PERSPECTIVES
of survival signals1. A generally accepted
OPINION
Immunotherapy and chemotherapy
a practical partnership
Richard A. Lake and Bruce W. S. Robinson
Abstract | This article discusses how recent
data have altered the way we understand
how dying tumour cells, particularly those
killed by chemotherapy, engage with
antitumour immune responses. These data
have significant implications for the
development of new protocols combining
chemotherapy with immunotherapy,
indicating an exciting potential for
therapeutic synergy with general
applicability to many cancer types.
Chemotherapy remains the treatment
modality of choice for most advanced can-
cers. However, for solid tumours in particu-
lar, it is rarely curative. Immunotherapy is a
less conventional form of therapy and is
also rarely curative. Chemotherapy and
immunotherapy have usually been regarded
as unrelated or, more commonly, antagonis-
tic forms of therapy, so relatively few studies
have investigated the relationship between
these treatments. Two a priori assumptions
have contributed to this state of affairs. First,
most chemotherapies kill target cells by
apoptosis and this mode of cell death has
been regarded immunologically as either
non-stimulatory or able to produce immune
tolerance a state where T cells can no
longer respond to the presented antigen by
mounting an immune response. Second,
lymphopaenia is a common side effect of
many anticancer drugs and this has also been
assumed to be detrimental to any potential
immune response. Although some studies
have probed the relationship between
chemotherapy and immune function in vivo,
NATURE REVIEWS | C ANCER
there has been a paucity of methods to accu-
rately analyse any changes. With increasingly
sophisticated models and the development of
a range of tools to scrutinize the progress of
any anticancer immune responses to growing
tumours, several recent studies have produced
unexpected results. When taken together, they
indicate that there is a strong and develo-
ping case for combining chemotherapy and
immunotherapy in cancer treatment.
Chemotherapy and cell death
Different chemotherapies kill tumour cells in
different ways and in the process they can
modulate the host immune system with con-
sequences that are only now beginning to be
fully elucidated. Here, we make the case for
adjuvant immunotherapy for patients under-
going chemotherapy and we hypothesize how
different approaches to stimulating the
immune system could be used as the preferred
adjuvant therapy, depending on the particular
type of cancer and the choice of chemother-
apy. As tumour-cell death is the goal of most
chemotherapy, we will discuss how different
drugs kill tumour cells. We will then describe
how each of the six key steps in the induction
of an effective antitumour immune response
(FIG. 1) might be altered by chemotherapy,
highlighting how these approaches could
enable synergistic combinations of the two
forms of therapy to be used clinically.
Death by apoptosis. Apoptosis is an intrinsic
mechanism by which cells die and it is
widely accepted that the willingness of cells
to die is usually countered by the provision
2005 Nature Publishing Group
current biochemical definition would
include the activation of a conserved series
of caspases2, a family of cysteine proteases
that facilitate the efficient dismantling of
the dying cell. When cells die by apoptosis,
phosphatidylserine, which in normal cells is
located on the inner leaflet of the plasma
membrane, translocates to the outer leaflet,
where it acts as a molecular flag, interacting
with receptors expressed by a range of differ-
ent cell types, including macrophages. In the
absence of inflammatory signals, the
macrophages phagocytose the apoptotic
remnants3. Apoptosis has long been consid-
ered as non-immunogenic or even tolerizing,
occurring in the absence of any inflamma-
tion4. Although the original definition of
apoptosis excluded inflammation, it is now
clear that innate immunity (BOX 1) can be
triggered by apoptosis and there has been
much speculation as to whether all forms of
apoptosis are equivalent. Therefore, Restifo
postulated that apoptosis occurring during
development or tissue turnover is immuno-
logically bland, whereas apoptosis follow-
ing viral infections or ligation of the death
receptor FAS (also known as CD95) is
intrinsically coupled to the production of
inflammatory signals that can trigger pow-
erful immune responses5. Molecular flags
which might be of either cellular or
microbial origin (BOX 1) differentiate
effete and harmless dying cells from those
that are associated with danger so that
harmless but potentially immunogenic
material is sequestered away from the
immune system, whereas antigens associ-
ated with infection or danger are presented
in an immunogenic context3. The death of a
tumour cell, either naturally or induced by
chemotherapy, where there is no inflamma-
tion might be expected to appear like nor-
mal tissue turnover, generating either no
immune response (ignorance) or toler-
ance. Some experiments support this
hypothesis, showing that tumour-derived
antigens from a lymphoma and a solid
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PERSPECTIVES

positive outcome for this drug in combination

Lymph node
with a dendritic cell (DC)- (antigen-presenting
b CD8+ T cell
cell (APC))-based vaccination therapy20. Other
chemotherapy agents such as paclitaxel induce
an abnormal metaphase by damaging micro-
tubules and disrupting the mitotic spindle.
c Cyclin-dependent kinase 1 activation is abnor-
a Tumour cell
mally prolonged in paclitaxel-treated cells,
Tumour resulting in a bypassing of the G2 checkpoint
APC and cell death by mitotic catastrophe21,22. The
Antigen
CD8+ T cell
finding that paclitaxel is less effective than the
apoptosis-inducing drug doxorubicin when
coupled to a vaccination protocol for the treat-
Circulation
ment of aggressive breast cancer23 might indi-
cate that mitotic catastrophe errs on the side of
being immunologically bland.
e
Traffic Apoptosis and the immune response
It is evident that some mechanistic under-
f
d standing of the essential principles of
Tumour
blood vessel immune recognition is required before we
can discuss how dead and dying tumour
cells alter this process (BOX 1). It is generally
Memory T cell
accepted that the sort of immune response
that would be favourable to tumour elimina-
Figure 1 | The six steps necessary for an effective antitumour CD8+ T-cell response. Effective
destruction of tumours by antigen-specific CD8+ T cells is a multistep process. Each of these six steps is
tion will include the generation of large
required and can be modulated by a range of factors: tumour antigens must be present (a); these antigens numbers of interferon- (IFN)- and TNF-
must reach/load professional presenting cells antigen-presenting cells (APCs) in the draining lymph secreting CD8+ T cells with the capacity to
node (b); specific T cells must respond by proliferation (c); the circulating T cells must enter the tumour (d); directly lyse tumour-cell targets. Therefore,
once in the tumour the T cells must be able to overcome local immune-suppressive molecules to the key issues to be aware of in pairing
recognize and kill targets (e); memory cells should be generated (f). Cancer immunotherapy can fail at any chemotherapy with immunotherapy relate
of these steps, so the development of assays to analyse each of these steps in vivo (TABLE 1) has been
to the molecular flags associated with differ-
the key to beginning to understand how chemotherapy and immunotherapy interact. MHC; major
histocompability complex. ent forms of cell death and how they are
contextually interpreted.
There is increasing evidence that some
part of the apoptotic process is required for
tumour induce tolerance during tumour pro- been shown to increase the number of apop- generating an immune reaction. In a concep-
gression6,7. However, there is now increasing totic blasts in leukaemia11. Importantly, the tually simple series of experiments, Bonotte
evidence that, under the right circumstances, degree of apoptosis was found to correlate and colleagues showed that immunogenic
chemotherapy-induced tumour-cell death with clinical outcome for several different colon carcinoma cells were sensitive to apop-
can set the stage for an effective antitumour tumour types1214. tosis and died in vitro if they were starved of
immune response. growth factors. When these cells were made
Anticancer drugs can induce apoptosis Death by non-apoptotic mechanisms. resistant to apoptosis by overexpressing the
both by death-receptor-dependent and -inde- Apoptosis is not the only mechanism by which anti-apoptotic protein BCL2, they gave rise to
pendent pathways. Some anticancer drugs cells die. Non-apoptotic death pathways progressive tumours in vivo. Interestingly, the
increase the expression of death receptors, include necrosis, autophagy and mitotic cata- antigenicity of the BCL2-expressing apopto-
including FAS, tumour-necrosis factor strophe. These alternate forms of cell death are sis-resistant cells was not altered, because
(TNF) receptors and TNF-related apoptosis- differentiated by particular combinations of animals that had been pre-immunized with
inducing ligand receptors. Tumour cells morphological and biochemical changes15,16. parental immunogenic cells were able to
commonly show abnormalities to various Significantly, different chemotherapeutic drugs reject the BCL2-expressing cells24.
components of these pathways8, so tumours have been shown to induce different forms of Apoptosis might also be a necessary com-
are differentially sensitive to these drugs. cell death. Temozolomide, for example, is a rel- ponent of some vaccine strategies. The degree
Other drugs do not alter expression of death atively new alkylating agent that induces G2/M of apoptosis might help to explain the finding
receptors, but trigger apoptosis by inducing arrest and autophagy with no apoptosis17,18. that Alphavirus-based vaccines, which induce a
release of cytochrome c from mitochondria. There have been no systematic analyses of the high level of apoptosis, are generally highly
Although chemotherapeutic drugs induce effects of this drug on the immune system, but immunogenic despite the fact that they pro-
their primary damage in many different it is a powerful inhibitor of lymphocyte prolif- duce less antigen than conventional DNA-
ways, most of them induce apoptosis not eration, with only marginal effects on the activ- based vaccines. Leitner and colleagues switched
only in vitro but also in vivo 9,10. For example, ity of the natural-killer lymphocyte subset19. off apoptosis by overexpressing the anti-apop-
a diverse set of agents, including cytarabine, Indeed, a single case report focusing on a child totic gene BCL-XL in an Alphavirus-based
mitoxantrone, etoposide and topotecan, have with recurrent malignant glioma describes a immunization approach. As expected, they

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2005 Nature Publishing Group

found that cells that were transfected with
BCL-XL lived longer and produced more anti-
gen, but in the absence of apoptosis the vaccine
was significantly less protective25. Conversely,
DNA-based vaccination is enhanced by
delivering pro-apoptotic signals26,27.
So what is the key feature of apoptosis that
drives the acquired immune response?
Phosphatidylserine is probably the best-stud-
ied molecular flag, but is almost certainly a
downregulator of the immune response.
Phosphatidylserine will become accessible to
macrophages after non-apoptotic forms of cell
death when the plasma membrane loses its
integrity. But it is likely that there are separate
signals associated with these dead cells, because
the intracellular components that would oth-
erwise be packaged into the apoptotic bodies
are released. Phosphatidylserine stimulates the
production of a set of anti-inflammatory
mediators, including transforming growth
factor-, prostanoids and interleukin-10
(IL-10)28. It is now clear that recognition of
phosphatidylserine, in the absence of any other
signal, has the capacity to suppress the release of
pro-inflammatory cytokines, including IL-12.
This occurs by direct transcriptional repression
of the p35 IL-12 subunit, leading to a loss of
IL-12 production29. It had been assumed that,
because phagocytosis of cells dying by apopto-
sis in vivo is efficient, tumour-cell apoptosis
would be likely to result in antigen sequestra-
tion or tolerance induction. However, when
massive apoptosis occurs, the tolerogenic
system might be overwhelmed, resulting in
secondary necrosis and release of pro-inflam-
matory mediators30. Some of the known
activators of immune function include heat-
shock proteins (HSPs). The HSPs that are
induced by stressing apoptotic leukaemia cells
increases their capacity to activate DCs31.
Heat-stressed tumour cells induce changes in
DCs, including an upregulation of co-stimula-
tory molecules (CD40, CD80 and CD86)
accompanied by an increase in IL-12 secre-
tion. Under some circumstances, phagocytosis
of apoptotic cells leads to secretion of growth
and survival factors, including vascular
endothelial growth factor32. Nevertheless, uric
acid is probably the most powerful endoge-
nous pro-inflammatory signal released from
injured cells33. So it is clear that a series of
mediators interacting on a range of cell types
take part in a set of complex feedback interac-
tions to determine the consequences of dead
cells for the immune system. And there is no
single response to apoptotic cells, but, rather,
the response to such cells depends on the way
in which apoptosis has been induced, the
amount of associated cellular stress and the
pattern of regulatory cytokines.
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PERSPECTIVES
Box 1 | Alerting the immune system: difference, dose, danger and duration
There are many ways that the host can eliminate unwanted cells. Probably the most effective
immunologically mediated host strategy is the deployment of cytotoxic T lymphocytes
(CTLs) that can directly lyse targets and have the capacity to secrete effector cytokines such
as interferon- and tumour-necrosis factor-. The conceptual problem for immunologists is
to understand how these cells can be targeted appropriately to discriminate between healthy
tissue, infected tissue and tumours.
Since Burnet defined the paradigm, immunologists have been comfortable with the view
that the immune system works by having the capacity to discriminate between self and non-
self 60. This concept of difference is of fundamental importance to the shape of the T-cell
repertoire61. Tumours carry many mutations and it is now clear that most tumours express
neo-antigens against which the host has a capacity to react. Of course, these antigens must
achieve a threshold concentration, that is, dose, before they will trigger any response.
The paradigm of self was challenged by Matzinger and others, who suggested that the
primary drive to elicit an immune response was to protect against danger62,63. In the
absence of danger signals there is either no immune response or tolerance might be
induced. Danger signals are thought to act principally at the level of antigen presentation
to T cells to induce the professional
antigen-presenting cells such as dendritic
cells to mature and express stimulatory Cell and virus Cell and Cell and self
tumour antigen proteins only
ligands and cytokines. The simplest of the
three main types of danger signal are the
conserved molecular motifs that are
ubiquitously carried by microorganisms64.
Heat-shock proteins and uric acid are key
among the mediators released by stressed
or damaged cells and can mark such cells
for destruction by either T cells or natural-
killer cells. Missing self brings an
important supplementary concept an
immune response can be activated by the 'Dangerous' Weak Tolerizing
Cell damage No danger None of
loss of inhibitory signals that would (e.g. uric acid) signals these signals
normally block the initiation of immune Innate immunity No CD40
responses against self . 65 activated signals
Toll-like Pro-
The fourth important D is duration receptors inflammatory
how long a particular antigen is around has triggered cytokines
Inflammation
profound implications for the induction of Cytokines
immunological memory58. CD40 signals
These concepts are illustrated in the figure. Others
Non-apoptotic death and the immune The tumour immune response in vivo
response. Few studies have investigated the The interaction between chemotherapy and
immune response to non-apoptotic death immunotherapy has not been extensively
caused by chemotherapy, largely because studied in vivo because the necessary tech-
there are few such drugs; some of these nology has only recently been developed
studies are cited above. Several studies (TABLE 1) . In vivo studies are necessary
based on in vitro work have found that because in vitro studies cannot accurately
cell death by necrosis induces DCs to reflect the biology and dynamics of these
act as potent APCs, whereas apoptotic cell complex interactions. This technology
death does not have this effect34. Therefore, includes the development of appropriate
in the steady state of normal tissue animal models and the ability to analyse
turnover, apoptotic death seems to be tumour-antigen-specific immune responses
tolerogenic, whereas necrotic death is at each of the six key checkpoints of the
more likely to be associated with danger antitumour response (FIG. 1). These assays
and the induction of an immune response can help to determine where in the
(reviewed in REF. 35 and see BOX 1). The sequence of events a particular therapy
immunological environment endured fails. For example, if the dose of antigen is
following massive apoptotic cell death not limiting but the capacity of T cells to
after effective chemotherapy is likely to be expand is limited, then increasing the dose
different, as discussed below. of antigen is unlikely to improve responses,
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PERSPECTIVES

Table 1 | Six key steps for specific CD8-mediated antitumour immunity mean that these antigens will induce a
response. The factors that determine whether
Step In vivo assay system for measuring response
an available antigen elicits an immune
Antigen threshold Quantification of levels of marker antigens by ELISA or western blot
response are explored in BOX 1.
Antigen presentation CFSE dye dilution assays66
T-cell response In vivo CTL assay using CFSE- and peptide-loaded target cells67; How might chemotherapy affect the dose of
tetramer analysis of tumour-specific T-cell numbers68; ELISPOT or antigens delivered for presentation? One of
intracellular cytokine staining for cytokine production can be
combined with other assays69 the key reference points for the generation of
a particular immune response is antigen dose
T-cell traffic Staining for T-cell infiltration; flow cytometry of extracted cells;
trafficking of dye-labelled cells (BOX 1). In a series of experiments using one

Target destruction Reduction in size of tumour; cytokine staining in tumour; function of of the above-described mouse models, we
cells extracted from the tumour were able to show that a tumour neo-antigen
Generation of memory Flow cytometry of extracted cells; adoptive transfer of extracted was constitutively and efficiently delivered to
cells; tumour growth after rechallenge the lymph nodes that drain the tumour, and
CSFE, 5,6-carboxyfluorescein diacetate succinimidyl ester; CTL, cytotoxic T lymphocyte; ELISA; enzyme- only those nodes, by a process known as
linked immunosorbance assay, ELISPOT: enzyme-linked spot recognition of specific T-cell function. cross-presentation39,40. Cross-presented anti-
gens cross from an exogenous source, which
typically delivers these antigens into the
whereas delivery of agents that support T-cell some will be efficiently delivered to the major histocompatibility complex class II
expansion is more likely to prove successful. immune system whereas others will not reach pathway, into the class I pathway, a pathway
These models and the tools to study them that threshold. When tumours regress with previously thought to be restricted to
have already produced new insights into anti- chemotherapy, increasing amounts of these endogenous antigens expressed exclusively by
tumour immune responses. We and others antigens are released from the dead and dying the cell itself. Despite this, little activation of
have transfected antigens into tumour cell lines cells and could be delivered into the antigen- antitumour CD8+ T-cell responses resulted
to act as markers to analyse tumour-specific presentation pathways, making them avail- except for low constitutive levels of cytotoxic
responses. These antigens do not alter the able to induce immune responses. It is likely T lymphocyte (CTL) activity restricted to the
immune response to the tumour, they simply that in this process some antigens that would draining lymph node. It is still not known
report back to the investigator what is hap- otherwise be below the threshold of availabil- whether live or dead or dying tumour cells
pening in vivo. The other crucial reagents in ity could now be presented to the hosts constitutively deliver these antigens into the
this system are tumour-antigen-specific T cells. immune system. This does not necessarily cross-presentation pathway.
Transgenic mice that express an identical T-cell
receptor on each functional T cell enable large
numbers of cells to be isolated and used to Table 2 | How chemotherapy could augment immunotherapy
analyse antigen presentation in vivo. These cells Essential steps in the Potential effects of chemotherapy on References
can be used as the cellular equivalent of induction of an antitumour the capacity of immunotherapy to
tumour-specific monoclonal antibodies. Using immune response destroy tumours
these tools, we can now begin to answer six key Antigen threshold Delivery of a broader range of different *
questions on how chemotherapy impacts on tumour antigens
the tumour immune response (TABLE 2). Antigen presentation Increased antigen cross-presentation 41
Partial activation of dendritic cells 41
How might chemotherapy affect the range of Priming of APCs for CD40 signal 50
antigens delivered for presentation? There
Killing subsets of APC 43
are large numbers, possibly tens or even
hundreds, of potential tumour antigens in T-cell response No tolerance induction by apoptotic tumour cells 41
any particular cancer3638. These are of two Lymphopaenia-related proliferation increases 57
tumour-specific T-cell response
types, neo and self, tumour antigens. Neo-
antigens are antigens that the host immune T-cell traffic Increased T-cell accumulation within tumour 50
system has never seen, so no tolerance has Target destruction Increased local tumour-antigen cross-presentation *
been induced. These are strong antigens and (permitting CD8 re-stimulation)
examples are mutated proteins and onco- Tumour debulking (less systemic suppression, 70
smaller target, less chance for escape variants etc.)
genic viruses. Self tumour antigens are
unmutated self proteins, but they are able to Partial sensitization of tumour cells for CTL lysis 55,56
induce immune responses because they have Generation of memory Promotion of long-term antigen-independent 58
limited expression in the host and therefore memory
limited opportunity for induction of toler- External regulation of Increased delivery of exogenous antigen 41
these steps
ance. Examples are proteins that have
expression limited to tumours and testes Increased CD4 help (for example, delivery of 50
CD40 signals)
(known as cancer-testis antigens) and dif-
ferentiation antigens that are not expressed Reduction in function of negative regulatory cells 44,46
in early life. These tumour antigens will be Induction of homeostatic proliferation 59
present at different concentrations so that *Not yet demonstrated in tumour models. APC, antigen-presenting cell; CTL, cytotoxic T lymphocyte.

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We hypothesized that if live cells were the
main source of cross-presented antigens, one
would expect to see a relative loss of antigen
presentation in the draining lymph node fol-
lowing effective chemotherapy. In fact, not
Forward scatter
only was there no reduction in tumour-anti-
gen cross-presentation (FIG. 2), but when the
data were analysed by correcting for tumour
size, the amount of cross-presentation, as
determined by proliferation of the cells used
as markers of cross-presentation, approxi-
mately doubled41. Because these assays are
functional, it is impossible to accurately quan-
tify how this change in proliferation equates
to the precise increase in the number of
molecules of antigen that appear in the cross-
presentation pathway, but, based on other
studies, this might correspond to a tenfold
increase in the dose of antigen42. Certainly, it
is clear that chemotherapy-induced apoptosis
in vivo does not sequester tumour antigens.
Rather, apoptotic tumour cells are a good
source of cross-presented tumour antigens.
Our published work on the effects of
chemotherapy-induced tumour-cell death
in vivo has largely concentrated on the
anti-metabolite gemcitabine. This drug can
induce massive tumour-cell apoptosis both
in vitro and in vivo, is widely used in
human cancers, and in tumour-bearing
animals can reduce the volume of estab-
lished tumours by over 80%. The observa-
tion that a gemcitabine-resistant tumour
did not show any increase in tumour-anti-
gen cross-presentation indicated that the
capacity of chemotherapy-induced tumour-
cell death to load the draining lymph nodes
with antigen was entirely attributable to the
induction of tumour-cell death and not
related to any non-specific effects of the
drug either on the cross-presenting func-
tions of APCs or on the endogenous
immune response41. Therefore, we hypothe-
size that any drug that kills tumour cells by
apoptosis will result in an increase in the
amount of cross-presented antigen.
Other ways that chemotherapy could
affect antigen presentation include a differen-
tial toxicity for particular subsets of cells.
Gemcitabine differentially depletes B cells and
induces a profound suppression of antitu-
mour antibody responses43. B cells are
bona fide APCs and might drive immune
responses towards the generation of antibod-
ies and away from the generation of CTLs.
Removing them with gemcitabine does not
seem to be detrimental to specific antitumour
cellular immunity and might be useful in
combination chemoimmunotherapy proto-
cols, the aim of which is to generate CTLs. By
contrast, vaccination protocols requiring a
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PERSPECTIVES
a No tumour b Tumour only c Tumour and gemcitabine
Undivided cells Dividing cells Dividing cells
CFSE
Figure 2 | Apoptosis delivers increased antigen loads into the antigen-presentation pathway.
BALB/c mice bearing tumours were treated with gemcitabine and antigen presentation was analysed by
adoptively transferring tumour-antigen-specific lymphocytes that were labelled with the marker dye 5,6-
carboxyfluorescein diacetate succinimidyl ester (CFSE). Three days later, fluorescence-activated cell
sorting was used to determine the proliferation status of the labelled lymphocytes. When no tumour
antigen is present there is no division and the peak of CFSE-loaded lymphocytes (marked with an arrow)
retains a high dye concentration (a). During normal tumour growth there is clear evidence of antigen
delivery, as additional peaks demonstrating a halving of CFSE concentration become visible (arrows in b).
After drug-induced apoptosis there is increased antigen presentation manifest as increased numbers of
dividing cells (arrows in c).
humoral (B-cell-mediated antibody) response are extracted and transferred to animals
for maximal efficacy might be compromised with immunogenic tumours, these tumours
in patients treated with gemcitabine. start to grow progressively. A single admin-
istration of CTX to animals bearing tolero-
How might chemotherapy affect the antitu- genic, progressively growing tumours
mour T-cell response in the draining lymph depletes regulatory T cells and delays the
node? Despite increasing tumour-antigen growth of the tumour. When CTX is com-
presentation in the draining lymph node, bined with an immunotherapy that is not
chemotherapy does not usually induce an curative by itself, the combination has the
effective antitumour response by itself. In capacity to eradicate established tolerogenic
the clinic, it is rare for cancers that have par- tumours in animal models46.
tially regressed in response to chemotherapy Given that an organism has a T-cell
to then continue to regress through repertoire with the capacity to recognize a
immune mechanisms when the treatment given antigen (the issue of difference), two
stops. Animal experiments support the view fundamental questions operationally define
that chemotherapy, as a single protocol that whether the immune system will respond
is not curative, does not usually induce an to it. The first is whether the antigen is pre-
immune response (see the section on mem- sent above a threshold dose level and the
ory below). The notable exception to this second is whether the antigen is dangerous.
observation is cyclophosphamide (CTX). These questions are of course put and
The immunomodulatory effects of CTX answered in biochemical terms (BOX 1).
have been known for a long time. In 1974, Duration of exposure to antigen also has
when the vogue for describing immune profound consequences for the develop-
responses in terms of suppressor circuits ment of immunological memory and is dis-
was at its height, it was found that some cussed below. Understanding the specific
forms of tolerance could be reversed by molecular events that transmit dose and
CTX. Appropriate doses of CTX have no danger signals is important for understand-
major direct effect on the number of lym- ing how antitumour chemotherapy and
phocytes but act by removing T-suppressor- immunotherapy could interact.
cell activity 44. Suppressor cells are now more Cross-presented antigens will be ignored
usually characterized as regulatory T cells45. if there are no T cells with receptors of
Above, we discussed how immunogenic appropriate specificity. When there are
colon carcinoma cells could be manipu- T cells that can respond, cross-presented
lated and made tolerogenic by inhibiting antigen can lead to their activation and pro-
their tendency to apoptose. Tolerogenic liferation or can induce tolerance28,47. Cross-
colon carcinoma cells grow progressively tolerance has been described for the antigen
in vivo with a corresponding expansion of used in our studies influenza virus
regulatory T cells. If these regulatory T cells haemagglutinin (HA) when it is expressed
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PERSPECTIVES

as a self-antigen rather than as a tumour
neo-antigen. Transgenic Ins-HA mice
express HA in the islet cells of the pancreas
as a normal self-protein. These animals do
not develop immune-mediated insulitis or
diabetes when exposed to HA-specific CTLs,
because cross-presentation of HA is followed
by rapid activation and deletion of respon-
der T cells48. This process is known as
peripheral tolerance and is a well-estab-
lished mechanism for avoiding destructive
autoimmune reactions49.
Tumour tissue might have been
expected to behave like self-tissue and so
cross-tolerize antitumour T cells in the
same way. It does not. Furthermore,
chemotherapy does not reduce the fre-
quency of tumour-antigen-specific T cells.
Viral signals can also drive this process,
as post-chemotherapy administration of
tumour-antigen-containing virus slows
tumour growth, whereas in the absence of
chemotherapy there is no effect41.
The reason why chemotherapy-induced
cell death primes rather than tolerizes host
immune responses is not yet known. It is
possible that tumours create a partial
inflammatory environment, characterized
by the accumulation of macrophages and
the release of some pro-inflammatory
cytokines like TNF and IL-6 with the asso-
ciated symptoms of weight loss, anorexia,
fevers and thrombocytosis. This balance
between pro- and anti-inflammatory medi-
ators might block CTL induction, but
additional chemotherapy could increase the
tumour-infiltrating T cells50. We speculate
that this occurs because of changes in the
balance of inflammatory mediators in the
local milieu. Overall, the increase in the
number of tumour-infiltrating T cells could
be a direct effect of the drug on the tumour
stroma or indirect, as a consequence of
increased phagocytosis. It is also possible
that chemotherapy primes a tumour-
specific CD4+ T-helper-cell response, as we
a Anti-CD40 therapy without apoptosis
100
80

Although it increases the rate of cross-pre-
sentation and therefore the amount of anti-
gen available to potentially cross-tolerize, it
does not substantially reduce the activity of
CTLs in vivo. Overall, the data do not sup-
port the view that the induction of tumour-
cell apoptosis tolerizes the tumour-specific
CD8+ T-cell response.
However, chemotherapy-induced anti-
gen cross-presentation is not a null event.
Rather, it provides a fertile environment for
priming the host immune system for other
immunostimulatory signals. Constitutive
tumour-antigen cross-presentation results
in cross-arming of effector CTLs, but these
cells remain in the lymph node that drains
the tumour. This pathway is the default
process. What then determines whether an
Percent survival
pro-inflammatory mediators such as HSPs 60
to sufficient levels to induce CD8+ T-cell
responses51. The increased number of dead
40
and dying tumour cells is also likely to
invoke an increase in phagocytosis, and if
these cells are activated, particularly by the 20
release of intracellular contents, they might
release more pro-inflammatory cytokines
0
and so increase the responsiveness of APCs 0 20 40 60 80 100 120 140
to cross-presented tumour antigens52. Days after tumour injection
Another possible direct effect of
Control
chemotherapy on cross-priming has been Anti-CD40
attributed to alkylating agents. In fact, any
drugs that modify DNA might have partic- b Anti-CD40 therapy with apoptosis
ular effects on the host immune response.
100
For example, co-culture of immature DCs
with tumour cells treated with melphalan
and chlorambucil caused the DCs to upreg- 80

APC that is loaded with antigen activates or ulate co-stimulatory molecules, secrete IL-12
Percent survival

tolerizes any T cells that it encounters?
One molecule on the APC that, when
triggered, can cause a switch from tolerance
to activation is CD40. CD40 signals deliv-
ered to antigen-loaded APCs drive the
process of T-cell priming and expansion
and also induce peripheral dissemination of
these CTLs so that they leave the lymph
node and circulate. In the process, they
retain strong antitumour killing capacity.
Importantly, these T cells are now enabled
to destroy established tumours40. Whether
these CD40 signals occur only in the drain-
ing lymph node or at the tumour target site
is not yet clear. However, when CD40 signals
are delivered to hosts bearing large tumours
it is no longer effective. It is clear though
that induction of apoptosis in large tumours
not only loads the APCs with tumour anti-
gens but also sensitizes them to CD40 sig-
nals, curing most mice studied in this way.
This immunotherapy is much more effec-
tive if delivered after apoptosis-induced
antigen loading rather than before50 (FIG. 3).
and efficiently activate T cells 53. These
effects could be recapitulated using DNA
purified from the killed tumour cells, sup-
porting the hypothesis that DNA damage is
itself recognized as inflammatory 53. The
precise mechanisms whereby chemother-
apy partially primes APCs and antitumour
T-cell responses is now under investigation,
as are the ways in which this can be aug-
mented by agents other than CD40 and
tumour-antigen-containing viruses.
How might chemotherapy affect T-cell traf-
fic? One of the stumbling blocks in tumour
immunology is the observation that it is
possible to generate an antitumour
response that is measurable in the circula-
tion but these T cells never enter the
tumour. Why would they? Entry of T cells
into any tissue is a highly orchestrated event
and, as discussed above, untreated tumours
are probably bland and anti-inflammatory.
We have found that single-protocol
chemotherapy caused an increased influx of
60
40
20
0
0 20 40 60 80 100 120 140
Days after tumour injection
Anti-CD40 before gemcitabine
Control (gemcitabine alone)
Anti-CD40 after gemcitabine
Figure 3 | Established tumours can be
cured when immunotherapy is delivered
following apoptosis induction. Mice with
established tumours were treated with
immunotherapy (anti-CD40 antibody) without
chemotherapy (a) before a full course of the
apoptosis-inducing agent gemcitabine (b) or
following the same chemotherapy (c).
Phosphate-buffered saline injections were
used as controls. KaplanMeier survival curves
are shown.

402 | MAY 2005 | VOLUME 5 www.nature.com/reviews/cancer

2005 Nature Publishing Group

have previously reported that these T cells
can increase the infiltration or retention of
CD8+ T cells into tumour sites39.
How might chemotherapy affect target-cell
destruction? Even if tumour antigens are effi-
ciently presented, and even if T-cell responses
are induced and these cells traffic into
tumour sites, this still might not be enough
to cause tumour destruction. There is some
evidence to indicate that in order to work
efficiently, CTLs require re-stimulation by
professional APCs located in the tumour40.
This process might invoke further rounds of
proliferation, so increasing the CTL fre-
quency, and it might affect the threshold sen-
sitivity at which these cells are triggered as
well as qualitatively affect the secretion of
IFN and TNF. How and if chemotherapy
alters this process is not known, but the
capacity of chemotherapy to deliver
increased antigen loads to APCs is not likely
to be restricted to the draining lymph nodes
the APCs within tumours are also likely to
receive increasing amounts of antigen, pro-
viding at least some of the signals required
for CD8+ T-cell re-stimulation within
tumours. Effective chemotherapy results in
tumour debulking, which will change the
effector T cell to tumour-target ratio. This
could result in a non-linear amplification of
the ability of CTLs to kill targets.
Another way that chemotherapy could
augment the capacity of tumour-infiltrating
lymphocytes to deliver their effector response
is by upregulating death receptors54. Because
T cells can use this pathway to kill targets, this
can make the tumour cells more susceptible
to T-cell-mediated destruction55. These same
drugs also have the capacity to sensitize can-
cer cells to lysis by weak, or low-avidity, CTLs
that would not otherwise kill them56. Drugs
such as doxorubicin and methotrexate pro-
mote apoptosis in some tumour cells by
inducing an upregulation in transcription of
the gene encoding FAS ligand54. Other
chemotherapy agents alter apoptosis path-
ways in different ways, thereby altering the
threshold of apoptosis sensitivity. Where dif-
ferent pathways to cell death synergize, we
can hypothesize that these effects are likely to
increase the sensitivity of cancer cells to
immunologically mediated killing.
How might chemotherapy affect the genera-
tion of immunological memory? In addition
to the antitumour effector responses
described above, the development of long-
term immunological memory could pro-
vide protection from both recurrence and
metastases. Previously, we have noted that
NATURE REVIEWS | C ANCER
single-protocol chemotherapy does not
induce an immune response if the tumour
continues to grow progressively. We became
aware of the potential of chemotherapy to
facilitate memory by rechallenging the occa-
sional long-term survivors of chemotherapy
(<2% of single-protocol-treated animals)
with tumorigenic numbers of cells. They
all survived.
It has now been demonstrated that CD8+
T-cell memory evolves differently if antigen
persists. When antigen is removed, such as
after an acute infection or successful
chemotherapy, CD8+ T cells undergo anti-
gen-driven proliferation and then differen-
tiate into effector CD8+ T cells and a small
number of these cells develop into memory
CD8 + T cells. These cells persist for long
periods in the absence of antigen. They
undergo homeostatic proliferation in
response to IL-7 and IL-15, and they
respond vigorously to antigen 57. By con-
trast, if antigen persists such as during a
chronic infection or during tumour growth,
even after a partial response to chemother-
apy, the antigen-independent phase of
memory CD8+ T-cell differentiation does
not occur and memory CD8+ T cells are not
properly induced 58. Obviously, antigens
from a growing tumour would continue to
be presented to the immune system, at least
partially recapitulating the environment of
a chronic infection. Chemotherapy offers
the opportunity to modulate this process in
two ways that might prove beneficial to the
induction of an effective immune response.
First, chemotherapy might reduce the
threshold of antigen delivery to the draining
node to a sufficiently low level to achieve
the antigen-independent rest period that is
required for the development of memory.
Second, if the chemotherapy induces some
level of lymphocyte loss (lymphopaenia),
the curious homeostatic response that is
triggered to try to restore lymphocyte num-
bers and fill the space might increase the
frequency of tumour-reactive T cells in the
process. These processes might have a role
in the effects of chemotherapy that have
been observed in some experimental proto-
cols. For example, increased responses were
observed in melanoma patients who
received non-myeloablative chemotherapy
before the adoptive transfer of activated
tumour-reactive T cells 59. Chemotherapy
might have increased the cross-presentation
of tumour cells in this trial; however, the
increased engraftment might have been
independent of antigen stimulation and
might have occurred primarily as a result
of homeostasis.
2005 Nature Publishing Group
PERSPECTIVES
Clinical implications
Measuring antitumour immune responses
in humans has been difficult because of the
lack of defined tumour antigens and the
lack of available tools to study the six key
steps in vivo.
However, the observations from animal
models have several implications for plan-
ning future studies combining immunother-
apy with chemotherapy in human clinical
cancer trials. First, as the level of cross-pre-
sented tumour antigen from established
tumours is increased by chemotherapy, it is
likely that the tumour itself will be a good
source of tumour antigens. This indicates
that there is no a priori requirement to
define, clone and purify individual tumour
antigens. Immunotherapy could then be
aimed at boosting responses to endogenous
cross-presented tumour antigens rather than
delivering more antigen by, for example, vac-
cination. Second, it cannot be assumed that
all drugs, even those that are known to
induce apoptosis, will have the same effects
on the immune system as gemcitabine. At
present, such data are not available for most
drugs and future drug evaluation will
require careful analysis. Third, individual
tumours will vary in their resistance to dif-
ferent chemotherapy agents and to apopto-
sis, and we do not know whether there is a
direct relationship between death and prim-
ing in this regard. It will be important to
determine which tumour characteristics
make them suitable and this might require
individualization for each patient, by
microarray analysis of apoptosis pathways.
Fourth, as post-chemotherapy delivery of
immunotherapy was always more effective
than pretreatment, the timing of such
immunotherapy is likely to be crucial. Also,
we noted that if the immunotherapy was
delayed following chemotherapy all the ben-
efits disappeared, presumably because either
the antigen-loaded APCs were cleared or the
partial priming of those APCs for CD40 sig-
nals disappeared. In cancer immunotherapy
trials it is usual to leave about 1 month
between cessation of chemotherapy and
commencement of immunotherapy. The
data discussed above indicate that a protocol
in which immunotherapy immediately fol-
lows chemotherapy, probably in repeating
cycles, might be more effective. Finally the
most appropriate adjuvant immunotherapy
to be used in such studies can only be
determined empirically.
As described above, immunotherapy in
the form of an APC-directed CD40 signal
following chemotherapy-induced apop-
tosis cures most tumour-bearing mice.
VOLUME 5 | MAY 2005 | 4 0 3
PERSPECTIVES
Humanized versions of CD40 antibodies are
now becoming available for clinical trials,
and studies combining chemotherapy with
this antibody can be undertaken. Another
way to deliver a CD40 signal is through
CD4+ T cells, so protocols that induce strong
antitumour CD4 responses could also be
combined with chemotherapy in the same
way. Viruses might also be used as adjuvants,
but in our studies the effects were not as
powerful as those seen with CD40.
At least some of the past failures of
immunotherapy can now be explained
based on our current ability to analyse anti-
tumour immune responses in vivo. For
example, we might have used IL-2 in situa-
tions where not enough antigen was avail-
able in the lymph nodes, we might have
waited too long before commencing
immunotherapy after chemotherapy and
we might have picked the wrong adjuvants
to combine with chemotherapy. Certainly,
the capacity we now have to analyse the
precise way in which each component of
the host antitumour immune system
engages with specific tumour antigens that
are destroyed by chemotherapy drugs will
enable any new clinical trials to be designed
on rational scientific foundations. A
first encouraging example of the clinical
reality of combining chemotherapy and
immunotherapy in melanoma patients has
now been published, as discussed above59.
Although the specific protocol was techni-
cally challenging, the clinical outcome was
extremely promising.
Richard A. Lake is at the Tumour
Immunology Group, School of Medicine and
Pharmacology, Western Australian Institute for
Medical Research, Perth, 6009, Australia.
Bruce W. S. Robinson is at the School
of Medicine and Pharmacology,
4th Floor, G-block,
Sir Charles Gairdner Hospital, Nedlands, Perth,
6009, Australia.
Correspondence to R.A.L.
e-mail: rlake@cyllene.uwa.edu.au
doi: 10.1038/nrc1613
Published online 20 April 2005
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Acknowledgements (FTase) or protein geranylgeranyltrans- sembly21,22. Therefore, it is clear that CAAX
We thank the current and former members of the Tumour
Immunology Group, but we are particularly grateful to R. van der ferase-I (GGTase-I), respectively3. Following proteins have a diverse range of functions
Most for proof reading the evolving manuscript and consistently the attachment of the isoprenoid, the AAX inside cells, but one common theme is that
thought-provoking debate. We apologize for our failure to fully
acknowledge many important contributions to this area. This tripeptide is removed in a reaction that is many of these proteins are involved in intracel-
research was supported by grants from the National Health and catalysed by a prenyl-protein-specific pro- lular regulatory processes that are important
Medical Research Council of Australia and the Cancer Council of
Western Australia. R.L. is supported by the Insurance tease known as RCE1, whereas in the third for tumorigenesis.
Commission of Western Australia. processing step a methyl group is trans- The most widely documented function of
Competing interests statement ferred to the now C-terminal prenylcysteine prenylation is to direct CAAX proteins to
The authors declare no competing interests. by the enzyme isoprenylcysteine carboxyl cellular membranes, although, in many
methyltransferase4,5 (ICMT; FIG. 1). cases, the modified C terminus is important
Online links Although many proteins are probably in proteinprotein interactions as well1,23.
DATABASES subject to the CAAX-processing pathway Regardless of how the protein uses its modi-
The following terms in this article are linked online to: (BOX 1), members of the RAS family of fied cysteine residue, one aspect is very clear
Entrez Gene:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene GTPases, which almost all contain the CAAX the modifications are crucial for the bio-
BCL2 | BCL-XL | CD40 | FAS | IFN | IL-10 | IL-12 | IL-6 | TNF motif, are particularly interesting because of logical activities of the proteins2427. For this
National Cancer Institute: http://cancer.gov/
melanoma
their well-established role in oncogenesis6,7. reason, the CAAX protein prenyltransferases,
Access to this interactive links box is free online. Mutational activation of RAS is associated most notably FTase, have been the focus of

NATURE REVIEWS | C ANCER VOLUME 5 | MAY 2005 | 4 0 5

2005 Nature Publishing Group