Immunotherapy___Chemotherapy[1] by Wang Dafeng

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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.

there has been a paucity of methods to accurately 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 developing case for combining chemotherapy and immunotherapy in cancer treatment. Chemotherapy and cell death

Chemotherapy remains the treatment modality of choice for most advanced cancers. However, for solid tumours in particular, 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, antagonistic 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,

Different chemotherapies kill tumour cells in different ways and in the process they can modulate the host immune system with consequences that are only now beginning to be fully elucidated. Here, we make the case for adjuvant immunotherapy for patients undergoing 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 chemotherapy. 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

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of survival signals1. A generally accepted 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 different cell types, including macrophages. In the absence of inflammatory signals, the macrophages phagocytose the apoptotic remnants3. Apoptosis has long been considered as non-immunogenic or even tolerizing, occurring in the absence of any inflammation4. 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 immunologically ‘bland’, whereas apoptosis following 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 powerful 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 associated 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 inflammation might be expected to appear like normal tissue turnover, generating either no immune response (‘ignorance’) or tolerance. Some experiments support this hypothesis, showing that tumour-derived antigens from a lymphoma and a solid VOLUME 5 | MAY 2005 | 3 9 7

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Lymph node CD8+ T cell

Tumour cell

a Tumour

Antigen

APC CD8+ T cell Circulation

e Traffic

Tumour blood vessel

Apoptosis and the immune response d

Memory T cell

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 required and can be modulated by a range of factors: tumour antigens must be present (a); these antigens must reach/load ‘professional’ presenting cells — antigen-presenting cells (APCs) — in the draining lymph node (b); specific T cells must respond by proliferation (c); the circulating T cells must enter the tumour (d); once in the tumour the T cells must be able to overcome local immune-suppressive molecules to recognize and kill targets (e); memory cells should be generated (f). Cancer immunotherapy can fail at any of these steps, so the development of assays to analyse each of these steps in vivo (TABLE 1) has been the key to beginning to understand how chemotherapy and immunotherapy interact. MHC; major histocompability complex.

tumour induce tolerance during tumour progression6,7. However, there is now increasing evidence that, under the right circumstances, chemotherapy-induced tumour-cell death can set the stage for an effective antitumour immune response. Anticancer drugs can induce apoptosis both by death-receptor-dependent and -independent pathways. Some anticancer drugs increase the expression of death receptors, including FAS, tumour-necrosis factor (TNF) receptors and TNF-related apoptosisinducing ligand receptors. Tumour cells commonly show abnormalities to various components of these pathways8, so tumours are differentially sensitive to these drugs. Other drugs do not alter expression of death receptors, but trigger apoptosis by inducing release of cytochrome c from mitochondria. Although chemotherapeutic drugs induce their primary damage in many different ways, most of them induce apoptosis not only in vitro but also in vivo 9,10. For example, a diverse set of agents, including cytarabine, mitoxantrone, etoposide and topotecan, have

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positive outcome for this drug in combination with a dendritic cell (DC)- (antigen-presenting cell (APC))-based vaccination therapy20. Other chemotherapy agents such as paclitaxel induce an abnormal metaphase by damaging microtubules and disrupting the mitotic spindle. Cyclin-dependent kinase 1 activation is abnormally prolonged in paclitaxel-treated cells, resulting in a bypassing of the G2 checkpoint and cell death by mitotic catastrophe21,22. The finding that paclitaxel is less effective than the apoptosis-inducing drug doxorubicin when coupled to a vaccination protocol for the treatment of aggressive breast cancer23 might indicate that mitotic catastrophe errs on the side of being immunologically bland.

been shown to increase the number of apoptotic blasts in leukaemia11. Importantly, the degree of apoptosis was found to correlate with clinical outcome for several different tumour types12–14. Death by non-apoptotic mechanisms. Apoptosis is not the only mechanism by which cells die. Non-apoptotic death pathways include necrosis, autophagy and mitotic catastrophe. These alternate forms of cell death are differentiated by particular combinations of morphological and biochemical changes15,16. Significantly, different chemotherapeutic drugs have been shown to induce different forms of cell death. Temozolomide, for example, is a relatively new alkylating agent that induces G2/M arrest and autophagy with no apoptosis17,18. There have been no systematic analyses of the effects of this drug on the immune system, but it is a powerful inhibitor of lymphocyte proliferation, with only marginal effects on the activity of the natural-killer lymphocyte subset19. Indeed, a single case report focusing on a child with recurrent malignant glioma describes a

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It is evident that some mechanistic understanding of the essential principles of immune recognition is required before we can discuss how dead and dying tumour cells alter this process (BOX 1). It is generally accepted that the sort of immune response that would be favourable to tumour elimination will include the generation of large numbers of interferon-γ (IFNγ)- and TNFαsecreting CD8+ T cells with the capacity to directly lyse tumour-cell targets. Therefore, the key issues to be aware of in pairing chemotherapy with immunotherapy relate to the molecular flags associated with different forms of cell death and how they are contextually interpreted. There is increasing evidence that some part of the apoptotic process is required for generating an immune reaction. In a conceptually simple series of experiments, Bonotte and colleagues showed that immunogenic colon carcinoma cells were sensitive to apoptosis and died in vitro if they were starved of growth factors. When these cells were made resistant to apoptosis by overexpressing the anti-apoptotic protein BCL2, they gave rise to progressive tumours in vivo. Interestingly, the antigenicity of the BCL2-expressing apoptosis-resistant cells was not altered, because animals that had been pre-immunized with parental immunogenic cells were able to reject the BCL2-expressing cells24. Apoptosis might also be a necessary component of some vaccine strategies. The degree of apoptosis might help to explain the finding that Alphavirus-based vaccines, which induce a high level of apoptosis, are generally highly immunogenic despite the fact that they produce less antigen than conventional DNAbased vaccines. Leitner and colleagues switched off apoptosis by overexpressing the anti-apoptotic gene BCL-XL in an Alphavirus-based immunization approach. As expected, they

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PERSPECTIVES found that cells that were transfected with BCL-XL lived longer and produced more antigen, 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-studied 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 otherwise be packaged into the apoptotic bodies are released. Phosphatidylserine stimulates the production of a set of anti-inflammatory mediators, including transforming growth factor-β, 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 apoptosis in vivo is efficient, tumour-cell apoptosis would be likely to result in antigen sequestration or tolerance induction. However, when massive apoptosis occurs, the tolerogenic system might be overwhelmed, resulting in secondary necrosis and release of pro-inflammatory mediators30. Some of the known activators of immune function include heatshock 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-stimulatory molecules (CD40, CD80 and CD86) accompanied by an increase in IL-12 secretion. 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 endogenous 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 interactions 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.

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-γ and tumour-necrosis factor-α. 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 nonself 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 Cell and virus Cell and Cell and self cells to mature and express stimulatory 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 naturalkiller cells. Missing self brings an important supplementary concept — an Tolerizing Weak 'Dangerous' immune response can be activated by the • None of • No danger • Cell damage loss of inhibitory signals that would these signals signals (e.g. uric acid) normally block the initiation of immune • Innate immunity • No CD40 65 signals activated responses against self . • Pro• Toll-like The fourth important ‘D’ is duration — inflammatory receptors cytokines triggered how long a particular antigen is around has • Inflammation profound implications for the induction of • Cytokines immunological memory58. • CD40 signals • Others These concepts are illustrated in the figure.

Non-apoptotic death and the immune response. Few studies have investigated the immune response to non-apoptotic death caused by chemotherapy, largely because there are few such drugs; some of these studies are cited above. Several studies based on in vitro work have found that cell death by necrosis induces DCs to act as potent APCs, whereas apoptotic cell death does not have this effect34. Therefore, in the steady state of normal tissue turnover, apoptotic death seems to be tolerogenic, whereas necrotic death is more likely to be associated with danger and the induction of an immune response (reviewed in REF. 35 and see BOX 1). The immunological environment endured following massive apoptotic cell death after effective chemotherapy is likely to be different, as discussed below.

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The tumour immune response in vivo

The interaction between chemotherapy and immunotherapy has not been extensively studied in vivo because the necessary technology has only recently been developed (TABLE 1) . In vivo studies are necessary because in vitro studies cannot accurately reflect the biology and dynamics of these complex interactions. This technology includes the development of appropriate animal models and the ability to analyse tumour-antigen-specific immune responses at each of the six key checkpoints of the antitumour response (FIG. 1). These assays can help to determine where in the sequence of events a particular therapy fails. For example, if the dose of antigen is not limiting but the capacity of T cells to expand is limited, then increasing the dose of antigen is unlikely to improve responses, VOLUME 5 | MAY 2005 | 3 9 9

PERSPECTIVES Table 1 | Six key steps for specific CD8-mediated antitumour immunity Step

In vivo assay system for measuring response

Antigen threshold

Quantification of levels of marker antigens by ELISA or western blot

Antigen presentation

CFSE dye dilution assays66

T-cell response

In vivo CTL assay using CFSE- and peptide-loaded target cells67; tetramer analysis of tumour-specific T-cell numbers68; ELISPOT or intracellular cytokine staining for cytokine production — can be combined with other assays69

T-cell traffic

Staining for T-cell infiltration; flow cytometry of extracted cells; trafficking of dye-labelled cells

Target destruction

Reduction in size of tumour; cytokine staining in tumour; function of cells extracted from the tumour

Generation of memory

Flow cytometry of extracted cells; adoptive transfer of extracted cells; tumour growth after rechallenge

CSFE, 5,6-carboxyfluorescein diacetate succinimidyl ester; CTL, cytotoxic T lymphocyte; ELISA; enzymelinked immunosorbance assay, ELISPOT: enzyme-linked ‘spot’ recognition of specific T-cell function.

whereas delivery of agents that support T-cell expansion is more likely to prove successful. These models and the tools to study them have already produced new insights into antitumour immune responses. We and others have transfected antigens into tumour cell lines to act as markers to analyse tumour-specific responses. These antigens do not alter the immune response to the tumour, they simply ‘report back’ to the investigator what is happening in vivo. The other crucial reagents in this system are tumour-antigen-specific T cells. 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 analyse antigen presentation in vivo. These cells can be used as the cellular equivalent of tumour-specific monoclonal antibodies. Using these tools, we can now begin to answer six key questions on how chemotherapy impacts on the tumour immune response (TABLE 2). How might chemotherapy affect the range of antigens delivered for presentation? There are large numbers, possibly tens or even hundreds, of potential tumour antigens in any particular cancer36–38. These are of two types, ‘neo’ and ‘self’, tumour antigens. ‘Neoantigens’ are antigens that the host immune system has never ‘seen’, so no tolerance has been induced. These are strong antigens and examples are mutated proteins and oncogenic viruses. ‘Self ’ tumour antigens are unmutated self proteins, but they are able to induce immune responses because they have limited expression in the host and therefore limited opportunity for induction of tolerance. Examples are proteins that have expression limited to tumours and testes (known as ‘cancer-testis’ antigens) and differentiation antigens that are not expressed in early life. These tumour antigens will be present at different concentrations so that

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some will be efficiently delivered to the immune system whereas others will not reach that threshold. When tumours regress with chemotherapy, increasing amounts of these antigens are released from the dead and dying cells and could be delivered into the antigenpresentation pathways, making them available to induce immune responses. It is likely that in this process some antigens that would otherwise be below the threshold of availability could now be presented to the host’s immune system. This does not necessarily

mean that these antigens will induce a response. The factors that determine whether an available antigen elicits an immune response are explored in BOX 1. How might chemotherapy affect the dose of antigens delivered for presentation? One of the key reference points for the generation of a particular immune response is antigen dose (BOX 1). In a series of experiments using one of the above-described mouse models, we were able to show that a tumour neo-antigen was constitutively and efficiently delivered to the lymph nodes that drain the tumour, and only those nodes, by a process known as ‘cross-presentation’39,40. Cross-presented antigens ‘cross’ from an exogenous source, which typically delivers these antigens into the major histocompatibility complex class II pathway, into the class I pathway, a pathway previously thought to be restricted to endogenous antigens expressed exclusively by the cell itself. Despite this, little activation of antitumour CD8+ T-cell responses resulted except for low constitutive levels of cytotoxic T lymphocyte (CTL) activity restricted to the draining lymph node. It is still not known whether live or dead or dying tumour cells constitutively deliver these antigens into the cross-presentation pathway.

Table 2 | How chemotherapy could augment immunotherapy Essential steps in the induction of an antitumour immune response

Potential effects of chemotherapy on the capacity of immunotherapy to destroy tumours

Antigen threshold

Delivery of a broader range of different tumour antigens

Antigen presentation

Increased antigen cross-presentation

Partial activation of dendritic cells

T-cell response

References

Priming of APCs for CD40 signal

Killing subsets of APC

No tolerance induction by apoptotic tumour cells

Lymphopaenia-related proliferation increases tumour-specific T-cell response

T-cell traffic

Increased T-cell accumulation within tumour

Target destruction

Increased local tumour-antigen cross-presentation (permitting CD8 re-stimulation) Tumour debulking (less systemic suppression, smaller target, less chance for escape variants etc.) Partial sensitization of tumour cells for CTL lysis

* 70 55,56

Generation of memory

Promotion of long-term antigen-independent memory

External regulation of these steps

Increased delivery of exogenous antigen

Increased CD4 help (for example, delivery of CD40 signals)

Reduction in function of negative regulatory cells Induction of homeostatic proliferation

44,46 59

*Not yet demonstrated in tumour models. APC, antigen-presenting cell; CTL, cytotoxic T lymphocyte.

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PERSPECTIVES a No tumour

b Tumour only Undivided cells

c Tumour and gemcitabine Dividing cells

Dividing cells

Forward scatter

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 following effective chemotherapy. In fact, not only was there no reduction in tumour-antigen 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, approximately doubled41. Because these assays are functional, it is impossible to accurately quantify how this change in proliferation equates to the precise increase in the number of molecules of antigen that appear in the crosspresentation 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 established tumours by over 80%. The observation that a gemcitabine-resistant tumour did not show any increase in tumour-antigen cross-presentation indicated that the capacity of chemotherapy-induced tumourcell 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 functions of APCs or on the endogenous immune response41. Therefore, we hypothesize 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 differential toxicity for particular subsets of cells. Gemcitabine differentially depletes B cells and induces a profound suppression of antitumour antibody responses43. B cells are bona fide APCs and might drive immune responses towards the generation of antibodies 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’ protocols, the aim of which is to generate CTLs. By contrast, vaccination protocols requiring a

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 5,6carboxyfluorescein 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 for maximal efficacy might be compromised in patients treated with gemcitabine. How might chemotherapy affect the antitumour T-cell response in the draining lymph node? Despite increasing tumour-antigen presentation in the draining lymph node, chemotherapy does not usually induce an effective antitumour response by itself. In the clinic, it is rare for cancers that have partially regressed in response to chemotherapy to then continue to regress through immune mechanisms when the treatment stops. Animal experiments support the view that chemotherapy, as a single protocol that is not curative, does not usually induce an immune response (see the section on memory below). The notable exception to this observation is cyclophosphamide (CTX). The immunomodulatory effects of CTX have been known for a long time. In 1974, when the vogue for describing immune responses in terms of suppressor circuits was at its height, it was found that some forms of tolerance could be reversed by CTX. Appropriate doses of CTX have no major direct effect on the number of lymphocytes but act by removing T-suppressorcell activity 44. Suppressor cells are now more usually characterized as regulatory T cells45. Above, we discussed how immunogenic colon carcinoma cells could be manipulated and made tolerogenic by inhibiting their tendency to apoptose. Tolerogenic colon carcinoma cells grow progressively in vivo with a corresponding expansion of regulatory T cells. If these regulatory T cells

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are extracted and transferred to animals with immunogenic tumours, these tumours start to grow progressively. A single administration of CTX to animals bearing tolerogenic, progressively growing tumours depletes regulatory T cells and delays the growth of the tumour. When CTX is combined with an immunotherapy that is not curative by itself, the combination has the capacity to eradicate established tolerogenic tumours in animal models46. Given that an organism has a T-cell repertoire with the capacity to recognize a given antigen (the issue of difference), two fundamental questions operationally define whether the immune system will respond to it. The first is whether the antigen is present above a threshold dose level and the second is whether the antigen is dangerous. These questions are of course ‘put and answered’ in biochemical terms (BOX 1). Duration of exposure to antigen also has profound consequences for the development of immunological memory and is discussed below. Understanding the specific molecular events that transmit dose and danger signals is important for understanding how antitumour chemotherapy and immunotherapy could interact. Cross-presented antigens will be ignored if there are no T cells with receptors of appropriate specificity. When there are T cells that can respond, cross-presented antigen can lead to their activation and proliferation or can induce tolerance28,47. Crosstolerance has been described for the antigen used in our studies — influenza virus haemagglutinin (HA) — when it is expressed VOLUME 5 | MAY 2005 | 4 0 1

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How might chemotherapy affect T-cell traffic? 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 circulation 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

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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 tumourspecific CD4+ T-helper-cell response, as we

a Anti-CD40 therapy without apoptosis 100

Percent survival

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 TNFα and IL-6 with the associated symptoms of weight loss, anorexia, fevers and thrombocytosis. This balance between pro- and anti-inflammatory mediators might block CTL induction, but additional chemotherapy could increase the pro-inflammatory mediators such as HSPs to sufficient levels to induce CD8+ T-cell responses51. The increased number of dead and dying tumour cells is also likely to invoke an increase in phagocytosis, and if these cells are activated, particularly by the release of intracellular contents, they might release more pro-inflammatory cytokines and so increase the responsiveness of APCs to cross-presented tumour antigens52. Another possible direct effect of chemotherapy on cross-priming has been attributed to alkylating agents. In fact, any drugs that modify DNA might have particular effects on the host immune response. For example, co-culture of immature DCs with tumour cells treated with melphalan and chlorambucil caused the DCs to upregulate co-stimulatory molecules, secrete IL-12 and efficiently activate T cells 53. These effects could be recapitulated using DNA purified from the killed tumour cells, supporting the hypothesis that DNA damage is itself recognized as inflammatory 53. The precise mechanisms whereby chemotherapy partially primes APCs and antitumour T-cell responses is now under investigation, as are the ways in which this can be augmented by agents other than CD40 and tumour-antigen-containing viruses.

0 0

100

120

140

120

140

Days after tumour injection Control Anti-CD40

b Anti-CD40 therapy with apoptosis 100

Percent survival

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 responder T cells48. This process is known as ‘peripheral tolerance’ and is a well-established 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 frequency of tumour-antigen-specific T cells. Although it increases the rate of cross-presentation and therefore the amount of antigen available to potentially cross-tolerize, it does not substantially reduce the activity of CTLs in vivo. Overall, the data do not support the view that the induction of tumourcell apoptosis tolerizes the tumour-specific CD8+ T-cell response. However, chemotherapy-induced antigen 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 APC that is loaded with antigen activates or 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 delivered 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 draining 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 antigens but also sensitizes them to CD40 signals, curing most mice studied in this way. This immunotherapy is much more effective if delivered after apoptosis-induced antigen loading rather than before50 (FIG. 3).

0 0

100

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. Kaplan–Meier survival curves are shown.

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PERSPECTIVES 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 efficiently 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 frequency, and it might affect the threshold sensitivity at which these cells are triggered as well as qualitatively affect the secretion of IFNγ and TNFα. 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, providing 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 cancer cells to lysis by weak, or low-avidity, CTLs that would not otherwise kill them56. Drugs such as doxorubicin and methotrexate promote apoptosis in some tumour cells by inducing an upregulation in transcription of the gene encoding FAS ligand54. Other chemotherapy agents alter apoptosis pathways in different ways, thereby altering the threshold of apoptosis sensitivity. Where different 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 generation of immunological memory? In addition to the antitumour effector responses described above, the development of longterm immunological memory could provide protection from both recurrence and metastases. Previously, we have noted that

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 occasional 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 antigen-driven proliferation and then differentiate 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 contrast, if antigen persists such as during a chronic infection or during tumour growth, even after a partial response to chemotherapy, 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 numbers 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 protocols. 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.

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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 planning future studies combining immunotherapy with chemotherapy in human clinical cancer trials. First, as the level of cross-presented 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, vaccination. 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 different chemotherapy agents and to apoptosis, and we do not know whether there is a direct relationship between death and priming 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 benefits disappeared, presumably because either the antigen-loaded APCs were cleared or the partial priming of those APCs for CD40 signals 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 follows 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 apoptosis 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 antitumour immune responses in vivo. For example, we might have used IL-2 in situations where not enough antigen was available 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 technically 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 1. 2. 3. 4.

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Acknowledgements We thank the current and former members of the Tumour Immunology Group, but we are particularly grateful to R. van der Most for proof reading the evolving manuscript and consistently thought-provoking debate. We apologize for our failure to fully acknowledge many important contributions to this area. This research was supported by grants from the National Health and Medical Research Council of Australia and the Cancer Council of Western Australia. R.L. is supported by the Insurance Commission of Western Australia.

Competing interests statement The authors declare no competing interests.

Online links DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene BCL2 | BCL-XL | CD40 | FAS | IFNγ | IL-10 | IL-12 | IL-6 | TNFα National Cancer Institute: http://cancer.gov/ melanoma Access to this interactive links box is free online.

OPINION

Post-prenylation-processing enzymes as new targets in oncogenesis Ann M. Winter-Vann and Patrick J. Casey Abstract | RAS and many other oncogenic proteins undergo a complex series of post-translational modifications that are initiated by the addition of an isoprenoid lipid through a process known as prenylation. Following prenylation, these proteins usually undergo endoproteolytic processing by the RCE1 protease and then carboxyl methylation by a unique methyltransferase known as isoprenylcysteine carboxyl methyltransferase (ICMT). Although inhibitors that have been designed to target the prenylation step are now in advanced-stage clinical trials, their utility and efficacy seem to be limited. Recent findings, however, indicate that the inhibition of these post-prenylationprocessing steps — particularly that of ICMT-catalysed methylation — might provide a better approach to the control of cancer-cell proliferation.

A broad class of eukaryotic proteins contain a carboxy-terminal CAAX motif, in which the ‘C’ denotes cysteine, the ‘A’ residues are usually aliphatic, and ‘X’ represents any amino acid1,2. The CAAX motif directs proteins through a series of post-translational modifications that are initiated by the attachment of a 15-carbon farnesyl or a 20-carbon geranylgeranyl lipid to the cysteine residue by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase-I (GGTase-I), respectively3. Following the attachment of the isoprenoid, the AAX tripeptide is removed in a reaction that is catalysed by a prenyl-protein-specific protease known as RCE1, whereas in the third processing step a methyl group is transferred to the now C-terminal prenylcysteine by the enzyme isoprenylcysteine carboxyl methyltransferase4,5 (ICMT; FIG. 1). Although many proteins are probably subject to the CAAX-processing pathway (BOX 1), members of the RAS family of GTPases, which almost all contain the CAAX motif, are particularly interesting because of their well-established role in oncogenesis6,7. Mutational activation of RAS is associated

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with various human cancers7, and is a principle step in a mechanism that can efficiently transform tissue-explant human cells8. In addition, many cancers contain alterations in elements that lie upstream of RAS in signalling cascades — such as the amplified expression or mutational activation of tyrosine kinases — and the resultant hyperactivation of RAS is thought to contribute to tumorigenesis7,9,10. The targeting of RAS signalling pathways is therefore important for research into the development of therapeutics. In addition to the RAS proteins themselves, several other CAAX proteins are involved in the initiation and progression of cancer (TABLE 1). The RHO family of GTPases, which includes RAC and CDC42, is implicated in both oncogenesis and metastasis11,12. Increased signalling by yet another GTPase, RAP1A, has been associated with myeloproliferation13. Constitutive activation of G-proteincoupled receptor (GPCR) pathways can also contribute to transformation14–18, and the γ-subunits of heterotrimeric G proteins are all CAAX proteins16. The list of CAAX proteins also includes many phosphatases and kinases — mutations in several of these are associated with cancer19,20. Finally, in both normal and transformed cells, CAAX proteins — including the nuclear lamins A and B, and the centromeric proteins CENP-E and CENP-F — are involved in processes that are important for cell division and nuclear-envelope assembly/disassembly21,22. Therefore, it is clear that CAAX proteins have a diverse range of functions inside cells, but one common theme is that many of these proteins are involved in intracellular regulatory processes that are important for tumorigenesis. The most widely documented function of prenylation is to direct CAAX proteins to cellular membranes, although, in many cases, the modified C terminus is important in protein–protein interactions as well1,23. Regardless of how the protein uses its modified cysteine residue, one aspect is very clear — the modifications are crucial for the biological activities of the proteins24–27. For this reason, the CAAX protein prenyltransferases, most notably FTase, have been the focus of VOLUME 5 | MAY 2005 | 4 0 5