Immune surveillance of tumors (original) (raw)
Spontaneous tumor development in immunodeficient mice. The simplest approach for testing the role of the immune system in controlling tumor development is to remove specific components of the mouse immune system and monitor the mice for the development of tumors. Predominantly through the use of gene-targeted mice, this approach has demonstrated that a number of immune effector cells and pathways are important for suppression of tumor development (Table 1).
Immunodeficient mouse strains that develop spontaneous tumors
A role for the adaptive immune system in suppressing tumor growth was revealed when it was shown that 129/Sv RAG2-deficient mice, which lack both B and T cells, develop spontaneous adenocarcinoma of the intestine and lung (35% and 15%, respectively, of all mice analyzed) at 15–16 months of age, and an additional 50% of mice develop intestinal adenomas (3). Interestingly, when _Rag2–/–_mice were also deficient for STAT1, an important mediator of signaling induced by both type I and type II IFN, tumor incidence is further increased, and the spectrum of tumors broadens to include breast adenocarcinomas (~40% of mice), colon adenocarcinomas (~10% of mice), or both (~20% of mice) (3). Together, these results suggest that both the innate and the adaptive arms of the immune system are involved in the prevention of tumors, as mice lacking both IFN signaling and an adaptive immune system develop a broader spectrum of tumors than mice only lacking an adaptive immune system.
Mice lacking T cell and NK cell cytotoxic effector pathways have also been shown to develop spontaneous tumors. For example, mice that lack perforin, a cytotoxic molecule used by cytotoxic cells such as CD8+ T cells and NK cells to form membrane pores in target cells, develop lymphomas with age. These spontaneous lymphomas are of B cell origin, develop in older mice (>1 year of age) regardless of the mouse strain (4, 5), and, when transplanted into WT mice, are rejected by CD8+ T cells (4). B cell lymphomas also arise in mice lacking both perforin and β2m, and tumor onset is earlier and occurs with increased prevalence compared with mice lacking only perforin. In addition, B cell lymphomas derived from mice lacking both perforin and β2m are rejected by either NK cells or γδ T cells following transplantation to WT mice, rather than by CD8+ T cells (as in tumors derived from mice lacking only perforin), demonstrating that cell surface expression of MHC class I molecules by tumor cells can be an important factor in determining which effector cells mediate immune protective effects (6). Intriguingly, mutations in the gene encoding perforin have also been identified in a subset of lymphoma patients (7), although it is not clear whether this contributes to disease. Mice lacking the death-inducing molecule TNF-related apoptosis-inducing ligand (TRAIL) or expressing a defective mutant form of the death-inducing molecule FASL have also been shown to be susceptible to spontaneous lymphomas that develop with late onset (8, 9). These aging studies have clearly demonstrated a critical role for cytotoxic pathways in immunoregulation and/or immunosuppression of spontaneous tumor development in mice.
Several cytokine-deficient mice also develop spontaneous malignancies (5, 10, 11). In one study, approximately 50% of IFN-γ–deficient C57BL/6 mice were found to develop T cell lymphomas that are predominantly disseminated lymphomas, although some cases of thymic lymphoma are also noted; interestingly, the susceptibility of _Ifng_–/– mice to T cell lymphomas is strain dependent (5). Furthermore, the spectrum of tumors observed in IFN-γ– and STAT1-deficient mice do not overlap, despite STAT1 being a crucial signaling molecule downstream of the IFN-γ receptor, indicating either that these molecules have some nonoverlapping activities or that the background strain has a modifying influence on tumor type. In addition, C57BL/6 mice lacking both IFN-γ and perforin display accelerated B cell lymphoma onset compared with perforin-deficient mice (5), indicating that IFN-γ has an important role in modifying the progression to B cell lymphoma in perforin-deficient mice. IL-12 and IL-18 are important IFN-γ–inducing cytokines; however, studies of aging have demonstrated that neither IL-12– nor IL-18–deficient mice display increased incidence of tumor development compared with WT mice (5).
A possible link between tumor immunity and autoimmune or infection-induced inflammation has been raised by several studies. Curiously, with age, 50% of mice lacking the β2 subunit of the IL-12 receptor (IL-12Rβ2) develop plasmacytomas or lung carcinoma in the context of the autoimmune disease immune complex mesangial glomerulonephritis (10). It is presently unclear why IL-12–deficient mice on the same genetic background as the IL-12Rβ2–deficient mice do not display either autoimmunity or spontaneous tumor development. Furthermore, mice deficient for both IFN-γ and GM-CSF have also been found to develop tumors with age; in this case, tumor development is associated with acute or chronic inflammatory lesions in a range of organs, and maintaining mice on the antibiotic enrofloxacin prevents (or at least delays) tumor onset (11). Collectively, these findings demonstrate that the immune system can suppress tumor development, but they do not constitute proof for tumor immunoediting per se. The finding that antibiotic treatment could prevent tumor development in _Gm-csf–/–Ifng_–/– mice raises the possibility that rather than directly eliminating tumor cells, the immune system might prevent tumor growth by the timely elimination of infections, thereby limiting inflammation, which is known to facilitate tumor development (12). However, this finding cannot be generalized, as _Rag2_–/– and _Rag2–/–Stat1_–/– mice maintained on the same antibiotics and housed under strict specific pathogen–free conditions still display heightened tumor incidence despite testing negative for common pathogens with known links to malignancy and showing no signs of idiopathic inflammation (3).
Carcinogen-induced tumors in immunodeficient mice. In order to more readily study the role of the immune system in shaping tumorigenesis, researchers have turned to inducible tumor models, including carcinogen-induced tumors. The 2 most commonly employed carcinogen-induced tumor models are fibrosarcomas induced using methylcholanthrene (MCA) and skin papillomas induced by a combination of 7,12-di-methylbenz[a]-anthracene (DMBA) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA). To date, a number of mice with defined immunodeficiencies have been tested for their susceptibility to carcinogens (Table 2).
Carcinogen-induced tumors in immunodeficient mice
Rag2–/– and SCID mice, both of which lack an adaptive immune system, display a heightened susceptibility to tumor induction with MCA (3, 13), and similar findings have been made in nude mice (14), which lack most T cells. Both αβ and γδ T cells were subsequently found to be important in suppressing MCA-induced tumors, as mice deficient for either of these T cell subsets display increased susceptibility to tumor induction (15). Interestingly, 40% of tumors derived from _Rag2_–/– mice are rejected when transplanted into WT recipients but grow progressively in either _Rag2_–/– hosts or mice depleted of CD4+ and CD8+ T cells, whereas tumors derived from WT mice grow readily when transplanted into either WT or _Rag2_–/– hosts (3). These observations are important because they not only show that carcinogen-induced tumors arise more frequently in immunodeficient mice, but also that tumors derived from these immunodeficient mice are more immunogenic than those arising in mice with a functional immune system. CD1d-restricted T cells, which bridge the innate and adaptive arms of the immune system, also have a role in suppressing MCA-induced fibrosarcomas. Mice that lack the potential TCR component Jα18 are unable to generate the semi-invariant Vα14-Jα18–containing TCR expressed by NKT cells and show increased susceptibility to fibrosarcoma induction (16, 17). Interestingly, similar to immunogenic tumors from _Rag2_–/– mice, a portion of tumors arising in mice lacking Jα18 are rejected when transplanted into WT hosts (18), indicating that they are immunogenic and that the absence of NKT cells might contribute to the enhanced tumor development in _Rag2_–/– mice. Evidence that the innate immune system is also important in suppression of MCA fibrosarcomas was provided in mice chronically depleted of NK cells, since these mice display increased tumor incidence (16).
To further understand how the immune system suppresses fibrosarcoma growth, a number of mice deficient for specific immune effector molecules and pathways have been examined, including mice lacking perforin (19), TRAIL (20), IL-12 (16), IFN-γ (21), IFNAR1 (a component of the type I IFN receptor) (22), and NK group 2, member D (NKG2D) (23). Each of these mouse strains demonstrates enhanced susceptibility to fibrosarcoma induction, suggesting that cytotoxic cells (NK cells and CD8+ CTLs) use these pathways to suppress tumor growth in vivo. Interestingly, further investigation of this phenomenon revealed that tumor cells are important targets for the antitumor effects of IFN-γ (21), whereas the host hematopoietic system is the target of the antitumor effects of type I IFN (22), suggesting that the ability of type I IFNs to induce antitumor activity in immune cells might be the critical mode of action for this cytokine family.
This interpretation of the results regarding the role of IFN-γ in prevention of MCA-induced fibrosarcomas has not been universally accepted, however, with other researchers proposing that IFN-γ contributes to an inflammatory response that results in the encapsulation of injected MCA (a process referred to as a foreign body reaction), limiting its spread and thereby reducing its carcinogenic effects (24). However, the finding that _Ifng_–/– mice are more susceptible to lymphomas induced by the soluble carcinogen N-methyl-N-nitrosourea (25), where encapsulation of the carcinogen is not possible, is at odds with this concept. The finding that restoration of IFN-γ receptor 1 (IFN-γR1) expression in MCA-induced tumor lines from _Ifngr1_–/– mice leads to a delay in tumor growth or complete tumor rejection when such tumors are transplanted into WT mice also strongly suggests IFN-γ is not merely a driver for encapsulation of MCA.
A role for the immune system in regulating the development of DMBA/TPA-induced papillomas has also been investigated (Table 2). With the DMBA/TPA model, skin carcinomas are induced by the topical application of DMBA (the tumor initiator), followed by repetitive doses of TPA (the tumor promoter). In this model, lesions progress from benign papillomas through to metastatic squamous cell carcinomas, and the number and progression of the lesions is dependent on the mouse strain. While γδ T cells confer protection from DMBA/TPA-induced papillomas (15), αβ T cells seem to promote tumor progression in this model of carcinogenesis (26). One mechanism by which γδ T cells might regulate tumor development is through NKG2D recognition of the stress ligand retinoic acid early transcript 1 (RAE1), expression of which is induced in the skin by DMBA/TPA treatment. NKG2D-expressing dendritic epidermal γδ T cells can kill RAE1-expressing targets in vitro (15), but in transgenic mice expressing RAE1 in the skin, NKG2D expression is downmodulated on lymphocytes and consequently these mice are more susceptible to papilloma induction than are WT mice (27). Collectively these data indicate that the NKG2D pathway is important in the control of carcinogen-induced tumors. IL-23 and IL-12 are functionally related heterodimeric cytokines that both contain the IL-12β subunit (although paired with distinct subunits) and activate distinct receptors that each contain the IL-12Rβ1 subunit. Recently, Langowski et al. induced papillomas in mice that lack either the IL-23–specific subunit that pairs with IL-12β or the IL-12–specific subunit that pairs with IL-12β (28). Interestingly, mice that lack functional IL-23 are resistant to tumor development, whereas mice that lack functional IL-12 develop increased numbers of papillomas compared with WT mice. In a broad panel of human tumors, the authors also found substantial upregulation of the mRNAs encoding both subunits of IL-23 and hypothesized that expression of IL-23 in human tumors has a causative role in promoting tumor development. Although the mechanism by which IL-23 promotes tumor growth requires further clarification, it has been found that carcinogen-treated IL-23–deficient mice produce less IL-17 (a cytokine with tumor growth–promoting activity; refs. 29, 30) than do WT controls. Moreover, since the DMBA/TPA model of cancer is known to be dependent on a strong inflammatory response, more work is needed to explore the relative importance of inflammation versus immunoediting in other primary tumor models and whether these are distinct or overlapping processes. Tumors induced by physical carcinogens such as UV radiation also seem to be controlled by the immune system (31), and it is thought that UV-induced immune suppression is an important factor in the development of UV-induced tumors, as UV-induced tumors are often immunogenic when transplanted into naive hosts but grow in immunosuppressed recipients or recipients depleted of CD8+ T cells (32).
Genetic tumor models in immunodeficient mice. Data supporting the ability of the immune system to suppress tumor development in genetic models of mouse cancer is less extensive. Several studies have demonstrated that the immune system can protect mice from malignancy induced by genetic means, whereas others have shown no effect (Table 3). Of the genetic tumor models, mice heterozygous for the tumor suppressor p53 (p53+/– mice) have provided the most convincing evidence for tumor immune surveillance, indicating a role for perforin (4), TRAIL (8), and IFN-γR1 (21) in immune control of tumor development. Transgenic expression of oncogenes under the control of tissue-specific promoters has become a common technique to investigate the process of tumorigenesis; however, few of these studies have addressed the role of the immune system in delaying or preventing the development of tumors. A role for IFN-γ in suppressing tumor development has been observed in mice expressing the human T cell leukemia virus (HTLV) type 1–derived oncogene Tax under the control of a granzyme B promoter (HTLV-Tax transgenic mice); these mice develop a lymphoproliferative disease similar to human adult T cell leukemia/lymphoma. When HTLV-Tax mice are crossed with _Ifng–/–_mice, tumor onset is accelerated, suggesting that IFN-γ has a protective effect in this model (33). In a second transgenic tumor model, using the oncogene encoding the SV40 large T antigen (Tag), IFN-γ deficiency has no effect on tumor onset or the spectrum of tumors that develop (34); instead, tumor growth causes the skewing of T cell responses, with tumor-specific T cells producing TGF-β rather than IFN-γ. Similarly, CD8+ T cells have no effect on tumor development in the rat insulin promoter–Tag4 (RIP-Tag4) pancreatic tumor model, where Tag is expressed in the pancreas under the control of RIP, resulting in the development of pancreatic tumors (35). These results again confirm that the role of the immune system in suppressing tumors is likely to be dependent on the natural history of the individual tumor and the oncogenic changes that it has acquired.
Genetic mouse tumor models and immunodeficiency
Summary of mouse models. In summary, various cell types, including αβ and γδ T cells, NKT cells, and NK cells, have been implicated in the processes of elimination and immunoediting, along with a number of effector molecules, including perforin and TRAIL, and cytokines, including IFN-γ, type I IFNs, and IL-12. It is important to note that the effector cells and cytokines thought to be involved in elimination and immunoediting differ among models, demonstrating that the success of immunoediting and the evidence of its occurrence varies among experimental systems. Indeed, there are models in which the immune system seems to have little influence on the rate of tumor onset or progression (34) and models in which the immune system has a distinct protective role, such as the carcinogen-induced tumor models outlined above. The level of immune regulation and tolerance (a state of nonresponsiveness to specific antigens) imparted by the tumors in each of these models might explain, at least in part, why in some cases the effect of subtracting immune elements on tumor progression is less overt. Blocking these tolerance mechanisms might reveal the true mechanisms of tumor suppressor immunity. Therefore, to date, observations in mouse models are an indirect readout of tumor elimination, and it has not yet been possible to observe the establishment and subsequent immune-mediated regression (i.e., elimination) of an autochthonous tumor.
Evidence for the equilibrium phase of tumor immunoediting, during which the tumor and antitumor immune response coevolve, is least advanced and again indirect. MCA-induced fibrosarcomas from mice lacking IFN-γ, IFNAR1, TRAIL, or T cells as well as lymphomas from mice lacking perforin can be eliminated when transplanted into immunocompetent recipients but grow progressively when transplanted into mice of the same genotype as that in which they arose. These data suggest that the immune system sculpts the immunogenic profile of evolving tumors, the exact process thought to occur during the equilibrium phase of immunoediting. In contrast to the equilibrium phase, abundant evidence exists for the escape phase of immunoediting (see below), and the growth of tumors in immunocompetent hosts is the most basic evidence that tumors can escape immune control. More specific evidence for the escape phase comes from studies in which an abortive, concurrent antitumor immune response is detected in mice with progressive tumor growth.