Interferons, Immunity and Cancer Immunoediting

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TUMOUR IMMUNOLOGY Interferons, immunity and cancer immunoediting

Gavin P. Dunn, Catherine M. Koebel and Robert D. Schreiber Abstract | A clear picture of the dynamic relationship between the host immune system and cancer is emerging as the cells and molecules that participate in naturally occurring antitumour immune responses are being identified. The interferons (IFNs) — that is, the type I IFNs (IFNα and IFNβ) and type II IFN (IFNγ) — have emerged as central coordinators of tumour–immune-system interactions. Indeed, the decade-old finding that IFNγ has a pivotal role in promoting antitumour responses became the focus for a renewed interest in the largely abandoned concept of cancer immunosurveillance. More recently, type I IFNs have been found to have distinct functions in this process. In this Review, we discuss the roles of the IFNs, not only in cancer immunosurveillance but also in the broader process of cancer immunoediting.

Transformation As research into tumour immunology continues at tumour growth, tumour cells might escape or attenuate The process of conversion of a an incredible pace, a considerable amount of work is this immune pressure, similar to the way in which these normal cell into a cancer cell. aimed at exploring the mechanisms that underlie the cells evade classic mechanisms of tumour suppression8. immunological recognition and elimination of cancer This concept evolved from the observation that tumours Cancer immunosurveillance and the downstream consequences of these processes. from immunocompetent hosts and immunodeficient Immunological protection 6,9–11 of the host against the It is now clear that the capacity of the immune system hosts have different immunogenic phenotypes . development of cancer, for recognition is not limited solely to the classic mod- Specifically, tumour cells that developed in the pres- resulting from immune effector els of self versus pathogen or self versus non-self but ence of an intact immune system — as usually occurs in functions stimulated by encompasses the more-subtle differences that exist humans — could form progressively growing tumours immune recognition of either transformed stress ligands or antigens between self and self. This conclusion when transplanted into immunocompetent recipients, expressed on transformed provided the argument for reconsidering the largely whereas tumour cells from immunodeficient mice were cells. discarded hypothesis of cancer immunosurveillance. This rejected when transplanted into wild-type hosts but not hypothesis, proposed by Burnet and Thomas1,2 about immunodeficient hosts. Therefore, the immunogeni city five decades ago, holds that the immune system protects of a tumour reflects the immunological environment the host against the development of cancers of non- from which it was derived, indicating that the relation- viral origin. Whereas studies that were carried out on ship between the immune system and cancer is more partially immuno deficient mice in the 1970s provided complex than was previously thought. arguments against this hypothesis, many contemporary The dual opposing functions of immunity — host studies of mice with molecularly defined inactivat- protection and tumour promotion — formed the ing defects in innate and/or adaptive immunity have conceptual basis for a process that we named cancer shown that immunodeficient mice do indeed develop immunoediting3–6,9 (FIG. 1). The cancer-immunoediting more spontaneous and carcinogen-induced tumours hypothesis emphasizes that extrinsic immune pressure than their immunocompetent counterparts3–7 (TABLE 1). either can block tumour growth, development and sur- Washington University School These data, therefore, now overwhelmingly support the vival or can facilitate tumour outgrowth by sculpting of Medicine, Department of Pathology and Immunology, existence of an effective cancer-immunosurveillance tumour immunogenicity or by inhibiting host-protective Box 8118, 660 South Euclid process in mice. Moreover, clinical data have led to a antitumour responses. In this manner, the acquired Avenue, St. Louis, growing appreciation that cancer immunosurveillance capacity of developing tumours to escape immune control Missouri 63110, USA. also occurs in humans4,5. is a seventh hallmark of cancer4,8. As depicted in FIG. 1, it is Correspondence to R.D.S. e-mail: schreiber@ Our understanding of natural immune responses envisaged that the cancer-immunoediting process consists immunology.wustl.edu to cancer has further improved following the realiza- of three phases: elimination (also known as protection), doi:10.1038/nri1961 tion that, as the immune system attempts to constrain equilibrium (persistence) and escape (progression).

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Table 1 | Increased susceptibility of immunodeficient mice to carcinogen-induced and spontaneous tumours Mouse immunodeficiency Immune status Tumour susceptibility relative to wild-type mice Refs Rag1–/– or Rag2–/– Lacks T cells, B cells and NKT cells ↑ MCA-induced sarcomas 6,27 ↑ Spontaneous intestinal neoplasias Rag2–/–Stat1–/– Lacks T cells, B cells and NKT cells; ↑ MCA-induced sarcomas 6 insensitive to IFNα, IFNβ and IFNγ ↑ Spontaneous intestinal and mammary neoplasias SCID BALB/c Lacks T cells, B cells and NKT cells ↑ MCA-induced sarcomas 27 Tcrb–/– Lacks αβ T cells ↑ MCA-induced sarcomas 100 Tcrd –/– Lacks γδ T cells ↑ MCA-induced sarcomas 100 ↑ DMBA- plus TPA-induced skin tumours Tcrb–/–Tcrd –/– Lacks αβ T cells and γδ T cells ↑ DMBA- plus TPA-induced skin tumours 101 Jα281 TCR gene-segment deficiency Lacks NKT-cell subset ↑ MCA-induced sarcomas 19,27,102 Lmp2–/– Lacks LMP2 subunit ↑ Spontaneous uterine neoplasms 103 Asialo-GM1-specific antibody Lacks NK cells, monocytes and ↑ MCA-induced sarcomas 27,102 treatment macrophages NK1.1-specific antibody treatment Lacks NK cells and NKT cells ↑ MCA-induced sarcomas 27,102 Thy1-specific antibody treatment Lacks T cells ↑ MCA-induced sarcomas 27,102 Immunization with self antigen Increased regulatory T-cell activity ↓ Latency of MCA-induced sarcomas 104 Stat1–/– Insensitive to IFNα, IFNβ and IFNγ↑ MCA-induced sarcomas 6,18 Wider tumour range in Stat1–/–Tp53–/– mice Ifngr1–/– Insensitive to IFNγ↑ MCA-induced sarcomas 6,18 Wider tumour range in Ifngr1–/–Tp53–/– mice Ifnar1–/– Insensitive to IFNα and IFNβ↑ MCA-induced sarcomas 9 Ifng–/– Lacks IFNγ↑ MCA-induced sarcomas 19,20,105 C57BL/6 mice: ↑ Spontaneous disseminated lymphomas; ↓ latency of tax-transgene-induced leukaemia BALB/c: ↑ Spontaneous lung adenocarcinomas Gmcsf–/–Ifng–/– Lacks GM-CSF and IFNγ↑ Spontaneous lymphomas 21 ↑ Non-lymphoid solid cancers Pfp–/–Ifng–/– Lacks perforin and IFNγ↑ MCA-induced sarcomas 19,20 ↑ Spontaneous disseminated lymphomas Pfp–/–b2m–/– Lacks perforin, MHC class I ↑ Spontaneous disseminated lymphomas 106 molecules and CD8+ T cells Pfp–/– Lacks perforin ↑ MCA-induced sarcomas 19,102,107 ↑ Spontaneous lymphomas ↑ Spontaneous lymphomas and sarcomas in Pfp–/–Tp53+/– mice Trail–/– Lacks TRAIL ↑ MCA-induced sarcomas 108,109 ↑ Spontaneous lymphomas ↑ Spontaneous lymphomas and sarcomas in Trail –/–Tp53+/– mice TRAIL-specific antibody treatment Blockade of TRAIL ↑ MCA-induced sarcomas 10 ↑ Spontaneous lymphomas and sarcomas NKG2D-specific antibody treatment Blockade of NKG2D ↑ MCA-induced sarcomas 39 Il12a–/– Lacks IL-12 ↑ DMBA- plus TPA-induced papillomas 68 Il23a–/– Lacks IL-23 ↓ DMBA- plus TPA-induced papillomas 68 Il12b–/– Lacks IL-12 and IL-23 ↑ MCA-induced sarcomas 40,68 ↓ DMBA- plus TPA-induced papillomas IL-12 treatment Exogenous IL-12 ↓ MCA-induced sarcomas 26 α-GalCer treatment Exogenous NKT-cell activation ↓ MCA-induced sarcomas 110 Tnf–/– Lacks TNF ↓ DMBA- plus TPA-induced papillomas 69 Conditional Socs1–/– SOCS1 expressed only by ↑ Spontaneous colitis-associated colorectal 70 T cells and B cells adenocarcinomas Conditional Socs1–/– plus SOCS1 expressed only by ↓ Spontaneous colitis-associated colorectal 70 IFNγ-specific antibody treatment T cells and B cells; IFNγ depletion adenocarcinomas β α α b2m, 2-microglobulin; DMBA, 7,12-dimethylbenz(a)anthracene; -GalCer, -galactosylceramide; GM1, a ganglioside; Gmcsf, granulocyte/macrophage colony- stimulating factor; Ifnar1, type I IFN receptor 1; Ifn, interferon; Ifngr1, IFNγ receptor 1; Il, interleukin; Jα281, joining gene segment Jα281 of TCRα; Lmp2, low-molecular- mass protein 2; MCA, 3-methylcholanthrene; NK, natural killer; NK1.1, NK-cell-associated antigen 1.1; NKG2D, NK group 2, member D; NKT, natural killer T; Pfp, perforin; Rag, recombination-activating gene; SCID, severe combined immunodeficient; Socs1, suppressor of cytokine signalling 1; Stat1, signal transducer and activator of transcription 1; Tcr, T-cell receptor; Tnf, tumour-necrosis factor; TPA, 12-O-tetradecanoyl phorbol 13-acetate; Tp53, tumour-suppressor protein p53; Trail, TNF-related apoptosis-inducing ligand.

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Figure 1 | Cancer immunoediting. The process of cancer immunoediting is envisaged as three phases: elimination, equilibrium and escape. The first phase, the elimination phase (formerly known as cancer immunosurveillance), consists of the recognition of transformed cells by the innate and the adaptive immune system, leading to the killing of these cells. One recently identified mechanism by which the innate immune system discriminates between self cells and transformed self cells involves the interaction of the natural killer (NK)-cell activating receptor NKG2D (NK group 2, member D) with specific tumour-cell-expressed ligands, which are induced by genotoxic stress, viral infection or chemically mediated transformation89. In addition, CD4+ and CD8+ αβ T cells recognize tumour-specific or tumour- associated antigens in the context of MHC class II and class I molecules, respectively, and B cells produce antibodies that recognize antigens at tumour-cell surfaces (not shown). Recognition by innate and adaptive immune mechanisms leads Hallmarks of cancer to direct killing of tumour cells, as well as production of chemokines and other cytokines that facilitate killing of tumour A specific set of features cells. If some tumour cells are not killed in the elimination phase, then the process can progress to the second phase: that must be acquired by a the equilibrium phase, a subclinical phase in which the tumour persists but is prevented from expanding by immune malignant cell. These features pressure. The third phase, escape, begins when the balance between the immune response and the tumour tilts towards are self-sufficiency in growth tumour growth as a result of immune exhaustion or inhibition or as a result of the emergence of tumour-cell variants signals, insensitivity to (shown in purple) that enable the tumour to evade immune pressure. Non-immunogenic transformed cells directly enter growth-inhibitory signals, the escape phase. This phase concludes with the appearance of clinically detectable, progressively growing tumours. evasion of apoptotic cell BRCA, breast cancer, early onset; ECM, extracellular matrix; IDO, indoleamine 2,3-dioxygenase; IFN, interferon; death, limitless replicative MIC, MHC-class-I-polypeptide-related sequence; NK, natural killer; NKT, natural killer T; p53, tumour-suppressor protein potential, sustained angiogenesis, tissue invasion p53; RAE1, retinoic acid early transcript 1; Rb, retinoblastoma protein; TRAIL, tumour-necrosis-factor-related apoptosis- + + and evasion of the antitumour inducing ligand; TReg, CD4 CD25 regulatory T; ULBP, cytomegalovirus UL16-binding protein. This figure is modified, with immune response. permission, from REF. 5 © (2004) Elsevier.

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It is thought that many different immune cells might responses are well established13. The key similarities interact with tumour cells from the earliest stages of and differences between type I IFNs and IFNγ that are transformation to the terminal phase of widespread important to our broader discussion of these cytokines metastasis. These interactions are controlled by end- in the context of cancer immunoediting are summarized in ogenously produced interferons (IFNs), and recent work TABLE 2, and details of the IFN-mediated signalling path- indicates that type I IFNs (IFNα and IFNβ) and type II ways are presented in TABLE 2 and FIG. 2. Detailed reviews IFN (IFNγ) might have non-redundant functions in the of IFN biology have been published elsewhere14–16. cancer-immunoediting process by affecting distinct target-cell populations. This Review focuses on the recent IFNγ in cancer immunosurveillance. Although the data that link the IFNs to the immune-mediated elimina- role of IFNγ in promoting host immune responses to tion of cancer and sculpting of developing tumours (that microorganisms is well recognized, the finding that IFNγ is, cancer immunoediting). also promotes host antitumour immunity reinvigorated interest in the cancer-immunosurveillance process. The IFNs in cancer immunosurveillance original data that pointed to an important role for end- In the past, the term cancer immunosurveillance was ogenously produced IFNγ in promoting tumour elimi- used to describe the protective function of immunity in nation came from experiments that sought to identify the control of tumour growth. However, it is now rec- the cytokines required for the lipopolysaccharide (LPS)- ognized that the scope of the tumour–immune-system dependent rejection of transplanted Meth A cells (which interaction is much broader, thereby necessitating are 3-methylcholanthrene (MCA)-induced fibrosarcoma expansion of the concept of cancer immunosurveil- cells of BALB/c mice)17. Neutralization of IFNγ with a lance to become cancer immunoediting. The concept specific monoclonal antibody, but not neutralization of cancer immunoediting incorporates the host- of tumour-necrosis factor (TNF) with a specific mono- protective cancer-immunosurveillance functions of the clonal antibody, abrogated the LPS-dependent rejection immune system as the first phase in a multistage process, of Meth A cells. In addition, other sarcomas induced and these functions are now embodied in the term by the carcinogen MCA grew more aggressively when elimination (protection) (FIG. 1). transplanted into naive mice that had been treated with The IFN family was originally recognized for its neutralizing IFNγ-specific monoclonal antibody. capacity to protect naive cells against viral infection, but The antitumour activity of IFNγ observed in it is now known to have important roles in regulating these studies was subsequently confirmed in primary and linking both the innate and the adaptive arms of tumorigenesis models. The initial report showed that immunity and has recently been shown to have obli- IFNγ-insensitive 129/Sv mice — lacking either the gate roles in the elimination phase of cancer immuno- IFNGR1 subunit of the IFNγ receptor or the transcrip- editing. Although type I IFNs were originally thought tion factor STAT1 (signal transducer and activator of to be mainly antiviral agents, recent studies have shown transcription 1) — developed MCA-induced sarcomas their importance as immunomodulators12. By contrast, more rapidly and 3–5-fold more frequently than did the immunomodulatory activity of IFNγ has long been their wild-type counterparts18. A similar observation was appreciated, and its roles in the control of host immune made using C57BL/6 mice that lack the gene that encodes

Table 2 | Comparison of type I and type II interferon production and signalling Property Type I IFNs Type II IFN References IFNα and IFNβ IFNγ Stimuli • Viruses • Antigen–MHC complexes 13,111 • Other microorganisms • Activating NK-cell ligands • IL-12 plus IL-18 • TLRs Cells producing IFN • All nucleated cells (especially IPCs) • NK cells 111,112 • NKT cells • T cells Number of proteins • IFNα: 12 (mice) and 12–13 (humans) • 1 (mice and humans) 14,97 • IFNβ: 1 (mice and humans) Cells expressing IFN receptors • All nucleated cells • All nucleated cells 14,96 Type of IFN receptor • IFNAR: IFNAR1–IFNAR2 • IFNGR: IFNGR1–IFNGR2 14,96 Signalling molecules • JAK1 and TYK2 • JAK1 and JAK2 14,96 Primary tumorigenesis • STAT1–STAT2–IRF9 complexes • STAT1–STAT1 complexes Transformation and tumour • STAT1–STAT1 complexes formation that occurs entirely in the original host, in contrast Transcription-factor-binding • ISRE • GAS 14,96 to tumour formation in a host sites • GAS owing to transplantation of GAS, IFNγ-activated site; IFN, interferon; IFNAR, type I IFN receptor; IFNGR, IFNγ receptor; IL, interleukin; IPC, IFNα-producing cells that were transformed cell; IRF9, IFN-regulatory factor 9; ISRE, IFN-stimulated response element; JAK, Janus kinase; NK, natural killer; NKT, natural killer T; in another host or in vitro. STAT, signal transducer and activator of transcription; TLR, Toll-like receptor; TYK2, tyrosine kinase 2.

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IFNγ19. IFNγ-insensitive mice were also more susceptible to spontaneous tumour formation. Specifically, mice that lack both the tumour-suppressor protein p53 and sensitivity to IFNγ (as a result of deficiency in either IFNGR1 or STAT1) formed tumours significantly more rapidly than did IFNγ-sensitive p53-deficient mice, and these mice also developed a broader range of tumour types18. In another study, IFNγ-deficient C57BL/6 mice developed disseminated lymphomas more frequently than did wild-type mice. By contrast, a small propor- tion of IFNγ-deficient BALB/c mice developed lung adenocarcinomas but not disseminated lymphomas, whereas wild-type BALB/c mice remained tumour free20. Another study showed that IFNγ cooperates with other cytokines to prevent tumour formation. Mice deficient in both granulocyte/macrophage colony-stimulating factor (GM-CSF) and IFNγ, as well as mice deficient in these cytokines and interleukin-3 (IL-3), developed lymphoma and non-lymphoid solid cancers at a higher rate than did mice deficient in GM-CSF alone, both IL-3 and GM-CSF, or IFNγ alone21. Compared with the other experimental models that have been discussed here, interpretation of this study is complicated by the dependence of neoplasia on infection; therefore, more • • • • work is needed to understand the relationships between • chronic infection, unopposed inflammation, cancer and • • immunosurveillance. Nevertheless, taken together, the • • • • findings show that IFNγ participates in a cancer elimina- • • tion (immunosurveillance) process that protects mice • • against chemically induced, spontaneously arising and transplantable tumours. Figure 2 | Interferon-induced signalling and transcription. Interferon-γ (IFNγ; Additional studies revealed that the antitumour type II IFN) induces reorganization of the IFNγ-receptor subunits, IFNGR1 and activity of IFNγ overlaps with that of recombination- IFNGR2, activating the Janus kinases JAK1 and JAK2, which are constitutively activating gene (RAG)-protein-expressing lymphocytes. 14,16 associated with each subunit, respectively . The JAKs phosphorylate a crucial Specifically, 129/Sv mice that lack IFNγ responsiveness tyrosine residue of IFNGR1, forming a STAT1 (signal transducer and activator (as a result of IFNGR1 or STAT1 deficiency), 129/Sv of transcription 1)-binding site; they then tyrosine phosphorylate receptor- bound STAT1, which homodimerizes through SRC homology 2 (SH2)-domain– mice that lack lymphocytes (as a result of RAG2 defi- phosphotyrosine interactions and is fully activated by serine phosphorylation. STAT1 ciency) and 129/Sv mice that lack both (as a result of homodimers enter the nucleus and bind promoters at IFNγ-activated sites (GASs) and STAT1 and RAG2 deficiency) showed similar increases induce gene transcription in conjunction with co-activators, such as CBP (cyclic- in the incidence of MCA-induced sarcomas compared AMP-responsive-element-binding protein (CREB)-binding protein), p300 and with wild-type controls6. So these data show that the minichromosome maintenance deficient 5 (MCM5)90,91. Disruption of the gene that IFNγ-dependent, STAT1-dependent and lymphocyte- encodes IFNγ, IFNGR1, IFNGR2, JAK1, JAK2 or STAT1 has shown that each of these dependent pathways of tumour suppression overlap 92 molecules is required for signalling through the IFNγ receptor . Further studies have considerably. This conclusion is also supported by other γ revealed that IFN -mediated signalling is controlled by several mechanisms: by studies of carcinogenesis in which mice that lack both dephosphorylation of IFNGR1, JAK1 and STAT1 (mediated by SH2-domain-containing perforin and IFNγ showed only a small increase in the protein tyrosine phosphatase 2, SHP2)92; by inhibition of the JAKs (mediated by suppressor of cytokine signalling 1, SOCS1)93,94; by proteasomal degradation of the incidence of tumours compared with mice that lack 19 JAKs92; and by inhibition of STAT1 (mediated by protein inhibitor of activated STAT 1, either factor . PIAS1)95. The type I IFNs (IFNα and IFNβ) bind a receptor that consists of the subunits Work has begun to identify the sources of IFNγ IFNAR1 and IFNAR2, which are constitutively associated with tyrosine kinase 2 (TYK2) during the cancer-elimination phase. A study by Gao and JAK1, respectively96,97. Type-I-IFN-induced JAK–STAT signalling is propagated et al.22 showed that γδ T cells are an important source of similarly to IFNγ-induced JAK–STAT signalling. Activated TYK2 and JAK1 IFNγ during the development of protective antitumour phosphorylate STAT1 or STAT2. Type-I-IFN-induced signalling then induces immune responses. This study used two groups of bone- homodimerization of STAT1 and heterodimerization of STAT1 and STAT2. STAT1 and marrow-chimeric mice in which the only difference was STAT2 associate with the cytosolic transcription factor IFN-regulatory factor 9 (IRF9), the inability of γδ T cells in one group to produce IFNγ. forming a trimeric complex known as IFN-stimulated gene factor 3 (ISGF3). On entering When challenged with MCA, mice with IFNγ-deficient the nucleus, ISGF3 binds IFN-stimulated response elements (ISREs). Studies of gene- γδ targeted mice have shown that JAK1, STAT1, STAT2 and IRF9 are required for signalling T cells were significantly more susceptible to tumori- through the type I IFN receptor97. TYK2 is required for optimal type-I-IFN-induced genesis than were control mice and showed a tumour signalling98,99. CTL, cytotoxic T lymphocyte; DC, dendritic cell; NK, natural killer; incidence equivalent to that seen in IFNγ-deficient + + mice. Additional studies will be necessary to identify TH1, T helper 1; TReg, CD4 CD25 regulatory T. This figure is modified, with permission, from Nature Reviews Immunology REF. 96 © (2005) Macmillan Publishers Ltd. other physiologically relevant sources of IFNγ in the

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cancer-immunosurveillance process and to elucidate A more recent study directly showed the require- the pathways that lead to IFNγ production. ment for endogenously produced type I IFNs in Studies of the contribution of IL-12 to antitumour protecting hosts against transplanted and primary immunity provide further insight into the physiologi- syngeneic tumours9. Mice that had been treated cally relevant stimuli for IFNγ production during the with blocking monoclonal antibody specific for the cancer-elimination phase. IL-12 regulates the induction type I IFN receptor34 failed to reject a panel of highly of IFNγ expression in both the innate and the adaptive immunogenic, unedited, MCA-induced sarcomas that immune compartments13,23,24. A role for the IL-12–IFNγ were rejected by wild-type mice that had been treated axis was first shown in tumour therapy models involving with a control monoclonal antibody. This result shows administration of exogenous IL-12 to tumour-bearing that type I IFNs have an obligate role in the elimina- mice. In one study, IL-12-dependent rejection of MC-38 tion of immunogenic syngeneic tumours. This study sarcoma cells was abrogated by injection of neutralizing also shows that type I IFNs participate in naturally IFNγ-specific monoclonal antibody25. This observation occurring, protective immune responses to primary was extended to primary tumorigenesis models in which tumours. Specifically, IFNAR1-deficient 129/Sv mice mice that were challenged with MCA and treated with that were challenged with MCA were significantly more exogenous IL-12 formed fewer sarcomas than did con- susceptible to tumour formation and formed tumours trol mice26,27. Other experiments showed that mice that with faster kinetics than their wild-type counterparts. lack the p40 subunit of the IL-12 heterodimer developed These findings have been independently corroborated 2–3-fold more MCA-induced sarcomas than did their using IFNAR1-deficient C57BL/6 mice and IFNAR2- wild-type counterparts27. These data show that endo- deficient C57BL/6 mice (M. J. Smyth, personal genously produced IL-12 also promotes cancer elimina- communication). tion and mediates some of its antitumour effects through The findings that both type I IFNs and IFNγ have induction of IFNγ expression. crucial roles in promoting host antitumour immunity clearly identify these cytokines as pivotal compo- Type I IFNs in cancer immunosurveillance. In the late nents in the cancer-elimination phase of the cancer- 1960s, it was shown that mice that were transplanted immunoediting process. Therefore, together with the with syngeneic tumours had significantly increased establishment of roles for γδ T cells, αβ T cells, natural survival times when treated with crude preparations killer (NK) cells, natural killer T (NKT) cells, perforin and of type I IFNs28,29. Subsequently, similar results were TNF-related apoptosis-inducing ligand (TRAIL) in obtained when type I IFNs were administered to mice protection of the host against tumour formation, the with Friend leukaemia cell (FLC) tumours or AGS cancer-elimination phase is beginning to be elucidated melanomas (which lack STAT1), or when type I IFNs at the molecular and cellular levels (TABLE 1). In defining were ectopically overexpressed in several tumour types30. the antitumour machinery of the host, further work Although this work, using exogenously delivered type I is needed to understand how the crucial cells and IFNs, clearly showed the therapeutic antitumour poten- molecules in different mouse tumour models work tial of this cytokine family, studies of endogenously together to protect individuals against a broad range produced type I IFNs have only recently assessed its of cancers. Therefore, organism-wide immune elimina- involvement in the cancer-elimination phase. tion of cancer might be more relevant to some tissues γδ T cell Gresser and colleagues31 were the first to address than others, because it requires the integration of anti- T cells express either a T-cell this question by showing that neutralizing polyclonal tumour processes that are specific to the anatomical receptor (TCR) composed of an α-subunit and a β-subunit goat antisera specific for mouse type I IFNs increased location, the original cell type and the transformation (αβ-TCR) or a TCR composed the growth and metastasis in mice of several different programme of a tumour. of a γ-subunit and a δ-subunit tumour-cell xenografts (human HeLa cells and baby (γδ-TCR). T cells that express hamster kidney cells). Subsequent work showed that Cancer immunoediting αβ -TCRs mainly recognize mice that were challenged with either allogeneic or syn- Classically, tumour suppression has been considered to antigenic peptides bound to conventional MHC class I or geneic mouse tumours showed higher tumour growth be a cell-intrinsic programme mediated by pathways that class II molecules. T cells that rates and significantly decreased survival times when involve proteins such as p53 and Rb (retinoblastoma express γδ-TCRs are less treated with serum that contained IFN-specific antibod- protein). These pathways coordinately defend cells from abundant, and the ligands for ies than when treated with control serum32. However, the oncogenic and/or genotoxic stimuli that can lead to these receptors are less well 35,36 characterized. in this study, the FLC tumours that were reported to malignant transformation . However, the data that be syngeneic to the DBA/2 recipient mice actively shed support the existence of a cancer-immunosurveillance Natural killer T cell the Friend leukaemia virus, introducing the potentially process illustrate that suppression of tumour growth (NKT cell). A T cell that confounding variable of antiviral immunity to this study might also be mediated by extrinsic forces, including the expresses both natural killer of antitumour immunity. Additional efforts have been immune system. Additional work has shown that tumour (NK)-cell receptors and an αβ T-cell receptor. NKT cells made to extend these data using tumour-transplantation cells that are under the pressure of immunosurveillance are characterized by cytolytic approaches in mice that lack the IFNAR1 subunit of the behave in a way that is central to tumour biology: they activity and by rapid type I IFN receptor33. However, because none of these escape the mechanisms that operate to suppress them. production of cytokines tumours was rejected by wild-type mice, it is difficult to The realization that this occurs was the main motiva- (including interferon-γ and interleukin-4), and they might draw firm conclusions about the specific roles of type I tion in changing our thinking about tumour–immune- regulate the function of other IFNs in the cancer-elimination phase on the basis of system interactions and led to the development of the T cells. these models. term cancer immunoediting to describe this process3–5.

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The first phase of cancer immunoediting is elimination, IFNs in cancer immunoediting which is the host-protective cancer-immunosurveillance Both type I IFNs and IFNγ have been shown to be crucial process and represents one possible outcome of immuno- components of the cancer-immunoediting process. This editing. Because this mechanism inhibits tumour develop- is because IFN-unresponsive hosts show an increased ment and/or growth, it influences the number of tumours tumour incidence, and the tumours that arise in IFN- that arises. If incomplete, the cancer-immunoediting unresponsive environments are highly antigenic and process can advance to the second phase — equilibrium are therefore qualitatively distinct from sarcomas from — in which residual cancer cells that are highly mutable wild-type mice. One of the first studies to document persist in the immuno competent host. Therefore, equi- the contributions of IFNs to cancer immunoediting librium might be similar to the older concept of tumour centred on IFNγ18. Specifically, tumours that developed dormancy37,38, and it represents either a second possible in IFNGR1-deficient mice were found to grow aggres- outcome of cancer immuno editing or a period of tran- sively in immunocompetent recipients. However, when sition to the third phase of the cancer-immunoediting sensitivity to IFNγ was conferred on the tumour cells by process — escape. Cancer cells that develop the capacity introducing the receptor subunit IFNGR1, the tumour to circumvent immunological suppression of tumours, cells became highly immunogenic and were rejected in by averting immune responses and/or by directly attenu- a CD4+ and CD8+ T-cell-dependent manner. Therefore, ating the protective antitumour functions of immune tumours from IFNGR1-deficient mice represent sarco- effectors, grow progressively and therefore enter the mas that cannot display their inherently high antigen- escape phase, which is a third possible outcome of cancer icity because of their inability to respond to IFNγ. These immunoediting. In this manner, immunity influences findings indicate that IFNγ has a key role in promoting the immunogenic quality of the tumours that grow. tumour immunogenicity. Whereas the evidence for cancer immunosurveil- More recently, type I IFNs were also shown to lance has come from studies of tumour incidence, the participate in tumour-sculpting effects9. Ninety-five evidence for cancer immunoediting has come from percent of sarcoma cell lines (35 of 36) from wild- studies of the immunogenicity of tumours that arise in type mice grew progressively when transplanted into immunocompetent versus immunodeficient hosts. The naive syngeneic mice that were immunocompetent. By clearest demonstration of cancer immunoediting came contrast, ~45% of MCA-induced sarcoma cells from from studies showing that sarcomas from mice defi- IFNAR1-deficient mice were highly immunogenic and cient in RAG2 (which are therefore immunodeficient) were rejected when transplanted into wild-type mice. were significantly more immunogenic (as a group) IFNAR1-deficient MCA-induced sarcomas therefore than tumours from wild-type mice (which arose in the show an immunogenic phenotype similar to sarcomas presence of an intact immune system)6. In that study, from RAG2-deficient mice6. all MCA-induced sarcomas, from either wild-type mice Supporting evidence for the physiological relevance or RAG2-deficient mice, grew progressively when trans- of IFNs in the cancer-immunoediting process comes planted into immuno deficient (RAG2 deficient) hosts6. from the identification of a subset of naturally arising However, whereas all MCA-induced sarcomas from human tumours that have lost the capacity to signal wild-type mice grew progressively when transplanted through the IFN receptors and therefore cannot induce into wild-type (naive immunocompetent) mice, 40% genes that are classically regulated by IFNs. So these of tumours from RAG2-deficient mice were rejected by tumour cell lines might represent edited tumours that wild-type hosts6. Independent studies have shown that were selected for in vivo outgrowth (escape) by an IFN- lymphomas and sarcomas from mice deficient in perf- dependent cancer-immunoediting process. Specifically, orin20,39, as well as sarcomas generated in mice deficient 4 of 17 lung adenocarcinoma cell lines were unable to in the joining gene segment Jα281 of the T-cell receptor signal through the IFNγ receptor due to identifiable α-chain11,40 (which lack NKT cells), were also highly defects in the proximal IFNγ-receptor signalling cascade: immunogenic and were rejected by wild-type mice. one tumour lacked expression of IFNGR1; two tumours Studies are ongoing to determine whether the same expressed abnormal forms of Janus kinase 2 (JAK2); and immune components that constitute the host-protective one tumour lacked expression of JAK1 (REF. 18). Another cancer-immunosurveillance network also participate in study extended this observation to prostate cancers42. the editing functions of immunity. The well-studied cell line LNCaP, which is known to be Recently, one group reported that tumours that insensitive to IFNγ 43, was found to be unresponsive to develop in immunocompetent mice expressing a type I IFNs as well, owing to repression of JAK1 expres- cryptic oncogene were immunogenic and were there- sion by epigenetic mechanisms. Other studies have fore unsculpted41. They used these results to argue identified a set of melanomas that are unresponsive to against the idea that cancer immunoediting has physio- IFNs as a result of negligible expression of STAT1 or logical relevance. However, these oncogene-induced overexpression of STAT5, as well as squamous-cell car- tumour cells caused potent immunosuppression in cinomas that are resistant to IFNs as a result of reduced the tumour-bearing host. Because tumour-induced STAT1 expression (owing to methylation of the STAT1 Epigenetic immunosuppression is an escape mechanism that is promoter)44–46. In addition, many tumours that lack Any heritable influence on the function of a chromosome or integral to cancer immunoediting, we think that the expression of immunologically relevant genes that are gene that is not caused by a results support the validity of this process rather than regulated by IFNs — such as MHC molecules, TAP1 change in DNA sequence. refute it. (transporter associated with antigen processing 1) and

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LMP2 (low-molecular-mass protein 2) — have been naturally occurring antitumour immune responses. identified47–49. Moreover, another study showed that This work has shown that the ability of IFNγ to promote overexpression of SOCS1 (suppressor of cytokine signal- tumour rejection is mediated, at least in part, through ling 1), a negative regulator of IFN-mediated signalling, its capacity to upregulate the MHC class I pathway of correlates with melanoma progression50. Taken together, antigen processing and presentation in tumour cells. these data show that, in addition to their involvement in Specifically, enforced expression of the MHC class I cancer immunosurveillance (that is, in the elimination pathway components TAP1 (REF. 6) or H2-Db (A. T. Bruce phase of cancer immunoediting), IFNs can provide the and R.D.S., unpublished observations) to levels similar selective pressure that drives the cancer-immunoediting to those in IFNγ-sensitive tumour cells that have been capacity of the immune system, leading to progression treated with IFNγ was found to convert RAD–gR.28 cells into the equilibrium and escape phases. (which are IFNγ insensitive) into cells that were rejected by wild-type mice in a CD8+ T-cell-dependent manner, Cancer immunoediting and IFNγ despite their unresponsiveness to IFNγ. Subsequent work A role for IFNγ in increasing tumour immunogenicity. showed that selective overexpression of TAP1 or H2-Db At present, more is known about the cellular targets of increased the susceptibility of RAD–gR.28 cells to in vitro IFNγ during cancer immunoediting than the targets of killing by RAD–gR.28-specific cytotoxic T lymphocytes type I IFNs. Studies of Meth A cells were the first to show (CTLs). These findings show that augmented expression that tumour cells are obligate targets of IFNγ17. Whereas of components of the MHC class I pathway is sufficient treatment with LPS induced rejection of parent Meth A to induce the rejection of an otherwise IFNγ-insensitive cells (which are IFNγ sensitive) growing in syngeneic tumour. BALB/c mice, it had no effect on IFNγ-unresponsive A recent finding by Bui et al.54 points to a distinct Meth A cells overexpressing a dominant-negative mechanism by which the effects of IFNγ on tumour- IFNGR1 mutant (denoted IFNGR1.∆IC) that lacked cell immunogenicity might coordinate the progression all but three amino-acid residues of the intracellular of the antitumour immune response. In this study, the domain17,51. Moreover, unlike parent Meth A cells, molecule H60 was identified as the most prevalent IFNγ-insensitive Meth A cells expressing IFNGR1.∆IC ligand for the NK-cell receptor NKG2D (NK group 2, failed to induce protection against the parent Meth A member D) on a range of MCA-induced sarcomas from cell line when transplanted into wild-type mice and 129/Sv mice. The in vitro or in vivo exposure of these then surgically resected. Finally, overexpression of sarcoma cells to IFNγ or IFNα transcriptionally down- IFNGR1.∆IC in the MCA-207 sarcoma cell line gener- regulated H60 expression (resulting in less H60 at the ated more-aggressive tumours than did the same tumour cell surface) while concomitantly upregulating MHC cell line transfected with empty vector. Subsequently, it class I expression. Furthermore, in vitro treatment of was shown that several rejectable (highly immunogenic tumour targets with IFNγ decreased their killing by and poorly tumorigenic) sarcomas from RAG2-deficient NK cells in a manner that was independent of IFNγ- mice6 were converted into progressively growing (poorly induced increases in MHC class I expression. However, immunogenic and highly tumorigenic) tumours when increased expression of these MHC molecules enhanced rendered insensitive to IFNγ through overexpression of the sensitivity of a tumour to recognition and/or lysis IFNGR1.∆IC (C.M.K., G.P.D. and R.D.S., unpublished by CTLs. These data indicate that the actions of IFNγ observations). Together, these results show that antigenic on tumour cells might coordinate the progression of the tumours become non-immunogenic in the absence of immune response to growing tumours so that the early IFNγ sensitivity. recognition and/or elimination of cancer cells by the Using the converse approach, Kaplan et al.18 showed innate immune system transitions to immune attack by that RAD–gR.28 cells, which are MCA-induced sarcoma the adaptive immune system. cells from an IFNGR1-deficient mouse (and therefore Because IFNγ regulates various biological pro- are IFNγ unresponsive), grew aggressively in syngeneic grammes14,15 that, a priori, could participate in abro- 129/Sv mice, even when injected at low cell numbers. By gating tumour growth, additional work is required contrast, RAD–gR.28 cells that were engineered to be to identify the full range of IFNγ-induced effects IFNγ responsive through enforced expression of IFNGR1 on a tumour that contribute to tumour rejection. were rejected after transplantation into wild-type mice. Given the scope of this Review, it is not possible to Similarly, introduction of IFNGR1 into MCA-induced discuss every antitumour mechanism that might be sarcoma cells from a mouse that lacked both IFNGR1 induced by IFNγ; however, some of the processes that and IFNAR1 converted these cells into highly immuno- might be important include the capacity of IFNγ to genic tumour cells that were rejected by wild-type mice9. inhibit cellular proliferation55,56, to promote apoptosis These results show that a tumour needs to be responsive through effects on the expression of caspases, CD95 to IFNγ to be immunogenic. This relationship between (also known as FAS) and TRAIL10,57–59, and to inhibit the IFNγ sensitivity of a tumour cell and its tumorigenicity angiogenesis60. Because it is probable that the IFNγ- has also been noted in models of immunotherapy and induced processes that are responsible for eliminating angiogenesis52,53. cancer cells might differ by tumour type, it will be Additional work from our laboratory has begun important to use in vivo models of primary tumori- to shed light on the mechanisms by which tumour- genesis to establish the physiological relevance of these cell responsiveness to IFNγ contributes to successful, IFNγ-induced processes.

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γ Host targets of IFN in antitumour immunity. Compared cell types (such as IL-17-producing TH cells, TH17 cells) with the role of IFNγ in increasing tumour immuno- can promote tumour development and growth, because genicity, less is known about how the effects of IFNγ on these tumour processes are decreased in the absence of host cells might contribute to the antitumour immune such cytokines68,69. Furthermore, dysregulation of IFNγ response. One demonstration of how IFNγ regulates host production and IFNγ-mediated signalling has been antitumour function came from the observation that shown to promote spontaneous carcinogenesis in the IFNγ was required for the eradication of IFNγ-insensitive colon of mice that express SOCS1 only in T cells and 6132A-PRO tumour cells by lymph-node cells from mice B cells70. Further studies are needed to determine whether that had been immunized with tumour-antigen-derived the main role of chronic inflammation in tumorigenesis is peptide61. In other studies, STAT1-deficient mice, which to increase transformation or to increase tumour growth are insensitive to IFNs and have a deficiency in polariza- in cells that have been transformed by other mechanisms,

tion of T helper (TH) cells towards TH1 cells, failed to and to identify whether these processes are linked to eliminate IFN-responsive tumour cells that were rejected chronic infection. by wild-type mice62. Furthermore, STAT6-deficient Cancer immunoediting and type I IFNs mice, which default to TH1-cell immune responses as a

result of a block in the differentiation of TH cells into Immune elimination of type-I-IFN-insensitive tumours.

TH2 cells, rejected tumour cells that grew progressively The accumulating data that support the involvement in wild-type mice63. These studies are consistent with an of endogenously produced type I IFNs in host anti- important role for IFNγ in inducing development of the tumour immunity have fuelled efforts to elucidate the

TH1-cell lineage, rather than the TH2-cell lineage, in turn mechanisms that underlie their effects. Type I IFNs promoting cell-mediated antitumour immune responses have been studied in many disease models and are by facilitating CTL maturation and macrophage activa- reported to regulate a panoply of immunological and tion13,23. However, because these studies used mice def- non-immunological processes71. However, as most of icient in STAT1 (and therefore unresponsive to IFNs), these studies monitored the physiological effects additional experiments in mice that lack host-cell sensi- of exogenously delivered type I IFNs, rather than endog- tivity to only IFNγ are necessary to define the host-cell enously produced type I IFNs, it is unclear which of the targets of IFNγ in antitumour immunity. many functions that have been ascribed to type I IFNs Another mechanism by which the effects of IFNγ are relevant to naturally occurring antitumour immune on host immune cells promote effective antitumour responses. immune responses was elucidated by a more recent study A recent study from our laboratory assessed the showing that IFNγ inhibits the generation and/or activa- physiologically relevant cellular targets of type I IFNs tion of naturally occurring (CD4+CD25+) regulatory T during the tumour-rejection process9. This study showed 64 (TReg) cells . Previous work from the same research that type I IFNs do not act on tumour cells directly during group showed that immunization of wild-type mice the antitumour immune response, thereby delineating with plasmids encoding specific tumour-expressed self an important difference between type I IFNs and IFNγ.

antigens (such as DnaJ-like 2) elicits a potent TReg-cell First, the study found that, despite insensitivity to type I response65. In both tumour-transplantation studies and IFNs, four IFNAR1-deficient MCA-induced sarcomas MCA-induced carcinogenesis studies, mice that received were rejected when transplanted into naive syngeneic plasmids encoding tumour-expressed self antigen and wild-type recipients. Second, several other IFNAR1- IFNγ were found to develop fewer tumours, with slower deficient sarcomas that grew progressively in wild-type regressor tumours kinetics, and to mount a less potent TReg-cell response mice were not converted into when than mice that received plasmids encoding only tumour- their sensitivity to type I IFNs was restored by enforced expressed self antigen64. These results indicate that early expression of IFNAR1. These findings are in contrast production of IFNγ blocks the development and/or to our observation that progressively growing IFNGR1- + immunosuppressive actions of TReg cells. CD8 T cells deficient MCA-induced sarcomas are converted into were reported to be the probable source of the IFNγ that highly immunogenic tumour cells that are rejectable (REF. 18) was suppressing the TReg cells. when transduced with DNA encoding IFNGR1 . Third, IFNAR1-deficient sarcoma cells, which are usu- A role for IFNγ in controlling transformation. Ongoing ally rejected by wild-type mice, formed progressively work in several laboratories is only now beginning growing tumours when engineered to be insensitive to to address how immunological influences (including IFNγ through introduction of the dominant-negative chronic inflammation) that occur throughout malignant receptor subunit IFNGR1.∆IC. Last, sarcoma cells from transformation ultimately determine whether the pro- mice deficient in both IFNAR1 and IFNGR1 (which lack tumour or antitumour functions of the immune system sensitivity to both types of IFN) were rejected when sen- Regressor tumour γ A cell line derived from a dominate. Indeed, chronic inflammation has been associ- sitivity to IFN was restored but not when sensitivity to primary tumour that is ated with an increased risk of developing cancer, possibly type I IFNs was restored. Together, these data show that rejected when transplanted in response to the chronic production of reactive oxygen tumour-cell responsiveness to IFNγ, but not type I IFNs, into naive syngeneic hosts and nitrogen species leading to DNA damage, tissue is required for the development of antitumour immune that are immunocompetent 66,67 but grows progressively in remodelling and, ultimately, transformation . Recent responses, thereby highlighting a clear difference in how syngeneic hosts that are studies have shown that certain pro-inflammatory cyto- these cytokines contribute to the elimination phase of immunocompromised. kines (such as IL-23 and TNF) and pro-inflammatory cancer immunoediting.

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Host targets of type I IFNs in antitumour immunity. physiologically relevant targets of their antitumour activ- Additional work from our laboratory showed that host ity. For example, type I IFNs have been found to activate cells (rather than tumour cells) are the crucial targets dendritic cells, to increase the cytolytic activity of macro- of the antitumour activity of type I IFNs9. Specifically, a phages and NK cells, to induce the production of IL-15, panel of highly immunogenic sarcomas were rejected by to prime T cells, to increase the survival of T cells and to wild-type mice but grew progressively when transplanted increase the production of stromally derived angiostatic into IFNAR1-deficient mice. Bone-marrow-chimera molecules74,75. Therefore, it is clear from many studies that approaches also showed that host haematopoietic cells type I IFNs can promote numerous immune functions, need to be sensitive to type I IFNs for tumour rejec- but further work using appropriate in vivo tumour mod- tion to occur. Highly immunogenic sarcoma cell lines els is required to reveal which of these immune functions were rejected by wild-type mice but formed progres- are physiologically relevant for antitumour immunity. sively growing tumours in IFNAR1-deficient mice or RAG2-deficient mice. By contrast, these tumour cells A role for type I IFNs in controlling transformation. In were rejected by lethally irradiated IFNAR1-deficient addition to the functions of type I IFNs during the devel- mice or RAG2-deficient mice that had been reconsti- opment of immune responses to established tumours, tuted with wild-type bone marrow. These studies, which recent findings have raised the possibility that type I show the requirement for host-cell responsiveness to IFNs might also have a role at an even earlier stage, endogenously produced type I IFNs, are consistent with in the inhibition of the process by which normal cells findings that were obtained using exogenously admin- are transformed into tumour cells. Specifically, type I istered type I IFNs. Gresser’s research group29,72 showed IFNs were reported to upregulate expression of the that treatment with type I IFNs increased the survival tumour-suppressor protein p53, and IFNβ was found to of DBA/2 mice that were challenged with either synge- inhibit the in vitro transformation of wild-type mouse neic IFN-sensitive or syngeneic IFN-insensitive tumour embryonic fibroblasts. Therefore, type I IFNs provide cells, indicating that host cells were the crucial targets of cells with an increased potential to control the transfor- type I IFNs. More recently, the administration of exog- mation process76. Another line of evidence that type I enous IFNα was shown to increase the survival of wild- IFNs might have a role in blocking cellular transforma- type C57BL/6 mice, but not of STAT1-deficient mice tion comes from the finding that the endoribonuclease (which are unresponsive to IFNs), that were challenged RNaseL, which is regulated by type I IFNs, can func- with AGS tumour cells from a STAT1-deficient mouse73. tion as a tumour suppressor in human cells77. Studies Taken together, these data indicate that endogenously of microdissected prostate tumours from patients with produced or therapeutically administered type I IFNs germline mutations in RNASEL showed that there was a mediate their antitumour effects mainly through acting loss of heterozygosity at the RNASEL locus. In addition, on the haematopoietic cells of the host. germline mutations in RNASEL have been linked to the Owing to the broad range of powerful immunoregu- prostate-cancer-susceptibility locus HPC1 (hereditary latory effects of type I IFNs, many cell types are probably prostate cancer 1)78 and to a significantly increased risk

Box 1 | Interferons and cancer immunotherapy Type I interferons Type I interferons (IFNs) — that is, IFNα and IFNβ — have been extensively used for the treatment of several cancers. Type I IFNs are approved by the US Food and Drug Administration (FDA) for the management of hairy-cell leukaemia, AIDS- related Kaposi’s sarcoma, follicular lymphoma, chronic myeloid leukaemia and melanoma (see List of approved oncology drugs with approved indications, in Further information). For example, in the Eastern Cooperative Oncology Group Trial EST 1684, 287 patients with melanoma were treated by surgical excision, then randomized to groups of either follow-up observation or treatment with high doses of IFNα for 48 weeks81. High-dose IFNα therapy significantly increased both relapse-free time and overall survival time compared with untreated patients. Other clinical trials are reviewed in REF. 30. A recent study supported the hypothesis that IFNα augments the antitumour immune response82. In an ongoing clinical trial, individuals who developed autoantibodies or clinical manifestations of autoimmunity had significantly longer overall and relapse-free survival compared with individuals who did not develop symptoms or signs of autoimmunity. The development of autoimmunity and the well-established parameter of lymph-node involvement were found to be the only independent prognostic indicators of overall and relapse-free survival. Clinical trials of IFNα are ongoing. Type II interferon Unlike type I IFNs, type II IFN — that is, IFNγ — is not approved by the FDA for the treatment of any cancers. Previous studies showed that treatment with IFNγ had no benefit for patients with metastatic renal-cell carcinoma83, advanced colon cancer84 or small-cell lung cancer85. However, improved survival was observed when IFNγ was used as an adjunct to therapy for individuals with stage-Ic–IIIc ovarian cancer86, as well as when administered intravesically to individuals with transitional-cell bladder carcinoma87 or when used in isolated-limb perfusion treatments of individuals with some non- melanoma cancers of the extremities88. Despite the proven pivotal role of endogenously produced IFNγ in animal models Angiostatic molecule of antitumour immunity, the limited success of this cytokine in cancer-immunotherapy trials in humans might be A molecule that blocks or explained by the following factors: tumour-cell insensitivity to IFNγ9,18,44, an inability to deliver IFNγ locally (given the opposes the growth of blood ubiquitous expression of its receptor) or an inability to therapeutically recapitulate the natural periodicity of IFNγ vessels and the formation of production. Clinical trials of IFNγ are ongoing. new blood vessels.

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of developing the disease79. However, it remains unclear by the IFNs, to sharpen the acuity of our picture of the whether the tumour-suppressor functions of RNaseL cancer-immunoediting process. depend on IFNs80. Additional studies are needed to Nevertheless, one could predict that the lessons that determine whether type I IFNs contribute to the func- have been learned about the roles of the IFNs in prom- tion of tumour-suppressor proteins in physiological oting natural immune responses to developing tumours models of transformation and whether the targets of should be useful in therapeutic settings. However, except the antitumour activity of type I IFNs change during the for a few types of cancer (BOX 1), this prediction has not progression from cellular transformation to malignancy. yet been realized. The limited success of the therapeutic use of IFNs might reflect our inability to target the Conclusions right IFNs to the right place at the right dose. This issue In the past 12 years, there has been a reversal in is particularly relevant to the IFNs, because nearly all how the interaction of a developing tumour with the cells constitutively express functional IFN receptors, otherwise-unperturbed immune system is viewed. so systemic administration of these cytokines might From both mouse models and human clinical trials, not necessarily augment immune effector functions at there is now overwhelming evidence in support of the the tumour site. We speculate that this could lead to a conclusion that the immune system does indeed protect state of immunological ‘confusion’, resulting in either the host against tumour development, thereby carrying a lack of appropriate cellular infiltration into the tumour out a cancer immunosurveillance (tumour elimination) or a state of host-cell or tumour-cell unresponsiveness. function. However, there is also a substantial amount Ultimately, we hope that future studies of IFN-dependent of evidence showing that the immune system can cancer immunoediting will provide insights that lead to promote tumour growth, either by sculpting tumour the development of more-effective IFN-based cancer immunogenicity or by suppressing host-protective immunotherapies. immune effector mechanisms. These diverse effects of immunity on developing tumours form the basis of the Note added in proof cancer-immunoediting hypothesis. A recent study adds strong support for the existence Although, in this Review, we have discussed the of a cancer-immunoediting process in humans. Using strong evidence that supports an obligatory role for a combination of histological and gene-expression IFNs in this process, we have only a ‘low-resolution analyses, Galon et al.113 showed that the presence, picture’ of their functions at present. It is clear that location and density of T cells and their products within endogenously produced type I IFNs and IFNγ do not colorectal tumours was a much better predictor of the function in a completely overlapping manner and that survival of patients witu cancer than the commonly used they exert their effects on different host-cell popula- tumour-staging criteria, which are based on the size and tions. It is also clear that IFNγ, but not IFNα and IFNβ, spread of a tumour. These data therefore not only show controls the immunogenicity of tumour cells, possibly that naturally occurring immune responses to tumours as a result of the selective production of IFNγ in the take place in humans but also support the concept that tumour microenvironment. Future efforts need to the immune system can maintain a subclinical tumour define better the molecular pathways that are affected in an equilibrium state.

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