direct effects of type i interferons on cells of immune system (stubbs et al. 2011)

Molecular Pathways

Direct Effects of Type I Interferons on Cells of the Immune System

Sandra Hervas-Stubbs1, Jose Luis Perez-Gracia2, Ana Rouzaut3, Miguel F. Sanmamed1,2,Agnes Le Bon4,5, and Ignacio Melero1,2

AbstractType I interferons (IFN-I) are well-known inducers of tumor cell apoptosis and antiangiogenesis via

signaling through a common receptor interferon alpha receptor (IFNAR). IFNAR induces the Janus

activated kinasesignal transducer and activation of transcription (JAK-STAT) pathway in most cells,

along with other biochemical pathways that may differentially operate, depending on the responding cell

subset, and jointly control a large collection of genes. IFNs-I were found to systemically activate natural

killer (NK) cell activity. Recently, mouse experiments have shown that IFNs-I directly activate other cells of

the immune system, such as antigen-presenting dendritic cells (DC) and CD4 and CD8 T cells. Signaling

through the IFNAR in T cells is critical for the acquisition of effector functions. Cross-talk between IFNAR

and the pathways turned on by other surface lymphocyte receptors has been described. Importantly, IFNs-I

also increase antigen presentation of the tumor cells to be recognized by T lymphocytes. These IFN-driven

immunostimulatory pathways offer opportunities to devise combinatorial immunotherapy strategies. Clin

Cancer Res; 17(9); 261927. 2011 AACR.

Background

Types of interferonInterferons (IFN) were discovered in 1957 by Isaacs and

Lindenmann as soluble proteins able to inhibit virus repli-cation in cell cultures (1). There are 3 types of IFNs: type I(IFN-I), type II (IFN-II), and type III (IFN-III; refs. 2, 3). IFNsbelonging to all IFN classes are very important for fightingviral infection. In humans, IFNs-I include IFN-a subtypes,IFN-b, IFN-e, IFN-k, and IFN-w (2). From an immunologicperspective, IFN-a and IFN-b are themain IFN-I subtypes ofinterest. Both are produced by almost every cell of the bodyin response to viral infection and share important antiviralproperties. The IFN-a family is amultigenic group of highlyhomologous polypeptides encoded by more than 13intronless genes in humans. IFN-b, in contrast, exists as asingle gene in most species. IFN-g is the only IFN-II. Thiscytokine does not have marked structural homology withIFNs-I and binds a different cell-surface receptor (IFNGR),also composed of 2 different subunits (IFNGR1 andIFNGR2; ref. 2). The recently classified IFN-III consists of

3 IFN-l molecules (IFN-l1, IFN-l2, and IFN-l3) and sig-

nals through a receptor complex consisting of interleukin-10 receptor 2 subunit (IL-10R2) and IL-28 receptor alphasubunit (IL-28Ra; ref. 3). It is well established that IFNsinduce the expression of hundreds of genes, which mediatevarious biological responses. Some of these genes are regu-lated by the 3 classes of IFNs, whereas others are selectivelyregulated by distinct IFNs.Although the role of IFN-II and IFN-III is very important

in immune responses, IFN-II has not yet shown any clinicalactivity for human cancer treatment (4), and IFN-III is notcurrently developed for any indication (3). However, sincethe discovery of IFNs, the use of IFN-I in therapy has beenvery widespread. In fact, systemic injections of IFN-I areapproved for the treatment of a variety of diseases, whichinclude solid and hematologic malignancies, multiplesclerosis, and, above all, chronic viral hepatitis. In thisreview, we focus on the role of IFN-I as a direct immunos-timulatory agent directly modifying functions of immunesystem cells and how these functions can be applied tobetter therapeutic strategies.

IFN-I signaling pathwaysAll types of IFN-I signal through a unique heterodimeric

receptor, interferon alpha receptor (IFNAR), composed of 2subunits, IFNAR1 and IFNAR2, which are expressed inmost tissues (5). IFNAR1 knockout mice have not onlyconfirmed the role this subunit plays in mediating thebiological response to IFNs-I, but have also establishedthe pleiotropic role that these IFNs play in regulating thehost response to viral infections and in adaptive immunity(68).Receptor occupancy rapidly triggers several signaling

cascades (Fig. 1), which culminate in the transcriptionalregulation of hundreds of IFN-stimulated genes (ISG). The

Authors' Affiliation: 1Division of Gene Therapy andHepatology, Centre forApplied Medical Research (CIMA), University of Navarra, Pamplona,2Medical Oncology Department and Cell Therapy Unit, University Clinic,University of Navarra, and 3Division of Oncology, CIMA, University ofNavarra, Pamplona, Spain; 4Institut Cochin, Universite Paris Descartes,CNRS (UMR 8104), and 5INSERM, U1016, Paris, France

Corresponding Author: Ignacio Melero, CIMA and Facultad de MedicinaUniversidad de Navarra, Av. Pio XII, 57, 31008 Pamplona, Spain. Phone:34-948194700; Fax: 34-948194717; E-mail: [email protected] or [email protected]

doi: 10.1158/1078-0432.CCR-10-1114

2011 American Association for Cancer Research.

ClinicalCancer

Research

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first signaling pathway shown to be activated by IFNs-I wasthe Janus activated kinasesignal transducer and activationof transcription (JAK-STAT) pathway (Fig. 1A; ref. 9), whichis active in almost all cell types. IFNAR1 is constitutivelyassociated with tyrosine kinase 2 (TYK2), whereas IFNAR2is associated with JAK1. Receptor binding results in trans-phosphorylation of JAK1 and TYK2. The activated JAKsthen phosphorylate conserved tyrosine residues in thecytoplasmic tails of the IFNAR. These phosphotyrosyl resi-dues subsequently serve as docking sites for the recruitmentof src-homology 2 (SH2)containing signaling molecules,such as STAT1 and STAT2. Once at the receptor, the STATproteins themselves become JAK substrates. STAT1 and

STAT2 are both phosphorylated on a single tyrosine(Y701 for STAT1 and Y690 for STAT2). Once phosphory-lated, STATs dimerize and move to the nucleus, where theyassociate with the IFN-regulatory factor 9 (IRF9) to formthe ISG factor 3 (ISGF3) transcription factor complex,which binds to IFN-stimulated response elements.Although STAT1 and STAT2 are the most important

mediators of the response to IFNs-I, other STATs, namelySTAT3 and STAT5, have been found to be phosphorylated(activated) by IFNs-I (Fig. 1A; refs. 1013). STAT4and STAT6 can also be activated by IFN-a; however, thisactivation seems to be restricted to certain cell types, suchas endothelial or lymphoid cells (1417). Following

2011 American Association for Cancer Research

Tyk2

IFN-/(A) JAK-STAT

STAT

STAT STAT

STAT

STAT3

STAT3

C3G

IRF9

IRF9

ISG15, IP-10,IRF-7, PKR,2-5-OAS,etc

IRF-1, IRF-2,IRF-8, IRF-9,etc

Regulation of expression of GTP-binding protein Ag processing/presentation proteinSurvival signals

Apoptosis Cellular growthDifferentiationSer phosphorylationof STAT1

c-MycSAP-1P53SP1SMADs

NFATEtsElk-1MEF2Stat1/3

JunElk-1NFATATF2

Ras

Raf1

MEK1/2

ERK1/2JNKp38

Rac1Rap1

ProliferationDifferentiationSurvival....

mTORInhibition ofGSK-3/CDKN1A/CDKN1B

PKCIKKIKK

NFB

MEF2MAPKAPRsk

FosElk-1Sap-1

ISG15Histone acetylationAntiviral effectGrowth inhibitory effectsHematopoietic suppressive signals

STAT1

STAT5

STAT5

CRKL

CRKL

CRKL

STAT2

STAT1

STAT2

VAV

JAK1 JAK1 JAK1 JAK1

VAV

VAV

STAT1, STAT3STAT4, STAT5STAT6

STAT1/2,3,4 or 5STAT2/3STAT5/6

STATSTAT

IFNAR

1IFN

AR2

IFN-/(B) CRK

IFN-/ IFN-/ IFN-/

Type I IFNs signaling pathways(C) PI3K and NFB

TCR complexCD45

CD4/8

(D) MAPK

IFNAR

1IFN

AR2

Homodimers

STAT STAT

STAT STAT

Homodimers

Heterodimers

Heterodimers

ISRE GAS GAS

Regulatedgenes orfunctions

NFB Rel ... ... ...

Tyk2

IFNAR

1IFN

AR2

IFNAR

1IFN

AR2

IFNAR

1IFN

AR2

Tyk2

NIK

Tyk2

RS1/2PI3K

AKT

Zap70

Lck

LA

TSLP76TRAFSTAT

5C3G

Figure 1. Signaling pathways activated by the IFN-I receptor. Schematic of signaling pathways downstream of the IFNAR, emphasizing cross-talk withdifferent signaling cascades, which depends on the identity and activation status of the cell responding to IFN-I. A, JAK-STAT signal pathway activated byIFNs-I. B, CRKL pathway. Upon JAK activation, CRKL associates with TYK2 and undergoes tyrosine phosphorylation. The activated form of CRKLforms a complex with STAT5, which also undergoes TYK2-dependent tyrosine phosphorylation. The CRKLSTAT5 complex translocates to the nucleusand binds specific GAS elements. C, PI3K and NF-kB pathways. Phosphorylation of TYK2 and JAK1 results in activation of PI3K and AKT, whichin turn mediates downstream activation of mTOR, inactivation of GSK-3, CDKN1A, and CDKN1B, and activation of IKKb, which results in activation ofNF-kB. IFNs-I also activate NF-kB by an alternative pathway that involves the linkage of TRAFs, which results in activation of NF-kB2. PI3K/AKT can alsoactivate NF-kB through an activation loop via PKCq. D, MAPK pathway. Activation of JAK results in tyrosine phosphorylation of Vav, which leads to theactivation of several MAPKs such as p38, JNK, and ERK1/2. The different MAPKs activate different sets of transcription factors, such as Fos, ELK1, Sap-1,MEF2, MAPKAP, and Rsk (activated by p38); Jun, ELK1, NFAT, and ATF2 (activated by JNK); andNFAT, ETS, ELK1,MEF2, STAT1/2, c-Myc, SAP-1, p53, SP1,and SMADs (activated by ERK1/2).

Hervas-Stubbs et al.

Clin Cancer Res; 17(9) May 1, 2011 Clinical Cancer Research2620

phosphorylation, activated STATs form homo- (STAT1,STAT3, STAT4, STAT5, and STAT6) or heterodimers(STAT1/2, STAT1/3, STAT1/4, STAT1/5, STAT2/3, andSTAT5/6; refs. 10, 11, 18, 19), which translocate to thenucleus and bind other regulatory sequences known asIFN-gactivated sites (GAS).Interestingly, distinct STATs have opposing biological

effects. Thus, STAT3 stimulates growth, whereas on thecontrary, STAT1 is a growth inhibitor. On the other hand,IFNs-Imediated activation of STAT4 is required for IFN-gproduction, whereas surprisingly, STAT1 negatively regu-lates the IFNs-Idependent induction of IFN-g (20). Therelative abundance of these dimeric transcription factors,which may vary substantially depending on cell type andactivation and/or differentiation state, is likely to have amajor impact on the overall response to IFNs-I (2123).Stimulation of the IFNAR/JAK/STAT1 pathway can also

lead to the upregulation of the TAM receptor Axl, whichaccumulates at the cell surface where it physically associateswith the IFNAR and usurps the IFNAR-STAT1 pathway bystimulating the transcription of suppressors of cytokinesignaling (SOCS; refs. 24, 25). The SOCS proteins extin-guish IFN-I signaling in a negative feedback loop. Forinstance, SOCS1 directly interacts with JAKs blocking theircatalytic activity, whereas SOCS3 seems to inhibit JAKs aftergaining access by receptor binding. In addition, SOCSproteins interact with the cellular ubiquitination machin-ery through the SOCS-box region and may redirect asso-ciated proteins, such as JAKs or IFNAR chains, toproteasomal degradation. It has been shown that SOCS1knockout mice develop lupus-like disease symptoms (26)and that SOCS1 silencing enhances the antitumor activityof IFNs-I (27), suggesting that SOCS are key elements inkeeping IFNs-I effects under control.

Alternative signaling pathways and cross-talk withother receptorsEvidence is accumulating that several other signaling

elements and cascades are required for the generation ofmany of the responses to IFNs-I, such as the v-crk sarcomavirus CT10 oncogene homolog (avian)-like (CRKL) path-way, the mitogen activated protein kinase (MAPK) path-way, the phosphoinositide 3-kinase (PI3K) pathway, andeither the classical or the alternative NF-kB cascade(Fig. 1B-D; refs. 9, 28). Some of these pathways operateindependently of the JAK-STAT, whereas others cooperatewith STATs to tune the response to IFNs-I. Interestingly,MAPK, PI3K, and NF-kB pathways involve immunorecep-tor tyrosine-based activation motives used by classicalimmunoreceptors, suggesting a cross-talk among IFNARand multiple receptors on immune system cells.The PI3K and NF-kB pathways. Upon activation, TYK2

and JAK1 phosphorylate insulin receptor substrate 1 (IRS1)and 2 (IRS2), which provide docking sites for the PI3K (9).STAT3 acts as an adapter to couple PI3K to the IFNAR1subunit. PI3K subsequently activates the serine-threoninekinase AKT, which in turn mediates downstream the acti-vation of mTOR and the inactivation of several proteins,

such as glycogen synthase kinase 3 (GSK-3) and cyclindependent kinase inhibitors 1A and 1B (CDKN1A andCDKN1B), all of them involved in the control of celldivision and proliferation. The AKT pathway also resultsin the activation of IkB kinase beta (IKKb), which givesrise to NF-kB activation. IFNs-I also activate NF-kB by analternative pathway, which involves the linkage of TNFreceptorassociated factors (TRAF) to the activation ofthe NF-kBinducing kinase (NIK), which in turn resultsin activation of NF-kB2. This alternative pathway isstrictly dependent on the activity of IKKa. In addition,PI3K/AKT can also activate NF-kB through an activationloop via protein kinase C zeta (PKCq). Overall, NF-kBactivated by IFNs-I regulates prosurvival signals andenhances the expression of several GTP-binding proteinsas well as molecules involved in antigen processing and/or presentation.The MAPK pathway. Activation of JAK results in tyro-

sine phosphorylation of the guanine nucleotide exchangefactor Vav, which leads to downstream activation of Ratsarcoma protein (Ras) and ras-related C3 botulinum toxinsubstrate 1 (Rac1), which subsequently leads to the activa-tion of several MAPKs: p38, c-Jun N-terminal kinases(JNK), and extracellular signal regulated kinases (ERK) 1and 2 (9). Vav can also activate PKCq, leading to theactivation of theNF-kB cascade. Several studies have shownthat IFN-amediated activation of ERK1/2 in T lympho-cytes requires proteins involved in the T-cell receptor (TCR)early signaling complex, such as CD45, lymphocyte-speci-fic protein tyrosine kinase (Lck), Zeta-chain-associatedprotein kinase 70 (Zap70), SH2 domain-containing leu-kocyte protein of 76 kDa (SLP76), and Vav1, and possiblylinker for activation of T cells (LAT), indicating cross-talkbetween pathways (2931). Moreover, it has been shownrecently that TCR deletion in Jurkat cells abrogated IFNAR-stimulated MAPK activity, whereas the canonical JAK-STATpathway remained unaffected (31). A hypothetical path-way that involves all these molecules and results in Vavactivation is depicted in Fig. 1D. The different MAPKsactivate different sets of transcription factors. Importantly,p38 functions are essential for the antiviral and antileuke-mic properties of IFNs-I, it plays a role in the hematopoie-tic-suppressive signals and is required for the growth-inhibitory effects of IFNs-I observed in certain lymphocytecultures. IFNs-Iactivated ERK cascades regulate cellulargrowth, differentiation, and serine phosphorylation ofSTAT1. The search for biochemical signals to explain theeffects of IFN-I on immune system cells is far from over.

Effects of IFNs-I on cells of the immune systemIt has long been established that the main mechanism

accounting for the efficacy of the IFN-Ibased therapies wasdue to their direct effect on malignant or virus-infectedcells. In fact, IFNs-I directly inhibit the proliferation oftumor and virus-infected cells and increase MHC class Iexpression, enhancing antigen recognition. Moreover,IFNs-I repress oncogene expression and induce that oftumor suppressor genes, which may contribute to the

IFN-a on Immune Cells

www.aacrjournals.org Clin Cancer Res; 17(9) May 1, 2011 2621

inhibitory effects of IFN-I on malignant cells in conjunc-tion with the antiangiogenic effects.IFNs-I have also proven to be involved in immune

system regulation (7, 8, 32). IFNs-I exert their effects onimmune cells either directly, through IFNAR triggering, orindirectly (i) by the induction of chemokines, which allowthe recruitment of immune cells to the site of infection; (ii)by the secretion of a second wave of cytokines, which couldfurther regulate cell numbers and activities (as for exampleIL-15, which plays a critical role in proliferation andmaintenance of NK cells and memory CD8 T cells; refs.33, 34); or (iii) by the stimulation of other cell types criticalfor the activation of certain immune cells, such as DC forthe activation of nave T cells.One of the earliest described immunoregulatory func-

tions of IFNs-I was their ability to regulate NK functions(32). IFNs-I enhance the ability of NK cells to kill targetcells and to produce IFN-g by indirect and direct mechan-isms (33, 35, 36). Furthermore, IFNs-I promote the accu-mulation and/or survival of proliferating NK cells by theIFN-I/STAT1dependent induction of IL-15 (33). IFNs-Ialso enhance the production or secretion of other cytokinesby the NK cell through the autocrine IFN-g loop (37).IFNs-I also affect monocyte and/or macrophage function

and differentiation. Thus, IFNs-I markedly support thedifferentiation of monocytes into DC with high capacityfor Ag presentation, stimulate macrophage antibody-dependent cytotoxicity, and positively or negatively regu-late the production of various cytokines (e.g., TNF, IL-1, IL-6, IL-8, IL-12, and IL-18) bymacrophages (38). In addition,autocrine IFN-I is required for the enhancement of macro-phage phagocytosis by macrophage colony-stimulatingfactor and IL-4 (39) and for the lipopolysaccharide-,virus-, and IFN-ginduced oxidative burst through thegeneration of nitric oxide synthase 2.The main function of DCs is to uptake and process

antigens for presentation to T cells. DCs are a unique celltype able to prime nave T cells and, therefore, are criticalantigen presenting cells (APC). IFNs-I have multiple effectson DCs, affecting their differentiation, maturation, andmigration. Thus, human monocytes cultured in granulo-cyte-macrophage colony-stimulating factor (GM-CSF) IFN-a/b, rather than the more commonly used combina-tion of GM-CSF IL-4, differentiatemore rapidly into DCs,exhibiting the phenotype of partially mature DCs, whileshowing a strong capability to induce a primary humanantibody response and CTL expansion when pulsed withantigen and injected into humanized severe combinedimmunodeficiency (SCID) mice (4042).

In vitro treatment of immature conventional DCs withIFN-a/b has been shown to upregulate surface expressionof MHC class I, class II, CD40, CD80, CD86, and CD83molecules in the human system (4345), associated with aheightened capacity to induce CD8 T-cell responses.Accordingly, it has been observed in mice that IFN-a actingon DC plays a key role in achieving efficient cross-primingof antigen-specific CD8 T lymphocytes (7, 8, 32). IFNs-Ialso affect the ability of DC to secrete IL-12p70. Low

concentrations of IFNs-I are essential for the optimal pro-duction of IL-12p70 (46), whereas higher levels of IFN-Isuppress IL-12p40 expression, thus dampening IL-12p70production (47).A key requisite to initiate the adaptive immune response

is the arrival of professional APC from infected tissue intolymph nodes. It has been shown that human DCs differ-entiated from monocytes in the presence of IFN-a exhibitupregulation of CC chemokine receptor type 7 (CCR7),correlating with an enhanced chemotactic response in vitroto CCR7 natural ligand (CCL19) and with strongmigratorybehavior in SCID mice (48). Interestingly, experiments inmice have also shown that IFN-a/b is required for plasma-cytoid DCs (pDC) to migrate from the marginal zone intothe T-cell area, where they form clusters (49). We have alsorecently shown that IFNs-I enhance the adhesion andextravasations of DCs across inflamed lymphaticendothelium in a lymphocyte function-associated anti-gen 1 (LFA-1) and very late antigen-4 (VLA4)depen-dent fashion (50). IFNs-I may also favor the encounterbetween DCs and specific lymphocytes in lymph nodesby promoting lymphocyte trapping in lymph nodes upondownmodulation of sphingosine 1-phosphate (51).IFNs-I have also been shown to potently enhance the

primary antibody response to soluble antigen, stimulatingthe production of all subtypes of immunoglobulin G (IgG),and inducing long-lived antibody production and immu-nologic memory (8). Direct effects of IFN-a on DCs (8), Bcells (52, 53), and CD4 T cells (53) all contribute to itsadjuvant activities on humoral immune responses. Inter-estingly, a direct effect of IFNs-I on B cells seems to becrucial for the development of local humoral responsesagainst viruses (54).Asmentioned above, CD4 T cells are direct targets of IFN-

I in the enhancement of antibody responses (53). Inhumans, it has also been described that direct effects ofIFN-I on naive CD4 T cells favor their differentiation intoIFN-gsecreting Th1-like T cells (55).Several studies suggested that IFNs-I might exert a direct

effect on CD8 T cells. Thus, it was reported that IFNs-Ipromote IFN-g production by CD8 T cells in a STAT4-dependent manner (56) and promote survival of CD8 Tcells from wild-type (WT) but not IFNAR-deficient(IFNAR/) mice (57). The definitive report showing thatCD8 T cells represent direct targets of IFN-Imediatedstimulation in vivo came from Kolumam and colleagues(58). By adoptively transferring virus-specific naive CD8 Tcells from IFNAR/ or IFNAR-sufficient mice into normalIFNAR WT hosts, IFNs-I were shown to act directly onmurine CD8 T cells, allowing their clonal expansion andmemory differentiation. Subsequent studies confirmed thiswork (5961). Elegant experiments in mice by Curtsingerand colleagues (62) have shown that, in addition to signalsvia TCR (signal-1) and CD28 (signal-2), nave CD8 T cellsrequired a third signal to differentiate into effector cells.cDNA microarray analyses showed that IFN-a couldregulate critical genes involved in CTL functions (63),providing evidence that IFNa promoted activation and



differentiation of CD8 T cells by sustaining the expressionof T-bet and Eomes through chromatin remodeling.Recently, we have shown that IFN-a provides a strongand direct signal to human CD8 T cells, thereby resultingin upregulation of critical genes for cytotoxic T-cell activity(cytolysis and IFN-g secretion) and for the production ofchemokines that would coattract other effector lympho-cytes. IFN-a was absolutely critical in the case of humannave CD8 T cells for effector function acquisition (64). Inmany instances, T cells may sense IFN-I before the cognateantigen is actually presented to them. As a result of apreexposure to IFN-I, CD8 T cells become preactivatedand acquire much faster effector functions upon antigenrecognition (65). This preactivation may reflect the upre-gulation by IFN-I of several mRNAs in antigen-naive CD8 Tlymphocyte, albeit without changes in the expression of thecorresponding protein, unless antigen-specific activationensues (64).

Clinical-Translational Advances

The contracting current indications of IFN-a as ananticancer agentIFN-a has received approval for treatment of several

neoplastic diseases (66). The most frequent indicationsfor IFN-a are chronic viral hepatitis. For the treatment ofthese infectious conditions, stabilized forms of IFN-a havebeen created by directed conjugation of polyethylene gly-col, which, by increasing molecular weight, retards renalclearance and, thereby, extends plasma concentrations withsustained receptor occupancy (67). However, PEG-conju-gated IFN is not widely used in oncology in the absence ofcomparative clinical studies with the unconjugated form.In oncology, the main indication of IFN-a is for patients

with resected stage II and III melanoma, in whom IFN-aprolongs disease-free survival and shows a trend towardincreased overall survival (68). However, the advent ofCTLA-4 antagonist antibodies will very likely displacethe use of IFN-a for melanoma therapy (69).Table 1 shows the spectrumofneoplastic diseases inwhich

IFN-a has been used. As shown, its role in cancer therapy issteadily decreasing because of its limited efficacy and theadvent of new, more effective, and/or safer treatments.

Future perspectivesIFNs-I are powerful tools to directly and indirectly

modulate the functions of the immune system. Evolutionhas shaped these cytokines as a key sign of alarm in caseof viral infection. The type of immune response that wewould like to induce and sustain against cancer antigensis identical to the one we commonly observe upon acuteviral infections. Obviously, the effects of IFN-I are inte-grated with many other key molecules that need to act in aconcerted fashion.As described in Table 1, side effects of systemic long-

term treatments and lack of sufficiently high efficacy havedampened the interest of IFN-a for clinical use in oncol-ogy. However, we believe that IFN-a is likely to be more

efficacious when acting locally and intermittently at themalignant tissue and tumor-draining lymph node thanwhen administered to achieve systemic bioavailability.Local delivery can be achieved with pharmaceutical for-mulations and gene therapy approaches (70). In ouropinion, the clinical use of recombinant IFN-a as a vaccineadjuvant should be reconsidered and new investigationscarried out with an eye to clinical applications (71).The rationale for using intermittent delivery arises from

observations indicating that IFN-I turns on signaling desen-sitizing mechanisms (i.e., because of SOCS1 induction).Intermittency at an optimized pace may help to avoid thesenegative feedback mechanisms in the responding immunecells. These ideas contrast with stabilized formulations,such as the widely used polyethylene glycol-conjugatedIFN-a and fusion proteins with albumin that extend thehalf-life of the cytokine.It is of much interest that a gene expression signature

induced by IFN-I in melanoma lesions predicts benefitfrom vaccines and may have a key role in recruitingeffector T cells to tumors by inducing chemokine genes(72). IFN-a has been recently used in promising combi-natorial immunotherapy approaches alongside DC vac-cination for glioblastoma, precisely to promote T-cellinfiltration and DC activation (73). In this regard, usingendogenous IFN-I inducers instead of recombinant cyto-kines may have advantages, because substances such asthe viral RNA analog poly I:C will also elicit other cyto-kines that may act in concert with IFNs-I to enhance theantitumor immune response.Creative combination strategies are building on the

knowledge that IFN-a effects on the immune systemare likely to improve its therapeutic profile against malig-nant diseases. For instance, combinations with immu-nostimulatory monoclonal antibodies, IL-15, IL-12, andIL-21, may hold much promise. Indeed, intratumoralrelease of mouse IFN-a along with systemic treatmentwith anti-CD137 mAb results in synergistic therapeuticeffects (74). Macrophages homing to tumor, transducedto express IFN-a, exert therapeutic effects without thesystemic side effects (75). These studies suggest that localdelivery as opposed to systemic administration should betested. Of great importance is the notion that the che-mokines induced by IFN-I (i.e., CXCL9, CXCL10) attractactivated lymphocytes, thereby calling more immunecells to infiltrate the malignant focus.When reflecting on the use of IFN-I for cancer, we must

think about reprogramming the tumor microenviron-ment, frequently devoid of any stimulatory signals. LocalIFN-I release with concomitant irradiation and chemo-therapy may help to turn some of the macroscopic tumorlesions into immunogenic vaccines. Factors such as CpGoligonucleotides or poly I:C that induce the release ofendogenous IFNs-I at a given tumor lesion (76) arepromising in this regard. These agents may constitutean advantageous alternative because these agents alsoinduce an additional array of proinflammatory mediatorsacting in synergy with IFNs-I.



Table 1. Contracting Indications of IFN-a for Malignant Conditions

Indication Stage Comment Standard Current Therapy Reference

Melanoma II/III Prolongs disease-freesurvival and shows a trendtoward increased overallsurvival.

IFN-a is the standard option. 68

IV Combination withIL2: Increased responserates but no survivalimprovement.

Chemotherapy. The adventof CTLA-4 antagonist antibodywill displace the use of IFN-afor melanoma inmunotherapy.

77, 78

RCC II/III Two phase III trials showednegative results.

Novel targeted agents suchas sunitinib, temsirolimus,sorafenib, or everolimushave decreased the use ofIFN-a to a minimum in bothresectable and advanced RCC.

79, 80

IV Widely studied as a singleagent or in combination withIL-2, chemotherapy, or both:modest or negative results.

81

Hairy cell leukemia First treatment that showedclinical benefit in this disease.

Purine analogs are nowconsidered standardtreatment for thisdisease.

82, 83

Recommended, in lowdose, as a therapeuticoption for maintenancetherapy and as analternative for frailpatients.

Multiple myeloma Extensively studied asmaintenance treatment,as single treatment, or incombination withchemotherapy: slightefficacy.

Largely replaced by newerand more effective agentssuch as thalidomide,lenalidomide, or bortezomib.

84

CMS PV Optional treatment forcytoreduction (94.6%complete responses).

The mainstay of therapyremains repeated phlebotomy.

8587

ET Treatment of choicein women withchildbearing potentialand optional treatmentin young patientsresistant tohydroxyurea.

Hydroxyurea. Anagrelide. 86

PM Cytoreduction therapy. Hydroxyurea. Thalidomide. 77CML First-line therapy in

combination withcytarabine: 71% clinicalresponse rate and 39% majorcytogenetic response rate.

Imatinib, in a phase III trial,has been superior intolerability, completehematologic and cytogeneticresponse rates, andprogression-free survival.

8892

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In conclusion, the notion of key direct effects of IFNs-Ion immune system cells and detailed knowledge on theelicited signaling pathways should help biomarker discov-ery to identify the small number of patients who couldactually benefit from current treatments. More importantly,these ideas might change the way we use IFN-a for cancertherapy, suggesting new strategies and combinations withother agents.

Disclosure of Potential Conflicts of Interest

I. Melero, A. Rouzaut, and S. Hervas-Stubbs receive laboratory reagentsand research funding fromDigna Biotech. I. Melero has acted as a consultantfor Bristol-Myers Squibb and Pfizer Inc.

Acknowledgments

We are grateful for continuous scientific collaboration on IFNs-I withJesus Prieto, Esther Larrea, Jose I. Riezu-Boj, Iranzu Gonzalez, and Juan Ruiz.

Grant Support

MEC/MCI (SAF200503131, SAF200803294, and TRA20090030), Departa-mento de Educacion y Departamento de Salud (Beca Ortiz de Landazuri) delGobierno de Navarra. Redes tematicas de investigacion cooperativa RETIC(RD06/0020/0065), Fondo de investigacion sanitaria (FIS PI060932), Europeancommission VII framework program (ENCITE), Immunonet SUDOE. FundacionMutua Madrilea, and "UTE for project FIMA". S. Hervas-Stubbs was supportedby AECC and by MCI (RYC-200700928).

Received October 22, 2010; revised January 17, 2011; accepted January17, 2011; published OnlineFirst March 3, 2011.

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Table 1. Contracting Indications of IFN- for Malignant Conditions (cont'd )

Indication Stage Comment Standard Current Therapy Reference

In comparison withchemotherapy IFNahas been shown tobe superior.

In imatinib resistance, IFNahas been precluded by theappearanceof dasatinib.

Hemangioma Complicate hemangiomas thatdo not respond to steroids.

Steroids. 93

Rate regression in58% of the patients.

AIDS-related Kaposisarcoma

Evidence for clinical activity. Liposomal anthracyclines arepreferred as the firsttreatment option.

94

PV, polycythemia vera; ET, essential thrombocythemia; PM, primary myelofibrosis; RCC, renal cell cancer; CMS, chronic myelo-proliferative syndrome; CML, chronic myeloid leukemia



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direct effects of type i interferons on cells of immune system (stubbs et al. 2011)

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