Review the Immunobiology of Cancer Immunosurveillance and Immunoediting

Immunity, Vol. 21, 137–148, August, 2004, Copyright 2004 by Cell Press The Immunobiology Review of Cancer Immunosurveillance and Immunoediting

Gavin P. Dunn,1 Lloyd J. Old,2 tective versus tumor-sculpting actions of the immune and Robert D. Schreiber1,* system in cancer (Shankaran et al., 2001; Dunn et al., 1Department of Pathology and Immunology 2002, 2004). Herein, we summarize recent work on the Center for Immunology cancer immunosurveillance and immunoediting pro- Washington University School of Medicine cesses—underscoring a new optimism that an enhanced 660 South Euclid Avenue understanding of naturally occurring immune system/ Box 8118 tumor interactions will lead to the development of more St. Louis, Missouri 63110 effective immunologically based cancer therapies. 2 Ludwig Institute for Cancer Research New York Branch at Memorial Sloan-Kettering From Cancer Immunosurveillance Cancer Center to Cancer Immunoediting 1275 York Avenue In 1909, Paul Ehrlich predicted that the immune system New York, New York 10021 repressed the growth of carcinomas that he envisaged would otherwise occur with great frequency (Ehrlich, 1909), thus initiating a century of contentious debate over immunologic control of neoplasia. Fifty years later, The last fifteen years have seen a reemergence of as immunologists gained an enhanced understanding interest in cancer immunosurveillance and a broaden- of transplantation and tumor immunobiology and immu- ing of this concept into one termed cancer immuno- nogenetics, F. Macfarlane Burnet and Lewis Thomas editing. The latter, supported by strong experimental revisited the topic of natural immune protection against data derived from murine tumor models and provoca- cancer. Burnet’s thinking was shaped by a consideration tive correlative data obtained by studying human can- of immune tolerance; he believed that tumor cell-spe- cer, holds that the immune system not only protects cific neo-antigens could provoke an effective immuno- the host against development of primary nonviral can- logic reaction that would eliminate developing cancers cers but also sculpts tumor immunogenicity. Cancer (Burnet, 1957, 1964, 1971). Alternatively, Thomas’s early immunoediting is a process consisting of three phases: view was evolutionary in nature; he theorized that com- elimination (i.e., cancer immunosurveillance), equilib- plex long-lived organisms must possess mechanisms rium, and escape. Herein, we summarize the data sup- to protect against neoplastic disease similar to those porting the existence of each of the three cancer im- mediating homograft rejection (Thomas, 1959). With the munoediting phases. The full understanding of the functional demonstration of mouse tumor-specific anti- immunobiology of cancer immunosurveillance and im- gens supporting the ideas of Ehrlich, Burnet, and munoediting will hopefully stimulate development of Thomas (Old and Boyse, 1964), the cancer immunosur- more effective immunotherapeutic approaches to veillance hypothesis, which stated that sentinel thymus- control and/or eliminate human cancers. dependent cells of the body constantly surveyed host tissues for nascently transformed cells (Burnet, 1970), gained recognition. Despite subsequent challenges to Comments made decades ago by the architects of the this hypothesis over the next several decades (Stutman, cancer immunosurveillance hypothesis, Burnet and 1974, 1979), new studies in the 1990s—fueled by tech- Thomas, that “there is little ground for optimism about nologic advances in mouse genetics and monoclonal cancer” (Burnet, 1957) and “the greatest trouble with the antibody (mAb) production—reinvigorated and ulti- idea of immunosurveillance is that it cannot be shown to mately validated the cancer immunosurveillance con- exist in experimental animals” (Thomas, 1982), reflect cept (Smyth et al., 2001b; Dunn et al., 2002, 2004) and the problems that, until recently, fomented intense de- expanded it to incorporate the contributions of both bate over whether natural immune defense mechanisms innate and adaptive immunity. can protect the host against the development of can- However, there has been a growing recognition that cers of nonviral origin. The difficulty was clear: if im- immunosurveillance represents only one dimension of munosurveillance of developing tumors in immunocom- the complex relationship between the immune system petent hosts was indeed successful, then how could and cancer (Dunn et al., 2002, 2004; Schreiber et al., such an apparently invisible process be experimentally 2004). Recent work has shown that the immune system revealed? With the development of mouse tumor models may also promote the emergence of primary tumors with using inbred mice with molecularly defined immunodefi- reduced immunogenicity that are capable of escaping ciencies, it has become possible to demonstrate the immune recognition and destruction (Shankaran et al., existence of a cancer immunosurveillance process that 2001). These findings prompted the development of the can prevent primary tumor growth. Moreover, there is an cancer immunoediting hypothesis to more broadly en- emerging recognition that cancer immunosurveillance compass the potential host-protective and tumor-sculpt- represents only one step of a broader process, termed ing functions of the immune system throughout tumor cancer immunoediting, that stresses the dual host-pro- development (Dunn et al., 2002, 2004). Cancer immunoediting is a dynamic process composed of three phases: *Correspondence: [email protected] elimination, equilibrium, and escape (Figure 1). Elimina- Immunity 138

Figure 1. The Three Phases of the Cancer Immunoediting Process Normal cells (gray) subject to common oncogenic stimuli ultimately undergo transformation and become tumor cells (red) (top). Even at early stages of tumorigenesis, these cells may express distinct tumor-specific markers and generate proinflammatory “danger” signals that initiate the cancer immunoediting process (bottom). In the first phase of elimination, cells and molecules of innate and adaptive immunity, which comprise the cancer immunosurveillance network, may eradicate the developing tumor and protect the host from tumor formation. However, if this process is not successful, the tumor cells may enter the equilibrium phase where they may be either maintained chronically or immunologically sculpted by immune “editors” to produce new populations of tumor variants. These variants may eventually evade the immune system by a variety of mechanisms and become clinically detectable in the escape phase. tion represents the classical concept of cancer immuno- sor and protects the immunocompetent host from the surveillance, equilibrium is the period of immune-medi- development of neoplasia. In this section, we discuss ated latency after incomplete tumor destruction in the current work on the elimination phase of the cancer elimination phase, and escape refers to the final out- immunoediting process that specifically addresses the growth of tumors that have outstripped immunological following three central questions. (1) What cells protect restraints of the equilibrium phase. the host from tumor development? (2) What are the critical effector functions of the immune system in cancer The Elimination Phase: Assembling the Cancer immunosurveillance? (3) How does the immune system Immunosurveillance Network distinguish between a transformed cell and its normal progenitor? Pivotal studies on IFN-␥ (Dighe et al., 1994; Kaplan et al., 1998; Shankaran et al., 2001; Street et al., 2001, 2002) and perforin (van den Broek et al., 1996; Smyth et al., Adaptive and Innate Immune Cells Play Critical 2000a, 2000b; Street et al., 2001, 2002) have shown that Roles in Cancer Immunosurveillance deficiencies in key immunologic molecules enhanced Rapidly accumulating data have begun to elucidate the host susceptibility to both chemically induced and spon- cellular basis of cancer immunosurveillance and demon- taneous tumors, demonstrating for the first time a critical strate that lymphocytes of both the adaptive and innate prediction of the prescient, but previously unsubstanti- immune compartments prevent tumor development. ated, cancer immunosurveillance hypothesis. Further- One of the most definitive studies performed to date more, other compelling data suggest that immunosur- employed gene-targeted mice lacking the recombinase veillance is not restricted to mouse models but also activating gene (RAG)-2 (Shankaran et al., 2001) and exists in humans (Dunn et al., 2002, 2004). Thus, today demonstrated that lymphocytes expressing rearranged the question is not if—but, rather, how—cancer immu- antigen receptors play critical roles in the cancer im- nosurveillance functions as an extrinsic tumor suppres- munosurveillance process. Mice lacking RAG-2 (or its Review 139

obligate partner RAG-1) cannot somatically rearrange gression of developing papillomas to more aggressive lymphocyte antigen receptors and therefore cannot pro- carcinomas. Together, these results reveal that ␣␤ and duce peripheral ␣␤ T cells, B cells, NKT cells, or ␥␦ ␥␦ T cell subsets make critical, but distinct, contributions T cells (Shinkai et al., 1992). Since RAG-2 expression is to host antitumor defense mechanisms. limited to cells of the lymphoid system, RAG-2Ϫ/Ϫ mice NK and NKT cells also participate in cancer immuno- provided an appropriate model to exclusively study the surveillance. C57BL/6 mice, depleted of both NK and effects of host lymphocyte deficiency on tumor develop- NKT cells by using the anti-NK1.1 mAb, were two to ment. Unlike other genetic models of immunodeficiency three times more susceptible to MCA-induced tumori- (such as SCID mice), the absence of RAG-2 does not genesis than wild-type controls (Smyth et al., 2001a). A affect DNA damage repair pathways in nonimmune cells similar effect was observed in C57BL/6 mice treated undergoing transformation. Following subcutaneous in- with anti-asialo-GM1, which selectively depletes NK but jection of the chemical carcinogen 3Ј-methylcholan- not NKT cells. Although anti-asialo-GM1 can also de- Ϫ Ϫ threne (MCA), 129/SvEv RAG-2 / mice developed sar- plete activated macrophages, the latter observation comas at the injection site faster and with greater nevertheless supports the involvement of innate immune frequency than strain-matched wild-type controls cells in blocking primary tumor development. NKT cells Ϫ Ϫ (Shankaran et al., 2001). After 160 days, 30/52 RAG-2 / were also implicated in immunosurveillance by two mice formed tumors, compared with 11/57 wild-type additional observations. First, J␣281Ϫ/Ϫ mice, lacking mice (p Ͻ 0.0001). Similar findings were obtained in V␣14J␣281-expressing invariant NKT cells, developed Ϫ Ϫ C57BL/6 RAG-1 / mice treated with MCA (Smyth et MCA-induced sarcomas at a higher frequency than wild- al., 2001a). type controls (Smyth et al., 2000a). Second, mice treated Ϫ Ϫ In addition, Helicobacter-negative RAG-2 / 129/ with the NKT cell-activating ligand ␣-galactosylceramide SvEv mice aged in a specific pathogen-free mouse facil- (␣-GalCer) throughout MCA-induced tumorigenesis ex- ity and maintained on broad-spectrum antibiotics ad- hibited a reduced incidence of tumors and displayed a ministered every other month developed significantly longer latency period to tumor formation than control more spontaneous epithelial tumors than did wild-type mice (Hayakawa et al., 2003). counterparts (Shankaran et al., 2001; A.T. Bruce and A recent provocative study by Cui et al. (Cui et al., Ϫ/Ϫ R.D.S., unpublished data). Specifically, 31/32 RAG-2 2003) provides further evidence that innate immune cells mice (13–28 months old) developed spontaneous neo- comprise an important arm of the immunosurveillance plasia, predominantly of the intestine. Eight had prema- network. A single BALB/c mouse was serendipitously lignant intestinal adenomas, 22 had intestinal adenocar- found that failed to form ascites when injected intraperi- cinomas, and one had an intestinal adenoma and a lung toneally with the extremely aggressive S180 sarcoma adenocarcinoma. In contrast, 21/33 wild-type mice re- cell line. Subsequent breeding revealed that the ob- mained neoplasia free to the end of their lives. Of the served cancer resistance trait was germline transmissi- wild-type mice that developed neoplastic disease, six had ble and was likely controlled by a single autosomal domi- adenomas of the Harderian gland, lung, intestine, stom- nant locus. In addition, the resulting “spontaneous ach, or liver, while six others developed unrelated can- regression/complete remission” (SR/CR) mice were able cers that predominantly appeared late in life. Thus, lym- to kill a range of both syngeneic and allogeneic tumor phocytes protect mice against both chemically-induced cells derived from multiple tissue sites. Despite their and spontaneous tumor formation. avid antitumor resistance, SR/CR mice did not exhibit Subsequent studies have extended these findings by autoimmune pathology or shortened lifespan. Interest- identifying which of the possible RAG-dependent lym- ingly, resistance to S180 was also observed when the phocyte subsets contribute to host antitumor defense. SR/CR trait was bred onto a nude genetic background, This work highlighted roles for ␥␦ T cells, ␣␤ T cells, suggesting that the SR/CR phenotype is predominantly and NKT cells in the immunosurveillance process. Mice mediated by innate immune cells. Characterization of lacking either ␣␤ T cells (TCR ␤Ϫ/Ϫ mice) or ␥␦ T cells the locus controlling the SR/CR phenotype should pro- (TCR ␦Ϫ/Ϫ mice) are more susceptible to MCA-induced vide new insights into innate immune control of tumor tumor formation than wild-type mice (on either an FVB growth. or C57BL/6 genetic background) (Girardi et al., 2001; Taken together, these data not only highlight roles for Gao et al., 2003). In contrast, using a carcinogenesis model involving initiation with 7, 12-dimethylbenz both innate and adaptive immune components in the [a]anthracene (DMBA) and promotion with 12-O-tetra- elimination phase of cancer immunoediting but also decanoylphorbol 13-acetate (TPA), host protection underline the complexity of the host’s immune response against tumor formation was found to be more depen- to developing tumors. Specifically, cancer immunosur- dent on the action of ␥␦ T cells than ␣␤ T cells. Whereas veillance appears to be a multivariable process in which FVB strain TCR ␦Ϫ/Ϫ mice formed significantly more immunologic responses are influenced by a tumor’s cel- DMBA/TPA tumors than wild-type FVB controls, FVB lular origin, mode of transformation, anatomic location, strain TCR ␤Ϫ/Ϫ did not (Girardi et al., 2001, 2003). How- stromal response, cytokine production profile, and in- ever, TCR ␤Ϫ/Ϫ ϫ␦Ϫ/Ϫ mice were significantly more sus- herent immunogenicity. Thus, it remains critical to ceptible to DMBA/TPA carcinogenesis than singly-defi- assess the effects of a wide range of immunologic cient TCR ␦Ϫ/Ϫ mice (Girardi et al., 2003), revealing a components on tumor development in many different host-protective role for ␣␤ T cells in the setting of ␥␦ models—both chemically induced and spontaneous— T cell deficiency. Interestingly, in the same study TCR to determine whether the immunosurveillance of all ␦Ϫ/Ϫ mice exhibited the highest ratio of carcinomas to cancer-susceptible tissues of the body is globally similar papillomas, suggesting that ␥␦ T cells regulate the pro- or locally distinct. Immunity 140

Effector Functions Underlying Immunosurveillance: IFN-␥ϩ/ϩ and TCR ␦Ϫ/Ϫ IFN-␥ϩ/ϩmice, whereas the exper- IFN␥ Production and Cytotoxicity imental group consisted of lethally irradiated IFN-␥Ϫ/Ϫ IFN␥ Production mice reconstituted with a mixture of bone marrow from The studies that identified physiologically relevant cellu- TCR␤Ϫ/Ϫ ϫ IFN-␥Ϫ/Ϫ and TCR ␦Ϫ/Ϫ IFN-␥ϩ/ϩ mice. Thus, lar effectors of immunosurveillance have been comple- the only deficiency in the reconstituted experimental mented by studies that defined two of the critical tasks group was that mature ␥␦ T cells could not produce these immune cells must effect to eradicate developing IFN-␥. When challenged with MCA, the control bone tumors: the production of IFN-␥ and the ability to kill. marrow chimera group displayed a susceptibility to tu- Endogenously produced interferon-␥ (IFN-␥) was shown mor formation similar to that of wild-type mice. In con- to protect the host against the growth of transplanted trast, chimeric mice with ␥␦ T cells that could not tumors and also the formation of primary chemically produce IFN-␥ were significantly more susceptible to induced and spontaneous tumors (Dighe et al., 1994; MCA-induced tumor formation than wild-type counter- Kaplan et al., 1998; Shankaran et al., 2001; Street et al., parts and displayed an increased MCA susceptibility, 2001, 2002). Injection of neutralizing mAbs for IFN-␥ into indistinguishable from that of IFN-␥Ϫ/Ϫ mice. Thus, ␥␦ mice bearing transplanted, established Meth A tumors T cells are one physiologically relevant source of IFN-␥ blocked LPS-induced tumor rejection (Dighe et al., in the cancer immunosurveillance process. Additional 1994). In addition, transplanted fibrosarcomas grew work is required to identify if there are other cellular faster and more efficiently in mice treated with IFN-␥- sources of IFN-␥ during tumor development and to de- specific mAbs. In models of primary tumor formation, termine whether other IFN-␥-producing cells participate IFN-␥-insensitive 129/SvEv mice lacking either the in responses to different types of tumors. IFNGR1 ligand binding subunit of the IFN-␥ receptor or More is known about the physiologically relevant tar- STAT1, the transcription factor that mediates much of gets of IFN-␥’s actions. Host cells are important targets IFN-␥’s biologic effects on cells (Bach et al., 1997), were of IFN-␥ during development of protective antitumor found to be 10–20 times more sensitive than wild-type immune responses. STAT1Ϫ/Ϫ mice with generalized mice to MCA tumor induction (Kaplan et al., 1998). These IFN-␥/IFN-␣␤ insensitivity are more sensitive to MCA mice developed more tumors, more rapidly, and at lower primary tumor induction (Kaplan et al., 1998) and also carcinogen doses than did wild-type controls. Similar cannot reject highly immunogenic transplanted IFN-␥- results were obtained in independent experiments by sensitive tumor cells (Fallarino and Gajewski, 1999; V. using C57BL/6 mice lacking the gene encoding IFN-␥ Shankaran and R.D.S., unpublished data). At least in (Street et al., 2001). In models of genetically driven tu- part, these results are explained by a requirement for morigenesis, mice lacking both the p53 tumor suppres- IFN-␥ sensitivity at the level of the host immune compart- sor gene and either IFNGR1 or STAT1 formed a wider ment. Through its capacity to promote the generation tumor spectrum compared to IFN-␥-sensitive mice lack- of tumor-specific CD4ϩ Th1 T cells and cytolytic T cells ing p53 only (Kaplan et al., 1998). In another study, 16/ (CTL) and to activate cytocidal activity in macrophages, 32 of IFN-␥Ϫ/Ϫ C57BL/6 mice developed disseminated IFN-␥ facilitates development of powerful anti-tumor ef- lymphomas compared to 0/39 wild-type C57BL/6 mice fector functions mediated by both adaptive and innate (Street et al., 2002). immunity (Bach et al., 1997). However, the tumor cells The overlap between the IFN-␥- and lymphocyte- themselves have also been shown to represent a critical dependent tumor suppressor pathways was revealed cellular target of IFN-␥. Highly immunogenic MCA-in- by comparing tumor formation in 129/SvEv mice lacking duced tumor cells derived from IFN-␥ sensitive RAG- IFN-␥ responsiveness (IFNGR1Ϫ/Ϫ or STAT1Ϫ/Ϫ mice), 2Ϫ/Ϫ mice are rejected when injected into naive synge- lymphocytes (RAG-2Ϫ/Ϫ mice), or both (RAG-2Ϫ/Ϫ x neic immunocompetent recipients (Shankaran et al., STAT1Ϫ/Ϫ) (RkSk mice) (Shankaran et al., 2001). Each 2001; G.P.D., C.M. Koebel, and R.D.S., unpublished group of mice formed three times more chemically in- data). However, when the IFN-␥ sensitivity of these cells duced tumors than syngeneic wild-type mice when in- is ablated by overexpression of a dominant-negative jected with 100 ␮g of MCA. Since no significant differ- IFNGR1 mutant, they become poorly immunogenic and ences were detected between any of the gene-targeted form aggressively growing tumors in wild-type mice mice, it was concluded that the IFN-␥/STAT1- and lym- (Dighe et al., 1994; G.P.D., C.M. Koebel, and R.D.S., phocyte-dependent tumor suppressor mechanisms were unpublished data). Conversely, poorly immunogenic largely overlapping. However, RkSk mice developed MCA-sarcoma cells derived from IFNGR1Ϫ/Ϫ mice be- spontaneous breast tumors that were not observed in come highly immunogenic cells and are rejected in im- wild-type or RAG-2Ϫ/Ϫ mice, therefore demonstrating munocompetent mice after ectopic expression of wild- that the overlap between the two pathways was incom- type IFNGR1 and restoration of IFN-␥ sensitivity (Kaplan plete. et al., 1998). IFNGR1Ϫ/Ϫ tumor cells are also rendered Additional work has begun to identify the relevant highly immunogenic by ectopic expression of IFN-␥- cellular sources and targets of IFN-␥ in immunosurveil- inducible components of the MHC class I antigen pro- lance. Recent work suggests that ␥␦ T cells are an impor- cessing and presentation pathway (Shankaran et al., tant source of IFN-␥ during the development of protec- 2001; A.T. Bruce and R.D.S., unpublished data), thus tive antitumor responses (Gao et al., 2003). To directly revealing that IFN-␥’s ability to upregulate tumor immu- test if ␥␦ T cells were a physiologically important source nogenicity is sufficient to explain the effects on tumor of IFN-␥ in blocking primary tumor formation, two sets detection and elimination in immunocompetent hosts. of bone marrow chimeras were generated. In the control One key remaining question is whether type I interfer- group, IFN-␥Ϫ/Ϫ mice were lethally irradiated and recon- ons (IFN-␣/␤) also participate in cancer immunosurveil- stituted with a mixture of bone marrow from TCR ␤Ϫ/Ϫ lance. Longstanding work from Gresser and colleagues Review 141

has shown that in vivo neutralization of endogenously and CD8ϩ ␣␤ T cells recognize tumor antigens in the produced IFN-␣/␤ enhances growth of transplanted tu- context of MHC class II and class I proteins, respec- mors in wild-type mice (Gresser and Belardelli, 2002). tively. Since the first human tumor antigen was identified Moreover, administration of IFN-␣/␤ has positive thera- in 1991 (van der Bruggen et al., 1991), many tumor anti- peutic actions on certain types of murine and human gens have been cloned and can be segregated into five cancers (Belardelli et al., 2002). More recent in vitro categories: (1) differentiation antigens, e.g., melanocyte work has pointed to a role for IFN-␣/␤ in preventing differentiation antigens, Melan-A/MART-1, tyrosinase, cellular transformation through mechanisms involving and gp-100; (2) mutational antigens, e.g., abnormal enhanced cellular expression of the p53 tumor suppres- forms of p53; (3) overexpressed/amplified antigens, e.g., sor gene in cells exposed to type I IFN (Takaoka et HER-2/neu; (4) cancer-testis (CT) antigens, e.g., MAGE al., 2003). Further work is needed to define the precise and NY-ESO-1; and (5) viral antigens, e.g., EBV and HPV cellular targets of IFN-␣/␤ in the cancer immunosur- (Boon and van der Bruggen, 1996; Rosenberg, 1999; veillance process and to determine whether IFN-␣/␤ Old, 2003). The molecular definition of tumor antigens functions in a manner that is identical to or distinct has revolutionized the field of tumor immunology by from IFN-␥. providing a firm basis for how the adaptive immune Cytolytic Capacity system discriminates between normal and neoplastic The second critical effector function of cancer immuno- cells. surveillance is the immune system’s ability to kill tumor In addition to tumor antigens presented on MHC mole- cells. Early studies identified perforin (pfp) as a critical cules, transformed cells may overexpress other mole- cytolytic molecule in the primary host antitumor re- cular signposts that can function as recognition targets sponse. After challenge with MCA, pfpϪ/Ϫ mice formed in the immunosurveillance process. Several studies two to three times more tumors than wild-type mice (van have pointed to the NKG2D-activating receptor, ex- den Broek et al., 1996; Smyth et al., 2000a, 2000b; Street pressed on NK cells, ␥␦ T cells, and CD8 ␣␤ T cells et al., 2001). In addition, 50% (10/20) of aging pfpϪ/Ϫ (Bauer et al., 1999; reviewed in Raulet, 2003), as one C57BL/6 mice developed spontaneous disseminated important component that is used by both adaptive and lymphomas as compared to 1/16 wild-type mice (Smyth innate immune cells to distinguish cancer cells from et al., 2000b). The kinetics of lymphoma development normal cells. Functional NKG2D receptors complexes were accelerated in pfpϪ/Ϫ mice also lacking p53 (Smyth consist of the NKG2D ligand binding polypeptide and et al., 2000b) or ␤2 microglobulin (Street et al., 2004). either the DAP10 or DAP12 signaling polypeptide (Gilfil- Subsequent studies revealed an important role for the lan et al., 2002). In humans, NKG2D binds to the MHC TNF-related apoptosis-inducing ligand (TRAIL) and have class I chain-related proteins A and B (MICA/B), as well underscored the importance of cytotoxicity manifest by as the UL16 binding proteins (ULBPs) (Cosman et al., innate immunity in immunosurveillance. A member of 2001; Pende et al., 2002) and the recently discovered the TNF superfamily that induces apoptosis through en- lymphocyte effector cell toxicity-activating ligand (Letal) gagement of the TRAIL-R2 (DR5) receptor in mice, TRAIL (Conejo-Garcia et al., 2003) (first reported as RAET1E is expressed constitutively on a subset of liver NK cells [Radosavljevic et al., 2002] and also termed ULBP4 and is induced by either IFN-␥ or IFN-␣/␤ in monocytes, [Chalupny et al., 2003]). The MICA/B proteins are highly NK cells, and dendritic cells (Smyth et al., 2003). When polymorphic, nonclassical MHC cell surface glycopro- ␤ injected with low doses of MCA, C57BL/6 mice treated teins that do not associate with 2m or require TAP1 with neutralizing antibodies to TRAIL (Takeda et al., for expression (Groh et al., 1996; Bahram, 2000). Inter- estingly, while MIC expression in normal tissues has only 2002) or lacking the TRAIL gene (Cretney et al., 2002) been documented on the gastrointestinal epithelium of developed fibrosarcomas at a higher incidence than the stomach and large intestines, MICA/B proteins are wild-type controls. Moreover, p53ϩ/Ϫ C57BL/6 mice often expressed in primary carcinomas of the lung, kid- treated with the neutralizing TRAIL-specific antibody de- ney, prostate, ovary, colon (Groh et al., 1999) and liver veloped more spontaneous sarcomas and disseminated (Jinushi et al., 2003b), as well as in melanomas (Vetter lymphomas over a two-year period than control IgG- et al., 2002). In addition, ULBPs (Pende et al., 2002) and treated mice (Takeda et al., 2002). Further study is re- Letal (Conejo-Garcia et al., 2003) are also frequently quired to identify the specific innate cell subsets that expressed on tumor cells. In mice, NKG2D binds to the manifest the TRAIL-dependent antitumor effects. Con- retinoic acid early transcript 1 (Rae-1) family proteins sidering that the TRAIL-R2 receptor is upregulated by Rae-1␣-⑀, the minor histocompatibility antigen H60 (Die- p53 in response to DNA damage (Wu et al., 1997, 1999), fenbach et al., 2000; Cerwenka et al., 2001), and mouse TRAIL killing may be a critical link between target cell UL16 binding protein-like transcript (MULT-1) (Carayan- genotoxic distress and immune-mediated destruction. nopoulos et al., 2002; Diefenbach et al., 2003). NKG2D ligand expression has been observed on a wide range Patrolling Transformation: Mechanisms that Adaptive of murine tumors (Diefenbach et al., 2000), and ectopic versus Innate Immunity Use to Distinguish Tumor expression of Rae-1, H60, or MULT-1 was sufficient to Cells from Normal Cells induce the rejection of several progressively growing, The third central question concerns how cells of the transplantable tumors (Cerwenka et al., 2001; Diefen- immunosurveillance network distinguish nascent trans- bach et al., 2001, 2003). formed or established tumor cells from normal cells. It will be important to characterize the regulation of Work over the last decade has begun to reveal the mo- NKG2D ligand expression in both human and murine lecular basis of this crucial distinction particularly within cells. These molecules are often described as “stress the adaptive immune compartment. Specifically, CD4ϩ molecules,” but to date no cancer-relevant signaling Immunity 142

pathways have been causally linked to their expression. (Burnet, 1970). Moreover, the surveillance functions of In human cells, MICA/B gene expression has been in- the immune system were thought to be executed only duced in several nontransformed human cell lines by at the earliest stages of tumorigenesis. However, recent heat shock at 42ЊC (Groh et al., 1999), infection with studies have started to explain what happens when tu- human cytomegalovirus (Groh et al., 2001), or exposure mors develop in immunocompetent hosts, as they do to E. coli (Tieng et al., 2002), although in dendritic cells in individuals with cancer, and what has emerged from MICA/B is upregulated by type I interferon (Jinushi et this is the realization that even when immunosurveil- al., 2003a) or M. tuberculosis infection (Das et al., 2001). lance fails, the relationship between immunity and can- However, it remains unclear how these conditions over- cer is far from over. lap the molecular cascades that underlie neoplastic An appreciation of the complexity of the immune sys- transformation (Hahn and Weinberg, 2002a; Hahn and tem/tumor interaction is based on work that compared Weinberg, 2002b). In mice, Rae-1 is upregulated by reti- the immunogenicities of tumors derived from immuno- noic acid in F9 cells (Nomura et al., 1994) and is also compromised versus immunocompetent mice. In one expressed early in development (Raulet, 2003). In addi- study, 17/17 tumors derived from wild-type 129/SvEv tion, one study assessed the expression of the NKG2D mice and 20/20 tumors derived from immunodeficient ligands H60 and Rae-1 after topical application of DMBA RAG-2Ϫ/Ϫ mice grew progressively when transplanted and TPA (Girardi et al., 2001). While no expression of into RAG-2Ϫ/Ϫ hosts (Shankaran et al., 2001). Further- these molecules was observed by RT-PCR in normal more, all 17 wild-type tumors grew progressively when skin, Rae-1 and H60 expression became detectable 24 transplanted into immunocompetent recipients. How- hr after carcinogen treatment. Strikingly, expression of ever, 8/20 tumors from RAG-2Ϫ/Ϫ mice were rejected both molecules was significantly increased in papillo- when transplanted into wild-type mice. These findings mas and carcinomas generated by DMBA/TPA treat- showed that tumors formed in an immunodeficient envi- ment. It is possible that the transformation process itself ronment are, as a group, more immunogenic than tu- induces molecules such as the NKG2D ligands so that mors that develop in immunocompetent hosts. Similarly, the genomic upheaval of tumorigenesis is directly trans- MCA-induced sarcomas derived from nude (Svane et lated into enhanced immune recognition. Further study al., 1996) or SCID mice (Engel et al., 1996) were rejected on the immunology of transformation will be necessary more frequently than similar tumors derived from wild- to detail when—and how—in the course of tumorigene- type mice when transplanted into wild-type hosts. More- sis a cancer cell becomes immunogenic. over, sarcomas from J␣281Ϫ/Ϫ mice grew progressively One aspect of the cancer immunosurveillance pro- when transplanted into J␣281Ϫ/Ϫ recipients, but their cess that has been the subject of much controversy is growth was significantly impaired when transplanted whether the unmanipulated immune system can detect a into wild-type mice (Smyth et al., 2000a). In addition, developing tumor, even one that may express distinctive lymphomas from pfpϪ/Ϫ mice grew avidly when trans- recognition molecules on its surface or contain tumor- planted into pfpϪ/Ϫ recipients, but most were rejected specific antigens. In the past, it was argued that cellular when transplanted into wild-type mice (Street et al., transformation did not provide a sufficient proinflamma- 2002). tory or “danger” signal to alert the immune system to Together, the functional demonstration that immunity the presence of a developing tumor (Matzinger, 1994; shapes tumor immunogenicity has laid the foundation Pardoll, 2003). However, it was recently realized that (1) for the development of the cancer immunoediting hy- danger signals, such as uric acid (Shi et al., 2003), may pothesis. This concept not only incorporates the original arise from the inherent biology of the tumor itself (Seong notion of cancer immunosurveillance but also recognizes and Matzinger, 2004) and (2) induction of proinflamma- that even after escaping immunosurveillance, a tumor’s tory responses through the generation of potential Toll- immunogenic phenotype is continuously shaped by the like receptor ligands, such as heat shock proteins immunological forces in its environment. In trying to (Ohashi et al., 2000; Asea et al., 2002; Srivastava, 2002), understand how tumors may ultimately be sculpted by or extracellular matrix derivatives, such as hyaluronic immunity, we propose that a tumor that has breached acid (Termeer et al., 2002) or heparan sulfates (John- the elimination phase of the immunoediting process may son et al., 2002), may share similarities to the events experience two subsequent phases in its interactions that underlie activation of innate immune responses to with the host’s immune system: equilibrium, followed microbial pathogens (Janeway, 1989). Importantly, by escape (Dunn et al., 2002, 2004). whereas locally controlled inflammation may be involved in initiating responses to tumors, excessive inflamma- The Equilibrium Phase of Cancer Immunoediting tion may facilitate the transformation process (Balkwill Based on the experimental systems described above, and Mantovani, 2001; Coussens and Werb, 2002; Dra- cancer immunosurveillance—i.e., the elimination phase noff, 2004). It is therefore important to better define how of the cancer immunoediting process—can eradicate a inflammation inhibits or promotes tumor development significant percentage of transformed cells. However, in vivo. some tumor cells may withstand the formidable pressure exerted by cancer immunosurveillance’s arsenal. Immunologic Sculpting of Cancer: When Therefore, we envision that there exists a period of la- Tumors Escape Immunosurveillance tency extending from the end of the elimination phase Originally, cancer immunosurveillance was envisaged to the beginning of the escape phase and the emergence as a binary process: the immune system either protected of clinically detectable malignant disease. This poten- the host from the development or cancer, or it did not tially protracted period in the course of the immune Review 143

system/tumor interaction that probably occurs prior to Likewise, we envision three possible outcomes for a the detection of clinically apparent tumors constitutes tumor that has entered the latent period of equilibrium: the equilibrium phase. The events that occur in the equi- (1) eventual elimination by the immune system, (2) per- librium phase of cancer immunoediting are likely quite manent maintenance in the equilibrium phase by the similar to those previously envisaged to occur in a pro- cellular and molecular controls of immunity, or (3) es- cess termed tumor dormancy (Wheelock et al., 1981; cape from immune pressure and transit to the final es- Uhr et al., 1991). In both cases, while the immune system cape phase of the immunoediting process. Importantly, initially constrains the growth of heterogeneous tumors the first two phases of the cancer immunoediting pro- composed of unstable and rapidly mutating cells, it may cess represent potential goals for immunotherapy: tu- approach an asymptote that Darwin could have pre- mor elimination or the durable control of cancer in equi- dicted; although many of the original tumor cells are librium. Currently, the equilibrium phase is the most destroyed, new variants arise carrying more mutations hypothetical of the three phases, and more data are that provide them with increased resistance to immune needed to prove its existence. Thus, it is critical to estab- attack. Ultimately, the dynamic interaction between im- lish new tumor models that will stringently test for the munity and cancer in the equilibrium phase produces existence of the equilibrium phase during primary tumor new populations of tumor cells vetted for survival in the development and subsequently define the effects of ex- immunocompetent host. perimentally controlled variable periods of equilibrium The tumor substrate on which immune cells continu- on the immunogenicities of established cancers in im- ously act can contain cancer cells that harbor thousands munocompetent hosts. of mutations (Loeb, 1991; Loeb et al., 2003). The enor- mous plasticity of the cancer cell genome is thought to The Escape Phase of Cancer Immunoediting arise from several types of genetic instability, including Edited tumor cells surviving the equilibrium phase of nucleotide-excision repair instability, microsatellite in- the cancer immunoediting process enter the escape stability, and chromosomal instability (Lengauer et al., phase where tumor growth proceeds unrestrained by 1998), the latter of which may induce gains or losses of immune pressure. To become clinically detectable in the whole chromosomes. Thus, the tumor cell’s constant immunocompetent host, cancer cells must circumvent genomic metamorphosis may eventually give rise to new both innate and adaptive immunologic defenses. The phenotypes that display reduced immunogenicity. Im- degree to which a tumor’s immunogenicity is shaped portantly, while the genesis of variation in tumor cell by its interaction with the host immune system may immunogenicity may be stochastic, the tumor that ulti- be determined by the identities of the immune editors mately emerges from the equilibrium phase is instruc- operative during the equilibrium phase. It is possible tively shaped by the repertoire of immune “editors” in that tumor escape from each different tissue site of its local environment and is thus conditioned to progress origin may be mechanistically distinct. It therefore fol- into the escape phase of the immunoediting process. lows that metastatic lesions may experience the most A clinical scenario that likely demonstrates the exis- significant immunologic sculpting—hewn by immune tence of the equilibrium phase in humans is the transmis- pressure from both primary tissue sites of origin as well sion of cancer from organ transplant donors to recipi- as distant sites of manifestation. ents. In these situations, transplanted organs appear Many studies have documented that tumor escape grossly cancer free at harvest. While some organ donors can be a direct consequence of alterations occurring in are subsequently found to harbor disease in other ana- edited tumor targets themselves. For example, some tomic sites, other transplant donors either have no clini- tumor cells develop direct or indirect lesions in antigen cal history of cancer or have been in durable remission from cancer prior to transplantation. One recent study processing and presentation pathways that facilitate reported the occurrence of metastatic melanoma 1–2 evasion from adaptive immune recognition. Analysis of years posttransplant in two allograft recipients who had human tumor specimens has shown that many display each received kidneys from the same donor (MacKie et losses of HLA class I proteins (Algarra et al., 2000; Marin- al., 2003). Upon subsequent investigation, it was found cola et al., 2000). In addition, other components of this that the donor had been treated for primary melanoma pathway, including TAP1 and the immunoproteasome 16 years before her death, but was considered tumor subunits LMP2 and LMP7, are frequently deficient in free at the time of organ donation. This study, together human tumors (Seliger et al., 2000). Moreover, other with others in the literature (Penn, 1991, 1996; Elder et lesions were identified that indirectly lead to antigen al., 1997; Suranyi et al., 1998), suggests that the pharma- processing or presentation defects. In one study, 4/17 cologic suppression of the immune systems of these (25%) human lung adenocarcinoma cell lines were found transplant recipients facilitated the rapid and progres- to be unresponsive to IFN-␥ due to the absence or ab- sive outgrowth of occult tumors that were maintained normal function of components of the IFN-␥ receptor in the equilibrium phase by the donor’s intact immune signaling pathway (Kaplan et al., 1998). Tumor cells ex- system. pressing these lesions failed to upregulate MHC class I We stress here that the cancer immunoediting pro- pathway activity when exposed to IFN-␥. Similar lesions cess may not always represent the linear progression were found in other studies that reported deficiencies of of a tumor from the elimination phase through the equi- IFN receptor signaling pathway components in prostate librium phase and into the final escape phase of clinical cancer (G.P.D. and R.D.S., unpublished data) and mela- detection. Indeed, this process may be terminated in noma (Wong et al., 1997). the elimination phase if the cancer immunosurveillance Tumor escape has also been observed as one out- process is successful at destroying a developing tumor. come of specific immunotherapeutic approaches. In one Immunity 144

study, ten patients harboring metastatic melanoma re- mice to reject tumors that grew progressively in con- ceived adoptively transferred T cells specific to the tu- trol mice. mor antigens MART-1/MelanA or gp100 with adjuvant These observations have stimulated interest in defin- IL-2 therapy (Yee et al., 2002). Although 8/10 patients ing the tumor antigens recognized by Tregs and in de- experienced stable, minor, or mixed clinical responses, termining whether an inhibitory T cell subset operates 3/5 patients studied exhibited specific loss of the tumor similarly in human cancer patients. By using SEREX antigen that was targeted during treatment. Similar find- analysis (Sahin et al., 1995), an expression cloning tech- ings were made in two other melanoma vaccine trials nique wherein IgG class antibodies present in the sera in which multiple tumors from individual patients under- of tumor bearing hosts are used to identify tumor anti- going vaccination with peptides from gp-100, MART-1, gens, a set of normal, nonmutated proteins, including and tyrosinase lost expression of either the targeted Dna J-like 2, were identified as tumor antigens. When melanoma antigens or all HLA molecules on which the challenged intravenously with fibrosarcoma cells, naive antigens were presented (Jager et al., 1997; Khong et mice preimmunized with a subset of these SEREX- al., 2004). These studies clearly show that tumors may defined antigens displayed a remarkable enhancement evade both naturally occurring or therapeutically in- of pulmonary metastases compared to control mice duced immune responses and suggest that at least (Nishikawa et al., 2003). This effect was inhibited if Dna some of the dynamics underlying the cancer immuno- J-like 2-immunized mice were first depleted of CD25ϩ editing process in the unmanipulated host are also at cells. Moreover, permissiveness for tumor growth could work when the three phases of cancer immunoediting be transferred to naive mice by passive transfer of are initiated therapeutically. CD4ϩCD25ϩ T cells derived from Dna J-like 2-immu- Other work has shown that inhibition of the protective nized hosts. More striking were the subsequent obser- functions of the immune system may also facilitate tu- vations that (1) Dna J-like 2-immunized mice developed mor escape. In this scenario, immunologically sculpted MCA-induced primary tumors more rapidly and with tumor cell variants may overproduce immunosuppres- greater frequency compared to control mice and (2) this sive cytokines, such as TGF-␤ or IL-10 (Khong and effect was abrogated if the immunized mice were pre- Restifo, 2002), or inhibit immune responses through treated with a depleting CD25-specific mAb (H. Nishi- other mechanisms. One study documented that soluble kawa, T. Kato, L.J.O., and H. Shiku, unpublished data). forms of the MIC NKG2D ligands, secreted by certain Together, these results suggest that CD4ϩCD25ϩ Tregs human tumors, downregulated the NKG2D receptor on play an important role in suppressing protective immune immune effector cells and attenuated lymphocyte-medi- responses against both primary and transplanted tumors. ated cytotoxicity (Groh et al., 2002). Other studies dem- Recent studies have suggested that Tregs can also onstrated that some tumor cells overproduce inhibitors be detected in a variety of human cancers. Specifically, of T cell responses, such as galectin-1 (Rubinstein et individuals with nonsmall cell lung cancer or cancers of al., 2004) and indoleamine 2,3-dioxygenase (IDO) (Uytten- the ovary (Woo et al., 2001), breast, or pancreas (Liya- hove et al., 2003). Finally, other developing tumors sup- nage et al., 2002) all displayed elevated levels of press induction of proinflammatory danger signals CD4ϩCD25ϩ Tregs. Moreover, another study has identi- through mechanisms involving activated STAT3, leading fied the CT antigen LAGE-1 as the first human tumor to impaired dendritic cell maturation that, in turn, pro- antigen specifically recognized by Tregs (Wang et al., vides the developing tumor with a potential mechanism 2004). These studies not only complement those in ani- to escape immune detection (Wang et al., 2003). Thus, mal models but also point to the probable clinical rele- a tumor may directly inhibit antitumor immune re- vance of regulatory T cell activity in human cancer. Fur- sponses by multiple mechanisms. ther studies will be necessary to characterize the Significant interest has recently focused on the phenotypes of Tregs, elucidate the molecular basis of premise that tumors may also facilitate the generation, their suppressive functions and identify more fully their activation, or function of immunosuppressive T cell pop- physiologic ligands. ulations (Terabe and Berzofsky, 2004), such as IL-13- producing NKT cells (Terabe et al., 2000) or CD4ϩCD25ϩ regulatory T cells (T regs). The latter have attracted con- Conclusion siderable attention due to their involvement in control- In his writings about cancer immunosurveillance, Lewis ling both pathologic and protective immune responses. Thomas reflected that, “It seemed to me then, and still Originally identified as a CD4ϩ T cell subset (comprising does, that some such built-in immunologic mechanism 5%–10% of all peripheral T cells) constitutively express- ought to exist for natural defense against cancer” ing CD25 that controls the behavior of autoreactive (Thomas, 1982). In this review, we have summarized T cells in vivo and suppresses T cell responses in vitro the functional evidence showing that such immunologic (Sakaguchi et al., 1995; Read et al., 1998; Thornton and defenses do, in fact, exist in the immunocompetent host Shevach, 1998), CD4ϩCD25ϩ Tregs were subsequently and have discussed the nature of the known cellular and suggested to play important roles in inhibiting naturally molecular components that inhibit tumor development. occurring and therapeutically induced protective im- Furthermore, we have pointed out several remaining mune responses against tumors. Two studies showed critical questions whose answers will ultimately frame that CD4ϩCD25ϩ Tregs are often responsible for the a full understanding of the host-protective cancer immu- failure of naive murine hosts to eliminate transplanted nosurveillance network. We have also discussed the tumors (Onizuka et al., 1999; Shimizu et al., 1999). Col- studies demonstrating that cancer immunosurveillance lectively, these studies documented that depletion of is not the whole story, thus prompting the development CD4ϩCD25ϩ Tregs by using an anti-CD25 mAb enabled of the cancer immunoediting hypothesis—i.e., the ac- Review 145

knowledgment that immunity’s powers to protect the Cerwenka, A., Baron, J.L., and Lanier, L.L. (2001). Ectopic expres- host from cancer may also drive the generation of tu- sion of retinoic acid early inducible-1 gene (RAE-1) permits natural mors better suited to survive in an immunologically in- killer cell-mediated rejection of a MHC class I-bearing tumor in vivo. Proc. Natl. Acad. Sci. USA 98, 11521–11526. tact environment. The most significant clinical implica- Chalupny, J.N., Sutherland, C.L., Lawrence, W.A., Rein-Weston, A., tion of this hypothesis is that most, if not all, tumors that and Cosman, D. (2003). ULBP4 is a novel ligand for human NKG2D. develop in humans may have undergone immunologic Biochem. Biophys. Res. Commun. 305, 129–135. sculpting as a result of a cancer immunoediting process, Conejo-Garcia, J.R., Benencia, F., Courreges, M.C., Khang, E., with the most dramatic consequences of the process Zhang, L., Mohamed-Hadley, A., Vinocur, J.M., Buckanovich, R.J., probably occurring before the tumor is clinically detect- Thompson, C.B., Levine, B., and Coukos, G. (2003). Letal, A tumor- able. Ultimately, if the cancer immunoediting process associated NKG2D immunoreceptor ligand, induces activation and indeed emerges as one of the leitmotifs of cancer pro- expansion of effector immune cells. Cancer Biol. Ther. 2, 446–451. gression, then an improved understanding of the immu- Cosman, D., Mullberg, J., Sutherland, C.L., Chin, W., Armitage, R., nobiology of cancer immunoediting and a molecular Fanslow, W., Kubin, M., and Chalupny, N.J. (2001). ULBPs, novel definition of how tumors are shaped by this process MHC class I-related molecules, bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor. Immunity will undoubtedly bring us closer to tumor immunology’s 14, 123–133. capstone: the use of immunotherapy to control and/or Coussens, L.M., and Werb, Z. (2002). Inflammation and cancer. eradicate neoplastic disease in the human cancer pa- Nature 420, 860–867. tient. Cretney, E., Takeda, K., Yagita, H., Glaccum, M., Peschon, J.J., and Smyth, M.J. (2002). Increased susceptibility to tumor initiation and Acknowledgments metastasis in TNF-related apoptosis-inducing ligand-deficient mice. J. Immunol. 168, 1356–1361. The authors are grateful to Drs. Ravindra Uppaluri and Jack Bui of Cui, Z., Willingham, M.C., Hicks, A.M., Alexander-Miller, M.A., How- Washington University School of Medicine for helpful comments ard, T.D., Hawkins, G.A., Miller, M.S., Weir, H.M., Du, W., and during the preparation of this manuscript. Work in the authors’ labo- DeLong, C.J. (2003). Spontaneous regression of advanced cancer: ratories quoted in this review was supported by grants from the identification of a unique genetically determined, age-dependent National Cancer Institute, the Ludwig Institute for Cancer Research trait in mice. Proc. Natl. Acad. Sci. USA 100, 6682–6687. and the Cancer Research Institute. Das, H., Groh, V., Kuijl, C., Sugita, M., Morita, C.T., Spies, T., and ␥ ␦ Received: July 20, 2004 Bukowski, J.F. (2001). MICA engagement by human V 2V 2 T cells Revised: July 27, 2004 enhances their antigen-dependent effector function. Immunity 15, Accepted: July 28, 2004 83–93. Published: August 17, 2004 Diefenbach, A., Jamieson, A.M., Liu, S.D., Shastri, N., and Raulet, D.H. (2000). Ligands for the murine NKG2D receptor: expression References by tumor cells and activation of NK cells and macrophages. Nat. Immunol. 1, 119–126. Algarra, I., Cabrera, T., and Garrido, F. (2000). The HLA crossroad Diefenbach, A., Jensen, E.R., Jamieson, A.M., and Raulet, D.H. in tumor immunology. Hum. Immunol. 61, 65–73. (2001). Rae1 and H60 ligands of the NKG2D receptor stimulate tu- Asea, A., Rehli, M., Kabingu, E., Boch, J.A., Bare, O., Auron, P.E., mour immunity. Nature 413, 165–171. Stevenson, M.A., and Calderwood, S.K. (2002). Novel signal trans- Diefenbach, A., Hsia, J.K., Hsiung, M.Y., and Raulet, D.H. (2003). A duction pathway utilized by extracellular HSP70: role of toll-like novel ligand for the NKG2D receptor activates NK cells and macro- receptor (TLR) 2 and TLR4. J. Biol. Chem. 277, 15028–15034. phages and induces tumor immunity. Eur. J. Immunol. 33, 381–391. Bach, E.A., Aguet, M., and Schreiber, R.D. (1997). The IFN␥ receptor: Dighe, A.S., Richards, E., Old, L.J., and Schreiber, R.D. (1994). En- a paradigm for cytokine receptor signaling. Annu. Rev. Immunol. hanced in vivo growth and resistance to rejection of tumor cells 15, 563–591. expressing dominant negative IFN␥ receptors. Immunity 1, 447–456. Bahram, S. (2000). MIC genes: from genetics to biology. Adv. Immu- Dunn, G.P., Old, L.J., and Schreiber, R.D. (2004). The Three Es of nol. 76, 1–60. Cancer Immunoediting. Annu. Rev. Immunol. 22, 329–360. Balkwill, F., and Mantovani, A. (2001). Inflammation and cancer: Dunn, G.P., Bruce, A.T., Ikeda, H., Old, L.J., and Schreiber, R.D. back to Virchow? Lancet 357 539–545. (2002). Cancer immunoediting: from immunosurveillance to tumor Bauer, S., Groh, V., Wu, J., Steinle, A., Phillips, J.H., Lanier, L.L., escape. Nat. Immunol. 3, 991–998. and Spies, T. (1999). Activation of NK cells and T cells by NKG2D, Dranoff, G. (2004). Cytokines in cancer pathogenesis and cancer a receptor for stress-inducible MICA. Science 285, 727–729. therapy. Nat. Rev. Cancer 4, 11–22. Belardelli, F., Ferrantini, M., Proietti, E., and Kirkwood, J.M. (2002). Ehrlich, P. (1909). Ueber den jetzigen Stand der Karzinomforschung. Interferon-alpha in tumor immunity and immunotherapy. Cytokine Ned. Tijdschr. Geneeskd. 5, 273–290. Growth Factor Rev. 13, 119–134. Elder, G.J., Hersey, P., and Branley, P. (1997). Remission of trans- Boon, T., and van der Bruggen, P. (1996). Human tumor antigens planted melanoma–clinical course and tumour cell characterisation. recognized by T lymphocytes. J. Exp. Med. 183, 725–729. Clin. Transplant. 11, 565–568. Burnet, F.M. (1957). Cancer—a Biological Approach. BMJ 1, 841–847. Engel, A.M., Svane, I.M., Mouritsen, S., Rygaard, J., Clausen, J., and Burnet, F.M. (1964). Immunological factors in the process of carcino- Werdelin, O. (1996). Methylcholanthrene-induced sarcomas in nude genesis. Br. Med. Bull. 20, 154–158. mice have short induction times and relatively low levels of surface Burnet, F.M. (1970). The concept of immunological surveillance. MHC class I expression. APMIS 104, 629–639. Prog. Exp. Tumor Res. 13, 1–27. Fallarino, F., and Gajewski, T.F. (1999). Cutting edge: differentiation Burnet, F.M. (1971). Immunological surveillance in neoplasia. Trans- of antitumor CTL in vivo requires host expression of Stat1. J. Immu- plant. Rev. 7, 3–25. nol. 163, 4109–4113. Carayannopoulos, L.N., Naidenko, O.V., Fremont, D.H., and Yoko- Gao, Y., Yang, W., Pan, M., Scully, E., Girardi, M., Augenlicht, L.H., ␥␦ yama, W.M. (2002). Cutting edge: murine UL16-binding protein-like Craft, J., and Yin, Z. (2003). T cells provide an early source of ␥ transcript 1: a newly described transcript encoding a high-affinity interferon in tumor immunity. J. Exp. Med. 198, 433–442. ligand for murine NKG2D. J. Immunol. 169, 4079–4083. Gilfillan, S., Ho, E.L., Cella, M., Yokoyama, W.M., and Colonna, M. Immunity 146

(2002). NKG2D recruits two distinct adapters to trigger NK cell acti- Lengauer, C., Kinzler, K.W., and Vogelstein, B. (1998). Genetic insta- vation and costimulation. Nat. Immunol. 3, 1150–1155. bilities in human cancers. Nature 396, 643–649. Girardi, M., Glusac, E., Filler, R.B., Roberts, S.J., Propperova, I., Liyanage, U.K., Moore, T.T., Joo, H.G., Tanaka, Y., Herrmann, V., Lewis, J., Tigelaar, R.E., and Hayday, A.C. (2003). The distinct contri- Doherty, G., Drebin, J.A., Strasberg, S.M., Eberlein, T.J., Goedege- butions of murine T cell receptor (TCR)␥␦ϩ and TCR␣␤ϩ T cells to buure, P.S., and Linehan, D.C. (2002). Prevalence of regulatory different stages of chemically induced skin cancer. J. Exp. Med. T cells is increased in peripheral blood and tumor microenvironment 198, 747–755. of patients with pancreas or breast adenocarcinoma. J. Immunol. 169, 2756–2761. Girardi, M., Oppenheim, D.E., Steele, C.R., Lewis, J.M., Glusac, E., Filler, R., Hobby, P., Sutton, B., Tigelaar, R.E., and Hayday, A.C. Loeb, L.A. (1991). Mutator phenotype may be required for multistage (2001). Regulation of cutaneous malignancy by ␥␦ T cells. Science carcinogenesis. Cancer Res. 51, 3075–3079. 294, 605–609. Loeb, L.A., Loeb, K.R., and Anderson, J.P. (2003). Multiple mutations Gresser, I., and Belardelli, F. (2002). Endogenous type I interferons as and cancer. Proc. Natl. Acad. Sci. USA 100, 776–781. a defense against tumors. Cytokine Growth Factor Rev. 13, 111–118. MacKie, R.M., Reid, R., and Junor, B. (2003). Fatal melanoma trans- Groh, V., Bahram, S., Bauer, S., Herman, A., Beauchamp, M., and ferred in a donated kidney 16 years after melanoma surgery. N. Spies, T. (1996). Cell stress-regulated human major histocompatibil- Engl. J. Med. 348, 567–568. ity complex class I gene expressed in gastrointestinal epithelium. Marincola, F.M., Jaffee, E.M., Hicklin, D.J., and Ferrone, S. (2000). Proc. Natl. Acad. Sci. USA 93, 12445–12450. Escape of human solid tumors from T-cell recognition: molecular mechanisms and functional significance. Adv. Immunol. 74, Groh, V., Rhinehart, R., Secrist, H., Bauer, S., Grabstein, K.H., and 181–273. Spies, T. (1999). Broad tumor-associated expression and recognition by tumor-derived ␥␦ T cells of MICA and MICB. Proc. Natl. Matzinger, P. (1994). Tolerance, danger, and the extended family. Acad. Sci. USA 96, 6879–6884. Annu. Rev. Immunol. 12, 991–1045. Groh, V., Rhinehart, R., Randolph-Habecker, J., Topp, M.S., Riddell, Nishikawa, H., Kato, T., Tanida, K., Hiasa, A., Tawara, I., Ikeda, H., S.R., and Spies, T. (2001). Costimulation of CD8␣␤ T cells by NKG2D Ikarashi, Y., Wakasugi, H., Kronenberg, M., Nakayama, T., et al. ϩ ϩ via engagement by MIC induced on virus-infected cells. Nat. Immu- (2003). CD4 CD25 T cells responding to serologically defined nol. 2, 255–260. autoantigens suppress antitumor immune responses. Proc. Natl. Acad. Sci. USA 100, 10902–10906. Groh, V., Wu, J., Yee, C., and Spies, T. (2002). Tumour-derived solu- Nomura, M., Takihara, Y., and Shimada, K. (1994). Isolation and ble MIC ligands impair expression of NKG2D and T-cell activation. characterization of retinoic acid-inducible cDNA clones in F9 cells: Nature 419, 734–738. one of the early inducible clones encodes a novel protein sharing Hahn, W.C., and Weinberg, R.A. (2002a). Modelling the molecular several highly homologous regions with a Drosophila polyhomeotic circuitry of cancer. Nat. Rev. Cancer 2, 331–341. protein. Differentiation 57, 39–50. Hahn, W.C., and Weinberg, R.A. (2002b). Rules for making human Ohashi, K., Burkart, V., Flohe, S., and Kolb, H. (2000). Cutting edge: tumor cells. N. Engl. J. Med. 347, 1593–1603. heat shock protein 60 is a putative endogenous ligand of the toll- Hayakawa, Y., Rovero, S., Forni, G., and Smyth, M.J. (2003). like receptor-4 complex. J. Immunol. 164, 558–561. ␣-Galactosylceramide (KRN7000) suppression of chemical- and Old, L.J. (2003). Cancer vaccines 2003: opening address. Cancer oncogene-dependent carcinogenesis. Proc. Natl. Acad. Sci. USA Immun. 3 (Suppl 2), 1. 100, 9464–9469. Old, L.J., and Boyse, E.A. (1964). Immunology of Experimental Tu- Jager, E., Ringhoffer, M., Altmannsberger, M., Arand, M., Karbach, mors. Annu. Rev. Med. 15, 167–186. J., Jager, D., Oesch, F., and Knuth, A. (1997). Immunoselection Onizuka, S., Tawara, I., Shimizu, J., Sakaguchi, S., Fujita, T., and in vivo: independent loss of MHC class I and melanocyte differentia- Nakayama, E. (1999). Tumor rejection by in vivo administration of tion antigen expression in metastatic melanoma. Int. J. Cancer anti-CD25 (interleukin-2 receptor ␣) monoclonal antibody. Cancer 71, 142–147. Res. 59, 3128–3133. Janeway, C.A., Jr. (1989). Approaching the asymptote? Evolution Pardoll, D. (2003). Does the immune system see tumors as foreign and revolution in immunology. Cold Spring Harb. Symp. Quant. Biol. or self? Annu. Rev. Immunol. 21, 807–839. 54, 1–13. Pende, D., Rivera, P., Marcenaro, S., Chang, C.C., Biassoni, R., Jinushi, M., Takehara, T., Kanto, T., Tatsumi, T., Groh, V., Spies, T., Conte, R., Kubin, M., Cosman, D., Ferrone, S., Moretta, L., and Miyagi, T., Suzuki, T., Sasaki, Y., and Hayashi, N. (2003a). Critical Moretta, A. (2002). Major histocompatibility complex class I-related role of MHC class I-related chain A and B expression on chain A and UL16-binding protein expression on tumor cell lines IFN-␣-stimulated dendritic cells in NK cell activation: impairment in of different histotypes: analysis of tumor susceptibility to NKG2D- chronic hepatitis C virus infection. J. Immunol. 170, 1249–1256. dependent natural killer cell cytotoxicity. Cancer Res. 62, 6178– Jinushi, M., Takehara, T., Tatsumi, T., Kanto, T., Groh, V., Spies, T., 6186. Kimura, R., Miyagi, T., Mochizuki, K., Sasaki, Y., and Hayashi, N. Penn, I. (1991). Donor transmitted disease: cancer. Transplant. Proc. (2003b). Expression and role of MICA and MICB in human hepatocel- 23, 2629–2631. lular carcinomas and their regulation by retinoic acid. Int. J. Cancer Penn, I. (1996). Malignant melanoma in organ allograft recipients. 104, 354–361. Transplantation 61, 274–278. Johnson, G.B., Brunn, G.J., Kodaira, Y., and Platt, J.L. (2002). Recep- Radosavljevic, M., Cuillerier, B., Wilson, M.J., Clement, O., Wicker, tor-mediated monitoring of tissue well-being via detection of soluble S., Gilfillan, S., Beck, S., Trowsdale, J., and Bahram, S. (2002). A heparan sulfate by Toll-like receptor 4. J. Immunol. 168, 5233–5239. cluster of ten novel MHC class I related genes on human chromo- Kaplan, D.H., Shankaran, V., Dighe, A.S., Stockert, E., Aguet, M., some 6q24.2-q25.3. Genomics 79, 114–123. Old, L.J., and Schreiber, R.D. (1998). Demonstration of an interferon Raulet, D.H. (2003). Roles of the NKG2D immunoreceptor and its ␥-dependent tumor surveillance system in immunocompetent mice. ligands. Nat. Rev. Immunol. 3, 781–790. Proc. Natl. Acad. Sci. USA 95, 7556–7561. Read, S., Mauze, S., Asseman, C., Bean, A., Coffman, R., and Powrie, ϩ ϩ Khong, H.T., and Restifo, N.P. (2002). Natural selection of tumor F. (1998). CD38 CD45RBlow CD4 T cells: a population of T cells with variants in the generation of “tumor escape” phenotypes. Nat. Immu- immune regulatory activities in vitro. Eur. J. Immunol. 28, 3435–3447. nol. 3, 999–1005. Rosenberg, S.A. (1999). A new era for cancer immunotherapy based Khong, H.T., Wang, Q.J., and Rosenberg, S.A. (2004). Identification on the genes that encode cancer antigens. Immunity 10, 281–287. of multiple antigens recognized by tumor-infiltrating lymphocytes Rubinstein, N., Alvarez, M., Zwirner, N.W., Toscano, M.A., Ilarregui, from a single patient: tumor escape by antigen loss and loss of J.M., Bravo, A., Mordoh, J., Fainboim, L., Podhajcer, O.L., and Rabi- MHC expression. J. Immunother. 27, 184–190. novich, G.A. (2004). Targeted inhibition of galectin-1 gene expres- Review 147

sion in tumor cells results in heightened T cell-mediated rejection; in immunologically deficient athymic-nude mice. Science 183, A potential mechanism of tumor-immune privilege. Cancer Cell 5, 534–536. 241–251. Stutman, O. (1979). Chemical carcinogenesis in nude mice: compari- Sahin, U., Tureci, O., Schmitt, H., Cochlovius, B., Johannes, T., son between nude mice from homozygous and heterzygous matings Schmits, R., Stenner, F., Luo, G., Schobert, I., and Pfreundschuh, and effect of age and carcinogen dose. J. Natl. Cancer Inst. 2, M. (1995). Human neoplasms elicit multiple specific immune re- 353–358. sponses in the autologous host. Proc. Natl. Acad. Sci. USA 92, Suranyi, M.G., Hogan, P.G., Falk, M.C., Axelsen, R.A., Rigby, R., 11810–11813. Hawley, C., and Petrie, J. (1998). Advanced donor-origin melanoma Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M., and Toda, M. in a renal transplant recipient: immunotherapy, cure, and retrans- (1995). Immunologic self-tolerance maintained by activated T cells plantation. Transplantation 66, 655–661. ␣ expressing IL-2 receptor -chains (CD25). Breakdown of a single Svane, I.M., Engel, A.M., Nielsen, M.B., Ljunggren, H.G., Rygaard, mechanism of self-tolerance causes various autoimmune diseases. J., and Werdelin, O. (1996). Chemically induced sarcomas from nude J. Immunol. 155, 1151–1164. mice are more immunogenic than similar sarcomas from congenic Schreiber, R.D., Old, L.J., Hayday, A.C., and Smyth, M.J. (2004). normal mice. Eur. J. Immunol. 26, 1844–1850. Response to ‘A cancer immunosurveillance controversy’. Nat. Im- Takaoka, A., Hayakawa, S., Yanai, H., Stoiber, D., Negishi, H., Ki- munol. 5, 4–5. kuchi, H., Sasaki, S., Imai, K., Shibue, T., Honda, K., and Taniguchi, Seliger, B., Maeurer, M.J., and Ferrone, S. (2000). Antigen-pro- T. (2003). Integration of interferon-␣/␤ signaling to p53 responses cessing machinery breakdown and tumor growth. Immunol. Today in tumour suppression and antiviral defence. Nature 424, 516–523. 21, 455–464. Takeda, K., Smyth, M.J., Cretney, E., Hayakawa, Y., Kayagaki, N., Seong, S.Y., and Matzinger, P. (2004). Hydrophobicity: an ancient Yagita, H., and Okumura, K. (2002). Critical role for tumor necrosis damage-associated molecular pattern that initiates innate immune factor-related apoptosis-inducing ligand in immune surveillance responses. Nat. Rev. Immunol. 4, 469–478. against tumor development. J. Exp. Med. 195, 161–169. Shankaran, V., Ikeda, H., Bruce, A.T., White, J.M., Swanson, P.E., Terabe, M., and Berzofsky, J.A. (2004). Immunoregulatory T cells in Old, L.J., and Schreiber, R.D. (2001). IFN␥ and lymphocytes prevent tumor immunity. Curr. Opin. Immunol. 16, 157–162. primary tumour development and shape tumour immunogenicity. Terabe, M., Matsui, S., Noben-Trauth, N., Chen, H., Watson, C., Nature 410, 1107–1111. Donaldson, D.D., Carbone, D.P., Paul, W.E., and Berzofsky, J.A. Shi, Y., Evans, J.E., and Rock, K.L. (2003). Molecular identification (2000). NKT cell-mediated repression of tumor immunosurveillance of a danger signal that alerts the immune system to dying cells. by IL-13 and the IL-4R-STAT6 pathway. Nat. Immunol. 1, 515–520. Nature 425, 516–521. Termeer, C., Benedix, F., Sleeman, J., Fieber, C., Voith, U., Ahrens, Shimizu, J., Yamazaki, S., and Sakaguchi, S. (1999). Induction of T., Miyake, K., Freudenberg, M., Galanos, C., and Simon, J.C. (2002). tumor immunity by removing CD25ϩCD4ϩ T cells: a common basis Oligosaccharides of Hyaluronan activate dendritic cells via toll-like between tumor immunity and autoimmunity. J. Immunol. 163, 5211– receptor 4. J. Exp. Med. 195, 99–111. 5218. Thomas, L. (1959). Discussion. In Cellular and Humoral Aspects of Shinkai, Y., Rathbun, G., Lam, K.P., Oltz, E.M., Stewart, V., Mendel- the Hypersensitive States, H.S. Lawrence, ed. (New York: Hoeber- sohn, M., Charron, J., Datta, M., Young, F., Stall, A.M., and Alt, F.W. Harper), pp. 529–532. (1992). RAG-2-deficient mice lack mature lymphocytes owing to Thomas, L. (1982). On immunosurveillance in human cancer. Yale inability to initiate V(D)J rearrangement. Cell 68, 855–867. J. Biol. Med. 55, 329–333. Smyth, M.J., Crowe, N.Y., and Godfrey, D.I. (2001a). NK cells and Thornton, A.M., and Shevach, E.M. (1998). CD4ϩCD25ϩ immunoreg- NKT cells collaborate in host protection from methylcholanthrene- ulatory T cells suppress polyclonal T cell activation in vitro by inhib- induced fibrosarcoma. Int. Immunol. 13, 459–463. iting interleukin 2 production. J. Exp. Med. 188, 287–296. Smyth, M.J., Godfrey, D.I., and Trapani, J.A. (2001b). A fresh look Tieng, V., Le Bouguenec, C., du Merle, L., Bertheau, P., Desreumaux, at tumor immunosurveillance and immunotherapy. Nat. Immunol. P., Janin, A., Charron, D., and Toubert, A. (2002). Binding of Esche- 2, 293–299. richia coli adhesin AfaE to CD55 triggers cell-surface expression of Smyth, M.J., Takeda, K., Hayakawa, Y., Peschon, J.J., van den Brink, the MHC class I-related molecule MICA. Proc. Natl. Acad. Sci. USA M.R., and Yagita, H. (2003). Nature’s TRAIL–on a path to cancer 99, 2977–2982. immunotherapy. Immunity 18, 1–6. Uhr, J.W., Tucker, T., May, R.D., Siu, H., and Vitetta, E.S. (1991). Smyth, M.J., Thia, K.Y., Street, S.E., Cretney, E., Trapani, J.A., Tani- Cancer dormancy: studies of the murine BCL1 lymphoma. Cancer guchi, M., Kawano, T., Pelikan, S.B., Crowe, N.Y., and Godfrey, D.I. Res. 51, 5045s–5053s. (2000a). Differential tumor surveillance by natural killer (NK) and NKT Uyttenhove, C., Pilotte, L., Theate, I., Stroobant, V., Colau, D., Par- cells. J. Exp. Med. 191, 661–668. mentier, N., Boon, T., and Van den Eynde, B.J. (2003). Evidence Smyth, M.J., Thia, K.Y., Street, S.E., MacGregor, D., Godfrey, D.I., for a tumoral immune resistance mechanism based on tryptophan and Trapani, J.A. (2000b). Perforin-mediated cytotoxicity is critical degradation by indoleamine 2,3-dioxygenase. Nat. Med. 9, 1269– for surveillance of spontaneous lymphoma. J. Exp. Med. 192, 1274. 755–760. van den Broek, M.F., Kagi, D., Ossendorp, F., Toes, R., Vamvakas, Srivastava, P. (2002). Interaction of heat shock proteins with pep- S., Lutza, W.K., Melief, C.J.M., Zinkernagel, R.M., and Hengartner, tides and antigen presenting cells: chaperoning of the innate and H. (1996). Decreased tumor surveillance in perforin-deficient mice. adaptive immune responses. Annu. Rev. Immunol. 20, 395–425. J. Exp. Med. 184, 1781–1790. Street, S.E., Cretney, E., and Smyth, M.J. (2001). Perforin and inter- van der Bruggen, P., Traversari, C., Chomez, P., Lurquin, C., De feron-␥ activities independently control tumor initiation, growth, and Plaen, E., Van den Eynde, B., Knuth, A., and Boon, T. (1991). A gene metastasis. Blood 97, 192–197. encoding an antigen recognized by cytolytic T lymphocytes on a Street, S.E., Trapani, J.A., MacGregor, D., and Smyth, M.J. (2002). human melanoma. Science 254, 1643–1647. Suppression of lymphoma and epithelial malignancies effected by Vetter, C.S., Groh, V., thor Straten, P., Spies, T., Brocker, E.B., and ␥ interferon . J. Exp. Med. 196, 129–134. Becker, J.C. (2002). Expression of stress-induced MHC class I re- Street, S.E., Hayakawa, Y., Zhan, Y., Lew, A.M., MacGregor, D., lated chain molecules on human melanoma. J. Invest. Dermatol. Jamieson, A.M., Diefenbach, A., Yagita, H., Godfrey, D.I., and Smyth, 118, 600–605. M.J. (2004). Innate immune surveillance of spontaneous B cell lym- Wang, H.Y., Lee, D.A., Peng, G., Guo, Z., Li, Y., Kiniwa, Y., Shevach, ␥␦ ϩ phomas by natural killer cells and T cells. J. Exp. Med. 199, E.M., and Wang, R.F. (2004). Tumor-specific human CD4 regulatory 879–884. T cells and their ligands: implications for immunotherapy. Immunity Stutman, O. (1974). Tumor development after 3-methylcholanthrene 20, 107–118. Immunity 148

Wang, T., Niu, G., Kortylewski, M., Burdelya, L., Shain, K., Zhang, S., Bhattacharya, R., Gabrilovich, D., Heller, R., Coppola, D., et al. (2003). Regulation of the innate and adaptive immune responses by Stat-3 signaling in tumor cells. Nat. Med. 10, 48–54. Wheelock, E.F., Weinhold, K.J., and Levich, J. (1981). The tumor dormant state. Adv. Cancer Res. 34, 107–140. Wong, L.H., Krauer, K.G., Hatzinisiriou, I., Estcourt, M.J., Hersey, P., Tam, N.D., Edmondson, S., Devenish, R.J., and Ralph, S.J. (1997). Interferon-resistant human melanoma cells are deficient in ISGF3 components, STAT1, STAT2, and p48-ISGF3␥. J. Biol. Chem. 272, 28779–28785. Woo, E.Y., Chu, C.S., Goletz, T.J., Schlienger, K., Yeh, H., Coukos, G., Rubin, S.C., Kaiser, L.R., and June, C.H. (2001). Regulatory CD4ϩCD25ϩ T cells in tumors from patients with early-stage nonsmall cell lung cancer and late-stage ovarian cancer. Cancer Res. 61, 4766–4772. Wu, G.S., Burns, T.F., McDonald, E.R., 3rd, Jiang, W., Meng, R., Krantz, I.D., Kao, G., Gan, D.D., Zhou, J.Y., Muschel, R., et al. (1997). KILLER/DR5 is a DNA damage-inducible p53-regulated death receptor gene. Nat. Genet. 17, 141–143. Wu, G.S., Burns, T.F., Zhan, Y., Alnemri, E.S., and El-Deiry, W.S. (1999). Molecular cloning and functional analysis of the mouse homologue of the KILLER/DR5 tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor. Cancer Res. 59, 2770–2775. Yee, C., Thompson, J.A., Byrd, D., Riddell, S.R., Roche, P., Celis, E., and Greenberg, P.D. (2002). Adoptive T cell therapy using antigen- specific CD8ϩ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells. Proc. Natl. Acad. Sci. USA 99, 16168–16173.