A. Historical Background
In the early twentieth century, Ehrlich first proposed the existence of immune surveillance for eradicating nascent transformed cells before they are clinically detected (Ehrlich, 1909). Almost 50 years later, Burnet and Thomas postulated that the control of nascent transformed cells may represent the actions of an ancient immune system, which played a critical role in preventing malignant transformation (Burnet, 1957). The idea was supported by experimental results showing strong immune-mediated rejection of transplanted tumors into mice. Although there was excellent evidence in support of the belief that immune surveillance mechanisms prevent the outgrowth of tumor cells induced by horizontally transmitted, ubiquitous, potential oncogenic viruses, there was much less evidence for immune surveillance acting against chemically induced tumors in syn-geneic mice (Klein, 1976). The use of genetically identical mice, however, generated tumor-specific protection from methylcho-lantrene (MCA) and virally induced tumors (Prehn and Main, 1957; Old and Boyse, 1964). These results from mouse models strongly suggested the existence of TAAs and immune surveillance for protection from transformed cells in the host, which was postulated by Burnet and Thomas (Burnet, 1957; Burnet, 1971; Thomas, 1982). Despite the fact that several lines of evidence from experimental mouse models showed the immune system played a critical role in dealing with transformed cells, it was argued that there was no increased incidence of spontaneous or chemically induced tumors in athymic nude mice compared to wild-type animals (Rygaard and Povlsen, 1974; Stutman, 1974). This evidence suggested that immune surveillance in mice targeted transforming viruses in tumors rather than the tumors themselves (Burnet, 1957). It is now known that athymic nude mice have NK cells and fewer T cells than wild-type mice, which can contribute to immune surveillance. Further, athymic mice have detectable populations of functional yS-T cell receptor-bearing lymphocytes (Ikehara et al, 1984; Maleckar and Sherman, 1987). Years later, titration of the MCA dosage revealed that nude mice actually did form more tumors than the control mice population (Engel et al., 1996). Similarly, tumor formation induced by MCA was greater in severe combined immuno-deficient (SCID) mice than in wild-type BALB/c mice (Engel et al, 1997). These observations put to rest one of the widely cited skepticisms about whether the immune system had any role in suppressing cancers that were not induced by transforming viruses.
The emergence of gene-targeted mice, such as knockout mice, has facilitated the gathering of evidence for immune-mediated surveillance of spontaneous epithelial tumors (Dunn et al, 2002). Strikingly, mice with a variety of immunodeficiencies produced by specific genetic ablations have been shown to be more susceptible to MCA-induced tumors and spontaneous lymphomas (Dunn et al., 2004).
During the mid-1970s to the 1990s, many investigators sought evidence to illustrate the immune surveillance concept. The discovery of NK cells provided a considerable stimulus for the possibility that they functioned as the effectors of immune surveillance (Herberman and Holden, 1978) even though a precise definition and understanding of these cells had not been confirmed. However, it was not until the 2000s that gene-targeted and lymphocyte subset-depleted mice were used to definitively establish the relative importance of NK and NK1.1+ T cells in protecting against tumor initiation and metastasis. In these models, CD3 + NK cells were responsible for tumor rejection and protection from metastasis in models where control of major histocompatibility complex (MHC) class I-deficient tumors was independent of interleukin 12 (IL-12) (Smyth et al, 2000a). C57BL/6 mice that were depleted of both NK and NKT cells by the anti-NK1.1 monoclonal antibody (mAb), which can eliminate both NK and NKT cells, were two to three times more susceptible to MCA-induced tumor formation than control mice (Smyth et al., 2001). Further, a similar result was observed in C57BL/6 mice treated with antiasialo-GM1, which selectively eliminates NK but not NKT cells, even though antiasialo-GM1 can also eliminate activated macrophages. A protective role for NKT cells was only observed when tumor rejection required endogenous IL-12 activity. In particular, T cell receptor (TCR) Ja281 gene-targeted mice confirmed a critical function for NKT cells in protecting against spontaneous tumors initiated by the chemical carcinogen, MCA. Ja281-/- mice, lacking Vp14Ja281-expressing invariant NKT cells, formed MCA-induced sarcomas at a higher frequency than did wild-type mice (Smyth et al, 2000a). Another study showed that mice treated with the NKT cell-activating ligand a-galactosylceramide throughout MCA-induced tumorigenesis exhibited a reduced incidence of tumors and displayed a longer latency period to tumor formation than did control mice (Hayakawa et al., 2003).
Mice lacking y8-T cells were also shown to be highly susceptible to multiple regimens of cutaneous carcinogenesis. After exposure to carcinogens, skin cells expressed Rae-1 and H60, MHC-related molecules structurally resembling human MHC class I chain-related A (MICA). Each of these is a ligand for NKG2D, a receptor expressed by cytolytic T cells and NK cells. In vitro, skin-associated NKG2D+ y8-T cells killed skin carcinoma cells by a mechanism that was sensitive to blocking NKG2D attachment (Girardi et al, 2001). The localization of y8-T cells in epithelia may therefore contribute to helping prevent epithelial malignancies.
Endogenously produced IFN-y protected the host against transplanted tumors and the formation of chemically induced and spontaneous tumors. When the mice were treated with neutralizing monoclonal antibody to IFN-y the growth rate of immuno-genic sarcomas transplanted into mice was greater than the growth rate in the control mice (Dighe et al, 1994). Further, overexpression of the truncated dominant negative form of the murine IFN-y receptor y subunit (IFNGR1) in Meth A fibrosarcoma completely abrogated tumor sensitivity to IFN-y, and the tumors showed enhanced tumorigenicity and reduced immunogenic-ity when they were transplanted into syn-
geneic BALB/c mice (Dighe et al., 1994). These results showed that IFN-y had direct effects on tumor cell immunogenicity and played an important role in promoting tumor cell recognition and elimination. In the experiment with MCA-induced tumor formation, compared with wild-type mice, mice lacking sensitivity to either IFN-y (IFNGR-deficient mice) or all IFN family members (signal transducers and activators of transcription [Stat]1-deficient mice; Statl is the transcription factor that is important in mediating IFNGR signaling) developed tumors more rapidly and with greater frequency when challenged with different doses of the chemical carcinogen MCA. In addition, IFN-y-insensitive mice developed tumors more rapidly than wild-type mice when bred onto a background deficient in the p53 tumor-suppressor gene (Kaplan et al, 1998). IFN-y-insensitive p53-/- mice also developed a broader spectrum of tumors compared with mice lacking p53 alone. The importance of this experiment lay in the finding that certain types of human tumors become selectively unresponsive to IFN-y. Thus, IFN-y forms the basis for an extrinsic tumor-suppressor mechanism in immunocompetent hosts. Using experimental (B6, RM-1 prostate carcinoma) and spontaneous (BALB/c, DA3 mammary carcinoma) models of metastatic cancer, mice deficient in both pfp and IFN-y were significantly less proficient than pfp- or IFN-y-deficient mice in preventing metastasis of tumor cells to the lung. The pfp and IFN-y-deficient mice were equally susceptible as mice depleted of NK cells in both tumor metastasis models; hence, IFN-y appeared to play an early role in protection from metastasis (Street et al., 2001). Further analysis demonstrated that IFN-y, but not pfp, controlled the growth rate of sarcomas arising in these mice; in addition, the host IFN-y and direct cytotoxicity mediated by cytotoxic lymphocytes expressing pfp independently contributed antitumor effector functions that together controlled the initiation, growth, and spread of tumors in mice.
In another study, both IFN-y and pfp were critical for suppression of lymphomagenesis, but the level of protection afforded by IFN-y was strain specific. Lymphomas arising in IFN-y-deficient mice were very nonim-munogenic compared with those derived from pfp-deficient mice, suggesting a comparatively weaker immune selection pressure by IFN-y (Street et al, 2002). A significant incidence of late onset adenocarcinomas observed in both IFN-y- and pfp-deficient mice indicated that some epithelial tissues were also subject to immune surveillance.
3. Perforin and Fas/FasL System
The cytotoxic factors perforin (pfp) and Fas/FasL are additional important factors involved in immune surveillance. In general, cell-mediated cytotoxicity attributed to cytotoxic T lymphocytes (CTLs) and NK cells is derived from either the granule exocytosis pathway or the Fas pathway. The granule exocytosis pathway utilizes pfp to direct the granzymes to appropriate locations in target cells, where they cleave critical substrates that initiate apoptosis. Granzymes A and B induce death via alternate, nonoverlapping pathways. The Fas/FasL system is responsible for activation-induced cell death but also plays an important role in lymphocyte-mediated killing under certain circumstances (Russell and Ley, 2002). The interplay between these two cytotoxic systems provides opportunities for therapeutic interventions to control malignant disease, but oversuppression of these pathways also leads to decreased tumor cell killing. In fact, C57/BL/6 mice lacking pfp-/- were more susceptible to MCA-induced tumor formation. In MCA-induced tumor formation, pfp-/- mice developed significantly more tumors compared with pfp-sufficient mice treated in the same manner (Street et al, 2001; Street et al., 2002). In addition, a previous study showed that pfp-dependent cytotoxicity is not only a crucial mechanism of both CTL-and NK-dependent resistance to injected tumor cell lines but that the cytoxicity also operates during viral and chemical carcinogenesis that were induced by MCA or 12-O-tetradecanoylphorbol-13-acetate (TPA) plus 7,12-dimethylbenzanthracene (DMBA) or induced by injection of oncogenic Moloney sarcoma virus in vivo (van den Broek et al, 1996). Experiments addressing the role of Fas-dependent cytotoxicity by studying resistance to tumor cell lines that were stably transfected with Fas did not provide evidence for a major role of Fas and did not exclude a minor contribution of Fas in tumor surveillance. Another study showed that pfp-/- mice have a high incidence of malignancy in distinct lymphoid cell lineages (T, B, NKT), indicating a specific requirement for pfp in protection against lymphomagenesis (Smyth et al, 2000a). The susceptibility to lymphoma was enhanced by the simultaneous lack of expression of the p53 gene. The pfp-/- mice were at least 1000-fold more susceptible to these lymphomas when transplanted, compared with immunocompetent mice in which tumor rejection was controlled by CD8+ T lymphocytes (Smyth et al., 2000a). Taken together, these results indicate that components of the immune system were involved in controlling primary tumor development and showed the differential role of pfp and IFN-Y in protecting tumor formation between lymphoid and epithelial malignancies.
Although evidence suggested that the immune surveillance of cancer is dependent on both IFN-y and lymphocytes, the critical demonstration for the involvement of lymphocytes came from the use of gene-targeted mice lacking the recombinase-activating gene 1 (RAG-1) or RAG-2. Homozygous mutants of RAG-2 are viable but fail to produce mature B or T lymphocytes (Shinkai et al, 1992). Loss of the RAG-2 function in vivo results in the total inability to initiate VDJ rearrangement, leading to a novel SCID phenotype. RAG-2 function and VDJ recombinase activity, per se, are not required for development of cells other than lymphocytes. Since nude mice do not completely lack functional T cells and the two components of the immune system, IFN-y and pfp, to prevent tumor formation in mice, an elegant study using RAG-2-/- and Stat1-/- mice model showed for the first time that lymphocytes and IFN-Y collaborate to prevent the formation of carcinogen-induced sarcomas and spontaneous epithelial carcinomas (Shankaran et al., 2001). In detail, both the wild-type and RAG-2-/- mice had a pure 129/SvEv genetic background, and they were injected with MCA and monitored for tumor formation. RAG-2-/- mice formed tumors earlier than wild-type mice and with greater frequency. After 160 days, 9 out of 15 RAG-2-/- mice but only 2 out of 15 wild-type mice formed MCA-induced tumors. The increased tumor formation in RAG-2-/- was comparable to that in IFN-Y-insensitive mice that lacked either the IFNGR1 (12 out of 20) or Stat1 (17 out of 30) versus 11 out of 57 wild-type mice. In the collaboration between the lymphocytes- and IFN-Y/Stat1-dependent tumor suppressor mechanisms, mice lacking both genes (i.e., RAG-2-/- x Stat1-/- mice [RkSk mice]) showed increased susceptibility to MCA-induced tumors with 13 out of 18 mice compared to 11 out of 57 wild-type mice. However, RkSk mice did not show a significant increased incidence compared to mice that lacked either RAG-2-/- or Stat1-/-. Thus, these findings indicated that T, NKT, and/or B cells are essential to suppress the formation of chemically induced tumors and to allow the presence of an extensive overlap between lymphocytes and Stat1-dependent IFN-Y-signaling.
As for the effect of tumor suppressor mechanisms on spontaneous tumors, 9 out of 11 wild-type mice were free of malignant disease, 2 had adenomas, but none had cancer. In contrast, 12 out of 12 RAG-2-/-mice showed malignant lesions in the intestinal tract and elsewhere. Half of these mice formed malignant diseases: 3 cecal adeno-carcinoma, 1 ileocecal adenocarcinoma, 1
small intestinal adenocarcinoma, and 1 lung adenocarcinoma. In addition, 6 out of 11 RkSk mice developed mammary carcinomas, including 2 with adenocarcinomas and 1 with a distinct adenocarcinoma in the breast and cecum. The other 5 RkSk mice did not show palpable masses but the following were found at necropsy: 2 cecal adenocarcinomas, 1 cecal and lung adeno-carcinoma, and 2 intestinal adenomas. Overall, 82% of the RkSk mice formed spontaneous cancers. Thus, these findings suggest the lack of lymphocytes, either alone or in combination with the IFN-y-signaling defect, and indicate that the RAG-2-/- and RkSk mice are significantly more susceptible for spontaneous epithelial tumor formation than their wild-type counterparts. Moreover, RkSk mice form more spontaneous cancers than RAG-2-/- mice, suggesting that the overlap of the tumor suppressor mechanisms mediated by lymphocytes and IFN-y/Stat1-signaling may only be partially effective (Shankaran et al., 2001).
In another report, the relative contributions of ap- and yS -T cells in blocking tumor formation by chemical carcinogens, such as MCA, DMBA, and TPA, were studied; this report also noted the effect of injecting the squamous cell carcinoma cell line phocine distemper virus (PDV) into TCRp-/-, TCRS-/-, and TCRp-/-S-/- mice, which lack aP-T cells, yS-T cells, and all T cells, respectively (Girardi et al., 2001). Comparing tumor formation using PDV cells between wild-type and TCRp-/- mice, 41 out of 110 sites developed tumors in TCRp-/-mice, whereas 13 out of 134 sites developed tumors in wild-type mice. Although tumor latency accounted for a minor reduction, ap-T cells reduced the number of tumors formed. In contrast, in TCRS-/- and TCRP-/-S-/- mice, nearly 100% of sites showed tumor formation, and the latency was substantially reduced. These findings indicate that aP-T cells and yS-T cells regulate the tumor growth of PDV cells in a distinct fashion and that the lack of aP-T cells is not compensated by the presence of yS-T cells and NK cells. In addition, the role of yS-T cells in the development of MCA-induced sarcomas and spindle cell carcinomas was studied, and an increase in the number of tumors formed in TCRP-/- and TCRS-/- mice after MCA injection was observed compared to FVB mice (Girardi et al, 2001). Further, in naturally occurring human carcinomas induced by DMBA and TPA, 67% of TCRS-/- mice showed tumor formation with increased tumor burden compared to 16% of wild-type mice. In contrast, TCRP-/- and wild-type mice were equally susceptible to DMBA- and TPA-induced carcinogenesis. In addition, TCRS-/-mice also showed a higher incidence of progression of papillomas into carcinomas. These findings indicate a distinct additional contribution in the regulation of tumor growth in aP-T cells and yS-T cells. In turn, it seems that aP-T cells act to inhibit initial tumor formation that converts to malignant progression, whereas yS-T cells directly inhibit tumor progression by using their cytotoxic mechanisms to kill tumor cells. Thus, the previous and recent data support the following basic concept of cancer immune surveillance originally proposed by Burnet and Thomas: the naturally existing immune system can recognize nascent transformed cells and can eliminate primary tumor formation by lymphocytes and secreted cytokines as important protective mechanisms in the host. The studies that used inbred mouse lines targeting disruptions in genes encoding critical components of the immune system are listed in Table 2.1, and these data support the control of tumor formation by the immune systems of both innate and adaptive immune compartments in cancer immune surveillance.
5. Type I IFNs
Much less is known about the involvement of type I interferons, IFN-a/p, which regulate immunological functions and
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