All in the head: obstacles for immune rejection of brain tumours

Tumour immunology and immunotherapy is a highly active field, a clinical testing ground for cutting edge immunological techniques and concepts. But this is after many years of fundamental advances in basic immunology. In this article we suggest that immunotherapy for brain tumours cannot be rationally advanced as rapidly as that for tumours in other sites. Our understanding of anti-tumour immune responses in the brain is sketchy and frequently extrapolated from other tissues having little in common with the central nervous system (CNS). The result is that current clinical trials are built upon shakier foundations, with the somewhat naive optimism that what is looking hopeful for other tumours will also be applicable to cerebral malignancies. But of course it is easy to criticise such well-meaning attempts to treat currently incurable cancers. In the basic and preclinical domain, brain tumour models that are readily applicable to the design of future immunotherapies are only in their infancy. The ideal transplantable tumour that reiterates the key features of a malignant primary astrocytoma (poorly immunogenic, infiltrative but non-metastatic, expressing multiple mechanisms mediating immune escape) has yet to be discovered. In the meantime, we must use individual model tumours and limit the scope of the conclusions that we make from each system. Moreover, we must overcome the significant technical difficulties encountered as we strive to preserve brain integrity, whilst implanting tumours in this unique site. Or we can look to genetic models, in which there is the development of 'spontaneous' brain tumours (in some cases aided by the intracerebral delivery of a viral vector) incorporating many of the genetic features and heterogeneity typical of spontaneous human cancer.1 However, these models have generally been constructed to address genetic and pathological issues and they present a significant challenge for interpretable immunological studies. With these difficulties in mind, perhaps we need to accept that brain tumour models and clinical immunotherapies are currently in their first generation. To progress to a more successful second generation of therapies, there is a need to abandon the idea that an automatic one-way progression exists from rodent models to the clinic. We need better models to make better therapies, but how to choose and design the models can be greatly guided by data from clinical trials, if the trial design actually permits the generation of useful biological data. To date, most brain tumour immunotherapies have borrowed technologies and approaches already explored for tumours in other sites, principally melanoma. Thus, most of the now 'classical' tumour immunology approaches have been explored for brain tumour immunotherapy: cytokine immune enhancement, whole tumour cell vaccines, cytokine-modified tumour cell vaccines, gene therapies with immune bystander effects, dendritic cell therapies (reviewed in refs. 2–4). A few notable and exciting novel approaches unique to the CNS should not be passed aside, for example, exploiting the migratory properties of neural stem cells to deliver immunoactive molecules efficiently to the tumour site.5 The overall conclusions from these studies are that, depending upon the stringency of the models utilized, tumours that are pre-established in the brain are generally more difficult to eliminate than those in other sites, and may require different effector mechanisms. For example, studies in which multiple cytokines have been tested as modulators of immune responses gave different results according to the tumour model (SMA-560, B16) and site of implantation.6,7 Other attempts to create a cellular vaccine by overexpression of intercellular adhesion molecule-1 on a glioma cell line, resulted in growth inhibition of glioma cells implanted subcutaneously, but not in the CNS.8 Another recent study using a recombinant Listeria monocytogenes tumour vaccine revealed more stringent T-cell subset requirements for protection against an intracerebral challenge compared with the same tumour implanted by the subcutaneous route.9 These examples, together with the fact that no convincingly successful clinical brain tumour immunotherapy has been demonstrated to date, should force us to reassess what we understand about brain tumour immunology, rather than just brain tumour immunotherapy. It is from this perspective that we will discuss the issues pertinent to the problem by drawing from both clinical and experimental situations. Both primary brain tumours and intracranial metastases pose a serious clinical problem, although their involvement with the host immune system will presumably have followed a different evolution. Concerning primary brain neoplasms, those derived from astrocytes are the most frequent, and the anaplastic astrocytomas and glioblastomas (grade III and IV astrocytomas, according to the WHO designation) are the most lethal. Indeed, despite some advances in surgical resection, radiotherapy and chemotherapy,10 it is unlikely that long-term survival rates can be significantly extended beyond the current median survival of less than 12 months for glioblastomas.11 Furthermore, although for low-grade astrocytomas the outlook is more favourable, not all can be adequately treated and they may progress to malignant lesions.1 Malignant astrocytomas infiltrate normal tissue, which renders total surgical resection virtually impossible without extensive neurological damage. It is thus essential to consider novel treatments such as immunotherapy in the hope of attacking the residual radioresistant and chemoresistant tumour cells. Other tumours metastasizing to the brain, such as melanoma, pose similar problems,12 and for these tumours it would also be useful to propose a therapeutic option that is applicable to the CNS. Whilst certain criteria for acceptable anti-tumour responses are applicable to all tumours, there are other requirements that are more stringent for cerebral malignancies. Indeed, the categorization of the CNS as an immune privileged site has perhaps retarded the development of immunotherapies for brain tumours, with the temptation to anticipate ineffective immune function in the brain. However, this prejudgement of the domain has arisen from a degree of misunderstanding of immune privilege. The term originally arose to describe the results of transplantation experiments in which there was extended survival of tissues transplanted to the CNS, compared with their survival in other sites.13,14 Features of the CNS were identified (certain of which are discussed individually in the sections below) that were proposed to explain this apparent lack of immune reactivity. These included the presence of the blood–brain barrier (BBB), low major histocompatibility complex (MHC) expression in the brain parenchyma, the absence of organized lymphatic drainage and a lack of dendritic cells in the normal brain parenchyma. Nevertheless, it is now apparent that immune reactions can and do occur in the CNS: autoimmune diseases of the CNS;15 immune responses to neurotropic viruses16 and parasites;17 and, as will be discussed herein, anti-tumour responses. Immune privilege is thus a term that requires an updated definition: it should remind us that although immune responses in the brain are often qualitatively and quantitatively different to those found in other sites, they are not absent. As elegantly phrased by Fabry et al.,18 we have to consider immune responses in the CNS as having a certain 'dialect'. Those aspects of immune privilege that impinge on afferent immune responses are clearly of fundamental interest, but may be less critical for brain tumour immunotherapy. For most vaccine strategies, immune responses will be induced at sites remote from the tumour, with the aim that effector cells can then recirculate to mediate their anti-tumour effects in the brain. The discussions that follow will therefore concentrate on those particularities of the CNS that influence the efferent arm of anti-tumour immune responses (Fig. 1), with a bias towards those that are presumed to be T-cell-mediated. Overview of the obstacles encountered by effector T cells primed in the periphery as they migrate towards a tumour situated in the brain parenchyma. After extravasation through the intact or locally compromised blood–brain barrier (BBB) (1),1 primed T cells encounter CNS-resident cells such as microglia and astrocytes (2), capable of antigen presentation, tolerance induction, or cytokine-mediated immunomodulation. Constitutive immunosuppressive factors present in the brain parenchyma (3) may also impede full differentiation or expression of effector molecules. As infiltrating T cells reach the tumour bed, they will be exposed to high concentrations of tumour-secreted factors that may synergize with tumour cell surface molecules to down-regulate effector function (4). Finally, efficient immune recognition of the tumour cell may be compromised by passive mechanisms of immune escape, such as low or absent MHC molecule expression and inefficient processing and presentation of tumour-associated antigens (5). Further details and references for these mechanisms are in the text. The first requirement for an effector T cell is that it must reach its target, the tumour. The problem of adequate tumour infiltration is applicable to all solid cancers.19,20 but when the tumour is located in the brain parenchyma, the T cell must also penetrate the tight junctions between the endothelial cells of the cerebral vasculature constituting the BBB.21,22 The integrity of this barrier is maintained by cells in intimate contact with the abluminal surface of the endothelium. Pericytes, perivascular cells and particularly astrocytes are implicated, the latter cells almost totally surrounding the vessel with their foot processes.23 Whilst the brain microvessels constituting the BBB appear impermeable compared with other microvessels, the barrier is conditional and selective. There is molecular and cellular traffic in both directions, but this is tightly regulated. Activated T cells can extravasate, but their trafficking may be more limited than for other sites. For example, the relative number of activated T cells found in the brain parenchyma after intravenous adoptive transfer of labelled T cells in rats was six times less than in muscle and more than 140 times less than that found in liver, for the same weight of tissue.24 For CD4+ T cells, migration away from the perivascular space into the parenchyma is inhibited in mice depleted of macrophages, suggesting that there are essential interactions with a perivascular cell after extravasation.25 The crux of the issue for tumour rejection is whether sufficient T cells reach their target to exert their anti-tumour effect. It is very difficult to quantify this for any tumour, nevertheless, under optimized conditions in different animal models, CD8+ T-cell-dependent immune responses are able to mediate anti-tumour effects in the brain.6,9,26–33 For spontaneous malignant astrocytoma in humans, the integrity of the BBB is locally compromised, and tumour-induced angiogenesis will not incorporate BBB characteristics. T-cell infiltration frequently occurs,34–36 but has only occasionally been correlated with a favourable prognosis.37 However, to date, the specificity and function of astrocytoma-infiltrating lymphocytes remains to be defined. If we consider the CD8+ cytotoxic T lymphocyte (CTL) as the prime effector T cell for tumour rejection, its fine specificity is of critical importance for the brain. A degree of collateral damage is considered acceptable for tumours in certain extracerebral locations38 and many defined experimental vaccines aim to induce CTLs that recognize differentiation antigens, such as Melan-A, expressed in melanoma cells and normal melanocytes.39 Nevertheless, the severity of the autoimmune component of experimental melanoma vaccines, compared with their anti-tumour efficacy is being closely monitored.40,41 For the CNS, autoimmune reactions in the brain can be very serious, because most tissue is indispensable and has limited capacity for self-renewal. Indeed, early tumour immunology studies noted that lethal allergic encephalomyelitis was induced in different species by immunization with human glioma tissue.42 It is clear that a better defined tumour vaccine will have a greater chance of avoiding such unacceptable results. However, this necessitates identifying antigens for T cells expressed by brain tumours. For malignant astrocytomas, identification of antigens recognized by T cells is far less advanced than for melanomas. However, certain antigenic similarities may be expected between melanomas and astrocytomas because their normal tissue counterparts derive from the neuroectoderm. Most studies addressing this possibility have analysed antigen expression at the mRNA level by reverse transcription–polymerase chain reaction. An initial study detected a proportion of tumours expressing one of several melanoma-associated antigens including MAGE family members, tyrosinase, TRP-1, TRP-2, gp100 and p97.43 However, subsequent publications found a much lower proportion of tumours expressing MAGE antigens, although occasional expression of other cancer-testis antigens was noted (SSX-1, SSX-2, SSX-4, SCP-1, TS85, and MAGE and GAGE family members).44,45 Although these results underline the potential antigenicity of certain brain tumours, some of the antigens that have a relatively high frequency of expression in malignant astrocytomas may be less useful as targets for immunotherapy because of their high homology to self-antigens (e.g. tumour-expressed GAGE-3 to GAGE-6 and GAGE-8 that are homologous to normal-brain-expressed GAGE-2 and GAGE-7).45 Furthermore, the only confirmation that epitopes from any of these antigens are presented at the cell surface for recognition by T cells comes from the study of certain astrocytoma cell lines that can be recognized by MAGE-specific CTL.46 However, this result reflects MAGE-1 antigen expression in cultured cells, whereas in vivo, astrocytomas are generally found to be negative for the MAGE-1 protein.45,47 This can probably be explained by a different level of DNA methylation induced by culture, because this regulates MAGE expression.48 A further candidate astrocytoma antigen that warrants further investigation is SART1259, originally identified in epithelial cancer cells and now shown to be expressed in various brain tumour lines and biopsies, including malignant astrocytoma.49 Astrocytoma cell lines expressing high levels of HLA-A24 and SART1259 could be recognized and killed by specific CTL derived from an oesophageal cancer patient, although no evidence of autologous responses was presented. Another possibility for an astrocytoma-associated antigen expression may be from neoepitopes present in epidermal growth factor receptor variant III (EGFR-vIII), expressed in a large proportion of malignant brain tumours.50 This antigen has until recently been explored as a target for monoclonal antibodies (initially for diagnostic purposes, but more recently for therapeutic application in preclinical mouse models.51 However, the possibility that EGFR-vIII-encoded T-cell epitopes may contribute to the anti-tumour response has been investigated in mouse models.52,53 and a vaccine incorporating a peptide from EGFR-vIII is currently under clinical trial. For most primary brain tumours, the issue of MHC expression is rather different to that in tumours derived from tissues of non-CNS origin, because the normal tissue counterparts of astrocytes and oligodendrocytes are essentially MHC negative or low.54,55 It is thus essential that MHC molecules are induced either during tumorigenesis or during immunotherapy-induced anti-tumour responses if classical CTL-mediated cytotoxicity is to be operational. For tumour cells of astrocytic origin, these uniformly express MHC class I molecules after in vitro culture, and can be induced to express MHC class II after interferon-γ (IFN-γ) treatment (refs 46, 56–58 and our own unpublished observations). The situation in vivo is far from clear, with contradictory reports in the literature.54,55,59,60 This may be because of differences in the immunohistochemistry protocols employed60 and difficulties in obtaining optimal staining for tumours such as high-grade astrocytomas that are characterized by zones of necrosis. The generally low MHC expression by normal tissue may actually be an advantage during immunotherapy in that it may spare normal tissue from immune attack. However, normal astrocytes MHC particularly after with vivo, as for cells, the situation is more on data from mouse models, some studies have demonstrated that astrocytes can be induced to express MHC class I molecules after viral or to However, other have that in the absence of the and the cell are the cells of the It is clear that this issue of MHC expression of both normal and tissue, and at various during will be in attempts to both and any effects for of in the CNS rapidly to a of intracranial because of the the of the This may and the cerebral by the presence of intracerebral tumour neurological function and of critical cells for which cannot be Furthermore, may and contribute to the or of autoimmune Indeed, in a recent rodent gene model for there was evidence that intracerebral immune responses may have or and is often the to immune responses induced by successful tumour vaccines, with some evidence that this may be without in a mouse model in which there is rejection of an tumour implanted In is often by of a treatment with of immune responses. However, there may be a and of for immunotherapy whilst tumour is and when are not future immunotherapies will be for the CNS to incorporate limited To date, of intracerebral tumours is such a major in animal models, that there has been little to of the component of the advances in understanding how cells can contribute to CNS suggest that successful future immunotherapies will these factors into For example, on in vitro astrocytes are proposed to more efficiently T T cells whereas activated cells, the of the brain, a It remains to be whether lymphocytes in vivo are to subset after contact with astrocytes or cells, if they were primed in by other cells The of astrocytes is such that various are including a fine of brain Thus, depending upon the presence of factors such as or tumour factor and family members, astrocytes can cytokines such as and or of these in vivo remains to be However, data are from mouse models in which the targets expression of various cytokines and to astrocytes (reviewed in Although such expression may appear the levels of cytokine were similar to those detected in or With these mice and neurological Although we have that T cells are able to infiltrate the brain, can effector T cells their function as they penetrate the brain parenchyma and the tumour from different models that this will upon several such as the of the induced immune the presence of other cell and the subset of the effector For brain infiltrating T cells, certain of normal brain have been to CTL effector For example, T cells capable of mediating were inhibited by although this was in the and has not been in For anti-tumour effector T cells, in the intracerebral tumour CD8+ T cells were to into effector cells in the brain a that was to a to growth present in the and the tissue However, these results were in to those with the similar which been with a model lymphocytes tested vivo from these mice full cytotoxic effector between these was that in the this was a response to a defined peptide whereas in the model most of the experiments were in or the contact with the tumour the infiltrating T cell may have other with of the brain. This has been most clearly defined for MHC class CD4+ T cells, although not as yet for cells with specificity for a tumour-expressed The cell is one of the few MHC class cells that are in the brain and is thus a prime candidate brain As already CD4+ T cells require contact with a perivascular cell to into the brain contact with cells has been to to tolerance or the of immune effects were particularly with the of the cells are in antigen presentation to CD8+ T cells is not they may influence CD8+ T-cell function through their influence on CD4+ T cells. CTL responses in the CNS have principally been in responses that are often CD4+ with of cytotoxic function in the absence of However, for anti-tumour immune from CD4+ T cells has been demonstrated in several different although it is difficult to because few studies have at this in that the of specific CTL responses. The to overcome for an anti-tumour effector cell is to function when in intimate contact with the tumour It is at this that active mechanisms of tumour immune will be most the of immunosuppressive molecules is and may synergize with potential immunosuppressive There has been a of publications the immunological in astrocytoma These low of T cells, responses as a result of T cell and T-cell cytotoxicity (reviewed by et It is to that these will be at their most in the of the tumour, and that they can explain lymphocytes are However, until it can be that the are specific for a tumour-expressed the remains Nevertheless, many advances have been in the for this apparent It that T cells from astrocytoma particularly express a or have in their of were also noted in a glioma This may at in the in vitro of T lymphocytes infiltrating malignant despite the of recombinant their low after and the in T-cell with cytotoxicity against tumour factors the tumour have been to be many of the immune Indeed, T lymphocytes from normal similar immunological when cultured in the presence of astrocytoma The most factor is originally T-cell which was first identified in the of a human cell that T-cell multiple and complex immunosuppressive effects, such as the inhibition of and antigen presentation by dendritic cells or other inhibition of T-cell and differentiation towards effector cells cytotoxic cells expressing or or The of in was further demonstrated in experiments in which inhibited by cell the and cytotoxic of autologous These in vitro data attempts to in vivo, which has probably because not only immune but also on the tumour cell Other using a of the growth of astrocytoma in but these experiments are also complex to because may also be in a the of the it has but whether it will be or to this cytokine in brain tumour remains The immunosuppressive properties of malignant demonstrated in vitro cannot be totally for by A of immunosuppressive molecules detected in astrocytomas or astrocytoma lines and all of which can certain immunosuppressive in However, whether these factors are a major in anti-tumour immune responses in vivo is far less either because of that sufficient factor is by tumour cells in vivo in particularly for because in vivo function is not but may actually anti-tumour through molecules such as are also proposed to contribute to tumour immune to the family and is in several biological through its with a of the growth factor receptor the of and a subsequent complex of to of cells, a central to immune Although was to be expressed by cells of origin, it was shown to be expressed by other normal and including malignant astrocytomas that we have analysed in our of in a of human astrocytoma lines and was as as in the and mouse glioma using a of techniques and reverse transcription–polymerase chain the were data demonstrated that astrocytoma cells lines and also astrocytoma cells tested can and efficiently target cells. Moreover, an early human astrocytoma cell was able to induce in CD4+ and CD8+ T cell lines derived from the autologous It has been that the use of T-cell targets is to function by tumour cells, because T cells can also express However, the of of our T cells to are correlated to their this the first after then rapidly and is at the when T cells were tested for these data suggest that astrocytoma cells express that can induce in targets and that whilst capable of through or can also be in of a from astrocytoma cells. Other have expression of by and, T cells were in the of astrocytoma However, the in vivo importance of this molecule for and tumours in remains Indeed, tumour expression of in models has been correlated either with tumour or with tumour probably factors clearly influence the of expression by tumour cells, for example, we that tumours expressing both and may be particularly to CTL effector this was in an in vivo model in which a could rejection if was also These data to explain the of in this model to the situation in the but it is that individual of factors to different tumours or models are for the of these issues in the to the of molecules with immunosuppressive potential are and although the in vivo of their function has yet to be is a MHC class I molecule expressed by a limited of particularly the but also certain cancers. in and T-cell immune responses have been but they are brain tumours, a proportion of astrocytoma cell lines and tumour expressed data inhibition of CD4+ and CD8+ T-cell responses in but this was only tested after of cell lines with high concentrations of or after gene transfer of into glioma For the studies have expression of this molecule on activated T and cells, with in immune responses with expressed on studies have now expression of by human glioma cell as as in vitro a of when tested on cells this was with expression levels of when tumour cells were However, under certain T cells after by tumour cells, a protection the CNS present an impossible course for immune effector cells induced during any future brain tumour There are sufficient results from certain models to suggest that this will not be the because it may not be to overcome all of the mechanisms of immune or by the CNS to some clinical However, with a better understanding of immune responses in the brain, we can make to key in which to for future brain tumour The fundamental importance of understanding immune of the brain in and cannot be It can be that the features of CNS immune whilst an to the cancer have to this most critical of the We the at our However, the optimal that has for a species may not be optimal for an We should therefore not be from the towards a immune response in as as the are and The in our is by from the for the and the