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MECHANISM OF A2A ADENOSINE RECEPTOR
MEDIATED IMMUNOSUPPRESSION IN INFLAMED
TISSUE MICRO-ENVIRONMENT
DOCTORAL THESIS PRESENTED
BY
RADHIKA GOPALKRISHNA KINI
ADVISORS: DR. MICHAIL SITKOVSKY AND DR. AKIO OHTA
TO
THE BOUVE΄ GRADUATE SCHOOL OF HEALTH SCIENCES
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY IN PHARMACEUTICAL
SCIENCES WITH SPECIALIZATION IN
IMMUNOLOGY
NORTHEASTERN UNIVERSITY
BOSTON, MA-02115
AUGUST, 2012
ii
Northeastern University
Bouve’ Graduate School of Health Sciences
Thesis title: Mechanism of A2A adenosine receptor mediated immunosuppression
in inflamed tissue micro-environment
Author: Radhika Gopalkrishna Kini
Program: Pharmaceutical Sciences (Specialization in Immunology).
Approval for thesis requirements of the Doctor of Philosophy Degree in
Pharmaceutical Sciences
Thesis Committee:
Dr. Akio Ohta (Chairman) Date:
Date:
Date:
Date:
Date:
Director of the Graduate School Date:
Dean Date:
Copy Deposited in Library Date:
iii
Table of Contents
Abstract…………………………………………………………………………... v
Abbreviations…………………………………………………………………… vii
List of figures…………………………………………………………………... viii
Acknowledgements……………………………………………………………… xi
1. Introduction...………………………………………………………………… 1
2. Inhibition of T cell effector functions by adenosine-A2AR signaling……... 6
2.1 Background and Significance……………………………………………. 6
2.2 Specific aim 1…………………………………………………………….. 12
2.3 Materials and methods…………………………………………………... 13
2.4 Results.………………………………………………………………….... 17
2.5 Discussion………………………………………………………………… 26
3. Adenosine-A2AR signaling promotes T regulatory cell dependent
immunosuppression…………………………………………………………. 28
3.1 Background and Significance…………………………………………... 28
3.2 Specific aim 2…………………………………………………….…….... 30
3.3 Materials and methods………………………………………………….. 31
3.4 Results…………………………………………………………………..... 35
3.5 Discussion………………………………………………………………... 52
4. Adenosine-A2AR mediated effect on induction of Foxp3 expression via
oxidative stress pathway………………………………………………......... 56
4.1 Background and Significance……………………………………….….. 56
4.2 Specific aim 3………………………………………………….………… 58
iv
4.3 Materials and methods……………………………………………….…. 59
4.4 Results……………………………………………………………………. 60
4.5 Discussion………………………………………………………………... 63
5. Conclusion……………………………………………………………………. 65
6. Future Directions………………………………………………………….…. 67
6. Bibliography……………………………………………………………....….. 69
7. List of Publications……………………………………………………….….. 81
v
ABSTRACT
The pathogenesis of many diseases involves uncontrolled inflammation. Overactive
inflammatory response to remove pathogens sometimes leads to undesirable outcome of
collateral tissue damage. The adenosine-A2A adenosine receptor (A2AR) pathway plays an
important role in protecting tissues from excessive damage during inflammation by suppressing
immune responses including T cell activation. Local levels of extracellular adenosine are known
to increase in tissues receiving insufficient oxygen supply (hypoxia). Tumors and local
inflammation sites are hypoxic and contain high levels of extracellular adenosine. Therefore, in
patho-physiological conditions such as infection and cancer, T cells may have to get activated in
an adenosine rich environment. The current study was designed to characterize effector functions
of the T cells that are activated in the presence of elevated levels of adenosine. The data
demonstrates that T cell activation in the presence of A2AR signaling strongly inhibited
development of cytotoxicity and cytokine-producing activity in T cells, whereas the inhibition of
T cell proliferation was only marginal. Both CD4+ and CD8
+ T cell subsets showed similar
results and were found to be susceptible to A2AR-mediated immunosuppression. Importantly,
the impaired effector functions persisted in long term culture even after removal of the A2AR
agonist. Thus, although the adenosine-rich microenvironment may allow for the expansion of T
cells, the functional activation of T cells may be critically impaired. This mechanism could
explain the inefficiency of anti-tumor T cells in the tumor micro-environment.
The persistence of T cell suppression after adenosine treatment might involve a possible
enhancement of professional immunosuppressive cell population. Regulatory T cells (Tregs)
play an important role in prevention of autoimmune disorders by suppressing the immune
vi
system. A2AR-mediated immunosuppression has been shown to be T cell autonomous in studies
of effector T cells, but it is not clear how A2AR stimulation affects Tregs. Interestingly, T cell
activation in the presence of A2AR stimulation resulted in the expansion of Tregs, which are
defined as CD4+CD25
hiFoxp3
+ cells. Treatment with A2AR agonist upregulated CTLA-4
expression on Treg cells and enhanced their immunosuppressive activity. The difference was not
just qualitative in nature but also quantitative with an increase in Treg cell number after A2AR
stimulation. The CD4+ Foxp3
+ cells were predominantly derived from CD4
+ CD25
+ natural Treg
although CD4+ Foxp3
+ cells were also induced from CD4
+ CD25
- cells. These data suggest that
A2AR-mediated stimulation of lymphocytes using A2AR agonists should be considered in
protocols for ex-vivo expansion of Treg before the transfer to patients in different medical
applications such as for the treatment of auto-immune disorders and allogenic reaction after
transplantation. Taken together, extracellular adenosine directly inhibits T cell activation and
impairs effector functions but adenosine may further establish immunosuppression by an indirect
and long term T cell inhibitory effect through the enhancement of Treg.
vii
List of Abbreviations
A2AR – A2A adenosine receptor
BSO - Buthionine sulfoximine
cAMP – cyclic adenosine monophosphate
CRE – cAMP response element
CREB – cAMP response element binding protein
CFSE - Carboxyfluorescein succinimidyl ester
CGS - CGS21680
CTLA-4 – Cytotoxic T lymphocyte antigen – 4
DCs – Dendritic cells
Foxp3 - Forkhead box P3
GSH - Glutathione
HIF-1α – Hypoxia inducible factor - 1α
HRE – Hypoxia response element
IFN-γ – Interferon gamma
IL - Interleukin
NAC – N-Acetylcysteine
NECA - 5’-N-ethylcarboxamidoadenosine
TCR – T cell receptor
Tregs – T regulatory cells
Tresp – T responder cells
ZM - ZM241385
viii
List of Figures
Figure 1 Schematic diagram representing the events of inflammatory response……………..… 2
Figure 2 Schematic diagram shows initiation and expansion of inflammatory response during
infection………………………………………………………………………………………….. 3
Figure 3 Schematic diagram shows inflammation can be a double-edged sword and it is
important to regulate the inflammation for the survival of host organism………………………. 5
Figure 4 Subtypes of adenosine receptors………………………………………………………. 7
Figure 5 Mechanisms involved in formation of extracellular adenosine….……………………. 8
Figure 6 Adenosine-A2AR anti-inflammatory mechanism……………………………………. 10
Figure 7 A2AR agonists suppress T cell activation, but the magnitude of inhibition is stronger
for IFN- γ production than proliferation………………………………………………………... 18
Figure 8 Activated T cells proliferated well in the presence of A2AR agonist, but the number of
IFN--producing T cells was largely decreased………………………………………………… 19
Figure 9 Both CD4+ and CD8
+ T cells respond well to A2AR agonists and are susceptible to
immunosuppressive effects……………………………………………………………………... 21
Figure 10 IL-2 supplementation did not reverse A2AR-mediated immunosuppression………. 22
Figure 11 Decreased cytotoxicity in activated T cells developed under influence of A2AR
agonist…………………………………………………………………………………………... 23
ix
Figure 12 Impaired IFN- producing activity in activated T cells developed in the presence of
A2AR agonist…………………………………………………………………………………… 24
Figure 13 Mixed lymphocyte culture (MLC) was set up in the presence of A2AR agonist, CGS
or NECA………………………………………………………………………………………... 35
Figure 14 Adenosine receptor mediated increase in frequency of Foxp3 expressing CD4+ T
cells……………………………………………………………………………………………... 37
Figure 15 Time-dependent changes of Treg increase during MLC with A2AR agonist…... 38, 39
Figure 16 Increase of CD39, CD73 and CTLA-4 expression on CD4+ cells cultured in the
presence of A2AR agonist………………………………………………………….………. 40, 41
Figure 17 A2AR stimulation increased regulatory activity of Treg…………………………… 43
Figure 18 Statistically significant increase in suppressive activity of Treg cultured in presence of
A2AR agonists………………………………………………………………………………….. 44
Figure 19 Increase in number of Treg after T cell activation in the presence of A2AR
agonists…………………………………………………………………………………………. 45
Figure 20 A2AR stimulation can enhance the induction of CD4+ Foxp3
+ cells from CD4
+ CD25
-
cells……………………………………………………………………………………………... 47
Figure 21 The expansion of Treg in the presence of A2AR agonist was due to the proliferation
of preexisting nTreg…………………………………………………………………………….. 50
Figure 22 Schematic representation of the hypothesis - Adenosine-A2AR mediated effect on
induction of Foxp3 expression via oxidative stress pathway……………………...…………… 57
x
Figure 23 Depletion of glutathione causes an increase in proportion of Treg cells……….…… 60
Figure 24 Addition of anti-oxidant cannot block the A2AR mediated increase in proportion of
Treg cells………………………………………………………………………………………... 62
xi
Acknowledgements
I would like to thank, Dr. Michail Sitkovsky for giving me the golden opportunity to explore the
world of scientific research. His enthusiasm and energy towards research and life is truly
infectious.
I am truly indebted and thankful to my mentor, Dr. Akio Ohta for the support and guidance that
he gave me throughout my academic program. I have learnt a lot from him but his passion,
dedication and discipline towards scientific research are the qualities that I would like to emulate
in my future career. It has been an honor and pleasure to work with him.
I would like to thank my thesis committee members, Dr. Richard Deth, Dr. John Gatley and Dr.
Simon Robson for their encouragement and valuable suggestions that assisted towards
completion of my thesis.
I owe sincere and earnest thankfulness to Akiko Ohta who has helped me tremendously
throughout the project and also otherwise. I would like to express my gratitude to all the present
and past lab senior scientists for their support and my sincere thanks to Susan for always being
my cheerleader.
I am obliged to many of my colleagues, Manasa, Dr. Steve, B cell Bob, Peter, Shalini and Molly
for their support and for providing a healthy and fun working environment in the lab.
A special thanks to my decade old partner in crime, Meenakshi without her this journey would
be incomplete.
xii
Last, but not the least, the people who made this dream possible, my parents and my sister,
Deepika who have been a constant source of strength and support. Without their selfless love this
would have been impossible. I dedicate my thesis to Amma, Anna and Deepu.
1
1. INTRODUCTION:
Exogenous agents such as infection or toxins trigger a host defense response which is
represented by inflammation. The inflammatory process is very crucial in removal of causative
agents such as pathogens and virus-infected cells and also to clear out the resulting dead cells
and tissue debris to initiate the healing process.
‘Inflammation’ which derived its name from the latin verb “inflammare”, means to set on fire, is
one of the oldest recorded medical conditions. This term was coined by the Roman physician
Aulus Cornelius Celsus in the 1st century AD and was the first one to document what we today
recognize as the four cardinal signs and symptoms of inflammation – rubor (redness), tumor
(swelling), with calor (heat) and dolor (pain). The fifth sign, function laesa (loss of function)
was added by another Roman physician, Aelius Galenus in the 2nd
century AD [3].
The signs and symptoms of inflammation can be explained by the changes happening in the
local vasculature of an injured tissue. The blood vessels in the affected area constrict which
results in engorged capillaries (vasodilation) leading to tissue redness and increase in tissue
temperature. It also leads to accumulation of protein rich exudate resulting from an influx of
fluids and cells from the engorged permeable capillaries into the tissue contributing to the tissue
swelling (edema). The increased permeability of the capillaries causes influx of phagocytes such
as neutrophils and monocytes which reach to the site of injury/infection through multiple steps.
The endothelial walls of the blood vessels start expressing adhesion molecules to which the
immune cells adhere and start their migration along the walls and then into the tissues to the site
of inflammation (Fig. 1)[4].
2
Figure 1: Schematic diagram representing the events of inflammatory response. Tissue damage
caused by bacterial infection leads to release of various chemotactic and vasoactive factors
which then triggers increased blood flow, capillary permeability and influx of fluids and
phagocytic cells. These cells then migrate from blood to the tissue, to the site of inflammation
and destroy the bacteria [4].
The activated immune cells secrete various cytokines and cytotoxic molecules such as IL-1, IL-
6, tumor necrosis factor–α (TNF-α), interferon–γ (IFN-γ) as well as chemokines which initiate
and expand the inflammatory process (Fig. 2). The inflammatory response expands rapidly and
the immune cells such as macrophages, basophils and eosinophils start clearing out the offending
stimulus. Once the offending stimulus is removed, the inflammatory response subsides, the
phagocytic cells begin clearing out the debris and the process of tissue repair and regeneration
begins. Hence, inflammation is very important process for healing the wounds and infection [4].
3
Figure 2: Schematic diagram shows initiation and expansion of inflammatory response during
infection [1].
There are two main types of inflammation - acute inflammation and chronic inflammation.
Acute inflammatory response occurs immediately within hours to days of injury. It is an initial
effort made by the immune system to clear the harmful stimuli and begin the resolution of
inflammation. The outcome of acute inflammation can be complete resolution with limited tissue
damage or fibrosis (scarring). Also, it may cause abscess formation after infections [4].
However, when acute inflammation cannot be resolved due to various reasons such as persistent
injury or infection, prolonged exposure to toxic substance or autoimmune disorders, it results in
chronic inflammation. As opposed to acute inflammation, chronic inflammation usually persists
4
for months or sometimes years. Since chronic inflammation occurs over a prolonged period of
time, the exposure time of the activated immune cells at the site of injury increases, increasing
the damage to surrounding healthy tissues. Intense and very extensive inflammation in tissues
can cause acute tissue dysfunction, which is often fatal. If the cells of the organ have little or no
regenerative ability such as neurons and cardiac cells, it will ultimately lead to decline or loss of
organ function. Extensive fibrosis in chronic inflammation also seriously impairs normal
functions of the tissue. It has been well documented that inappropriate inflammation or a long-
term inflammation leads to pathogenesis of many diseases including cancer, heart disease, and
atherosclerosis. In the case of chronic inflammation, inflammatory responses may be implicated
in inducing DNA damage in the proliferating cells resulting in point mutations, deletions or
rearrangements leading to cancer.
Thus, proper regulation of inflammatory responses is extremely important for survival of the host
organism. Inadequate inflammatory response may lead to overwhelming, potentially fatal
infection, whereas excessive response can cause autoimmune disorder and organ dysfunction,
which may also result in death [Fig. 3].
5
Figure 3: Schematic diagram shows inflammation can be a double-edged sword and it is
important to regulate the inflammation for survival of the host organism [2].
Hence, it is very important to identify and understand endogenous mechanisms that regulate
inflammatory pathways. Knowledge of the endogenous pathways involved in the down-
regulation of inflammation will help us in identifying novel treatments which can selectively
suppress or enhance the natural anti-inflammatory pathway [5].
One such physiological non-redundant pathway that has been identified is the adenosine-A2AR
anti-inflammatory pathway [6]. Understanding the molecular mechanisms involved in the
adenosine-A2AR pathway will help us modulate them to our advantage in development of novel
drugs and therapeutic strategies.
Therefore, the objective of my study was to investigate the mechanism of adenosine-A2AR
mediated immunosuppression in the inflamed tissue micro-environment.
6
2. Inhibition of T cell effector functions by adenosine-A2AR signaling
2.1 BACKGROUND AND SIGNIFICANCE:
Role of adenosine in regulating inflammation:
The pharmacological effects of adenosine in various physiological processes such as
neurotransmission, modulatory functions of heart and kidney, vascular smooth muscle dilation
was first documented over 80 years ago and its role in modulating inflammatory processes by
immune cells such as mast cells and neutrophils was described about 25 years ago. Adenosine
receptors expressed on the cell surface are G-protein coupled receptors with four subtypes A1,
A2A, A2B and A3 [8]. The A1 and A3 receptor subtypes couple predominantly to Go and Gi
proteins and inhibit adenylyl cyclase whereas A2A and A2B receptor subtypes couple
predominantly to Gs protein and activate adenylyl cyclase, thereby increasing intracellular levels
of cAMP [5]. A2B receptors also interact with Gq protein in some cells. Affinity binding studies
have shown that the A2A receptor subtype is a high affinity receptor whereas A2B is low affinity
receptor (Fig. 4) [5]
7
Figure 4: Subtypes of adenosine receptors. Adenosine receptors belong to the family of seven
transmembrane G-protein coupled receptors [5].
A2AR is predominantly expressed on T cells [9, 10]. A2AR agonists strongly suppress T cell
activation via induction of highly immunosuppressive intracellular cAMP [5-8, 11]. Genetic
deletion and use of antagonists of A2AR has shown increased inflammatory tissue damage [2,
12, 13]. These observations demonstrate that the adenosine-A2AR pathway is non-redundant and
plays an important role in down-regulation of inflammation [5-7].
Adenosine is a purine nucleoside which is ubiquitously present in human tissues. Adenosine
plays an important role in many physiological processes such as energy generation and protein
metabolism [14]. For the metabolism of extracellular adenosine, adenosine triphosphate (ATP) is
converted to adenosine diphosphate (ADP) to adenosine monophosphate (AMP) by the enzyme
ecto-apyrase (CD39). This AMP is further broken down to adenosine by the action of enzyme 5’-
nucleotidase (CD73). Accumulation of extracellular adenosine is transient since it is converted to
8
inosine by ecto-adenosine deaminase (ADA). Also, with the help of bidirectional transporters
present on many cells, adenosine is able to travel across the membrane to maintain equilibrium
between intracellular and extracellular adenosine concentrations [15]. Increase in the
intracellular concentration of adenosine leads to passive diffusion to the outside. A large amount
of ATP is also released by cells whose membrane integrity has been compromised such as
apoptotic and necrotic cells (Fig. 5).
Figure 5: Mechanisms involved in formation of extracellular adenosine. Extracellular adenosine
can be formed from conversion of ATP to ADP/AMP to ADP by ecto enzymes, CD39 and
CD73. It can be also exported outside the cell by means of equilibrative transporters (Ent). The
nature of ATP transporter is unclear although ATP is also exported from cells by exocytosis.
Also, ATP is released into extracellular space by damaged or necrotic cells. The enzyme
adenosine kinase is responsible for reversible reaction between adenosine and AMP [17].
9
Under normal conditions extracellular adenosine is maintained at low levels (<50nM) but tissue
insult due to excessive inflammatory collateral damage to the tissue microvasculature leads to an
increase in levels of extracellular adenosine of more than 200-fold. Damage to the
microvasculature, in turn, leads to a decrease in the oxygen tension in the tissue [5, 7]. Tissue
hypoxia induces up-regulation of extracellular adenosine-generating enzymes 5’-nucleotidase
(CD73) and apyrase (CD39) [18-20]. Also, the enzyme responsible for degradation of adenosine
to inosine, adenosine deaminase, may be down-regulated by hypoxia [21, 22].
All the above mentioned factors support the view that hypoxia in the local tissue is responsible
for the increase in extracellular adenosine during tissue inflammation. This is also recently
demonstrated by Choukѐr et al., who showed that whole body hypoxia plays a critical role in
reducing tissue damage during inflammation via the adenosine-A2AR pathway [23]. Therefore,
increased extracellular adenosine subsequent to hypoxia can reduce tissue injury through
interaction with A2AR (Fig. 6) [15, 24].
The same mechanism that protects normal tissues from excessive damage by inflammation may
also play a role in protecting tumors from anti-tumor T cells. Cancerous cells divide rapidly,
making the cancerous tissues hypoxic in nature. A chronic state of hypoxia has been
demonstrated in tumors [25, 26]. The state of hypoxia correlates with the increased gradient of
extracellular adenosine from the periphery to the core/center of the tumor [27]. The presence of
adenosine in tumors might play a role in suppressing anti-tumor immune responses. Ohta et al.
demonstrated that the mice lacking A2AR could reject the tumor, while the wild type mice failed
to do so [27]. Also, A2AR antagonists prevented tumor growth by enhancing the effects of
adoptively transferred antitumor T cells [27]. The above results support the hypothesis that a
10
hypoxia-adenosinergic mechanism might play a role in inhibition of anti-tumor T cells, thereby
protecting the tumors.
Figure 6: Adenosine-A2AR anti-inflammatory mechanism: In response to inflammatory stimuli
such as pathogen, injury or cell death, immune cells are activated to initiate immune responses.
Activated immune cells kill the pathogen by release of pro-inflammatory cytokines and cellular
cytotoxicity and cause inflammation. Once inflammatory responses expand, it causes collateral
tissue damage and also results in damage to blood vessels causing hypoxia. This leads to
production of the anti-inflammatory molecule, adenosine by damaged tissues which then acts on
adenosine receptors present on T cells to inhibit the inflammatory response [7].
The above evidences indicated that tissues under inflammatory insult tend to form a self-
protective environment by elevating the levels of extracellular adenosine, thereby preventing
11
damage to tissues of vital organs by the attack of immune cells [5, 7]. Therefore, for the T cells
to act in an inflamed tissue to clear out the pathogens and virus infected cells or tumor cells, they
may have to get activated in an adenosine-rich environment. Since adenosine is strongly
suppressive to T cells [5-8, 11], it was speculated that T cells activated in the presence of
elevated levels of adenosine may be functionally different from T cells activated in the normal
environment. Hence, the primary aim of this study was to compare the effects of A2AR agonists
on T cell proliferation and its effector functions, cytokine production and cytotoxicity.
12
2.2 SPECIFIC AIM 1
To investigate inhibition of T cell effector functions by adenosine-A2AR signaling:
1. To determine the susceptibility of T cell proliferation and cytokine production to A2AR
agonists.
2. To compare the susceptibility of CD4+
and CD8+
T cells to A2AR agonists.
3. To measure cytotoxic activity in T cells activated in the presence of A2AR agonist.
4. To determine the long term effect on effector functions of T cells activated in the presence of
A2AR stimulation.
13
2.3 MATERIALS AND METHODS
2.3.1 Mice
C57BL/6 mice were obtained from Charles River Laboratories (Wilmington, MA). The mice
used were of 8-10 weeks old and were housed in the animal facility of Northeastern University
as per the institutional animal care guidelines.
2.3.2 Cytokine production and cell proliferation
Spleen cells (5 x 105 cells) from C57BL/6 mouse were stimulated with anti-CD3 mAb (145-
2C11; BD Biosciences, San Diego, CA) at 0.1 g/ml in a 96-well plastic flat bottom plate.
A2AR agonists, 5’-N-ethylcarboxamidoadenosine (NECA; Sigma, St. Louis, MO) or CGS21680
(CGS; Tocris, Ellisville, MO), were added at different concentrations of 10-9
, 10-8
, 10-7
, 10-6
and
10-5
M. Culture supernatant was collected after 24 h for subsequent measurement of cytokine
levels. IFN- and IL-2 levels were quantified by ELISA (R&D Systems; Minneapolis, MN) as
per the manufacturer’s instructions. For proliferation assay, after collecting supernatant, cells
were incubated with 1 Ci [3H] thymidine (American Radiolabeled Chemicals, St. Louis, MO)
for an additional 4 h. After the incubation, the cells were harvested using cell harvester and
allowed to air dry for about 1h. Then the samples were collected into plastic tubes and filled with
5ml of scintillation fluid. Radioactivity of incorporated thymidine was counted using a beta-
radiation counter.
2.3.3 Purification of T cells
Purification of CD4+ and CD8
+ T cell populations was performed using an Auto MACS
separator (Miltenyi Biotec, Auburn, CA). Spleen cells were labeled with FITC-conjugated anti-
CD4 or anti-CD8 mAb (BD Biosciences), and followed by anti-FITC microbeads (Miltenyi
14
Biotech). After cell sorting, purity of the cells was more than 98 %. The purified cells (2 x 105
cells) were stimulated with anti-CD3 mAb (0.1 g/ml) in the presence of feeder cells (5 x 105
cells). The feeder cells were prepared from spleen cells depleted of CD3-positive cells using
Auto MACS, and were pretreated with 50 g/ml mitomycin C for 30 min at 37 ºC.
2.3.4 Apoptosis assay
Spleen cells were activated for 24 h in the presence of various concentrations of CGS (10-9
, 10-8
,
10-7
, 10-6
and 10-5
M) and examined for the induction of apoptotic cell death. After incubation,
the cells were washed twice with cold PBS and then apoptotic cells were detected by Annexin V-
FITC apoptosis detection kit (BD Biosciences).
2.3.5 Proliferation assay using CFSE
Spleen cells were washed with PBS and labeled with carboxyfluorescein succinimidyl ester
(CFSE) (Molecular Probes, Eugene, OR) at 1 M for 8 min. To remove excess CFSE, the cell
suspension was diluted with fetal calf serum and centrifuged. This washing step was repeated
twice. The CFSE-labeled cells (2 x 105) were stimulated with anti-CD3 (0.1 μg/ml) mAb for 36h.
After the culture, the cells were stained with PE-conjugated anti-CD8 and allophycocyanin-
conjugated anti-CD4 mAbs, and cell division was analyzed by flow cytometer.
2.3.6 Intracellular cytokine staining
Cytokine production from T cells was analyzed by intracellular staining as described [28]. After
the stimulation with anti-CD3 mAb for 24 h, cells were further incubated in the presence of
brefeldin A (10 g/ml) for 2 h. Cells were then surface-stained with PE-conjugated anti-CD8
mAb and allophycocyanin-conjugated anti-CD4 mAb for 10 min. The cells were then further
fixed with 4% paraformaldehyde-PBS for 15 min, permeabilized with permeabilization buffer
15
(50 mM NaCl, 5 mM EDTA, 0.02% NaN3, 0.5% Triton X-100, 10 mM Tris-HCL, pH 7.5) for 15
min. After fixation and permeabilization, intracellular IFN- was stained with FITC-labeled anti-
IFN- mAb and incubated for 45 min, and then analyzed by FACSCalibur (BD Biosciences). All
antibodies were obtained from BD Biosciences.
2.3.7 cAMP assay
Measurements of cAMP levels were performed as described previously [29]. Cells (2 x 105)
were prepared in 160 μl of media and then incubated with 40 μl of NECA, CGS or ZM in an
1.5ml eppendorf tube for 15 min at 37 C. The concentration of A2AR agonists was 5 M.
A2AR antagonist, ZM241385 (Tocris) was added at 1 M. The reaction was stopped by addition
of 25 μl of 1N hydrochloric acid. It was mixed and frozen at -80oC till the cAMP levels were
measured. cAMP levels were then determined by ELISA (GE Healthcare, Buckinghamshire,
UK) as per the manufacturer’s instructions.
2.3.8 Cytotoxicity
Cytotoxicity of T cells against P815 mastocytoma (H-2d) was determined by
51Cr release assay.
P815 cells (2 x 106) were incubated with 100 μCi [
51 Cr] sodium chromate for 1h at 37
oC. After
incubation, those cells were washed with culture medium for 3 times by centrifugation to remove
excess chromium. Effector cells were prepared 36 h after stimulation of C57BL/6 spleen cells
with anti-CD3 mAb, and incubated with the labeled target cells (1 x 104
cells) for 4 h at 37oC in
the presence of anti-CD3 mAb (1 g/ml). Effector-target ratio was 10:1 and 5:1. Brief
centrifugation is required for sedimentation of the cells in a 96-well v bottom plate. After 4 h,
supernatant was collected to count the [51
Cr] release from the target cells using gamma-radiation
counter. Spontaneous 51
Cr release and maximum 51
Cr release was measured by culturing target
16
cells alone and by addition of 1 N hydrochloric acid to the same number of target cells
respectively. Cytotoxicity was calculated as percentage cell lysis when spontaneous 51
Cr release
and maximum 51
Cr release were considered to be 0% and 100%, respectively.
2.3.9 Statistics
All experiments were independently repeated at least three times. Data represent mean SD.
Statistical calculations were performed using Student’s t-test. Statistical significance was
accepted for p values less than 0.05.
17
2.4 RESULTS:
2.4.1 Expansion of T cells with impaired IFN-γ production in the presence of A2AR
agonists
The purpose of this experiment was to determine the susceptibility of T cell proliferation and
cytokine production in the presence of various concentrations of A2AR agonists. The agonists,
NECA (non-specific adenosine receptor agonist) and CGS21680 (A2AR-specific agonist)
inhibited T cell proliferation and IFN- levels in a concentration-dependent manner (Fig. 7A, B).
The suppression of T cell proliferation and IFN- production by A2AR agonists were not seen in
A2AR knockout mice confirming that the immunosuppressive effects were via A2AR
stimulation (Fig. 7C, D). At 10 nM concentration of CGS or NECA, there was a decrease in T
cell proliferation by 10% (Fig. 7A). T cells retained 80 % of their proliferative activity even at
the highest concentration of A2AR agonists. The previous result was confirmed using CFSE
staining of spleen cells. The cells showed high T cell proliferation in presence of CGS which can
be seen by dilution of fluorescence in the newly divided cells (Fig. 8A). A decrease in left peaks
of both CD4+ and CD8
+ shows that CGS inhibits proliferation to a certain extent. In this
experimental setting, CGS was not able to completely suppress T cell proliferation, even at its
highest concentration (Fig. 7A, 8A). The inhibition that we see in cell proliferation is not due to
CGS induced apoptotic cell death (Fig. 8B).
In contrast to its effect on cell proliferation, IFN-γ production was inhibited to a great extent,
even at 10 nM of CGS and NECA. At the highest concentration of CGS and NECA, more than
90% of IFN-γ production was inhibited (Fig. 7B).
18
Figure 7: A2AR agonists suppress T cell activation, but the magnitude of inhibition is stronger
for IFN- γ production than proliferation. (A, B) Inhibition of T cell proliferation and IFN-γ levels
in a concentration dependent manner by both CGS and NECA. † P < 0.01,
‡ P < 0.001 for both
control vs CGS and control vs NECA. Data represent average SD of triplicate samples. (C, D)
Suppression of T cell proliferation and IFN- γ production by CGS was not observed in T cells
from A2AR-deficient mice in comparison to wild type mice. Data represent average SD of
triplicate samples. b P < 0.01,
c P < 0.001 wild type vs A2AR
-/-.
This dramatic fall in IFN-γ production was not simply because of the decrease in the number of
proliferated T cells. In Fig. 8C, it can be seen from attenuating fluorescence intensity that the
IFN-γ producing T cells are strongly inhibited by CGS in a concentration-dependent manner.
19
These results show that even under the highest concentration of A2AR agonists, T cells are able
to expand well, but their effector function of IFN-γ production is severely impaired.
Figure 8: Activated T cells proliferated well in the presence of A2AR agonist, but the number of
IFN--producing T cells was largely decreased. (A) Spleen cells were labeled with CFSE and
cultured with anti-CD3 mAb for 36 h. The cells were analyzed by flow cytometry after surface
staining of CD4 and CD8. The decreased fluorescence intensity indicates dilution of CFSE in
proliferated cells. (B) There was no significant increase of apoptotic cells in CGS-treated cells.
Spleen cells were activated and stained with FITC-annexin V and propidium iodide (PI). The
20
numbers represent percentage of cells in quadrants. CGS did not increase apoptotic cells at any
concentrations. Only results from control and the highest concentration of CGS were shown as
representative. (C) Decrease of IFN--producing cells by CGS. The numbers represent
percentage of CD8+ IFN-
+ cells.
2.4.2 Both CD4+
and CD8+ T cells are sensitive to A2AR mediated immunosuppression
T cell subsets, CD4+ and CD8
+ cells were purified to determine whether both are equally
susceptible to A2AR mediated immunosuppression. Post A2AR stimulation, cAMP production
was measured in order to confirm the presence of functionally active A2AR on T cells. Purified
CD4+ and CD8
+ cells showed increased levels of cAMP in the presence of CGS or NECA
(Fig.9A). The reversal of elevated cAMP levels by A2AR antagonist ZM241385 confirmed this
effect to be A2AR mediated response. Although CD4+ cells produced higher levels of cAMP
when compared to CD8+ cells, the concentration of CGS required for the significant induction of
cAMP was similar in both of the cells (Fig. 9B). B cells showed even lower levels of cAMP
when compared to CD8+ cells (Fig 9A). cAMP production by both CD4
+ and CD8
+ T cells
implies that A2AR-mediated immunosuppressive mechanism might be targeting both T cell
populations. Upon studying the effects of A2AR agonist on the activation of purified CD4+ and
CD8+ T cells, similar responses were observed to those seen in unseparated cell cultures. CGS
slightly decreased proliferation in both CD4+ and CD8
+ T cells (Fig. 9C). In contrast to the slight
suppression of T cell proliferation, IFN-γ production from CD4+ and CD8
+ T cells was strongly
suppressed by CGS (Fig. 9D). These results reiterate the observations made with unseparated
cell culture that IFN-γ production is inhibited strongly whereas cell proliferation is resistant to
A2AR stimulation in both CD4+ and CD8
+ T cells.
21
Figure 9: Both CD4+ and CD8
+ T cells respond well to A2AR agonists and are susceptible to
immunosuppressive effects. (A) NECA- or CGS-inducible cAMP levels in purified CD4+ &
CD8+ T cells. ZM241385 (ZM) is an A2AR antagonist. (C, D) Suppression of proliferation and
IFN- production by purified CD4+ and CD8
+ cells stimulated in the presence of CGS. Data
22
represent average SD of triplicate samples. * P < 0.05, † P < 0.01 vs control in both CD4
+ and
CD8+ cells.
2.4.3 Effects of A2AR agonist on cytokine production
Next, the effect of A2AR agonist on other cytokines such as IL-2 which plays an important role
in activated T cells was examined. IL-2 production was suppressed in the presence of CGS. This
inhibition was not as strong as that seen in IFN-γ. IL-2 levels were decreased by 40% by CGS
(Fig. 10A). Since, inhibition of IL-2 could be responsible for the immunosuppressive action of
A2AR agonist, recombinant IL-2 was added along with CGS at the start of the culture. However,
addition of IL-2 failed to reverse the A2AR-mediated inhibition of T cell proliferation and IFN-γ
production (Fig. 10B, C).
23
Figure 10: IL-2 supplementation did not reverse A2AR-mediated immunosuppression. (A)
Decrease of IL-2 levels by A2AR agonists. †
P < 0.01 vs control in both CGS- and NECA-treated
cells. (B, C) Recombinant mouse IL-2 (5 ng/ml) was added to compensate decrease of
spontaneous IL-2 production. There was no significant improvement in T cell proliferation and
IFN- production by IL-2 supplementation. Data represent average SD of triplicate samples.
2.4.4 Evaluation of Cytotoxicity
The inhibition of effector function by A2AR agonist was not restricted to cytokine production.
Cytotoxic activity was also suppressed in T cells in the presence of CGS. The inhibition of
cytotoxicity was approximately half of control (Fig. 11). The extent of inhibition was weak as
compared to IFN-γ, similar to IL-2 but stronger than proliferation.
Figure 11: Decreased cytotoxicity in activated T cells developed under influence of A2AR
agonist. T cell activation was induced as in Fig. 1 with various concentrations of CGS. After
extensive wash to remove CGS, cytotoxicity against P815 cells was examined by 4-h standard
24
51Cr release assay. Data represent average SD of triplicate samples. * P < 0.05,
† P < 0.01,
‡ P <
0.001 vs control.
2.4.5 Long term effect on effector functions of T cells activated in presence of A2AR
stimulation
Suppression of T cell effector functions by A2AR agonist could be a temporary change which
happens only in the presence of A2AR agonist or a persistent impairment. To examine the IFN-γ
producing potential of the cells in the absence of A2AR agonist, the activated cells were washed
to remove CGS and then the same number of cells was subsequently restimulated with anti-CD3
and anti-CD28 mAbs immediately after the removal of CGS (Day 2). As a result, T cells
activated in the presence of CGS could produce a significantly smaller amount of IFN-γ (Fig.
12A).
Figure 12: Impaired IFN- producing activity in activated T cells developed in the presence of
A2AR agonist. T cell activation was induced by anti-CD3 mAb with various concentrations of
CGS. Cells were then restimulated by immobilized anti-CD3 and anti-CD28 mAbs either
25
immediately after removal of CGS (Day 2) or after culturing for 4 more days in the absence of
CGS (Day 6). When cells were rested for 4 days, IL-2 (5 ng/ml) was added to the culture. IFN-
levels in the supernatant were quantified 24 h after restimulation.
To examine whether suppression of IFN-γ production would persist even longer, the activated T
cells were rested for 4 days and then restimulated on the 6th
day. IFN-γ production from T cells
activated in the presence of CGS was still lower, indicating the development of less efficient
effector T cells in an adenosine-rich environment (Fig. 12B).
26
2.5 DISCUSSION:
The adenosine-mediated pathway plays an important role in protecting tissues during
inflammation [5, 7], as shown by worsening of damage incurred by the tissue in A2AR knockout
mice [6, 12, 13, 30]. Since T cells predominantly express A2AR on their surface, they are the
targets of this mechanism [9, 10]. Both T cell populations of CD4+ and CD8
+ were susceptible to
adenosine-A2AR stimulation (Fig. 8).
In the adenosine-A2AR signaling pathway, adenosine binds to its receptor and activates
adenylate cyclase which in turn increases the intracellular cAMP levels. The increase in cAMP
levels leads to activation of protein kinase A (PKA) [6, 8]. PKA then phosphorylates COOH-
terminal Src kinase (Csk) which then interrupts Lck activation, thereby inhibiting T cell
activation [31]. Prior studies of cAMP and PKA mediated inhibition of T cell functions show
that A2AR agonists by increasing cAMP levels inhibit cytotoxic activity via down-regulation of
Fas ligand and granule exocytosis [32-35]. These data explain how the A2AR-mediated increase
of cAMP may inhibit general T cell responses such as proliferation [36] and cytokines
production [37, 38].
To see whether different functions of T cells are equally susceptible to inhibition by A2AR
signaling was the most intriguing question. The effect of A2AR-mediated inhibition on T cell
proliferation, cytokine production and cytotoxicity was tested and compared in T cells from the
same cell culture. Down regulation of effector functions such as IFN-γ/IL-2 production and
cytotoxicity was observed whereas T cell proliferation was not affected much. This suggests that
adenosine does not totally suppress T cell functions but when T cells are activated in presence of
adenosine, the proliferated T cells will have limited effector functions (Fig. 7, 8, 10 & 11).
27
The present data helps us in understanding about immunosuppression occurring due to the
adenosine-rich microenvironment near and within tumor [39-41]. Anti-tumor T cells isolated
from the tumors of cancer patients are incapable of killing tumor cells. But interestingly, these
ineffective anti-tumor T cells regain their tumor specific toxicity upon culturing them with IL-2
in vitro [42]. This suggests that in cancer patients, T cells get activated when they come across
the tumor antigen but the anti-tumor T cells lose their effector functions in the tumor
environment [43]. It is important to note that though T cells respond very well to tumor antigen,
in the tumor microenvironment they become tolerized, which can be seen by their loss of effector
functions [44, 45].
The present study demonstrates that T cell activation in the presence of A2AR agonists has
detrimental effect on their effector functions though their proliferation is not affected as much as
their effector functions. Thus, it suggests that blocking of the adenosine-A2AR pathway may
ameliorate the effector functions of anti-tumor T cells, thereby helping fight the tumor.
Combining this anti-adenosinergic treatment with other immunotherapies such as adoptive T cell
transfer and tumor vaccine that will promote the number and/or function of anti-tumor T cells
will be beneficial in treating cancers.
The adenosine-A2AR immunoregulatory mechanism may have evolved for the fundamental
reason of protecting normal tissues from collateral damage during inflammation [5-7, 12, 13, and
30]. This study suggests that this mechanism may have evolved to limit excessive damage in the
tissue milieu, without overall inhibition of T cell expansion. Also, since we did not see strong
inhibition of proliferation or apoptosis of activated T cells by A2AR stimulation, it suggests that
this mechanism may play a role in preserving antigen-specific T cells reflecting T cell “memory”
(Fig. 7, 8).
28
3. Adenosine-A2AR signaling promotes T regulatory cell-dependent
immunosuppression
3.1 BACKGROUND AND SIGNIFICANCE:
In autoimmune disorders, our immune system fails to discriminate between self / non-self
antigens and destroys cells and tissues of the body. This ensuing inflammation because of an
overactive immune response must be suppressed in order to treat autoimmune disorder [46].
Recent studies have shown that immunosuppressive functions of regulatory T (Treg) cells play
an important role in the prevention of autoimmune disorders. Treg is a subset of the T cell
population that acts as negative regulator of immune responses, thereby preventing excessive
tissue damage. Tregs are characterized by their expression of CD4, CD25 (IL-2 receptor alpha
chain) and Forkhead box P3 (Foxp3). They have an ability to suppress proliferation of
conventional T cells [47, 48]. Expression of Foxp3 is important for development of Tregs [48].
Treg are also of great interest due to their potential to treat immunological diseases and control
physiological and pathological immune responses. However, there are still important and yet to
be answered questions about the influence of the microenvironments in lymphoid and inflamed
tissues in the development and immunoregulatory functions of Treg.
The relationship between Treg cells and the immunosuppressive effect of extracellular adenosine
has not been clearly elucidated yet. There are several evidences suggesting that Treg activity is
mediated by the accumulation of extracellular adenosine. Studies report that high levels of
adenosine generating enzymes, CD39/CD73 are expressed on the surface of Treg cells indicating
their capability to increase local extracellular adenosine concentration by ATP catabolism [19,
29
20]. Treg cells have been shown to use adenosine as one of their immunosuppressive
mechanisms [20].
However, there is still lack of data that may firmly implicate the adenosine and A2AR in
functions of Treg. A2AR stimulation was reported to up-regulate Foxp3 mRNA [38] but the
effects of A2AR agonist on the number and immunosuppressive activity of Treg are not known.
Hence, there is a possibility that T cell activation in adenosine-rich environment may enhance
generation and/or regulatory activities of Tregs. Since immunosuppressive effect of adenosine
might be not only a direct inhibition of T cell activation but also through the enhanced Treg
activities, Tregs in the presence of A2AR stimulation will be examined in terms of their
expansion and their regulatory activities.
30
3.2 SPECIFIC AIM 2
To investigate the role played by adenosine in generation/expansion of immunosuppressive
Tregs and their regulatory activities:
1. To determine the changes in Treg population (Foxp3+ cells) after culturing with A2AR
agonist.
2. To determine changes in the immunosuppressive activity of Treg when cultured with A2AR
agonist.
3. To determine whether the increase in proportion of Foxp3+ cells activated in presence of
adenosine analogs is expansion of natural Tregs or generation of new Tregs.
31
3.3 MATERIALS AND METHODS
3.3.1 Mice
C57BL/6 (Thy1.2+) and BALB/c mice were purchased from Charles River Laboratories
(Wilmington, MA). B6.PL-Thy1a/CyJ mice (Thy1.1+ C57BL/6 mice) were purchased from
Jackson Laboratory. Mice were used at 8-12 weeks of age. The experiments were approved by
the Northeastern University Institutional Animal Care and Use Committee and were carried out
in accordance with the institutional animal care guidelines.
3.3.2 Mixed lymphocyte culture (MLC)
Antigen-specific effector T cells were induced by allogenic mixed lymphocyte culture using
spleen cells from C57BL/6 (H-2b) and Balb/c (H-2
d) mice. Spleen cells from Balb/c mice were
pre-treated with 50 μg ml-1
mitomycin C for 30 min at 37°C. After being washed three times by
centrifugation, 2 x 106 Balb/c spleen cells (stimulator) were mixed with 6 x 10
6 C57BL/6 spleen
cells (responder) in 2 ml of RPMI1640 medium containing 10% foetal calf serum. The cells were
cultured for 5 days in a 12-well plastic plate in presence or absence of A2AR agonists, 5’-N-
ethylcarboxamidoadenosine (NECA; Sigma, St. Louis, MO) or CGS21680 (CGS; Tocris,
Ellisville, MO) and A2AR antagonist, ZM241385 (Tocris) at 1 μM. On day 5, the activated
effector cells (2 x 106 cells) were re-stimulated using mitomycin C-treated Balb/c spleen cells (4
x 106
cells). The re-stimulated cells were cultured for additional 2 days, and were analyzed on
day 7.
3.3.3 Flow cytometric analysis
The resulted cells after MLC were analyzed by flow cytometry. Following antibodies were used
to label surface molecules: PE- and allophycocyanin (APC)-conjugated anti-CD4, FITC-
32
conjugated anti-CD8, PE-conjugated anti-CD25 and FITC-conjugated anti-H-2Kb antibodies. For
the analysis of Treg, the cells were subsequently fixed and permeabilized using Foxp3 staining
buffer set (eBioscience, San Diego, CA), and were labeled with APC-conjugated anti-Foxp3 and
PE-conjugated anti-CTLA-4 antibodies. All antibodies were from BD Biosciences (San Diego,
CA) except for anti-CD25 (Miltenyi Biotec, Auburn, CA) and anti-Foxp3 (eBioscience)
antibodies. The data were acquired using FACSCalibur (BD Biosciences).
3.3.4 MLC in the absence of CD8+ cells
In later experiments, this system was modified to suit the purpose of our experiment. To enrich
Treg after the culture, MLC was set up using CD8+-depleted C57BL/6 spleen cells. CD8
+ T cells
were depleted before starting MLC using Auto MACS separator (Miltenyi Biotec, Auburn, CA).
For sorting, the whole spleen cells were labeled with FITC-conjugated anti-CD8 mAb (BD
Biosciences), followed by anti-FITC microbeads (Miltenyi Biotech). These magnetically labeled
CD8+ T cells were then depleted from the other spleen cells and used for the culture. After cell
sorting, more than 98 % of the CD8+ T cells were depleted. These responder cells were cultured
with mitomycin C-treated stimulator cells as described above.
3.3.5 Cell proliferation assay using CFSE-labeled cells
The extent of T cell proliferation was monitored by the stepwise dilution of fluorescence in
CFSE labeled cells. The cells were labeled with CFSE as described above (Section 2.3.5).
3.3.6 Regulatory activity of Treg
After MLC using CD8+-depleted responders for 7 days (2 days after restimulation), the
regulatory activity was evaluated according to the inhibition of effector T cell proliferation.
CD8+-depleted spleen cells from Thy1.1-expressing C57BL/6 mouse were labeled with CFSE
33
and used as the source of responder T cells (Tresp). Tresp (2.5 x 104 CD4
+ cells) were co-
cultured with the product of MLC, which contains Treg, so that the ratio of CD4+ cells in Tresp
and CD4+ FoxP3
+ cells in the MLC would be constant between groups. Tresp cell proliferation
was induced with anti-CD3 mAb (0.1 μg/ml 145-2C11; BD Biosciences) for 2 days in a round-
bottomed 96-well plate, and the extent of Tresp proliferation was monitored by their stepwise
dilution of fluorescence and analyzed after gating for Thy1.1+ CD4
+ cells.
3.3.7 Treg from CD4+
CD25- cells
To start MLC in the absence of natural Treg, CD25+ cells were removed from the responder cells
prior to the culture. Spleen cells were labeled with PE-conjugated anti-CD25 and anti-CD8
mAbs, and the labeled cells were depleted using anti-PE microbeads (Miltenyi Biotec) and
AutoMACS. After 7-days MLC as described above, the appearance of CD4+
Foxp3+ cells was
tested by flow cytometry.
3.3.8 Natural Treg
CD4+
CD25+ cells were purified from spleen cells of Thy1.1-expressing C57BL/6 mice as
described [49]. CD24+ cells and CD8
+ cells were removed from the spleen cells using FITC-
conjugated antibodies and anti-FITC microbeads. Subsequently, CD25+ cells were retrieved by
positive selection using PE-conjugated anti-CD25 antibody and anti-PE microbeads. This
procedure achieves 95-98% pure CD4+
CD25+ cells. Responder cells of MLC were reconstituted
by mixing Thy1.1+
CD4+
CD25+ cells (6 x 10
4) with Thy1.2
+ spleen cells depleted of CD8
+ and
CD25+ cells (3 x 10
6). After 7-days MLC as described above, the origin of CD4
+ Foxp3
+ cells
were separately analyzed for natural Treg-derived Thy1.1+ cells and CD4
+ CD25
- cells-derived
Thy1.1- cells.
34
3.3.9 cAMP induction in Treg
Purified CD4+
CD25+ cells (1.6 x 10
5) were incubated with CGS or NECA for 15 mins at 37
0C.
The concentration of A2AR agonists was 10 μM and 1uM for A2AR antagonist, ZM241385.
cAMP levels were determined by ELISA (GE Healthcare, Buckinghamshire, UK)
3.3.10 Statistics
Data represent mean SD. Statistical calculations were performed using Student’s t-test.
Statistical significance was accepted for p values less than 0.05.
35
3.4 RESULTS:
3.4.1 Adenosine mediated down-regulation of activation marker, CD25 on CD8+ T cells
Up-regulation of the T cell activation marker CD25 was tested in T cells activated in the
presence of adenosine receptor agonists. Whole spleen cells from C57BL/6 mice (H-2b) as
responders and those from Balb/c mice (H-2d) as stimulators were used to setup mixed
lymphocyte culture in the presence of CGS and NECA at 1 μM concentration. These cells were
stimulated for 5 days and then restimulated with Balb/c spleen cells. The culture was maintained
for 2 more days and then the cells were analyzed on day 7 for the expression of T cell activation
marker, CD25.
It was found that in the presence of the adenosine analogs, CGS and NECA, there was a down-
regulation in the expression of CD25 on CD8+ T cells. When the A2AR selective antagonist ZM
was added along with A2AR agonists, ZM inhibited CGS and NECA mediated down-regulation
of CD25 expression on T cells (Fig. 13, top panel). This result corresponds to the A2AR-
mediated suppression of T cell activation described above.
36
Figure 13: Mixed lymphocyte culture (MLC) was set up in the presence of A2AR agonist, CGS
(1M) or NECA (1M). After 5 days, the cultured cells were restimulated with the same
allogenic stimulator cells for 2 more days in the same condition. A2AR stimulation inhibited
CD25 expression in CD8+ cells (top row), whereas the population of CD4
+ CD25
+ cells was
rather increased in the same culture (bottom row). The addition of A2AR antagonist ZM (1M)
reversed the changes. Numbers in the panels represent percentages in each quadrant. The data
represents four independent experiments with reproducible results.
3.4.2 Adenosine increased CD25 expression on CD4+ T cells
Since, the proportion of CD25+
CD8- cells was found to rather increase when CGS or NECA was
added to the culture; the same culture was analyzed for CD25 expression on CD4+ T cells.
Contrary to CD8+ cells, CD25 expression on CD4
+ T cells was found to increase in the presence
of A2AR agonists, CGS and NECA. The use of ZM in the culture with CGS or NECA caused
reversal of the up-regulation of CD25 expression on CD4+ T cells (Fig 13, Bottom panel).
3.4.3 Increase in frequency of Treg in the presence of adenosine receptor agonists
A2AR stimulation is generally immunosuppressive; therefore, the increase of CD4+ CD25
+ cells
was not likely to represent activation of CD4+ effector T cells. Since CD4
+ CD25
+ phenotype
represents not only activated T cells but also Tregs, the observed increase in CD25 expression on
CD4+ T cells might be explained by increase in Tregs. Foxp3 was used here to identify T
regulatory cells. It was found that T cell activation in the presence of A2AR agonists, CGS and
NECA lead to increase in the frequency of Foxp3 expressing CD4+
T cells. The reversal by ZM
shows that the increase in the frequency of Foxp3 expressing CD4+
T cells is mediated through
A2AR (Fig. 14).
37
Figure 14: Adenosine receptor mediated increase in frequency of Foxp3 expressing CD4+ T
cells. On day 7 the cells were collected and labeled for CD4 and Foxp3 as described previously.
The change in CD4+
CD25+ cells correlated with an increase of Foxp3-expressing CD4
+ cells.
The data represents four independent experiments with reproducible results.
The increase in Foxp3 cells in the presence of A2AR stimulation was statistically significant on
day 5 and even more impressive on day 7 (Fig 15A, B).
To confirm the role played by adenosine-A2AR stimulation, the experiments were done using
A2ARKO responder cells. CGS and NECA treated cells failed to block activation of CD8+ cells
and also to induce CD25 and Foxp3 expression on CD4+ cells (Fig. 15C).
38
39
Figure 15: Time-dependent changes of Treg increase during MLC with A2AR agonist. Cell
culture was done as described in fig 13. Spleen cells from wild-type (A, B) and A2ARKO mice
(C) were used as responder cells. Cells were analyzed by flow cytometry on day 5 (A) and Day 7
(B, C). A2AR agonists inhibited CD8+ T cell activation and enhanced CD25/Foxp3 expression in
CD4+ cells from wild-type mice, but not A2ARKO mice. Data represents average ± SD of 3-4
independent experiments, * P < 0.05, **P < 0.01, ***P < 0.001 vs Control.
3.4.4 Characterization of A2AR stimulated increase of CD4+CD25
+ cells
The increased CD4+ cells not only expressed CD25 and Foxp3 but also CD39, CD73 (Fig 16A)
and CTLA-4 molecules (Fig 16B). These molecules play an important role in immunoregulatory
activity of Tregs [19, 20, 48]. Interestingly, the analysis of Foxp3+ cells revealed higher levels of
CTLA-4 expression on CGS and NECA treated cells (Fig. 16B, C). Tregs are known to
constitutively express CTLA-4 and up-regulate it upon activation [48]. CTLA-4 on Tregs plays
an important role in the suppression of T cell activation [48, 50, 51]. This result implies a
possibility that Tregs induced in the presence of A2AR stimulation may have pronounced
immunosuppressive activities.
40
41
Figure 16: Increase of CD39, CD73 and CTLA-4 expression on CD4+ cells cultured in the
presence of A2AR agonist. MLC was done as explained in the Figure 12. (A) CD39 and CD73
expression in CD4+
Foxp3+ cells. H-2K
b+ CD4
+ cells were gated for analysis. (B) CTLA-4
expression was analyzed by intracellular staining together with Foxp3. H-2Kb+
CD4+ cells were
gated for analysis. Numbers in the panels represent percentages in each quadrant. (C) Histogram
plots of CTLA-4 intensity in CD4+
Foxp3+ cells. Numbers represent mean fluorescence intensity
(MFI) of CTLA-4. This data represents 5 independent experiments with reproducible results. (D)
Statistically significant increase in expression of CTLA-4 in presence of A2AR agonists. Data
represents average ± 5 independent experiments, * P < 0.05 vs Control.
3.4.5 The increase in proportion of Foxp3+ cells activated in presence of adenosine analogs
have increased ability to suppress T cell activation
Immunoregulatory activity of these CD4+Foxp3
+ cells was measured by their suppressive effect
on proliferation of CFSE labeled responder T cells (Tresp). The extent of Tresp inhibition
42
changes dependent on the ratio of Treg to Tresp. In regular MLC, CD8+ T cells mainly expand in
response to allogenic MHC thereby diluting the number of Tregs, which makes regular MLC
inconvenient as the source of Treg in this assay. Therefore, CD8+ T cells were depleted before
setting up the MLC. The subsequent culturing conditions remained same. On day 7, the cells
were analyzed for the expression of Foxp3 and similar to our previous results, there was
increased Foxp3-expressing cells upon CGS/NECA treatment (Fig. 17 top panels).
To maintain the uniformity in the assay, same number of CD4+
Foxp3+ cells from the culture was
added to the constant number of CFSE-labeled CD4+ T responder (Tresp) cells. When compared
to Tresp alone, the addition of CD4+
Foxp3+ cells inhibited T cell proliferation in a dose
dependent manner. Modest decrease in Tresp cell proliferation was seen when the cells from
control MLC was added at 2:1 (Tresp:Treg) ratio (Fig. 17, left). Similar degree of T cell
suppression was observed when the cells from CGS or NECA treated MLC was added at 8:1
ratio (Fig 17 middle and right, Fig 18). Also, addition of control Treg at 1:1 ratio caused more
significant reduction of proliferation and similar degree of reduction was achieved with the cells
from CGS or NECA treat MLC at 4:1 ratio (Fig 17 and 18). A stronger inhibition in T cell
proliferation was seen when higher number of cells from CGS or NECA treated MLC (2:1 and
1:1) was added. This result confirmed that A2AR stimulation resulted in emergence of Treg that
are more suppressive in nature and are approximately 4 times stronger when compared to control
Treg.
43
44
Figure 17: A2AR stimulation increased regulatory activity of Treg. To enrich Treg in MLC,
CD8+ cells were depleted from responder cells prior to the culture. It was confirmed that the
treatment with CGS and NECA increased Treg population in this culture condition (top panels).
The regulatory activity on T cell proliferation was determined by CFSE assay. CD8+-depleted
spleen cells from Thy1.1-expressing C57BL/6 mouse were labeled with CFSE and used as
responder cells (Tresp). Tresp containing 2.5 x 104 CD4
+ cells were co-cultured with the product
of MLC, which contains Treg. Tresp:Treg in the figure is the ratio of CD4+ cells in Tresp to
CD4+
Foxp3+ cells in the MLC. The extent of CD4
+ Tresp cell proliferation was analyzed 2 days
after the stimulation with anti-CD3 mAb. The histograms were gated for Thy1.1+
CD4+ cells.
Broken lines indicate the same peak (Peak 3). This data represents 3 independent experiments
with reproducible results.
Figure 18: Statistically significant increase in suppressive activity of Treg cultured in presence
of A2AR agonists. The data from the CFSE assay is shown as a proportion of cells that entered
into extensive proliferation. Numbers represent combined percentages of peak 3 and 4 from the
45
Fig 17. This data represents 3 independent experiments with reproducible results.* P < 0.01, **P
< 0.001 for both control vs CGS and control vs NECA.
3.4.6 A2AR stimulation increased not only regulatory activity of Treg but also the number
of Treg
The increase in proportion of Treg in cultures treated with A2AR agonist does not necessarily
indicate a numerical increase of Treg. Since, A2AR agonists can be suppressive to the activation
of effector T cell; it might indicate a proportional increase of Treg. Therefore to evaluate
numerical increase, total cell number in the culture was counted. Then numbers of CD4+
Foxp3+
and CD4+
Foxp3- cells were calculated from their proportions in the flow cytometric analysis.
The result showed a statistically significant increase of CD4+
Foxp3+ cells and a statistically
insignificant decrease of CD4+
Foxp3- cells by A2AR agonists (Fig. 19).
Figure 19: Increase in number of Treg after T cell activation in the presence of A2AR agonists.
MLC was set up in the absence of CD8+ cells. Two days after restimulation (7 days after initial
46
stimulation), total live cells were counted under a microscope. Numbers of H-2Kb+
CD4+ Foxp3
+
and H-2Kb+
CD4+ Foxp3
- cells were estimated from the result of flowcytometric analysis. The
number of H-2Kb+
CD4+ cells in the beginning of MLC was approximately 1 x 10
6 cells
including approximately 1 x 105 H-2K
b+ CD4
+ Foxp3
+ cells. Data represent average SD of 3
independent experiments. * P < 0.01 vs control MLC.
3.4.7 The increase in proportion of Foxp3+ cells activated in presence of adenosine analogs
could be generation of inducible Tregs
Above results confirmed that A2AR stimulation results in Tregs that are not only qualitatively
different as shown by their increased ability to suppress T cell activation but also quantitatively
different in Treg population. It is known that there are different types of Tregs present. They are
either generated naturally in the thymus or they are differentiating from naïve T cells in the
periphery. Tregs that are generated naturally are known as natural Tregs (nTregs), while those
that develop in the periphery are known as adaptive or inducible Tregs (iTregs). This led to the
next part of the study to determine whether the increase in proportion of Foxp3+ cells activated in
presence of adenosine analogs is an expansion of natural Tregs or generation of new Tregs.
To look at the generation of new Tregs, MLC was started without nTreg which was achieved by
depleting CD25+ cells. This got rid of most of the nTregs. Those that remained (CD25
-
CD4+Foxp3
+) were only 0.5-0.6% of CD4
+ cells (Fig 20A). After analysis on day 7, there was an
induction of CD4+Foxp3
+cells to 4.5% of CD4
+ cells in control MLC whereas A2AR agonists
treated responders saw an induction of CD4+Foxp3
+ cells to 12% of CD4
+ cells (Fig 20B). Also,
the increase was not just in proportion but there was a numerical increase in the number of
47
CD4+Foxp3
+ cells in this culture (Fig 20C). These results suggest that Tregs can be induced from
CD4+Foxp3
- cells during MLC and A2AR stimulation enhances this induction.
48
Figure 20: A2AR stimulation can enhance the induction of CD4+ Foxp3
+ cells from CD4
+
CD25- cells. (A) MLC responder cells after the depletion of CD25
+ cells. The numbers indicate
percentages of CD4+
CD25+
and CD4+ Foxp3
+ cells. The percentage of CD4
+ cells was
approximately 30% because of co-depletion of CD8+ cells. (B) Induction of CD4
+ Foxp3
+ cells
from the cells in (A). After 5 (primary stimulation) plus 2 (restimulation) days of MLC, the
increase of CD4+ FoxP3
+ cells in control was further enhanced in the presence of A2AR
agonists. The data were gated for CD4+ cells. The numbers represent percentages of Foxp3
+ cells
in CD4+ population. This data represents 3 independent experiments with reproducible results
(C) Increase in number of CD4+
Foxp3+ cells from CD4
+ Foxp3
- responder cells. Total live cells
were counted under a microscope after 7 days of culture. Numbers of H-2Kb+
CD4+ Foxp3
+ cells
were estimated from the result of flowcytometric analysis as in Fig 16. Data represent average
SD of 3 independent experiments. * P < 0.05 vs control.
3.4.7 A2AR stimulation may promote induction of CD4+CD25
- cell derived Treg but it
mainly promotes expansion of natural Treg in the culture
Next, the contribution from nTreg and CD4+
CD25- derived Treg in the A2AR-mediated increase
of CD4+
FoxP3+ cells was to be examined. To distinguish preexisting nTreg from CD4
+ CD25
-
derived Treg produced during culture, CD4+
CD25+ cells were purified from Thy1.1
+ mice. The
purity of CD4+
CD25+ cells was approximately 95-98% and up to 96% of these cells were
expressing Foxp3 (Fig. 21A). cAMP production was measured in these purified Tregs to confirm
the presence of functionally active A2AR on Tregs. Purified Tregs showed increased levels of
cAMP in the presence of CGS and NECA and the reversal of elevated cAMP levels by A2AR
antagonist ZM confirmed this effect to be A2AR mediated response (Fig 21B). The purified
49
Thy1.1-expressing nTreg were reconstituted with CD8+, CD25
+-depleted Thy1.2
+ spleen cells as
MLC responders. MLC of these responder cells in normal condition yielded CD4+ cells
predominant with Foxp3- effectors (Fig. 21C). These CD4
+ Foxp3
- cells were mostly from
Thy1.1- cells as expected, while most of CD4
+ Foxp3
+ cells were Thy1.1
+. There were also some
Thy1.1- CD4
+ Foxp3
+ cells, but these accounted for only a minor portion (20%) of total CD4
+
Foxp3+ cells (Fig. 21C). Treatment with A2AR agonists strongly reduced the proportion of
Foxp3- effectors and increased CD4
+ Foxp3
+ cells. CD4
+ CD25
- cell-derived Treg emerged from
Thy1.1- cells were found to increase by A2AR stimulation (FoxP3
+ within Thy1.1
- cells: 7% in
control, 29% in CGS and 16% in NECA); however, induction of CD4+
CD25- cell derived Treg
had a limited contribution to the increase of total CD4+
Foxp3+ cells (Fig. 21C). The CD4
+
Foxp3+
population after A2AR stimulation was again mostly Thy1.1+ cells, which accounted for
almost 90% of total CD4+
Foxp3+ cells. These data suggest that A2AR stimulation may promote
induction of CD4+
CD25- cell derived Treg and expansion of nTreg, but the latter mechanism
may play a major role in the numerical increase of Treg.
To further analyze the proliferation of nTreg, Thy1.1+ CD4
+ CD25
+ cells were labeled with
CFSE. In the reconstituted MLC, nTreg were found to enter massive proliferation (Fig 21D).
CD4+
CD25+ cells proliferated well even in the presence of CGS and NECA but these A2AR
agonists did not further promote proliferation. Interestingly, a large proportion of nTreg lost
Foxp3 expression in control MLC, while nTreg with A2AR agonists maintained Foxp3
expression better (Fig 21D). These results suggest that A2AR stimulation can, at least in part,
increase the number of Treg by preventing down-regulation of Foxp3.
50
Figure 21: The expansion of Treg in the presence of A2AR agonist was due to the proliferation
of preexisting nTreg (A) Purified CD4+
CD25+ cells represent nTreg. The numbers indicate
51
percentages of CD4+
CD25+
and CD25+ Foxp3
+ population in the purified cells. These CD4
+
CD25+
cells were obtained from Thy1.1-expressing mice and mixed with Thy1.2-expressing
MLC responder cells depleted of CD4+
CD25+
cells, which were prepared as described for Fig.
5A. Therefore, MLC responder cells were reconstituted so that only nTreg were expressing
Thy1.1. (B) Treg expresses functional A2AR. Incubation of purified CD4+
CD25+ cells with
A2AR agonist induced cAMP production, which was blocked by ZM, A2AR antagonist. Data
represent average SD of 3 independent experiments. * P < 0.001 vs control. (C) A large part of
A2AR-mediated increase of Treg was derived from nTreg. On day 7, the MLC was analyzed for
the expression of Foxp3. The data were gated for CD4+ cells. The numbers represent percentages
of each quadrant. Thy1.1+ (upper quadrants) and Thy1.1
- (lower quadrants) cells were mostly
CD4+
CD25+
nTreg and CD4+
CD25- non-Treg cells in the beginning of culture, respectively. (D)
Active proliferation of nTreg in MLC and well-maintained Foxp3 expression by treatment with
A2AR agonist. MLC responders were reconstituted as in (C) expect that CD4+
CD25+ cells from
Thy1.1-expressing mice were labeled with CFSE. The panels show CFSE fluorescence and
Foxp3 expression in Thy1.1+ cells on day 4. This data represents 3 independent experiments with
reproducible results.
52
3.5 DISCUSSION:
In MLC, massive expansion of T effector cells along with increase of Treg was observed in the
same culture. This may be similar to the physiological immune responses in vivo where boosting
of pro-inflammatory activities also give rise to compensatory anti-inflammatory responses to
limit excessive tissue damage. Such responses are not seen in the culture where T cells are
activated using artificial stimulation, anti-CD3 mAb as T effector cells expand strongly and
overwhelm the culture.
The results from previous study (Specific Aim # 1) demonstrated that the immunosuppressive
action on effector functions such as proliferation, cytokines production and cytotoxicity by
adenosine and its analogs on T cells is a direct action through A2AR expressed on them. In
addition to the inhibitory effect on T cell priming, A2AR stimulation produced activated T cells
with impaired effector function. Indeed, T cells activated in the presence of A2AR agonist
showed persistently lower cytokine-producing activity even after the removal of A2AR agonist.
The study demonstrated that A2AR stimulation not only inhibited effector T cell activation but
also at the same time increased the number of Treg (Fig 13-15 and 19). Also, CD4+
Foxp3+ cells
arising from this culture express molecules like CD25, CTLA-4, CD39 and CD73 and have
immunoregulatory activities similar to the Tregs induced by other methods (Fig. 16-18). This
data implies that A2AR inhibits T cell activation and also creates immunosuppressive
environment by inducing expansion of Treg. Therefore, extracellular adenosine when present at
immunosuppressive concentrations may be regulating the immune response even after it
disappears from the environment subsequently.
53
The resultant increase in number of Tregs was due to an increase in CD4+
CD25- derived Treg
and nTreg cells. Development of CD4+
CD25- derived Treg was shown in the culture that was
depleted of nTreg and the increase in number of CD4+
CD25- derived Treg in presence of A2AR
agonists (Fig. 20). It was important to determine which of the cells, nTreg and CD4+
CD25-
derived Treg were contributing to the increase in CD4+
CD25+ cells in the normal MLC. Since, it
is otherwise difficult to distinguish between them, Thy1.1 expressing nTreg were reconstituted
with MLC responders and the increase of CD4+
CD25- derived Treg in presence of A2AR
agonist was confirmed in the regular MLC (Fig. 21). This result is consistent with the findings of
previous study where CGS treatment in the T cell culture resulted in mRNA upregulation of
Foxp3 and LAG-3 [38]. This significant increase in CD4+
CD25- derived Treg only accounted
for a minor portion of A2AR mediated increase of Treg with major portion being contributed by
nTreg (Fig. 21). Previous study has reported that the purified nTreg expand when cultured with
allogenic dendritic cells and IL-2 [52]. Although IL-2 was not added exogenously in our culture
system, the activated cells are capable of producing IL-2 which might have contributed in the
expansion of Treg. The massive proliferation of nTreg was observed in Fig. 21D.
There can be other possible reasons for an increase of nTreg which includes prevention of down-
regulation of Foxp3 and cell death during Treg culture [53, 54]. Also, A2AR agonists have
shown to prevent activation induced cell death of CD4+ T cells [55]. Similar to the previous
reports, a number of nTreg were found to lose their Foxp3 expression after activation whereas
A2AR stimulation considerably prevented their loss of Foxp3 expression (Fig. 21D).
A2AR stimulation not only resulted in a numerical increase of CD4+
CD25+
Treg but also an
increase in their immunoregulatory activities. There is a significant increase, approximately 4-
fold, in the regulatory activity of CGS/NECA treated cells compared to control Treg. The
54
mechanism behind their immunoregulatory activity is not clear. However, CTLA-4 and CD25
can be the possible molecules involved in the regulatory activity of Tregs. Previous studies have
shown the importance of CTLA-4 in the regulatory activity of Tregs where the deficiency of
CTLA-4 on the Tregs in mice resulted in auto-immune disorders [48, 50]. Another study has
shown that Tregs express high levels of CD25, the IL-2 receptor alpha chain and it competes
with the effector T cells for IL-2, thereby depriving it of the cytokine resulting in cell death of
effector T cells [51, 56].
As discussed earlier, the tumor micro-environment is rich in extracellular adenosine [27].
Adenosine in the tumor microenvironment may be one of the important immunosuppressive
mechanisms, which discourages anti-tumor immune responses. A direct action at A2AR on T
cells can suppress anti-tumor T cells. The present data suggests that adenosine-A2AR
stimulation might play a role in enhancing Tregs in tumor microenvironment making it further
suppressive to anti-tumor T cells. Several studies have reported that the tumor micro-
environment contains Tregs which suppresses effector T cells [57-60]. In addition, the possibility
of enhanced regulatory activity by A2AR stimulation might play a role in further suppressing the
anti-tumor T cells. The role played by adenosine in the in vivo physiological control of Treg
activity needs to be determined.
While promotion of Treg number and activity is detrimental to tumor immunotherapy but
transfusion of Treg is a promising approach for the treatment of autoimmune diseases and
allogenic reaction after transplantation [61, 62]. Because large doses of Treg are necessary to
suppress graft vs host disease (GvHD), Treg require massive expansion ex vivo before transfer,
but the expansion of Treg is somewhat challenging. It is difficult to start the expansion from a
large number of Treg because of the low frequency of Treg in peripheral blood. In addition, since
55
both Treg and activated effector T cells share CD4+
CD25+ phenotype, polyclonal activation of T
cell could result in considerable contamination by effector T cells [61]. Treatment with A2AR
agonist induces expansion of Treg, while it suppresses activation of effector T cells. Such a
culture condition favoring Treg outgrowth may be suitable for the expansion of Treg.
56
4. Adenosine-A2AR mediated effect on induction of Foxp3 expression via
oxidative stress pathway
4.1 BACKGROUND AND SIGNIFICANCE:
The previous chapter demonstrates that the development and immunosuppressive functions of
Tregs are under the influence of the adenosine-A2AR signaling pathway. However, the
mechanism behind Foxp3 up-regulation via A2AR needs to be investigated.
Caffeine, an A2AR antagonist, has shown to induce neuronal glutathione (GSH) synthesis by
promoting cysteine uptake [63]. Therefore, there is a possibility that A2AR stimulation in the T
cells might be affecting cysteine uptake. Intracellular levels of cysteine acts as a rate limiting
step for GSH synthesis. GSH provides a reducing environment which is important for T cell
activation and proliferation. Reduction in the levels of antioxidant GSH causes imbalance in the
redox status which then significantly affects T cell proliferation, DNA synthesis, and cytotoxic T
cell activity [64, 65] and leads to oxidative stress within the cell. Tregs have also been shown to
suppress activation and proliferation of T cells by interfering with glutathione metabolism in
dendritic cells (DCs) [66].
Oxidative stress has shown to rapidly inhibit the enzyme methionine synthase which controls
global DNA methylation [67]. Inhibition of methionine synthase reduces DNA methylation
activity. Reduced DNA methylation interferes with epigenetic regulation of gene expression. In
epigenetic regulation, histones or the DNA themselves are the target for modifications where
gene transcriptions can be affected by altering the accessibility of the specific DNA regions to
57
the transcription factors [68]. Thus, decreased DNA methylation might lead to increase in
expression of Foxp3 by allowing access of transcription factors to Foxp3 promoter region.
Thus, the main hypothesis of this study was that adenosine-A2AR stimulation might increase the
Foxp3 expression via an oxidative stress pathway (Fig. 22).
Figure 22: Schematic representation of the hypothesis - Adenosine-A2AR mediated effect on
induction of Foxp3 expression via oxidative stress pathway
58
4.2 SPECIFIC AIM 3
To investigate the role played by Adenosine-A2AR mediated effect on induction of Foxp3
expression via oxidative stress pathway
1. To determine the changes in Treg population (Foxp3+ cells) after culturing with oxidative
agent, Buthionine sulfoximine (BSO).
2. To use an antioxidant, N-acetylcysteine (NAC) in presence of CGS and then look for
induction of Tregs and measure their regulatory activity.
59
4.3 MATERIALS AND METHODS
4.3.1 Mice
C57BL/6 and BALB/c mice were purchased from Charles River Laboratories (Wilmington,
MA). Mice were used at 8-12 weeks of age. The experiments were approved by the Northeastern
University Institutional Animal Care and Use Committee and were carried out in accordance
with the institutional animal care guidelines.
4.3.2 Mixed lymphocyte culture (MLC)
Mixed lymphocyte culture was set-up as described previously (Section 3.3.2). The cells were
cultured for 5 days in a 12-well plastic plate in presence or absence of A2AR agonist, CGS21680
(CGS; Tocris, Ellisville, MO) at 1 μM, Thiol depleting agent, buthionine sulfoximine (BSO;
Sigma, St. Louis, MO ) at various concentrations of 0.5mM, 0.25mM, 0.125mM and 0.05mM
and anti-oxidant N-acetylcysteine (NAC; Sigma, St. Louis, MO) at 5mM and 1mM. On day 5,
the activated effector cells (2 x 106 cells) were re-stimulated using mitomycin C-treated Balb/c
spleen cells (4 x 106
cells). The re-stimulated cells were cultured for additional 2 days, and were
analyzed on day 7.
4.3.3 Flow cytometric analysis
The resulted cells after MLC were analyzed by flow cytometry as described above (Section
3.3.3).
4.3.4 Statistics
Data represent mean SEM. Statistical calculations were performed using Student’s t-test.
Statistical significance was accepted for p values less than 0.05.
60
4.4 RESULTS:
4.4.1 Addition of oxidative agent causes an increase in proportion of Treg cells in the
culture
To determine whether oxidative stress can increase Treg population, the oxidative agent BSO, an
inhibitor of GSH synthesis, was added to MLC (Fig. 23). On day 7, the cells were analyzed for
Foxp3 expression. In comparison to control, BSO treated cells showed an increase in the
proportion of Foxp3 expression within CD4+ cells, with 0.125mM concentration showing a
statistically significant increase in expression of Foxp3. This result supports the theory that there
is a possibility for adenosine-A2AR signaling to act through the oxidative stress pathway thereby
leading to increase in expression of Foxp3+ cells.
Figure 23: Depletion of glutathione causes an increase in proportion of Treg cells. Cell culture
was done as described in Fig 13 in the presence of oxidative agent, buthionine sulfoximine
(BSO). Cells were analyzed by flow cytometry on Day 7. BSO enhanced Foxp3 expression in
61
CD4+ cells with statistical significance achieved at the 0.125mM concentration. CGS alone was
used as a positive control. Data represents average ± S.E.M of 3-4 independent experiments, * P
< 0.05, **P < 0.01 vs control.
4.4.2 Addition of antioxidant does not block the increase in proportion of Treg cells in the
presence of A2AR stimulation
To confirm the theory of adenosine-A2AR signaling utilizing the oxidative stress pathway to
increase the expression of Foxp3, an antioxidant, NAC, was used in the culture to prevent
oxidative stress. NAC is a precursor for formation of the antioxidant glutathione within the cell.
The thiol group of NAC in glutathione provides a proton and is able to reduce free radicals
thereby reducing the oxidative stress in the cell. The culture was set up as described above to
examine whether NAC can block Treg increase by A2AR agonist. Day 7 analysis revealed that
NAC treatment alone did not affect Foxp3+ population at 1 mM but increased Foxp3
+ proportion
at 5 mM. At both concentrations NAC failed to reverse or block the CGS induced up-regulation
of Foxp3 expression. These results suggest that adenosine-A2AR signaling induces Foxp3
expression independent of the oxidative stress pathway.
62
Figure 24: Addition of anti-oxidant cannot block the A2AR mediated increase in proportion of
Treg cells. Cell culture was done as described in Fig 13 in presence of anti-oxidant, NAC. Cells
were analyzed by flow cytometry on Day 7. NAC alone treatment showed two different
responses with enhanced Foxp3 expression at 5mM concentration and no effect at 1mM
concentration. CGS induced up-regulation of Foxp3 expression in CD4+ cells was not blocked at
both concentrations of NAC. CGS alone was used as a positive control. Data represents average
± S.E.M of 3 independent experiments, * P < 0.05 vs Control.
63
4.5 DISCUSSION:
Oxygen is very important for the survival of an organism. However, it is crucial to protect them
from over oxidation, which has lead to the evolution of antioxidant systems to counteract the
harmful effects of reactive oxygen species (ROS) [69]. Although at low levels, ROS are used in
biological redox signaling, their increased levels are implicated in many diseases such as cancer
and cardiovascular and neurodegenerative diseases [70]. The antioxidant systems play an
important role in maintaining equilibrium between intra- and extracellular redox status, thereby
protecting the cells from oxidative stress. Antioxidant systems include glutathione, cysteine and
thioredoxin that are used to maintain redox homeostasis and control various cellular functions,
i.e. gene expression, cell cycle progression and apoptosis [69]. Disturbances in the normal redox
status of cells results in oxidative stress. During oxidative stress, there is increased production of
oxidizing species and/or failure of the body’s antioxidant system to neutralize the reactive
species. The most destructive aspect of oxidative stress is the damage caused by ROS to the
cellular DNA through genetic mutations and also through epigenetic alterations [70].
Based on previous studies, the main hypothesis of this study was that adenosine-A2AR
stimulation increases the Foxp3 expression via oxidative stress pathway which affects DNA
methylation. The study was conducted to understand the underlying mechanism involved in the
regulation of the Foxp3 gene at the molecular level.
The present findings suggest that oxidative stress by itself resulted in an increase in proportion of
Foxp3+ cells (Fig. 23). This led to the assumption that NAC as an antioxidant might be able to
block the increase in proportion of Foxp3+ cells. However, the NAC treatment to block the
oxidative stress pathway in the presence of A2AR stimulation failed to prevent the increase in
64
Foxp3 expression (Fig. 24). Also, the data showed that NAC has different responses in Foxp3
up-regulation at two different concentrations. This can be explained by previous studies that have
indicated that NAC may have dual and opposing effects on the immune system depending upon
the different concentrations [71]. Since there was no down-regulation in Foxp3 expression after
NAC treatment in presence of A2AR agonist, the results suggests that the adenosine-A2AR
mediated Foxp3 induction is not mediated by its effects on DNA methylation through oxidative
stress pathway.
However, oxidative stress has shown to have effects on T cell activation and proliferation. T
cells for their activation and proliferation requires a reducing microenvironment. This reducing
environment is provided by the antigen presenting cells i.e. DCs [72]. The DCs have shown to
increase cystine uptake through the xc- cystine transporter present on them and increase GSH
synthesis. The GSH is then converted to extracellular cysteine, thereby providing a reducing
environment for T cell proliferation [73]. T cells lack the ability to import cystine and to make
their own cysteine. Cysteine is very important for T cells to synthesize GSH. Therefore, they are
dependent on DCs to provide the cysteine for their metabolic needs. Thus, the redox status is
very crucial for the T cells [73].
One of the mechanisms employed by Tregs to suppress the T cell activation is by interfering in
the accumulation of extracellular cysteine by consuming cysteine, thereby starving the effector T
cells of cysteine [73]. Also, Tregs affect the GSH synthesis in the DCs by decreasing the
expression of the rate limiting enzyme, γ-glutamylcysteine synthetase in GSH synthesis [66].
Thus, Tregs have been shown to modulate the redox potential by interfering in GSH metabolism
in DCs and T cells.
65
5. CONCLUSIONS:
Understanding about immunosuppression occurring due to adenosine rich microenvironment in
the inflamed tissue or tumor tissue will be important to develop strategies to immunologically
treat the diseases.
The first study (Specific Aim #1) highlights the comparison of A2AR-mediated inhibition of T
cell proliferation, cytokine production and cytotoxicity in the T cells from the same cell culture.
Effector functions such as IFN-γ/IL-2 production and cytotoxicity were sensitive to the A2AR-
mediated immunosuppression, whereas T cell proliferation was much more resistant. This
suggests that adenosine does not totally suppress T cell functions but when T cells are activated
in the presence of adenosine, the proliferated T cells will have limited effector function. Thus,
blocking of the adenosine-A2AR pathway may ameliorate the effector functions of anti-tumor T
cells thereby helping fight the tumor. Combining this anti-adenosinergic treatment with other
immunotherapies such as adoptive T cell transfer and tumor vaccine that will promote the
number and/or function of anti-tumor T cells will be beneficial in treating cancers.
The second study (Specific Aim #2) showed that A2AR stimulation increased the number of
Foxp3+ T regulatory cells. The Treg population expanded in the presence of adenosine analogs
has increased ability to suppress T cell activation. Thus, adenosine-A2AR causes not just a
quantitative difference in Treg population but the Tregs are qualitatively different with increased
ability to suppress T cell activation. The A2AR-mediated increase of Treg was not because of
oxidative stress subsequent to A2AR stimulation (Specific Aim #3). The quantitative and
qualitative enhancement of Treg by the adenosine-A2AR pathway may be relevant to the
66
establishment of longer-lasting immuno-modulation. This mechanism may be utilized in the
expansion of Treg for treatment of autoimmune diseases and GvHD.
67
6. FUTURE DIRECTIONS:
As discussed earlier in Specific Aim #1, hypoxia in inflamed and cancerous tissues is known to
result in accumulation of extracellular adenosine which then signals through A2AR on the T
cells. Since, hypoxia is upstream of A2AR signaling, it will be interesting to look at the effect of
hypoxia in promotion of Treg expansion through adenosine signaling. Hypoxia induced hypoxia
inducible factor-1α (HIF-1α) and adenosine-A2AR induced elevated cAMP results in activation
of HIF-1α→hypoxia response element (HRE) pathway and cAMP→CREB/ATF→cAMP
response element (CRE) pathway. Together this pathway might trigger increased transcription of
immunosuppressive molecules in Treg cell of HRE/CRE containing genes [74]. Previous studies
have shown that hypoxia and/or A2AR induced cAMP to stimulate secretion of TGF-β, IL-10
and Galectin-1 in non-lymphoid, myeloid and T lymphoid cells [75, 76]. These cytokines are
implicated in the immunosuppressive functions of Treg cells and are under the control of HRE
and CRE. However, experiments need to be done to show the direct effect of HRE and CRE in
Tregs.
Also, previous study has shown that TCR signaling causes activation of the downstream
transcription factor, cAMP response element binding protein/activating transcription factor
(CREB/ATF) resulting in their binding to the Foxp3 promoter region and regulating the
expression of Foxp3 [77]. A2AR signaling has shown to activate CREB via activation of PKA
by cAMP. Thus, A2AR activation might lead to transcription of Foxp3 directly by activation and
binding of CREB to Foxp3 locus. Future studies exploring this pathway in Tregs needs to be
performed.
68
The second study (Specific Aim #2) showed that A2AR stimulation led to the development and
expansion of immunosuppressive CD4+ CD25
+ Foxp3
+ Tregs. However, it would be interesting
to further study the stability of CD4+ Foxp3
+ T cells arising after A2AR stimulation. Several
studies have shown that Foxp3 expression is not always stable. The exposure to pro-
inflammatory cytokines, IL-6 and IL-1 has shown to down-modulate Foxp3 expression in Treg
and promote their conversion to IL-17 producing Th17 cells [78]. Also, adoptive transfer studies
have shown that approximately 10% of purified Treg when transferred into WT mice lose their
Foxp3 expression and are converted to conventional T cells [79, 80]. In a different study by
Williams and Rudensky, have shown that the ‘ex-Treg’ after losing their Foxp3 expression are
converted into effector T cells producing cytokines, i.e. IFN-γ, IL-4 and IL-17 [81]. In the same
study, the pathogenic potential of these newly converted ‘ex-Treg’ into effector T cells was seen
upon their transfer to lymphopenic recipients where the recipients were characterized by severe
tissue lesions and wasting disease. Therefore, more experiments are needed to evaluate the
stability of Foxp3 expression after A2AR stimulation in the patho-physiological conditions in
vivo and also for their ex vivo expansion.
69
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8. List of Publications based on the studies in the current thesis:
1. Ohta, A., A. Ohta, M. Madasu, R. Kini, M. Subramanian, N. Goel, and M. V. Sitkovsky.
2009. A2A adenosine receptor may allow expansion of T cells lacking effector functions in
extracellular adenosine-rich microenvironments. J. Immunol. 183: 5487-5493.
2. Ohta, A., R. Kini, A. Ohta, M. Subramanian, M. Madasu, and M. V. Sitkovsky. 2012. The
development and immunosuppressive functions of CD4+ CD25+ FoxP3+ regulatory T cells
are under influence of the adenosine-A2A adenosine receptor pathway. Front. Immun. 3:
190.
