key: cord-0765970-vv31uh8t
authors: Borden, Ernest C.; Sen, Ganes C.; Uze, Gilles; Silverman, Robert H.; Ransohoff, Richard M.; Foster, Graham R.; Stark, George R.
title: Interferons at age 50: past, current and future impact on biomedicine
date: 2007
journal: Nat Rev Drug Discov
DOI: 10.1038/nrd2422
sha: 760d51895422ff5246bbd33c0308164e2dae4395
doc_id: 765970
cord_uid: vv31uh8t

The family of interferon (IFN) proteins has now more than reached the potential envisioned by early discovering virologists: IFNs are not only antivirals with a spectrum of clinical effectiveness against both RNA and DNA viruses, but are also the prototypic biological response modifiers for oncology, and show effectiveness in suppressing manifestations of multiple sclerosis. Studies of IFNs have resulted in fundamental insights into cellular signalling mechanisms, gene transcription and innate and acquired immunity. Further elucidation of the multitude of IFN-induced genes, as well as drug development strategies targeting IFN production via the activation of the Toll-like receptors (TLRs), will almost certainly lead to newer and more efficacious therapeutics. Our goal is to offer a molecular and clinical perspective that will enable IFNs or their TLR agonist inducers to reach their full clinical potential.

The discovery and molecular understanding of the cell ular mechanisms and clinical use of interferons (IFNs) have been a major advance in biomedicine over the past 50 years. This family of secreted autocrine and para crine proteins stimulates intracellular and intercellular networks that regulate resistance to viral infections, enhance innate and acquired immune responses, and modulate normal and tumour cell survival and death. After their discovery in 1957 (Ref. 1), it was soon appre ciated that IFNs were critically important to the health of animals and humans and that the IFN system had potential as therapies for infections for both RNA and DNA viruses (TIMeLINe).

However, advances in molecular biology a decade later and into the 1970s were required before the promise could be realized. The 1980s saw their introduction into the clinic as the first pharmaceutical products of the budding biotechnology industry, and, importantly, as a demonstration of the effectiveness of IFNs not only for viral diseases and cancer but also for multiple sclerosis (MS). The 1990s were marked by an expansion in their clinical applications with regulatory approvals world wide and a further understanding of molecular events influencing biological actions. Current studies have led to new insights into IFNs as a fundamental component of the innate immune system. Additionally, studies have revealed how IFN production is induced through Toll like receptors (TLRs), the actions of IFNstimulated genes (ISGs), the identification of viral mechanisms that resist actions of this potent protein, and how mutation and suppression of gene products of the IFN system in and by malignant cells may affect the initiation and progression of cancer.

After binding to highaffinity receptors, IFNs initiate a signalling cascade through signalling proteins that can also be activated by other cytokines, which were first identified through studies of IFNs. Cellular actions are mediated through specific ISGs, which underlie the antiviral effects, as well as immunoregu latory and antitumour effects. Future drugs that could act as molecular activators for ISGs, many of which exist in a latent state or as agonists for TLRs, might be expected to have potent antiviral, antitumour and/or immunomodulatory effects.

From more than 100,000 published papers, we offer a perspective with a focus on human IFNs to stimulate the future investigation of important questions. Although IFNs function as an integrated system, conceptually it helps to consider their production, which is medi ated through TLR activation, and their action, which is mediated through JAK/STAT (Janus kinase/Signal Transducers and Activators of Transcription) and other signalling pathways. The production of IFNs is important for understanding the role of IFNs in innate immunity, while their effects relate to dissecting under lying and future mechanisms of action and application. Highlighting initial pivotal discoveries and more recent findings of conceptual importance, we review how IFNs are induced, the cellular actions of IFNs and ISGs, human therapeutic applications, and summarize important ques tions for biomedicine and drug and clinical development initiatives.

How synthesis is induced Production of IFNs, both in vitro and in vivo, is transient and requires stimulation by viruses, microbial products or chemical inducers. In the course of the discovery of IFNs, either live or heatinactivated influenza viruses were initially identified as inducers 1 . Subsequently other microbial products, including those of bacteria, proto zoa, and RNA and DNA viruses, were also recognized to induce IFN 2 . It was also shown that microbial nucleic acids, lipids, polysaccharides or proteins trigger induc tion of IFNs through activation of TLRs (fIG. 1 ). An early pivotal discovery identified doublestranded (ds) RNAs, both natural and synthetic, as potent inducers 3 , leading to the simplistic paradigm that viruses induce IFNs by producing dsRNA; in reality, it is only one of the viral gene products responsible for induction. Nonetheless, dissection of cellular responses that lead to induction were spearheaded by the analysis of dsRNAmediated signalling pathways.

dsRNA is recognized by TLR3, which is present mostly in endosomal membranes 4 , and also by two cytoplasmic RNA helicases, retinoic acidinducible gene I (RIGI) and melanoma differentiation associated protein 5 (MDA5) 5 (fIG. 2) . The cytoplasmic proteins can also recognize singlestranded (ss) RNAs but only if they have 5′triphosphates. Mice in which the TLR3 (Ref. 6), the RIG-I 7 or the MDA5 (Ref. 8) gene has been disrupted are more susceptible to virus infection; however the relative importance of the three proteins for inhibitory activity varies for the immune defence against different viruses. Surprisingly, the presence of TLR3 can enhance pathogenesis in mice infected with influenza A virus 9 or west Nile virus 10 . An adaptor protein for TLR3 signalling is TLR adapter molecule 1 (TRIF), whereas the mitochondrial protein IFNβpromoter stimulator 1 (IPS1; also known as vISA) is an adaptor for RIGI and MDA5; both TRIF and IPS1 recruit inhibitor of nuclear factorκb (NFκb) kinase (IKK) and TANKbinding kinase (TbK1), the common activator kinases 11 . other nucleic acids such as ssRNA, acting through TLR7 and TLR8, and bacterial oligodeoxyribonucleotides, acting through TLR9, are also potent inducers 12 . bacterial lipopolysaccharides induce IFNs through TLR4 and also recruit TRIF; viral glycoproteins bind and activate dif ferent TLRs. An additional cytoplasmic receptor exists for recognizing viral DNA 13 .

IFN genes, which are normally transcriptionally silent, are induced by the binding of TLRactivated transcription factors to their promoters. Transcriptional induction of IFNβ has been a model experimental sys tem for defining interactions of transcription factors as an enhanceosome multiprotein complex with DNA 14 . The most important transcription factors for induction are proteins of the IFN regulatory factor (IRF), specifi cally IRF3 and IRF7, and NFκb families 15, 16 . IRFs are activated by the kinases TbK1 or IKKε; activated IRFs then dimerize and translocate to the nucleus 15 . The IKK protein kinase complex phosphorylates Iκb and releases it from NFκb; NFκb is then further activated by phosphorylation by other kinases 17 .

To evade the IFN system, viruses have evolved many mechanisms to block IFN synthesis and actions -acting at almost every step of the signalling pathway 18, 19 . For example, a hepatitis C virus (HCv)encoded protease can cleave IPS1 off the mitochondrial membrane and block RIGI/MDA5mediated signalling 20 

An antiviral unit is the concentration of an interferon required to inhibit virus replication in vitro by 50%; an international WHO standard provides a reference base for each major interferon type.

establishment of an antiviral state through the inter action with RIGI 22, 23 . Conversely, patients with defects in the production of type I IFNs, due to mutation of the UNC93B gene, are highly susceptible to herpes simplex virus 1 (HSv1) encephalitis 24 . Development of smallmolecule activators of induction are only beginning; however, delineation of the responsible signalling pathway has identified many target proteins. CpG oligonucleotides are activators of TLR9 (Ref. 25); the quinolinamine imiquimod 26 and its analogues activate TLR7 (Refs 25, 26) ; and DMXAA induces IFN synthesis through a TLRindependent pathway 27 . Development of chemical modulators that selectively activate IFN synthesis or block the synthesis of inflammatory cytokines could have a broad therapeutic potential 28, 29 .

The realization that IFNs constitute a protein family arose initially from the definition of antigenic differences between human fibroblast (IFNβ) and leukocyte IFNs (IFNα) 30 . Although protein purification studies sug gested the potential multigenic nature of IFNα, this was only firmly established by the cloning of IFNα1, IFNα2 and IFNβ [31] [32] [33] , which, when accomplished, helped solidify the financial future of the nascent biotechnology industry. The number of functional genes identified that encode type I IFNs has grown subsequently: 17 nonallelic genes have now been described in humans. All lack introns and cluster on chromosome 9 (Refs 34, 35) . of the type I IFNs, there are 13 IFNαs (plus an additional synthetic concen sus sequence and also additional minor allelic variants), whereas there is only one type of IFNβ, IFNω, IFNε or IFNκ (fIG. 3) . Among mammals, the number of type I IFN genes is variable; some have unique types (for example, IFNδ occurs only in pigs and IFNτ only in ruminants) and others are devoid of a particular type (for example, IFNω in mice). Consistent with the universal biological definition of IFNs (that is, proteins inducing relatively speciesspecific antiviral effects), all type I IFNs, which are mostly nonglycosylated pro teins of 165-200plus amino acids, share homologies that range from 30-85% within a species. essentially all have relatively high specific potencies (1 × 10 7 to 1 × 10 9 antiviral units per mg protein).

Although type I IFNs have qualitative and quantita tive differences in their antiviral and other actions [33] [34] [35] [36] [37] [38] [39] [40] , the reason for origin and maintenance through evolu tion of these related proteins is unknown. All mamma lian species have retained, however, at least one IFNα and one IFNβ 41 . In humans, expression of IFNκ and IFNε seems tissue specific 35, 42 , but all cells are able to produce other IFNs. In monocytederived dendritic cells, in which viral infection induces expression of the 15 IFNα/IFNβ/IFNω subtypes, stimulation of TLR3 or TLR4 induces mostly IFNβ and IFNα1, which emphasizes the differences in the promoter sequences for the IFNαs, IFNω, and IFNβ genes that govern the response to different inducers 43 .

Type I IFNs belong to the helical cytokine family with secondary structures of a five αhelix bundle held in position by two disulphide bonds 44 . They act through a cellsurface receptor composed of two ubiquitously expressed transmembrane proteins, IFN (α, β and ω) receptor 1 (IFNAR1) and IFNAR2 (the genes for which are clustered on chromosome 21), and are associated with two cytoplasmic tyrosine kinases, TYK2 and JAK1 (Ref. 39) (fIG. 3) . Formation of the IFN-receptor complex involves one side of the IFN protein interacting with IFNAR2 in a region forming the hinge between the two fibronectin type III (FnIII) domains (fIG. 3) ; binding affinity is in the nanomolar range 39 . IFNAR1 binds IFNs with an affinity 1,000fold weaker than that of IFNAR2, with a binding site located opposite to the IFNAR2 bind ing site. binding studies are consistent with the ternary complex between IFNAR1, IFN and IFNAR2 having a 1:1:1 stoichiometry, and a similar if not identical archi tecture for all type I IFNs. Ternary complex assembly is a twostep process; the ligand binds first to one IFNAR and then recruits the second with no identified inter action between the two IFNARs 39 .

As affinities for IFNAR2 are generally much higher than for IFNAR1, a binding pathway in which IFNs bind first to IFNAR2 and then IFNAR1 should have a higher probability. However, with IFNα1 having a low affinity for IFNAR2, the relevance of the reverse binding pathway, which could lead to differing cellular effects, has been confirmed 45 . If differences in the struc tures of the IFN-receptor complexes cannot account for the differential activities of type I IFNs, then a body of argument -which includes studies on the activities of engineered IFNs -suggests that differential affinities for IFNARs and thus, ternary complex stability, govern differential biological activities 39, [46] [47] [48] . The cellsurface concentration of IFNARs and their lateral organization into microdomains could also be important cellular parameters that shape responsiveness to individual IFNs 49 . Similar or other changes in receptor organiza tion may also account for the increased susceptibility to Hbv infection that occurs with polymorphisms in class II cytokine receptor genes 50 . IFNγ is a single glycosylated protein of 140 amino acids that is designated as a type II IFN because of its dis tant aminoacid sequence homology with type I IFNs, and its production by natural killer (NK) or activated T cells. Like type I IFNs, it binds to two class II cytokine receptor proteins; when ligand bound it forms a complex of two of each of the receptor proteins linked to an antiparallel homodimer of IFNγ 51 (fIG. 3) . IFNγ receptor 1 (IFNGR1) maps to chromosome 6 and has a JAK1 binding domain and a STAT1 docking site. IFNGR2 contains a JAK2 bind ing domain and maps to chromosome 21q22.1 in a cluster that also contains IFNAR1, IFNAR2 and interleukin 10 receptor 2 (IL10R2; also known as IL10Rb) 52 . Although IFNGR1 is constitutively present on all cells, IFNGR2 is tightly regulated and less widely expressed. Promoter polymorphisms and/or mutations within both chains have been associated with increased susceptibility to malaria and mycobacteria -in a few patients these defects have been reconstituted by marrow transplantation 53 . The type III IFN family with three subtypes of IFNλ, which are coproduced with IFNβ, activate the same main signalling pathway as type I IFNs but have evolved a completely different receptor structure 54 (fIG. 3) .

Initial studies identified several genes that were induced by type I IFNs and analysis of their promoters identified conserved DNA elements [55] [56] [57] [58] . Proteins bound to these elements after treatment with type I IFNs were purified and identified as STAT1, STAT2 and IRF9 .

To identify other components of IFNdependent signal ling cascades, the promoter element of the 6-16 gene was used to drive IFNdependent expression of guanine phosphoribosyl transferase; cells that did not respond to IFNs were then selected with 6thioguanine. Following chemical mutagenesis, several mutant cell lines were obtained, each lacking a protein essential for signalling. For example, mutant U1A was shown by complemen tation to lack the tyrosine kinase TYK2 (Refs 60,62). Subsequently seven STAT and four JAK family members were identified; these transcription factors and tyrosine kinases have been shown to be essential for responses not only to IFNs but also to other cytokines and growth factors as well 61 .

Minimum requirements for a response to type I IFNs are the heterodimeric IFN receptor; the tyrosine kinases TYK2 and JAK1, which reciprocally transphosphorylate the receptor chains when activated; STAT1 and STAT2, which are phosphorylated in response to signalling; and the unphosphorylated IRF9 (fIG. 3) . Transcription in response to IFNdependent signalling is initiated by highaffinity binding to specific palindromic promoter sequences of the trimeric complex of STAT1, STAT2 and IRF9. The response to IFNγ requires only the two receptor proteins, the kinases JAK1 and JAK2, and STAT1. STAT1 and STAT3 bind competitively to the same phosphotyrosine residue of IFNGR1, with the binding of STAT1 greatly favoured 63 . Although initial work was carried out primarily in human fibroblasts, recent studies have identified additional complexity that allows individual cell types to respond by activating different STATs in response to the same IFN (reviewed in Refs 64, 65) . Analysis of defects in the IFN system has identified germline mutations in humans that result in deficiencies of STAT1 or TYK2, with enhanced suscepti bility to infection by viruses 61, 66 . Although the mouse has been a useful model, the human defects are not always identical to the effects that result from the targeted dele tions of these genes in mice 61 .

upon activation of receptors, the JAKs undergo autophosphorylation and transphosphorylation to increase their activity, and then phosphorylate the IFN receptors and finally STATs. However, the kinase activities of the JAKs are not sufficient to explain all nuances of signalling. Tissuespecific differences in activating additional protein kinases probably con tribute to the differential responses of various cells to a single type of IFN. In at least some cell types, the p85 subunit of phosphatidylinositol 3kinase (PI3K) is associated with IFNAR1. The activation of p85 by IFN leads to AKT phosphorylation and expression of the chemokine (CXC motif) ligand 11 (CXCL11) gene, encoding an important chemokine 67 . Type I IFNs also activate p38, and inactivation of p38 blocks induction by IFNβ of CXCL11 and TNFSF10 (encoding tumour necrosis factorrelated apoptosisinducing ligand, APo2L/TRAIL) 68 and of CXCL10 (encoding the chemo kine IP10; also known as IFNγinduced peptide, 10 kDa) in primary leukocytes 69 . An important function of the activation of protein serine kinases such as p38 and protein kinase C (PKC) in response to IFNdependent signalling is phosphorylation, directly or indirectly, of transcription factors 70 . Serine 727 of STAT1 is phosphorylated in response to IFNγ by the kinase cascade PI3K-AKT-PKC-MKK4-p38 (MKK4, mitogenactivated protein kinase kinase 4; also known as MAP2K4), with some variation in the acti vation of different PKC or MKK proteins in different cells 65 . IFNdependent activation of PI3K, extracellular response kinases (eRKs) and p38 stimulates the phos phorylation of NFκb (but not Iκb), AP1 and possibly Pu.1, respectively. These activated transcription factors may then either drive gene expression independently of activated STATs or cooperate with activated STATs on certain promoters (fIG. 4) . Conversely, transcription initia ted by phosphorylated STATs does not proceed indefi nitely; homeostasis and balance result from the actions of phosphatases such as SHP1 and SHP2 and a family of ISGs, the suppressor of cytokine signalling (SoCS) proteins 71, 72 . SoCS inhibit receptor signalling both by directly inhibiting JAKs and by targeting the receptor complex for proteasomal degradation 71 .

Prior exposure to other cytokines conditions how a cell will respond and, conversely, IFNs condition responses to other cytokines 64, 65, 73 . An excellent example of such an effect is prior exposure of human macrophages to IFNγ, which changes the response to IL10 from activation of STAT3 to activation of STAT1 (Ref. 74) . Although, as reductionist scientists, we tend to study the responses of cells in culture to treatment with IFNs alone, the situation in vivo is obviously much more complex. also known as DDX58) or melanoma differentiation associated protein 5 (MDA5; also known as IFIH1). a | TLR3 recognizes dsRNA in the lumen of the endosome, which causes phosphorylation of specific tyrosine residues in TLR3 by an unidentified protein tyrosine kinase (PTK). TLR3 dimerizes, binds to CD14 and activates the signalling complex assembled by TLR adaptor molecule 1 (TRIF). Two major pathways bifurcate from TRIF. One, composed of tumour necrosis factor (TNF) receptor-associated factor 3 (TRAF3) and TANK-binding kinase (TBK1/IKKE), leads to phosphorylation of the transcription factor IFN regulatory factor 3 (IRF3). IRF3 requires further phosphorylation by the phosphatidylinositol 3-kinase (P13K)/AKT pathway for its full activation, which is initiated by binding PI3K to phosphorylated TLR3. The other branch acts through TRAF6 and transforming growth factor-β-activated kinase 1 (TAK1; also known as MAP3K7) leading to the activation of nuclear factor-κB (NFκB), JUN and activating transcription factor 2 (ATF2) transcription factors. The activated transcription factors translocate from the cytoplasm to the nucleus, bind to the cognate sites in the promoters of the target genes and singly or in combinations induce their transcription. b | The cytoplasmic RNA helicases RIG-I and MDA5 recognize dsRNA or 5′ triphosphorylated single-stranded (ss) RNA and use the mitochondrial membrane-bound protein IFNβ-promoter stimulator 1 (IPS1; also known as VISA) as the specific adaptor. IPS1 functions like TRIF and activates the same transcription factors leading to the induction of similar genes. In addition, they cause apoptosis by activating caspases 8 and 10 through the interaction of FADD with IPS1. Solid arrows denote steps that have been fully delineated, stippled arrows show steps that contain as yet unknown intermediaries. AIP3, atrophin-1 interacting protein 3; CCL5, chemokine (C-C motif) ligand 5; CXCL10, chemokine (C-X-C motif) ligand 10; IFIT1/2, interferon-induced protein with tetratricopeptide repeats 1/2; IKK, inhibitor of NFκB kinase; SELE, selectin E (endothelial adhesion molecule 1).

Type I: IFN-αs (13 types) β/ω/κ/ε/δ/τ both STAT1 and STAT3 have activities in addition to their roles as cytokineactivated transcription factors. STAT1 is activated in response to both type I and type II IFNs and STAT3 is activated in response to gp130 cytokines such as IL6. As STAT1 and STAT3 drive essen tially opposite biological responses, large signaldependent changes in their concentrations will affect their relative acti vation by a further signal. Indeed, an increase in the ratio of STAT1-STAT3 after IFNα2 treatment of patients with melanoma correlated with survival 75 . Another consequence of cytokinedependent increases in STAT expression is that unphosphorylated STAT1 and STAT3 have important func tions that are quite distinct from those of the phosphoryl ated proteins 76, 77 . For example, unphosphorylated STAT3 activates a subset of κbdependent genes by forming a complex with NFκb 78 . Thus the role of STATs in signalling after receptor binding has expanded from kinaseactivated transcription factors to proteins that, even in the absence of ligand activation, activate transcription and partici pate in celltype specificity, resulting in diverse patterns of ISG induction in different cell types in response to a single IFN.

ISGs: molecular mechanisms of antiviral action ISGs are a diverse group of more than 300 genes (which and how many are a function of celltype signalling variations as discussed above) that mediate the bio logical and therapeutic effects of IFN stimulation 79, 80 (TABLe 1) . Studies of their mode of action have resulted in fundamental discoveries concerning translational control, regulation of RNA stability and editing, and protein transport and turnover 18 . Furthermore, proteins that are induced upon IFN stimulation, especially those that can be activated or inhibited in vivo, are targets for highthroughput screening for identification of new modulators of the IFN system. examples for this are 2′,5′oligoadenylate syn thetases (oASs) and ribonuclease L (RNASeL), which inhibit a broad range of RNA viruses 81 . viral dsRNA can directly activate one of several human oAS proteins to produce a unique 2′to5′ linked oligoadenylate of 3-6 bases (2-5A) from ATP 82 . The only wellestablished function of 2-5A is activation of the ubiquitous, latent enzyme, RNASeL 83 . 2-5A binding to RNASeL induces monomeric, inactive RNASeL to dimerize into a potent endoribonuclease that cleaves singlestranded regions of RNA on the 3′ side of upup and upAp dinucleotides [84] [85] [86] . The oAS-RNASeL pathway can inhibit the replication of encephalomyocarditis virus, Coxsackie virus b4, west Nile virus, some retroviruses and HCv 81 . Furthermore, degradation of cellular mRNA and rRNA by RNASeL damages the host cell machinery that is required for viral replication and can result in apoptosis, contrib uting to both antiviral and antitumour actions [87] [88] [89] . RNASeL also cleaves selfRNA into small degradation products that activate the recognition receptors, RIGI and MDA5, to induce IFNβ, similar to that of non self viral RNA 90 , thus perpetuating and amplifying the production of IFNβ. A highthroughput screen has resulted in the identification of small molecules that can activate RNASeL and produce broadspectrum antiviral effects 91 .

The dsRNAactivated protein kinase (PKR) and oAS were the first enzymes identified that uniquely respond to IFNs 92, 93 . PKR is a serine/threonine kinase that mediates translational and transcriptional control in response to dsRNA and other signals [92] [93] [94] [95] [96] . In addi tion, the cellular protein PACT (also known as PRKRA) activates PKR in the absence of dsRNA 97 . PKR mediates translational control by phosphorylating the protein synthesis initiation factor eIF2α, resulting in an inac tive complex between eIF2-GDP and the recycling factor, eIF2b. These events produce global inhibition of protein synthesis that blocks further viral replication and full amplification of the viralinduced cellular stress response. Many viruses, however, evade PKR through a range of strategies such as binding and sequestering dsRNA, thus depriving PKR of its activator or inhibition of its kinase activity 98 .

Another ISG family that influences translation is the strongly induced p56related proteins (IFIT gene prod ucts). Two of these, p56 and p54, inhibit protein synthesis by blocking the action of the translation initiation factor eIF3 (Ref. 99 ). p56 and p54 bind to different subunits Type I interferons (IFNs) (α, β ω, κ, ε, δ (pigs), τ (ruminants)) interact with IFN (α, β and ω) receptor 1 (IFNAR1) and IFNAR2; type II IFNγ with IFNγ receptor 1 (IFNGR1) and IFNGR2; and type III IFN-λs with IFNλ receptor 1 (IFNLR1; also known as IL28RA) and interleukin 10 receptor 2 (IL10R2; also known as IL10RB). Type II IFNγ is an antiparallel homodimer exhibiting a two-fold axis of symmetry. It binds two IFNGR1 receptor chains, assembling a complex that is stabilized by two IFNGR2 chains. These receptors are associated with two kinases from the JAK family: JAK1 and TYK2 for type I and III IFNs; JAK1 and JAK2 for type II IFN. All IFN receptor chains belong to the class 2 helical cytokine receptor family, which is defined by the structure of the extracellular domains of their members: approximately 200 amino acids structured in two subdomains of 100 amino acids (fibronectin type III modules), themselves structured by seven β-strands arranged in a β-sandwich. The 200 amino-acids domain usually contain the ligand binding site. IFNAR2, IFNLR1, IL10R2, IFNGR1 and IFNGR2 are classical representatives of this family, while IFNAR1 is atypical as its extracellular domain is duplicated. GAS, IFNγactivated site; IRF9, IFN regulatory factor 9; ISGF3, IFN-stimulated gene factor 3, refers to the STAT1-STAT2-IRF9 complex; ISRE, IFN-stimulated response element; P, phosphate; STAT1/2, signal transducers and activators of transcription 1/2. IFNR2  IFNR1  IFN   JAK   Cytokine   TYK2 JAK JAK Caspase A group of enzymes that have a role in promoting apoptosis (that is, programmed cell death). Inhibition of such enzymes might be useful for combating cell and tissue damage in conditions such as myocardial infarction, stroke, inflammatory diseases and neurodegenerative diseases. Augmentation of such enzymes, through the production of proapoptotic proteins, might be useful for combating proliferative conditions, such as cancer.

of eIF3 and block some of its diverse functions. HCv mRNA translation is inhibited more strongly by p56 than by cellular mRNAs, because its initiation is internal ribo some entry site (IReS)mediated, and not CAPmediated; thus it selectively inhibits viral protein synthesis 100 .

The ISGencoded antiviral protein Mx was identi fied because of the resistance of mouse strain A2G to influenza A viruses (Mx1, orthomyxovirus resistance gene 1) [101] [102] [103] . Mx proteins are large (~80 kDa) GTPases in the dynamin superfamily that selfassemble and bind viral nucleocapsids. This interferes with intracellular trafficking and activity of viral polymerases, thus inhibiting replication of many RNA viruses including influenza and measles viruses 104 . The human homo logue, MXA, is a cytoplasmic protein that associates with intracellular membranes.

ISG15 encodes a ubiquitinlike, 15 kDa protein that modifies more than 100 proteins through a process known as ISGylation [105] [106] [107] . ISG15 inhibits HIv1 release from cells, mimicking the effect of IFN 108 and infections by influenza, herpes and Sindbis viruses 109 . The antiviral mechanism of ISG15 in vivo is unknown but could relate to its cytokinelike properties or to its ability to conju gate and modify the function of cellular or viral proteins. ISG15 is also a target gene for dysregulation of the p53 and ISG pathways that occurs in many types of cancer, suggesting an additional role in tumorigenesis 107 .

Phospholipid scramblase 1 (PLSCR1), a protein implicated in Ca 2+ dependent reorganization of plasma membrane phospholipids 110 , either inserts into the plasma membrane or binds DNA in the nucleus depending on its palmyitoylation 111 . PLSCR1 has anti viral activity that possibly results from the enhanced transcription of a subset of ISGs 112 . TRAIL/APo2L is an ISG that contributes to apoptosis and therefore probably to both the antiviral and antitumour effects of IFNs [113] [114] [115] . Although many ISGs promote apoptosis, some promote cell survival. For example, the ISG G1P3 (or 6-16) 116,117 localizes to mitochondria and has anti apoptotic actions, including inhibition of caspase3 (Ref. 118) . A related ISG, encoded by IFI27 (ISG12) 119 , promotes an agedependent resistance to alphavirus encephalitis in mice without affecting either levels of apoptosis or viral yields 120 . Additional ISGs with f o c u s o n a n t i v i r a l s Inducible nitric oxide synthase (iNOs). An inducible haem containing enzyme that produces nitric oxide in response to inflammatory signals.

Proteins that function in the nuclear transport of protein and RNA.

probable or confirmed antiviral activities include the guanylatebinding protein 1 (GbP1); a 3′,5′exonucle ase encoded by ISG20; the promyelocytic leukaemia protein (PML); adenosine deaminase (ADAR1); the endoplasmic reticulumassociated protein viperin (cig5) that can inhibit human cytomegalovirus; induc ible nitric oxide synthase (iNoS); and the nucleoporins Nup98 and Nup96 (Refs 121-128). Finally, many IFNpathway signalling proteins are themselves ISGs, thus providing an autocrine loop that amplifies IFN responses; examples are IRF7, RIGI, MDA5 and STAT1. As ISGs with high levels of transcriptional induction are still poorly characterized functionally 79, 80, 128 , some could prove to be critical mediators of antiviral and other actions. because all biological effects of IFNs are mediated through the action of ISGs (TABLe 1) , further research into understanding the functions of the protein products of these may lead to more efficacious antiviral and other therapeutics.

In addition to direct inhibition of viral replication by ISGs, a second level of IFN action augments adaptive and acquired immune responses. early warning of pathogen presence is delivered by tissueassociated and circulating dendritic cells, one type of which, the plasmacytoid dendritic cell, is the circulating type I IFNproducing cell 129 . In addition to TLR activation on cells at the sites of pathogen invasion or replication, this response culminates with dendritic cellmediated presentation to CD4 + T cells of pathogenderived peptide fragments that are bound to surface major histocompatibility complex (MHC) class II molecules. MHC class II proteins are selectively upregu lated by IFNγ, whereas type I IFNs fail to do so owing to the STAT2dependent induction of SoCS1 (Ref. 130 ). Infected cells that display peptide fragments associated with MHC class I molecules on the surface are recognized and subsequently eliminated by CD8 + T cells, thereby clearing the virus. either type I IFNs or IFNγ can mark edly upregulate MHC class Idependent antigen presenta tion. In addition to MHC molecules, other ISGs involved in antigen processing include the lysosomal membrane permeabilization (LMP) components of proteasomes and transporters for antigen processing (TAPs), which shuttle peptides into the endoplasmic reticulum for loading onto nascent MHC class I proteins [131] [132] [133] [134] [135] .

IFNs also promote accumulation of leukocytes at sites of pathogen invasion; specifically, IFNs (along with cytokines such as TNFα and IL1β), strongly pro mote the expression of vascular adhesion molecules including intracellular adhesion molecule 1 (ICAM1). Furthermore, IFNs induce the production of chemotactic DDX58) ; RNASEL, ribonuclease L; STAT1, signal transducer and activator of transcription 1, 91 kDa; TRAIL/APO2L, tumour necrosis factor-related apoptosis-inducing ligand (also known as TNFSF10); Viperin (cig5), also known as RSAD2; XAF1, X-linked inhibitor of apoptosis-associated factor 1; XIAP, X-linked inhibitor of apoptosis protein (also known as BIRC4).

cytokines (chemokines), which participate in leukocyte recruitment. As examples, three closely related chemo kines involved in accumulation of activated T cells and macrophages are the ISGs CXCL9 (also known as MIG, monokine induced by IFNγ); CXCL10 (also known as IP10, IFNγ 10 kD inducible protein); and CXCL11 (also known as ITAC, interferoninducible Tcell αchemo attractant) [136] [137] [138] [139] . True to their names, these chemokines are not expressed in the absence of IFN signalling.

In the development of an adaptive immune response, IFNγ is produced by an early warning NK cell, or by activated T cells 140 . IFNγ governs expression of class II transactivator (CIITA), a master regulator of transcrip tion of the MHC class II molecules themselves, as well as the associated invariant chain, which helps stabilize MHC class II heterodimers. HLADM catalyses the dis placement of the invariant chain from the MHC class II peptide binding site as the mature MHC class IIpeptide conjugate is finalized for insertion into the plasma membrane [141] [142] [143] [144] [145] [146] [147] [148] [149] . Finally, of substantial importance in the host response to the virus is the IFNmediated activa tion of cytotoxic effector function among cells of innate and adaptive immunity including NK cells, dendritic cells, macrophages and T cells. Indeed, the property of stimulating macrophages contributed substantially to the recognition of IFNγ as a biologically important lymphokine and as a 'different' IFN 150 .

Human therapeutic applications based on preclinical studies of broad spectrum inhibi tion of virus replication, IFNs were initially investigated as antivirals with activity against RNA and DNA viruses. Clinical effectiveness for both has now been established. but development of relatively specific, low molecular mass antivirals has largely supplanted broad application except for Hbv and HCv chronic infections. The first uS Food and Drug Administration (FDA) approval for IFN α2, however, was not for virus infection but for cancer, which was driven by interest created by publicity result ing from its effectiveness in American Cancer Society trials. Subsequently, placebocontrolled randomized trials established the effectiveness of IFNβ for relaps ing, remitting MS -an apparent paradox in terms of the mechanistic understanding of IFN actions, as IFNs, as discussed above, are generally immune augmenting rather than immunosuppressive. Presently, a number of drugs are being or have been designed to target different components of the IFN system for different therapeutic indications (fIG. 5) .

Viruses. The recognition that Hbv often caused a chronic infection leading to cirrhosis and hepatocellular carcinoma suggested that infected patients might benefit from IFNs 151 . Initial clinical trials of impure IFNα 152 suggested benefit but studies with impure IFNβ were less promising 153 . These lowdose studies were followed, however, by higher doses of recombinant IFNs, when they became available, which then confirmed beneficial effects 154 .

Hbv chronic infection evolves with hepatitis e antigen (HbeAg)positive quiescent viruses escaping inhibition during conversion of an immunotolerant to an immunoactive phase, with enhanced immune elimination of infected hepatocytes 155, 156 . In many, this immune response causes suppression of viral replication and HbeAg loss. A quiescent phase or 'healthy carriage' may ensue but disease reactivation is common (HbeAg negative disease 157 ). In HbeAgpositive early Hbv infec tion, IFNs have not been particularly effective. However, in the immunoactive chronic phase, Hbv is sensitive to IFNα2 and the ongoing immune response is aug mented, leading to quiescent HbeAgnegative disease in up to 40% of patients. IFNα2 (RoferonA, Hoffmann LaRoche; IntronA, Schering-Plough), now usually in the form of a longacting pegylated version, has been widely used to treat HbeAgpositive Hbv infections 158 .

IFNα2 has also been used in the HbeAgnegative disease that develops when viral mutations permit viral reactivation following HbeAg loss. IFNs reduce viraemia (usually by over 90%) and induce host responses, but drug withdrawal often leads to disease recurrence; how ever, a proportion of patients (approximately 10-15%) have a prolonged period of viral suppression 159 . Therapy for chronic Hbv infection illustrates the two comple mentary activities of IFNs: in HbeAgpositive disease IFN increases an immune response, whereas in HbeAg negative disease IFNs act as direct antivirals.

In the late 1980s 'nonA, nonb hepatitis' or 'post transfusion hepatitis' was effectively treated with IFNs 160 . Subsequent studies identified the causative agent as HCv 161 . Initial clinical studies of IFNα2 resulted in sustained and curative virological responses in up to 20% of patients 162 . Response rates to monotherapy with IFNα2 for chronic HCv infection were transformed in the mid1990s by combined use with the weak antiviral ribavirin -over 40% of patients responded 163 . These results mimic studies for HSv keratitis in which therapy with a combination of IFNs and a weak antiviral agent (acyclovir) were synergistic 164 . Therapy for chronic HCv infection has now evolved and current regimes com monly use a longacting pegylated IFNα2 plus ribavirin with cure of up to 60% of patients. Possibly as a result of selection against RNASeL cleavage sites in its genome, or inhibition of PKR, effectiveness is much less for geno type 1 than for genotypes 2 and 3 of HCv 162, 271, 272 .

In addition to Hbv and HCv infections, other chronic viral infections have been effectively treated. both sys temic and topical IFNαs and IFNβ have reduced virus titres and decreased clinical manifestations of herpes zoster, HSv and cytomegalovirus infections [165] [166] [167] [168] [169] . Almost simultaneous introduction of acyclovir and its analogues, however, which proved to have greater clinical efficacy and reduced side effects, ended clinical develop ment of IFNs for these indications. Papilloma virus infec tions of skin, larynx and genitals were found to respond with regression of warts upon either intralesional or systemic administration of IFNαs and IFNβ [170] [171] [172] [173] [174] [175] . when compared with placebo, useful therapeutic effects resulted for patients with extensive or refractory disease, but permanent eradication was infrequent. These studies did, however, establish a basis for the use of the TLR7 IFNinducing agonist imiquimod topically for genital warts with decreases in HPv DNA and with complete [176] [177] [178] . In studies of HIv, virus recovery and clinical manifestations of both early and late stages were reduced 179 . However, effectiveness of azidothymidine or protease inhibitors was not enhanced when IFNs were used in combination 180 .

once adequate quantities of human IFNs became avail able through recombinant DNA production, prophylaxis and treatment of acute respiratory virus infections were assessed. Reduction in virus yield, infection frequency and symptom scores resulted from administration of intranasal IFNs in experimental challenge infections with rhinoviruses, influenza viruses and coronaviruses [181] [182] [183] [184] . Prophylactic efficacy for natural rhinovirus colds in fam ily and work settings was also identified [185] [186] [187] . However, as a result of the nasal erosions and bleeding resulting from mucosal irritation by IFNα2 and IFNβ, symptom scores under field conditions were significantly higher in treated patients when compared with placebo. Case based use in the setting of the severe acute respiratory syndrome (SARS) epidemic suggested clinical effective ness of the consensus sequence type I IFN against the coronavirus aetiological agent 188 . These findings and the preclinical and clinical antiviral effectiveness with topical and/or highdose administration suggest that IFNs might have prophylactic or therapeutic effectiveness in SARS or in an influenza or other virus pandemic.

In about 85% of patients with MS, an inflammatory demyelinating disorder of the cen tral nervous system, disease begins with approximately annual episodes of transient neurological dysfunction (relapsing-remitting MS or RRMS). Initial studies of IFNs in the 1970s followed tissueculture studies sug gesting that cells from MS patients secreted less IFN like activity following viral induction than did controls. These findings, combined with a notion that a slow or chronic viral infection might be causative, resulted in the evaluation of using an intrathecal, impure IFNβ as therapy that identified a reduction in relapses 189 ; however, subsequent clinical trials were either inconclusive (IFNα2) or detrimental (IFNγ) [190] [191] [192] . but in 1993, recombinant IFNβ given subcutane ously in a randomized placebocontrolled trial for RRMS reduced relapses by about a third and resulted in marked reductions in subclinical disease, as assessed by magnetic resonance imaging (MRI) 193, 194 . This report ushered in the modern age of MS therapeutics: by show ing that the natural history of MS could be modified; by documenting that IFNβ was clinically beneficial; and by the demonstration of MRI lesions as a useful surrogate of clinical effectiveness, now widely used in MS drug development. It is now common clinical practice to initiate IFNβ (betaferon/betaserom, bayer Schering/Chiron; Avonex, biogen Idec; Rebif, Merck Serono (or medications of comparable efficacy)) at the time of diagnosis 195 . Attacks decrease by about 30%, numbers of new and active MRI lesions (which reflect inflammation) are often reduced as soon as 1month after initiation, and longterm clinical benefits are now considered plausible 196 . Although it has unequivocally represented a breakthrough, improvements on IFNβ are needed as it is only partially effective and is expen sive for the lifelong, noncurative use 195, 197 .

Pathogenesis of MS remains unknown but evi dence implicates genetic-environmental interactions with critical timing of exposures to initiating factors. epidemiological studies highlight epstein-barr virus and low plasma levels of vitamin D, and genetic studies implicate several polymorphic variants of immune response genes [198] [199] [200] [201] . The most obvious clinical outcome from IFNβ is a reduction in MRI lesions [202] [203] [204] , and pro tein products of ISGs probably mediate these effects.

As one example, an IFNβ ISG product, CD69, forms an inhibitory association with a sphingosine 1phosphate receptor (S1PR). The consequence in vivo is suppressed lymphocyte exit from lymph nodes and restriction in numbers of circulating lymphocytes available to cross the blood-brain barrier 205 . Reduction in expression of matrix metalloproteinase 9 (MMP9) in activated lymphocytes and increased soluble vascular cell adhesion molecule (svCAM) levels in plasma have also been identified and assigned putative roles in the beneficial effects of IFNβ for patients with MS 202, 206, 207 . expressionarray studies and candidate gene evaluations have been applied, without 

Regions of abnormal signals in the brain or spinal cord, detected by magnetic resonance imaging (MRI), and indicative of tissue changes related to the multiple sclerosis pathogenic process. Depending on the imaging technique, these MRI changes can reflect inflammation, demyelination, axonal destruction or scarring.

A group of compounds that belongs to the corticosteroid family. These compounds can either be naturally produced (hormones) or synthetic. They affect metabolism and have antiinflammatory and immunosuppressive effects. Many synthetic glucocorticoids (for example, dexamethasone) are used in clinical medicine as antiinflammatory drugs.

success, in attempts to identify molecular biomarkers of the therapeutic effect of IFNβ in MS 208 . Development of validated outcome measures for treatment success (and failure) will aid in this process 209 .

Cancer. based on the reduction in disease morbidities 210, 211 , initial regulatory approvals for the marketing of IFNα2 for a chronic b (hairy) cell leukaemia occurred within 5 years from clinical introduction as a result of close collaboration between academic institutions, govern ment and industry. In hairy cell leukaemia and chronic myelogenous leukemia (CML), IFNα2 decreased mar row infiltration with malignant cells and normalized peripheral haematological parameters 210, [212] [213] [214] . In CML, in addition to reductions in leukaemic cell mass, a decrease resulted in cells with the abnormal, activated bCR-AbL kinase [212] [213] [214] . over 90% of patients with CML with complete cytogenetic response were in remission at 10 years 213 . However, the survival advantage for IFNα2, when compared with chemotherapy for CML, has now been exceeded by the even greater effectiveness of the targeted inhibitor of the activated bCR-AbL kinase, such as imatinib (Gleevec; Novartis) and now other newer tyrosine kinase inhibitors.

In addition to hairy cell leukaemia and CML, thera peutic effectiveness of IFNα2 in causing at least partial disease regression has been identified in more than a dozen other malignancies including myeloma, lym phomas, melanoma, renal cell and bladder carcinoma, and Kaposi's sarcoma 215 . For example, in lymphomas of various histologies and of both bcell and Tcell pheno types, IFNα2 has been effective in inducing tumour regressions in almost half of the patients involved in the study, and even in patients previously treated with chemotherapy 215, 216 . Prolonged diseasefree and overall survival in intermediate prognosis lymphomas has resulted from IFNα2 in combination with chemo therapy, even given for limited periods, in randomized multicenter trials 215, 216 . International Phase III trials have been conducted with survival impact confirmed in meta static renal carcinoma, but like in CML, the orally active, targeted tyrosine kinase inhibitors have changed the natural history of renal carcinoma, extending survival in metastatic disease more than the injectable IFNα2.

Cure of metastatic malignancies can result when micrometastases are eliminated in patients at highest risk for recurrence after surgical removal of a primary tumour. effectiveness as a surgical adjuvant for murine tumours provided the rationale leading to pioneering clinical studies that suggested benefit of impure IFNα when given after surgery for osteosarcoma 217, 218 . This surgical adjuvant approach was the basis for evalua tion of IFNα2 in patients at high risk for recurrence of melanoma. Initial beneficial effects of significant pro longation of diseasefree survival have now largely been validated by combined analyses of multiinstitutional trials, by subsequent studies that have included evalua tion of pegylated IFNα2 and by metaanalyses [219] [220] [221] .

Like other potent physiological mediators such as glucocorticoids, IFNs have toxicities when administered with pharmacological intent 222, 223 . These have been dose related and particularly difficult at the high dose used for melanoma. with the initial dose, malaise, fever and chills, which last for a few hours, dominate but tachy phylaxis occurs with subsequent injections. Fatigue and anorexia, the aetiology of which is not understood, are often doselimiting with chronic administration for can cer or MS; at higher doses weight loss occurs and may be significant (>10%). Reversible elevation of hepatic transaminases may occur, as may haematological effects, most markedly granulocytopaenia.

Like in MS, failure to fully understand the mecha nism(s) of antitumour action has slowed further devel opment. Suppression, mutation and polymorphisms of IFNs and their signalling mechanisms in and by malig nant cells are emerging as important contributors to can cer development [224] [225] [226] [227] [228] [229] [230] [231] [232] [233] [234] . Mutations in RNASEL have been associated with prostate carcinoma and with presence of the retrovirus XMRv 235,273-275 . epigenetic and genetic silencing of IFNsignalling or ISG expression may also influence tumour development 236 . Reversal of these effects are likely to be the basis for effectiveness of IFNs and/or inducers in murine carcinogeninduced tumours, and may contribute to effectiveness in advanced disease, and provides a rational for developing TLR agonists for Box 1 | Important research questions for the future • What specific roles do the multiple isoforms of interferons (IFNs) play in host defences?

• What new roles will be identified for the IFN system and their protein products in the crosstalk between innate and cellular immunity?

• How do cellular gene products avoid activating Toll-like receptor (TLR) pathways while microbial gene products activate them very efficiently?

• What are cell type differences in other alternative signalling pathways interacting with JAK/STAT?

• What are cell and tissue specific differences in molecular responses?

• How do responses differ in cells of different maturities (for example, stem cells, senescent cells)?

• What are the functions of the still many uncharacterized IFN-stimulated genes (ISGs)?

• Which of the ISGs are most important for the specific cellular and clinical effects?

• Will ways of activating the IFN system prove therapeutically useful for the newly human papilloma virus (HPV)-associated squamous neoplasms of upper airways?

• What mechanisms cause the difficult spectrum of clinical side effects of fatigue and anorexia?

• Do IFNs play a bystander or pathogenic role in lupus erythematosis, rheumatoid arthritis or aplastic anaemia?

• Do low or high-affinity antibodies to IFNs or their protein products influence innate immunity?

• What are the molecular causes of resistance to IFNs in viral diseases? Multiple sclerosis? Cancer?

• Will helicases or proteases augment activity of IFNs for hepatitis B virus infections?

• Will anti-angiogenic or promoter methylation inhibitors increase antitumour activity?

• Can novel receptor agonists and antagonists be engineered for specific functions?

• Can a physicochemical rather than biological standard be developed for scientific and regulatory comparison?

• Will small-molecule activators of IFN-inducible proteins have antiviral and/or antitumour effects?

• How effective will components of the IFN system prove as immunological adjuvants?

• Can effective oral systemic inducers be identified? f o c u s o n a n t i v i r a l s chemoprevention [237] [238] [239] . Indeed, TLR agonists appear to be effective and are already establishing a role in treat ment of malignancy with the proven effectiveness of the TLR7 agonist imiquimod used topically for basal cell carcinomas as an example. Furthermore, relative clinical safety has been established for phosphorthioate oligoribonucelotide agonists for TLR9. Induction of apoptosis by the ISG products APo2L/ TRAIL and Fas has been identified in many malignant cell types, as has induction of APo2L/TRAIL on immune effector cell surfaces, thus sensitizing tumour cells to Tcell, NK cell and macrophagemediated cytotoxicity [240] [241] [242] [243] (TABLe 1) . Intralesional administration of IFNα into basal cell carcinomas increased Fas expression and correlated with regression 244 . IFNγ has increased susceptibility to apoptosis by Fas activators and cytotoxic chemotherapies in many cell types including melanoma and colorectal carcinoma 245, 246 . Through interactions with p53 and the inhibitor of apoptosis, XIAP, the ISG product XAF1 may allow APo2L/TRAIL to fully activate downstream caspases 247, 248 . In addition, the ISG product IRF1 can suppress another antiapoptotic protein, Survivin 249 .

Antitumour activity in vivo may also be mediated by augmented lytic activity of immune effector cells and by enhanced immunogenicity of tumour cells. both Tcell and NKcell trafficking, expansion and lytic activity can be promoted by IFNs and ISGs; furthermore, IFNγ is secreted from these activated cells into the tumour micro environment [250] [251] [252] [253] [254] [255] . In addition to stimulating immune effector cells, IFNs have critical roles in antigen process ing and presentation, as discussed above, both by T cells and dendritic cells 256 . In addition, IFNγ can upregulate the tumourassociated antigens, carcinoembryonic anti gen and TAG72, both in vitro and in vivo 257 .

IFNs can also inhibit angiogenesis by altering the stimuli from tumour cells and by directly inhibiting endothelial cells -indeed, they were the first angiogenic inhibitor identified 258 . endothelial cells are inhibited in motility 259 , undergo coagulation necrosis in vitro and inhi bition of angiogenesis occurs in vivo within 24 hours of tumour cell inoculation 260, 261 . Suppression of basic fibrob last growth factor (bFGF; also known as FGF2) correlated with reduced vascularization and tumour growth 262, 263 . IFNs also inhibit vascular endothelial growth factor (veGF) mRNA and protein expression by regulating its promoter 264 . IL8, a mediator of angiogenesis, was inhibited in vitro and in vivo by IFNα2b and IFNβ; other angiogenesis inhibitory members of the chemokine family, CXCL9, CXCL10 and CXCL11, are ISGs 265-267 .

In endothelial cells, the ISG product guanylate bind ing protein 1, interferoninducible, 67 kDa (GbP1), functioned as an inflammatory response factor inhib iting endothelial cell proliferation and angiogenesis in part through MMPs 268 . Clinically, IFNα2 has proved effective in the treatment of infantile haemangiomas, haemangioblastomas, giant cell tumour of the mandible and Kaposi's sarcoma 215, 269 . Thus, induction of ISGs that function as angiostatic inhibitors, coupled with second ary downregulation of angiogenic factors, may contribute to antitumour effects 269 .

IFNs provide fundamental cellular defence mechanisms against viral infections and cancer and are thus critically important to the health of animals and humans. because of their clinical effectiveness in limiting virus replication, reducing tumour cell mass, controlling disease symptoms and prolonging survival, IFNs are now licensed world wide for the treatment of various viral, malignant and immune disorders; market sales approach uS$4 billion. As part of the innate immune response, IFNs are not only a principal cytokine that blocks viral replication through the action of specific ISGs, but also (particularly IFNγ) mediate critical elements of the cellular immune response for recurring bacterial infections in chronic granuloma tous disease and for mycobacteria. because all biological effects of IFNs are mediated through the action of ISGs, understanding the functions of these genes may lead to more efficacious anticancer and antiviral therapeutics. For example, certain IFNregulated proteins, such as oAS, RNASeL and PKR, exist in either latent inactive or active states, which could be targeted for potent anti tumour and/or antiviral effects (fIG. 5) .

IFNs have therefore more than reached the effective ness anticipated by early virologists: they are not only an antiviral with a spectrum of clinical effectiveness against both RNA and DNA viruses, but have been the prototypical biological response modifiers for oncology, and have proved to have effectiveness in suppressing manifestations of MS. The study of IFNs has resulted in fundamental insights into cellular signalling mecha nisms and innate and acquired immunity. In addition, their therapeutic use has improved the quality and quan tity of life for millions of patients worldwide. However, to fully realize their potential, many questions remain unanswered (BOX 1). As exemplified by recent publica tions 276-278 , further investigations will only enable IFNs to have even greater impacts in biomedicine.

Isaacs 

Antiviral signaling through pattern recognition receptors

Cytoplasmic doublestranded DNA sensor

An atomic model of the interferon-β enhanceosome

Type I interferon gene induction by the interferon regulatory factor family of transcription factors

Multiple functions of the IKKrelated kinase IKKε in interferon-mediated antiviral immunity

Indentification of specific protein kinases, which are distantly related to the more well-known IKK kinases

Manipulation of the nuclear factor-κB pathway and the innate immune response by viruses

Control of antiviral defenses through hepatitis C virus disruption of retinoic acid-inducible gene-I signaling

Type 1 interferons and the virus-host relationship: a lesson in detente

Anti-immunology: evasion of the host immune system by bacterial and viral pathogens

RIG-I mediated antiviral responses to single-stranded RNA bearing 5′ phosphates

Herpes simplex virus encephalitis in human UNC-93B deficiency

A Toll-like receptor recognizes bacterial DNA

Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway

The chemotherapeutic agent DMXAA potently and specifically activates the TBK1-IRF-3 signaling axis

Targeting signal transduction as a strategy to treat inflammatory diseases

Therapeutic potential of Toll-like receptor 9 activation

Two antigenically distinct species of human interferon

At least three human type α-interferons: structure of α 2

Human leukocyte and fibroblast interferons are structurally related

The structure of eight distinct cloned human leukocyte interferon cDNAs

Interferons, interferon-like cytokines, and their receptors

Characterization of the type I interferon locus and identification of novel genes

Interferon-α and -β differentially regulate osteoclastogenesis: role of differential induction of chemokine CXCL11 expression

IFN-α subtypes differentially affect human T cell motility

Differential responses to IFN-α subtypes in human T cells and dendritic cells

The receptor of the type I interferon family

Modification of TLR-induced activation of human dendritic cells by type I IFN: synergistic interaction with TLR4 but not TLR3 agonists

New and atypical families of type I interferons in mammals: comparative functions, structures, and evolutionary relationships

Interferon-κ, a novel type I interferon expressed in human keratinocytes

Viral infection and Toll-like receptor agonists induce a differential expression of type I and λ interferons in human plasmacytoid and monocytederived dendritic cells

Elucidation of the basic threedimensional structure of type I interferons and its functional and evolutionary implications

Determination of the two-dimensional interaction rate constants of a cytokine receptor complex

Identification of the structural effects that lead to the differential activities of α and β IFNs. The bioactivity profile of a given type I IFN subtype is shown to be related to the stability of its complex with the receptor

Differential receptor subunit affinities of type I interferons govern differential signal activation

An interferon α2 mutant optimized by phage display for IFNAR1 binding confers specifically enhanced antitumor activities

Differential responsiveness to IFN-α and IFN-β of human mature DC through modulation of IFNAR expression

Class II cytokine receptor gene cluster is a major locus for hepatitis B persistence

The IFN γ receptor: a paradigm for cytokine receptor signaling

Full house: 12 receptors for 27 cytokines

Listeria monocytogenes and recurrent mycobacterial infections in a child with complete interferon-γ-receptor (IFNγR1) deficiency: mutational analysis and evaluation of therapeutic options

IL-28 and IL-29: newcomers to the interferon family

Identification of two distinct regulatory regions adjacent to the human β-interferon gene

Molecular cloning and sequence of partial cDNA for interferon-induced (2′-5′)oligo(A) synthetase mRNA from human cells

Transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells

α-Interferon-induced transcription of HLA and metallothionein genes containing homologous upstream sequences

The proteins of ISGF-3, the interferon α-induced transcriptional activator, define a gene family involved in signal transduction

How cells respond to interferons

This paper identifies a JAK family member that is necessary for the IFN response, thus showing how type I IFN stimulates the phosphorylation of tyrosine residues in the receptor and the STATs

Alternative activation of STAT1 and STAT3 in response to interferon-γ

Complex modulation of cell type-specific signaling in response to type I interferons

Cell typespecific signaling in response to interferon-γ

Human primary immunodeficiencies of type I interferons

Requirement of phosphoinositide 3-kinase and Akt for interferon-β-mediated induction of the β-R1 (SCYB11) gene

Alternative and accessory pathways in the regulation of IFN-β-mediated gene expression

Differential responses to IFN-α subtypes in human T cells and dendritic cells

Mechanisms of type-I-and type-IIinterferon-mediated signaling

The role of suppressors of cytokine signaling (SOCS) proteins in regulation of the immune response

Anticancer activity of sodium stibogluconate in synergy with IFNs

Role of STAT3 in type I interferon responses. Negative regulation of STAT1-dependent inflammatory gene activation

Sensitization of IFN-γ Jak-STAT signaling during macrophage activation

Modulation of STAT1 and STAT3 signaling in melanoma by high-dose IFN-α2b

Defective TNF-α-induced apoptosis in STAT1-null cells due to low constitutive levels of caspases

Novel roles of unphosphorylated STAT3 in oncogenesis and transcriptional regulation

Unphosphorylated STAT3 accumulates in response to IL-6 and activates transcription by binding to NFκB

Identification of genes differentially regulated by interferon α, β, or γ using oligonucleotide arrays

Functional classification of interferon-stimulated genes identified using microarrays

Viral encounters with OAS and RNase L during the interferon antiviral response

pppA2′p5′A2′p5′A: an inhibitor of protein synthesis synthesized with an enzyme fraction from interferon-treated cells

Expression cloning of 2-5A-dependent RNAase: a uniquely regulated mediator of interferon action

2-5A-dependent RNase molecules dimerize during activation by 2-5A

Interferon action -sequence specificity of the ppp(A2′p)nA-dependent ribonuclease

Interferon action: RNA cleavage pattern of a (2′-5′)oligoadenylatedependent endonuclease

Interferon action and apoptosis are defective in mice devoid of 2′, 5′-oligoadenylate-dependent RNase

The role of 2′-5′ oligoadenylateactivated ribonuclease L in apoptosis

A study of the interferon antiviral mechanism: apoptosis activation by the 2-5A system

Small self RNA generates by RNase L amplifies antiviral innate immunity

Small-molecule activators of RNase L with broad-spectrum antiviral activity

Interferon-mediated protein kinase and low-molecular-weight inhibitor of protein synthesis

Isolation of two interferon-induced translational inhibitors: a protein kinase and an oligo-isoadenylate synthetase

Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon

Signal integration via PKR

PKR; a sentinel kinase for cellular stress

PACT, a protein activator of the interferon-induced protein kinase, PKR

Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase

Novel functions of proteins encoded by viral stress-inducible genes

α Interferon induces distinct translational control programs to suppress hepatitis C virus RNA replication

Inheritance of resistance to influenza virus in mice

Interferon induces a unique protein in mouse cells bearing a gene for resistance to influenza virus

Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus

Interferon-induced Mx proteins in antiviral host defense

A human 15-kDa IFN-induced protein induces the secretion of IFN-γ

In vitro and in vivo secretion of human ISG15, an IFN-induced immunomodulatory cytokine

The interferon regulated ubiquitin-like protein, ISG15, in tumorigenesis: friend or foe?

Innate antiviral response targets HIV-1 release by the induction of ubiquitin-like protein ISG15

IFN-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and Sindbis viruses

Molecular cloning of human plasma membrane phospholipid scramblase. A protein mediating transbilayer movement of plasma membrane phospholipids

Phospholipid scramblase 1 is imported into the nucleus by a receptor-mediated pathway and interacts with DNA

Phospholipid scramblase 1 potentiates the antiviral activity of interferon

Type I interferons (IFNs) regulate tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression on human T cells: a novel mechanism for the antitumor effects of type I IFNs

Preferential induction of apoptosis by interferon (IFN)-β compared with IFN-α2: correlation with TRAIL/ Apo2L induction in melanoma cell lines

Apo2L/TRAIL and Bcl-2-related proteins regulate type I interferon-induced apoptosis in multiple myeloma

Characterization of a human gene inducible by α-and β-interferons and its expression in mouse cells

Small ISGs coming forward

G1P3, an interferon inducible gene 6-16, is expressed in gastric cancers and inhibits mitochondrial-mediated apoptosis in gastric cancer cell line TMK-1 cell

The interferon α induced protein ISG12 is localized to the nuclear membrane

Age-dependent resistance to lethal alphavirus encephalitis in mice: analysis of gene expression in the central nervous system and identification of a novel interferon-inducible protective gene, mouse ISG12

Interferon-induced guanylate binding protein-1 (GBP-1) mediates an antiviral effect against vesicular stomatitis virus and encephalomyocarditis virus

ISG20, a new interferon-induced RNase specific for single-stranded RNA, defines an alternative antiviral pathway against RNA genomic viruses

PML mediates the interferon-induced antiviral state against a complex retrovirus via its association with the viral transactivator

Functionally distinct double-stranded RNA-binding domains associated with alternative splice site variants of the interferon-inducible double-stranded RNA-specific adenosine deaminase

Viperin (cig5), an IFNinducible antiviral protein directly induced by human cytomegalovirus

Inhibition of vaccinia virus DNA replication by inducible expression of nitric oxide synthase

Role of nucleoporin induction in releasing an mRNA nuclear export block

Samuel CE Antiviral actions of interferons

The nature of the principal type 1 interferon-producing cells in human blood

Stat2-dependent regulation of MHC class II expression

Intracellular surveillance: controlling the assembly of MHC class I-peptide complexes

A review of the roles of constitutive and IFNinducible components in MHC class I-peptide complex assembly

Expression of MHC II genes

Antigen presentation by MHC class I and its regulation by interferon γ

γ-Interferon and expression of MHC genes regulate peptide hydrolysis by proteasomes

The ins and outs of intracellular peptides and antigen presentation by MHC class I molecules

Interferon-inducible T cell alpha chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3

A macrophage mRNA selectively induced by γ-interferon encodes a member of the platelet factor 4 family of cytokines

Characterization of β-R1, a gene that is selectively induced by interferon β (IFN-β) compared with IFN-α

γ-Interferon transcriptionally regulates an earlyresponse gene containing homology to platelet proteins

Reciprocal regulation between natural killer cells and autoreactive T cells

The class II transactivator CIITA is a transcriptional integrator

Specificity and expression of CIITA, the master regulator of MHC class II genes

Epigenetic regulation of MHC-II and CIITA genes

A review of the regulation of MHC class II genes, the IFN-inducible factor CIITA, chromatin modification and constitutive transcription factors

HLA-DM interactions with intermediates in HLA-DR maturation and a role for HLA-DM in stabilizing empty HLA-DR molecules

Assembly and intracellular transport of HLA-DM and correction of the class II antigenprocessing defect in T2 cells

The structure and function of the novel MHC class II molecule, HLA-DM

HLA-DM acts as a molecular chaperone and rescues empty HLA-DR molecules at lysosomal pH

An essential role for HLA-DM in antigen presentation by class II major histocompatibility molecules

HLA-DM -an endosomal and lysosomal chaperone for the immune system

Similarities of murine γ interferon and the lymphokine that renders macrophages cytotoxic

Australia antigen and the biology of hepatitis B

Effect of human leukocyte interferon on hepatitis B virus infection in patients with chronic active hepatitis

Fibroblast interferon in HBsAgpositive chronic active hepatitis

Randomized, controlled trial of recombinant human α-interferon in patients with chronic hepatitis B

Role of hepatitis B virus specific cytotoxic T cells in liver damage and viral control

Mutation preventing formation of hepatitis B e antigen in patients with chronic HBV infection

Peginterferon Alfa-2a, lamivudine, and the combination for HBeAg-positive chronic hepatitis B

Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-negative chronic hepatitis B

Treatment of chronic nonA nonB hepatitis with recombinant human α interferon: a preliminary report

Isolation of a cDNA clone derived from a blood borne non-A, non-B viral hepatitis clone

Treatment of hepatitis C

Past, present, and future hepatitis C treatments

Combination therapy for dendritic keratitis with acyclovir and α-interferon

Topical therapy of ulcerative herpetic keratitis with human interferon

Human leukocyte interferon for the treatment of herpes zoster in patients with cancer

Prevention of reactivated herpes simplex infection by human leukocyte interferon after operation on the trigeminal root

Recombinant interferon α-2a for treatment of herpes zoster in immunosuppressed patients with cancer

Double-blind, placebo-controlled trial of human lymphoblastoid interferon prophylaxis of cytomegalovirus infection in renal transplant recipients

Interferon therapy in juvenile laryngeal papillomatosis

Effects of interferon-α on human warts

Interferon therapy for condylomata acuminata

Demonstrates clinical effect for these HPV-related neoplasms, with implications for treatment of HPV-related upper respiratory malignancies

Interferons in the treatment of genital human papillomavirus infections

Use of interferon-α in laryngeal papillomatosis: eight years of the Cuban national programme

Imiquimod, a patient-applied immune-response modifier for treatment of external genital warts

A randomized, controlled, molecular study of condylomata acuminata clearance during treatment with imiquimod

Imiquimod for the treatment of genital warts: a quantitative systematic review

Interferon-α in patients with asymptomatic human immunodeficiency virus (HIV) infection. A randomized, placebo-controlled trial

Phase II, randomized, open-label, community-based trial to compare the safety and activity of combination therapy with recombinant interferon-α2b and zidovudine versus zidovudine alone in patients with asymptomatic to mildly symptomatic HIV infection. HIV Protocol C91-253 Study Team

Inhibition of respiratory virus infection by locally applied interferon

Prevention of rhinovirus colds by human interferon α-2 from Escherichia coli

Intranasal interferon as protection against experimental respiratory coronavirus infection in volunteers

Intranasally administered interferon as prophylaxis against experimentally induced influenza A virus infection in humans

Intranasal recombinant α-2b interferon treatment of naturally occurring common colds

Prophylactic efficacy of intranasal α 2-interferon against rhinovirus infections in the family setting

Intranasal interferon-α 2 for prevention of natural rhinovirus colds

Interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study

A study using intrathecal natural IFNβ, showing a potent reduction in relapse rate

Systemic α-interferon therapy of multiple sclerosis

Exacerbations of multiple sclerosis in patients treated with γ interferon

Treatment of multiple sclerosis with γ interferon: exacerbations associated with activation of the immune system

Interferon β-1b is effective in relapsing-remitting multiple sclerosis. I. Clinical results of a multicenter, randomized, double-blind, placebo-controlled trial

Interferon β-1b is effective in relapsing-remitting multiple sclerosis. II. MRI analysis results of a multicenter, randomized, double-blind, placebo-controlled trial. UBC MS/MRI Study Group and the IFNB Multiple Sclerosis Study Group

Interferon-β treatment for multiple sclerosis

Interferon-β for multiple sclerosis: long-term benefits?

This review describes studies that, after almost 15 years of use, strongly suggested that IFNβ treatment might be efficacious in the way that investigators had hoped: to delay or preclude the onset of progressive MS

Recombinant therapeutics: from bench to bedside (if your health plan concurs)

Environmental risk factors for multiple sclerosis. Part II: noninfectious factors

Environmental risk factors for multiple sclerosis. Part I: the role of infection

A predominant role for the HLA class II region in the association of the MHC region with multiple sclerosis

This report established that the MHC association, by a wide margin the strongest genetic susceptibility trait, resided in the HLA class II region

Old suspects found guilty -the first genome profile of multiple sclerosis

Increases in soluble VCAM-1 correlate with a decrease in MRI lesions in multiple sclerosis treated with interferon β-1b

Serial contrast-enhanced magnetic resonance imaging in patients with early relapsing-remitting multiple sclerosis: implications for treatment trials

The effect of interferon-β on blood-brain barrier disruptions demonstrated by contrast-enhanced magnetic resonance imaging in relapsing-remitting multiple sclerosis

CD69 acts downstream of interferon-α/β to inhibit S1P1 and lymphocyte egress from lymphoid organs

Interferon β-1b decreases the migration of T lymphocytes in vitro: effects on matrix metalloproteinase-9

Interferon β-1b inhibits gelatinase secretion and in vitro migration of human T cells: a possible mechanism for treatment efficacy in multiple sclerosis

Genome-wide network analysis reveals the global properties of IFN-β immediate transcriptional effects in humans

Defining interferon β response status in multiple sclerosis patients

Cytokine therapeutics: lessons from interferon α

A review of how IFNs were developed and their importance as a prototype for other biologicals as antitumour therapeutics

Hairy cell leukemia: review of treatment

Long-term follow-up of the Italian trial of interferon-α versus conventional chemotherapy in chronic myeloid leukemia

Interferon-α-based treatment of chronic myeloid leukemia and implications of signal transduction inhibition

Chronic myeloid leukemia and interferon-α: a study of complete cytogenetic responders

An expanse on the clinical effectiveness of IFNs in both haematological malignancies and solid tumours

Meta-analysis to evaluate the role of interferon in follicular lymphoma

Anti-tumor effects of interferon in mice injected with interferonsensitive and interferon-resistant Friend leukemia cells. VI. Adjuvant therapy after surgery in the inhibition of liver and spleen metastases

Interferon-α as the only adjuvant treatment in high-grade osteosarcoma: long term results of the Karolinska Hospital series

A pooled analysis of Eastern Cooperative Oncology Group and intergroup trials of adjuvant high-dose interferon for melanoma

Does adjuvant interferon-α for high-risk melanoma provide a worthwhile benefit? A meta-analysis of the randomised trials

Randomized adjuvant therapy trials in melanoma: surgical and systemic trials

Safety profile of interferon-α therapy

Mechanisms and management of toxicities associated with high-dose interferon α-2b therapy

Deletions of interferon genes in acute lymphoblastic leukemia

Homozygous deletions within human chromosome band 9p21 in melanoma

Homozygous loss of the interferon genes defines the critical region on 9p that is deleted in lung cancers

Cloning a novel member of the human interferon-inducible gene family associated with control of tumorigenicity in a model of human melanoma

Interferon-inducible protein IFIXα1 functions as a negative regulator of HDM2

Down-regulation of the transporter for antigen presentation, proteasome subunits, and class I major histocompatibility complex in tumor cell lines

Expression profiling of a human cell line model of prostatic cancer reveals a direct involvement of interferon signaling in prostate tumor progression

Characterization of human lymphocyte antigen class I antigen-processing machinery defects in renal cell carcinoma lesions with special emphasis on transporter-associated with antigen-processing down-regulation

Points to the potential role of suppression of constitutive of ISG expression, possibly by promoter methylation in the evolution of cell transformation

Microarray analyses uncover UBE1L as a candidate target gene for lung cancer chemoprevention

STAT3 polymorphism predicts interferon-α response in patients with metastatic renal cell carcinoma

RNASEL Arg462Gln variant is implicated in up to 13% of prostate cancer cases

Augmentation of effects of interferonstimulated genes by reversal of epigenetic silencing: potential application to melanoma

IFN-α prevents the growth of preneoplastic lesions and inhibits the development of hepatocellular carcinoma in the rat

Protection from carcinogen-induced murine bladder carcinoma by interferons and an oral interferon-inducing pyrimidinone, bropirimine

A preclinical example of the potential for IFNs or inducers to treat high-risk individuals

Can therapy of hepatitis C affect the development of hepatocellular carcinoma?

Apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand cooperates with anticancer drugs to overcome chemoresistance in antiapoptotic Bcl-2 family members expressing jurkat cells

Antiviral response by natural killer cells through TRAIL gene induction by IFN-α/β

Virus or TLR agonists induce TRAILmediated cytotoxic activity of plasmacytoid dendritic cells

Regression of basal cell carcinoma by intralesional interferon-α treatment is mediated by CD95 (Apo-1/Fas)-CD95ligand-induced suicide

Heterogenous susceptibility to CD95-induced apoptosis in melanoma cells correlates with Bcl-2 and Bcl-x expression and is sensitive to modulation by interferon-γ

Modulation of the Fas signaling pathway by IFN-γ in therapy of colon cancer: Phase I trial and correlative studies of IFN-γ, 5-fluorouracil, and leukovorin

Identification of X-linked inhibitor of apoptosis-associated factor-1 (XAF1) as an interferon-stimulated gene that augments TRAIL/ Apo2L-induced apoptosis

Promoter CpG hypermethylation and downregulation of XAF1 expression in human urogenital malignancies: implication for attenuated p53 response to apoptotic stresses

Ectopic expression of interferon regulatory factor-1 promotes human breast cancer cell death and results in reduced expression of survivin

Biological properties of recombinant α-interferons: 40th anniversary of the discovery of interferons

Immuneinflammatory mechanisms in IFNγ-mediated antitumor activity

A review of the immunoregulatory effects of IFNs

Recent developments in the transcriptional regulation of cytolytic effector cells

STAT1 in peripheral tissue differentially regulates homing of antigen-specific Th1 and Th2 cells

Another excellent and complimentary review of the immunoregulatory effects of IFNs

Type I IFN contributes to NK cell homeostasis, activation, and antitumor functions

Interferon-γ, the functional plasticity of the ubiquitin-proteasome system, and MHC class I antigen processing

Intraperitoneal administration of interferon-γ to carcinoma patients enhances expression of tumor-associated glycoprotein-72 and carcinoembryonic antigen on malignant ascites cells

Angiogenesis: an organizing principle for drug discovery

Inhibition of cell motility by interferons

Microvascular injury in pathogenesis of interferon-induced necrosis of subcutaneous tumors in mice

Inhibition of angiogenesis by interferons: effects on tumor-and lymphocyte-induced vascular responses

Inhibition of basic fibroblast growth factor expression, angiogenesis, and growth of human bladder carcinoma in mice by systemic interferon-α administration

Evidence for the causal role of endogenous interferon-α/β in the regulation of angiogenesis, tumorigenicity, and metastasis of cutaneous neoplasms

The authors are indebted to Sandya Rani and Kristin Kraus for careful reading of parts of the manuscript. This work was supported in part by grants to the authors from NIH R01 CA90914, CA115,494, CA044059, CA103943, CA089132, CA62220, CA68782, M01 RR018390, NS32151, NS38667, ARC 3158.

A review emphasizing the potential role of antiangiogenic effects of IFNs. 264