Hindawi Publishing Corporation
Clinical and Developmental Immunology
Volume 2012, Article ID 927240, 13 pages
J. G.J. V.Aerts,1and J. P. J. J. Hegmans1
1Department of Pulmonary Medicine, Erasmus Medical Center-Daniel den Hoed Cancer Center, P.O. Box 2040,
3000 CA Rotterdam, The Netherlands
2Department of Thoracic Surgery, Erasmus Medical Center-Daniel den Hoed Cancer Center, P.O. Box 2040,
3000 CA Rotterdam, The Netherlands
Correspondence should be addressed to R. Cornelissen, email@example.com
Received 26 January 2012; Accepted 17 April 2012
Academic Editor: Masoud H. Manjili
Copyright © 2012 R. Cornelissen et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Treatment options for malignant mesothelioma are limited, and the results with conventional therapies have been rather
disappointing to this date. Chemotherapy is the only evidence-based treatment for mesothelioma patients in good clinical
condition, with an increase in median survival of only 2 months. Therefore, there is urgent need for a different approach to battle
this malignancy. As chronic inflammation precedes mesothelioma, the immune system plays a key role in the initiation of this
type of tumour. Also, many immunological cell types can be found within the tumour at different stages of the disease. However,
mesothelioma cells can evade the surveillance capacity of the immune system. They build a protective tumour microenvironment
to harness themselves against the immune system’s attacks, in which they even abuse immune cells to act against the antitumour
immune response. In our opinion, modulating the immune system simultaneously with the targeting of mesothelioma tumour
cells might prove to be a superior treatment. However, this strategy is challenging since the tumour microenvironment possesses
numerous forms of defence strategies. In this paper, we will discuss the interplay between immunological cells that can either
inhibit or stimulate tumour growth and the challenges associated with immunotherapy. We will provide possible strategies and
discuss opportunities to overcome these problems.
Links between cancer and inflammation were first noted
by Rudolf Virchow in 1863, on observations that tumours
often arose at sites of chronic inflammation and that
inflammatory cells were present in biopsy samples from
tumours . In a severe combined immunodeficiency
(SCID) mouse xenograft model, it has recently been shown
that inflammation precedes the development of human
malignant mesotheliomas . Also, epidemiological studies
have revealed that chronic inflammation caused by chemical
and physical agents, autoimmune and by inflammatory
reactions of uncertain aetiology, predisposes for certain
forms of cancer [3, 4]. Recently our group demonstrated
a significantly shorter survival in patients with lung can-
cer in subjects with a history of pulmonary tuberculosis
than patients without tuberculosis , revealing even a
more complex interplay between inflammation and cancer.
Increasing evidence indicates that the inflammation-cancer
connection is not only restricted to the initiation of the
cancer process, since all types of clinically manifested cancers
appear to have an active inflammatory component in their
microenvironment. These experimental findings and clinical
observations have led to cancer-related inflammation being
acknowledged as an important hallmark of cancer .
2.1. Tumour-Immune Surveillance. Old, Klein, and others
investigated murine tumour transplantation models and
showed that the immune system of healthy recipient mice
2Clinical and Developmental Immunology
was able to distinguish transformed malignant cells from
normal cells [7, 8]. Even preceding these publications, Frank
MacFarlane Burnet and Lewis Thomas formulated their
cancer immunosurveillance hypothesis: “It is by no means
inconceivable that small accumulations of tumour cells may
develop and because of their possession of new antigenic
potentialities provoke an effective immunological reaction
with regression of the tumour and no clinical hint of its
existence” . At that time this hypothesis was controversial;
however, with the current knowledge and ongoing research,
it is apparent their premise seems to be correct because
there is strong evidence from animal studies that cells of
the immune system carry out surveillance and can eliminate
nascent tumours .
acquired by tumour cells in the process of neoplastic
transformation that can elicit a specific immune response
by the host. It is known that several immunological cell
types are involved in the recognition and destruction of
tumours during early stages of development. These include
cells and factors of the innate immune system, including
macrophages, neutrophils, complement components, γδ
T cells, natural killer (NK) cells, NKT cells and certain
cytokines (IL-12, IFN-γ) and cells of the adaptive immune
system, including B lymphocytes, helper T cells (Th cells),
and cytotoxic T lymphocytes (CTLs).
TAAs need to be presented to the cells of the adaptive
immune system. Dendritic cells (DCs) are widely acknowl-
edged for their potent antigen presenting capacity and play a
activation and modulation of lymphocyte subsets . DCs
originate from bone marrow precursor cells and are found
at low frequencies in peripheral tissues where they maintain
an immature phenotype and search their surroundings for
foreign substances. Immunogenic TAAs are secreted or shed
by tumour cells or released when tumour cells die and can
be taken up by DCs or other antigen presenting cells (APCs).
Upon encountering an antigen, DCs mature and migrate to
regional draining lymphoid organs. The captured antigen
is processed and presented by major histocompatibility
complex (MHC) class I and class II molecules on their
cell membrane leading to the activation of antigen-specific
lymphocytes. This results in antibody production by B
lymphocytes and tumour-specific CTLs to assist the innate
immune responses in the killing of tumour cells.
2.2. Tumour-Immune Escape. Increasing evidence reveals
that when tumours progress in time, tumour cells undergo
changes to escape immune surveillance. This process encom-
passes three phases: elimination, equilibrium, and escape.
During the first phase, tumour cells have to escape the
immune surveillance to survive. Then these surviving
equilibrium between tumour growth and tumour killing by
cellsof the immune system.In this stage,tumourscanpersist
for years without progressing to more severe tumour stages.
However, during this period, tumour cells may undergo
mutations caused by their genetic instability, potentially
generating variants that can escape the immune system, by
either evading the induction of an immune response or by
inhibiting antitumour responses via a variety of immune
2.3. Immune Suppressive Mechanisms. The tumour immune
escape mechanism can be greatly enhanced by the induction
of an immune suppressive tumour microenvironment. In
this microenvironment, inflammatory cells and molecules
have a major influence on cancer progress. Effective adaptive
immune responses are suppressed through the activation
of several pathways. For example, the differentiation and
activation of dendritic cells, which are the key initiators
of adaptive immune responses, are inhibited by signals
(such as IL-10 and VEGF) present in the tumour microen-
vironment. In addition, tumours, peripheral blood, and
lymph nodes contain increased amounts of regulatory T
cells (Tregs), which suppress both the adaptive and innate
immune responses . Also, a heterogeneous population
of myeloid-derived suppressor cells (MDSCs) are induced
in tumour-bearing hosts; these cells, as well as tumour-
associated macrophages (TAMs or M2 macrophages), are
potent suppressors of antitumour immunity. Not only do
MDSCs and TAMs suppress the antitumour response, but
they also assist the malignant behaviour of tumour cells
by secreting cytokines, growth factors, matrix-degrading
enzymes, and proteases, which promote tumour progression
or enhance metastasis.
In conclusion, immune cells can either protect the
host against cancer development or promote the emergence
of tumours with reduced immunogenicity leading to a
complex interplay of tumour growth and tumour regression
mechanisms (Figure 1) .
Cancer immunotherapy attempts to activate or enhance the
antitumour effects of the immune system of the patient, or it
may assist in the capabilities of the immune system to fight
cancer. Multiple approaches for immunotherapy have been
developed over the years, and many are in various stages of
(pre-)clinical research. Immunotherapy can be divided into
3.1. Passive Immunotherapy. Passive immunotherapy makes
use of in vitro produced immunologic effectors that are
capable of influencing tumour cell growth. The most com-
mon form of passive immunotherapy is called monoclonal
antibody therapy. It consists of humanized monoclonal
antibodies that are investigated in several human malignan-
cies. Monoclonal antibodies can target cells directly  or
indirectly. Monoclonal antibodies are also used as immune
modulators to inhibit immune suppressive molecules/cells
or activate immune stimulatory molecules. Efficacy of this
approach can sometimes be enhanced by linking a toxin to
these antibodies (e.g., radionucleotides or anticancer drugs).
amatuximab (previously known as MORab-009) were
Clinical and Developmental Immunology3
Figure 1: Interplay between immunological cells that inhibit tumour growth on the right of the tumour and cells that aid in tumour
progression on the left. (Tumour is depicted as black cells with a red nucleus in the middle.) iDC: immature dendritic cell, Treg: regulatory
T cell, MDSC: myeloid-derived suppressor cell, TAM: tumour-associated macrophage, mDC: mature dendritic cell, B: B cell lymphocyte,
CTL: cytotoxic T lymphocyte, M1 MØ: M1 macrophage, NK(T): natural killer (T) cell, Th17: helper T lymphocyte 17, FB: fibroblast.
promising [16–18] and therefore progressed to clinical trials.
CAT-5001, administered to mesothelioma patients, among
other cancer types, showed only modest clinical responses
[17, 18]. Amatuximab failed to demonstrate any radiological
responses in a phase I trial in mesothelioma and other cancer
types ; however preclinical studies demonstrated signif-
icant antitumour efficacy using combination of amatuximab
and chemotherapy treatment  justifying a multicenter
phase II clinical trial utilizing cisplatin/pemetrexed with
amatuximab in mesothelioma patients. This trial has been
completed and results are expected soon. More recently a
phase I study of SS1(dsFv)PE38, a recombinant antime-
sothelin immunotoxin, was commenced which is ongoing at
this moment (ClinicalTrials.gov Identifier: NCT00575770).
Another method of passive immunotherapy uses adap-
tive transfer of (autologous or allogeneic) antigen-specific
effector cells (like T cells and NK cells) that can be expanded
and/or activated ex vivo and subsequently administered
to the patient to attack the tumour . This approach
showed the potential to reconstitute host immunity against
pathogens, like Epstein-Barr virus (EBV) in immune sup-
pressed patients, but more importantly also provides evi-
dence that adaptive T cell transfers can prevent the induction
of EBV-associated lymphomas . This led to the concept
that antigen-specific T cell transfer can be used as an
antitumour therapy to eradicate established tumours. The
is challenging .
approaches aim at inducing or boosting immune effector
cells in vivo against tumour cells, through the administration
Active Immunotherapy. Activeimmunotherapeutic
of immune mediators capable of activating the immune
Several cytokines are capable of activating and recruit-
ing specific immune cells that can enhance antitumour
immunity (e.g., IL-2, IL-12, IL-15, TNF-α, GM-CSF). These
cytokines can be used as single agent or in combination with
other immunotherapeutic strategies.
Defined TAA epitopes have been used to vaccinate
cancer patients ; however this approach is limited by
the relatively low number of identified specific peptides
and by the requirement of MHC typing. By using the
whole TAA protein for immunization, the need of peptide
identification can be circumvented. These proteins can be
taken up by APCs and endogenously processed into epitopes
for presentation to T cells. Adjuvants need to be added to
induce APCs activation and avoid tolerance induction .
DNA sequences coding for specific TAAs can be directly
injected into the skin. DNA then needs to be taken up,
transcribed into mRNA, translated into a protein, and
processed into peptides by APCs.
In mesothelioma, the TAA’s mesothelin and Wilms
tumour-1 (WT-1) are highly expressed and thought to
be physiologically relevant to this tumour type . In
the Memorial Sloan-Kettering Cancer Center a phase I
peptide vaccination clinical trial in mesothelioma patients
is ongoing (ClinicalTrials.gov Identifier: NCT01265433). In
these patients, inoculation with WT-1 peptide elicited WT-
1-specific CD4 and CD8 T-cell responses, with minimal
toxicity . TroVax has been shown to stimulate an
immune response to a particular protein widely found on
mesothelioma cells called 5T4; a clinical trial testing the
Trials Unit (ClinicalTrials.gov Identifier: NCT01569919).
4Clinical and Developmental Immunology
An important restriction of this method is the relatively
inefficient delivery into APCs. Viruses engineered to express
TAAs can be injected directly into the patient. The virus then
infects the host cell, leading to cell death and presentation of
antigenic epitopes to the immune system. A wide variety of
administration of a vaccine with a measles virus strain is
performed at the Mayo Clinic (ClinicalTrials.gov Identifier:
NCT01503177). However there are concerns regarding the
immune dominance of viral antigens over TAAs, resulting in
a strong anti-virus response leading to virus eradication and
attenuation of the antitumour immune response .
DCs have emerged as the most powerful initiators of
immune responses. In the natural activation of the adaptive
immune system against tumour cells, DCs play a crucial role
since they are capable to engulf tumour antigens and activate
lymphocytes in an antigen-specific manner. Therefore, the
application of DCs to therapeutic cancer vaccines has been
The research group of Dr. Robinson published a very
interesting trial, in which they used an autologous tumour
mesothelioma material and administered subcutaneously
together with granulocyte-macrophage colony stimulating
factor (GM-CSF). GM-CSF facilitates APCs recruitment and
survival in vivo which in turn may generate tumour-specific
immunity after uptake of the TAA from the lysate. Twenty-
oped positive delayed type hypersensitivity skin tests, and
ern blotting, proving that GM-CSF could induce tumour-
of the patients developed at least one type of anti-MM
immune response. Furthermore, the therapy was safe and
regressions were observed .
While this study showed potential for GM-CSF as
immunotherapeutical approach, in vivo stimulation of
APCs is also a very attractive method. Sipuleucel-T is
an active cellular immunotherapy consisting of autologous
peripheral-blood mononuclear cells (PBMCs), including
APCs. Recently, Kantoff et al. published a phase III trail
where they used ex vivo activated Sipuleucel-T with a
recombinant fusion protein (PA2024). PA2024 consists of a
prostate antigen, prostatic acid phosphatase that is fused to
GM-CSF, an immune-cell activator. Sipuleucel-T prolonged
survival among men with asymptomatic or minimally
symptomatic metastatic castration-resistant prostate cancer
, providing evidence for cell-based immunotherapeutic
agents in solid tumours.
In mesothelioma, the source of the TAA for DC loading
remains a critical issue that will determine the efficacy of
the DC-based vaccination. A careful identification and char-
acterization of antigenic epitopes is needed when peptides
want to be used. However, the ideal source of TAAs may be
the tumour itself, since it expresses all the TAAs that can be
Incubating DCs with dead tumour cells (necrotic or
apoptotic cells), as was shown in a pioneering article by
the research group of Dr. Gregoire, DCs were exposed to a
full array of antigenic peptides that rapidly gain access to
both MHC Class I (cross-presentation) and MHC Class II
involving cytotoxic T lymphocytes (CTLs) as well as CD4+
T cells. In their paper they successfully demonstrated in
vitro culture and antigen loading in a human mesothelioma
model, resulting in a specific CTL response .
One of the advantages of an ex vivo culture model
is that DCs can be generated in large amounts and
pulsed with tumour antigens under optimal conditions.
In mesothelioma, we previously investigated the effect of
DC-based immunotherapy on the outgrowth of mesothe-
lioma in a murine model . We established that DC-
responses leading to prolonged survival in mice. The efficacy
of immunotherapy was dependent on the tumour load; most
beneficial effects were established at early stages of tumour
On the basis of these preclinical animal studies, we
have performed the first clinical trial in which autologous
tumour lysate-pulsed DCs were administrated in mesothe-
lioma patients . Patients were eligible for the study
when sufficient tumour cells could be obtained from pleural
effusion or tumour biopsy material at the time of diagnosis.
DC-immunotherapy was planned after completion of the
cytoreductive therapy provided that during chemotherapy
no major side effects occurred and there was no progressive
disease. Patients received three immunizations with mature
DCs, loaded with autologous tumour lysate. Each immu-
nization, consisting of 50 × 10e6 cells, was administered
intradermally and intravenously (Figure 2). Overall, the
vaccination regimen with loaded DCs was well tolerated,
and a successful immune reaction was induced by the DC
The University Hospital of Antwerp has started a similar
protocol in mesothelioma and several other solid tumours
but is using WT-1 as antigen loading for the DCs (Clinical-
for patient’s tumour material.
Another method to load DCs is to make use of measles-
virus-infected mesothelioma cells. It was shown that this
ing of autologous T cells by DCs loaded with measles-virus-
infected mesothelioma cells led to a significant proliferation
of tumor-specific CD8 T cells .
While immunotherapy was proven safe and feasible, it has
not established its place yet in mesothelioma treatment.
Partly, this is due to the presence of immunosuppressive
cells in peripheral blood, lymphoid organs, and within
the tumour environment that hamper immunotherapeutic
treatments. Several strategies have been performed or are
currently tested that target the immunosuppressive cells
aiming to improve the efficacy of immunotherapy. In the
following sections, we will focus on three populations of
Clinical and Developmental Immunology5
Ex vivo generated
Figure 2: Schematic drawing showing the administration of ex vivo cultured mature dendritic cells into a patient (1), resulting in antigen
presentation in the lymph node (2) and a specific antitumour cytotoxic antitumour response (3). Tumour cells are depicted as dark cells.
suppressive cells, the MDSCs, Tregs, and TAMs, that are
clear that these populations contribute to the impaired
tion of these cell types will be an important key to increase
the efficacy of immunotherapy and should lead to better
prognosis for cancer patients.
4.1. Myeloid-Derived Suppressor Cells. MDSCs are a hetero-
geneous population of bone-marrow-derived myeloid cells,
comprising of immature monocytes/macrophages, granulo-
cytes, and DCs at different stages of differentiation . A
subset of MDSCs, mononuclear MDSCs (MO-MDSCs), is
mainly found at the tumour site while polymorph nuclear
MDSCs (PMN-MDSCs) subset is found in blood, lymphoid
organs, and at the tumour site. They express a number
of surface markers, that are on themselves not unique but
in combination can define MDSCs. MDSCs are increased
in cancer patients, and it is anticipated that they play a
suppressive role during the innate and adaptive immune
responses to cancer but have also been described in the
course of other pathologic processes such as thermal injury,
various infectious diseases, sepsis, trauma, after bone mar-
row transplantation, and in some autoimmune disorders.
Activation of MDSCs not only requires tumour-derived
factors (e.g., tumour-derived prostaglandin E2 (PGE2)) but
also IFN-γ produced by T cells and factors secreted by
tumour stromal cells (like IL-1β, IL-4, IL-6, IL-10, IL-13).
Activation of cytokine receptors on MDSCs leads to activa-
of immune suppressive substances (like TGF-β, reactive
oxygen species (ROS), and nitric oxide synthetase (NOS)).
MDSCs can inhibit the antitumour immune response in
(i) MDSCs are capable of producing reactive oxygen
species (ROS) and peroxynitrite, which is responsible
for most of the adverse effects on T cells, linked
to ROS. Changes caused by nitration of the T cell
receptor make T cells incapable of interacting with
the MHC complex on APCs, which is necessary to
obtain T cell-specific stimulation [36, 37].
(ii) MDSCs can inhibit the antitumour response in an
the enzyme inducible nitric oxide synthetase (iNOS),
leading to the generation of NO. NO can suppress T
cell function though various mechanisms including
the inhibition of the cell signalling pathways and
inducing DNA damage to T cells.
(iii) Arginase-I activity by MDSCs depletes L-arginine
from the environment, contributing to the induction
expression of the T cell receptor [38, 39].
(iv) MDSCs block T-cell activation by sequestering cys-
teine and thus limiting the availability of the essential
amino acid cysteine .
6Clinical and Developmental Immunology
(v) MDSCs can inhibit T cell proliferation by producing
IL-10 and TGF-β .
(vi) Antitumour cells, like NK- and NKT-cells, can be
inhibited by MDSCs via TGF-β1-dependent mech-
anisms. MDSC can bind to the TGF-β receptor on
target cells via membrane-bound TGF-β, leading
to activation of intracellular pathways resulting in
downregulation of NK-specific receptors .
(vii) The plasma membrane expression of enzyme
ADAM17 on MDSCs cleaves L-selectin on na¨ ıve T
cells, decreasing their ability to home to sites where
they could be activated .
(viii) MDSCs can indirectly enhance immune suppression
via the induction of Tregs [43–45].
(ix) MDSCs differentiate under certain biological condi-
tions into mature functionally competent macropha-
ges or to DCs influencing tumoural responses .
4.2. Targeting MDSCs. Both gemcitabine and 5-fluorouracil
(5FU) have shown to be selectively cytotoxic on MDSC in
murine tumour models . The treatment of tumour-
bearing mice with 5FU led to a decrease in the number of
MDSC in the spleens and tumour beds of animals whereas
no significant effect on T cells, NK cells, DCs, or B cells was
noted. 5FU showed a superior efficacy over gemcitabine to
deplete MDSC and selectively induced MDSC apoptotic cell
Gene expression profile analysis of multiple tumour
types identified SCF (c-kit ligand) as a candidate tumour
factor involved in MDSC accumulation. Inhibiting c-kit
using the tyrosine kinase inhibitor sunitinib resulted in a
decrease of the number of MDSC and Treg in advanced
tumour-bearing animals .
The production of ROS by MDSCs, which is responsible
for most of the adverse effects on T cells, is highly depending
upon cyclooxygenase-2 (COX-2) enzyme activity . The
inducible COX-2 enzyme is essential in the biosynthesis
of prostaglandins. Celecoxib is a selective COX-2 inhibitor.
Therefore, we investigated the effect of celecoxib treatment
on the four MDSC subsets that were identified in the
spleen of tumour-bearing mice . When combining DC-
based immunotherapy and celecoxib treatment, a significant
to no or single modality treatment. Treatment of tumour-
bearing mice with dietary celecoxib prevented the local and
systemic expansion of all MDSC subtypes, and also their
suppressive function was impaired. At the National Cancer
Institute, allogeneic tumour cell vaccine is combined with
celecoxib and metronomic oral cyclophosphamide as adju-
vants in thoracic malignancies (ClinicalTrials.gov Identifier:
NCT01143545); the rationale for using cyclophosphamide is
discussed further in this paper.
4.3. Tumour-Associated Macrophages. Macrophages are a
major component of the leukocyte infiltrate in the tumour
microenvironment  and have even been described
as key orchestrators of cancer-related inflammation .
Classically activated (M1) macrophages, following exposure
to IFN-γ, have antitumour activity and tissue-destructive
activity. In response to IL-4 or IL-13, macrophages undergo
alternative (M2) activation. M2 macrophages are oriented
to tissue repair, tissue remodelling, and immune regulation.
TAMs generally have the phenotype and functions similar to
M2 macrophages and display a defective NF-κB activation in
response to different proinflammatory signals .
TAM recruitment in tumours is mediated by several
cytokines, of which CCL2 seems to be the main player; other
chemokines involved in monocyte recruitment are CCL5,
CCL7, CXCL8, and CXCL12, as well as cytokines such as
VEGF, PDGF, and the growth factor M-CSF . It has
been shown that MO-MDSCs are capable of differentiating
towards TAMs. Therefore, similar recruitment factors are
described that contribute to the infiltration of TAMs and
MDSCs into tumour tissue . In addition, dynamic
changes of the tumour microenvironment occur during the
transition from early neoplastic events toward advanced
tumour stages resulting in local hypoxia, low glucose level,
and low pH. These events in the tumour microphysiology
drive the switch from a M1 macrophage toward the M2 type.
through various mechanisms and contribute to angiogenesis
and tumour invasiveness.
(i) TAMs are able to produce immune suppressive
cytokines, like CCL17, CCL18, CCL22, IL-1β, IL-6,
IL-10, and TGF-β. IL-10 in combination with IL-
6 can lead to upregulation of molecules in TAMs,
which are implicated in suppression of tumour-
specific T cell immunity .
ase (IDO), a well-known suppressor of T cell acti-
vation. IDO catalyzes the catabolism of tryptophan,
an essential amino acid acquired for T cell activation
(iii) TAMs contribute to immune suppression via indirect
ways. Secretion of CCL18 leads to recruitment of
native T cells. Attraction of naive T cells into the
tumour microenvironment is likely to induce T cell
anergy . Besides CCL18, CCL17 and CCL22 are
abundantly expressed. These cytokines interact with
CCR4 receptor expressed by Tregs and induce T-
helper 2 polarization . Via expression of VEGF,
TAMs can block antigen uptake by APCs and attract
MDSCs, which can function as TAM precursors but
are also actively suppressing T cell function. MDSCs
are depending on prostaglandin E2 (PGE2) for their
function. PGE2 is secreted by many types of cancer;
however, TAMs are also capable of producing PGE2
and therefore assist MDSC function .
(iv) In tumour stroma, TAMs produce matrix metallo-
proteases (MMP) and other proteases, leading to
degradation of the extracellular matrix. During this
process, several cytokines, chemokines, and growth
factors are released from the matrix that promotes
Clinical and Developmental Immunology7
and facilitates endothelial cell survival and migration
and thereby enhances angiogenesis .
(v) Besides indirect mechanisms, angiogenesis is also
directly stimulated by TAMs. TAMs can pro-
duce proangiogenic factors like vascular endothelial
growth factor (VEGF), transforming growth fac-
tor (TGF)-β, and platelet-derived growth factors
vascularisation, especially in hypoxic regions within
the tumour [53, 62].
(vi) In addition to angiogenesis, TAMs are also strongly
involved in lymphangiogenesis, a process mediated
D via VEGFR3 .
(vii) Outside the scope of the tumour microenvironment,
but a pivotal step in general tumour biology, TAMs
cooperate on tumour dissemination by promoting
invasion characteristics. One of the main factors
involved significantly is TNF-β: coculture of neo-
plastic cells with macrophages enhances invasiveness
of malignant cells through TNF-dependent MMP
induction by macrophages .
4.4. Targeting TAMs. There is accumulating evidence sup-
porting the hypothesis that effects on TAMs may contribute
to the antitumour effect of bisphosphonates . We inves-
tigated the effect of zoledronic acid (ZA) in mesothelioma-
inoculated mice. Our data showed that the addition of
ZA to macrophage-inducing culture conditions significantly
inhibits the upregulation of F4/80, MHCII, and CD11c. In
addition, these data reveal that adding tumour supernatant
leads to polarization of the macrophage phenotype towards
M2 subtype and that ZA can prevent this polarization in
vitro, leading to a significant reduction in the CD206 expres-
sion on macrophages cultured in the presence of ZA. In vivo,
however, no significant differences on tumour progression
and survival could be observed between untreated mice and
mice treated with ZA, because the reduction in TAMs was
associated with an increase in MDSC .
IL-6 stimulates tumour macrophage infiltration in ovar-
ian cancer, and recently it has been shown that this action
can be inhibited by the neutralizing anti-IL-6 antibody
siltuximab in preclinical and clinical studies .
A recent study revealed that activation of macrophages
by the infusion of antibodies against CD40 may induce
macrophage-mediated tumour regression in 30% of cases in
both a mouse model for pancreatic cancer and in patients
with pancreatic cancer [66, 67].
Since TGF-β is responsible for skin tumour infiltration
by macrophages enabling the tumours to escape immune
destruction , TGF-β seems to be a major player in the
formation of the suppressive tumour microenvironment.
Blockade of TGF-β has been shown to enhance tumour
not been unravelled yet . Since CCL2 plays a major role
in the recruitment of TAMs, anti-CCL2 would be a logical
step in preventing this recruitment. However, it seems that
anti-CCL2 does not prevent the influx of TAMs ; this
could be due to the inability to reach an adequate dosage of
anti-CCL2 in the tumour microenvironment to counteract
the influx of TAMs.
4.5. Regulatory T Cells. Tregs are a population of CD4+
T cells with a central role in the prevention of autoim-
munity and the promotion of tolerance via their sup-
pressive function on a broad repertoire of cellular targets
. Characteristic of human Tregs is the expression of
CD25 (IL-2 receptor-α chain), forkhead box P3 (Foxp3)
transcription factor, glucocorticoid-induced TNF-receptor-
3), cytotoxic T-lymphocyte-associated antigen 4 (CTLA4),
and a downregulation of CD127 (IL-7R); however, all these
markers are not truly Treg specific . Tregs can be divided
into natural Tregs and adaptive Tregs. Natural Tregs are
important in the suppression of autoreactive T cells that
slip through the selection processes, and therefore natural
Tregs maintain peripheral tolerance against self-antigens
preventing autoimmunity. In humans, these cells represent
2–5% of total circulating CD4+ T cells in peripheral blood
. Adaptive Tregs arise from naive T cells and are
triggered by suboptimal antigen stimulation and stimulation
with TGF-β. Adaptive Tregs can be subdivided into IL-10
secreting Tregs type I (Tr1 cells), TGF-β producing Tregs
(Th3 cells), or IL-35 secreting Tregs (iTr35 cells). These cells
are characterized by the secretion of immune suppressive
cytokines directly inhibiting T cells and converting DCs into
capacity, mainly mediated by IL-35 from Tregs, to other T
cells is called infectious tolerance .
Tregs infiltrate human cancers, and their prevalence in
tumour-infiltrating lymphocytes is much higher than their
proportion in peripheral blood, constituting 20% or more of
tumour-infiltrating CD4+ lymphocytes . Elevated levels
of Tregs have been identified in blood of cancer patients,
compared with normal individuals, and their presence
predicts for poor survival . In mesothelioma patients,
elevated levels of Tregs have also been identified in pleural
fluid, with a clear patient-to-patient variability .
Natural Tregs are derived in the thymus and migrate
into the periphery. It has been proposed that Tregs need
to be activated and/or expanded from periphery and bone
marrow if needed. Since 25% of CD4+ T cells in the bone
marrow plays an active role in humoral and cellular immune
TAA-specific Tregs accumulate in the peripheral lym-
phoid organs and at the tumour side. However TAA-specific
activation Tregs can migrate back to the bone marrow and
induce T cell tolerance before these cells enter the circulation
. Although exact mechanisms are not fully explored,
it has been shown that CCR4+ (receptor for CCL22)
CCL22 . Also CD62L and CCR7 have been described as
important homing markers on Tregs . CD62L is critical
for the migration of Tregs to draining lymph nodes. CCR7 is
8Clinical and Developmental Immunology
expressed by a majority of Tregs and is essential in homing to
lymphoid organs and microenvironments expressing CCL19
(the ligand for CCR7) .
As MDCSs and TAMs, Tregs have several pathways that
limit antitumour responses.
(i) Direct cell-cell interaction between Tregs and tar-
get cells is important for tolerance induction by
Tregs . These target cells include CD4+ and
CD8+ effector cells, B cells, NK, T cells, DCs,
and monocytes/macrophages. The cell-cell binding
leads to apoptosis by activation of programmed cell
death ligands (PDLs), the release of perforin 
and granzyme-A or B  and by reducing the
proliferation through upregulation of intracellular
cyclic AMP [84, 85].
(ii) Tregs produce themselves or induce other cells to
secrete immunosuppressive cytokines such as IL-10,
IL-35, and TGF-β to blunt immune responses ,
but also other molecules produced by Tregs like car-
bon monoxide  and galectins  are reported
to play roles in suppression. However, the relative
importance of the individual inhibitory factors is
dependent on the target disease and experimental
(iii) Tregs can inhibit antitumour effector NK and NK T
cells via membrane-bound TGF-β . The binding
of membrane-bound TGF-β on Tregs to TGF-β-
receptor on target cells leads to the activation of
intracellular pathways, which eventually leads to the
downregulation of the NKG2D-receptor on NK and
(iv) CTLA4+ Tregs induce the expression of indoleamine
2,3-dioxygenase (IDO) in APCs, a potent regulatory
molecule mediating the catabolism of the essen-
tial amino acid tryptophan into the proapoptotic
kynurenine, which is toxic to neighbouring T cells
(v) Tregs are forming aggregates around DCs to prevent
contact between DCs and T cells and in this way dis-
turb the induction of the adaptive immune response
by preventing proper antigen presentation [91, 92].
(vi) Treg aggregation leads to decreased upregulation of
CD80 and CD86 on immature DCs and downregula-
tion of these molecules on mature DCs . These
phenomena are antigen specific and dependent on
lymphocyte function-associated antigen 1 (LFA-1)
and CTL-associated protein 4 (CTLA-4) .
(vii) Tregs induce B7-H4 expression by APCs, a member
of the B7 family that negatively regulates T-cell
(viii) Expression of both ectoenzymes CD39 and CD173
on Tregs can hydrolyse pericellular ATP/AMP into
the cAMP or the immunosuppressive nucleoside
(ix) Binding of lymphocyte activation gene 3 (LAG3) on
Tregs to the MHC class II molecules expressed on
immature DC suppresses DC maturation .
(x) Activated Tregs, which express more high-affinity IL-
2R than conventional T cells, may absorb IL-2 from
cells that need IL-2 to survive .
However, none of these mechanisms can explain all
aspects of suppression. It is probable that various combina-
tions of several mechanisms are operating, depending on the
milieu and the type of immune responses.
4.6. Targeting Tregs. Owing to the significant role of Tregs
in the failure of immune surveillance and immunotherapy,
many attempts to deplete Tregs or inhibit their function
in cancer patients have been studied. Many of the strate-
gies to reduce Tregs target CD25, which makes up the
alpha subunit of the IL-2R, that is present on the surface
of Tregs and activated cells. An engineered recombinant
fusion protein of IL-2 and diphtheria toxin (denileukin
diftitox [Ontak]) and other CD25-directed immunotoxins
(daclizumab, LMB-2, RFT5-SMPT-dgA) have been inves-
tigated for Treg depletion, which seems to kill selectively
lymphocytes expressing the IL-2 receptor. However, early
human trials have not proven that this approach results
in tumour regression and have shown that these strategies
may not adequately deplete Foxp3+ Tregs and may also
deplete antitumour effector cells [98–101]. Other possible
approaches to reduce immunosuppression of Tregs are via
CTLA-4 blockade (e.g., ipilimumab) [102, 103], anti-GITR
agonism , and vaccination against Foxp3 , and
some other suggested approaches, such as the inhibition
of IDO, TGF-β, ectonucleotidase (expressed by Tregs and
generates immunosuppressive adenosine), or the activation
of other agents such as OX40 or Toll-like receptor 8 have not
yet proven to be beneficial. IL-7 administration was shown
to increase T cell numbers and decrease the Treg fraction in
humans ; on the contrary, other reports have shown
that IL-7 leads to the development of Tregs [107, 108]. In
conclusion, there are many conflicting results in abrogating
the action of Tregs, and thus it is unclear which approach
holds promise for cancer treatment.
development and functionality of the Tregs ; the
mechanism behind this effect, however, is not completely
understood. We investigated the effect of CTX on immune
suppression, and the combination of CTX and DC-based
immunotherapy was studied in a murine MM model .
Our data showed that metronomic administration of low-
dose CTX has a strong immune-modulating effect in vivo.
This is currently tested in a clinical trial in mesothelioma
Tregs can be significantly reduced in mice with antimurine
reductions in Treg cells in the spleens and tumours. Using
these antibodies, the tumour microenvironment was
also drastically altered. This resulted in a significant
improvement of immunotherapy . Sorafenib has been
Clinical and Developmental Immunology9
Time of death
Figure 3: Tumour growth is a dynamic biologic process, that is,
the net result of cells dividing and other cells dying. Intrinsic
tumour biology, as well as extrinsic factors such as therapies,
affects the tumour’s growth rate. However, chemotherapy only
affects the tumour growth rate while it is being administered,
which may result in a dramatic but transient response. Following
discontinuation of chemotherapy, the growth rate returns to its
Immunotherapy (red line), on the other hand, can alter the biology
of the host by inducing an active antitumour immune response
including a memory response. This may not cause an immediate
or dramatic change in tumour burden, but continued cumulative
slowing pressure on tumour growth rate, especially if started early
in the disease course, which may lead to substantially longer
overall survival. The arrow indicates the initiation of treatment;
cross indicates time of death from cancer . (Figure used with
permission from author.)
proven cytotoxic for Tregs, although the pathway is not
fully understood. Sorafenib treatment is associated with a
decrease in frequency of Treg cells without influencing the
function of peripheral immune effector cells . Recently,
p300 was found to be an important target for modulation of
host Foxp3+ Treg functions, and an inhibition of p300 using
a small molecule inhibitor, C646 (p300i), impaired Foxp3
acetylation and inhibited Treg function .
4.7. Immune-Adjuvant Therapies. An alternative approach
to immunotherapy is to enhance the intrinsic activity of
the immune system. In this field, ipilimumab was proven
to be active in metastatic melanoma . Ipilimumab
is a monoclonal antibody against cytotoxic T-lymphocyte
antigen (CTLA)-4. It is normally expressed at low levels on
the surface of na¨ ıve effector T cells but is upregulated on the
cell surface when there is a long-lasting and strong stimulus
via the T cell receptor (TCR). CTLA-4 then competes with
CD28 for CD80/CD86 on APCs, effectively shutting off TCR
signalling, and thereby serves as a physiologic “brake” on
the activated immune system . Ipilimumab prevents
this feedback inhibition, resulting in an unabated immune
response against the tumour. The side effects of this therapy,
however, can be significant due to the downregulation of
tolerance to patient’s own normal tissue, and colitis is often
seen in patients . In mesothelioma, preclinical models
have been well described, and a phase II trial is currently
ongoing in Italy .
Other preclinical approaches are the Toll-like receptor
(TLR) ligands to activate DCs  or TLR7 agonist to
induce systemic, CD8+ T-cell, and type-I IFN antitumour
5.Need for RevisingResponse Evaluation
Immunotherapy represents a new class of agents in the treat-
ment of mesothelioma. As seen for Sipuleucel-T in prostate
cancer and ipilimumab in melanoma, improvement in the
overall survival of patients was seen; however, the agents
did not change initial disease progression. Even a transient
worsening of disease manifested either by progression of
known lesions or the appearance of new lesions can be seen,
before disease stabilizes or tumour regresses.
Commonly accepted treatment paradigm, however, sug-
gests that treatments should initially decrease tumour
volume, which can be measured using CT scan. Also,
progression-free survival is increasingly used as an alterna-
tive end-point of studies. This seems to be unfortunate for
immunotherapy, which may initiate an immune response
that ultimately slows the tumour growth rate, resulting in
longer survival, but not a decrease in tumour volume on CT
or an increased progression-free survival (Figure 3). Future
trials are currently planned to investigate these hypotheses;
however, clinicians at this moment may need to recon-
sider how to measure success of their immunotherapeutic
In conclusion, the role of the immune system in mesothe-
lioma is vast. The tumour uses villainous tricks to evade
system. Immunotherapy tries to modulate this immune
system to strengthen the antitumour effect, which is unfor-
tunately hampered by these defence mechanisms from the
tumour. At this moment, MSDCs, TAMs, and Tregs seem to
be the key players in this process, but undoubtedly extended
research will eventually unravel this complex interplay of
cells and will reveal more cell types and/or subtypes.
Targeting these defence mechanisms could be the key to
fully unleash the potential of immunotherapy. Since several
cell types are responsible for tumour survival, probably
combination therapy targeting multiple cell types will be
necessary. It is thrilling that the immunotherapy has been
established in several tumour types as a proven therapy in
recent years and that many trials are ongoing with promising
results. In mesothelioma, the first steps have been made,
and, using the accumulating knowledge, immunotherapy
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