced
proliferation to allogeneic and polyclonal stimulus. Moreover, their hypoproliferative
capacity could not be overcome by the addition of exogenous IL-2 or by recall
stimulation. [Feger et al, 2007]. Thereby, we wondered whether the whereby azainduced CD3+/HLA-Gpos T cells have similar functional properties.
Isolated aza-treated CD3+/HLA-Gpos cells show a reduced proliferation to allogeneic
stimuli in all responder to stimulator ratios in comparison with their negative
counterparts (aza-treated CD3+/HLA-Gneg) (figure 3.17).This experiment shows that
aza-treated CD3+/HLA-Gpos T cells are hypoproliferative. However, low levels of
CFSE intensity (indicating high proliferation activity of cells) was also detected at
both populations after culture without allogeneic stimuli. These low levels of CFSE
could be due to spontaneous proliferation of the cells. In that case aza-treated
CD3+/HLA-Gpos cells showed a low proliferative capacity in contrast to their
negative counterparts (figure 3.18).
67
Results
Figure 3.17 : This figure shows a representative analysed result. After FACS sorting
aza-treated CD3+/HLA-Gneg and aza-treated CD3+/HLA-Gpos were used as
responders cells (R) which were stimulated with different ratios of allogeneic PBMC
(Stim). Proliferation was determined according to CFSE after 6 days.
68
Results
Figure 3.18: The graph show summary of 3 independent experiments done at
different responder to stimulator ratios (1:0, 1:1, 1:2). The percentage of proliferation
of isolated aza-treated CD3+/HLA-Gneg (black columns) and aza-treated
CD3+/HLA-Gpos (grey columns) are presented. Results are shown as mean +/- SEM.
Asterisks indicate statistical significant decrease.
69
IV. Discussion
70
Discussion
4. Discussion and perspectives
Human leukocyte antigen G (HLA-G) is a tolerogenic molecule, initially found
to be expressed in extravillous cytotrophoblast contributing to maternal-fetal
tolerance. Although its expression in adults was considered to be highly
restricted, it recently appeared to be broader than initially thought. Thus HLA-G has
a direct inhibitory effect, acting as a shield against immune aggression. However there
is increasing evidence that it also has long-term inhibitory effects. Indeed HLA-G is
involved in the generation of suppressor cells and could be a new marker of
suppression (Carosella et al, 2008).
Besides this highly tissue restricted expression, it was recently found that thymus
generates a discrete (1.6% of CD4+ T cells and 3.3% of CD8+ T cells) subset of
naturally occurring HLA-Gpos regulatory T-cells with regulatory properties in the
peripheral blood of healthy adults (Feger et al, 2007). Specifically, these naturally
occurring HLA-G-expressing regulatory CD4 T cells have suppressive function which
is IL-10 dependent and reversible. Moreover, they do not require the presence of other
cell types to exert their regulatory function (Huang Yu-Hwa et al,2009).We have also
identified a discrete subset (median 1.6%, n=14) of HLA-G expressing T cells
circulating in the peripheral blood of healthy individuals (Lazana et al, 2011). HLA-G
expression is under tight regulation and involves mechanisms acting partially at the
transcriptional level. Although several factors (such as GM-CSF, IFNs, IL-10, TNF-a,
TGF-b) have been found to regulate HLA-G expression at either the
transcriptional or posttranscriptional level, these factors have no effect on HLAG gene transcription in cells in which the HLA-G gene is strongly repressed.
Recent studies have shown that HLA-G gene activity includes
epigenetic
mechanisms such as DNA demethylation or histone acetylation. Ex vivo treatment
with the hypomethylating agent has been found to induce the HLA-G
transcription and cell surface expression in different cell lines and in primary
leukemia cells. Surprisingly, DNA methylation has been considered to play a
role in the regulation of T-cell effector function and cytokine gene expression,
indicating a promising role of hypomethylating agents for immunmodulation.
Indeed, the administration of the hypomethylating agent 5-aza-dC prevented
the development of GvHD and expanded the numbers of immunomodulatory T
regulatory cells (Tregs) in animal models (Goodyear et al, 2012). The expanded
regulatory T cells (FOXP3+) following treatment with 5-aza-dC show very
different functional characteristics to naturally occurring Tregs, despite their immuno
phenotype similarities. In vitro treatment of conventional T-cells with aza
inhibited their activation, proliferation and secretion of proinflammatory
cytokines and after longer exposure converted them to immunosuppressive T71
Discussion
cells (Sanchez-Abarca et al,2010). Interestingly, although those cells treated with
hypomethylating agent 5-aza-dC express FOXP3 their suppressor function is
independent of it suggesting that other genes responsible for the suppressor function
are also regulated by DNA methylation. Choi et colleagues have identified 48
candidate genes for future studies without including HLA-G gene( Choi et al, 2010).
Based on these reports, we hypothesized that the hypomethylating agent aza could be
used to induce HLA-G expression in cells such as CD3+ T cells (no HLA-G
expression) via epigenetic modification and probably conversion of these T cells into
T regulatory cells. In this study we confirmed that the addition of the DNA
demethylating 5-aza-dC activated HLA-G expression in T cells (CD3+) obtained by
healthy individuals. Importantly, the highest HLA-G protein synthesis was achieved
with demethylating treatment rather than with the addition of the histone
deacetylation inhibitor (TSA). It was possible to specify the optimal concentration
with the lowest toxic effects and the maximum expression in which we could obtain a
sizeable population of HLA-G+ T cells in order to further characterize them as a
possible regulatory - suppressor population. We also exclude the possibility of any
positive selection of natural occurring HLA-G+ T cells.
In this field there is a need to correlate the increase of HLA-G protein synthesis
following 5-aza-dC treatment with up-regulation of HLA-G gene transcription. Given
the fact that the effect of the DNA demethylating agent occurs in cells actively
undergoing cellular division and in order to exclude any pro-apoptotic effect of it,
cells should be stimulated before treatment. Moreover the expression of the different
HLA-G transcripts after induction needs to be investigated. Finally it would be of
interest to phenotypic characterize those cells and distinguish them from other
induced T regulatory populations with specific regulatory markers or by indentifying
their cytokine profile.
HLA-G has been considered to be a key mediator in immune tolerance. Experiments
with HLA-G transfected antigen presenting cell lines revealed that the HLA-G
inhibits the cytolytic function of both NK and cytotoxic T cells, the
alloproliferative response of CD4+ cells and the maturation and function of
dendritic cells (Carosella, Favier et al. 2008). We used MLR model, which is
considered to be the in vitro equivalent of allogeneic transplantation, has been
the standard assay used to identify allogeneic lymphocyte proliferation.
Our data suggest that HLA-G+ induced T cells after treatment with the
hypomethylating agent 5-aza-dC were able to function as suppressor cells at
responder to third party ratio 1:5 which could partially be antagonized by blocking
HLA-G with a neutralizing antibody (mAb 87G). Addition of their 5-aza-dC treated
negative counterparts increased cell proliferation at all ratios. These results
72
Discussion
demonstrate that the HLA-G expressed by 5-aza-dC treated T cells played a key role
in their immunomodulatory properties. Suppression of 5-aza-dC treated HLA-G+T
cells was not depending on cell contact, as we could show using transwell
experiments. This results was and in line with other studies using HLA-G
transfected cell lines. Moreover, it is therefore assumed that soluble factors, such
as soluble HLA-G (sHLA-G) may also mediate their suppressive function. In order to
strong their suppressor activity it is crucial to investigate the role of activating them
by adding IL-2 or use them as “third party – suppressor” cells in a system where
stimulatory allogeneic antigen presenting cell (APCs) would be present.
Moreover similar to the CD4+CD25+ Tregs 5-aza-dC treated HLA-GposT cells show
reduced proliferation to allogeneic stimuli. However, it is crucial to further investigate
this hypoproliferative nature and if this can be overcome by the addition of exogenous
IL-2.
HLA-G–dependent suppressor cells may be cells whose main characteristic is to
express the immunotolerogenic molecule HLA-G and therefore are better
characterized by their function than by a common phenotype. The exact cellular and
molecular mechanisms how HLA-Gpos T cells exert autologous immune suppression
remain elusive at present. One hypothesis is that suppressive function is mediated by
HLA-G that directly interacts with respective receptors on HLA-G–negative T cells.
The only described HLA-G receptor present on T cells is ILT-2 (CD85j, LIR-1), thus
making this immunoglobulin-like transcript a possible candidate underlying the
molecular interaction of HLA-G–mediated T-cell suppression. HLA-G–positive cells
could induce or trigger HLA-G–negative cells to promote anti-inflammatory or
antiproliferative cytokine milieu. These changes might contribute to the production of
“suppressive” soluble factors, which might include more than HLA-G.
It is therefore assumed that HLA-G is a key molecule characterizing phenotype and
function of the 5-aza-dC induced HLA-Gpos T-cell population and that HLA-G–
dependent suppressor cells are diverse by nature, function, and significance.
In this report, we describe a novel and simple possible approach to ex vivo generating
induced HLA-G+ T lymphocytes with immunoregulatory properties by using the DNA
demethylation agent 5-aza-dC. This approach may help to overcome the current
obstacles for the routine use of nTregs in the human clinical trials including limiting
numbers of nTregs in peripheral blood, loss of suppressor properties after in vitro
expansion, and lack of cell surface markers necessary for efficient affinity
purification. These results sustain the hypothesis that HLA-G is an important
mediator of immunotolerance, opening up new therapeutic strategies of alloHSCT
patients.
73
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