(Ph.D.) 2007 Dysfunction: Mechanism and Therapeutic Strategies
Transcript of (Ph.D.) 2007 Dysfunction: Mechanism and Therapeutic Strategies
Thesis for doctoral degree (Ph.D.)2007
Tumor‐Induced Immune Dysfunction: Mechanism and Therapeutic Strategies
Mikael Hanson
Thesis for doctoral degree (Ph.D.) 2007 Tum
or‐Induced Immune D
ysfunction: Mechanism
and Therapeutic Strategies Mikael H
anson
From Department of Oncology‐Pathology, Cancer Centrum Karolinska,
Karolinska Institutet, Stockholm, Sweden
TUMOR-INDUCED IMMUNE DYSFUNCTION:
MECHANISM AND THERAPEUTIC STRATEGIES
Mikael Hanson
Stockholm 2007
Doctorial Thesis Tumor‐Induced immune Dysfunction: Mechanism and Therapeutic Strategies © Mikael Hanson, 2007 All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by Larserics Digital Print AB, Sundbyberg ISBN 978‐91‐7357‐356‐6
Abstract Cancer is one of the major causes of premature death in humans. Although standard
treatments, such as surgery and chemotherapy, are successful in many cases, there
are instances when their utility and efficacy are limited. Immunotherapy against
cancer has recently been developed as an example of a new generation of
“targeted” therapy. However, the immunosuppressive milieu associated with
tumors is an obstacle that needs to be overcome to improve the response rates of
immunotherapeutic approaches.
Several biological processes are involved in the induction and resolution of an
immune response and these need to be in perfect equilibrium to allow optimal
functioning of the immune system. However, this balance is skewed in cancer
patients creating a state of chronic inflammation that in turn results in a suppression
of the immune system leading to tumor‐induced immune dysfunction. Several
mechanisms have been suggested to contribute to this phenomenon, including
mechanisms that induce oxidative stress in cancer patients.
The aim of this thesis is to elucidate the effects of oxidative stress on lymphocytes
and define methods of reducing it. We have shown that oxidative stress affects
competent anti‐tumor cells, such as CD8+ T effector memory cells and CD56dim NK
cells, most as compared to other cells in the immune system. This may provide one
explanation for the limited clinical response noted with active immunotherapy
against cancer. To counteract oxidative stress, we have developed two approaches;
firstly, we have been able to increase NK cell function in cancer patients by oral
administration of the antioxidant vitamin E. Secondly, by transferring a gene
encoding for the antioxidant enzyme catalase we were able to increase the
antioxidant capacity in lymphocytes and improve their ability to resist oxidative
stress.
The work performed within this thesis furthers the understanding of oxidative
stress‐induced suppression of lymphocytes. This study has also contributed to the
development of approaches for reversing this suppression. Methods for reversing
oxidative stress‐induced immune dysfunction may potentially improve the clinical
outcome of subsequent active immunotherapy regimens against cancer.
List of Publications
I. Takahashi, A*., M. G. Hanson*, H. R. Norell, A. M. Havelka, K. Kono, K. J. Malmberg, and R. V. Kiessling. 2005. Preferential cell death of CD8+ effector memory (CCR7‐CD45RA‐) T cells by hydrogen peroxide‐induced oxidative stress. J Immunol 174:6080.
II. Harlin, H*., M. Hanson*, C. C. Johansson, D. Sakurai, I. Poschke, H. Norell,
K.‐J. Malmberg, and R. Kiessling. 2007. The CD16‐CD56bright NK Cell Subset Is
Resistant to Reactive Oxygen Species Produced by Activated Granulocytes
and Has Higher Antioxidative Capacity Than the CD16+CD56dim Subset.
J Immunol 179:4513
III. Hanson, M. G., V. Ozenci, M. C. Carlsten, B. L. Glimelius, J. E. Frodin, G. Masucci, K. J. Malmberg, and R. V. Kiessling. 2007. A short‐term dietary supplementation with high doses of vitamin E increases NK cell cytolytic activity in advanced colorectal cancer patients. Cancer Immunol Immunother 56:973.
IV. Hanson, M.*, K. Mimura*, C. Larsson, T. Palma, D. Sakurai, H. Norell, M. Li, M. Nishimura, and R. Kiessling. 2007. Transduction with the antioxidant enzyme catalase protects human T cells against oxidative stress. Submitted.
*Shared first authorship
Contents
INTRODUCTION ........................................................................................ 1
1. The Start and Stop of Inflammation ............................................................ 2
1.1. Onset of Inflammation .............................................................................. 3 1.2. Resolution of Inflammation ...................................................................... 5
2. Tumor Immunology ..................................................................................... 9
2.1. Cancer as a Chronic Inflammation .......................................................... 10 2.2. Tumor Escape .......................................................................................... 12
2.2.1. Immunoediting and Tumor Escape. ........................................................................... 15 2.2.2. Tumor Cell Immune Selection ................................................................................... 16 2.2.3. Tumor Cell Immune Subversion ................................................................................ 17
3. Lymphocyte Cell Death ............................................................................. 25
3.1. Oxidant‐induced Cell Death .................................................................... 27
AIMS OF THE THESIS ................................................................................ 31
RESULTS AND DISCUSSION......................................................................... 32
4. Part 1 ‐ Differences in Susceptibility to Cell Death of Lymphocyte Subsets 32
4.1. Implications of Preferential Cell Death of CD8+ TEM (Paper I) ................. 32 4.2. Antioxidants Rescue CD56bright NK Cells from Cell Death (Paper II) ........ 37 4.3. Conclusion Part 1 .................................................................................... 39
5. Part 2 – Therapies to Reverse Oxidative Stress in Lymphocytes ................ 41
5.1. Using Vitamin E to Reduce Oxidative Stress in Colon Cancer Patients (Paper III) ................................................................................................. 41 5.2. Arming T Cells with an Antioxidant Enzyme (Paper IV) .......................... 43 5.3. Conclusion Part 2 .................................................................................... 44
CONCLUDING REMARKS ............................................................................ 45
ACKNOWLEDGEMENTS ............................................................................. 48
REFERENCES .......................................................................................... 50
List of Abbreviations 4‐HNE 4‐hydroxynonenal IDO Indoleamine 2,3‐dioxygenase
8‐OxoGua 8‐oxo‐2‐deoxyguanosine IFN‐γ Interferon gamma
ACAD Activated cell autonomous death IL Interleukin
AICD Activation‐induced cell death iNOS Inducible nitric‐oxide synthase
Apaf‐1 Apoptotic protease activating factor 1 LMP Low molecular mass polypeptide
APM Antigen presentation machinery MCA Methylcholanthrene
ARG1 Arginase 1 MDSC Myeloid‐derived suppressor cells
ATP Adenosine 5'‐triphosphate MHC Major histocompatibility complex
Bad
Bak
Bcl2‐antagonist of cell death
Bcl‐2 Homologous Antagonist Killer
MIC Major histocompatibility complex class I chain‐related
Bax Bcl‐2–associated X protein NCR Natural cytotoxicity receptors
Bcl‐2 B‐cell lymphoma‐2 NF‐κB Nuclear factor‐kappa B
BH Bcl‐2 Homology NK Natural killer
Bid BH3 interacting domain death agonist NKG2A Natural killer group 2A
Bik Bcl‐2‐interacting killer NO Nitric oxide
Bim BCL‐2‐interacting mediator of cell death
NO2
PARP
Nitrogen dioxide
Poly(ADP‐ribose)polymerase
Caspase Cysteine‐dependent aspartate‐ directed proteases
PBL
PBMC
Peripheral blood lymphocytes
Peripheral blood mononuclear cells
CAT Catalase PD‐1 Programmed death‐1
CCR7 Chemokine receptor‐7 PGE2 Prostaglandin E2
CD Cluster of differentiation PMA Phorbol myristate acetate
CD95L CD95‐ligand PUMA p53‐upregulated modulator of apoptosis
cdk4 Cyclin‐dependent kinase 4 RAG Recombination activating gene
CTL Cytotoxic T lymphocytes RNS Reactive nitrogen species
CTLA‐4 Cytotoxic T‐lymphocyte‐ associated protein 4
ROS
SLPI
Reactive oxygen species
Secretory leukocyte protease inhibitor
DC
DIABLO
Dendritic cell
Direct inhibitor of apoptosis‐binding
Smac Second mitochondria‐derived activator of caspases
protein with a low isoelectric point, SOD Superoxide dismutase
DISC Death‐inducing signaling complex TAA Tumor‐associated antigens
DNA
FasL
Deoxyribonucleic acid
Fas ligand
TAP
TCM
Transporter associated with antigen processing
Central memory T cells
Foxp3
GM‐CFS
Forkhead box P3
Granulocyte macrophage‐colony
TCR
TEM
T cell receptors
Effector memory T cells
stimulating factor TGF‐β Tumor growth factor beta
GSH Glutathione TH1 T helper 1
GSSG Disulfide glutathione TIL Tumor‐infiltrating lymphocytes
H2O2 Hydrogen peroxide TNF‐α Tumor necrosis factor‐α
HIV
HLA
Human immunodeficiency virus
Human leukocyte antigen
TRAIL Tumor‐necrosis factor‐related apoptosis‐inducing ligand
HMGB1 High mobility group box 1 Treg Regulatory T cells
IAP Inhibitors of apoptosis proteins VEGF Vascular endothelial growth factor
ICAD Inhibitor of caspase‐activated Dnase
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Introduction Despite the great progress made in research, cancer still continues to be one of
the major causes of premature death in humans. In 2003, 48 867 new cases of
cancer were recorded in Sweden, which is twice as many as in the year 19701.
Many cancer types have a much better treatment prospect today compared to 20
– 30 years ago, while other more aggressive cancers, such as pancreatic cancer,
still have a very poor prognosis. Thus, there is a great need for new and innovative
cancer therapies to treat these cancers.
Current Therapies of Cancer Radical surgery is presently one of the first line
treatments of solid tumors. It is an effective, yet drastic, way to eliminate tumor
tissue. Unfortunately, many tumors are inoperable due to their anatomical
location or to the extent of dissemination to the surrounding tissue. In many cases,
the surgeon needs to remove healthy tissue, including lymph nodes surrounding
the tumor, giving rise to other complications. In the case of non‐solid tumors, e.g.
leukemia, surgery is not an option. The second most common approach of cancer
therapy is exposing the tumor to radiation. Radiotherapy has been used since the
late 19th century and constitutes 30 % of all treatments. This therapy is often used
in combination with surgery or chemotherapy. However, despite its potential
benefit, radiation can cause serious damage to the adjacent nonmalignant tissue.
As for surgery, the anatomical location and spread of the tumor may restrict the
potential utility of radiation in cancer therapy. The third major arm of cancer
treatment is chemotherapy using cytotoxic drugs that preferentially kill cancer
cells. Although the treatment with these drugs in several malignancies is effective,
the adverse effects are often severe. The side effects of certain chemotherapy
regimens are severe enough to preclude their use in the treatment of the elderly
or patients whose health is compromised. The limitations of existing treatment
approaches underline the need for novel therapies. The field of potential cancer
therapies is evolving and is now extended to involve many medical disciplines. For
example, the introduction of hormone and receptor antagonists for the treatment
of hormone‐dependent cancers has improved the prognosis of advanced cancers.
Tamoxifen, an antagonist of the estrogen receptor, has been successfully used in
treating breast tumors that are dependent on the estrogen receptor. However, a
significant number of patients develop recurrences that are resistant to tamoxifen
treatment2.
2
The progress in chemo/radiotherapy and surgery together with other approaches
like hormone antagonists have greatly improved the survival in many cancers, but
there is still considerable room for improvement. One approach of treating
cancers involves the use of immunological entities, also known as immunotherapy.
Passive immunotherapy entails the administration of therapeutic antibodies
whereas active immunotherapy, popularly known as cancer vaccines, relies on
stimulating the body’s own immune defense for the eradication of the tumor
cells3,4. Presently, at least seven antibodies are licensed for the treatment of
cancer; however, active immunotherapy approaches have not achieved the
success that the scientific community had hoped for.
The limited success of active immunotherapy against cancer may be attributed to
several factors5 (described in detail in section 2). In the progression of the disease,
cancer cells evolve strategies to avoid an immune response through loss of
components responsible for the presentation of cancer‐specific antigens and/or
loss or gain of cellular functions that renders the cancer cell resistant to cell death.
These mechanisms are intrinsic to the cancer cells and do not have an immediate
effect on the tumor microenvironment. Concurrently, cancer cells are in constant
interaction with their surroundings. The cancer cell progressively mutates, thereby
acquiring traits that enable the cell to utilize a variety of normal physiological and
biological processes to its advantage. These processes have been named tumor‐
induced immune dysfunction and can be summarized as the tumors ability to
manipulate the physiological functions of the tumor stroma (normal tissue
surrounding the tumor), the immune system, and the healing process. The focus of
this thesis is to investigate mechanisms and possible therapeutic strategies to
reverse tumor‐induced immune dysfunction. An awareness of the basics of
inflammation and resolution of inflammation is vital to understanding this thesis,
as these physiological events also play a critical role in tumor‐induced immune
dysfunction.
1. The Start and Stop of Inflammation
The inflammatory process has evolved to prevent an infection or a “xenobiotic
insult”6. Inflammation is induced in response to a traumatic tissue damage or an
infection and is a complex process involving cells and soluble factors7. The
regulation of inflammation is tightly governed by pro‐ and anti‐inflammatory
signals and, in normal condition, leads to the eradication of the infectious agents
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and to tissue repair7. For reasons of simplicity, this section describes the typical
inflammation caused by non‐infectious tissue damage (mild trauma) with respect
to the chronic inflammation seen in tumor tissues.
1.1. Onset of Inflammation
In response to mild trauma, tissue resident leucocytes (e.g. mast cells and
macrophages) perceive that a potential infectious agent may have penetrated the
skin and infected the tissue. To counteract this possible infection, mast cells,
activated by neuropeptides released by neurons in response to pain, secrete
several effector molecules, e.g. histamine, pro‐inflammatory leukotrienes,
prostaglandin E2 (PGE2), tumor necrosis factor‐α (TNF‐α), and chemokines8.
Histamine, leukotrienes, and PGE2 cause vasodilatation (responsible for the heat
and redness seen in inflammation) and extravasation of fluid (responsible for the
swelling seen in inflammation)8. The trauma also causes cell disruption, thus
releasing constitutively expressed intracellular proteins, such as heat‐shock
proteins9, the transcription factor high mobility group box 110 (HMGB1) and
mitochondrial peptides resembling prokaryotic components11. When detected by
tissue‐resident macrophages, these proteins trigger activation and the release of
TNF‐α and chemokines by the macrophage7.
Fig 1 ‐ Activation of the inflammatory response.In response to mild trauma mast cells and macrophages become activated and secretefactors that attract cells from the innate and adoptive immune system to the site ofinflammation. Hsps: heat‐shock proteins, HMGB1: high mobility group box 1, TNF‐α: tumor necrosis factor‐α, PGE2: prostaglandin E2. Adapted from Nathan7.
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The pro‐inflammatory molecules also activate the endothelium of blood vessels to
express various cell adhesion molecules, such as E‐ and P‐selectin, needed for
leukocyte rolling and transmigration from the peripheral blood into the damaged
tissue12. Once adhered to the endothelial wall of the activated blood vessel,
chemoattractants direct the leukocyte to the site of inflammation. The first cell
type to migrate to the inflamed tissue is the neutrophil7. The neutrophil is a cell
with a short lifespan and phagocytic capacity. It is the most abundant cell in the
blood and upon tissue damage will accumulate at the site of inflammation within
hours of insult12 (Fig 1). At the site of tissue damage, TNF‐α activated neutrophils
secrete various enzymes, such as myeloperoxidase, elastase, matrix
metalloproteases, and cathepsins that decompose extracellular matrix
components. Furthermore, the neutrophils secrete reactive oxygen species (ROS)
through an oxidative burst7,12,13. The events described above lead to further
neutrophil infiltration, thus potentiating the inflammatory process.
Peripheral blood monocytes are next to enter the site of inflammation, guided by
chemoattractants produced during the initial inflammatory response. Once
activated in the damaged tissue, monocytes differentiate into macrophages or
immature dendritic cells (DCs)14. The macrophages now act as the main source of
growth factors and cytokines that modulate both inflammation and resolution of
inflammation15 (see section 1.2 for details). Macrophage‐ and monocyte‐derived
TNF‐α and chemoattractants recruit and activate more neutrophils. The
combination of neutrophil‐derived products, e.g. anti‐bacterial peptides and mast
cell‐derived PGE2, attracts lymphocytes16 and leukotrienes recruit DCs to the site
of inflammation17, thus ensuring a response by the adaptive immune system.
Cytokines produced by lymphocytes together with microbial‐like products, e.g.
formyl‐peptides released from necrotic cells, trigger macrophages to release anti‐
microbial molecules, e.g. ROS and reactive nitrogen species (RNS)7.
In summary, the onset of an inflammation is a cascade of events initiated by cells
and soluble factors. It is an immediate response that ensures a quick and efficient
removal of infectious agents. As a simplification, one may consider the immediate
inflammatory response to have two purposes 1) to ensure an infiltration of
immune cells from peripheral blood into the damaged tissue and 2) to combat the
infectious agent at the site of inflammation. To facilitate cellular infiltration a
cascade of cytokines and chemokines are released through different means and to
execute an immediate removal of infectious agents, macrophages and neutrophils
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secrete various anti‐microbial molecules, e.g. ROS and anti‐bacterial peptides. In
addition, the initiation of inflammation also triggers an essential process, i.e.
resolution of inflammation and tissue repair.
1.2. Resolution of Inflammation
Normally, inflammation caused by mild trauma quickly declines and the tissue
heals. To this end, the switch from an anti‐bacterial, tissue‐damaging mode to a
tissue repair mode needs to be made at the closing stages of inflammation. The
onset of macrophage infiltration into the inflamed tissue coincides to the decline
in numbers of neutrophils at the site of inflammation15. As long as there are pro‐
inflammatory components, e.g. HMGB1 and formyl‐peptides, macrophages will
produce chemokines to attract peripheral blood neutrophils7. The transformation
from inflammation to tissue repair initiates when the amount of pro‐inflammatory
components decreases and when macrophages, at a late stage of inflammation,
start to produce secretory leukocyte protease inhibitor (SLPI), a serin protease
inhibitor with anti‐inflammatory18 and tissue repair‐promoting effects19. The
release of SLPI by macrophages 1) suppresses neutrophil infiltration into the
inflammatory site by inhibiting the secretion of chemoattractants, 2) inhibits ROS
production by neutrophils20 and 3) prevents tumor growth factor‐β (TGF‐β) break
down19. The remaining neutrophils undergo apoptosis and macrophages start to
ingest apoptotic neutrophils7. The phagocytosis of apoptotic bodies derived from
neutrophils initiates macrophages to produce TGF‐β7. In turn, the production of
TGF‐β starts the process of tissue repair.
Concurrent to the activation of the innate immune response, cells of the adaptive
immune response enter the site of inflammation and mount a humoral and/or
cellular adaptive immune response. For most infections, the cellular component of
the adaptive immune response needs to be reduced to minimize tissue damage
and autoimmunity once the infectious agent is cleared. This is mediated by several
physiological processes such as 1) regulation of T cells and natural killer (NK) cells
through cell death, 2) deprivation of essential nutrients necessary for lymphocyte
function at the site of inflammation, and 3) production of immune suppressive
cytokines.
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Regulation of Inflammation through Cell Death of T Cells and NK Cells
It has been shown that induction of cell death in T cells and NK cells regulates the
lymphocyte immune response. There are two distinct mechanisms in which
induction of cell death plays a key role, i.e. activation‐induced cell death21 (AICD)
and activated cell autonomous death22 (ACAD). Upon encounter and recognition of
antigens presented on DCs, T cells clonally expand and home to the site of
inflammation23. To counteract possible autoimmune reactions, T cells that are
activated without proper co‐stimulation are eliminated through AICD23. Over time,
the majority of the expanded T cells gain traits that make them susceptible to cell
death, while some T cells differentiate into a memory phenotype with an
increased resistance to AICD24. The induction of cell death in activated T cells acts
as a safety mechanism assuring that the effector phase of the adaptive immune
response is transient and declines when the infectious agent has been cleared. It
has been shown that the increased susceptibility of activated T cells to cell death is
due to increased expression of cluster of differentiation 95 (CD95), thus sensitizing
the cells to CD95‐mediated cell death by autocrine or paracrine CD95‐ligand
(CD95L) expression25. Other studies have also indicated a role for TNF‐α26 and
tumor‐necrosis factor‐related apoptosis‐inducing ligand27 (TRAIL) in the induction
of AICD. In addition, it has recently been shown that NK cells also commit to AICD
as interleukin‐2 (IL‐2) stimulated NK cells express and release CD95L upon natural
cytotoxicity receptors (NCR) engagement with target cells, thus mediating suicide
of NK cells expressing CD9528.
The absence of survival signals also contributes to ACAD in T cells22. ACAD is
regulated by the balance between intracellular anti‐ and pro‐apoptotic proteins
(mechanisms of cell death are explained in detail in section 3). It has been shown
that during expansion, T cells downregulate the expression of the anti‐apoptotic
protein B‐cell lymphoma (Bcl)‐XL29 and upregulate the expression of the pro‐
apoptotic proteins Bcl‐2‐interacting mediator of cell death (Bim) and p53‐
upregulated modulator of apoptosis (PUMA)30‐32. This shift in the balance renders
the T cells sensitive to cell death stimuli.
Deprivation of Essential Nutrients L‐arginine is an essential amino acid
needed for synthesis of proteins. L‐arginine is mainly catabolized in vivo by
arginase 1 (ARG1) and inducible nitric‐oxide synthase (iNOS) to produce urea and
L‐ornithine, and nitric oxide (NO) and L‐citrulline, respectively33,34. It has been
proposed that in higher organisms metabolism of L‐arginine regulates unwanted T
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cell expansion35. Recently, Rodriguez et al. showed that L‐arginine starvation
impairs the expression of cyclin D3 and cyclin‐dependent kinase 4 (cdk4) in T cells,
thus inhibiting downstream signaling leading to G0‐G1 cell cycle arrest36. In
parallel, catabolism of tryptophan has been proposed to downregulate the T cell
response at the maternal‐fetal interface. It has been shown that expression of
indoleamine 2,3‐dioxygenase (IDO), which catalyzes L‐tryptophan conversion into
kynurenine, is important for the establishment of the immune privilege site and
that starvation of L‐tryptophan leads to cell cycle arrest37.
Suppressive Cytokines In the late stages of the inflammatory process the
switch from inflammation to resolution and tissue repair is orchestrated by
immune suppressive (or pro‐tissue repair) cytokines. TGF‐β expressed in the later
phase of inflammation inhibits activation, effector function, proliferation and
differentiation of T cells38,39. TGF‐β is an important regulator of the immune
system as deficiency in TGF‐β results in lethal autoimmunity in mice40,41. TGF‐β has
also been shown to inhibit NK cells by inducing downregulation of the activating
NK cell receptors NKp30 and natural killer group 2D (NKG2D)42 and enhance the
expression of the inhibitory receptor CD94/NKG2A in T cells43. TGF‐β also drives
the expansion of regulatory T cells (Treg), a T cell subset responsible for controlling
the immune response44‐47. In addition, IL‐10 reduces the immune response by
suppressing the production of pro‐inflammatory cytokines, such as interferon‐γ
(IFN‐γ) and IL‐2, and impairs DC function, thus limiting priming of T cells48,49.
Furthermore, vascular endothelial growth factor (VEGF) has also been shown to
delimit DCs ability to present antigens to T cells50.
In conclusion, there are factors that induce the inflammatory process and other
factors that lead to the resolution of inflammation (see Table I for summary).
These factors are programmed to be produced following a particular time course.
Table I – Key molecules involved in inflammation and resolution of inflammation Induction of Inflammation Resolution of Inflammation Inflammation initiator molecules CytokinesExamples : Formyl peptides, bacterial products, HMGB1, neuropeptides, Hsps
Examples: TGF‐β, IL‐10, VEGF
Inflammatory chemoattractants/cytokines Lack of nutrientsExamples: TNF‐α, TGF‐β, PGE2, Histamine, leukotrienes
Examples: ARG1 (deprival of L‐arginine) and IDO(deprival of L‐ tryptophan) activity
Toxic molecules Examples: Antibiotic proteins, e.g. LL‐37,RNS and ROS
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Figure 2 provides a simplified summary of the different factors involved in
inflammation and resolution of inflammation and relates them to the time course
of the various processes. Many of these factors have been shown to be implicated
in tumor‐induced immune dysfunction.
Fig 2 ‐ Induction and resolution of inflammation leading to tissue repair.Upon insult, tissue‐resident mast cells and macrophages become activated by bacterial/viral‐derived products, e.g. LPS and fMLP, or by products produced after tissue damage, e.g. neuro‐peptides, HMGB1, and formyl‐peptides derived from mitochondria of damaged cells. The activation facilitates the inductionof inflammation during which the first wave of inflammatory cells entering the site of inflammation areneutrophils. Thereafter, monocytes enter the site and differentiate into macrophages. Neutrophils andmacrophages facilitate antibiotic activity through secretion of toxic molecules, such as ROS. The next waveof immune cells to infiltrate the site of inflammation is T cells previously primed in secondary lymph nodes by DC migrating from the inflamed tissue. T cells recognize cells that express the antigen that they wereprimed against, e.g. viral proteins, and the kill target cells. When the signals from the insult have diminished, e.g. the infectious agent is cleared, the inflammatory response is converted to a pro‐resolution state. For example, macrophages switch from pro‐ to anti‐inflammatory, thus secreting immune suppressive cytokines, such as TGF‐β. In parallel, lymphocyte activity is downregulated by induction of cell death and deprival of essential nutrients. The above described processes aid the subsequent tissue repair,e.g. angiogenesis, leading to healing of the damaged tissue.
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2. Tumor Immunology
In the 1960’s the first experiments testing the immunogenicity of tumors were
conducted. Mice immunized with attenuated, syngeneic tumors, were protected
from a subsequent challenge with live tumor51,52. This protection was tumor‐
specific as challenge with a different tumor, even with one induced by the same
carcinogen, did not protect the mice from developing tumors. In contrast, tumors
induced by virus, such as papilloma virus, share the same antigens and mice are
protected from a subsequent challenge using a different tumor induced by the
same virus53. These observations formed the basis of the idea that a tumor‐specific
adaptive immune responses against non‐virally‐induced tumors could occur.
Several tumor‐associated antigens (TAA) have been described54‐56. These TAAs can
be categorized into 1) differentiation antigens, i.e. antigens that are expressed
during some stage of the differentiation by normal cells in the same lineage, e.g.
the melanoma antigen MART‐1, 2) mutated antigens, i.e. antigens derived from
mutated proteins, e.g. mutated p53, 3) overexpressed antigens, i.e. antigens that
are expressed at low levels in normal cells, but are overexpressed in tumor cells,
e.g. Her‐2/neu, 4) cancer‐testis antigens, i.e. antigens that are normally expressed
only in male germ cells, e.g. NY‐ESO‐1, and 5) viral antigens, i.e. antigens that are
derived from viral proteins expressed by tumor–inducing viruses, e.g. human
papilloma virus protein E6.
Immunotherapy Against Cancer Several strategies to utilize the immune
system for eradicating tumors have been tested. As the immune response is
directed by various immune regulatory cytokines, investigators have used these in
clinical trials. The “prototype” cytokine for these efforts has been IL‐2, a cytokine
that activates both T cells and NK cells in vivo. In a series of clinical trials,
pioneered by Steven Rosenberg and colleagues, showed that administration of IL‐2
to cancer patients had beneficial effects in 16 % of the patients57. Although the
clinical effect was relatively low, patients that responded to the IL‐2 treatment
showed signs of long‐term protection from the disease compared to patients
treated with conventional strategies, e.g. chemotherapy. However, high dose IL‐2
treatment is associated with severe adverse effects, such as lung edema, which
makes this treatment difficult to manage58.
As described above, tumors display antigens that may be recognized by the
immune system. After the development of successful immunotherapies based on
10
vaccination using mouse tumor models, the scientific community had great
expectations on this approach to treat human cancers. However, after vaccination
against various TAAs the overall conclusion is that only very low clinical response
can be achieved in cancer patients. For example, a meta‐analysis comprising thirty‐
two phase I/II vaccination trials reporting on 527 patients with advanced or
metastatic colorectal cancer showed a very low frequency (< 1 %) of clinical
responses when immunotherapy was used as a treatment59.
Adoptive cell transfer therapy has recently been developed and clinical trials have
demonstrated that it has great potential of causing tumor shrinkage. This
treatment utilizes autologous tumor‐infiltrating lymphocytes (TIL) that have been
stimulated and expanded ex vivo and then transferred back to the patient60. In
conjunction with non‐myeloablative, lymphodepleting chemotherapy, this remedy
has a ~ 50 % clinical response rate in patients with melamona61. However, these
clinical protocols are very cost‐ and labor‐intensive and advanced techniques and
skills are needed for this therapeutic approach, making it unfeasible for treating
large numbers of patients.
In conclusion, there is a great need to improve active immunotherapy approaches
and to boost the immune response in cancer patients at the same time, in order to
overcome the tumor‐induced immune dysfunction.
2.1. Cancer as a Chronic Inflammation
Cancer development is a multistep process in which normal cells acquire a series
of mutations in tumor suppressor genes and proto‐oncogenes, i.e. genes that
control cell proliferation62. As first reported by Rous & Kidd, it is thought that viral
or chemical carcinogens induce somatic changes that initiate a pre‐neoplastic state
before the transformation into a cancer cell63. This initiation comprises
deoxyribonucleic acid (DNA)‐damages that are irreversible and induce no
phenotypic change of the pre‐cancer cell15. In order for the pre‐cancer cell into
transform to a cancer cell, further insult is needed. These insults, including
exposure to chemical irritants, factors secreted upon wound healing, hormones,
chronic irritation and inflammation that induces cell proliferation, facilitate
recruitment of inflammatory cells, which leads to additional DNA‐damage15. These
events induce additional mutations in tumor suppressor genes and proto‐
oncogenes, which ultimately lead to an immortal cancer cell.
11
Under physiological conditions inflammation is self‐limiting and controlled tightly
by the induction‐versus‐resolution balance (described in section 1.1 and 1.2).
However, chronic inflammation at the tumor site is either due to a persistence of
the initiating factors or a failure in the resolution of the inflammation15 . In a tumor
two compartments can be identified: cancer cells and tumor stroma (surrounding
tissue)64. The tumor stroma provides vasculature to ensure proper gas exchange,
supply of nutrients, and waste disposal, thus facilitating growth and sustenance of
the tumor. The tumor stroma has traditionally been divided into three categories:
blood vessels, inflammatory cells, and connective tissue. The inflammatory cells
that are present in the tumor stroma mainly consist of macrophages and
lymphocytes and these components of the stroma are seen in all cancers, but their
extent varies between cancer types15. For example, in carcinomas such as breast,
stomach, and pancreas cancer, the tumor stroma accounts for over 90 % of the
tumor mass. On the other hand, malignant melanoma and medullary carcinomas
have minimal tumor stroma. Several similarities have been observed between
tumor stroma generation and chronic inflammation: 1) initiation by leakage of
plasma proteins or injured blood vessels64, 2) extra‐vascular blood clotting64, 3)
recruitment of inflammatory cells64, 4) presence of pro‐inflammatory molecules,
such as TNF‐α and histamine, and pro‐resolution cytokines, such as TGF‐β, IL‐10
and VEGF15, and 5) oxidative stress at the tumor site65.
The Link Between Inflammation and Cancer Several lines of evidence
indicate that infection and inflammation may induce cancer progression15,66‐69
(Table II). Infection can induce chronic inflammation, thus facilitating oncogenesis
through mechanisms described above. For example, a strong positive correlation
between colon cancer development and inflammatory bowel disease, such as
chronic ulcerative colitis and Crohn’s disease, has been observed15. Hepatitis C
virus infection is known to be associated with liver carcinoma70, schistosomiasis
increases the risk of bladder carcinoma71, and Helicobacter pylori infection is the
leading cause of stomach cancer72. These observations indicate that inflammation
is a key factor in tumor development and one may argue that by interfering with
tumor‐induced inflammation/resolution it could be possible to induce a functional
immune response and in turn facilitate tumor eradication.
12
Table II – Inflammatory conditions associated with cancerInflammatory conditions Associated Cancer Asbestosis, silicosis Mesothelioma, lung carcinomaBronchitis Lung carcinomaGingivitis Oral squamous cell carcinomaInflammatory bowel disease Colorectal carcinomaLichen sclerosus Vulvar squamous cellcarcinomaChronic pancreatitis Pancreatic carcinomaReflux oesophagitis Oesophageal carcinomaSkin inflammation MelanomaGastritis/ulcers Gastric adenocarcinomaHepatitis Hepatocellular carcinomaMononucleosis B‐cell non‐Hodgkin’s lymphoma, Burkitts
lymphoma AIDS Non‐Hodgkin’s lymphoma, squamous cell
carcinomas, Kaposi’s sarcoma Modified from Coussens & Werb15
2.2. Tumor Escape
The “six hallmarks of cancer” is a concept of cancer progression where the cancer
cell gains six characteristics: the cancer cell is capable of providing autocrine
growth signals, ignoring growth‐inhibitory signals, avoiding cell death, replicating
without limits, sustaining angiogenesis, and invading tissues through basement
membranes and capillary walls73. It has now also been proposed that a seventh
hallmark should be included: avoidance of immune surveillance74. Numerous cell
types have been proposed to be involved in immune surveillance of cancer, i.e.
CD8+ and CD4+ T cells, NK cells, NKT cells, and IFN‐producing killer dendritic cells75.
The eradication of cancer cells can be mediated by direct perforin dependent lysis
of tumor cells (by triggering of specific T cell receptors (TCR):peptide/MHC class I
recognition by T cells or NK cell triggering by “missing‐self” signals and/or stress‐
induced NK cell receptor ligands), and/or by cytokines, e.g. IFN‐γ75. All of these
mechanisms can be counteracted by the tumor.
The Rise and Fall of the Immune Surveillance Concept Paul Ehrlich was
the first to formulate the idea that the immune system could repress the
frequency of carcinomas76. In the 1960s with the development of inbred strains of
mice, immunization against tumor‐transplants showed the existence of tumor‐
specific antigens51,52. Sir Frank Macfarlane Burnet and Lewis Thomas incorporated
this discovery into the formal hypothesis of ‘cancer immune surveillance’. Burnet
stated:
13
‘It is by no means inconceivable that small accumulations of tumor cells may develop and because of their possession of new antigenic potentialities provoke an effective immunological reaction with regression of the tumor and no clinical hint of its existence’ (cited from 76).
In 1970, Burnet defined the immune surveillance concept, based on speculations
made by Thomas:
‘In large, long‐lived animals, like most of the warm‐blooded vertebrates, inheritable genetic changes must be common in somatic cells and a proportion of these changes will represent a step toward malignancy. It is an evolutionary necessity that there should be some mechanism for eliminating or inactivating such potentially dangerous mutant cells and it is postulated that this mechanism is of immunological character’ (cited from 76).
After the introduction of the immune surveillance hypothesis many experiments
were conducted to investigate how suppression of the immune system in mice
affected the spontaneous or carcinogen‐induced tumor incidence. However, these
studies did not unequivocally prove or disprove the hypothesis. For example,
Rygaard et al. showed no differences between spontaneous tumor formations in
nude mice compared to normal mice indicating that immune surveillance did not
exist77,78. However, these experiments had pitfalls, which were mainly caused by
the limited understanding of the nude mouse. For example, it is now known that
nude mice are not completely immunodeficient as these mice have functional NK
cells79, thus, immune surveillance may occur in these animals.
The Return of the Concept From the mid 1970s the immune surveillance as a
concept gradually lost credibility. However, three key findings in the mid 1990s
sparked a renewal of the concept. Utilizing knock‐out mice that lack the IFN‐γ
receptor 1 (IFN‐γR1‐/‐ mice), it was shown that wild‐type mice had lower incidence
of chemically induced and spontaneous tumors80‐82. Secondly, mice lacking
perforin (perforin‐/‐ mice), a component of the cytolytic granules of cytotoxic T
cells and NK cells that is important in mediating lymphocyte‐dependent tumor
cytotoxicity, were more sensitive to methylcholanthrene (MCA)‐induced tumor
formation compared to wild‐type mice82‐86. Finally, studies using mice that lack
recombination activating gene 1 or 2 (RAG‐1‐/‐, RAG‐2‐/‐) and can therefore not
undergo TCR rearrangement further supported the existence of the immune
surveillance hypothesis. Experiments in this model showed that lymphocytes not
14
only protected the host against formation of chemically induced sarcomas, but
also prevented the development of spontaneous epithelial tumors87,88.
Furthermore, epidemiological studies have reported a higher probability of
developing non‐viral tumors in immune deficient humans89,90. A positive
correlation between lymphocyte infiltration and increased survival of cancer
patients has also been shown, arguing for a role of the immune system in the
eradication of human tumors91‐93.
Skepticism Still Remains So far, the studies supporting the role of immune
surveillance have not formally proven that it does exist. These studies, e.g.
increased susceptibility to cancer in mice lacking functional T cells and B cells (e.g.
RAG‐1‐/‐ mice), show the final outcome as tumor/no tumor. However, the formal
proof, i.e. the eradication of small, un‐palpable tumors by the immune system has
not been shown. Further, the possibility that the tumor is a consequence of a
secondary event, e.g. inflammation caused by an infection, cannot be ignored, as
these mice are very susceptible to bacterial or viral infection.
A recent study by Willimsky & Blankenstein argued against immune surveillance94.
In this study a mouse model of sporadic cancer based on rare spontaneous
activation of a dormant oncogene was developed. However, the tumors grew in
spite of expressing the rejection antigen and the outgrowth of the tumor was
paralleled with an increasing T cell tolerance to the rejection antigen. Thus, tumor
cells did not evade the immune system by altering recognition i.e. loss of antigen.
The outgrowth of tumor cells was initially due to tolerization of tumor‐specific T
cells and, in later stages of tumor development, tumor‐induced immune
suppression was the underlying process for cancer in this model.
Although several lines of evidence point towards the existence of an immune
surveillance system, it has not been formally proven. Presently, there is no
consensus on what would constitute unequivocal proof. One may also argue that
the murine model experiments should be conducted in germ‐free mice, enabling
the investigation of the role of immune surveillance in oncogenesis without
infections as a confounding factor.
The fact that immunocompromised mice develop more tumors than normal mice
can be regarded as an indication of immune surveillance, not a proof for
continuous eradication of cancer cells. In order to obtain proof‐of‐concept, one
15
must develop a mouse model in which newly formed cancer cells can be
visualized. For example, one may use a mouse model with a dormant oncogene
that, upon activation, will co‐express a reporter gene, such as the luciferase gene,
enabling detection of small, unpalpable tumors in living animals. If a causal
relationship between cancer formation, induction of an immune response, and
eradication of the newly formed cancer cells is observed, the model could pose a
valid argument for the existence of immune surveillance.
While immune surveillance against non‐virally‐induced tumors still remains to be
proven, evidence for immune surveillance against virally‐induced tumors exists.
For example, it has been shown that young women vaccinated against human
papillomavirus before first exposure to the virus have decreased incidence of
cervical intraepithelial neoplasia95, arguing for immune surveillance against virally‐
induced tumors.
2.2.1. Immunoediting and Tumor Escape.
Cancers develop despite a functional immune system. It has been shown that
tumors that develop in immune compromised hosts, e.g. RAG‐1‐/‐ mice, are
rejected upon transplantation into an immune competent recipient, but tumors
developed in wild‐type mice with a functional immune system grow when
transplanted into syngenic wild‐type mice87. Thus, tumors may be “sculptured” by
the immune system promoting the outgrowth of tumor cell variants that are
poorly recognized by immune cells. It is thought that the evolution of tumor cells
into less immunogenic clones is due to the inherent genetic instability of the tumor
cell. To conceptualize these new findings with the inclusion of the immune
surveillance theory, Dunn et al. proposed the term immunoediting74. In essence,
the model proposes that the immune system facilitates tumor progression by
exerting immunological pressure on the tumor as it develops (see Fig 3).
Immunoediting comprises of three phases74: 1) elimination, 2) equilibrium, and 3)
escape. In the elimination phase, tumor cells are continually eliminated by the
immune system. However, if a tumor cell acquires traits that enable survival, the
immune system and the tumor enter a state of equilibrium where the immune
system suppresses tumor growth, but is unable to eradicate the tumor. This causes
high immunological pressure on the tumor cells and favors survival of cells with
mutations that makes the cells less immunogenic, less sensitive to cell death, and
have traits that can induce immune suppression. This is called tumor escape and
16
several mechanisms have been shown to be involved. One may divide the
mechanisms into two categories; 1) immune selection and 2) immune subversion.
2.2.2. Tumor Cell Immune Selection
Tumor cells that have been able to survive the initial elimination process by the
immune system (immune surveillance) and entered the equilibrium phase are
constantly targeted by effector cells of the immune system. In a Darwinian
selection process, tumor cells that are not recognized by the immune system or
tumor cells that gain traits that renders them resistant to cell death will have a
growth advantage.
Lack of Tumor Recognition In order for a tumor cell to be recognized and
killed by CD8+ T cells (cytotoxic T lymphocytes (CTL)), interactions between
peptide:major histocompatibility complex class I (peptide:MHC I) on the tumor cell
surface and TCR on T cells must take place. Through the oncogenic process, cancer
cells can evade recognition by the T cell through mutations, gene deletions or gene
regulation, leading to downregulation of proteins which are important for the
antigen presentation machinery (APM). For example, it has been shown that the
transporter associated with antigen processing (TAP), which is responsible for
loading peptides on to the MHC I molecule, and essential components in the
immunoproteosome, i.e. the low molecular mass polypeptide (LMP)‐2 and LMP‐7,
are defective in patients with uveal melanomas96 and bladder cancer97 due to
mutations in their genes. The consequence of this may be total or partial loss of
Fig 3 ‐ The interactions between the tumor cells and the immune system.The figure depicts tumor progression in relation to the immune system. Adaptedfrom Zitvogel et al.75.
17
peptide:MHC I on the cell surface of the cancer cell. In addition, studies from our
group have shown total loss of the human leukocyte antigen (HLA) class I locus in
ovarian carcinoma98 which may contribute to disease progression resulting in
failure of active immunotherapy99. Antigenic loss, or “epitope” loss, is another way
by which tumor cells avoid recognition by the immune system100. The rapid
generation of new, genetically instable tumor cells creates an array of tumor cell
variants that may lack a tumor antigen. Thus, the antigen loss variants may be able
to proliferate unnoticed by the immune system.
Lack of Susceptibility to Cell Death Upon recognition, immune cells kill
tumor cells by inducing apoptosis. There are two main ways of inducing apoptosis;
1) the death receptor signaling pathway and 2) the perforin/granzyme B
pathway101. Both pathways result in intracellular cysteine‐dependent aspartate‐
directed proteases (caspase) activation, which leads to apoptosis. Tumor cells may
prevent apoptosis by modulating these pathways. For example, it has been shown
that the death receptor signaling pathway can be avoided by cells that release
factors, such as soluble Fas102, or autocrine secretion of Fas ligand (FasL)103. TRAIL‐
mediated apoptosis may be evaded through expression of the decoy receptors
TRAIL‐R3 and ‐R4, which lack the intracellular domain for correct apoptosis
signaling104. In the perforin/granzyme B pathway, T cells or NK cells release
granules containing the proteolytic enzyme granzyme B, which together with
perforin induces apoptosis. It has been demonstrated that cancer cells can escape
this type of killing by overexpressing an inhibitor of granzyme B105.
In addition, apoptosis is regulated by pro‐apoptotic and anti‐apoptotic molecules.
Bcl‐2, a proto‐oncogene encoding a mitochondrial protein, is a key element in
blocking programmed cell death. Dysregulation of Bcl‐2 function leads to an
increased ability to withstand apoptotic stimuli in cancer cells leading to cancer
progression. Bcl‐2 over‐expression in acute myeloid leukemia cells is associated
with an increased recurrence rate and a significantly lower survival after intensive
chemotherapy106.
2.2.3. Tumor Cell Immune Subversion
Upon further immune selection of tumor cells, the tumor can evolve to subvert the
immune system by utilizing normal physiological functions. Several mechanisms
have been proposed to explain this tumor‐induced immune dysfunction.
18
Inhibition of T cells by Tolerogenic DCs Mature DCs prime T cell responses,
thus allowing naïve T cells to be activated and become effector cells which in turn
eradicate infected107 or transformed cells108. Mature DCs also direct the immune
response to polarize into a T helper 1 (TH1) or TH2 type109‐111 and improve the
generation of T cell memory112. To perform these tasks, DCs have to undergo
differentiation and maturation113. Maturation of DCs is induced by both bacterial
products114,115 and pro‐inflammatory cytokines, especially TNF‐α116. If an antigen is
captured and presented by a DC that is not fully activated, thus lacking the proper
co‐stimulatory molecules for interaction with T cells, this results in the induction of
T cell tolerance113. It has been shown that DCs at the tumor site have an immature
phenotype113. In addition, tumor cells can modify immature DCs to induce Treg 117
and facilitate T cell unresponsiveness118. It has been suggested that tumor‐derived
factors, such as VEGF, granulocyte macrophage‐colony stimulating factor (GM‐
CFS), IL‐10, and TGF‐β, may induce the accumulation of immature DCs, thus
contributing to T cell tolerance towards tumor cells119.
Immune System‐derived Soluble Factors Associated with Tumor‐
induced Immune Dysfunction The induction and resolution of an immune
response is mainly regulated through soluble factors, such as cytokines (see
section 1 for details). Normally, factors mediate the end of an inflammatory
episode, e.g. TGF‐β production in late stage of an inflammatory process,
transforms the response from anti‐pathogenic and tissue‐damaging to promoting
angiogenesis and tissue repair. However, the chronic state of inflammation in
cancer patients facilitates the generation of immune suppressive soluble factors
when the immune system is triggered, thus suppressing a potentially tumoricidal
immune response.
CD4+ Treg have the ability to produce high levels of the immunosuppressive
molecules IL‐10 and TGF‐β and under normal circumstances are important to
counteract autoimmunity120. There is strong evidence that CD4+ Treg are involved in
the tumor‐induced suppression of the immune system121. CD4+ Treg that express
forkhead box P3 (FOXP3) and high levels of CD25, also known as naturally
occurring Treg, can be found in the tumor or in the circulation of cancer patients,
and have been shown to promote tumor‐induced immune dysfunction in several
cancers. For example, an inverse correlation between the presence of Treg and
survival has been reported in ovarian carcinoma122. In addition, depletion of Treg
with anti‐CD25 antibodies leads to increased tumor rejection by T cells in mice123.
19
The secretion of TGF‐β may be a major obstacle for effective immunotherapy as
recent studies show that TGF‐β acts on CTLs to repress the expression of perforin,
granzymes, FasL, and IFN‐γ, all important for CTL function124. It has also been
shown in mice that neutralization of TGF‐β with specific antibodies restores the
expression of IFN‐γ, which leads to tumor clearance in vivo. In addition, it has been
shown that TGF‐β down‐modulates the expression of the activating receptors
NKp30 and NKG2D on T cells and NK cells42 and induces expression of the
inhibitory receptors CD94/NKG2A43. Moreover, CD1d‐restricted NK cells may
induce immune suppression by mechanisms involving IL‐13 and TGF‐β125. In
humans, there seems to be evidence for a comparable paradigm. Decreased
frequency and function of CD8+ memory T cells and TILs was noted in TGF‐β rich
cultures from patients in a melanoma vaccine study, indicating an
immunosuppressive role of TGF‐β in cancer patients126. Additionally, Treg were
found in high frequencies in peripheral blood of cancer patients as compared to
healthy donor blood and at the tumor site in patients with invasive breast or
pancreas cancers, where they are likely to promote tumor growth by inhibiting
anti‐tumor immune responses127.
IL‐10 modulates the function of several cells in adaptive immunity and is
considered to be one of the main immunosuppressive molecules128. IL‐10 can
stimulate Tregs to secret TGF‐β, adding to the immune suppressive environment129.
It has also been suggested that IL‐10 plays a direct role in the differentiation of
naïve CD4+ T cells into Tregs120. Additionally, IL‐10 has been shown to be secreted by
melanoma cells leading to impaired function of DCs130. IL‐10 also favors a TH2
polarization by decreasing the production of IFN‐γ and IL‐12 by CD4+ T cells128. It
has been shown that high serum levels of IL‐10 correlate with poor survival in
renal cancer131. Additionally, it has been suggested that PGE2 induces Tregs,
potentiating the secretion of IL‐10 and TGF‐β132.
Induction of Lymphocyte Cell Death in Cancer Lymphocyte homeostasis is
regulated by the thymic output of naïve T cells and death of peripheral T cells that
have completed their functions133. It has been shown that this homeostasis is
disturbed in cancer patients leading to rapid lymphocyte turnover and loss of
tumor‐specific effector cells133. It has been observed that TIL and peripheral blood
lymphocytes (PBL) exhibit a high frequency of cell death compared to other
inflammatory sites133,134. Furthermore, studies have demonstrated signaling
defects in the T cell receptor complex molecule CD3ζ in cancer patients135‐137 and
20
absence or decreased expression of CD3ζ has been correlated with poor survival of
oral carcinoma patients138. It has also been shown that signs of cell death are
paralleled with a decrease in CD3ζ expression, suggesting a shared mechanism of
the two events139.
Deprival of Essential Nutrients in Cancer As described above, limitation of
essential nutrients is a frequently used mechanism to limit biological activity, e.g.
an immune response. However, due to the chronic inflammatory state in patients
with cancer, the deprivation of these nutrients leads to inhibition of tumor‐
reactive immune responses. Two enzymes, IDO and ARG1, have been suggested to
exert the immune inhibitory effect.
IDO, an enzyme involved in L‐tryptophan metabolism, catalyzes the rate limiting
step in the oxidative breakdown of L‐tryptophan. IDO has been shown to be
involved in immune tolerance in the placenta, which protects the fetus against
attack by maternal T cells and has lately been shown to exhibit
immunosuppressive functions in the tumor‐micro environments when expressed
in DCs140. Studies investigating L‐tryptophan and its metabolites indicate that IDO
is chronically activated in cancer patients141 and that IDO activation correlates with
progression of cancer 142. It has also been shown that low L‐tryptophan
concentration in the blood correlates with decreased survival in patients with T
cell leukemia or colon carcinoma142,143 and that several tumor types express
IDO144. The mechanism of immune suppression is probably a combination of two
factors; 1) overexpressed IDO in DC or tumor cells, depleting L‐tryptophan in the
tumor‐micro environment, thereby inhibiting cell cycle progression in
lymphocytes37 and 2) the toxic metabolite kynurenine produced in the reaction
mediated by IDO, which is capable of inducing apoptosis145. For example,
Uyttenhove et al. showed that overexpression of IDO in tumor cells intensified the
tumor cells’ ability to grow more aggressively and that this correlated with a
decrease of functional T cells at the tumor site144. The study also showed that
administration of IDO inhibitors reduced tumor mass and stimulated anti‐tumor
immune responses.
ARG1 is also involved in inhibiting anti‐tumor immune response by depleting L‐
arginine35,146. It has been shown that depletion of L‐arginine downmodulates CD3ζ
expression146,147 and impairs the expression of cyclin D3 and cdk4 in T cells,
inhibiting downstream signaling leading to G0‐G1 cell cycle arrest36. A study
21
analyzing the activity of ARG1 in peripheral blood mononuclear cells (PBMC) of
renal cancer patients showed a significant increase in ARG1 activity compared to
healthy controls148. The study also revealed a correlation between ARG1 activity
and a decrease in CD3ζ expression in T cells and NK cells, indicating ARG1‐
dependent immune suppression of these cells. Several cell populations have been
shown to be implicated in ARG1 activity in tumor‐bearing hosts; e.g. 1) increased
ARG1 activity has been detected in cancer cell lines of colon, breast, or prostate
cancer149, 2) CD11b+, CD14‐ myeloid‐derived suppressor cell150 (MDSC) and
granulocyte expression of ARG1151 in patients with cancer mediate CD3ζ‐
downregulation, thus inhibiting T cell proliferation and cytokine production.
Tumor Cell‐specific Immunosuppressive Mechanisms There are several
additional, tumor‐specific mechanisms that are utilized to suppress the immune
system in tumor bearing individuals. In addition to the mechanisms mentioned
above, these include expression of FasL, ligands for inhibitory receptors on T cells
and NK cells, and galectins on the tumor cells.
The Fas‐dependent cell death pathway is triggered in cells expressing Fas by the
interaction with its ligand, FasL152. FasL expression has been detected in several
cancer types, including colon153, lung154, and esophageal155 cancer. Interaction of T
cells and tumor cells induces Fas‐dependent cell death in T cells, thereby
facilitating a “Fas/FasL counter attack”152. However, the presence of Fas/FasL
counter attack is controversial, for example studies have shown pro‐inflammatory
functions of FasL expressed on tumor cells156.
Tumor cells express ligands for inhibitory receptors on T cells and NK cells that
block effector functions of these cells. For example, many tumors express major
histocompatibility complex class I chain‐related A (MICA), a stress‐induced
molecule with immune stimulatory function5. However, it has been shown that
tumor cells may shed MICA, which can bind to the NK cell or T cell activating
receptor NKG2D, thus inhibiting the function of this receptor157. Soluble MICA has
been detected in plasma of cancer patients in whom a downregulation of NKG2D
on T cells and NK cells was seen157,158.
Galectin‐1, a member of the lectin family, can hamper the T cell response159. It is
now evident that tumors, including breast, prostate, and colon cancer express
galectin‐1160. Furthermore, a negative correlation between galactin‐1 expression,
tumor progression161 and numbers of T cells in the tumor162 has been shown.
22
Studies indicate that galectin‐1 suppresses T cells through 1) sensitization of T cells
to Fas‐mediated cell death163, 2) inhibition of TCR signaling by downmodulation of
CD3ζ164, and 3) suppression of pro‐inflammatory cytokines165.
Immune Suppressive Inflammatory Effector Molecules in Cancer The
above mentioned mechanisms of tumor‐induced immune dysfunction originate
from physiological processes that aim to resolve an inflammatory response. It has
been shown that effector molecules in the inflammation phase mediate immune
suppression. ROS and RNS produced by myelomonocytic cells are two of the key
effectors in the anti‐microbial machinery166,167. However, these reactive molecules
have been shown to have adverse effects on the tumor‐specific response. It has
been shown that ROS are produced by splenic macrophages from tumor‐bearing
mice168, macrophages isolated from metastatic lesions of human melanomas169,
and activated granulocytes derived from peripheral blood of cancer patients170.
The secretion of ROS by these cells induces loss of T cell and NK cell functions,
including defects in receptor associated signaling molecules, such as CD3ζ, defects
in nuclear factor‐κB (NF‐κB) activation in T cells171,172, as well as a reduced cytokine
production in response to T cell stimulation171,173‐175. These phenomena are also
noted in T cells from cancer patients176. ROS‐producing cells may be monocytes,
macrophages, granulocytes, or immature myeloid cells and can be detected in the
tumor‐micro environment168,177‐179, or in the peripheral circulation170. In addition,
it has been shown that ROS may be produced by the tumor cells themselves,
adding to the oxidative stress in the tumor‐micro environment180‐182.
NK cells are extremely sensitive to ROS‐induced dysfunction183. ROS produced by
granulocytes and monocytes have been shown to hamper NK cell function and
viability as well as the expression of NCR on NK cells169,184‐187 . The down‐
modulation of NCRs and other NK cell receptors has been observed in NK cells
from patients with acute myeloid leukemia 188, as well as other chronic
inflammatory diseases such as hepatitis C189, and active human immunodeficiency
virus (HIV) infection190.
In murine models, MDSC inhibited IFN‐γ production by CD8+ T cells when peptide
epitopes were presented in conjunction with MHC class I on MDSC surface191. This
was not true for CD4+ cells, indicating the specific impairment of cytotoxic cells. In
some studies, this inhibition required direct cell‐cell contact and appeared to be
dependent on ROS, particularly hydrogen peroxide (H2O2)192. In patients with
23
advanced tumors, immature myeloid cells inhibited antigen‐specific T cell
responses, indicating an explanation for reduced T cell response in patients with a high frequency of MDSCs in the tumor environment177.
NO is a free radical that at low doses regulates several biological functions, such as
blood flow and smooth muscle relaxation. This molecule is also produced by
macrophages at high levels to function as an antimicrobial agent193. Studies
indicate that NO at higher doses is involved in chronic inflammation, such as in
cancer194,195. High concentrations of NO can generate RNS, such as nitrogen
dioxide (NO2●)196. These reactive molecules cause damage to DNA, proteins, and
lipids, leading to hampered cell functions, increased inflammatory response, and
cell death197. The main NO‐producing enzyme in macrophages is iNOS193. Several
lines of evidence indicate elevated levels of RNS at the tumor site. In breast
carcinoma, tumor‐infiltrating macrophages express iNOS198 and studies have found
the expression of iNOS in colon adenomas199, ovarian cancer200, and
melanomas201. NO has been shown to severely inhibit antitumor responses202 by
suppressing T cell function203. Additionally, NO produced by macrophages inhibits
activation of signal transduction, thereby suppressing T cell responses204.
In parallel, studies utilizing probes for oxidative stress have shown that patients
with cancer display increased levels of oxidative stress. For example, elevated
tissue levels of malondialdehyde and 4‐Hydroxynonenal (4‐HNE), which are
markers for the presence of lipid peroxidation by ROS, correlate with the clinical
staging of colorectal cancer65. Furthermore, increased oxidative stress‐induced
DNA damage, measured by increased levels of 8‐oxo‐2 ‐deoxyguanosine (8‐
OxoGua), has been detected in lymphocytes of colorectal cancer patients205. It has
also been suggested that oxidative stress is present in the blood, as studies show
decreased levels of antioxidants in the plasma of colon cancer patients65,205.
In conclusion, chronic inflammation associated with malignant tumors deregulates
the induction versus resolution phases of the inflammatory process, which in turn
facilitates disease progression. Several mechanisms are speculated to be
responsible for the immune suppression and figure 4 shows a simplified model of
cancer‐associated chronic inflammation. In consequence, in order to improve the
immunotherapy regimens available today, a combinatorial remedy aiming not only
to eradicate the tumor, but to reverse tumor‐induced immune dysfunction, is
needed.
24
Fig 4 ‐ Chronic inflammation caused by a tumor. Upon insult, in this case triggered by tumor progression, tissue‐resident mast cells andmacrophages become activated by products produced after tissue damage or necrosis of tumorcells, e.g. neuro‐peptides, HMGB1, and formyl‐peptides derived from mitochondria of damagedcells. As seen in physiological response to an insult, the activation leads to neutrophilinfiltration. Thereafter, monocytes enter the site and differentiate into macrophages whereneutrophils and macrophages secrete ROS and RNS. Next to enter the tumor site are tumor‐specific T cells that recognize cells that express tumor‐associated antigens (TAA) and kill thetumor cells. However, the tumor is not totally eradicated as tumor‐escape variants survive theinitial immune response. This in turn leads to further tumor growth and increased activation ofthe innate immune system triggered by the continued necrosis of tumor cells and tumorstroma. In consequence, neutrophils and macrophages are chronically activated and continueto secrete ROS and RNS. In parallel, tumor‐specific T cells continue to enter the tumor site, butlose effector functions and go into apoptosis due to the immune‐suppressive tumor‐microenvironment, including low levels of nutrients, accumulation of immune suppressivecytokines, tolerization by immature DCs, and oxidative stress. In conclusion, the processdescribed above leads to chronic inflammation characterized by an environment that is toxicand suppressive for cells that are capable of tumor eradication, such as T cells and NK cells. Inaddition, tissue repair processes, such as angiogenesis, will also be induced, thus facilitatingfurther tumor growth.
25
3. Lymphocyte Cell Death
Cell death is an essential physiological process in higher organisms as cell
homeostasis is maintained by a balance between proliferation and cell death206.
For example, selective cell death is crucial for proper organ development206 and, as
described above, cell death of lymphocytes and neutrophils is a necessity for the
resolution of the inflammation phase. Defects in the process will lead to severe
disease, such as autoimmunity, degenerative diseases and immunodeficiency206.
To ensure proper regulation of this vital process, hundreds of proteins are
committed to govern the pro‐ and anti‐cell death signaling within cells207.
As a simplification, cell death can be divided into 1) active cell death, i.e. apoptosis
and 2) passive cell death, i.e. necrosis208. Apoptosis is mediated by a cascade of
events, triggered by cellular stress, that leads to cell death and removal of the cell
without release of toxic substances into the surrounding tissue, thereby
preventing inflammation207. Apoptosis is characterized by morphological changes,
such as chromatin and cytoplasmic condensation, DNA fragmentation,
phosphatidylserine externalization and formation of apoptotic bodies208. On the
other hand, necrosis is a passive, accidental cell death that manifests by rapid loss
of plasma membrane integrity, which leads to an uncontrolled destruction of the
cell and evokes the inflammatory process207. Recently, it has become clear that cell
death in higher organism is more complex and cannot be completely explained
through the two processes mentioned above; however a detailed explanation of
these processes is beyond the scope of this thesis.
The machinery mediating apoptosis involves caspases209. Humans express at least
seven caspases that govern this process210. These molecules may be separated into
two categories 1) the initiator caspases 2, 8, 9 and 10, and 2) the effector caspases
3, 6 and 7211. Moreover, the apoptotic process is mainly mediated through two
pathways, 1) the mitochondrial pathway, i.e. intrinsic pathway and 2) the death‐
receptor pathway, i.e. extrinsic pathway (see Fig 5).
Intrinsic Pathway The intrinsic pathway of apoptosis is regulated through
proteins that interact in the vicinity of the mitochondria212. This pathway is
governed by the Bcl‐2 family of proteins. This protein family is divided into groups
as they have opposing roles in cell death due to differences in the number of Bcl‐2
Homology (BH) domains that the protein contains213. The anti‐apoptotic group,
26
having three or four BH domains, includes Bcl‐2, Bcl‐xL, and Bcl‐w. Upon activation
or overexpression, these proteins hinder the cell from implementing the cell death
machinery212. In contrast, the pro‐apoptotic Bax‐like proteins (Bcl‐2–associated X
protein (Bax), Bcl‐2 Homologous Antagonist Killer (Bak), etc), which have two or
three BH domains, and the BH3‐only group of proteins (Bcl2‐antagonist of cell
death (Bad), Bcl‐2‐interacting killer (Bik), BH3 interacting domain death agonist
(Bid) etc), which has one short BH3 domain, induce cell death212. When the cell is
exposed to stress, e.g. starvation or oxidative stress, the balance may be shifted
from the anti‐ to a pro‐apoptotic state through activation of genes encoding the
pro‐apoptotic proteins or activating post‐translational modifications of pro‐
apoptotic proteins214. This will lead to formation of pores in the mitochondria
triggering mitochondria depolarization214, which leads to permeabilization and
release of cytochrome c into the cytoplasm of the cell212.
To counteract unwanted pore formation in the mitochondria, the anti‐apoptotic
proteins stabilize the mitochondrial membrane215 by interacting with the pro‐
apoptotic proteins, thus blocking their apoptotic capacity216. Cytochrome c is
released into the cytosol and, together with apoptotic protease activating factor 1
(Apaf‐1) and pro‐caspase 9, forms the apoptosome that in turn activates the
executioner caspase 3208,217. Upon mitochondrial disruption, other effector
molecules, such as Second mitochondria‐derived activator of caspases
(Smac)/direct inhibitor of apoptosis‐binding (DIABLO), are released, boost the cell
death machinery after cytochrome c release by hindering the function of several
inhibitors of apoptosis proteins (IAP) 218.
Extrinsic Pathway Death receptors of the TNF receptor superfamily include Fas,
TNF receptor, and TRAIL receptor. Upon ligation, these receptors associate with
adaptor proteins containing death domains to form the death‐inducing signaling
complex (DISC), resulting in caspase 8 activation208. In turn, active caspase 8
cleaves and thereby activates caspase 3208. Caspase 8 also cleaves Bid, which
induces Bak and/or Bax incorporation into the mitochondrial outer membrane,
leading to mitochondrial membrane depolarization and release of cytochrome c as
in the intrinsic pathway219. The activation of caspase 3 leads to 1) cleavage of the
inhibitor of caspase‐activated DNase (ICAD), inducing chromatin and DNA
degradation220, 2) cleavage of lamins resulting in shrinkage of the nucleus, 3)
degradation of the cytoskeleton, and 4) membrane blebbing due to cleavage of
27
p21‐activated kinases221. The cumulative effect of all these processes is cell death
by apoptosis.
3.1. Oxidantinduced Cell Death
ROS have a dual function in cellular systems, as it has been demonstrated that
they can both stimulate cellular signaling pathways and trigger loss of cellular
functions and cell death222,223. It has been shown that redox signaling is important
in T cell function, as activation of the TCR induces ROS generation224, which is
necessary for downstream‐signaling pathways225. On the other hand, ROS may also
induce cell death in lymphocytes226. Thus, the outcome of exposure to ROS seems
Fig 5. The intrinsic and extrinsic pathway of apoptosis.The extrinsic apoptotic pathway is executed upon ligation of death receptors, e.g.triggering of Fas by FasL, and will lead to caspase 8 activation. Caspase 8 activationin turn activates caspase 3. In parallel, caspase 8 may also cleave Bid, thus inducingmitochondrial depolarization and cytochrome c release. The intrinsic apoptoticpathway is triggered by cellular stress, e.g. DNA‐damage or ROS exposure, andcauses a shift in the balance between pro‐ and anti‐apoptotic molecules favoringpro‐apoptotic signaling. This leads to mitochondrial membrane depolarization,cytochrome c release and activation of the apoptosome leading to caspase 3activation. Activation of caspase 3 directs downstream processes in the apoptoticmachinery leading to cell death. Adapted from Hengartner et al.212
28
Box 1 – The Basics of ROS and RNS
Upon activation, neutrophils and macrophages undergo respiratory burst. Respiratory burst is a rapid chemical reaction, facilitated by phagocyte oxidase (Phox), to produce superoxide (O●
2‐) according to the reaction227: NADPH (M(n‐1)+) + O2 → NADP+ (Mn+) + O●
2‐.
O●2‐ may convert, via super oxide dismutase (SOD), into hydrogen peroxide (H2O2)
228. In turn, myeloperoxidase (MPO) may convert H2O2 into singlet oxygen (
1O2), and further non‐enzymatically into ozone (O3). MPO may also assist in the conversion of H2O2 into hypochlorous acid (HOCl), hypobromous acid (HOBr), and hypoiodous acid (HOI)229,230. In parallel, O●
2‐ and H2O2 may non‐enzymatically produce hydroxyl radical (OH●). The above
mentioned molecules are examples of reactive oxygen species (ROS).On the other hand catalase (CAT) may decompose H2O2 into H2O and O2. Furthermore, the primary source of reactive nitrogen species (RNS) is NO produced by macrophages231. NO is generated by inducible nitric oxide synthase (iNOS) in the reaction231: L‐arginine → L‐citrulline + NO
●. Together with O●2‐, NO● may, non‐
enzymatically, produce peroxynitrite (ONOO‐). Chemical reactions involving NO● lead to oxidation, nitration (addition of NO2), nitrosation (addition of NO), and nitrosylation (addition of NO on cysteine residues) of cellular components231. Thus, activation of neutrophils and macrophages leads to the generation of ROS and RNS. These molecules are chemically unstable and may lead to damage of cellular structures, such as proteins, lipids, or DNA. Furthermore, these molecules may also act as second messengers in signal transduction, thereby affecting the cellular response to oxidative stress. Adapted from Ohshima et al.228
29
unpredictable and it may be argued that it leads to activation of different
processes, such as cell death or cell proliferation, depending on the pro‐ and anti‐
apoptotic balance within the cell and/or the antioxidant capacity of the cell.
When studying T cell lymphomas as a model for normal T cells, it was
demonstrated that T cell death involves caspase 3 activity as caspase inhibitors
prevented cell death232‐234. These studies also showed loss of the mitochondrial
membrane potential and release of cytochrome c upon H2O2 exposure234,235 and
demonstrated that the intrinsic cell death pathway was predominant upon
exposure to H2O2232. Furthermore, T cells that were activated in vivo induced
apoptosis via a Fas‐ and TNF‐α‐independent pathway and AICD of T cells was
characterized by caspase‐independent loss of mitochondrial membrane
potential223. In parallel, a caspase‐dependent DNA loss and enhanced generation
of ROS was observed. In addition, studies have shown that in the absence of
survival factors T cells accumulate ROS, increase expression of pro‐apoptotic
protein Bim and amplify iNOS expression236. In this study antioxidants were used
to verify ROS‐dependent cell death, BIM induction, and caspase activation. In NK
cells studies have shown that ROS induce late caspase activation, DNA
fragmentation, cellular condensation, and maintenance of intracellular Adenosine
5'‐triphosphate (ATP)187,237. Other studies have implicated poly(ADP‐
ribose)polymerase (PARP) as a key mediator of ROS‐induced cell death187. Thus,
there seems to be a narrow window between activation and inhibition of function
and cell death when lymphocytes are exposed to ROS.
Counteracting Oxidative Stress Under normal physiological conditions,
damage caused by ROS is counteracted by 1) several enzyme systems, such as
superoxide dismutase (SOD) and catalase (CAT), and 2) scavenger systems,
including the glutathione system238, the antioxidants vitamin C239, carotenoids240
and vitamin E241. SOD catalyzes the dismutation of O2− into O2 and H2O2
242, while
CAT decomposes 2 H2O2 into 2 H2O and O2243. Taken together theses enzyme
systems act synergistically by efficiently clearing ROS through the overall reaction:
→ 2 O2 4 O2
− + 4 H+ → 2 H2O2 → O2 + 2 H2O SOD CAT
The glutathione system is a complex antioxidant system that consists of
monomeric glutathione (GSH), disulfide glutathione (GSSG), and glutathione
30
peroxidase, which catalyzes the reaction 2GSH + H2O2 → GSSG + 2H2O, and
glutathione reductase, which converts GSSG to GSH. The glutathione system is a
key defense against H2O2 and other peroxides238.
In conclusion, oxidative stress may either 1) induce a positive signal leading to
lymphocyte activation, 2) have no significant effect on signaling pathways, or 3)
induce a negative signal, leading to loss‐of‐function or cell death of the
lymphocyte. The outcome could be dependent on several factors, including the
level of oxidative stress and the lymphocyte´s susceptibility to cell death. On the
other hand, the outcome may also be dependent on the antioxidant capacity of
the lymphocyte, protecting cells with higher levels of antioxidants from oxidative
stress‐induced loss‐of‐function or cell death.
31
Aims of the Thesis To improve the cancer immunotherapy treatments of today, a deeper
understanding of tumor‐induced immune dysfunction is needed. This knowledge
may lead to the development of new therapeutic strategies to reverse the immune
suppression seen in cancer patients.
Accordingly, the aims of this thesis are:
► To improve the understanding of mechanisms leading to tumor‐induced
immune dysfunction and its effects on the immune system; focusing on
oxidative stress as a source of immune suppression.
► To develop new treatment modalities to minimize tumor‐induced immune
dysfunction; focusing on prevention of oxidative stress.
32
Results and Discussion 4. Part 1 Differences in Susceptibility to Cell Death of Lymphocyte Subsets
ROS are known to be involved in phorbol myristate acetate (PMA)‐induced death
of neutrophils, HIV‐induced death of T cells, death of pancreatic β cells, and neural
cell death244‐248. However, the molecular events leading to oxidant‐induced cell
death of lymphocytes is not fully understood. ROS‐induced lymphocyte cell death
has primarily been studied using T cell lymphomas232‐234, but the effect of oxidative
stress on primary lymphocytes has not been extensively investigated. By
examining ROS‐induced cellular events within the various primary lymphocyte
subsets, one can distinguish differences in the responses of the lymphocyte cell
types to oxidative stress. The results are critical to a more detailed understanding
of the influence of oxidative stress on the individual components that constitute
the cell mediated immunity, particularly against tumors.
4.1. Implications of Preferential Cell Death of CD8+ TEM (Paper I)
T cells are one of the main effector cell types used in immunotherapy against
cancer. As described above, oxidative stress, caused by ROS that are produced by
granulocytes and macrophages systemically or at the tumor site, has detrimental
effects on T cell function and viability. It has been shown that ROS‐exposure of T
cells at physiological levels leads to impaired TCR signaling, inhibition of NF‐κB
function, and decreased cytokine production171,173,175. H2O2‐secretion has been
proposed as one of the sources of oxidative stress and may be one mechanism
leading to tumor‐induced immune dysfunction. We have previously demonstrated
that H2O2 suppresses TH1 cytokine production and that this suppression correlates
with inhibition of NF‐κB activity171. Of interest, the suppression of cytokines was
primarily seen in memory T cells.
T Cell Subsets T cells can be categorized into subsets according to their effector
functions and the expression of cell surface receptors249‐251 (Box 2). In essence, T
cells can be sorted into two main compartments, CD4+ and CD8+ T cells. CD4+ T
cells (T helper cells) support and coordinate the adaptive immune response, thus
facilitating the effector functions, i.e. antibody‐ or cell‐
33
Box 2 – Basics of T Cell Subsets
T cell memory is a requirement for a functional adaptive immune system. Through clonal expansion and differentiation of T cells specific for an antigen, the individual can acquire a life‐long protection against a pathogen. Naïve T cells that have undergone the positive and negative selection in the thymus enter the blood circulation, home to the secondary lymphoid organs, e.g. lymph nodes, and await activation by DCs carrying processed antigens from an inflammatory site252. If the T cell receptor on a naïve T cell recognizes the MHC:peptide complex (signal 1) on the surface of DC, the DC expresses proper co‐stimulatory receptors (signal 2), and the DC secretes stimulatory cytokines (signal 3), the T cell becomes activated and starts to proliferate. These T cells then enter the circulation and home to the inflamed tissue252. When the infection is cleared the majority of T cells go into apoptosis through AICD or ACAD. However, some T cells survive and become memory T cells. If the pathogen infects the individual again these memory T cells swiftly become reactivated and facilitate protection. T cells may be divided into subsets249‐251 according to 1) the existence of effector function of the T cell and 2) phenotypic expression of cell surface receptors that allow cells to home to secondary lymphoid organs versus non‐lymphoid tissues. Simplified, T cells (CD3+ CD56‐) can be divided into CD4+ and CD8+ T cells. CD4+ T cells mainly assist the adaptive immune response determining the appropriate effector functions to use: antibodies or CTLs. CD8+ T cells (CTL) can lyse target cells upon encountering a cell expressing the cognate antigen for its TCR. After clonal expansion of CD4+ or CD8+ T cells, some cells survive and become memory T cells. These memory T cells are divided into effector memory T cells (TEM) and central memory T cells (TCM)
253. Upon re‐activation TEM migrate to the inflammatory site and have the capacity for immediate effector function253. On the other hand, TCM have modest capacity for effector function and preferably home to T cell areas of secondary lymphoid organs. Upon antigen stimulation TCM have a high capacity to proliferate and differentiate into effector cells in response to antigenic stimulation253. Thus, T cells may be divided into different subsets using the phenotypic cell surface marker CD45RO, CD45RA and the functional cell surface marker chemokine receptor‐7 (CCR7)253. The functional consequence of CD45RA or CD45RO expression on T cells is not fully understood. The expression of CCR7 on T cells facilitates homing to secondary lymphoid organs253. By using these markers one may divide T cell subsets according to the following model:
34
mediated response. On the other hand, CD8+ T cells (CTL) are responsible for the
direct lytic activity against target cells. Activated CD4+ or CD8+ T cells expand and
subsequently the majority of T cells undergo apoptosis; however, some cells
survive and differentiate into memory T cells. By using cell surface markers and
correlating the expression of these markers to the function of T cells, Lanzavecchia
and co‐workers have functionally characterized the T cell subsets253. T cells may
be divided into naïve, early effector (expressing CD45RA) and memory subset
(expressing CD45RO). Memory T cells can be further divided into two subsets,
effector memory T cells (TEM) and central memory T cells (TCM), depending on their
ability to 1) migrate to the inflammatory site or to secondary lymphoid organs and
2) perform immediate effector function or proliferate and differentiate into
effector cells in response to antigenic stimulation253. The TEM subset, which
expresses CD45RO but lacks expression of chemokine receptor‐7 (CCR7) and
CD45RA, is characterized by the ability to home to an inflammatory site and
perform effector function. In contrast, the TCM subset, which expresses CD45RO
and CCR7 but lacks expression of CD45RA, is primarily located in secondary
lymphoid organs and has a potent ability to proliferate upon re‐activation.
In a previous study we observed that cytokine production in the memory T cell
subset was decreased upon oxidative stress, but naïve cells remained functional.
We proposed the hypothesis that the loss of function of memory T cells was due to
a “pre‐apoptotic” state caused by ROS‐exposure. To this end, we developed an
experimental setup resembling oxidative stress in vivo. In the previous study, H2O2
was added exogenously to T cells and after 10 minutes the cells were washed to
stop the ROS‐exposure. However, it may be speculated that ROS‐exposure in vivo
has a longer duration. Since we used a setup that had H2O2 present throughout the
entire incubation period in this study, we believe that this system resembles the in
vivo situation more accurately. Furthermore, we improved the model by including
purification of T cells using negative sorting techniques, enabling a more detailed
analysis of cellular events in the different T cell subsets.
Differences in Sensitivity to ROS‐induced Cell Death in T Cell Subsets
In this study, we examined the disparities in viability and function following
oxidative stress among the various T cell subsets. We conducted a detailed
phenotypic analysis, using fluorescently labeled antibodies specific for surface
markers together with viability staining using cell death markers. Large differences
were noted between the T cell subsets when comparing sensitivity to cell death
35
induced by increasing levels of H2O2. We concluded that CD8+ T cells are more
sensitive to oxidative stress than CD4+ T cells and that memory T cells, in each
subset, are more sensitive than naïve T cells. Furthermore, when investigating the
different memory T cell subsets, we found that the CD8+ TEM subset is particularly
sensitive to cell death caused by low levels of oxidative stress and that CD8+ TCM
were not as sensitive as CD8+ TEM, although substantial cell death in CD8+ TCM was
also seen.
By using a pan‐caspase inhibitor we were able to characterize the mode of ROS‐
induced cell death in memory T cells. We found that by inhibiting caspases we
were able to reduce H2O2‐induced cell death, indicating an important role for
caspases in the cell death process. After conducting detailed time‐kinetic
experiments analyzing the temporal relationship between depolarization of
mitochondrial membrane potential, caspase 8, and caspase 3 activity, we
concluded that the intrinsic cell death pathway is primarily responsible for cell
death, as mitochondrial depolarization was observed prior to caspase 8 and
caspase 3 activity. In summary, memory T cells, especially CD8+ TEM, are more
sensitive to oxidative stress‐induced cell death as compared to other T cell subsets
and cell death is characterized by caspase activity induced by mitochondrial
depolarization. These findings are schematically depicted in Figure 6.
Fig 6 ‐ Different susceptibility to H2O2‐induced cell death of T cell subsets Upon exposure to ROS, CD8+ T cells go into apoptosis at lower H2O2
concentrations as compared to CD4+ T cells. In addition, in the CD8+ T cellcompartment memory T cells, in particular TEM, are more sensitive tooxidative stress‐induced cell death and more rapidly undergo apoptosis ascompared to other T cell subsets.
36
Differences in Signal Transduction in the T Cell Subsets It is possible that
ROS produced by the T cell itself may have an important role in signal
transduction. The model of oxidative stress described in previous sections is an
example of oxidative stress supplied by external sources, e.g. ROS produced in the
oxidative burst of neutrophils. It is known that H2O2 can diffuse through the cell
membrane and one may speculate that exogenously added H2O2 can exert its
effects intracellularly by acting directly on intracellular components. It has been
shown that ROS can act as an internal messenger affecting cellular events, such as
cell death caused by T cell activation223. Although not investigated here, it is
possible that H2O2 used in this study acts as a signal transduction molecule
mimicking normal T cell activation signaling events, thus inducing AICD. In theory,
memory T cells may be more susceptible to AICD following H2O2‐exposure due to
factors connected to differentiation of T cells into a memory phenotype.
Differences in Pro‐ and Anti‐apoptotic Molecules Another possible
explanation for the disparity of sensitivity between the T cell subsets may be
related to differences in expression levels of anti‐apoptotic (e.g., Bcl‐2 and Bcl‐xL)
and pro‐apoptotic (e.g., Bax, Bak, and Bim) proteins. Indeed, it has been shown
that CD8+ T cells have elevated levels of Bcl‐xL and Bax as compared to CD4+ T
cells254; it is therefore possible to speculate that the differences in pro‐apoptotic
protein expression could explain the differences in susceptibility to cell death
between CD4+ and CD8+ T cells. In addition, Akbar et al. have shown that memory
T cells have decreased levels of the anti‐apoptotic protein Bcl‐2 as compared to
naïve T cells255. Other reports have demonstrated that Bcl‐2 and Bcl‐xL levels in T
cells decrease after activation31,256,257. It is possible that exogenously added H2O2
induces cellular stress or DNA‐damage, which leads to activation and/or
expression of pro‐apoptotic molecules. The increase in pro‐apoptotic components
may then be sufficient to override anti‐apoptotic components in memory T cells as
these cells have lower levels of counteracting anti‐apoptotic proteins compared to
naïve T cells, resulting in preferential cell death of memory T cells.
Implication for Current Immunotherapy Preferential cell death of CD8+ TEM
by oxidative stress may be a contributing factor to tumor‐induced immune
dysfunction and may explain the difficulties in developing effective
immunotherapy against cancer. For example, IL‐2 stimulated, tumor‐specific T cell
lines from tumor‐infiltrating lymphocytes used in an adoptive cell transfer settings
are predominantly of the CD8+ TEM subtype61,258 and it may be hypothesized that
37
these cells, upon homing to the tumor site following administration to the patient,
may encounter an oxidative milieu resulting in their elimination by the
mechanisms described in this study. These therapies might therefore benefit from
concomitant administration of oxidative stress antagonists, thereby rescuing T
cells from cell death and augmenting the anti‐tumor response.
4.2. Antioxidants Rescue CD56bright NK Cells from Cell Death (Paper II)
Like T cells, human NK cells can be divided into subsets. For NK cells, this division
is based on the levels of CD56 expression, which correlate with differences in their
biological function. The two NK cell subsets, CD56dim and CD56bright NK cells, have
different patterns of responding to stimuli. CD56dim NK cells have the ability to
exert immediate cytotoxic functions and CD56bright NK cells mainly influence the
immune system by secreting cytokines upon activation259. It has been shown that
NK cells protect from infection and may be of importance for the eradication of
tumor cells260. However, as described above, several studies have concluded that
NK cells are exceptionally sensitive to oxidative stress. Thus, the environment in
cancer patients is likely to hamper NK cell‐mediated anti‐tumor immunity.
A number of studies have reported an altered ratio of CD56dim to CD56bright NK cells
at the inflammatory site261,262. Members of our laboratory have observed a
decrease in CD56dim NK cells in ascites of ovarian cancer patients (manuscript in
preparation). It is, however, not known if this is due to aberrations in induction,
proliferation, or recruitment of CD56bright NK cells or due to a preferential death of
CD56dim NK cells at the tumor site.
Based on our findings of differential susceptibility to oxidative stress‐induced cell
death in T cell subsets, we hypothesized that the altered CD56dim/CD56bright ratio
might be due to differences in sensitivity of NK cells to cell death upon exposure to
oxidative stress. To address this question we developed an in vitro model
comprising co‐culture of purified NK cells with ROS‐producing neutrophils. In
parallel with the oxidative stress model that we developed in paper I, a detailed
investigation of possible differences between the NK cell subsets was conducted.
Altered CD56dim/CD56bright NK Cell Ratio due to Differences in
Antioxidant Capacity CD56dim NK cells undergo cell death upon co‐culture with
activated neutrophils, as a consequence of H2O2 secreted by the latter. In contrast,
38
Box 3 – Basics of NK Cell Activation
NK cells are lymphocytes that are regarded as a part of the innate immune system263. It has been shown that NK cells are important in the immune response against viruses264 and tumors265. NK cells do not possess recognition receptors, such as TCR on T cells; on the contrary, NK cells are governed by a balance of stimulatory and inhibitory signals mediated by receptor:ligand interaction with the target cell266. Thus, NK cell activation is regulated by which stimulatory and inhibitory ligands that are expressed by the target cell and what stimulatory and inhibitory receptors are expressed on the NK cell267. One important feature in the regulation of the NK cell is its ability to differentiate infected or tumor cells, that have low levels of MHC class I molecules, compared to normal cells267. This is made possible by expression of several of MHC class I‐binding receptors on NK cells267. If the target cell lacks the ligand for these receptors the NK cell becomes active due to a reduced inhibitory signal in the interaction with the target cell, which leads to lysis of the target cell267; i.e. missing‐self recognition by NK cells268. However, NK cells also express stimulatory and inhibitory receptors specific for ligands on the target cell that is expressed if the cell is subjected to stress, such as virus infection, DNA‐damage‐ or carcinogenic transformation of a cell266. If the target cell expresses high levels of these stress‐induced ligands, this may override the inhibitory signal facilitated by MHC class I molecules leading to executing of cell death of the target cell by NK cells267. Adapted from Lainer266
Adapted from Raulet & Vance267
39
CD56bright NK cells are almost resilient to cell death. The selective susceptibility of
CD56dim NK cells to cell death was verified in a series of experiments exposing NK
cells to exogenously added H2O2. Of note, the shift in CD56dim/CD56bright NK cell
ratio was only true for ROS as NO‐ or γ‐radiation‐induced cell death did not show
the same pattern, indicating a unique role for ROS. A possible explanation for the
differences in susceptibility to ROS‐induced cell death may be a difference in
antioxidant levels in the different NK cell subsets. Indeed, assessment of the ability
of ROS to enter the cell, as well as the antioxidant capacity exhibited by lysate
from the different NK cell subsets indicated lower levels of antioxidants in CD56dim
NK cells as compared to CD56bright NK cells.
Potential Explanation for Differences in Antioxidant Levels An
independent study in parallel also reported that CD56bright NK cells are less
sensitive to oxidative stress as compared to CD56dim NK cells269, substantiating the
findings of our study. Thorén et al.269 demonstrated that apoptosis in both NK cell
subsets is dependent on PARP activity and that the differences in antioxidant
levels are due to increased levels of surface thiols in the CD56bright NK cell
subset187,269. Furthermore, Hanna et al. have conducted a detailed mapping of
gene expression in the different NK cell subsets and found increased levels of
glutathione peroxidase 1 in CD56bright NK cells, an enzyme that catalyzes the
reduction of H2O2 and glutathione, thereby adding to the antioxidant capacity of
this subset270.
In conclusion, we suggest that the accumulation of CD56bright NK cells at
inflammatory sites, can be explained by a rapid loss of CD56dim NK cells due to
ROS‐induced cell death. The increased cell death of CD56dim NK cells as compared
to CD56bright NK cells results from lower antioxidant levels and leads to a
preferential survival and accumulation of CD56bright NK cells at the site of
inflammation.
4.3. Conclusion Part 1
In this series of experiments we show a disparity between T cell and NK cell
subsets in their sensitivity to oxidant‐induced cell death. Strikingly, in both T cells
and NK cells, the cells with the highest potential to mount an anti‐tumor response
are the most sensitive to oxidative stress, i.e. CD8+ TEM and CD56dim NK cells. This
skewed sensitivity to ROS‐induced cell death may have a great impact on the
40
success of immunotherapy approaches utilizing these cells as primary effector
cells.
The underlying mechanism for the differential sensitivity to ROS‐induced cell death
in subsets of T cells and NK cells is, however, not identical. Based on our finding
that NK cell subsets display altered levels of antioxidant molecules, we conducted
a similar analysis investigating the antioxidant capacity in T cell subsets
(unpublished data). The experiments performed thus far show no, or marginal,
differences in antioxidant levels between T cell subsets. The preferential cell death
of CD8+ TEM and CD56dim NK cells may have different mechanisms. Our data
suggest that, in T cells, a difference in sensitivity to H2O2‐induced cell death
signaling is the main reason for the different outcome of ROS‐induced cell death in
the T cell subsets (Figure 7A). This might be due to excess activation signaling, i.e.
H2O2 acting as a second messenger leading to AICD. Alternatively, H2O2 could
induce cellular stress leading to cell death as memory T cells have lower levels of
anti‐apoptotic proteins. In contrast, the differences in sensitivity of NK cell subsets
to ROS‐induced cell death may be due to differences in antioxidant levels, leading
to lower overall levels of oxidative stress in CD56bright NK cells as compared to
CD56dim NK cells on exposure to identical levels of oxidizing substances (Figure 7B).
Although not addressed in this study, there may nevertheless be differences in
sensitivity to cell death signaling in NK cell subsets as NK cells can also undergo
activation‐induced cell death.
In conclusion, these studies propose the use of treatment modalities aimed at
reducing oxidative stress in cancer patients in order to rescue CD8+ TEM and
CD56dim NK cells from ROS‐induced cell death upon encounter of the oxidative
milieu in the tumor‐micro environment, thus enabling a more efficient anti‐tumor
response.
41
5. Part 2 – Therapies to Reverse Oxidative Stress in Lymphocytes
It is now clear that tumor‐induced immune dysfunction substantially reduces the
efficacy of cancer immunotherapy. In addition to immunotherapy, one may
develop combinatorial approaches that target these immune suppressive
mechanisms in order to counteract immune suppression. With regard to oxidative
stress as a cause of lymphocyte cell death, there are two main approaches to
introduce molecules that can decompose ROS and decrease oxidative stress: 1)
administration of antioxidants or 2) utilizing antioxidant enzyme systems.
5.1. Using Vitamin E to Reduce Oxidative Stress in Colon Cancer Patients (Paper III)
The Use of Oral Administered Antioxidants Vitamin E is a lipid‐soluble
antioxidant that can be administered orally to cancer patients271. Vitamin E
protects cells from ROS and increases cell membrane stability when located in
cellular membranes271. We have previously conducted a small clinical trial
Fig 7 ‐ Different mechanisms mayexplain the differential susceptibility ofROS‐induced cell death in T cells and NKcell subsets. A) In T cells, the most likely mechanisticexplanation of the differences insusceptibility to cell death by naive andmemory T cells is the differences in thesensitivity to ROS‐induced cell deathsignaling between the subsets.B) CD56bright NK cells have elevated levelsof antioxidants as compared to CD56dim
NK cells, which might explain thedifferences in susceptibility to ROS‐induced cell death between the subsets.
42
comprising 13 colorectal cancer patients receiving a short‐term (2 weeks)
supplementation of 750 mg vitamin E with the aim to reduce oxidative stress and
thereby improve the status of the immune system272. Analysis of peripheral blood
T cells from these patients showed an enhanced production of IL‐2 and IFN‐γ and
an increase in the CD4/CD8 ratio. With regard to NK cells, studies have shown that
vitamin E may enhance NK cell activity273,274 and can protect lymphocytes from
ROS‐induced DNA damage when added to co‐cultures with PMA‐stimulated
monocytes275. Furthermore, vitamin E together with trace elements, has been
shown to enhance NK cell activity276. Studies have also shown that vitamin E levels
are decreased in colon cancer patients indicating a high turnover of
antioxidants65,205 and a possibility to restore antioxidant levels by supplementation
of vitamin E.
Supplementation of Vitamin E Enhances NK Cell Lytic Activity in
Cancer Patients We conducted a detailed analysis of the function, phenotype
and receptor expression of NK cells from seven patients with colorectal cancer
(Dukes stage C and D) that had received vitamin E during a period of two weeks.
Our data show a clear improvement in NK cell lytic activity after vitamin E
treatment. The mechanism for the improved activity could not be established
despite numerous experiments. We did however determine that the increased NK
cell activity was not due to 1) increased numbers of NK cells or an increase in the
proportion of the CD56dim NK cell subpopulation, 2) increase in perforin expression
or an enhanced ability of NK cells to produce IFN‐γ, 3) alteration in levels of CD4+
Treg, 4) alteration in viability of NK cells, or 5) alternations in the overall cytokine
milieu in blood plasma. An increase in the expression of the activating NKG2D‐
receptor was noted in NK cells from all patients. In theory, upregulation of the
NKG2D receptor may be a factor contributing to the increased cytolytic activity of
the NK cells. However, one patient who did not demonstrate increased NK cell
activity also had an increase in NKG2D expression reducing the possibility that
upregulation of NKG2D is exclusively responsible for the increased NK cell activity
observed in this study.
Potential Mechanism of Action of Vitamin E Supplementation Although
we were unable to define the mechanism of its protective effect, vitamin E can
reduce oxidative stress either through incorporation into the membranes of NK
cells or by reducing the overall oxidative status in peripheral blood, thus indirectly
43
decreasing the oxidative stress on NK cells. Other antioxidant modalities have
been shown to be effective in boosting the immune system. For example,
administration of the antioxidant histamine together with IL‐2 and IFN‐α showed
improvement in the anti‐tumor response in melanoma patients277,278 . In summary,
we demonstrate that oral administration of an antioxidant can improve NK cell
function and that cancer immunotherapy in conjunction with vitamin E may have a
greater clinical efficacy.
5.2. Arming T Cells with an Antioxidant Enzyme (Paper IV)
Using Gene Therapy to Introduce an Antioxidant Enzyme Another way
to increase the ability of lymphocytes to resist oxidative stress is to utilize
antioxidant enzymes that normally protect cells from ROS‐induced injury, such as
SOD, CAT and enzymes of the glutathione system238. Gene therapy approaches
have been used in other studies to improve lymphocyte function. For example,
gene transfer of a chimeric GM‐CSF–IL‐2 receptor279 or the CD28 molecule280 into
human CTLs have been shown to improve T cell function. Expression of
telomerase gene in T cells has been demonstrated to prolong the lifespan of
transduced cells281. Gene modification of lymphocytes is thus feasible and has
even been successfully used to introduce tumor‐specific TCR into primary T cells
for adoptive therapy of cancer patients282.
Several studies have shown that the exogenously added antioxidant enzyme CAT
can protect lymphocytes from oxidative stress169,170,192. We therefore developed a
gene therapy approach utilizing a retroviral system to insert the CAT gene into
primary T cells, based on the premise that CAT expression would rescue the T cells
from ROS‐induced suppression. This gene therapy approach has been successfully
utilized in other disease models, such as hyperoxia‐induced injury283, oxidative
stress‐induced pancreatic islet destruction284 and neural cell degeneration caused
by ROS285.
CAT Expression Rescues T Cells from Oxidative Stress We were able to
increase levels of functional CAT in CD4+ and CD8+ T cells by retroviral delivery of
cDNA encoding for the human wild‐type CAT gene. CAT transduced T cells were
less sensitive to oxidative stress‐induced loss of function and cell death as
compared to control cells when exposed to ROS. These experiments proved that
CAT expression in primary CD4+ or CD8+ T cells can rescue these cells from
oxidative stress, giving “proof‐of‐principle” that a gene therapy approach using an
44
antioxidant enzyme can potentially reverse tumor‐associated immune suppression
resulting from ROS.
Advantages of a Gene Therapy Approach Gene transfer based
immunotherapies in cancer patients is cost and labor intensive. However, this
approach has several benefits, as there are established clinical protocols using
retroviral systems to transduce tumor‐specific T cell receptors282 and concurrent
retroviral delivery of the CAT gene could be done without major modifications.
Furthermore, oral administration of antioxidants has several limitations including
inadequate permeation of the antioxidant into the tumor site, as well as adverse
effects related to the high antioxidant levels needed to achieve appropriate in situ concentrations. The use of CAT transduced T cells ameliorates some of these
issues since the cells can proliferate and actively home to the tumor site as well as
persist in the patients, protracting the anti‐tumor effect.
5.3. Conclusion Part 2
We have investigated two approaches for using antioxidants in an attempt to
decrease oxidative stress. In Paper III, we demonstrate that oral administration of
vitamin E induces an increase in NK cell lytic activity. In Paper IV we developed a
gene therapy approach enabling expression of the antioxidant enzyme CAT in
primary T cells. Both methods can be applied to reduce oxidative stress in cancer
patients. Systemic administration of vitamin E increased the antioxidant levels in
the patients, whereas CAT transduction of T cells specifically sustains T cells upon
encounter of an oxidative milieu, such as the tumor‐micro environment.
45
Concluding Remarks The immune system needs to be in perfect synchrony in order to be able to
perform its function. The immune system responds to infections with both innate
and adaptive components in order to rapidly eliminate pathogens. The immune
response is tightly regulated in order to avoid immunopathological consequences.
This regulation is mediated through pro‐ and anti‐inflammatory mechanisms that
govern the outcome of the immune response.
The balance of the immune system in cancer patients is skewed towards chronic
inflammation. This chronic inflammation is known to decrease the efficacy of
immunotherapy approaches. Tumor‐associated immune suppression results from
several mechanisms, such as secretion of anti‐inflammatory cytokines, increased
cell death of lymphocytes, deprivation of essential nutrients, and oxidative stress.
Studies have shown that oxidative radicals formed intracellularly may activate
lymphocytes224,225; however, at higher concentrations ROS may instead induce loss
of function and cell death of T cells and NK cells169,183. We have here shown that
oxidative stress targets the most efficient tumor reactive cells, i.e. CD8+ TEM and
CD56dim NK cells. Our findings might explain the limited clinical responses noted
for anti‐tumor therapy utilizing these cell types.
We have used two strategies in order to counteract ROS‐induced immune
suppression, 1) oral administration of antioxidants aimed at decreasing the
systemic as well as the intratumoral oxidative stress or at increasing antioxidant
levels in lymphocytes and 2) utilizing an enzyme, introduced by gene‐transfer into
lymphocytes, in order to increase their antioxidant capacity. Our results show that
both approaches may be beneficial in the reversal of the oxidative stress‐induced
immune dysfunction in cancer patients.
Other modalities have been suggested to counteract tumor‐mediated immune
suppression caused by mechanisms other than oxidative stress. For example
Cheng et al. were able to improve the function of tolerized CD4+ T cells286, whereas
Wang et al. were able to activate, instead of tolerize, tumor‐specific T cells287 by
manipulating the signaling pathways in DCs using tyrosine kinase inhibitors. In
46
Fig 8 ‐ The combination of im
munotherapy and treatm
ents aiming to reverse tum
or‐induced immune dysfunction m
ay lead to improved clinical outcom
e.Chronic inflam
mation in cancer patients creates a disadvantageous m
ilieu for immunotherapy approaches. There are several m
odalities to reduce the immune suppression.
Oxidative stress‐induced im
mune suppression can be reduced by utilizing antioxidants. The effect of suppressive cytokines can be decreased by either targeting the cytokine
itself, e.g. by administration of anti‐TG
F‐β mAb, or by targeting cell populations responsible for the secretion of the suppressive cytokines, e.g. depletion of T
regs by anti‐CD25
mAb. Inhibitory receptors on T cells, e.g. CTLA
‐4, can be targeted thereby relieving the suppressive effect and drugs reversing the tolerizing capacity of DC can be em
ployed.Drugs can block N
O production, thus reducing RN
S levels, or inhibit enzymes responsible for deprival of essential nutrients, thus im
proving lymphocyte function. The
combination of one or several of these approaches w
ith immunotherapy treatm
ents may lead to reversal of tum
or‐induced immune dysfunction and an im
proved clinicaloutcom
e.
47
addition, studies have shown that targeting inhibitory receptors, such as cytotoxic
T‐lymphocyte‐associated protein 4 (CTLA‐4)288 and programmed death‐1 (PD‐1)
receptor289, on T cells with antagonistic antibodies potentiates the anti‐tumor
immune response.
CD4+ Treg are implicated in the tumor‐induced immune dysfunction and one of the
main immune suppressive mechanisms that CD4+ Treg exert is secretion of TGF‐β.
Several studies have shown that depletion of CD4+ Treg with antibodies, such as
anti‐CD25, has beneficial effects on anti‐tumor activity123. Furthermore, it has
been suggested that neutralization of TGF‐β by methods such as administration of
an anti‐TGF‐β receptor antibody and drugs blocking the intracellular signaling of
the TGF‐β receptor290 may also lead to reversal of this suppression. Blocking
enzymes responsible for deprival of essential nutrients at the tumor site, i.e. IDO
and ARG1, may also improve T cell function. Indeed, Muller et al. have observed a
synergistic effect combining small molecule that inhibits IDO with
chemotherapy291. Studies have also shown that blockage of ARG1 inhibits tumor
growth and that this effect is mediated by the immune system146. NO production
by iNOS as a source of RNS has also been targeted in order to reduce tumor‐
induced immune dysfunction and it has been shown that administration of iNOS
inhibitor reverses T cell suppression292. It has further been shown that co‐
administration of ARG1 and iNOS inhibitors has synergistic effects on anti‐tumor
therapy292.
In conclusion, it is evident that there are ways to reverse tumor‐induced immune
dysfunction. As depicted in figure 8, it is conceivable that by combining existing
immunotherapy approaches with one or several methods of reversing tumor‐
induced immune dysfunction, an improved clinical response in cancer patients
could be achieved.
48
Acknowledgements The effort, summarized in this thesis, has been fun, frustrating, interesting, and a
lot of work. I perform at my best when I am surrounded by and collaborating with
talented co‐workers and when I have the support of my friends and family.
I have been blessed with the support of many people to whom I would like to
express my sincere gratitude.
Rolf Kiessling, my supervisor, for support, knowledge, and for providing an
excellent balance between scientific guidance and independence; for pushing me
to continue and enabling me to do “extra”‐scientific activities. Thank You!
Jelena Leviskaya, my co‐supervisor, for listening and for high‐quality advices
regarding science and non‐science.
Mike Nishimura, for a fruitful collaboration, your vast knowledge, and your
generosity.
Isabel, Christian J, and Raja for helping me with the writing of this thesis.
All former and present colleagues in the group: Maria, Simona, Isabel, Lena‐Maria,
Chiara, Andres, Kosaku, Takhashi, Eiji, Mitsue, Jan‐Alvar, Anita, Lars A, Andreas,
Kalle L, Max, Tomas, Silvia, Akihiro, Alvaro. A special thanks to: Kristian H for
convincing me that there is a “light in the tunnel”, Christian J for superb teamwork
and not despairing during the endless night shifts, Helena for being a super‐
collaborator (when the blizzard is coming you know who to call…), Volkan for your
kindness and Turkish food, Daiju for everlasting brainstorming and idea creation (I
hope I’ll visit you and your lovely family in Japan soon), Anna DG for always being
you (fun, talkative, energetic, positive (most of the time ☺ ), truly caring, and
helping me develop my computer‐teaching skills, Kalle M for being an inspiration
and the go‐to guy, Mattias C (lost to the dark side) for your enthusiasm and caring,
Håkan for making the group more interesting, Telma & Charlotte for making the
CAT‐project possible, and Carl Tullus; I miss you.
People at the floor: Thank you for creating a fun and inspiring work atmosphere. A
special thanks to Eva M, Marzia, Maxim, Ashley, Pavel, Fredrik E “the roomie”,
Anki, Kajsa, David, Ingrid for all your help, Barbro, Lena, Tina, Torbjörn, Peter,
Karin, Kalle A, Andrea, and Giuseppe.
49
People at CCK: Thank you for the many ways you have helped me. A special thanks
to: Dan G, Stig L, Bita, Aleksandra, Marianne, Jacob, Magnus B, Evi, Sören, Joe,
Emily, Elisabeth, Elle, Anders, Gunilla, Eva‐Lena, Mari, and Juan.
Class‐mates of Biomedicine: Thank you for making the first four years at KI so
much fun! Especially, Markus (also lost to the dark side) for sauna and laughs, Nina
R, for being genuine, driven and caring, Linda S for your kindness, and Jonas for
being my dear friend, idea creator, and source of inspiration.
Friends: Martin for being my friend and “brother”, Nina W for always caring and
adding new perspectives on life, Anneli & Peter, Lina, Anna G, Linus & Katarina,
Karolin for your long and true friendship.
Family: Bosse and Kaj for being my role models in medicine, Arne & Gudrun for
welcoming me into your family and always being there, Sanna & Fredrik for always
having your door open, for summers and for winters, Fabian & Josefin for being
the best cousins, Mormor for being an inspiration ‐ keep on dancing, Tian – my
wonderful brother, for your warmth, for always laughing and for showing me how
to be a kid. Eva, for being a great svägerska – you are always positive! Linda – my
darling sister, for all our talks, your honesty and love, for teaching me not to be a
robot, Mamma & Pappa for making me who I am, for your everlasting support,
generosity, and love.
Albin & Viktor skratt kärlek ömhet skoj brottas läsa saga spela boll jag älskar er.
Lena for being there, for believing in me, for letting me do what I wish, for having
dinner served when it is “just such a day”, for pushing to go further when I am
hesitating, for being a loving mother and my girl.
For me you are love laughs security trust best friend. Without you I am not me.
50
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