Primary tumours are currently treated by a combina-
tion of therapies, in most cases including surgery, local
radiotherapy and chemotherapy. Even when the tumour
has apparently been defeated, micrometastases of dor-
mant tumour cells (or cancer stem cells) frequently lead to
tumour relapse and therapeutic failure. To win the fight
against cancer, it is necessary not only to develop strate-
gies to kill all cancer (stem) cells efficiently, by using the
correct combination and schedule of chemotherapeutic
agents, but also to attempt to stimulate an immune
response so that the immune system can keep residual
tumour cells in check.
Cancer is widely considered to be a cell-autonomous
genetic disease that results from alterations in oncogenes,
tumour-suppressor genes and genome-stability genes.
However, the tumour-cell microenvironment, the stroma
and immunity also have a major role in cancer. Indeed,
for the development of full-blown neoplasia, cancer cells
have to overcome intrinsic (cell autonomous) and extrin-
sic (immune mediated) barriers to oncogenesis1. Only
when tumour cells overcome immune control can they
progress and kill the host. Accordingly, the increased
incidence of some solid tumours in immunosuppressed
patients, reports of spontaneous tumour regression,
and the positive prognostic impact of tumour-specific
cytotoxic T lymphocytes (CTLs) or antibodies support
the idea that the immune system has an effect on tumour
progression in humans1.
For historical reasons, drug discovery programmes
for cancer therapy have neglected the possibility that
immune reactions might contribute to the efficacy of
treatment. For example, since 1976, the National Cancer
Institute, USA, has used a drug screening and validation
strategy in which human tumour cells are xenotrans-
planted into immunodeficient mice2 (Timeline). Although
cancer chemotherapy and radiotherapy is often viewed
as a strategy that mainly affects tumour cells, accumulat-
ing evidence indicates that cytotoxic drugs also affect the
immune system to contribute to tumour regression.
In this Review, we summarize current knowledge on
the contribution of the immune system to conventional
cancer therapies. The immune system is elicited in
two ways by conventional therapies. Some therapeutic
programmes can elicit specific cellular responses that
render tumour-cell death immunogenic. Other drugs
may have side effects that stimulate the immune system,
through transient lymphodepletion, by the subversion
of immunosuppressive mechanisms or through direct
or indirect stimulatory effects on immune effectors.
Moreover, vaccination against cancer-specific antigens
can sensitize the tumour to subsequent chemotherapeu-
tic treatment. The challenge is to hijack the host immune
system so that it can control any residual disease, or as
stated by Prendergast and Jaffee, to stop “segregating
cancer immunology from cancer genetics and cell biol-
ogy”3. We anticipate that the physician of the future will
need to integrate parameters that pertain to the host and
the tumour in order to provide optimal management of
Theoretical bases for antagonistic effects
Many of the therapeutic procedures that are used in
oncology today can curtail the immune response against
tumour cells. For example, although it is well established
*INSERM, U805, Institut
Gustave Roussy, 39 Rue
Camille Desmoulins, F‑94805
Villejuif, Paris, France.
‡CIC BT507, Institut Gustave
Roussy, 39 Rue Camille
Desmoulins, F‑94805 Villejuif,
§Faculté de Médecine Paris
Sud‑Université Paris XI,
63 Rue Gabriel Péri, 94276
Le Kremlin Bicêtre Cedex,
Leclerc, 1 Rue du Professeur
Marion, 21000 Dijon, France.
¶INSERM, U848, Institut
Gustave Roussy, Pavillon de
Recherche 1, 39 Rue Camille
Villejuif, Paris, France.
Correspondence to L.Z.
Cancer stem cells
A small population of
undifferentiated cells from
which the differentiating cancer
cells originate. These cells are
suspected to account for
relapse after conventional
Immunological aspects of cancer
Laurence Zitvogel*‡§, Lionel Apetoh*§, François Ghiringhelli*‡§|| and
Abstract | Accumulating evidence indicates that the innate and adaptive immune systems
make a crucial contribution to the antitumour effects of conventional chemotherapy-based
and radiotherapy-based cancer treatments. Moreover, the molecular and cellular bases of
the immunogenicity of cell death that is induced by cytotoxic agents are being progressively
unravelled, challenging the guidelines that currently govern the development of anticancer
drugs. Here, we review the immunological aspects of conventional cancer treatments and
propose that future successes in the fight against cancer will rely on the development and
clinical application of combined chemo- and immunotherapies.
NATURE REvIEWS | immunology?
vOLUME 8 | JANUARy 2008 | 59
© 2008 Nature Publishing Group
A gene of which the
overexpression or gain-of-
function mutation contributes
A gene that, when eliminated
or inactivated, is permissive for
the development of cancers.
These genes often determine
cell-cycle checkpoints or
facilitate induction of
programmed cell death.
Genes that control cell-cycle
advancement and/or DnA
repair to allow for the
maintenance of genome
From the Greek for ‘new
formations’. new growths or
tumours, which can be
Host receptors (such as Toll-like
receptors) that are able to
molecular patterns and initiate
signalling cascades (involving
activation of nuclear factor-κB)
that lead to an innate immune
from animal studies that the lymph nodes provide the
optimal environment for T-cell priming, surgical oncolo-
gists frequently re-sect the tumour-draining lymph
nodes, with the goal of performing histological staging
of the tumour and removing local metastases4. More
importantly, many chemotherapeutic drugs have notable
immunosuppressive side effects, either directly, by inhib-
iting or killing effector cells, or indirectly, by provoking
anergy or immune paralysis (TABle 1).
Immunosuppressive side effects of chemotherapeutics.
Several of the cancer chemotherapeutics that are used
today are also used as immunosuppressants for the
treatment of severe systemic autoimmune diseases.
This applies to cyclophosphamide5 and methotrexate6
(BOX 1), which impair the proliferative and/or effector
functions of peripheral T cells. Protein tyrosine kinase
inhibitors may also affect the T-cell arm of adaptive
immunity. At high doses, imatinib mesylate (Gleevec;
Novartis), which specifically blocks signalling through
the receptors KIT, c-ABL (cellular Abelson leukaemia-
virus protein) and its oncogenic fusion derivative
BCR (B-cell receptor)–ABL, suppresses T-cell prolif-
eration and activation, presumably through inhibi-
tion of the protein tyrosine kinase LCK7. Studies that
were carried out in mouse models have revealed that
imatinib mesylate selectively curtails the expansion
of memory CTLs, but does not affect primary T- and
B-cell responses8. Treatment of patients with imatinib
mesylate may also suppress the graft-versus-leukaemia
effect from allogeneic transplantations and increase
susceptibility to viral and bacterial infections9.
Owing to their capacity to trigger lymphocyte apop-
tosis, glucocorticoids are also important components of
the chemotherapeutic cocktails that are used in treat-
ing several lymphoproliferative diseases. High doses of
glucocorticoids are prescribed to patients with cancer to
attenuate chemotherapy-associated nausea and vomit-
ing. Glucocorticoids suppress the production of pro-
inflammatory cytokines (such as interferon-α (IFNα),
IFNβ, interleukin-1α (IL-1α) and IL-1β) and chem-
okines (such as CXC-chemokine ligand 8 (CXCL8),
CC-chemokine ligand 7 (CCL7), CCL8, CCL11, CCL13,
CCL17, CCL19 and CCL20) by blood mononuclear cells
from healthy donors10. Although glucocorticoids induce
the expression of the pattern-recognition receptors Toll-like
receptor 2 (TLR2) and TLR4, they severely impair the dif-
ferentiation of and antigen presentation by dendritic cells
(DCs) in vitro and in vivo11. Moreover, glucocorticoids
are known to repress the expression of genes that are
associated with the adaptive immune response (includ-
ing those that encode components of the MHC class II
machinery, the T-cell receptor (TCR) genes TCRA,
TCRB, TCRD and TCRG, signal transducer and activa-
tor of transcription 1 (STAT1) and CD40), alter T-cell
development and function, suppress the development
of TH1 cells (T helper 1 cells) and bias responses towards
the TH2-cell type, thereby precluding the elicitation of
effector and memory antitumour immunity. Several
members of the transforming growth factor-β (TGFβ)
family that suppress T-cell and natural killer (NK)-cell
effector functions are upregulated by glucocorticoids.
Glucocorticoids inhibit the cell-surface expression of the
main natural cytotoxicity receptors (NKp30 and NKp44)
and reduce IL-2- and IL-15-triggered NK-cell prolifera-
tion while compromising the NK-cell cytotoxicity that is
mediated by the NKp46, nKG2D (natural-killer group 2,
member D) or 2B4 receptors12.
Timeline | Historical evolution of anticancer drug development and the concept of tumour immunosurveillance
First demonstration of the
antitumour effects of
nitrogen mustard in non-
Paul Ehrlich’s theory
Creation of the National Cancer
Chemotherapy Service Center:
establishment of mouse models
for in vivo drug screening.
FDA approval of
New NCI guidelines
for drug screening
of human cell lines into
FDA approval of
Sir F. Macfarlane Burnet’s
hypothesis of cancer
First use of
chemotherapy as an
Observation that the incidence
of chemically induced tumours
in wild-type and athymic nude
mice is comparable.
animals show higher
Black boxes indicate events and milestones that contributed in the clinical developmental programme of anticancer agents. Blue boxes indicate events identifying immune
effectors in the control of spontaneous or carcinogen-induced tumours. *Taxol; Bristol–Myers Squibb. ‡Gleevec; Novartis. §Avastin; Genentech/Roche. IINexavar; Onyx/Bayer.
DC, dendritic cell; FDA, Food and Drug Administration; IFNg, interferon-g; NK cell, natural killer cell; NCI, National Cancer Institute; Rag2, recombination-activating gene 2.
shown to be susceptible
T-cell transfer shown to lead
to tumour eradication28.
pathway shown to
protect mice from
1909 1942 1955 1957 1959 1974 1976 1977 1978 1982 1992 1996 1998 2001 2004 2006
Rag2–/– mice show
susceptibility to spontaneous
First use of molecular-targeted
protein tyrosine kinase inhibitor
Side effects of imatinib mesylate shown
to trigger DC-mediated NK-cell
activation and NK-cell-based tumour
First use of anti-angiogenic
Memory T cells in human
shown to predict clinical
SorafenibII in metastatic
FDA approval of
60 | JANUARy 2008 | vOLUME 8
© 2008 Nature Publishing Group
(T helper 1 cells). Among
the two well-described subsets
of activated CD4+ T cells,
TH1 cells produce interferon-g
and tumour-necrosis factor
and enhance cell-mediated
immunity. TH2 cells produce
interleukin-4 (il-4), il-5 and
il-13, supporting humoral
immunity and counteracting
(natural-killer group 2,
member D). A lectin-type
activating receptor encoded by
the natural killer (nK)-cell gene
complex and expressed on the
surface of nK, nKT, gδ T cells
and some cytolytic CD8+ αβ
T cells. nKG2D ligands are
related sequence A (miCA) and
miCB in humans, as well as
retinoic acid early transcript 1
(RAe1) and H60 in mice. Such
ligands are generally expressed
at the cell surface of infected,
stressed or transformed cells.
(eR stress). A response by the
eR that results in the disruption
of protein folding and in the
accumulation of unfolded
proteins in the eR.
A signal emitted by dying cells
to facilitate their recognition
and phagocytosis by
neighbouring healthy cells.
Isolated limb perfusion
(ilP). A surgical technique
consisting of injection of
chemotherapeutic agents into
the artery of an extremity while
the venous outflow is
recovered, thus avoiding
systemic drug effects.
Cell-death modalities: apoptosis and tolerance.
Chemotherapy-induced tumour-cell death occurs
frequently (but not exclusively) through apoptosis, a
cell-death modality that is reputedly non-immunogenic
or tolerogenic (BOX 2). Based on the assumption that
apoptosis, a morphologically defined phenomenon (with
nuclear condensation and fragmentation as prominent
hallmarks), is biochemically homogeneous, it is assumed
that chemotherapy would elicit non-immunogenic cell
death. Accordingly, cancer cells that are treated with
mitomycin C, which induces classical apoptosis, fail
to elicit an immune response when injected subcutan-
eously into histocompatible hosts in the absence of
adjuvant. Similarly, 20 apoptosis-inducing compounds
that operate through distinct modes of action failed to
induce immunogenic cancer-cell death. This applies
to drugs that kill cancer cells through mitochondrial,
lysosomal or endoplasmic reticulum stress (ER stress), as
well as protein tyrosine kinase inhibitors, proteasome
inhibitors or DNA-damaging agents (such as alkylating
agents or topoisomerase inhibition)13. One explanation
of the non-immunogenic nature of apoptosis resides in
the way in which cell corpses are handled by the immune
system. For example, apoptosis is accompanied by the
exposure of phosphatidylserine in the outer leaflet of
the plasma membrane. Phosphatidylserine exposure
functions as an ‘eat-me’ signal, which triggers phagocy-
tosis by macrophages, and stimulates the production of
anti-inflammatory cytokines. Likewise, the standard
apoptotic programme is linked to the absence of immuno-
stimulatory signals, as discussed in a later section.
Immunosuppression by massive tumour-antigen
release. The sudden and systemic release of numer-
ous dying tumour cells resulting from chemotherapy
might have deleterious consequences on subsequent
tumour-specific immune responses. Regionally
advanced melanomas of the limbs can be treated by
isolated limb perfusion (ILP) with tumour-necrosis
factor (TNF) and the drug melphalan (Alkeran;
Celgene) (BOX 1). This results in local responses that are
up to 80% complete, but does not prolong the survival
of the patients14. So, despite the marked cell death that
is induced by vasodilatation (vasoplegia), the induc-
tion of pro-inflammatory mediators, the expression of
the adhesion molecules on endothelial cells, and the
recruitment of lymphocytes, granulocytes and macro-
phages into infused tumour beds15, these patients fail
to mount a protective immune response. Instead, pro-
inflammatory cytokines (such as IL-1β, TNF and IL-6)
that are produced by either the tumour or host immune
cells may promote tumour progression through a
molecular signalling pathway that involves nuclear
In addition, although this is theoretical, the massive
release of tumour antigens during ILP may cause a sort
of high-dose antigen-mediated tolerance. Indeed, the
delivery of large amounts of antigen could be deleteri-
ous for mounting reactive effector T cells. It has been
shown that there is a direct correlation between the
amount of antigens that are expressed in the periphery
and the degree of T-cell proliferation and number of
tolerogenic antigen-specific CD8+ T cells in the drain-
ing lymph nodes17. Studies to evaluate melanoma-
specific CD8+ T-cell responses after ILP are underway
and may confirm these hypotheses.
Therefore, owing to their chemical nature (for
example, as alkylating agents and glucocorticoids),
their mode of action (for example, in which tumour-
cell death is not preceded by cellular stress, and vaso-
plegia) or their handling (for example, their dosing
and route of administration), several components
of the therapeutic arsenal can be detrimental to the
Accumulating evidence has indicated that radiotherapy
and some cytotoxic compounds promote specific
Table 1 | Deleterious effects of cytotoxic compounds
High doses of alkylating
agents and/or methotrexate
Upregulation of TGFβ production
Impairment of NK-cell functions
Suppression of production of pro-inflammatory cytokines and chemokines
Impairment of DC differentiation or activation
Removal of lymph nodes
Alteration of TCR-mediated T-cell activation
Impairment of CD8+ T-cell memory
Induction of tolerance by high doses of antigen?
Inhibition of T-cell and NK-cell proliferation and activation
*Gleevec; Novartis. DC, dendritic cell; ILP, isolated limb perfusion; NK, natural killer; TCR, T-cell receptor; TGFβ, transforming
growth factor-β; TH2, T helper 2; TNF, tumour-necrosis factor.
High doses of TNF (ILP)
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A major transcription factor
that is activated by numerous
genotoxic insults to induce
cell-cycle arrest, cellular
senescence or apoptosis.
p53 is frequently mutated or
functionally inactivated in
A cellular response that is
usually elicited by DnA-
damaging agents (such as
ionizing irradiation or
mutagenic chemicals) and
involves the activation of
DnA-damage foci (with
H2AX as a hallmark). The
DnA-damage response elicits
cell-cycle arrest, DnA repair
(natural-killer T cells).
A heterogeneous subset of
T cells that are characterized
by the co-expression of semi-
invariant T-cell receptor (TCR)
α-chains together with nK-cell
A nearly irreversible stage of
permanent G1 cell-cycle arrest,
linked to morphological
changes (flattening of the cells),
metabolic changes and
changes in gene expression
(with expression of senescence-
the induction of which depends
on p53 and cell-cycle-blockers
such as p21 and p16.
Distant antitumour effects seen
after local radiation therapy.
anticancer immune responses that contribute to the
therapeutic effects of conventional therapy.
DNA damage: a fast track to immunity? DNA-
damaging agents, such as ionizing irradiation or topo-
isomerase inhibitors, stimulate a complex response
that involves the activation of tumour-suppressor
proteins, such as the protein kinases ATM (ataxia-
telangiectasia mutated) and CHK1 (checkpoint
kinase 1), as well as the transcription factor p53.
This DnA-damage response induces the expression of
NKG2D ligands on tumour cells in an ATM-depend-
ent and CHK1-dependent (but p53-independent)
manner18. NKG2D is an activating receptor that is
involved in tumour immunosurveillance by NK cells,
nKT cells, gδ T cells and resting (in mice) and/or acti-
vated (in humans) CD8+ T cells.
Although p53 is not required for the expression of
NKG2D ligands in cells undergoing DNA damage, a
recent study has highlighted an important cooperation
between p53-induced tumour-cell senescence and the
innate immune system19. It was found that restora-
tion of p53 function in established liver cancers led
to tumour regression, but only when the mice had
functional NK cells, neutrophils and macrophages.
Reactivation of p53 in hepatocellular cancers induced
the expression of pro-inflammatory cytokines
(such as IL-15 and macrophage colony-stimulating
factor (M-CSF)), adhesion molecules (such as inter-
cellular adhesion molecule 1 (ICAM1) and vascular cell-
adhesion molecule 1 (vCAM1)) and chemokines (such
as CCL2 and CXCL1), which could contribute to the
p53-induced recruitment of neutrophils, macrophages
and NK cells into tumour beds19. This example illus-
trates how molecules that are activated during the
DNA-damage response (ATM, CHK1 and p53) can
alert the innate immune system to mediate an anti-
Distant effects of radiotherapy. Besides its direct
cytotoxic properties towards tumour cells, irradiation
mediates many effects on cells and tissues, some of
which may stimulate an immune response. Low doses
of ionizing irradiation upregulate the expression of
MHC class I molecules, tumour-associated antigens
(carcinoembryonic antigen and mucin 1)20 and CD95
(also known as FAS) by tumour cells, as well as adhe-
sion molecules by endothelial cells21, thereby boosting
CTL activity22 and T-cell trafficking towards irradiated
tumour sites23. Local irradiation of a single tumour
site can reduce the size of non-irradiated metastases
that are located at a distant site, a phenomenon known
as the ‘abscopal effect’, when mediated by the immune
system. In an elegant study, Reits et al.24 showed that
irradiation enhances tumour antigenicity in three
ways by modulating the repertoire of tumour-derived
peptides that are presented to the immune system.
First, irradiation enhances the degradation of existing
proteins, thereby increasing the intracellular pool of
peptides for MHC class I presentation. Second, activa-
tion of mammalian target of rapamycin (mTOR) in
irradiated tumour cells stimulates protein translation
and increases peptide production. Third, irradiation
stimulates the synthesis of new proteins and therefore
antigenic peptides, which can be presented for rec-
ognition by different T-cell repertoires24. Moreover,
Box 1 | Properties of cytotoxic compounds
Cytotoxic compounds that are used for the treatment of neoplastic diseases and that are discussed in this Review can be
classified into several groups.
Antimetabolites. These include 5‑fluorouracil, which is a fluoropyrimidine analogue of the endogenous
pyrimidine nucleoside that perturbs RNA and DNA synthesis. Gemcitabine (Gemzar; Eli Lilly) is another
synthetic pyrimidine nucleoside analogue. Another antimetabolite, methotrexate, inhibits dihydrofolate
reductase, thereby depleting cells of reduced folates, which are required for DNA synthesis.
Alkylating agents. These?include melphalan (Alkeran; Celgene) and cyclophosphamide, which is chemically related to
nitrogen mustard. In the liver, cyclophosphamide is converted to the active metabolites aldophosphamide and
phosphoramide mustard, which bind to DNA, thereby inhibiting DNA replication and initiating cell death.
Anthracyclines. These are chemotherapeutic agents that are based on samine and tetrahydronaphthacenedione.
Anthracyclines inhibit DNA and RNA synthesis by intercalating between base pairs of the DNA or RNA strand, thereby
preventing the replication of rapidly growing cancer cells. They also create iron‑mediated free oxygen radicals that
damage the DNA and cell membranes.
DNA methyltransferase inhibitors. 2′‑deoxy‑5‑azacytidine (DAC) is a chemical analogue of cytidine nucleoside. The
incorporation of DAC into DNA or RNA inhibits DNA methyltransferase, thereby causing demethylation, which in turn
leads to the reactivation of silenced genes, including some tumour‑suppressor genes.
Platinum compounds. These?include cisplatin and oxaliplatin (Eloxatin; Sanofi–Aventis). Oxaliplatin is a
diaminocyclohexane platinum agent that is active against human colon cancer and is synergistic with 5‑fluorouracil.
Active oxaliplatin derivatives form both inter‑ and intra‑strand platinum–DNA crosslinks, which results in inhibition of
DNA replication and transcription.
Spindle poisons. These include the taxanes paclitaxel (Taxol; Bristol‑Myers Squibb) and docetaxel (Taxotere; Sanofi–
Aventis). Their main mechanism is the stabilization of GDP‑bound tubulin in microtubules. Microtubules are essential to
cell division, and taxanes therefore stop cell division — a ‘frozen mitosis’. In contrast to the taxanes, the vinca alkaloids
destroy mitotic spindles.
62 | JANUARy 2008 | vOLUME 8
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(Regulatory T cell).
A specialized type of CD4+
T cell that can suppress the
responses of other T cells.
These cells provide a
crucial mechanism for the
maintenance of peripheral
self-tolerance and are
characterized by the
expression of CD25 (also
known as the α-chain of the
interleukin-2 receptor) and
the transcription factor
forkhead box P3 (FOXP3).
potent synergistic effects against established tumours
between passively transferred CTLs24 or TLR9 lig-
ands25 and ionizing radiation have been reported24.
High doses of alkylating agents for lymphoablation.
The therapeutic induction of lymphopaenia has raised
considerable interest in the context of adoptive trans-
fer therapies and vaccination against melanomas26.
Transient lymphopaenia is thought to enhance the
efficiency of these therapies by activating homeo-
static mechanisms that stimulate the tumour-reactive
effector T cells and by counteracting tumour-induced
suppression. Common methods to induce lym-
phopaenia include low-dose total body irradiation
for reversible myelosuppression, or treatment with
cyclophosphamide alone or in combination with
fludarabine, which promotes long-term lymphopaenia
in humans27. In addition to eradicating the cells that
may suppress antitumour responses, such as regula-
tory T cells28, lymphoid reconstitution could overcome
cancer-induced defects in T-cell signalling, endow host
antigen-presenting cells with co-stimulatory func-
tions, and increase the production and availability of
cytokines, such as IL-7, IL-15 and IL-21, resulting in
enhanced CD8+ T-cell activity28. Lymphodepletion also
enhances T-cell homing into tumour beds and intra-
tumoral proliferation of effector cells27,29. Finally, total
body irradiation could cause mucosal barrier injury,
resulting in microbial translocation and systemic
release of the TLR4 ligand lipopolysaccharide (LPS),
which has been shown to increase DC and T-cell acti-
vation and to promote tumour regression30. Animal
studies have shown that lymphoablation enhances the
effectiveness of adoptively transferred tumour-specific
CD8+ T cells31. This strategy has been successful in a
clinical trial that involved 35 patients with advanced
metastatic melanomas that were refractory to conven-
Lymphodepletion can also be combined with vac-
cination strategies that promote the differentiation
of central memory T cells that recognize self tumour
antigens32,33. Moreover, lethal myeloablation followed
by autologous haematopoietic stem cell (HSC) rescue
further enhances the efficacy of adoptive transfer thera-
pies for tumours33. Profound lymphodepletion results in
the expansion of both adoptively transferred T cells and
host naive T cells.
So, chemotherapy-induced or irradiation-induced
lymphodepletion, combined with additional manipu-
lations, such as adoptive cell transfer, vaccination or
HSC rescue, may be useful approaches for generating
antitumour immune responses.
Low-dose cyclophosphamide for suppression of regula-
tory T cells. Although high doses of cyclophosphamide
have direct tumoricidal effects and cause immuno-
suppression by lymphoablation (see above), low doses
of the same compound mediate immunostimulation.
Low doses of cyclophosphamide can potentiate delayed-
type hypersensitivity (DTH) responses34 by acting on a
cyclophosphamide-sensitive regulatory T-cell subset28.
Low doses of cyclophosphamide decrease the number
and inhibitory function of CD4+CD25+ regulatory
T cells (TReg cells) by downregulating the expression of key
functional markers of TReg cells, forkhead box P3 (FOXP3)
and glucocorticoid-induced TNF-receptor-related protein
(GITR)35. The effects of cyclophosphamide on TReg cells
and cyclophosphamide-stimulated IFNα production36
might account for the augmented antibody responses and
the persistence of memory T cells. All these effects con-
tribute to the eradication of immunogenic tumours in
synergy with specific immunotherapies37,38. However,
Box 2 | Death modalities and immunogenicity
Cancer cells can respond to therapy either by differentiation (which is often an irreversible process linked to proliferative
arrest and apoptotic removal), proliferative arrest (which in the case of senescence is indefinite) or cell death.
Tumour‑cell demise often occurs through apoptosis, a stereotyped pattern of morphological changes that involves
chromatin condensation (pyknosis), nuclear fragmentation (karyorhexis), shrinkage of the cytoplasm, blebbing of the
plasma membrane, and final disintegration of the cell into membrane‑surrounded apoptotic bodies. These changes are
rarely seen in vivo, because dying cells are recognized and engulfed by neighbouring cells before they enter the late
stages of the apoptotic process. In some cases, tumours can also die by necrosis (characterized by swelling of the cell and
the cytoplasmic organelles before the plasma membrane ruptures and the cellular content is spilled into the intercellular
space), by autophagy (sequestration of large parts of the cytoplasm in autophagic vacuoles, often before the cells
undergo apoptosis), or as a result of mitotic catastrophe (morphologically discernible by multi‑ or micronucleation
and/or mitotic arrest before apoptosis)95. Attempts to correlate these morphological death modalities with the absence
or presence of immunogenicity have largely failed96.
Although there are teleological arguments that propose that apoptosis must be non‑immunogenic or even tolerogenic
(because physiological cell death occurs through apoptosis, but does not lead to autoimmunity), and although necrosis
(which is often pathological) has been condemned as pro‑inflammatory, the theoretical equations in which apoptosis
equals non‑immunogenic and necrosis equals immunogenic do not withstand experimental verification, at least in
models of tumour vaccination. Rather, it seems that apoptosis is non‑uniform in biochemical terms, meaning that various
pathways can lead to cell death and induce stimulus‑specific changes (for example, in the composition of the proteome
that is associated with the plasma membrane). Moreover, the apoptotic execution phase can involve a variable degree of
caspase‑dependent and caspase‑independent catabolic reactions, meaning that similar morphologies may have been
acquired through distinct biochemical routes. As a result, the classification of cell‑death modalities must await further
refinement (with the progressive substitution of morphological characteristics by biochemical criteria) before cell‑death
types can be unambiguously correlated with immunogenicity or its absence.
NATURE REvIEWS | immunology?
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(Human epidermal growth-
factor receptor 2). A receptor
protein-tyrosine kinase that is
overexpressed in a subset of
human breast cancers.
3LL tumour model
3ll is a non-small-cell lung
cancer cell line that grows
in vivo after injection into
C57Bl/6 syngenic hosts.
Myeloid suppressor cells
A group of immature
CD11b+GR1+ cells (which
include precursors of
dendritic cells and myeloid
cells) that are produced in
response to various tumour-
derived cytokines. These cells
have been shown to induce
specific CD8+ T-cell tolerance.
the ablation of regulatory cells is likely to be of varying
importance, depending on the tumour type, stage and
In small clinical studies, the combination of low
doses of intravenous cyclophosphamide with vac-
cines has been shown to augment DTH responses40,41,
decrease the proportion of CD4+2H4+ (CD45+) sup-
pressor T cells42 and prolong the survival of patients
with metastatic cancer40. A daily dose of oral cyclo-
phosphamide, also referred to as a metronomic pro-
gramme, given for 1 month to patients with end-stage
cancer could suppress TReg-cell inhibitory functions,
restore the proliferative capacity of effector T cells
and restore the cytotoxicity of NK cells43,44. However,
the optimized schedule of cyclophosphamide yielding
immunostimulatory effects in patients with cancer
awaits further randomized investigation.
Anthracyclines as immunostimulators? Immune modu-
lation by the anthracycline doxorubicin has been studied
for decades45,46. Recently, it has been shown that doxoru-
bicin enhances the antitumour potency of the GM-CSF-
transfected tumour-cell vaccine when given before or
after immunization47. The antileukaemic effects of
a combination of IL-12 and various cytotoxic agents
(cisplatin, cyclophosphamide, paclitaxel or doxoru-
bicin) were evaluated in immunocompetent mice
with syngeneic L1210 leukaemia cells, and it was
demonstrated that significantly prolonged survival of
the mice is only achieved with a combination of IL-12
and doxorubicin48. The therapeutic effect of IL-12 plus
doxorubicin was lost when the mice were irradiated
or injected with silica, indicating that radiosensitive
immune cells (possibly T cells) and macrophages
are required for the therapeutic effect. As a possible
mechanism to account for the immune-mediated
anticancer effects of anthracylines, it has recently been
discovered that anthracyclines — unlike many other
cytotoxic agents — can elicit immunogenic cell death
Taxanes: TLR4 ligands and beyond. The taxanes
paclitaxel (Taxol; Bristol–Myers Squibb) and docetaxel
(Taxotere; Sanofi–Aventis) both bind to the β-subunit
of tubulin and affect microtubule polymerization, lead-
ing to cell-cycle arrest at the G2/M stage and subsequent
cell death. Paclitaxel binds to mouse TLR4 (but not to
human TLR4) and so can mimic bacterial LPS by acti-
vating mouse macrophages and DCs through a pathway
that requires TLR4, MD2 (a key component of the LPS
receptor complex) and myeloid differentiation primary-
response gene 88 (MyD88)50.
In mice that are transgenic for human epidermal
growth-factor receptor 2 (HeR2; also known as NEU
and ERBB2), which is overexpressed in some breast
cancers, and that are therefore tolerant to HER2, treat-
ment with paclitaxel increases the efficacy of tumour
vaccines that express HER2 and GM-CSF51. Similarly,
mice that have a mammary adenocarcinoma trans-
fected with a candidate tumour antigen respond more
efficiently to intratumour DC inoculations when
they receive paclitaxel52. The optimal scheduling
of docetaxel treatment combined with a GM-CSF-
producing tumour vaccine was assessed in the 3ll
tumour model53. When mice that have established 3LL
tumours were pre-treated with docetaxel, followed
by vaccination with irradiated GM-CSF-transfected
3LL tumour cells, significant tumour regression and
prolonged survival were observed in comparison with
chemotherapy alone. If docetaxel was administered after
tumour-cell vaccination, treatment with taxane abro-
gated the antitumour effects of the vaccine, presumably
by negatively affecting the dividing effector T cells. Mice
that survived the treatment developed memory CTLs
specific for a 3LL epitope (MUT-1) and resisted rechal-
lenge with live 3LL tumour cells. In humans, there is
circumstantial evidence that taxanes can stimulate the
anticancer immune response. T-cell proliferation and
NK-cell cytolysis assays have revealed that a cohort of
patients with breast cancer (stage II/III) treated with
taxanes showed enhanced T-cell and NK-cell func-
tions compared with patients treated without taxanes54.
Therefore, experimental data obtained in mice and
humans contravene traditional thinking that taxanes
suppress immune-cell functions.
Multiple immunostimulatory effects of gemcitabine.
Gemcitabine (Gemzar; Celgene) is a synthetic pyrimidine
nucleoside analogue that is efficient in the treatment of
pancreatic, breast and lung cancers (BOX 1). Its phospho-
rylated metabolites inhibit ribonucleotide reductase and
DNA polymerase-α, which results in the depletion of the
deoxynucleotide pool, thereby stopping DNA synthesis.
Gemcitabine inhibits B-cell proliferation and antibody
production in response to tumour antigens55, a phenom-
enon that may skew antitumour immunity towards bene-
ficial T-cell responses56. Moreover, gemcitabine reduces
the frequency of CD11b+GR1+ myeloid suppressor cells57.
Gemcitabine-induced apoptosis of established tumours
may enhance the DC-dependent cross-presentation of
tumour antigens to T cells58. Consistent with these data,
gemcitabine can function in synergy with CD40 stimu-
lation of T cells to cure established mouse tumours59. The
immunostimulatory effects of gemcitabine have been
confirmed in patients. In patients with pancreatic60, non-
small-cell lung61 or colon62 cancer, gemcitabine combined
with recombinant cytokines or vaccines could enhance
the frequency of tumour-specific CTL precursors and
result in objective response rates.
5-Fluorouracil. 5‑Fluorouracil is a fluoropyrimidine that is
commonly used against breast cancer and gastrointestinal
malignancies (BOX 1). In vitro, 5-fluorouracil induces the
expression of heat-shock proteins (HSPs) in tumour cells
and facilitates antigen uptake by DCs and subsequent
cross-presentation of tumour antigens63. Human colon
carcinoma cell lines that were treated with 5-fluorouracil
acquired CD95 and ICAM1 expression and became more
sensitive to lysis by CTLs. In a mouse model, intratumoral
inoculation of DCs after systemic chemotherapy that was
based on 5-fluorouracil induced the T-cell-dependent
eradication of the DC-treated site and, importantly, also of
64 | JANUARy 2008 | vOLUME 8
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A pre-neoplastic syndrome
characterized by a
hypercellular bone marrow
with reduced haematopoietic
in contrast to anti-angiogenic
approaches, which aim to
prevent the neovascularization
processes in tumours, vascular-
disrupting agents aim to cause
the rapid and selective
shutdown of the established
tumour vasculature, leading to
secondary tumour-cell death.
The development of new
blood vessels from
existing blood vessels. it is
frequently associated with
tumour development and
dendritic cells). isolated in
mouse models only, these cells
express both nK-cell and B-cell
markers but lack plasmacytoid
DC- and T-cell-specific, and
They react to a large variety
of tumour cells by producing
interferon-g and killing the
tumour cells without
A family of cell-surface
receptors capable of mediating
cell death on ligand-induced
trimerization. The best-studied
members include tumour-
necrosis factor receptor 1
(TnFR1), FAS (or CD95, which
binds FAS ligand) and two
receptors for TnF-related
(TRAilR1 and TRAilR2).
tumours located at a distant site, resulting in the long-term
survival of the mice. Such effects can only be obtained by
treatment with a combination of DCs and 5-fluorouracil,
not with each treatment alone64. In another model,
5-fluorouracil was also able to augment the immuniz-
ing efficacy of a peptide-based vaccine directed against
thymidilate synthase, its own molecular target65. These
mouse data have prompted the design of a sizeable
randomized clinical trial in patients with metastatic colon
cancer (X. Cao, personal communication).
5-Aza-2′-deoxycitidine. The DNA methyltransferase
inhibitor 5-aza-2′-deoxycitidine (DAC) is used in the
treatment of leukaemias and myelodysplastic syndrome66.
The antineoplastic effect of DAC is thought to involve
reversing the hypermethylation of cancer-associated
promoters, which inhibits gene transcription. Treatment
with DAC restores the expression of MHC class I
molecules and cancer testis antigens on tumour
cells, rendering the tumour cells susceptible to CTL
attack67. Moreover, hypermethylation of the tumour-
suppressor gene that encodes death-associated protein
kinase (DAPK) can be suppressed by treatment of
leukaemic cells with DAC, thereby restoring the IFNg-
mediated apoptotic-cell-death pathway66. Cytokines that
induce IFNg secretion could therefore potentially be in
synergy with DAC. Indeed, combining IL-12 with DAC
was synergistic in a T-cell-dependent manner against
L1210 leukaemia and B16F10 melanoma in mice68.
Antivascular flavonoids. Vascular-disrupting agents are
distinct from anti-angiogenic agents in that they target
pre-existing tumour vessels rather than neoangio-
genesis. Flavone acetic-acid derivatives are known to
have antivascular properties69. However, the flavonoid
5,6-dimethylxanthenone-4-acetic acid (DMXAA) has
been shown to have immunological side effects and
to elicit type-I-IFN-dependent NK-cell responses, as
well as CD4+ T-cell-dependent antitumour responses69.
In models of large thoracic tumours, DMXAA was able
to promote the infiltration of CD11b+ cells and CD8+
T cells into tumour beds that release immunostimulatory
cytokines and chemokines (CXCL10, RANTES, IL-6,
IFNg, TNF, CCL3, CCL2, CXCL9 and inducible nitric-
oxide synthase). As the antineoplastic effects of DMXAA
depend on perforin and CD8+ T cells, DMXAA could
induce the activation of tumour-associated macrophages
that contribute to the recruitment of other antitumour
effectors70. Recently, DMXAA has been shown to be
a novel and specific activator of the TANK-binding
kinase 1 (TBK1)–IFN-regulatory factor 3 (IRF3) signal-
ling pathway. Therefore, treatment of primary mouse
macrophages with DMXAA has resulted in robust IRF3
activation and a marked increase in the production of
the mRNA encoding IFNβ (ReF.71).
Immunostimulatory side effects of imatinib mesylate.
Imatinib mesylate is a clinically approved drug for the
treatment of gastrointestinal stromal tumours (GISTs),
which are associated with activating mutations in KIT
or PDGFRA (platelet-derived growth factor receptor-α).
This treatment is based on the rationale that imatinib
mesylate directly targets the pathognomonic mutations in
these receptor protein-tyrosine kinases. However, a sub-
set of patients with GISTs who have no KIT or PDGFRA
mutation still respond to imatinib mesylate72. These
antitumour effects were found to be related to NK-cell
activation that is promoted by the side effects of imatinib
mesylate on immune cells43,72. Indeed, imatinib mesylate
mediated NK-cell-dependent antitumour effects in vivo
in mice with cancer cells that did not respond to ima-
tinib mesylate in vitro73. Imatinib mesylate blocks KIT
signalling in host DCs, thereby triggering DC-mediated,
NK-cell activation and NK-cell-dependent antitumour
effects. In patients with GISTs, NK-cell IFNg secretion
that is induced by imatinib mesylate constitutes a positive
prognostic parameter, suggesting that NK cells also con-
tribute to the therapeutic effect of imatinib mesylate in
humans72. So, depending on the local concentrations and
the duration of exposure to imatinib mesylate, one might
expect either inhibitory functions of imatinib mesylate
on the differentiation of DCs and memory T cells or
stimulatory effects on NK cells.
In mice, the combination of imatinib mesylate plus
IL-2 induces the expansion of a unique population
of tumour-infiltrating effector cells. These effector
cells have been termed IFN-producing killer DCs
(iKDCs)74 because they share phenotypic and func-
tional properties with myeloid DCs and NK cells
and can produce IFNg. Unlike B220– NK cells, these
CD11c+B220+NK1.1+ cells can lyse various target cells
regardless of the expression of MHC class I molecules
or NKG2D ligands. IKDCs also upregulate MHC
class II expression on contact with transformed cells
and produce large amounts of IFNg, which is in con-
trast to B220– NK cells74. Adoptive transfer into B16F10
tumour-bearing immunodeficient (Rag–/–Il2rg–/–) mice
has revealed that IKDCs but not B220– NK cells sig-
nificantly delay tumour progression. Together, these
data indicate that imatinib mesylate may exert part
of its antitumour effects through the expansion and
stimulation of IKDCs.
Mechanisms of T-cell-mediated immunity
The immuno-adjuvant effects of cytotoxic com-
pounds discussed in this article may primarily rely
on the capacity of antigen-presenting cells to engulf
dying tumour cells and then to process and present
tumour antigens to naive and/or central memory
T cells. Therefore, and as previously reviewed75, the
signals that are delivered by stressed or dying tumour
or stromal cells under the influence of cytotoxic com-
pounds can be expected to regulate antigen uptake
(eat-me signals and antigen transfer), as well as antigen
processing and presentation, thereby influencing DC
maturation, co-stimulation, polarization and traffick-
ing (FiG. 1; TABle 2). In addition, stressed tumour cells
may upregulate stimulatory ligands for NK cells (for
example, NKGD2 ligands after DNA damage) or death
receptors (for example, CD95 and TRAILR), thereby
increasing their susceptibility to lysis by endogenous
immune effectors (FiG. 2; TABle 2).
NATURE REvIEWS | immunology?
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of calreticulin to
the cell surface
Release of IL-1β (?),
ATP (?), uric acid (?)
and type I IFNs (?)
Nature Reviews | Immunology
initiation of a CD8+ T-cell
response against an antigen
that is not present in antigen-
presenting cells (APCs). The
antigen must be taken up by
APCs and then re-routed to
the mHC class i presentation
Chaperones. A common response to cell stress, includ-
ing that induced by chemotherapy, is the transcriptional
activation of a series of molecular chaperones that belong
to the class of inducible HSPs. Such HSPs protect against
cell death by re-folding damaged proteins and by direct-
ing damaged proteins to proteasome-mediated degrada-
tion. In addition, they inhibit apoptosis at the pre- and
post-mitochondrial levels. HSPs can also stimulate the
immune system at several levels.
First, HSP70 and HSP90 (as well as gp96 and cal-
reticulin) can act on the scavenger receptor CD91 on
the surface of DCs, thereby transmitting a maturation
signal76. As well as binding damaged proteins, HSPs may
bind peptides, including tumour-specific antigens, and
may direct them to MHC class I and II pathways for
presentation to T cells. Purified peptide–HSP complexes
can induce protective immunity against the tumour from
which they derive77. Peptide–HSP complexes may be par-
ticularly efficient as a source of antigen for cross-priming,
as has been shown in vivo in suitable mouse models78.
Moreover, encouraging results from HSP-based vaccine
trials have been reported79.
Second, in addition to their function as vehicles for
peptide antigens, chaperones may have an important role
in DC activation as eat-me signals. For example, HSP90
appears on the surface of human myeloma cells after treat-
ment with the proteasome inhibitor bortezomib (velcade;
Millennium Pharmaceuticals)80 and then serves as a
contact-dependent activation signal for autologous DCs.
Of note, this HSP90 exposure depends on the cytotoxic
agent bortezomib and does not occur after g-irradiation
or treatment with steroids, indicating that there are
stimulus-dependent differences in the type of stress
Third, another chaperone, calreticulin, can also be
exposed on the surface of dying cells and act as an eat-
me signal for macrophages, presumably by interacting
with CD91 on the engulfing cell81. In response to some
cell-death inducers, and in particular anthracyclines
and ionizing irradiation, calreticulin can translocate
from the lumen of the ER to the surface of cells at
an early pre-apoptotic stage13,82. This early exposure
of calreticulin and its aggregation in discrete foci on
the cell surface (ecto-calreticulin) is not a general
feature of apoptosis (FiG. 1). When tumour cells are
treated for a few hours with anthracyclines and then
injected subcutaneously into mice, they are highly
efficient in inducing a specific DC and antitumour
T-cell response49. Ecto-calreticulin is crucial for the
recognition and engulfment of dying tumour cells
by DCs. Thus, anthracyclines and g-irradiation that
induce ecto-calreticulin cause immunogenic cell death,
whereas other pro-apoptotic agents (such as mitomy-
cin C and etoposide) neither induce ecto-calreticulin
nor immunogenic cell death. Depletion of calreticulin
expression by transfection with specific small interfering
RnAs (siRNAs) abolishes the immunogenicity of cell
death that is elicited by anthracyclines, whereas an
exogenous supply of calreticulin (as a recombinant
protein) or the use of pharmacological agents that
favour calreticulin translocation (such as PP1 phos-
phatase inhibitors) can enhance the immunogenicity
of cell death13,80. Such PP1 inhibitors (for example,
tautomycin and caliculin A) can increase the thera-
peutic efficacy of mitomycin C and etoposide in vivo
by eliciting a specific immune response83.
Therefore, calreticulin exposure by tumour cells may
allow prediction of the therapeutic outcome, and studies
are ongoing to apply this knowledge to human disease82.
The pharmacological reestablishment of calreticulin
Figure 1 | Sequential?events?that?link?tumour-cell?stress?with?activation?of?
antigen-presenting?cells.?Following tumour insult by cytotoxic agents, tumour cells
rapidly translocate intracellular calreticulin (CRT) to the cell surface (within 1 hour
in vitro), which acts as?a mandatory eat-me signal for dendritic cells (DCs) and induces
immunogenic cell death. By 12 hours, molecular chaperones, such as heat-shock
protein 90 (HSP90), can appear on the tumour-cell surface, contributing to tumour-cell–
DC adhesion and the first steps of DC maturation. Next, by 18 hours, the release by dying
tumour cells of the chromatin-binding protein high-mobility group box 1 protein
(HMGB1) is required for optimal Toll-like receptor 4 (TLR4)-dependent processing of the
phagocytic cargo by DCs. Finally, maturation signals (possibly type I interferons (IFNs) or
interleukin-1 (IL-1)) that are delivered by stressed tumour cells complete the maturation
programme that is elicited by the earlier events on DCs.
66 | JANUARy 2008 | vOLUME 8
© 2008 Nature Publishing Group
Small interfering RNAs
(siRnA). Synthetic RnA
molecules of 19–23
nucleotides that are used to
‘knock down’ (that is, to silence
the expression of) a specific
gene. This is known as RnA
interference (RnAi) and is
mediated by the sequence-
specific degradation of mRnA.
exposure may ameliorate the efficacy of chemotherapy,
and the development of non-toxic PP1-inhibitory
molecules that can induce calreticulin exposure is in
HMGB1 and TLR4. Therapies that are based on anthra-
cycline, irradiation and oxaliplatin (Eloxatin; Sanofi–
Aventis) are more efficient at inhibiting the growth of
established syngeneic tumours in immunocompetent
mice than in athymic (nude) littermates, indicating that an
intact immune system enhances the therapeutic efficacy
of conventional anticancer treatments84,85. This result has
prompted the search for a link between innate and adaptive
immune responses and has led to the systematic screening
of the role of TLRs in the efficacy of chemotherapy.
Surprisingly, the only TLR deficiency that compro-
mises the efficacy of chemotherapy or radiotherapy
in vivo is TLR4 and its downstream effector MyD88
(ReFS 84,85). Indeed, dying tumour cells can be cross-
presented by DCs to naive T cells in vitro and in vivo,
Table 2 | Immunostimulatory properties of cytotoxic drugs
Increased MHC class I expression
Increased tumour-associated antigen production Irradiation, DAC24,67
mTOR activation Irradiation24
HMGB1 releaseAnthracyclines, oxaliplatin‡, irradiation84
Antigen presentationDC activation (through TLR4 and MyD88)Paclitaxel§ (in mice), irradiation30,50,100
APC activation (through TBK, IRF3 and IFN)Vascular-disrupting agents71
Self-peptide–MHC class I complexes
Increased antigen cross-presentation
Myelosuppression and HSCs
Taxanes and intratumour DCs or GM-CSF-
Anthracyclines, oxaliplatin, irradiation
IL-12 with DAC
5-fluorouracil with vaccines
Low doses of total body irradiation
Cytokine sink removal?
Increased ICAM1, PECAM and VCAM1 expression
Increased RANTES and CXCL10 production
Increased CD95 expression
Increased HSP production
Decreased succinate dehydrogenase activity
Increased TRAIL expression
Self-peptide–MHC class I complexes
High doses of alkylating agents, fludarabine
Homing to tumours Irradiation
Cisplatin, irradiation, 5-fluorouracil and dacarbazine
Imatinib mesylate# with IL-2
Total body irradiation
Cyclophosphamide, fludarabine with or without
vaccines or HSCs
Generation of memory
Inhibition of regulatory
Inhibition of MSCs and B cells
Inhibition of TReg cells
Inhibition of M2 macrophages
Reduced TGFβ production
Type I IFNs
NK-cell triggeringImatinib mesylate
*Additional mechanisms are described in ReF. 75. ‡Eloxatin; Sanofi–Aventis. §Taxol; Bristol–Myers Squibb. IIVelcade; Millennium Pharmaceuticals. ¶Gemzar; Eli Lilly.
#Gleevec, Novartis. APC, antigen-presenting cell; CXCL10, CXC-chemokine ligand 10; DAC, 5-aza-2′-deoxycitidine; DC, dendritic cell; GM-CSF, granulocyte/
macrophage colony-stimulating factor; HMGB1, high-mobility group box 1 protein; HSC, haematopoietic stem cell; HSP, heat-shock protein; ICAM1, intercellular
adhesion molecule 1; IFN, interferon; IL, interleukin; IRF3, IFN-regulatory factor 3; MSCs, myeloid suppressor cells; mTOR, mammalian target of rapamycin;
NK, natural killer; PECAM, platelet/endothelial cell-adhesion molecule; TBK, TANK-binding kinase; TGFβ, transforming growth factor-β; TLR4, Toll-like receptor 4;
TReg cells, CD4+CD25+ regulatory T cells; TRAIL, tumour-necrosis-factor-related apoptosis-inducing ligand; VCAM1, vascular cell-adhesion molecule 1.
NATURE REvIEWS | immunology?
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Nature Reviews | Immunology
Lysis by activated
NK cells, NKT cells,
IKDCs, γδ T cells
and caliculin A
MHC class I
MHC class II
and they promote the differentiation of tumour-
specific CTLs only when host DCs harbour a func-
tional TLR4–MyD88 pathway. TLR4-deficient DCs
are unable to present antigen from dying cells (taken
up by phagocytosis) although they are normal in their
ability to present antigen from soluble proteins (taken
up by pinocytosis)84,85, which suggests a specific defect
in antigen presentation after phagocytosis. TLR4 has
been reported to inhibit the lysosome-dependent
degradation of phagosomes in macrophages86.
Accordingly, the kinetics of fusion between phago-
somes and lysosomes is slower in wild-type DCs than
in TLR4-deficient DCs loaded with dying tumour
cells, which suggests that the TLR4 defect causes
rapid lysosomal degradation of phagocytic material.
Antigen presentation by TLR4-deficient DCs can
be restored by inhibiting the activity of lysosomes,
either with chloroquine (a lysosomotropic alkaline)
or bafilomycin A1 (a specific inhibitor of the vacuolar
ATPase responsible for lysosomal acidification). In
addition, chloroquine can reverse the adverse effects
of TLR4 deficiency on chemotherapeutic responses
Further studies have revealed that the ligand of
TLR4 that is produced by the dying tumour cells
is high-mobility group box 1 protein (HMGB1), a
nuclear protein that is released from dying cells during
late-stage apoptosis84,85 (FiGS 1,2). Depletion of HMGB1
from dying tumour cells with siRNAs or neutraliza-
tion of HMGB1 with specific antibodies abolishes the
TLR4-dependent, DC-mediated presentation of anti-
gens from dying tumour cells in vitro and in vivo84. So,
HMGB1 release is required for the immunogenicity of
cell death through its effect on TLR4. However, neither
Figure 2 | linking?tumour-cell?stress?and?effector-cell?killing.?Tumour cells may be lysed by activated cytotoxic
cells, such as natural killer (NK) cells, NKT cells, interferon-producing killer dendritic cells (IKDCs), gδ T cells and
cytotoxic T lymphocytes (CTLs). However, this direct killing activity may be indirectly facilitated by the intervention of
various effector mechanisms that sensitize tumour cells to express stress or danger signals that promote their
recognition by particular effector cells. The DNA-damage response promotes the expression of ligands for NKG2D
(NK group 2, member D), while restoration of functional p53 might induce the recruitment and activation of innate
immune cells through the induction of CC-chemokine ligand 2 (CCL2) and interleukin-15 (IL-15). Inhibitors of histone
deacetylases (HDACs) and inhibitors of the phosphatase PP1 contribute to the expression of NKG2D ligands and of
cell-surface calreticulin, respectively, eliciting innate immune responses and phagocytosis, respectively. The release
of high-mobility group box 1 protein (HMGB1) by tumour cells can be promoted by anthracyclines, oxaliplatin and
irradiation, as well as by soluble TRAIL (tumour-necrosis factor-related apoptosis-inducing ligand) (and potentially
activated NK cells and IKDCs that express TRAIL), and is mandatory for dendritic cell (DC)-mediated cross-
presentation of apoptotic tumours to T cells. Proteasome inhibitors, such as bortezomib, induce cell-surface
expression of heat-shock protein 90 (HSP90) by tumour cells, facilitating antigen uptake and maturation of DCs. Many
chemotherapeutic agents can upregulate the expression of MHC class I molecules, tumour antigens and CD95 on
tumour cells, leading to enhanced susceptibility to CTLs. ATM, ataxia-telangiectasia mutated; CRT, calreticulin;
68 | JANUARy 2008 | vOLUME 8
© 2008 Nature Publishing Group
HMGB1 nor calreticulin (nor a combination of both)
can promote complete DC maturation, indicating
that the search for immunostimulatory molecules
produced by dying cells must continue.
Importantly, a polymorphism in TLR4 (rs4,986,790),
which leads to a single-nucleotide exchange (A896G)
and a single amino-acid substitution (Asp299Gly)
in the extracellular domain of TLR4, was found to reduce
the binding of HMGB1 to human TLR4. Accordingly,
DCs that are derived from patients who carry the
TLR4 Asp299Gly allele were far less efficient in cross-
presenting antigens from dying melanoma cells to
CTLs than were DCs from patients who carry the
normal TLR4 allele84,85. This DC defect could be
overcome by adding chloroquine. In a retrospective
study, we analysed the time to metastasis in a cohort
of 280 patients who had been treated for breast cancer
with local lymph-node invasion, following a stand-
ard protocol of local surgery, local radiotherapy and
systemic anthracycline. Patients who had the TLR4
Asp299Gly allele developed metastasis more quickly
than patients who had the normal TLR4 allele, estab-
lishing TLR4 Asp299Gly as an independent predic-
tive factor of early disease progression84. These results
support the concept that a selective immune defect (in
the DC-mediated presentation of antigen from dying
cells) can compromise the response to anticancer
Clinical trials of combination therapies
Numerous clinical studies have combined conventional
anticancer treatments with immunotherapies (TABle 3).
Pending confirmation through randomized, controlled
clinical trials, some of these studies suggest that chemo-
therapy and immunotherapy can create a synergy.
Two clinical studies in which DC vaccines were
administered before salvage chemotherapy have indicated
that synergistic antitumour effects between vaccination
and cytotoxic compounds can be achieved in late-stage
cancers. In one study, 29 patients with end-stage small-
cell lung cancer that was resistant to first-line platin-
based therapy were enrolled in a vaccination protocol
that involved autologous DCs infected with adenoviral
vectors encoding p53 (ReF. 87). The tumour progressed
in 23 patients, who then received salvage chemotherapy
with paclitaxel or carboplatin. The response rate to
these second-line chemotherapies was 61.5% and 38%,
respectively, survival at 1 year post-vaccination, a result
that was not expected from historical controls. Another,
retrospective study examined the impact of vaccination
with peptide-loaded or lysate-loaded DCs on the efficacy
of conventional therapy for glioblastoma. Chemotherapy
acted in synergy with previous therapeutic vaccination to
extend patient survival. The authors suggested that the
immunological targeting of tumour antigen tyrosinase-
related protein 2 (TRP2) expressed by glioblastoma cells
increased the sensitivity to inducers of cell death87.
Table 3 | Chemo-immunotherapy protocols reported since 2000*
Dacarbazine with cisplatin
• Group A: IFNα alone
• Group B: IFNα and IL-2
No significant effect for progression
— free survival and objective response
Dacarbazine with cisplatin
• Group A: placebo
• Group B: IFNα and IL-2
21% objective responses in group A versus
33% in group B; non-significant for time to
Renal carcinoma Gemcitabine‡
• Group A: IL-2, IFNα and
• Group B: IL-2, IFNα and
5-fluorouracil plus retinoic acid
• Group C: IFNα and vinblastine
Objective responses in ~20% and stable
disease in ~50% of patients
5-fluorouracil or vinblastineSignificant superiority of responses in
group A over groups B and C
Colon carcinomaGemcitabine, 5-fluorouracil
• GM-CSF and IL-2
Objective responses in ~70% and stable
disease in 95% of patients
MesotheliomaCisplatin and doxorubicinObjective responses in ~30% of the cases112
CisplatinSignificant superiority of combined
treatment over monotherapy to control
No clinical efficacy
*A list of chemo-immunotherapy protocols that were launched before 2000 is provided in Gomez et al.115 ‡Gemzar; Eli Lilly. §Eloxatin; Sanofi–Aventis.
GM-CSF, granulocyte/macrophage colony-stimulating factor; IFN, interferon; IL-2, interleukin-2.
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Nature Reviews | Immunology
• TGFβ, B7-H4,
• iNOS, arginase
• HLA-G, ILT4
• Lymph-node ablation
• Dose intensity
• Neoadjuvant versus
• Molecular pathways
• Molecular pathways
exposure or tumour
• KIRs and MHC class I
chemotherapy before surgical
resection of the tumour.
chemotherapy after surgical
resection of the primary
Similar intriguing synergistic effects were reported
when combining DNA vaccines and chemotherapy in
prostate cancers and other end-stage tumours88–90.
Patients who succeeded in mounting vaccine-specific
immune responses responded to salvage chemo-
therapy, whereas those who failed did not. The afore-
mentioned clinical studies and those summarized in
TABle 3 illustrate that the combination of chemother-
apy and immunotherapy may provide a valid thera-
peutic option for the treatment of neoplastic disease.
However, defining the appropriate doses and schedule
for the optimal synergy between the two strategies, as
well as the key indications and tumour types53, repre-
sents our current challenge.
Concluding remarks and future outlook
There is accumulating evidence that conventional ther-
apy for cancer may profit from the participation of the
immune system. This immune contribution is elicited in
two ways by conventional therapies. On the one hand,
some therapeutic programmes can elicit specific cellular
responses — beyond the stereotypical apoptotic pathway
— that render tumour-cell death immunogenic. These
immunogenic modifications include: pre-apoptotic
calreticulin translocation in response to anthracyclines;
induction of expression of MHC molecules, tumour-
specific antigens or death receptors in response to
epigenetic modifiers; HSP90 expression in response
to bortezomib; and post-apoptotic HMGB1 secretion
in response to anthracyclines and oxaliplatin. Other
chemotherapeutic agents that induce immunogenic
tumour-cell death may elicit yet more mechanisms.
On the other hand, some drugs may have side effects
(beyond their effect on the tumour itself) that stimu-
late the immune system, through transient lym-
phodepletion, the subversion of immunosuppressive
mechanisms and the direct or indirect stimulatory
effects of immune effectors.
In addition, many experimental therapies that have
been tested in mice and a few clinical trials suggest that
vaccination against cancer-specific antigens can sensitize
the tumour against subsequent chemotherapeutic treat-
ment. Although the mechanisms that underlie such a
synergistic effect have not been elucidated, we speculate
that the vaccination-induced increase in the frequency
of primed T cells, although by itself irrelevant for tumour
progression, may constitute a major advantage as soon as
the tumour is insulted by cytotoxic drugs.
Based on these premises, we anticipate that, in the
future, physicians will need to integrate four parameters
to design an optimal therapeutic programme (FiG. 3)
— the first three pertaining to the host and the last one
to the tumour.
First, the emerging field of cancer immuno-
epidemiology91 will determine which polymorphisms and
mutations dictate therapeutic outcome in patients with
cancer. As it stands, it seems that polymorphisms in the
genes that encode TLR4, IL-10 and IL-18 may affect the
therapeutic response in breast cancer84, lymphoma92 and
ovarian cancer93, respectively. Similarly, polymorphisms in
the Fc receptor for IgG affect the response to therapeutic
Second, mechanisms of tumour-induced tolerance,
including suppressor cells (such as TReg cells, T regula-
tory 1 cells, myeloid suppressor cells and tolerogenic DCs)
or their effector mechanisms (such as TGFβ, arginase and
IL-10) have to be overcome using specific therapies that
may include depleting antibodies, cytokine antagonists or
small molecules with immunostimulatory properties.
Third, deleterious effects that are provoked by
chemotherapy or radiotherapy on the host’s defence
system have to be prevented or avoided. For example,
the prescription of glucocorticoids during chemo-
therapy, as well as the ablation of tumour-draining
lymph nodes, may be counterproductive because they
suppress or avoid the immune responses. Similarly,
a neoadjuvant therapy of radiochemotherapy may be
immunologically more relevant than adjuvant thera-
pies, and chemotherapies may have to be scheduled so
that they liberate a maximum of tumour antigen while
avoiding unwarranted immunosuppression.
Fourth, the intrinsic characteristics of tumour cells
will have to be determined using ‘omics’ methods, not
only to find the tumour’s Achilles’ heel for an optimal
cytotoxic drug combination (or ideally a ‘targeted’ ther-
apy) but also to determine the immunological character-
istics of the tumour. Precise knowledge on the capacity of
the tumour to translocate calreticulin, to expose HSP90,
and to express antigen and ligands for recognition by
cytotoxic effector cells may yield invaluable information
for the optimal design of immunochemotherapies.
Figure 3 | Reconciling?host?and?tumour?requirements?for?optimal?management?of?
cancer?patients.?For improved clinical management of cancer, four parameters
should be integrated in cancer diagnosis and treatment. First, cancer immuno-
epidemiology indicates that polymorphisms in the genes that dictate immune
responses, such as Toll-like receptor 4 (TLR4) and interleukin-1β (IL1b), should
influence the design of the therapy. Second, tumour-induced tolerance relies on
suppressor cells or their mediators, such as transforming growth factor-β (TGFβ) and
IL-10, which could be antagonized by dedicated therapies that have the aim of
restoring antitumour immune responses. Careful immunomonitoring of patients with
cancer should reveal which among these immunosuppressive pathways needs to be
counterbalanced. Third, the deleterious effects of conventional therapies (including
the use of steroids, lymph-node resections and escalating doses of chemotherapeutic
agents with immunosuppressive and myelosuppressive side effects) should be
avoided. Fourth, by examining the intrinsic characteristics of tumour cells using
‘omics’ approaches, one should be able to identify the immunosuppressive
characteristics of the tumour, its antigenic profile and its capacity to succumb to
immunogenic cell death. FcgR, Fc receptor for IgG; IDO, indoleamine 2,3-
dioxygenase; ILT4, immunoglobulin-like transcript 4; iNOS, inducible nitric-oxide
synthase; KIR, killer-cell immunoglobulin-like receptor; NKG2D, natural-killer group 2,
member D; PDL1, programmed cell death ligand 1; STAT3, signal transducer and
activator of transcription 3.
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The authors are supported by grants from the Ligue Nationale
contre le Cancer (L.Z., G.K. and L.A.), the European Union
(ALLOSTEM, DC-THERA; L.Z.), Cancéropôle Île-de-France,
Institut National du Cancer and Agence Nationale pour la
Recherche (G.K.). F.G. is supported by a Poste d’acceuil
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
HMGB1 | IL-12 | TLR4
Institut Gustave-Roussy: http://www.igr.fr/
NATURE REvIEWS | immunology?
vOLUME 8 | JANUARy 2008 | 73
© 2008 Nature Publishing Group