Interferon-gamma at the crossroads of tumor immune surveillance or evasion

May 2018 | Volume 9 | A icle 8471 Re iew published: 04 May 2018 doi: 10.3389/ immu.2018.00847 F on ie s in Immunology | www. on ie sin.o g Edi ed by: Fab izio Ma ei, Is i u o Supe io e di Sani à, I aly Re iewed by: Alexand e Co hay, Oslo Uni e si y Hospi al, No way Ch is ine Susanne Falk, Hanno e Medical School, Ge many *Co espondence: Ma ia José Oli ei a [email p o ec ed] Special y sec ion: This a icle was submi ed o Cance Immuni y and Immuno he apy, a sec ion o he jou nal F on ie s in Immunology Recei ed: 06Decembe 2017 Accep ed: 05Ap il2018 Published: 04May2018 Ci a ion: Cas oF, Ca dosoAP, Gonçal esRM, Se eK and Oli ei aMJ (2018) In e e on-Gamma a he C oss oads o Tumo Immune Su eillance o E asion. F on . Immunol. 9:847. doi: 10.3389/ immu.2018.00847 in e e on-Gamma a he C oss oads o Tumo immune Su eillance o e asion Flá ia Cas o1,2,3, Ana Pa ícia Ca doso1,2, Raquel Madei a Gonçal es1,2,3, Ka ine Se e4 and Ma ia José Oli ei a1,2,5* 1 i3S – Ins i u o de In es igação e Ino ação em Saúde, Uni e sidade do Po o, Po o, Po ugal, 2 INEB – Ins i u o de Engenha ia Biomédica, Uni e sidade do Po o, Po o, Po ugal, 3 ICBAS – Ins i u o de Ciências Biomédicas Abel Salaza , Uni e sidade do Po o, Po o, Po ugal, 4 IMM – Ins i u o de Medicina Molecula João Lobo An unes, Faculdade de Medicina, Uni e sidade de Lisboa, Lisbon, Po ugal, 5 Depa amen o de Pa ologia e Oncologia, Faculdade de Medicina, Uni e sidade do Po o, Po o, Po ugal In e e on-gamma (IFN-γ) is a pleio opic molecule wi h associa ed an ip oli e a i e, p o-apop o ic and an i umo mechanisms. This e ec o cy okine, o en conside ed as a majo e ec o o immuni y, has been used in he ea men o se e al diseases, despi e i s ad e se e ec s. Al hough b oad e idence implica ing IFN-γ in umo immune su - eillance, IFN-γ-based he apies unde going clinical ials ha e been o limi ed success. In ac , ecen epo s sugges ed ha i may also play a p o umo igenic ole, namely, h ough IFN-γ signaling insensi i i y, down egula ion o majo his ocompa ibili y com- plexes, and up egula ion o indoleamine 2,3-dioxygenase and o checkpoin inhibi o s, as p og ammed cell-dea h ligand 1. Howe e , he IFN-γ-media ed esponses a e s ill posi i ely associa ed wi h pa ien ’s su i al in se e al cance s. Consequen ly, majo esea ch e o s a e equi ed o unde s and he immune con ex u e in which IFN-γ induces i s in ica e and highly egula ed e ec s in he umo mic oen i onmen . This e iew discusses he cu en knowledge on he p o- and an i umo igenic e ec s o IFN-γ as pa o he complex immune esponse o cance , highligh ing he ele ance o iden i y IFN-γ esponsi e pa ien s o he imp o emen o he apies ha exploi associa ed sig- naling pa hways. Keywo ds: ype ii in e e on, immuno egula ion, cance mic oen i onmen , immuno he apy, immune con ex u e iNTRODUCTiON In e e ons (IFNs) a e pleio opic cy okines wi h an i i al, an i umo and immunomodula o y p ope ies, being cen al coo dina o s o he immune esponse (1). The e m “in e e ons” comes om he desc ip ion o molecules p o ec ing cells by “in e e ing” wi h i al in ec ion (2, 3). Th ee majo ypes o IFNs a e dis inguished by hei sequence iden i y, gene ic loci, cell o o igin, na u e, and dis ibu ion o hei ecep o s and esul ing s imuli (Table1). The human ype I IFN amily comp ises 17 dis inc p o eins, mainly ep esen ed by IFN-α and IFN-β, which a e ubiqui ously exp essed and signal h ough hei cogna e ecep o , composed by IFNαR1 and IFNαR2 subuni s [ e iewed in Re . (4)]. IFN-γ is he lone membe o ype II IFN amily. I is mo e es ic i ely exp essed and is s uc u ally and unc ionally di e en om he o he ypes o IFNs. Mos ecen ly, a ype III IFN amily was desc ibed o be composed o ou homologous p o eins (IFNλ1–4), which bind he IFNλR1 and in e leukin (IL)-10Rβ he e odime ic ecep o [ e iewed in Re . (8)]. To da e, ype I and ype III IFNs ha e been mainly in ol ed in hos –pa hogen Table 1 | Compa ison o human ype I, ype II, and ype III IFN p oduc ion and signaling. P ope ies Type i iFN (iFN-α, iFN-β) Type ii iFN (iFN-γ) Type iii iFN (iFN-λ) Membe s 17 p o eins: 13 IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω 1 p o ein: IFN-γ4 p o eins: IFN-λ1, IFN-λ2, IFN-λ3, IFN-λ4 IFN-p oducing cells All nuclea ed cells Tcells, B cells, NKcells, NKTcells, and APCs All nuclea ed cells, mainly mDCs, pDCs, and epi helial cells IFN- esponding cells All nuclea ed cells All nuclea ed cells Lung, in es ine, and li e epi helial cells S imuli DAMPs and PAMPs IL-12, IL-15, IL-18, ype I IFN, and PAMPs DAMPs and PAMPs IFN ecep o IFN ype I ecep o (IFNαR): IFNαR1 and IFNαR2 subuni s IFN ype II ecep o (IFNγR): IFNγR1 and IFNγR2 subuni s IFN ype III ecep o (IFNλR): IFNλR1 and IL10Rβ Signaling molecules TYK2, JAK1, all STATs, CRKL, and IRS JAK1, JAK2, STAT1, and STAT3 TYK2, JAK1, STAT1, STAT2, and IRF9 T ansc ip ion ac o binding si es ISRE (canonical) GAS (non-canonical) GAS (canonical) ISRE (non-canonical) ISRE Func ions An i i al, an ip oli e a i e esponse, egula ion o cell su i al/apop osis, and immuno egula ion An i i al, an ip oli e a i e, immunomodula o y, and an i umo esponse An i i al esponse, mucosal immuni y Re e ence (4, 5) (6, 7) (8) APCs, an igen-p esen ing cells; CRKL, CT10 egula o o kinase-like; DAMPs, damage-associa ed molecula pa e ns; GAS, gamma-ac i a ed si e; IFN, in e e on; IFN-γ, in e e on- gamma; IFNαR1–2, ype I ecep o ; IFNγR, ype II ecep o ; IFNλR, ype III ecep o ; IL, in e leukin; IRF, in e e on- egula o y ac o ; IRS, insulin ecep o subs a e; ISRE, in e e on- sensi i e esponse elemen ; JAK, Janus kinase; mDCs, myeloid dend i ic cells; NK, na u al kille ; NKT, na u al kille Tcells; PAMPs, pa hogen-associa ed molecula pa e ns; pDCs, plasmacy oid dend i ic cells; STAT, signal ansduce and ac i a o o ansc ip ion; TYK, y osine kinase. 2 Cas o e al. Dual Role o IFN-γ in Cance F on ie s in Immunology | www. on ie sin.o g May 2018 | Volume 9 | A icle 847 in e ac ions, and hei exp ession is ac i a ed h ough immune sys em sen inel ecep o s, such as pa e n ecogni ion ecep o s. Despi e he simila unc ion o ype I and III on an i i al in ec ions, i is he i al opism ha dic a es he ela i e con ibu ion o each IFN (9). Mo eo e , whe eas almos all nuclea ed cells espond o ype I IFN, ype III IFNs esponse is es ic ed o issues wi h a high isk o i al exposu e and in ec ion, as he mucosal su aces. The ole o ype II IFN in p omo ing hos immune esponse o mic oo ganisms is simila ly well documen ed. No ably, i is also known o play a pi o al unc ion on cance immune su eillance, s imula ing an i umo immuni y and p omo ing umo ecog- ni ion and elimina ion (10–16). This e iew ocuses on ype II IFN signaling, cellula unc- ions, and di ec ed he apies and was encou aged by no el indings e ealing egula o y mechanisms o IFN-γ and i s p og- nos ic as well as he apeu ic po en ial. In ac , since Wheelock who epo ed ha IFN-γ inhibi ed i al eplica ion in 1965 (17), i ook a ound 30yea s o en isage his cy okine as a a ge o an i umo immuni y (18). In e e on-gamma is a homodime o med by he non- co alen associa ion o wo 17kDa polypep ide subuni s. Du ing syn hesis, a e mul iple N-glycosyla ion, bo h subuni s bind in an an ipa allel manne , cons i u ing a ma u e 50kDa molecule (19, 20). No ably, he IFN-γ symme y sugges s ha a single molecule can bind simul aneously o wo ecep o s, ampli ying he unde lying esponses. Cellula esponses induced by IFN-γ may also in ol e c oss-communica ion wi h IFN-α/β ecep o s, ampli ying IFN-γ signaling and i s e ec s (21, 22). In e e on-gamma is sec e ed p edominan ly by ac i a ed lymphocy es such as CD4 T helpe ype 1 (Th1) cells and CD8 cy o oxic Tcells (23–26), γδ Tcells (27–33), and na u al kille (NK) cells (34, 35) and, o a less ex en , by na u al kille Tcells (NKT), B cells (36–39), and p o essional an igen-p esen ing cells (APCs) (40–42). I s exp ession is induced by mi ogens and cy okines, such as IL-12 (43, 44), IL-15 (45), IL-18 (46, 47), and ype I IFN (48, 49). IFN-γ pleio opic unc ions a e media ed by cell-speci ic exp ession o hund eds o IFN-γ- egula ed genes ha encompass in lamma o y signaling molecules, apop osis and cell cycle egula o s, and ansc ip ional ac i a o s (50). Au oc ine IFN-γ p oduced by APCs can ac locally and con ib- u e o sus ain sel and neighbo cell ac i a ion (51–53), c ucial o ea ly con ol o pa hogen sp eading, while Tlymphocy es a e he majo pa ac ine sou ce o IFN-γ in adap i e immuni y. Unde physiological condi ions, he cons i u i e exp ession o ype I and II IFNs is igh ly con olled, emaining localized o issues, wi h- ou sys emic e ec s (54–56). Fo ins ance, cons i u i e exp ession o endogenous IFN-γ con ibu es o he homeos asis o immune cell unc ions (57), main enance o he hema opoie ic s em cell niche (58), and bone o ma ion (59). Combina ion app oaches o boos inna e immune ac i a ion ha e been explo ed o con e ge on o IFN pa hways. Howe e , IFN-γ- ela ed signaling can also ha e supp essi e immuno egula o y e ec s on an i i al (60, 61), au oimmune (62, 63), as well as on an i umo esponses (64, 65). Un eiling cellula a ge s o IFN-γ is c i ically impo an o i s he apeu ic applica ion, o p edic pa ien esponses, pa icula ly in cance s whe e his cy okine can exe p o umo igenic e ec s. The e o e, he cellula and molecula e ec s o IFN-γ, wi h pa - icula emphasis on i s dual ole on umo immuni y and how o o e come i s limi a ions, will be he majo ocus o his e iew. CaNONiCal SiGNaliNG aND ReGUlaTORY MeCHaNiSMS The iFN-γ Recep o The IFN-γ ecep o is composed o wo ligand-binding IFNγR1 chains associa ed wi h wo signal- ansducing IFNγR2 chains, which a e esponsible o connec ing o he cy oplasmic ans- duc ion machine y (see Figu e 1). The IFNGR1 and IFNGR2 a e localized in ch omosome 6 and 21, espec i ely, and hei FiGURe 1 | In e e on-gamma (IFN-γ) canonical signaling pa hway. Upon ligand binding, IFNγR1 and IFNγR2 oligome ize and ansphospho yla e, ac i a ing Janus ac i a ed kinase (JAK) 1 and JAK2. These, in u n, phospho yla e IFNγR1, c ea ing a docking si e o he signal ansduce and ac i a o o ansc ip ion (STAT) 1. Phospho yla ed STAT1 homodime izes in an an ipa allel con igu a ion, o ming a complex gamma-ac i a ed ac o (GAF), which ansloca es o he nucleus and binds o gamma-ac i a ed si e (GAS), loca ed a he p omo e s o p ima y esponse genes, inc easing hei ansc ip ion. Upon induc ion, ansc ip ion ac o in e e on- egula o y ac o 1 (IRF1) binds o in e e on-s imula ed esponse elemen (ISRE) and enhances he ansc ip ion o se e al seconda y esponse genes esponsible o se e al immunomodula o y unc ions. Supp esso o cy okine signaling (SOCS) p o eins nega i ely egula e he IFN-γ pa hway by inhibi ing JAKs and STAT1 phospho yla ion. Th ough dephospho yla ion and deace yla ion, he con igu a ion o STAT1 homodime s e e s o pa allel, igge ing hei exi om he nucleus. 3 Cas o e al. Dual Role o IFN-γ in Cance F on ie s in Immunology | www. on ie sin.o g May 2018 | Volume 9 | A icle 847 exp ession di e s signi ican ly. While IFNγR1 is cons i u i ely exp essed a mode a e le els on he su ace o almos all cells, IFNγR2 is cons i u i ely exp essed a low le els, and i s exp ession is igh ly egula ed, acco ding o he s a e o cellula di e en ia- ion o ac i a ion (66). Fo example, CD4 T helpe cell subse s di e in hei abili y o espond o IFN-γ (67, 68). Rema kably, IFN-γ ac i a es he signal ansduce and ac i a o o ansc ip- ion (STAT) 1 ha main ains he exp ession o T-be , he mas e ansc ip ion ac o ha con ols IFN-γ exp ession in Tcells (69). This signaling cons i u es a posi i e eedback loop ha maximizes Th1 immuni y (70–72). No ably, Th1cells a e mo e esis an o he an ip oli e a i e e ec s o IFN-γ han Th2 cells. This is likely due o lowe le els o exp ession o he IFNγR2 subuni ha allows Th1cells o con inue o p oli e a e du ing IFN-γ signaling. By con as , Th2 cells ha do no p oduce IFN-γ exp ess highe le els o he IFNγR2 subuni , ende ing hem pa icula ly sus- cep ible o he p esence o IFN-γ ha inhibi s hei p oli e a ion (67, 68, 73). Ne e heless, IFNγR2 down egula ion may be also induced in Th2 cells when hey a e exposed o IFN-γ (68). Thus, IFN-γ appea s o egula e he exp ession o i s own ecep o on speci ic cell ypes, ep esen ing a egula o y mechanism o cellula desensi iza ion in esponse o cy okines p esen a he local mic oen i onmen . As a esul , IFNγR2 exp ession can be a limi ing ac o in IFN-γ esponsi eness and unc ional ou come ha can dic a e he Th1–Th2 pheno ype swi ch and modula e he subsequen immune esponse. JaK/STaT Signaling Pa hway The biological e ec s o IFN-γ a e elici ed h ough ac i a ion o in acellula molecula signaling ne wo ks, mainly ia he JAK/ STAT pa hway, which modula es he ansc ip ion o hund eds o genes and media es di e se biological esponses (50, 74–76). Upon IFN-γ binding, he in acellula domains o IFNγR2 oligome ize and ansphospho yla e wi h IFNγR1, ac i a ing 4 Cas o e al. Dual Role o IFN-γ in Cance F on ie s in Immunology | www. on ie sin.o g May 2018 | Volume 9 | A icle 847 he downs eam signaling componen s, JAK1 and JAK2. The ac i a ed JAKs phospho yla e he in acellula domain o he ecep o ( y osine 440 on human IFNγR1), c ea ing binding si es o STAT1 (77). STAT1 is hen phospho yla ed in he C- e minus on y osine Y701 esidues by JAK, esul ing in he o ma ion o STAT1 homodime s complexes, known as gamma-ac i a ed ac o s (GAFs), which ansloca e o he nucleus and egula e gene exp ession h ough binding o gamma-ac i a ed si e (GAS) elemen s in he p omo e s o in e e on-s imula ed genes (ISGs) (78). One o he majo p ima y esponse genes induced by STAT1 signaling is he ansc ip ion ac o in e e on- egula o y ac o 1 (IRF1), a membe o he IFN egula o y ansc ip ion ac o am- ily (79). IRF1 unc ions as a ansc ip ion ac i a o o in e e on- s imula ed esponse elemen s (ISRE), leading o he ansc ip ion o a la ge numbe o seconda y esponse genes (Figu e1). Fo ins ance in b eas cance cells, a genome-wide iden i ica ion o IFN-γ-induced IRF1 ac i a ion e eals o e 17,000 binding si es, wi h “apop osis” o “cell dea h” as he mos en iched a ge p ocesses unde lying he di ec umo icidal p ope y o he cy okine (80). Howe e , umo cells also de elop esis ance o IFN-γ h ough di e en ial IRF1 esponsi eness, poin ing ou ha he JAK/STAT signaling pa hway needs o be igh ly egula ed o a oid de imen al consequences o excessi e s imula ion and highligh ing i s ole on immune esponses and umo igenesis (81). STAT1 a ge s o he IFN-γ-media ed signaling also include he SMAD amily membe 7 (SMAD7), and p o eins in ol ed in cell cycle egula ion, such as c-Myc and he cyclin-dependen kinase inhibi o 1A (82–84). The JAK/STAT signaling pa hway is egula ed a se e al le els by posi i e and nega i e mechanisms. In pa icula , de egula ion o inhibi ion o he JAK/STAT pa hway leads o lowe ed immu- ni y and is o en associa ed wi h inc eased umo igenesis (85, 86) o me as a ic dissemina ion (87). STATs a e also in ol ed in he de elopmen and unc ion o he immune sys em and play a ole in main aining umo su eillance [ e iewed in Re . (88)]. STAT1, as a umo supp esso , is deduc ed o i s exp ession in umo cells, modula es hei immunological s a us and consequen ly hei esponse o an i umo immune esponses. Indeed, STAT1- de icien umo cells we e mo e suscep ible o NK cells while STAT1-p o icien umo cells we e mo e sensi i e o CD8+ Tcells (89). In he same way, STAT1-de icien mice ha a e impai ed in Th1cell pola iza ion, exhibi ed educed IFN-γ exp ession and comp omised cy oly ic and NK ly ic ac i i y, ailing o con ol umo g ow h in con as wi h wild- ype mice (90). In addi ion, cell-au onomous umo -supp esso unc ions o STAT1 ha e also been epo ed in b eas cance (91). Howe e , he e is g owing e idence ha STAT1 also ac s as a umo p omo e (92–94) since i can enhance esis ance o chemo he apeu ic agen s and adia ion in ca cinoma (95). Impo an ly, STAT1 also pa ici- pa es in he signaling om di e en cy okines, including IL-21, IL-27, and IL-35. These cy okines ha e been p oposed o limi an i umo immuni y in speci ic cellula , molecula , and mic o- en i onmen al con ex s (96–101). Thus, STAT1 phospho yla ion e lec s no only he h eshold and magni ude o IFN-γ esponse bu also o o he immune media o s, highligh ing he impo - ance o he egula ion o STAT1 phospho yla ion. One o he mos impo an nega i e egula o s o he JAK/STAT signaling pa hway is he supp esso o cy okine signaling (SOCS) p o eins, which exp ession is inc eased in esponse o IFN-γ signaling h ough IRF1 (102, 103). SOCS blocks he ac i i y o JAKs by a nega i e eedback loop, bu also egula es o he cy okines down- s eam signaling. SH2 domains in SOCS p o eins di ec ly bind o phospho yla ed y osine esidues o ac i a ed JAKs, blocking he ec ui men o signal ansduce adap o s, such as STATs, and JAK ac i i y (102). Fu he mo e, SOCS p omo e in e ac ions ha lead o ubiqui ina ion and p o easome deg ada ion o compo- nen s o he JAK/STAT signaling (104, 105). SOCS1 e en p e en s egula o y T (T eg) cells om p oducing IFN-γ by supp ession o STAT1, a oiding he con e sion o T eg cells in o e ec o cells (106). In addi ion, SOCS2-de icien mice showed a educ ion in lung me as ases and an inc ease in su i al ollowing melanoma challenge (107). Al e na i ely, he ansc ip ional ac i i y o STAT1 can be posi i ely egula ed by o he signaling cascades igge ed by IFN-γ binding, such as he mi ogen-ac i a ed p o ein kinase pa hway, p o ein kinase C, and PI3K/AKT, which phospho yl- a e STAT1 in i s ansac i a ion domain (108). Adding o he complexi y, unde ce ain ci cums ances, IFN-γ also can ac i a e STAT1-independen pa hways h ough o he ansc ip ion ac- o s, namely STAT3 (109), STAT5 (110), nuclea ac o -kappa B (NF-κB) (111), and ac i a o p o ein 1 (112). In conclusion, he p ima y esponse o IFN-γ is media ed by GAF ha ac s on genes wi h GAS binding sequence in hei p omo e , while he p ima y esponse o ype I IFNs is media ed by ISGF3 (STAT1/ STAT2/IRF9 complex) ha induces genes ha ha e ISRE in hei p omo e . Thus, some o he ISGs a e egula ed by bo h ypes o IFNs, whe eas o he s a e selec i ely egula ed by each ype o IFN, consequen ly po en ia ing he di e si y o biological esponses. biOlOGiCal FUNCTiONS iFN-γ ac ions on immune Cells In e e on-gamma signaling pa hway coo dina es se e al bio- logical esponses, p ima ily in ol ed in hos de ense and immune su eillance bu also in he es ablishmen o adap i e immuni y (Figu e2) and in he egula ion o in lamma ion, apop osis and cell cycle. One o he i s desc ibed biological e ec s o IFNs was he up egula ion o he majo his ocompa ibili y complex (MHC) molecules (113, 114) as well as he up egula ion o he whole MHC I and II an igen p ocessing and p esen a ion machine y including anspo e associa ed wi h an igen p ocessing (TAP) 1/2, in a ian chain, and he exp ession and ac i i y o he p o easome (115–122). Fu he mo e, in some umo ypes, such as mul iple myeloma and melanoma cells, IFN-γ can also up egula e he MHC class II ansac i a o (CIITA) ha leads o MHC class II exp ession (123, 124). Thus, IFN-γ ini ia es an immune-an igenic exposu e p og am in he a ge cells, and his ensu es he apid ecogni ion o s essed issues. IFN-γ is a majo p oduc o Th1-media ed immune esponse and o ches a es Th1 e ec o mechanisms, as u he ac i a ion o inna e immu- ni y (mac ophages and NK cells) in a posi i e eedback loop. Up egula ion o cell su ace MHC class I by IFN-γ is c ucial o hos esponse o in acellula pa hogens and umo cells, due o cy o oxic Tcell ac i a ion, p omo ing cell-media ed immuni y. FiGURe 2 | Immunomodula o y e ec s o in e e on-gamma (IFN-γ). IFN-γ p oduced by immune cells a ec s he beha io o dis inc immune cells wi hin he umo mic oen i onmen . Speci ically, IFN-γ plays a majo ole in ac i a ing an icance immuni y, by p omo ing he ac i i y o CD4 T helpe ype 1 cells, CD8 cy o oxic T lymphocy e (CTL), na u al kille (NK) cells, dend i ic cells (DCs), and mac ophages, p omo ing he an igen p esen a ion. Addi ionally, IFN-γ ac i a es mac ophages owa ds a mo e p o-in lamma o y and umo icidal pheno ype (M1-like). Al e na i ely, IFN-γ inhibi s egula o y T (T eg) cells, Th2 and Th17 di e en ia ion and unc ions. 5 Cas o e al. Dual Role o IFN-γ in Cance F on ie s in Immunology | www. on ie sin.o g May 2018 | Volume 9 | A icle 847 IFN-γ di ec ly ac s as a cy o oxic CD8 Tcell di e en ia ion signal, and i is essen ial o he induc ion o cy o oxic Tcell p ecu - so p oli e a ion (125, 126). IFN-γ also up egula es cell su ace MHC class II on APCs, hus p omo ing pep ide-speci ic ac i a- ion o CD4 Tcells (25, 127–129). In addi ion, IFN-γ ac i a es mac ophages owa d a p o-in lamma o y p o ile, exhibi ing an inc eased phagocy ic abili y as well as enhanced mic obial kill- ing ac i i y (130). In ac , IFN-γ was ini ially shown o induce “classical” ac i a ion o mac ophages and pola iza ion owa d a umo icidal pheno ype (131). In e es ingly, he o iginal name o IFN-γ was mac ophage ac i a ion ac o (132, 133). IFN-γ con ols speci ic gene exp ession p og ams in ol ing mo e han 290 genes ela ed o cy okine and chemokine ecep o s, cell ac i a ion ma ke s, cellula adhesion p o eins, MHC p o eins, p o easome o ma ion, p o ein u no e , and signaling media- o s and egula o s (134). The abili y o IFN-γ o induce umo cell killing includes he ac i a ion o he NADPH-dependen phagocy e oxidase sys em, ni ic oxide p oduc ion, yp ophan deple ion and up egula ion o lysosomal enzymes (121, 135, 136). These e en s esul in ec ui men o e ec o cells o help in he in lamma ion esolu ion p ocess (137, 138). In addi ion, as a majo cy okine o Th1cells, IFN-γ main ains Th1 lineage commi men h ough a posi i e eedback loop ha s abilizes he Th cell pheno ype (72, 139–141) and c oss-inhibi s he di - e en ia ion o o he Th cell subse s (Figu e2). Indeed, IFN-γ inhibi s Th2 cell di e en ia ion (142, 143) and consequen ly IL-4 p oduc ion. This egula ion in ol es he inhibi ion o he IL-4/ STAT6 pa hway, equi ed o Th2 cell di e en ia ion, and i is media ed a leas by IFN-γ-induced SOCS1 ha inhibi s IL-4R signaling (144, 145). Fu he mo e, IFN-γ-induced T-be inhibi s Th2 cell di e en ia ion by di ec ly in e e ing wi h he ac i i y o Th2 cell-speci ic ansc ip ion ac o , GATA-3 (146). Hö e and colleagues, using ma hema ical models, p oposed ha IL-4 also ac s o p opaga e Th2 cell di e en ia ion (147). A high IL-4 le el p omo es inc eased GATA-3 exp ession ha u he enhances GATA-3 ansc ip ional imp in ing o Th2 di e en ia ion (147, 148). This model p oposed ha high exp ession s a e o GATA-3 can be supp essed by s ong inhibi ion o au oac i a ion, as obse ed in he p esence o Th1-pola izing condi ions (147, 149). IFN-γ was also desc ibed o down egula e he IL-4-inducible gene exp ession (150). The c oss- egula ion o Th1 and Th2 cells was also demons a ed in STAT6-de icien mice, which lack Th2 pheno ype and associa ed immune esponses. These animals displayed augmen ed umo -speci ic IFN-γ p oduc ion and cy o oxic Tcell ac i i y and, consequen ly ejec ed he umo cell line ha g ew p og essi ely in he wild- ype con ol (151). In e e on-gamma p oduced by Th1 cells also coun e ac s Th17cell de elopmen and hei e ec o unc ions (152–154). Se e al mechanisms can be conside ed as he inhibi ion o molecules in ol ed in he Th17 di e en ia ion (155, 156), he inhibi ion o STAT3 by STAT1 (157) and ecen ly, T-be was demons a ed o p e en di e en ia ion o Th p ecu so s in o Th17cells by blocking he exp ession o he Th17cell lineage- speci ic ansc ip ion ac o , RORγ (158). Fu he mo e, IFN-γ also exe s egula o y unc ions o limi issue damage associa ed wi h in lamma ion (63, 159–162) (Figu e 2). IFN-γ has been classically conside ed as a p o-in lamma o y cy okine, in ol ed in he egula ion o an i-in lamma o y esponses, by an agoniz- ing he IL-10 (157, 163) and TGF-be a (164) signaling pa hways. Consequen ly, IFN-γ inhibi s T eg cell di e en ia ion and unc- ions (165, 166). Howe e , in some ch onic in lamma ion condi- ions, IFN-γ plays a c ucial ole in a enua ing issue des uc ion. In his case, IFN-γ migh be p o ec i e (62, 167) by p omo ing he numbe and unc ion o T eg cells (168–170). In addi ion, IFN-γ p oduc ion by T eg cells hemsel es was shown o be a key ea u e o he T eg cells ha a e capable o dampening Th1cell esponses (171–174). Thus, IFN-γ dic a es he di e en ia ion o specialized Foxp3+T-be + T eg cells ha selec i ely supp ess Th1cells, and cons i u e a nega i e eedback loop o minimize he de imen al e ec o IFN-γ. IFN-γ also p omo es he di e en ia ion o 6 Cas o e al. Dual Role o IFN-γ in Cance F on ie s in Immunology | www. on ie sin.o g May 2018 | Volume 9 | A icle 847 myeloid-de i ed supp esso cells (MDSCs) ha es ain o e ac- i a ion o e ec o Tcells, main aining issue homeos asis (175, 176). O he egula o y mechanisms in ol ing IFN-γ signaling ha dampen he magni ude o he immune esponse ha e been epo ed, as he induc ion o indoleamine 2,3-dioxygenase (IDO) by T eg cells, monocy es and s omal cells (177–180), and o he p og ammed cell dea h 1 (PD-1) ligand (PD-L1) on immune and ans o med cells, inhibi ing Tcell esponses (181–183). iFN-γ ac ions on T ans o med Cells and on he Tumo Mic oen i onmen In e e on-gamma is in ol ed in an ip oli e a i e (18), an i- angiogenic (184) and p o-apop o ic e ec s es ablished agains neoplas ic cells. How IFN-γ induces he signaling pa hways ini ia ing and p opaga ing he apop o ic cascade emains o be elucida ed. The le el o complexi y is demons a ed by he ac ha he mechanism migh depend on he umo cells hemsel es. Fo example, while in a glioblas oma cell line he induc ion o apop osis was due o supp ession o he PI3K/AKT pa hway, in ano he glioblas oma cell line apop osis occu ed independen ly o he PI3K/AKT pa hway bu equi ed NF-κB (185). I was also shown ha IFN-γ induces apop osis o human panc ea ic ca - cinoma cells in a caspase-1-dependen manne (186). A e iew co e ed in de ail he mechanism o induc ion o p og ammed cell dea h (187). So a , he known biological unc ions o IFN-γ indica e ha , al hough i can ac as a po en induce o an i umo immuni y, i ac ually has a dual ole and may also a o umo immune e asion. iFN-γ iN CaNCeR The i s epo s poin ing o he ele ance o IFN-γ in an i umo immuni y came om s udies wi h he ib osa coma (Me h A) cell line, e ac o y o IFN-γ signaling, since i lacks he exp ession o he IFNγR1 subuni . IFN-γ-insensi i e Me h A cells displayed enhanced umo igenici y compa ed wi h con ol cells and we e no ejec ed in syngeneic umo mice models, sugges ing ha IFN-γ plays an impo an ole in umo cell elimina ion (18). This inding was u he suppo ed by expe imen s using 129/ SV IFN-γ insensi i e mice, lacking he IFNγR1 subuni o STAT1, which de eloped 3-me hylcholan h ene (MCA)-induced sa comas mo e apidly and mo e equen ly han hei wild- ype coun e pa s (12). Simila ly, hese IFN-γ-insensi i e mice lacking he umo -supp esso p o ein p53 o med spon aneous umo s mo e apidly han IFN-γ-sensi i e p53-de icien mouse (12). In addi ion, C57BL/6 mice ha lack he gene encoding IFN-γ also displayed highe suscep ibili y o expe imen al (B6, RM-1 p os a e ca cinoma) and spon aneous (BALB/c, DA3 mamma y ca cinoma) models o p ima y and me as a ic umo s (13, 14). No ably, u he s udies desc ibed ha IFN-γ may coope a e wi h o he molecules o p e en umo o ma ion. Mice de icien in bo h g anulocy e/mac ophage colony-s imula ing ac o (GM-CSF) and IFN-γ de eloped lymphoma and non-lymphoid solid umo s a a highe a e han did mice de icien in GM-CSF o IFN-γ alone (15). Addi ional s udies e ealed ha mice insen- si i e o IFN-γ, o ha lack he ecombina ion ac i a ing gene (RAG) p o ein ( ailing o p oduce ma u e B and Tlymphocy es), o ha lack bo h, showed simila incidence o MCA-induced sa comas, sugges ing ha he Tcell–IFN-γ axis is in ol ed in immune su eillance (10). The ole o IFN-γ on cance immunoedi ing eme ged om s udies assessing he immunogenici y o umo s om immuno- compe en e sus immunode icien mice. Kaplan e al. showed ha MCA-induced sa coma cells om IFNγR1-de icien mice (un esponsi e o IFN-γ signaling) g ow as agg essi ely in immu- nocompe en as in IFNγR1-de icien mice. Howe e , when IFN-γ esponsi eness was con e ed on he umo cells by in oducing he IFNγR1 subuni , hey became mo e immunogenic and we e ejec ed h ough a Tcell-dependen manne (12). This cons i- u es he i s demons a ion ha IFN-γ sensi i i y o he umo is undamen al o an e icien an i umo esponse. O he s udies e ealed ha wild- ype hos s ejec ed 40% o MCA-induced sa comas de i ed om RAG2-de icien mice, showing ha hese umo s we e mo e immunogenic han hose om wild- ype mice (10). In addi ion, human umo s we e e alua ed o hei abili y o up egula e MHC I exp ession in esponse o IFN-γ s imula ion. These s udies e ealed ha 33% o 33 melanoma umo cell lines showed a educ ion in IFN-γ sensi i i y while 4 o 17 lung adenoca cinoma cell lines we e o ally un esponsi e o IFN-γ (12). This lack o esponse esul ed om cellula de ec s on IFNγR1 and o JAK p o eins and may explain he abili y o many umo cells o e ade he immune esponse. Recen ly, JAK1/2 de iciency was demons a ed o p o ec melanoma cells om an i umo IFN-γ ac i i y and esul s in T-cell- esis an melanoma lesions (188). O he s epo ed he lack o STAT1 in melanoma cell lines and in some ch onic myeloid leukemia cells (189). Fu he mo e, DNA me hyla ion ha selec i ely ep esses CIITA, in colo ec al and gas ic cance cell lines, was associa ed wi h he absence o IFN-γ-induced HLA-DR, sugges ing ha his epigene ic al e a ion o CIITA enables some gas oin es inal cance cells o e ade he immune sys em (190). Concomi an ly, epigene ic al e a ions ep essing MHC2TA we e desc ibed in Tcell leukemias, Bcell lymphomas, and in se e al cance cells, such as small cell lung cance and neu oblas oma cells ha we e unable o exp ess MHC II upon IFN-γ s imula ion (191–194). Consis en ly, IFN-γ up egula es CIITA exp ession on mul iple myeloma and melanoma cells inc easing hei MHC II exp es- sion (123, 124). These indings indica e ha IFN-γ ac s on umo cells, enhancing hei ecogni ion by CD8 Tcells as well as by CD4 Tcells, and un eiling a key ole in he p omo ion o umo immunogenici y. Al oge he , hese wo ks pa e he way o he elabo a ion o he s epping-s one concep o immunoedi ing p omo ed by IFN-γ (195, 196). iFN-γ-Media ed Mechanisms Unde lying an i umo igenic e ec s As desc ibed ea lie , he mechanisms by which IFN-γ exe s i s an i umo e ec s depend on mul iple p ocesses. IFN-γ is desc ibed as an an ip oli e a i e agen ha egula es he exp es- sion o cyclin-dependen kinase inhibi o 1 (p21) h ough STAT1 ac i a ion in umo cells (84, 197). Mo eo e , IFN-γ is able o p omo e umo cells apop osis by up egula ing he exp ession o caspase-1, -3, -8 (198, 199) and by enhancing he sec e ion o FAS and FAS ligand (200) and TNF- ela ed apop osis-inducing 7 Cas o e al. Dual Role o IFN-γ in Cance F on ie s in Immunology | www. on ie sin.o g May 2018 | Volume 9 | A icle 847 ligand (201, 202). Recen s udies showed ha IFN-γ also induces i s umo icidal e ec s h ough a o m o egula ed nec o ic dea h (also named as nec op osis) ha elies on he ac i i y o he se ine– h eonine kinase RIP1 (203). Impo an ly, IFN-γ is also in ol ed in he inhibi ion o angiogenesis, impai ing he p oli - e a ion and su i al o endo helial cells, inducing ischemia in he umo s oma (184, 204, 205). In pa icula , IFNγR is exp essed on blood endo helial cells and engagemen o he ecep o esul s in blood essel des uc ion and nec osis, an impo an mecha- nism ha leads o umo ejec ion (206). Conside ing he e ec o IFN-γ on he hos immune cells p esen a he umo mic oen i onmen , majo e o s ha e been made o he de elopmen and es ablishmen o combined clinical he apeu ic applica ions (90, 151, 207). IFN-γ is c i ical o Tcell, NK and NKTcell a icking in o he umo s h ough CXCL9, CXCL10, and CXCL11 chemokine induc ion (208, 209). Acco dingly, Tcells ail o mig a e o umo si e in IFNγ-de icien mice (65). In commi men , dipep idylpep idase 4 inhibi ion, a p o ease ha inac i a es hese chemokines, enhanced umo ejec ion by inc easing lymphocy es a icking in o he umo (210). La ely, galec in-3 sec e ed by se e al umo s was dem- ons a ed o bind glycosyla ed IFN-γ a he umo ex acellula ma ix, a oiding IFN-γ di usion and he o ma ion o an IFN- γ-induced chemokine g adien equi ed o Tcell ec ui men and in il a ion (211). In addi ion, CXCL10 also p e en s umo angiogenesis by blocking endo helial cell p oli e a ion (212) and consequen ly a dec ease in mic o essel densi y as obse ed in melanoma umo xenog a s (213). Apop osis o endo helial cells by IFNs causes es ic ion o blood low wi hin he umo ascula u e, leading o umo sh inkage (214). This is an e ec o IFN-γ, no di ec ly a ge ed o he umo cell, bu o he umo ascula u e, wi h d as ic and desi able e ec s on umo g ow h. A ecen epo also showed ha IFN-γ was essen ial o he ini ial p iming and di e en ia ion o cy o oxic Tcells esiding in he pe iphe y o he eye, con ibu ing o he eg ession o in aocula umo s (215). Suppo ing da a om he apy models showed ha IFN-γ induces su i in and i i202, wo genes in ol ed in Tcell ma u a ion, su i al, and p oli e a ion, in umo -speci ic Tcells (216). O e all, hese s udies demons a ed he ele ance o IFN-γ on Tcell-media ed an i umo immuni y. In e e on-gamma is also in ol ed in mac ophages umo i- cidal ac i i y (217). This cy okine suppo s a CD4 T cell/ mac ophage e ec o axis which ac s as immune su eillance mechanism o MHC II-nega i e cance cells (25). Indeed, upon ecogni ion o umo an igens p esen in he con ex o MHC II by mac ophages, CD4 Tcells sec e e IFN-γ ha u he ac i a es mac ophages in he umo , leading o umo g ow h inhibi ion (25). This collabo a ion be ween CD4 Tcells and mac ophages was also essen ial o success ul cance immune su eillance in non-solid cance s, as myeloma and B-cell lymphoma. Indeed, Th1-sec e ed IFN-γ was shown o igge a cy o oxic ac i i y o umo -associa ed mac ophages (TAMs) and also induces CXCL9/MIG and CXCL10/IP-10 sec e ion by mac ophages, which may a ec he umo p og ession by angiogenesis inhibi ion (129). IFN-γ-ac i a ed mac ophages also acqui e a umo icidal pheno ype wi h he up egula ion o cy o oxici y- associa ed ma ke s including g anzyme A/B, and NKG2D (129). In addi ion, in STAT6-de icien mice, ha display inc eased le els o IFN-γ, ejec ion o me as a ic disease a e emo al o he p ima y umo in ol ed he gene a ion o p o-in lamma o y mac ophages, also e med M1-like mac ophages, and a dec ease in MSDCs ha accumula ed du ing p ima y umo o ma ion (218). S udies om APCMin/+ mice ( ha a e highly suscep ible o spon aneous in es inal adenoma o ma ion) lacking IFN-γ signaling showed an accumula ion o TAMs, mo e p one owa ds p o umo al (M2-like) pola iza ion, and up egula ion o ma ix me allop o eases. These esul s sugges ha IFN-γ un esponsi e- ness con ibu es o he c ea ion o an an i-in lamma o y mic o- en i onmen , a o able o in es inal umo igenesis (219). The p ope ies o IFN-γ o e e se he myeloid immunosupp essi e unc ions we e also demons a ed in p o umo ole o human o a ian TAMs (220) and human M2-like mac ophages (221). Impo an ly, IFN-γ has also a key ole on IL-12 p oduc ion, suppo ing he ac i i y o his la e cy okine in cance immune su eillance (222–225). Indeed, exogenous IL-12 adminis a ion in o ib osa coma-bea ing mice esul ed in a comple e umo eg ession (222). This obse a ion was ex ended o p ima y umo igenesis models ea ed wi h exogenous IL-12 (226, 227). Consis en wi h his, chime ic an igen ecep o - edi ec ed Tcells enginee ed o p oduce IL-12 whe e ound o sec e e inc eased IFN- γ le els and o display enhanced an i umo cell ac i i y (228–230). Rega ding he impo ance o IFN-γ in cance diagnos ics, IFN- γ-associa ed signa u es ha e a p edic i e alue in cance immune pheno ypes (81, 231, 232). In addi ion, IFN- ela ed gene signa- u e is a p edic i e ma ke o chemo he apy and adio he apy e iciency o b eas cance (94) as well as o PD-1 o cy o oxic Tlymphocy e an igen-4 (CTLA-4) blockade in a ious ypes o malignancies (233–235). Consis en ly, immuno he apy using immune checkpoin blocke s (an i-CTLA-4 and/o an i-PD-1) combined wi h an icance accines, clea ly associa e inhibi ion o umo g ow h wi h inc eased p opo ion o IFN-γ-p oducing e ec o Tcells (236, 237). This is also e i ied in clinical ials, h ough which he an i-CTLA-4 he apy was associa ed wi h an inc ease o IFN-γ-p oducing ICOS+ (inducible cos imula o y) CD4 Tcells and o T e ec o /T eg cell a io in bladde cance samples (238). In addi ion, PD-1 blockade was demons a ed o enhance T cell in il a ion by p omo ing IFN-γ-inducible chemokines (239). In o he way, i was ecen ly shown ha IFN-γ-induced T eg cell agili y (loss o supp essi e unc ion) is equi ed o esponse o an i-PD-1 he apy (240). Al oge he , he e sa ili y o IFN-γ and i s ine- uned biologi- cal e ec s highligh i s ele ance o he apeu ic applica ions, and some clinical ials ha e al eady encou aging esul s. In ac , 75% o me as a ic melanoma pa ien s we e non- esponde s o an i-CTLA-4 he apy, and his was associa ed wi h genomic de ec s o IFN-γ signaling genes on umo s (241). Recen ly, apelin ecep o (APLNR) was desc ibed o egula e JAK/ STAT signaling, modula ing IFN-γ esponses. Mul iple loss- o - unc ion mu a ions in APLNR we e iden i ied in pa ien umo s e ac o y o immuno he apy (242). The inclusion o IFN-γ in he i s -line ea men o o a ian cance esul ed in bene i ega ding p og ession- ee su i al, wi h accep able ox- ici y (243). IFN-γ ea men also appea s o be e ec i e agains bladde umo s by ec ui men and ac i a ion o in a umo al 8 Cas o e al. Dual Role o IFN-γ in Cance F on ie s in Immunology | www. on ie sin.o g May 2018 | Volume 9 | A icle 847 leukocy es (244). In a phase I clinical ial, which combined adop i e T cell he apy wi h in alesional adminis a ion o adeno i us exp essing IFN-γ in me as a ic melanoma, 38.5% o he pa ien s had an o e all objec i e esponse and 46% we e able o con ol he disease (245). iFN-γ-Media ed Mechanisms Unde lying P o umo igenic e ec s I is becoming inc easingly clea ha IFN-γ can exe ce ain e ec s suppo ing umo igenesis. Immune e asion can ope a e h ough umo cells losing esponsi e o IFN-γ signaling o a oid i s an ip oli e a i e, p o-apop o ic, and immuno egula- o y ac ions. This has been demons a ed wi h he umo cells losing he ecep o o IFN-γ o a componen o JAK/STAT signaling (12, 18). In addi ion, cons i u i e ac i a ion o inhibi- o y molecules o his pa hway, as SOCS1 and SOCS3, limi s he ac ions o IFNs on human melanoma cells (246) and a o s he ac i a ion o al e na i e signaling pa hways, as STAT3, which is associa ed wi h umo p og ession (247). These e idences sugges ha umo cells de elop IFN-γ-dependen s a egies o e ade he immune sys em, leading o he eme gence o e y agg essi e umo s, which a e on he basis o immunoedi ing. In 2011, Zaidi and Me lino p oposed ha IFN-γ ac ions migh play a physiological ole in p o ec ing cells om damage in a se ing o issue emodeling and epai , while on cells ha bo ing oncogenic mu a ions, he same mechanisms may p e en cell des uc ion and allow comple e ans o ma ion (248). Consis en wi h his, NF-κB in umo cells was shown o ac as a p o ec i e mechanism agains IFN-γ-induced nec op osis (203). Indeed, he e a e signi ican e idences ha umo cells can ake he ad an age o IFN-γ as an induce o an i-in lamma o y esponses and p o umo e ec s. The i s epo o he nega i e po en ial e ec s was in 1987 by Taniguchi and colleagues who p oposed ha IFN-γ changes he me as a ic abili y o he B16 melanoma cells in a cell-au onomous manne (249). Da a om expe imen s using he CT26 colon ca cinoma model showed ha IFN-γ p omo es umo escape h ough he down egula ion o he endogenous umo an igen gp70 (250). IFN-γ exp ession by human melanoma samples was associa ed wi h enhanced exp ession o MHC class II molecules and he acquisi ion o a mo e agg essi e pheno ype (251, 252). One o he p incipal mechanisms o umo immune escape is he supp ession o cy o oxic Tcells and o NKcell-media ed immune esponses. B ody and colleagues showed ha IFN-γ up egula es IDO in melanoma cells and ec ui s T eg cells o a oid immune ecogni ion (253). Cu iously, IFN-γ induced IDO compe ence on human monocy e-de i ed DCs bu had no e ec on p o-in lamma o y cy okine elease, sugges ing ha IFN-γ igge s IDO ac i i y and p o-in lamma o y cy okine elease as dis inc cellula p og ams. In addi ion, IDO-compe en DCs induced egula o y ac i i y on allogeneic Tcells (179). IFN-γ was also desc ibed o be in ol ed in he accumula ion o MDSCs in in lamed li e , which leads o Tcell supp ession (254). MDSCs p oducing ni ic oxide dec eased IFN-γ esponsi eness o immune cells, such as T and NKcells (255). One impo an aspec is he abili y o IFN-γ o induce PD-L1 exp ession in cance , s omal and myeloid cells o impai e ec o umo immuni y (181). Abiko and colleagues demons a ed ha he con ac be ween umo cells and CD8 Tcells is necessa y o he induc ion o PD-L1, unde lying he impo ance o pa ac ine exposu e o IFN-γ (256). Recen epo s sugges ha loss o IFN-γ pa hway genes, such as JAK1 and JAK2, is associa ed wi h esis - ance o an i-PD-1 he apy (257, 258). P olonged IFN-γ signaling in umo s was also shown o coo dina e PD-L1-dependen and PD-L1-independen esis ance o immune checkpoin blockade and o o he he apeu ic combina ions, such as adia ion and an i-CTLA-4, h ough a mul igenic esis ance p og am (259). In addi ion, o he inhibi o y pa hways a e ein o ced by IFN-γ, including CTLA-4 and CD86/CD80 in e ac ion (260). In e e on-gamma was used in clinical ials o melanoma bu no signi ican imp o emen o pa ien s was obse ed (261–264). In ac , IFN-γ ea men had no con ibu ion o he ou comes o pa ien s wi h me as a ic enal cell ca cinomas (265), leukemia (266), panc ea ic ca cinoma (267), b eas can- ce (268), o in o he pos ope a i e su gical he apy o colon cance (269). Fu he mo e, a phase 3 ial o IFN-γ plus s and- a d ea men wi h ca bopla in/pacli axel e sus ca bopla in o pacli axel alone, o ea ed ad anced o a ian umo s, was ea ly e mina ed due o a highe incidence o se ious hema ological oxici ies in pa ien s ecei ing combined he apy compa ed wi h chemo he apy alone (270). The ailed a emp s o ea cance pa ien s wi h exogenous IFN-γ aised se e al conce ns: he absence o umo immunogenici y, he lack o IFN-γ-signaling componen s, he up egula ion o IFN-γ signaling inhibi o s, he immunosupp essi e umo mic oen i onmen , he lack o e ec o Tcells, o p esence o ane gic Tcells and, in some cases oxici y. These accumula ing e idences ein o ce he impo ance o de e mine he g ade o pa ien s’ IFN-γ- esponsi eness. Fo example, in cases wi h low IFN-γ ac ions, ac i e immuniza ion ei he ia IFN-γ ea men o ia adju an s o he immune sys em, as oll-like ecep o ligands, should be conside ed, as demons a ed ecen ly by using bac e ial ou e memb ane esi- cles ha e adica e es ablished umo s in an IFN-γ-dependen mechanism (271). The combina ion wi h adio- and chemo- he apy is expec ed o be use ul h ough immunogenic cell dea h ha also elici s he inna e immune sys em. P omising esul s we e ob ained wi h combina ion o low-dose 5- luo ou acil wi h ecombinan in e e on-gamma (IFN-γ) in pa ien s wi h ad anced hepa ocellula ca cinoma (272). In cases wi h high le els o IFN-γ signaling, he he apy wi h an i-PD-1/an i-PD- L1 is expec ed o be impo an . O e all, hese indings indica e ha he local immune mic oen i onmen o umo s is complex and a iable and ha o an e ec i e he apy i is essen ial o e alua e, indi idually, he immune p o ile o pa ien s o immune con ex u e [ e iewed in Re . (232, 273)], aking in o accoun ha i may e ol e and modi y h oughou he an icance he apy (Figu e3). iFN-γ iN THeRaPY—wHeRe aRe we aND wHeRe aRe we GOiNG? In e e on-gamma he apy has ensued in clinical applica ions app o ed by he Food and D ug Adminis a ion in he ea - men o ch onic g anuloma ous disease, in 1999 and se e e FiGURe 3 | Dual ace o in e e on-gamma (IFN-γ) in umo immuni y. IFN-γ can display bo h an i umo and p o umo ac i i ies. Unde bo h ci cums ances, IFN-γ in luences umo cells di ec ly and indi ec ly, by ac i a ion o immune cells. The an i umo e ec s comp ise he de elopmen , ec ui men , and ac i a ion o inna e immune cells as well as he ac i a ion and main enance o e ec o Tcells. The an i umo e ec s o IFN-γ esul in di ec inhibi ion o umo p oli e a ion, ecogni ion, and elimina ion. In o he way, he p o umo igenic ole o IFN-γ in ol es p oli e a i e and an iapop o ic signals, as well as escape o he umo cells om ecogni ion and cy olysis by cy o oxic Tlymphocy es (CTLs) and na u al kille (NK) cells. The b oad ange o IFN-γ ac ions depends on he con ex o umo speci ici y, IFN-γ-signaling in ensi y, and o he mic oen i onmen condi ions. 9 Cas o e al. Dual Role o IFN-γ in Cance F on ie s in Immunology | www. on ie sin.o g May 2018 | Volume 9 | A icle 847 malignan os eope osis, in 2000. Despi e he p omising he apeu ic applica ions o IFN-γ in se e al se ings, i s limi ed success in cance -immuno he apy ials migh be due o cance cell un esponsi eness o his cy okine, he ailu e o deli e i locally o wi h he adequa e pe iodici y o achie e a he apeu ic e ec . Mo eo e , IFN-γ clinical use has also been es ic ed due o se e al limi a ions inhe en o i s molecula p ope ies. Essen ially, hese include s abili y p oblems, such as acid deg- ada ion, and also he endency o agg ega e i e e sibly unde mild dena u ing condi ions, wi h subsequen loss o biological ac i i y [ he pha macological aspec o IFN-γ is e iewed in Re . (274, 275)]. Fu he mo e, IFN-γ is apidly clea ed om he blood when adminis e ed in a enously (276), equi ing equen e-adminis a ions o high cy okine concen a ions, o elici an e ec i e esponse a he a ge si e, leading o sys emic oxici y and side e ec s, such as e e , a igue, nausea, omi ing, dia hea, neu o oxici y, and leukopenia (277). These ad e se e ec s a e caused mainly by high se um concen a ion o he p o ein, due o an unequal dis ibu ion be ween body luids and issues (276) and, addi ionally, o he ubiqui y o ecep o s which a e exp essed a he memb ane o he majo i y o human cells (278, 279) and also o he exis ence o a ci cula ing soluble o m (which unc ion emains elusi e) (280). 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