The adaptive immune response to sporadic cancer

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<ul><li><p>The adaptive immune response tosporadic cancer</p><p>Summary: Most of the current experimental cancer models do not reflectthe pathophysiology of real-life cancer. Cancer usually occurs sporadicallyand is clonal in origin. Between tumor initiation and progression,clinically unapparent pre-malignant cells may persist for years or decadesin humans. Recently, mouse models of sporadic cancer have beendeveloped. The mouse germ-line can be engineered with high precisionso that defined genes can be switched on and off in the adult organism in atargeted manner. Analysis of the immune response against sporadic tumorsrequires the knowledge of a tumor antigen. Ideally, a silent oncogene, forwhich the mice are not tolerant, is stochastically activated in individualcells. This approach offers the opportunity to analyze the adaptive immuneresponse throughout the long process of malignant transformation andmost closely resembles cancer in humans. In such a model with the highlyimmunogenic SV40 large Tantigen as a dormant oncogene, we discoveredthat sporadic cancer is recognized by the adaptive immune system at thepre-malignant stage, concomitant with the induction of tumor antigen-specific tolerance. These results demonstrated that even highly immuno-genic sporadic tumors are unable to induce functional cytotoxic Tlymphocytes. Based on this model, we conclude that immunosurveillanceplays little or no role against sporadic cancer and that tumors must notescape immune recognition or destruction.</p><p>Keywords: sporadic cancer, immunosurveillance, tolerance, rejection antigen, cytotoxicT lymphocytes, antibodies</p><p>Introduction</p><p>Spontaneous tumor-induced immune responses have been</p><p>analyzed for decades. As cancer is a heterogeneous disease and</p><p>experimental models are different, virtually all possibilities of</p><p>tumorimmune cell interactions have been demonstrated. It</p><p>has been postulated that immune cells recognize and eliminate</p><p>tumor cells, whereas other models suggest that tumor cells</p><p>recruit immune cells that they require for growth. While the</p><p>former has been firmly established for a variety of virus-</p><p>induced tumors in mice and humans (1, 2), the latter was first</p><p>observed by Virchow almost 150 years ago and has been</p><p>confirmed countless times in experimental carcinoma models,</p><p>commonly referred to as inflammation-induced cancer (36).</p><p>Because the immune response to virus-induced tumors is</p><p>fundamentally different from that against spontaneous tumors</p><p>(1), it is not reviewed here.</p><p>Gerald Willimsky</p><p>Thomas Blankenstein</p><p>Immunological Reviews 2007</p><p>Vol. 220: 102112</p><p>Printed in Singapore. All rights reserved</p><p>r 2007 The Authors</p><p>Journal compilationr 2007 Blackwell Munksgaard</p><p>Immunological Reviews0105-2896</p><p>Authors addresses</p><p>Gerald Willimsky1, Thomas Blankenstein1,2</p><p>1Institute of Immunology, Charite Campus Benjamin</p><p>Franklin, Berlin, Germany.2Max-Delbruck-Center for Molecular Medicine, Berlin,</p><p>Germany.</p><p>Correspondence to:</p><p>Thomas Blankenstein</p><p>Max-Delbruck-Center for Molecular Medicine,</p><p>Robert-Rossle-Str. 10, 13092 Berlin, Germany</p><p>Tel.:1149 30 9406 2816</p><p>Fax: 1149 30 9406 2453</p><p>e-mail:</p><p>Acknowledgements</p><p>The work described here was supported by grants from the</p><p>Deutsche Forschungsgemeinschaft (SFB633 and TR36)</p><p>and the European Community (FP6 program ATTACK).</p><p>102</p></li><li><p>Most experiments with non-viral tumors have been</p><p>performed by tumor transplantation. Through these studies,</p><p>important discoveries have been made. (i) Tumor cells differ</p><p>greatly in inherent immunogenicity (7, 8). (ii) If transplanted</p><p>tumor cells are rejected, it requires T cells, CD81 cytotoxic</p><p>and/or CD41 helper T cells. In most cases, tumor transplant</p><p>rejection requires antigen cross-presentation by activated</p><p>dendritic cells for T-cell activation and interferon-g (IFN-g)and perforin as effector molecules (9, 10). (iii) Tumor</p><p>transplantation rejection antigens are tumor specific, the result</p><p>of somatic mutation, and are usually not cross-protective</p><p>between different tumor lines (11). (iv) Under</p><p>immunological pressure, for example in response to effective</p><p>immunotherapy, tumor cells can escape immune recognition</p><p>or destruction by various mechanisms (12). (v) Under</p><p>rigorous experimental conditions, e.g. with established</p><p>tumors, immunotherapy approaches can be informative and</p><p>form a solid basis for clinical studies (13). (vi) Last but not</p><p>least, mouse tumor transplantation studies have led to the</p><p>discovery of the major histocompatibility locus, H-2 in the</p><p>mouse, and the development of inbred strains.</p><p>There are many more important discoveries that resulted</p><p>from tumor transplantation studies. However, the spontaneous</p><p>immune response against transplanted tumor cells, usually</p><p>injected as a single-cell suspension, is highly artificial.</p><p>Transplanted tumors may be more accessible for infiltrating</p><p>immune cells. Their growth is often analyzed at a site different</p><p>from its origin, and it is difficult to exclude phenotypic</p><p>changes occurring during in vitro cell culture. Transplanted</p><p>tumor cells usually grow very fast, because they have acquired</p><p>malignancy in their primary host. Successful tumor</p><p>transplantation requires a large number of cells to be injected.</p><p>Many cells rapidly die after injection, which can lead to</p><p>artificial T-cell recognition, and, in general, the host is</p><p>exposed to a large number of tumor cells at a single time-</p><p>point that does not reflect cancer development as it occurs in</p><p>humans. Importantly, the immune response to primary tumors</p><p>cannot be deduced from tumor transplantation experiments,</p><p>because tumors that progressively grew in an immune-</p><p>competent primary host were promptly rejected following</p><p>transplantation to a naive immune-competent recipient (14).</p><p>Various non-transgenic primary tumor models have been</p><p>used that better resemble typical tumor development. Most</p><p>often, chemical or physical carcinogens have been used to</p><p>induce autochthonous tumors. Examples are methyl-</p><p>cholanthrene (MCA) (15), a combination of DMBA (7,12-</p><p>dimethylbenzanthracene) and TPA (12-O-tetradecanoylphorbol</p><p>-13-acetate) (16, 17), or ultraviolet (UV) irradiation (7).</p><p>Although these carcinogens often contribute to cancer in</p><p>humans and therefore represent important experimental</p><p>models, the problem is that the potential rejection antigens</p><p>(18) are not known and are unique for each individual tumor</p><p>(11). Thus, the tumor-specific T-cell response cannot be</p><p>followed in the primary tumor-bearing host. This circumstance</p><p>makes it difficult, if not impossible, to judge the influence of</p><p>antigen-specific T cells on primary tumor development.</p><p>Recombination-activating gene-1 (Rag-1) knockout mice that</p><p>have no T cells, natural killer T (NKT) cells, and B cells do not</p><p>develop MCA-induced tumors significantly more frequently or</p><p>with shorter latency compared with control littermates (19).</p><p>Rag-2 knockout mice that have an identical phenotype as Rag-1</p><p>knockout mice also do not spontaneously develop tumors more</p><p>frequently compared with wildtype control mice (20). This</p><p>finding is not surprising, because nude mice that lack the</p><p>thymus and all thymus-dependent T cells also do not develop</p><p>MCA-induced or spontaneous tumors more frequently than</p><p>control mice (21, 22). Some investigators obtained different</p><p>results in the immune-deficient mice. Possible explanations for</p><p>the contradictory results have been reviewed elsewhere (23, 24).</p><p>In any case, tumor models that do not allow the analysis of</p><p>tumor-specific T-cell responses in the primary tumor-bearing</p><p>host have been widely misinterpreted with regard to a</p><p>spontaneous protective anti-tumor T-cell response (24).</p><p>Sporadic cancer models</p><p>Cancer in most cases occurs sporadically, is clonal in origin,</p><p>and arises through sequential accumulation of somatic muta-</p><p>tions and/or epigenetic changes in genes, whose gain or loss of</p><p>function is associated with malignant transformation. It is</p><p>thought that in a Darwinian selection process, continuously</p><p>more malignant clones emerge. It is the combined and</p><p>occasionally synergistic action between activated oncogenes</p><p>and inactivated tumor suppressor genes that cause malignancy.</p><p>This knowledge and techniques to modulate the mouse germ-</p><p>line with high precision and at the single-gene level allow</p><p>the construction of cancer models that better mimic the</p><p>pathophysiology of real-life cancer. Several excellent reviews</p><p>on the development of mouse cancer models toward sporadic</p><p>cancer have been published (2527). Only developments as</p><p>they are relevant for tumor immunology are briefly recapitu-</p><p>lated here.</p><p>In first-generation mouse models, transgenic mice were</p><p>generated, in which an oncogene (e.g. SV40 Tag, Myc)</p><p>was expressed in a cell type-specific fashion. In these</p><p>transgenic mice, the oncogene was expressed constitutively in</p><p>Immunological Reviews 220/2007 103</p><p>Willimsky &amp; Blankenstein The immune response to sporadic cancer</p></li><li><p>a specific tissue (2832). Alternatively, with the possibility of</p><p>gene-targeting by homologous recombination, tumor</p><p>suppressor genes have been inactivated in the germ-line by</p><p>use of genetically modified embryonic stem cells. By CreLoxP-</p><p>mediated recombination, these genes can be inactivated and</p><p>tumors can be induced in a cell type-specific fashion, e.g. by</p><p>application of Cre recombinase using adenoviruses (33), but</p><p>typically by crosses to a second transgenic mouse that carries</p><p>the recombinase gene (3436). For immunological analysis,</p><p>these models bear several disadvantages. Large numbers of</p><p>cells, almost a whole organ, are simultaneously transformed,</p><p>resulting in an untypical short tumor latency period and fast</p><p>non-clonal tumor growth (Fig. 1). The polyclonality probably</p><p>alters the microenvironment that is known to be able to either</p><p>stimulate or inhibit tumor growth. Furthermore, transgenic</p><p>oncogenes expressed by tissue-specific promoters are self-</p><p>antigens, so that it is very difficult to exclude tolerance, even</p><p>though occasionally such mice have retained immune</p><p>competence for the oncogene (3740). Tumor-specific</p><p>antigens that likely occur during multi-step carcinogenesis in</p><p>oncogene-transgenic or tumor suppressor gene-deficient mice</p><p>are not known.</p><p>To avoid tolerance, the oncogene can be activated in a cell</p><p>type-specific manner in the adult animal by temporally</p><p>controlled transgene expression (41). In this case, the</p><p>oncogene is regulated by a minimally active promoter that</p><p>includes a tetracycline-responsive element (TRE). Gene</p><p>expression is obtained by a second transgene, which encodes</p><p>a transactivator under the control of a cell type-specific</p><p>promoter, usually by intercrossing the respective single-</p><p>transgenic mice. Feeding double-transgenic mice with</p><p>doxycycline, a derivative of tetracycline, will then induce or</p><p>repress transgene expression, depending on the use of Tet-on</p><p>or Tet-off systems (4244). In addition to controlling</p><p>oncogene expression in a time-dependent manner, these</p><p>models also allow examination of whether malignancy</p><p>requires persistent oncogene expression (oncogene</p><p>addiction) (4245) or whether the initiating oncogene</p><p>becomes dispensable with progressive malignancy. Another</p><p>strategy to regulate gene expression conditionally is the</p><p>use of fusion proteins with the estrogen receptor hormone-</p><p>binding domain. The estrogen receptor is engineered in</p><p>such a way that exogenous administration of the synthetic</p><p>estrogen antagonist tamoxifen results in the transgene</p><p>product relocating into the nucleus. This approach has</p><p>been used successfully by directly fusing the c-Myc oncogene</p><p>to the estrogen receptor (46). Additionally, it can be used</p><p>to regulate the recombination activity of the recombinases</p><p>Cre and FLP.</p><p>Oncogene expression can also be induced by controlled</p><p>activation using a Cre- (or FLP-) mediated recombination</p><p>strategy. In these mice, an attenuator separates the promoter</p><p>from the oncogene. The attenuator can be any gene whose</p><p>expression prevents expression of the 30-located oncogene and,therefore, serves as a stop-cassette. It is flanked by LoxP</p><p>recognition sites for Cre site-specific recombinase. The</p><p>oncogene can be activated by Cre recombinase-mediated</p><p>deletion of the stop-cassette. A large number of Cre</p><p>recombinase gene transgenic mice are available that express the</p><p>recombinase under different promoters. In mice double</p><p>transgenic for the conditional oncogene and recombinase</p><p>genes, the oncogene can be expressed in a tissue-specific</p><p>fashion, which likely will result in tolerance for the oncogene.</p><p>Alternatively, Cre recombinase expression may be achieved</p><p>by transcriptional activation (tetracycline, IFN, polyinosinic</p><p>polycytidylic acid) or post-translational activation (4-hydro-</p><p>xytamoxifen, mifepristone) in the adult organism. The inducible</p><p>expression/activation models better mimic de novo expression of</p><p>the oncogene comparable to somatic mutations that typically</p><p>activate oncogenes (47). In principle, the mice should not have</p><p>developed tolerance for the oncogene at the time of Cre</p><p>recombinase expression and oncogene activation. However,</p><p>Fig. 1. Growth kinetics of sporadic versus non-sporadic cancer.A schematic drawing is shown of the growth kinetics of transplantedtumor cells, primary tumors in mice, in which an oncogene is expressedin a cell type-specific fashion (whole organ tumor), and primary tumorsin mice, in which an oncogene is activated in individual cells (sporadictumor). Sporadic tumors, as observed in LoxPTag mice, are characterizedby pre-malignant lesions at around 69 months of age and a variable butusually very long latency period, until tumors progress. The tissue-specific expression of SV40 T antigen in LoxPTagAlbCre mice resultsin transformation of a large number of cells and tumor progression aftera short latency period. As sporadic tumors are induced in a stochasticfashion, tumor initiation and progression can be variable from mouse tomouse. Therefore, each red line represents an individual LoxPTagmouse.</p><p>104 Immunological Reviews 220/2007</p><p>Willimsky &amp; Blankenstein The immune response to sporadic cancer</p></li><li><p>tumor development is not sporadic, because the oncogene is</p><p>activated in many somatic cells.</p><p>It is difficult to assess how tightly Cre recombinase</p><p>expression can be controlled or whether attenuated oncogene</p><p>expression can be prevented. Transgenic models with</p><p>temporally controlled and reversible systems could be leaky.</p><p>The leakiness could be due to spontaneous (tamoxifen-</p><p>independent) Cre recombinase expression in double-</p><p>transgenic mice (authors unpublished observations),</p><p>oncogene expression in the non-induced situation (e.g. in the</p><p>absence of tetracycline), or incomplete function of the stop-</p><p>cassette. Potentially, leakiness may lead to partial tolerance for</p><p>the oncogene before tumor development starts. If leakiness of</p><p>conditional Cre recombinase expression is a rare phenomenon</p><p>or attenuated oncogene expression is low, immune</p><p>competence for the oncogene might be comparable to that of</p><p>non-transgenic mice. Importantly, in this situation leakiness is</p><p>stochastic and occurs in single cells, which faithfully mimics</p><p>sporadic tumor development. A...</p></li></ul>


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