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ROLEOFNKCELLSINIMMUNOTHERAPYANDVIROTHERAPYOFSOLIDTUMORS.

ClaudiaCantoni(a,b,c),KorneelGrauwet(d),GabriellaPietra(a,e),MonicaParodi(a),Maria
CristinaMingari(a,b,e),AndreaDeMaria(b,e,f,),HermanFavoreel(d,),MassimoVitale(e,
)
(a)DepartmentofExperimentalMedicine(DIMES),UniversityofGenova,Genova,Italy
(b)CenterofExcellenceforBiomedicalResearch (CEBR),UniversityofGenova,Genova,
Italy
(c)IstitutoGianninaGaslini,Genova,Italy
(d)LaboratoryofImmunology,DepartmentofVirology,ParasitologyandImmunology,Facultyof
VeterinaryMedicine,GhentUniversity,Belgium
(e)IRCCSAOUSanMartinoISTGenova,Genova,Italy
(f)DepartmentofHealthSciences(DISSAL),UniversityofGenova,Genova,Italy
()Theseauthorsshareseniorauthorship

KEYWORDS:NKcellssolidtumortumormicroenvironmenttumorescapeimmunotherapy
virotherapy

Correspondence to:
MassimoVITALE:massimo.vitale@hsanmartino.it
ClaudiaCANTONI:claudia.cantoni@unige.it

ACKOWLEDGEMENTS:
ThisworkwassupportedbygrantsawardedbyAIRC:IGprojectn.15428(M.V.);Ministerodella
Salute: Ricerca Finalizzata RF20102316197 (A.D.M),RFIG20081200689 (M.C.M.);
ProgrammanazionalediricercasullAIDS,Accordidicollaborazionescientifica45G.11,40H69
(A.D.M.);MIURProgettodiAteneo2014(A.D.M);theSpecialResearchFundofGhentUniversity
(grants01J29110,01J11611and01G01311)(H.F.);F.W.O.Vlaanderen(grants1.5.077.11Nand

G.017615N) (H.F.); the Hercules Foundation (grant AUGE035) (H.F.), and an international
mobilitygrantfromF.W.O.Vlaanderen(H.F.).

ABSTRACT
AlthoughNKcellsareendowedwithpowerfulcytolyticactivityagainstcancercells,theirrolein
differenttherapiesagainstsolidtumorshasnotyetbeenfullyelucidated. Theirinteractionswith
variouselementsofthetumormicroenvironmentaswellastheirpossibleeffectsincontributingto
and/orlimitingoncolyticvirotherapyrenderthispotentialimmunotherapeutictoolstilldifficultto
exploitatthebedside.Herewewillreviewthecurrentliteraturewiththeaimofprovidingnewhints
tomanagethispowerfulcelltypeinfutureinnovativetherapies,suchastheuseofNKcellsin
combinationwithnewcytokines,specificmAbs(inducingADCC),TyrKinaseinhibitors(TKi),
ImmunomodulatoryDrugs(IMiDs),and/orthedesignofoncolyticvirusesaimedatoptimizingthe
effectofNKcellsinvirotherapy.

INTRODUCTION
Immediatelyaftertheirdiscovery,NKcellscaughttheattentionofmanyresearchersfortheirability
tokillaratherwiderangeoftumorcelltypes.Fromtheoutset,NKcellsappearedasareadyto
use cell population that could be employed against a variety of tumors without the need for
selectingspecificclones,asvirtuallyallNKcellswereendowedwithaMHCunrestrictedwide
specificityfortumorcells.Theexploitationofthesecellsforeffectiveimmunotherapyseemedan
achievablegoalandpromptlystimulatedtoinvestigatethemechanismsregulatingtheirfunction.
ThebrilliantintuitionofKlasKarre(whopostulatedthatNKcellswereabletokillonlywhenthey
missedtherecognitionofautologousMHCImoleculesontargetcells)andthepioneeringstudies
ofdifferentgroupsledquiterapidlytodefinetherecognitionmechanismofNKcells[14].Human
NKcellsweredemonstratedtorecognizeandkilltumorcellswithdownregulatedHLAImolecule
expressionbyusingacomplexgroupofHLAIspecificinhibitoryreceptors(KIRsandNKG2A)
andseveralmajoractivatingreceptors(includingNKG2D,DNAM1andtheNaturalCytotoxicity

Receptors NCRs: NKp46,NKp30,NKp44)[5,6].Thesubsequentdefinitionofthe panelof


activatingreceptorligands(currentlystillincomplete)confirmedthat,indeed,someofsuchligands
arecommonlyexpressedbyvarioustumorcelltypes(seeTable1).
InspiteofthesubstantialadvanceinourunderstandingofNKcellbiology,theattemptstoexploit
thesecellsinimmunotherapyhavebeenfrustratingformanyyears.The efficacyoftheadoptive
transfer of ex vivo activated NK cells and/or their in vivo activation by IL2 infusion was
challengedbytheshortlifespanoftransferredmatureNKcells,andbytheclinicalsideeffects
inducedbythecytokine[79].Inaddition,IL2couldalsohaveparadoxicalnegativeeffectsonthe
immunologicalresponsebyinducingtheexpansionofCD25+Tregulatorycells(Tregs)orthe
ActivationInducedCellDeath(AICD)onNKcells[10,11]. Finally,at variancewithinvitro
establishedtumorcelllines,inseveralinstances,primarytumorcellsmaintaintheexpressionof
HLAImoleculesandarethusprotectedfromtheattackofautologousNKcells.Importanthintsto
overcometheseproblemswereofferedbyAndreaVelardisgroup,whosuggestedthatallogeneic
NKcellscoulddevelopfromdonorsstemcellsfollowinghaploidenticalHematopoieticStemCell
Transplantation (HSCT) in patients with AML [12]. In the Tdepleted haploHSCT, the HSC
recipientcanexpressHLAIallelesthatarenotrecognizedbyKIRsofdonorNKcells(HLA/KIR
mismatch).SuchallogeneicNKcellsexpressingKIRsthatfailHLAIallelerecognitionexertan
effectiveantileukemiaactivityinthepatient.Thus,underthisparticularallogeneicsetting,NK
cellsderivedfromdonorsprogenitorsovercomethelimitationsthatinitiallyhinderedthedesignof
effectiveNKbasedimmunotherapies.Duringthepast15years,haploHSCTrevealeditselfasan
extremelyusefultooltocurehighriskleukemiapatientslackinganHLAmatcheddonor.However,
sofarthisapproachhasbeenmostlyconfinedtohematologicalmalignanciesbothinadultsandin
children.[13,14].
Insolidtumorsthepicturemaybemorecomplex.Thedevelopmentofatumororchestratedlocal
microenvironmentcanheavilyimpactontheavailabilityandfunctionofNKcellswithinthetumor

tissue[1517].Aplethoraofsolublefactorsandcellularnetworks,aswellaspeculiarphysical
and/orchemicalconditions,canaffecttheabilityofNKcellstorecognizeandkilltumorcells,or
influencetheirabilitytomigratefromthebloodtothetumorsite.Itshouldalsobeconsideredthat
NKcellspresentinthetumormicroenvironmentmaydirectlyoriginatefromthelocalperipheral
tissues.Inthiscontext,recentstudieshaveshownthatdifferentperipheraltissuesandsecondaryor
tertiarylymphoidorgansmaycontainmembersofanNKrelatedfamilyofinnatelymphoidcells
(ILCs).Thesecellscanusetheirreceptorstofulfillpeculiarfunctions,differentfromkillingtumor
cells[1821].ThisstillexpandingheterogeneousgroupofILCsinclude:1)conventionalcytotoxic
NKcells(recentlyproposedtobedefinedascytotoxicILC1),whichconstitutethelargemajorityof
peripheralblood(PB)NKcells(butarealsopresentintissues), releaseIFN andTNF,and
functionallyinteractwithdifferentAPC; 2)poorlycytolyticCD56brightCD16dim NKcells,which
constitutethelargemajorityoflymphnodal(LN)NKcells(butarealsopresentintissuesandPB),
releaseprimarilyIFNandGMCSF,andinteractwithDC,participatingtotheinductionofTh1
polarizationinLN;3)NKp44+NK22cells(includedintheILC3group),whicharelocatedinthe
MALT,releaseIL22andareinvolvedinthemucosalhomeostasisanddefense;4)decidualNK
cells,whichproduceIFN,IL8,IP10,SDF1andVEGF,andarethoughttoplayanimportant
roleintheplacentaldevelopmentandinductionofmaternalfetaltolerance[22].This complex
repertoireofNKcellsubsetswiththeirdifferentrelativefunctionsshouldalsobeconsideredto
betterexploittheirfunctionalcapabilitiesinoldandnewtherapeuticapproaches,eithertoidentify
newpossiblebeneficialsynergies,oreventorecognizestillunnoticedNKdrivenunwantedeffects.
AnotherwellknownfunctionofNKcellshasalsobecomeimportantfortumortherapy,namely
theirabilitytorecognizeandkillvirusinfectedcells[56].ThisaspectofNKcellbiologyhascome
intoplaybythedesignofstrategiestotargettumorcellsusingoncolyticvirotherapy[23].Inthis
context, NK cells may represent a double edged sword as the NKmediated killing of virally

infectedtumorcellsmayeitherpotentiatetheantitumoreffectofvirusesorlimittheinfection
spreadwithinthetumor[24,25]andthereforepreventsuccessfromoncovirotherapy.
Thus,incontrasttotheinitialidea,theexploitationofNKcellsforthecureofcancerisquite
complicatedandrequiresmultipleapproachesthatshouldbeadaptedtothetypeoftumorand/or
adoptedtherapeuticstrategy.

NKCELLSANDTHETUMORMICROENVIRONMENT
ThepresenceofanNKcellinfiltratehasbeenobservedtovariableextentinseveraltypesofsolid
tumorsincludingmelanomas,gastrointestinalstromaltumors(GIST),colorectal,renal,lungand
breastcancers [2634].Insomecases,GISTandrenalcarcinomalungmetastasesdisplayedhigh
levelsofNKcellinfiltration,whichcorrelatedtoabetterprognosis[29,32].Thetypeandthesize
oftheNKcellinfiltrateinneoplastictissuescandependonseveralfactors,includingthespecific
chemokinesproducedinthetumormicroenvironmentandthechemokinereceptorpatternexpressed
bydifferentNKcellsubsets.Inlungandbreastcancersithasbeenshownthat poorlycytotoxic
CD56brightNKcellsfrequentlyrepresentthemajorNKcellsubsetrecruitedatthetumorsite[31,33].
ThesetumorsexpressedhighlevelsofmRNAcodingforCXCL9andCCL19,twochemokinesare
recognizedbychemokinereceptorsselectivelyexpressedbyCD56bright cells(CXCR3andCCR7
respectively). Ontheotherhand,somestudiesalsoreportasignificantpresenceofCD56 dim NK
cellsinlungtumorsandinmelanomanodalmetastases[30,34].Inthelattercase,cellsfromtumor
tissueweredemonstratedtoproduceIL8,which,indeed,canpreferentiallyattractCD56 dim NK
cells.AnotherimportantissueisthelocationoftheNKcellinfiltratewithinthetumortissue. In
colorectalcancerstudiesithasbeenrecentlyshownthat,inspiteofthepresenceofchemokinesthat
areabletorecruitbothCD56brightandCD56dimNKcellsubsets,NKcellsarelocalizedwithinthe
stroma,ratherthanbeingindirectcontactwithneoplasticcells[35].AsimilarNKcelllocalization

hasbeendetectedinmelanoma andinGIST[32,36,37]. Thesefindingssuggestthatadditional


poorlyidentifiedfactorsmayinfluenceNKcellrecruitmenttothetumornest.
BesidesaffectingNKcellmigrationintotheneoplastictissue,thetumormicroenvironmentcanalso
influencetheefficacyofNKcellswithinthetumor.Differentmechanismscancontributetotumor
escapefromanNKcellmediatedattack:1)theinductionofanimmunosuppressivemilieu;2)the
inhibition of NK cell effector functions; and 3) the selection of tumor cells with an altered
expressionofligandsforNKcellreceptors[15,17,38].
DifferentimmunecellshavebeenshowntointeractwithNKcellsandregulatetheirfunction.Ina
TH1/M1polarizedcontext,thisinteractionresultsinanimprovedantitumorimmuneresponse[39,
40].Unfortunately,immunecellsinthetumormicroenvironmentarefrequentlyalteredorpolarized
towardsatype2response[41]andmayinhibit/modulatethecapacityofNKcellstointeractwith
andkilltumorcells[15,17].Recentstudiesperformedonhepatocellularcarcinomahaveshown
thatmyeloidderivedsuppressorcells(MDSCs)andtumorassociatedmacrophages(TAMs)from
patientsareabletomodulateNKcellfunctions[42,43].Oneimportantmechanismaccountingfor
thesuppressionofNKcellsistheproductionofseveralfactors,includingTGF,IDO,IL4,and
PGE2 [4447],whichcanhinderNKcellactivationorinducedownregulationofNKactivating
receptorsresponsibleforrecognitionandkillingoftumorcells.Forexample,TGF inhibitsthe
expressionofNKp30andNKG2Dactivatingreceptors[45],whileIL4decreasestheabilityofNK
cells to produce cytokines and to interact with DCs [46]. Both IDO and PGE2 can affect the
expressionofdifferentactivatingreceptorsonNKcells,withaconsequentdecreaseinNKcell
cytotoxicactivityagainsttumorcells[36,47].Thesesolublefactorscanbeproducedbyimmune
and/ornonimmunecellswithinthetumormicroenvironment.Forexample,TregscandampenNK
cellfunctionbycompetingforIL2availability,butalsobyexposingmembraneboundTGF[48,
49]. Among nonimmune cells, fibroblasts associated to tumor tissues (TAF), obtained from

melanoma metastases, have been shown to prevent the IL2induced upregulation of NKp44,
DNAM1andNKp30receptors.Thiseffectwasmediatedbothbydirectcelltocellcontactandby
PGE2release[36].AsimilarbehaviorhasbeenobservedinTAFsderivedfromcolorectalcancer
(HCC)andhepatocellularcarcinoma[50,51].TherelevanceofNKcellfibroblastinteractionsin
vivoisfurthersuggestedbythefindingthatinmelanomametastasesandGISTlesionsNKcellscan
localizeinproximitytofibroblastssurroundingtumornests[32,36,37].
Asubstantialsuppressiveroleisalsoexertedbytumorcellsthroughseveralmechanisms.Wehave
recentlydemonstratedthatmelanomaderivedprimarycelllines couldinhibittheexpressionof
several activating NK receptors, thus decreasing NK cellmediated killing of tumor cells. This
effectwasmediatedbyIDOexpressionand/orPGE2release[52].Differenttumorcelltypescan
releaseMHCclassIchainrelatedgeneA(MICA),aligandforNKG2Dactivatingreceptor,by
shedding,thusinducingdownregulationorunresponsivenessofNKG2D[53].Ovariancancercells
caninterferewithNKcellfunctionsbyinducingNKG2Ddownregulationthroughthereleaseof
macrophageinhibitoryfactor(MIF),orbyexpressingtheglycoproteinMUC16,whichcaninhibit
synapseformationbetweenNKcellsandtumorcells[54,55].Tumorcellscanalsoaffecttheability
of NK cells to infiltrate the tumor. It has been recently shown that neuroblastoma cells could
modulatethechemokinereceptorrepertoireofNKcellsthroughTGF production[56].Tumor
cellsfromdifferentcancerscanreleaseB7H6,aligandforNKp30activatingNKreceptor,through
ectodomainsheddingmediatedbycellsurfaceproteasesADAM10andADAM17,thusreducing
NKcellmediatedrecognition[57].Finally,hypoxia,aconditionfrequentlyassociatedtothetumor
microenvironment,caninfluencetheimmuneresponseagainstthetumor. AsfarasNKcellsare
concerned, we have recently demonstrated that the hypoxic status can deeply inhibit both the
expressionandthefunctionofdifferentactivatingreceptors.NKcells,however,werenotaffected
byhypoxiaintheirabilitytoperformAntibodyDependentCellmediatedCytotoxicity(ADCC)
[58].

TheantitumoractivityofNKcellsandtheirprolongedinteractionwithcancercellscanalsoexerta
selectivepressurethatmayfavorthegenerationoftumorcellsdisplayinganabnormalexpressionof
ligandsspecificforactivatingorinhibitoryNKcellreceptors.Ourgrouphasshownthatinvitroco
cultureofmelanomacellswithNKcellsresultedinanIFNdependentupregulationofHLAI
expressiononmelanomacells,whichwasresponsibleforthe(acquired)resistanceofthesecellsto
NKmediated killing [26]. Tumor cells may also escape from NKmediated recognition by
modulating cell surface expression of ligands for activating NK receptors, as in the case for
NKG2Dligands [53].ActivatingreceptorsexpressedonNK cells maybeimportantfortumor
immunoediting. Forexample,ithasrecentlybeenshownthat tumorsthatdevelopedinNKp46
deficientmiceexpressedhigherlevelsofNKp46ligandsascomparedtotumorsthatdevelopedin
wildtypemice[59].TheupregulationofligandsabletotransmitinhibitorysignalstoNKcells
representsanotherpossiblemechanismoftumorescapefromNKcellantitumoractivity.Inthis
context,ithasbeenrecentlyshownthatProliferatingNuclearCellAntigen(PCNA),frequently
overexpressedindifferenttumortypes,canengagetheactivatingNKp44receptorandinducea
paradoxicalinhibitorysignal[60].NKcellcytotoxicfunctioncanalsobeinhibitedthroughthe
engagementofCD300a,aninhibitoryreceptorrecognizingphosphatidylserineexposedoncertain
tumorcells[61].

ROLEOFNKCELLSINTHERAPIESTARGETINGSOLIDTUMORS
Althoughcancerimmunotherapystrategies havemainlyreliedontheactivityoftumorantigen
specificTcells,inrecentyearsthetherapeuticuseofNKcellstotreatmalignancieshasrapidly
movedforward[62,63].Inthissection,wewillreviewtheNKcellbasedtherapeuticapproaches
that are being currently explored in either clinical or preclinical contexts. These approaches
include: a) invivo stimulationof endogenousNKcellsagainsttumors(bytheuseofcytokines,

antibodiesorimmunomodulatorydrugs); b) adoptivetransferofinvitroexpandedandactivated
NKcells(bothinautologousandallogeneicsettings);c)geneticmodificationofNKcells.
ThestimulationofdifferentNKcellfunctions,includingcytotoxicityagainsttumorcellsandIFN
productioncanbeinducedbytheuseofseveralcytokinesincludingIL2,IL15,IL12,IL18and
interferons .TheadministrationofrecombinantIL2resultedinremarkablecytokinerelated
toxicity(suchasvascularleaksyndromeandhearttoxicity)andlimitedclinicalbenefits(possibly
duetotheinductionofTregs)[8,64],Theseissuespromptedthesearchforothercytokinesableto
effectivelyboostNKcells.IL15,IL12,andIL18arebeingevaluatedbothinpreclinical[65]and
clinicalcancermodels [6668].Whilethesecytokinesdisplaylimitedanticanceractivitywhen
usedassingleagents,arecentreportinmiceshowedthatpoorlyfunctionalNKcells,infiltrating
MHCclassIdeficienttumors,couldbeeffectivelyboostedbythecombineddeliveryofIL12and
IL18[69].Thisstudywarrantstheevaluationofthepossibleuseofthiscytokinecombinationin
humans.
TheefficacyofNKcellsintargetingtumorcellsmaybealsoincreasedbyinterveningwiththe
inhibitoryinteractionbetweenKIRsandHLAImolecules,thatarepossiblyexpressedontumor
cells.Inthiscontext,ahumanmonoclonalantibody(mAb)(17F9/IPH2101)abletoblockKIRs
that recognize HLAC molecules has been recently developed [70]. The use of lirilumab (a
recombinantversionof17F9)hasbeenshowntoincreaseNKcellmediatedkillingofHLAC
positivetumorcellsinananimalmodel[71].
The therapeutic potential of PB NK cells can also be exploited for the treatment of both
hematologicalandsolidtumors thankstotheirabilitytomediateADCCthroughcrosslinkingof
CD16.TheNKcellmediatedADCChasbeensuggestedtoberelevanttothetherapeuticeffectof
Rituximab or Cetuximab in mAbbased tumortargeted therapies [72]. Along this line, several

reports indicated that NK cell function could be harnessed by the administration of mAbs
recognizingtumorantigens[73].
Besidescytokinesandantibodies,severalimmunomodulatorydrugsareabletoreinstateNKcell
effector function either directly or indirectly. Among these drugs, Lenalidomide (a synthetic
derivativeofThalidomide)hasbeenextensivelytestedinpatients.Interestingly,Lenalidomidenot
onlydirectlyactsonNKcells(bypromotingboththeirnaturalcytotoxicityandADCCfunction)
butitcanalsosensitizetumorcellstoNKmediatedkilling(byupregulating theexpressionon
tumor cells of ligands recognized by activating NK receptors) [63]. Along this line, several
chemotherapeuticdrugs(suchasetoposideanddacarbazine),histonedeacetylasesinhibitors(such
asVPA),HSP90inhibitorsandproteasomeinhibitorsareabletoinducetheexpressionofTRAIL
andNKG2DLsontumorcells[74].Finally,alsotheTKiarebeingevaluatedfortheireffectson
theNKmediatedtumorcellkilling[reviewedin74].TheinterestontheTKiwasstimulatedbythe
initial observation that Imatinib therapy of GIST patients could induce an increased NK cell
functionthatcorrelatedwithapositivetherapyresponse.Thesubsequentstudiesledtoarather
complex picture showing that different members of the TKi family can modulate the NK cell
functionthroughcombined,evencontrasting,actions,rangingfromthesuppressionofTregsorthe
enhancementoftheNK/DCcrosstalk,tothereductionofNKG2DLexpressionontumorcells.
PerhapsthemostinterestingapproachtoincreasetheefficacyofNKcellsisthesearchforpossible
synergiesbetweentheabovedescribedstrategies.Forexample,thesynergisticeffectoftheKIR
blockade by Lirilumab and the Rituximabmediated ADCC has been recently tested in a KIR
transgenic murine model [71]. A clinical trial using IPH2101 antiKIR mAb combined with
lenalidomideisinprogressinmultiplemyeloma[75].
The early studies employing ex vivo lymphokineactivated killer (LAK)cells showed noclear
benefit of this form of therapy compared with IL2 administration [8, 76]. In a recent study,
Rosenberg and colleagues [77] reported that a lymphodepleting chemotherapy, consisting of

fludarabineandlowdosecyclophosphamide,mightpromotehomeostaticexpansionofautologous
adoptivelytransferredNKcells.Interestingly,thesecellsexpressedCD16andthuscouldmediate
ADCCwithoutcytokineactivation[77].
AutologousNKcellsmaybeinhibitedbyrecognitionofselfHLAImoleculesexpressedbytumor
cells. Therefore, the use of allogeneic NK cells in a KIR/HLAI mismatch setting may be
tentatively appealing, especially in view of the promising results obtained with HSCT in
hematologic malignancies [12, 13]. The adoptive transfer of allogeneic NK cells is under
investigationinsolidtumors,aswitnessedbyrecentclinicaltrialsevaluatingthesafetyandthe
efficacy of this approach [7880]. However, the suppressive effects related to the local
microenvironmentaswellastheuseofmatureNKcells(insteadofHSCT)maycomplicatethe
issue.
The poor ability of transferred NK cells to home to tumor sites represents another important
negativefactorthatmightexplain,atleastinpart,theweakantitumoreffectofadoptiveNKcell
therapyagainstsolidmalignancies.Recently,ithasbeenshownthatNKcells,whenexposedtoIL
15andglucocorticoids,expresshighlevelsofCXCR3thusincreasingtheirpotentialtoinfiltrate
CXCL10+tumors[81].
Finally,anotherintriguingnewtherapeuticstrategy,notyetarrivedinclinicaltesting,isthetumor
targeting with genetically modified NK cells engineered to express chimeric antigen receptors
specificfortumorantigens(CAR)[82]orcytokines.

ONCOLYTICVIROTHERAPYANDSOLIDTUMORS
Overthepasttwodecades,virotherapyhasreceivedincreasingattentionwithregardtoitspotential
application towards (particularly hard to treat) cancers with very poor prognosis, such as
glioblastoma.Thegeneralideabehindoncolyticvirotherapyistheuseof(geneticallyengineered)
virusesthatselectivelyreplicateinandlysetumorcells,whilesparinghealthytissue.Remarkably,

thisapproachmaybeparticularlyinterestingalsointhecureofsolidtumors,sincevirusreplication
andspreadmayenabletheoncolyticvectortopenetratethetumor,andtherebyenhanceimmune
andtherepauticdrugpenetrationincombinationtherapies(insertoldreference86and87;Woller,
2014;Chiocca,2014)..
Sincethefirstapprovalofanoncolyticvirus,adenovirusbasedoncolyticvirusH101(Oncorine),
forcancertreatmentinChinain2005,inparticularforheadandneckcancerincombinationwith
cisplatinbasedchemotherapy,thefieldis rapidlymovingforward(YuW.,2007,CancerDrug
targetsClinicaltrialswithoncolyticadenovirusinchina)(XiaZJ,AiZheng,2004)(GarberK,
2006Journalofnationalcancerinstitute)(insertoldreference83,PolJ.,2014).Thisisillustrated
bydifferentoncolyticvectorscurrentlyindifferentstagesofclinicaltrials,asshownintable2.
OncolyticadenovirusCG0070,modifiedtoexpressGMCSFundercontrolofthehumanE2F1
promotor,isbeingpreparedforphaseII/IIIclinicaltrialstotreatinvasivebladdercancerandhas
providedpromisingresultsinphaseItesting.InphaseItesting,atotalof81%oftreatedpatients
respondedtothetreatmentandthemaximumtolerateddosewasnotreached,demonstratingthe
slumberingpotentialofoncolyticvirotherapy(BurkeJM,2012,J.Urol).
A broad range of oncolytic viral vectors (based on HSV1, reovirus, adenovirus, rhabdovirus,
parvovirus,Newcastlesdiseasevirus,vesicularstomatitisvirus,vacciniavirusandmeaslesvirus)
arebeingassessedinrecentlylaunchedphaseI/IItrialstotreatavarietyofsolidcancers.The
oncolyticvirusesParvOryx,NDVandReolysinarenaturallyoccurringvirusesselectedduetotheir
oncolyticantitumorpotencyandabilitytopreferablytargetandreplicateintumorcells,whilstother
oncolyticviruses,suchasMVNIS,arebasedonattenuatedlaboratorystrainvirusesreportedto
preferably replicate in and lyse tumor cells (MVNIS: Dingli D., 2004, Blood )(ParvOryx:
Geletneky,BMCCancer,2012)(NDV:Mansour,M,JournalofVirology,2011)(Reolysin:Galanes
E. Molecular therapy, 2012). Several oncolytic viruses that are being developed to treat solid
tumorshavebeengeneticallymodifiedtoincreasetumorselectivity.TheoncolyticvirusColoAd1

was generated by passaging a mixture of different adenovirus serotypes on target tumor


representativesofbreast,colon,prostateandpancreaticcancertoselectfortumorspecificityand
oncolyticcapabilities (Kuhn,2008,PlosOne).TheoncolyticHSV1basedvirusHSV1716lacks
the viral virulence factor ICP34.5 which attenuates the virus since it loses the ability to
dephosphorylateeukaryotictranslationinitiationfactor2,and,thereby,failstosuppresshostcell
mediatedshutoffofproteinsynthesisinhealthycells(HSV17161: MacLeanAR,JGenVirol,
1991(Harland,2002,Genetherapy)(PapanastassiouV,GeneTher,2002)(LeibDA,PNAS,2000)
(ChouJ.,1995,PNAS).However, byyetunidentifiedfactors, HSV1716was foundto beless
attenuated in various tumors cells, thereby providing some tumor selectivity (Randazzo BP,
Virology,1996).
Althoughremovalofvirulencefactorscandrasticallyincreasesafety,severeattenuationofthe
oncolyticviruscanalsointerferewithitsoncolyticefficacy.Tocircumventthisproblem,oncolytic
viruseshavebeenconstructedthatarespecificallyattenuatedinnontumorcells.Forexample,the
oncolyticvirusRQNestin34.5,avectorbasedonanICP34.5deletedHSV1straincontainsthe
ICP34.5geneundercontrolofthetumorselectivenestinpromotor.Asaresult,thisvectorshows
specificallyincreasedreplicationintumorcellsandenhancedantitumorspecificityandpotencyin
mice, compared to the ICP34.5deleted HSV1 backbone (Kambara, Cancer Res, 2005). This
rQnNestin34.5 vector is currently being investigated in phase I clinical trials (Ning J, 2014,
frontiersinmicrobiology).
Severaloncolyticvirusescurrentlyassessedinclinicaltrialsexpressadditionalgeneswiththeintent
tomanipulatetheextracellulartumorenvironment(suchastheextracellularmatrix),tomodulate
thehostsimmunityortoanalyzetheoncolyticvirusspreadinvivobyreportergenes.Interestingly,
theVCN01virus,ahumanadenoviruscurrentlyassessedinclinicalphaseItrials,isparticularly
designedtoaddresscurrentsolidtumorspecificrestrictions.Thedenseextracellulartumormatrix
hasbeenidentifiedasapotentialadditionalhurdleinvirotherapyofsolidtumors (Reviewedin:

Wojton,2010,CytokinegrowthfactorRev.).Thisinterlockedmeshworkofmatrixproteinsformsa
physical barrier that not only suppresses access of NK cells and other immune cells, but also
hindersspreadandpenetrationoftheoncolyticvirus,leadingtosuboptimaltherapeuticeffects.In
linewiththis,virusspreadinsolidtumorswasreportedtobelimitedtodiscretetumorareasupon
exvivoinfectionofsolidtumorswithNewcastlesdiseasevirus,andinclinicaltissuesfrompatients
treatedwithG207,anHSV1basedoncolyticviruswithcompletedphaseI/IItrials(Yaakov,2012,
Journalgeneralvirology)(MarkertJM,2009,MolTher).Inthisrespect,theoncolyticadenovirus
basedvectorVCN01wasdesignedtodegradehyaluronicacid,amajorconstituentofextracellular
tumor matrix, via expression of the PH20 hyaluronidase. In mice with prestablished tumors,
expression of PH20 hyaluronidase by an oncolytic virus following intratumoral and systemic
administration has been reported to increase efficacy and improved spread within the tumor,
comparedtotheemptyvirusbackbone(GuedanS,MolTher,2010).PreclinicaltrialswithVCN01
inmiceandhamstersalsoprovidedpromisingresults,supportingselectivity,safetyandantitumor
activityofVCN01(RodriguezGarcia,2015,ClinCancer).

ONCOLYTICVIROTHERAPYANDNKCELLS
Theinteractionofoncolyticviruseswiththehostimmunesystemisparticularlydelicateandmay
have a critical impact on the therapeutic efficacy. On the one hand, oncolytic vectortriggered
immuneresponsesmaybeessentialelementsoftheantitumoreffectsofthetherapy.Ontheother
hand,prematureclearanceoftheselfreplicatingoncolyticvectorbythehostimmuneresponsemay
severelylimittherapeuticpotential[25].Hence,oneofthemostimportantchallengestowardsthe
generationofsuccessfulselfreplicatingoncolyticviralvectorsistofinetunetheinteractionofthe
vector with the host immune system in such a way that beneficial vectortriggered antitumor
immuneresponsesaremaximizedwhileallowingsufficienttimeforthevectortoreplicateand
spreadbeforebeingeliminatedbytheimmuneresponse.

ParticularlyimportantplayersinthisdelicatebalanceareNKcells,displayingbothpotentanti
tumorandantiviralactivity.

NaturalKillercellvirusinteraction.
TheinteractionofviruseswithNKcellsfindsitsframewithinthecoevolutionaryadaptationofthe
immunesystemtoinvadingpathogens.Afterthefirstcharacterizationoftheirmechanismofaction
andtheobservationoftheinvasive,severe,disseminatedclinicalcourseofotherwiselocalizedviral
infections(e.g.herpesviruses)inpatientswithNKcelldeficiencies[88,89],NKcellshavebeen
recognizedasarelevantfirstlinedefenseagainstviruses.Thisoriginalviewhasbeenradically
upgradedinrecentyears,leadingtotheadditionofseveralNKcellfunctionalfeatures including
memorylikeactivitythroughselectivesubsetexpansionuponviralinfection(HCMV,MCMV,
Chikungunya,Hantavirus)andinherentregulationofNCRexpression/function[90,91].
NK cells can sense infected cells either through direct interaction with pathogenassociated
molecularpatternsviaTolllikereceptors,orthroughrecognitionofviraland/or(virusinduced)
cellularligandsofmajoractivatingNKcellreceptors.Suchactivatinginteractions,combinedwith
thereducedengagementofinhibitoryNKreceptorsduetoafrequentlydecreasedsurfaceHLAI
expressiononinfectedcells,caninduceNKcellcytotoxicactivityandcytokinesecretion.
NKcellsdirectlyinteractwithandbindtoviralglycoproteinsthroughtheNCRs(NKp46,NKp30,
NKp44)asdescribed,forexample,forinfluenza,WestNile,anddengueviruses[9295](seeTable
3).Inaddition,NCRsrecognizestillundefinedvirusinducedcellularligands.Whethersomeof
theseligandscouldbesharedwithtumorcellsispoorlyknown,eveniftherearehintssuggesting
thatanewlyidentifiedNKp44ligand,21speMLL5,couldbebothexpressedbytumorcellsand
inducedonTcellsbyHIVgp41peptides.Alongthisline,alsoNKG2DandDNAM1receptors
recognizeligandsonvirallyinfectedcellsthatarecommontotumorcells:MICA/BandULBPs,
andPVRandNectin2respectively.Inthiscontext,ithasbeenshownthatseveralvirusesactivate

DNAdamageresponse,acommonpathwayinvolvedintheregulationofNKG2DandDNAM1
ligandexpressionininfectedandtumorcells[53,96,97].
ViruseshavedevelopedstrategiestoevadeNKcellcontrolontheirreplicationeithertargeting
activatingorinhibitoryreceptorexpression/recognitionortamperingwiththeexpressionofcellular
ligandsforNKcellactivatingreceptorsinthecellstheyarereplicating,orsimultaneouslyinducing
combinations of these mechanisms (see table 4). Accordingly, in HIV infection NK cells are
activated[98],showdecreasedNCRexpression[99],decreasedproductionofIFNandperforin,
increasedproportionsofnoncytolyticandnonsecretingCD56CD16+ NKcells[100],increased
expression of HLAspecific inhibitory receptors (KIRs) [101], reduced expression [102], or
increasedshedding[103,104],ofligandsforactivatingreceptors.Herpesvirusesmayinteractwith
and degrade ligands for activating NK cell receptors [105107] or may encode viral proteins
mimickingHLAclassImoleculesthatwillblockNKcellfunctionthroughinteractionwithKIRs
(orNKG2A).Alongthisline,HCVmayblockNKcellfunctionthroughtriggeringofCD81onNK
cells,ormaydecreasetheirproductionofIFN uponIL12signaling[108,109],ormayinduce
theirproductionofimmunosuppressivecytokinessuchasIL10[110].
GeneticindividualvariabilityhoweverplaysarelevantroleinmoldingthegeneralizedNK/virus
interaction into different individual responses to the same invading pathogen. Inherent/genetic
carriageofgivenHLAclassIspecificinhibitoryreceptorsmayimproveNKcellactivityagainst
viruses. In HCV infection, for example, the combination of specific KIRs and HLAC alleles
associatestohigherlikelihoodtoclearacuteinfectionandtorespondtopegylatedIFNa/ribavirin
treatment.InadditionchronicallyinfectedHCVpatients displayatleasttwodifferentNKcell
transcriptionalsignatureswhichareassociatedwithdifferentresponsestoantiviraltreatment[91],
thussuggestingthatinherent/geneticNKcellregulationsaffecttheresponsetoviruses.Thisis
furtherprovenbytheobservationofdifferentNKp30orNKG2Dsurfacemodulationinpatients
clearingacuteHCVinfectionorclearingchronicinfectionuponantiviraltreatment [111114].

Similarly,inHIV1infectionanonprogressiveclinicalcourseisassociatedwithKIR3DS1and
HLABw4carriage[115]andwithaninherent/geneticregulationofNKp44,NKp30andNKp46
expressionuponNKcellactivation[116].
Overall, therefore, the manipulation of virus/NK cell interaction for the purpose of oncolytic
virotherapy may profit from addressing and generally interfering with the so far highlighted
mechanisms, but also may require additional insight in individual NK cell responses to viral
triggers.Indeedindividualgeneticandinherentpatientdifferencesmayaffecttheintendedoutcome
thusbenefitingfromintegratedindividualizedstrategies.

NKcelldependentoncolyticvectors
Forsomeoncolyticviralvectors,theantitumoreffecthasbeenreportedtolargelydependonNK
cellactivity.Forexample,theoncolyticpotentialofOrfvirusinmurinemodelsoflungcancerand
ofSindbisvirusinSCIDmicebearinghumanovariancarcinomatumorsstronglylargelydependson
NK cell activity [117118]. Increased antitumor activity of NK cells may occur due to virus
inducedmodulationoftheNKcellactivating/inhibitoryligandprofileonthesurfaceofinfected
cancercells.Indeed,infectionofgliomacells withtheoncolyticagentmyxomavirus leads to
downregulation of MHC I surface expression via the viral M153R protein and consequent
increasedNKcellmediatedlysisofhumangliomatumorsinSCIDmiceandprolongedmouse
survival [119]. Also, parvovirus infection of colon carcinoma cells leads to increased NCR
dependenttumorcellkillingbyNKcells(Bhat&Rommelaere,2013)..Besidessuchdirecteffect
onNKcellmediatedlysisoftumorcells,viralinfectionmayalsoindirectlyleadtoincreasedanti
tumor activity of NK cells. In this context, the oncolytic rhabdovirus Maraba MG1 infects
conventionaldendriticcells,whichleadstomaturationofthesecellsandconsequentlyincreased
NKcellmediatedantitumorresponses[120].

Genetic engineering of oOncolytic virotherapy withvectors to trigger enhanced NK cell


activation
SeentheimportantcontributionofNKcellstotheantitumoreffectofdifferentoncolyticviruses,
severalgeneticallyengineeredoncolyticvectorshavebeendeveloped thatshowwiththeaimof
triggeringenhancedNKcellresponses,oftenbasedonthestrategiesexplainedhigherintheNK
cellbasedimmunotherapysection.Someofthesehaveledtopromisingresults.Forexample,a
strongantitumorresponsewasobservedinaxenograftmousemodelofhumanpancreaticductal
adenocarcinomausinggeneticallyengineeredoncolyticparvovirusesthatexpressIL2orMCP
2/CCL7,whichledtoefficientrecruitmentofNKcellsandmonocyticcellstothetumornest[121].
Beneficialeffectsofcombiningvirotherapywithbiological treatments usingIL2havealsobeen
reportedforoncolyticVSV[122,123].ForSindbisvirus,viralexpressionofIL12ledtoanNK
celldependentincreaseinanticancerefficacyinSCIDmicebearinghumanovariancarcinoma
tumors[118].Inlinewiththis,geneticallyengineeredoncolyticadenovirusesexpressingIL12or
coexpressingIL12andIL18resultedinincreasedinfiltrationandactivityofNKandTcellsin
tumortissuesinmurinemodelsofprostateadenocarcinomaandofmelanomarespectively,and
consequentlyincreasedmousesurvival[124,125].Anotheroncolyticadenovirusengineeredwitha
CpGenriched genome resulted in a significant and NK celldependent increase in antitumor
efficacyinaxenograftmodeloflungcancer [126]. Also,theuseofanoncolyticvacciniavirus
strain expressing an agonist antibody against the costimulatory molecule CD137 (41BB) in a
mousemodelofbreastcarcinomaresultedinincreasedantitumorefficacythatdependedonNK
cells,Tcellsandneutrophils[127].

NKcellsandoncolyticvirusesincancervaccination

Somegroupshavealsoharnessedtheabilityofoncolyticvirusestotriggertheantitumorimmune
response,includingtheNKcellresponse,inthedesignofcancervaccines.Immunizationofmice
with coloncarcinomacellsthatwerepreviouslyinfectedinvitrowithanoncolyticVSVvirus
protectedtheanimalsfromsubsequenttumorchallenge,aneffectthatwasfurtherenhancedby
expressionofGMCSFbythevirus[128].Therearealsoindicationsthatthereciprocalinteraction
betweenDCandNKcellsmaybebeneficialinthecontextofcancervaccinationusingviruses.DC
loadedwithreovirusinfectedhumanmelanomacells(butnotreovirusinfectedtumorcellsalone)
inducedchemotaxisofNKcellsandIFN productionandantitumorcytotoxicityinNKcells,
whichpossiblymaycontributetotumorregression [129]. Inaddition,vaccinationofmicewith
dendritic cells (DC) harboring a nontransmissible recombinant Sendai virus prevented lung
metastasisofneuroblastoma(butnotprostaticcancer).DepletionstudiesindicatedthatNKcells
werecrucialfortheprotectiveeffect[130].

InhibitoryeffectsofNKcellsononcolyticvirotherapy
Although,asindicatedabove,(enhanced)NKcellactivitymaycontributetothetherapeuticeffects
of particular oncolytic viruses, the antiviral activity of NK cells may also lead to unwanted
prematureclearanceofthevirus,reducingitstherapeuticpotential.SuchnegativeeffectofNKcell
activity is particularly relevant when the oncolytic virotherapy involves the administration of
relativelylowdosesofvirusthatneedstoundergomultipleroundsofreplicationtosufficiently
spreadwithinthetumorandreachmaximaltherapeuticeffectiveness.Insuchcases,ideally,the
antiviraleffectofNKcellsshouldbeminimal,especiallyduringtheearlystagesoftreatment,while
retaining the antitumor activity of NK cells [131]. One approach to reach this goal is to
genetically engineer oncolytic vectors so that they display an optimized activating/inhibitory

balancetowardsNKcells,whichmayinvolvetheintroductionof(viral)NKcellevasivegenesin
theoncolyticviralgenome.
In rats bearing multifocal hepatocellular carcinoma and treated with oncolytic VSV, NK cell
depletionresultedinalogarithmicelevationofintratumoralVSVtitersandanenhancedantitumor
response,indicatingasuppressiveeffectofNKcells onthetherapeuticeffectoftheoncolytic
vectorinthissetup[132].Interestingly,geneticengineeringofherpesvirusimmuneevasiongenes
intheVSVgenomewasabletocounteractNKcellactivityandenhancetheoncolyticpotentialof
VSVinthisanimalmodel.Indeed,introductionoftheequineherpesvirus1chemokinebinding
viralgGglycoproteininVSVreducedthenumberofNKandNKTcellsinthetumors,resultingin
a1logenhancementofintratumoralvirustiters,increasedtumornecrosisandprolongedanimal
survival without toxicities [133]. In a followup study, NK cell activity was more specifically
suppressedviaintroductionofthehumancytomegalovirusUL141geneinVSV.UL141encodesa
viralproteinthatdownregulatesCD155andCD112,ligandsfortheNKcellactivatingreceptor
DNAM1[133,134].Again,recombinantVSVexpressingUL141resultedinaonelogelevationof
intratumoralvirusreplication,enhancedtumornecrosisandprolongedsurvival[135].
AnoptimizedbalanceofNKactivation/inhibitionsignalsmaybeparticularlyrelevantfor
herpessimplexvirus1(HSV1)basedoncolyticvectors,asthesetypicallydependonseveral
viralreplicationroundsandefficientspreadwithinthetumortobeeffective [136]. Indeed,
AlvarezBreckenridge et al. elegantly demonstrated that NK cells are responsible for
prematureclearanceofanHSV1basedoncolyticvectorinamouseglioblastomamodel[24].
TheyshowedthatNKcellactivityistriggeredbyHSV1inducedupregulationofNCRligands,and
that NK cellmediated clearance of the viral vector represents an important limitation in
glioblastomavirotherapy[24].Inlinewiththesedata,thepositiveeffectsofepigeneticmodulators
likevalproicacidandhistonedeacetylaseinhibitorsonHSV1basedoncotherapyinglioblastoma

mousemodelswereas foundtocorrelatewithreducedrecruitmentofNKcellstotumorbearing
brainsandsuppressedNKcellcytotoxicity [136]. Hence,itwillbeinterestingtodeterminethe
oncolyticpotentialoffuturerecombinantHSV1oncolyticvectorsthatencodeNKcellsuppressing
proteins. A particularly interesting candidate may be the gD glycoprotein of the porcine
alphaherpesviruspseudorabiesvirus(PRV),whichiscloselyrelatedtoHSV1.PRVgDhas
beenreportedrecentlytointerferewithDNAM1dependentNKcellmediatedkillingthrough
downregulationoftheDNAM1ligandCD112bothinporcineandhumancells,including
humanglioblastomacells[107].

Combinationofviro/chemotherapyandNKcells
The future of oncolytic virotherapy, like that of NK cellbased oncotherapy, will probably
involve its integration in combination therapy with more conventional approaches like
chemotherapy[84].Hence,futureresearchwillneedtocarefullyinvestigatewhethersometherapy
combinationsmayreinforceorrathercounteractoneanother.Bothtypesofoutcomehavebeen
reportedandmaydependatleastinpartonsynergisticorcounteractingeffectsonNKcellactivity
bythecombinationtherapy.Ontheonehand,inamousemodelofaggressivehumanovarian
cancer,thecombinationofSindbisvirotherapywithtopoisomeraseinhibitorchemotherapyresulted
insynergisticNKcelldependenteffectivetherapy,whichwasnotobservedwitheithertreatment
alone [137]. On the other hand, although combining oncolytic VSV with cyclophosphamide
chemotherapy increased VSV titers in mesothelioma tumors in mice, it unexpectedly reduced
therapeutic efficacy compared to cyclophosphamide alone. This antagonistic effect appeared to
involve a VSVinduced TGFdependent suppression of cyclophosphamidetriggered NK cell
activity[138].

CONCLUSIONSANDFUTUREPERSPECTIVES
Attempts at exploiting NK cell adoptive use or NK cell manipulation for a successful
immunotherapy and/or virotherapy of solid tumours (Figure 1) overall have been so far
stymied by a combination of factors including too early clearance (and inefficient tumor
penetration/intratumoralspread)ofoncolyticviruses,poorNKcellpenetrationorlocalizationin
proximityoftumorcells andsusceptibilityofinfiltratingNKcellstobemanipulatedand
disarmed.Theinformationandlessonslearntfromthesefailures,togetherwithnewinsights
gatheredinNKcellbiologyindifferenthealthconditionswillprovideanimprovedbasisfor
futureImmuno/oncovirotherapy.Inparticular,theindepthknowledgeoftheroleofNKcells
inthepresentandfuturetherapeuticapproachesisaprerequisitetoidentifycrucialsynergies
(figure1).Inthiscontext,strategiestoincreaseNKcell:tumourinteractionanddirectkilling,
andtumourtargetingdrugsormAbsarebeingpresentlyevaluated.Manipulationofviruses
usedforoncotherapywillbetargetedatreducingtooearlyvirusclearancebyNKcellsthus
allowingspereadtothewholetumorthusallowingimprovedkilling.Allthesestrategiesand
new methods will need to carefully take into account the phenotypic and functional
characteristicsofNKcellsinindividualpatients.
Finally, evidences showing that a very high NK cell phenotype diversity exists in each
individual will also need to beconsidered in future work.Indeed, standardized therapies
using NK cell manipulation in any given group of patients may conflict with different
interindividualcharacteristicsinvolvingNKcellfunctionandphenotypeinthesepatientsand
therefore may lead to treatment failure independent from an intrinsic usefulness of the
immuno/virotherapyregimen.NewtechniquesofNKcellorvirusmanipulation(e.g.single

cell transcriptional analysis, masscytomery, etc.) will provide useful tools to optimally
individualizeimmunotherapeuticregimensexploitingNKcell:tumorinteraction.

EXECUTIVESUMMARY

NKcellsandthetumormicroenvironment.
o Tumor microenvironment can regulate the availability and location of NK cells
withinthetumortissue
o TumormicroenvironmentcanregulatethefunctionofNKcellswithinthetumor

tissue
o TumorcellscanedittheirsurfacephenotypetoescapeNKcellattack
RoleofNKcellsintherapiestargetingsolidtumors
o NKbasedimmunotherapy
o mAbmediatedtumortargeting
o DrugmediatedtumortargetingandIMiDs
RoleofNKcellsinoncolyticvirotherapy
o NKcellsvirusinteraction
o NKcelldependentoncolyticvectors
o OncolyticvirotherapywithenhancedNKcellactivation
o NKcellsandoncolyticvirusesincancervaccination
o InhibitoryeffectsofNKcellsononcolyticvirotherapy
o Combinationofviro/chemotherapyandNKcells

REFERENCES
1. *Krre K. How to recognize a foreign submarine. Immunol Rev. 155, 5-9
(1997).
*The story of how the Missing Self recognition hypothesis
has been conceived as told by the author himself.
2. *Moretta A, Vitale M, Bottino C, et al. P58 molecules as putative receptors
for majorhistocompatibility complex (MHC) class I molecules in human
natural killer (NK) cells. Anti-p58 antibodies reconstitute lysis of MHC
class I-protected cells in NK clones displaying different specificities. J.
Exp. Med. 178, 597-604 (1993).
3. *Litwin V, Gumperz J, Parham P, Philips J, Lanier LL. NKB 1: a natural killer
cell receptor involved in the recognition of polymorphic HLA-B molecules.
J. Exp. Med. 178, 597-604 (1993).
*The first two papers describing HLA-I specific NK
receptors.
4. *Sivori S, Vitale M, Morelli L, et al. p46, a novel natural killer cell-specific
surface molecule that mediates cell activation. J. Exp. Med. 186, 11291136 (1997).
*The first paper describing an activating receptor
specifically expressed by human NK cells.

5. **Vivier E, Raulet DH, Moretta A, et al. Innate or adaptive immunity? The


example of natural killer cells. Science 331, 44-49 (2011).
**a comprehensive review describing the most recent
insights on the NK cell functions and the panel of NK receptors
involved in their control.
6. Moretta L, Bottino C, Pende D, Castriconi R, Mingari MC, Moretta A.
Surface NK receptors and their ligands on tumor cells. Semin. Immunol.
18, 151-158 (2006).
7. Zhang Y, Wallace DL, de Lara CM, et al. In vivo kinetics of human natural
killer cells: the effects of ageing and acute and chronic viral infection.
Immunology 121, 258-265 (2007).
8. Kammula US, White DE, Rosenberg SA. Trends in the safety of high dose
bolus interleukin-2 administration in patients with metastatic cancer.
Cancer 83, 797-805 (1998).
9. Overwijk WW, Schluns KS. Functions of C cytokines in immune
homeostasis: current and potential clinical applications. Clin Immunol.
132, 153-165 (2009).
10.
Rodella L, Zamai L, Rezzani R, et al. Interleukin 2 and interleukin 15
differentially predispose natural killer cells to apoptosis mediated by
endothelial and tumour cells. Br J Haematol. 115, 442-450 (2001).
11.
Ghiringhelli F, Mnard C, Martin F, and Zitvogel L. The role of
regulatory T cells in the control of natural killer cells: relevance during
tumor progression. Immunol. Rev. 214, 229-238 (2006).
12.
*Ruggeri L, Capanni M, Casucci M, et al. Role of natural killer cell
alloreactivity
in
HLA-mismatched
hematopoietic
stem
cell
transplantation. Blood 94(1):333-339 (1999).
*The seminal paper showing the potential of alloreactive NK
cells coming from HSCT.
13.
Locatelli F, Pende D, Mingari MC, et al. Cellular and molecular basis
of haploidentical hematopoietic stem cell transplantation in the
successful treatment of high-risk leukemias: role of alloreactive NK cells.
Front Immunol. 4, 15 (2013).
14.
Knorr DA, Bachanova V, Verneris MR, Miller JS. Clinical utility of
natural killer cells in cancer therapy and transplantation. Semin Immunol.
26, 161-172 (2014)
15.
Stojanovic A, Cerwenka A. Natural killer cells and solid tumors. J
Innate Immun 3, 355-364 (2011).
16.
Baginska J, Viry E, Paggetti J, et al. The critical role of the tumor
microenvironment in shaping natural killer cell-mediated anti-tumor
immunity. Front Immunol. 4, 490 (2013).
17.
Vitale M, Cantoni C, Pietra G, Mingari MC, Moretta L. Effect of tumor
cells and tumor microenvironment on NK-cell function. Eur. J. Immunol.
44, 1582-1592 (2014).
18.
Shi FD, Ljunggren HG, La Cava A, and Van Kaer L. Organ-specific
features of natural killer cells. Nat. Rev. Immunol. 11, 658-671 (2011).
19.
Montaldo E, Vacca P, Moretta L, Mingari MC. Development of human
natural killer cells and other innate lymphoid cells. Semin Immunol. 26,
107-113 (2014).

20.
Eberl G, Di Santo JP, Vivier E. The brave new world of innate
lymphoid cells. Nat Immunol. 16, 1-5 (2015).
21.
Sivori S, Olive D, Lopez-Botet M, Vitale M. NK receptors: tools for a
polyvalent cell family. Front Immunol 5, 617 (2014).
22.
Vacca P, Moretta L, Moretta A, and Mingari MC. Origin, phenotype
and function of human natural killer cells in pregnancy. Trends Immunol.
32, 517-523 (2011).
23.
Chiocca EA, Rabkin SD. Oncolytic viruses and their application to
cancer immunotherapy. Cancer Immunol Res. 2, 295-300 (2014).
24.
*Alvarez-Breckenridge CA, Yu J, Price R, et al. NK cells impede
glioblastoma virotherapy through NKp30 and NKp46 natural cytotoxicity
receptors. Nat. Med. 18, 1827-1834 (2012).
* Review paper covering the beneficial and unwanted
effects of NK cells in oncolytic virotherapy
25.
**Alvarez-Breckenridge CA, Yu J, Kaur B, Caligiuri MA, Chiocca EA.
Deciphering the Multifaceted Relationship between Oncolytic Viruses and
Natural Killer Cells. Adv. Virol. 2012, 702839 (2012).
** Landmark paper showing that NK cells may be a limiting
factor in oncolytic virotherapy by premature clearance of
the viral vector.
26.
Balsamo M, Vermi W, Parodi M, et al. Melanoma cells become
resistant to NK-cell-mediated killing when exposed to NK-cell numbers
compatible with NK-cell infiltration in the tumor. Eur. J. Immunol. 42,
1833-1842 (2012).
27.
Burke S, Lakshmikanth T, Colucci F, Carbone E. New views on
natural killer cell-based immunotherapy for melanoma treatment. Trends
Immunol. 31, 339-345 (2010).
28.
Delahaye NF, Rusakiewicz S, Martins I, et al. Alternatively spliced
NKp30 isoforms affect the prognosis of gastrointestinal stromal tumors.
Nat. Med. 17, 700-707 (2011).
29.
Remark R, Alifano M, Cremer I, et al. Characteristics and clinical
impacts of the immune environments in colorectal and renal cell
carcinoma lung metastases: influence of tumor origin. Clin Cancer Res.
19, 4079-4091 (2013).
30.
Platonova S, Cherfils-Vicini J, Damotte D. et al. Profound coordinated
alterations of intratumoral NK cell phenotype and function in lung
carcinoma. Cancer Res. 71, 5412-5422 (2011).
31.
Carrega P, Bonaccorsi I, Di Carlo E, et al. CD56(bright)perforin(low)
noncytotoxic NK cells are abundant in both healthy and neoplastic solid
tissues and recirculate to secondary lymphoid organs via afferent lymph.
J. Immunol. 192, 3805-3815 (2014)
32.
Rusakiewicz S, Semeraro M, Sarabi M, et al. Immune infiltrates are
prognostic factors in localized gastrointestinal stromal tumors. Cancer
Res. 73, 3499-3510 (2013).
33.
Mamessier E, Sylvain A, Thibult ML, et al. Human breast cancer
cells enhance self tolerance by promoting evasion from NK cell antitumor
immunity. J. Clin. Invest. 121, 3609-3622 (2011).
34.
Ali
TH,
Pisanti
S,
Ciaglia
E,
et
al.
Enrichment
of
CD56(dim)KIR+CD57+ highly cytotoxic NK cells in tumour-infiltrated
lymph nodes of melanoma patients. Nat. Commun. 4,5, 539 (2014)

35.
Halama N, Braun M, Kahlert C, et al. Natural killer cells are scarce in
colorectal carcinoma tissue despite high levels of chemokines and
cytokines. Clin. Cancer Res. 17, 678-689 (2011).
36. Balsamo M, Scordamaglia F, Pietra G, et al. Melanoma-associated
fibroblasts modulate NK cell phenotype and antitumor cytotoxicity. Proc.
Natl. Acad. Sci. U S A. 106, 20847-20852 (2009).
37.
Messaoudene M, Fregni G, Fourmentraux-Neves E, et al. Mature
cytotoxic CD56(bright)/CD16(+) natural killer cells can infiltrate lymph
nodes adjacent to metastatic melanoma. Cancer Res. 74, 81-92 (2014).
38.
Vesely MD, Kershaw MH, Schreiber RD, Smyth MJ. Natural innate
and adaptive immunity to cancer. Annu. Rev. Immunol. 29, 235-271
(2011).
39.
Moretta A, Marcenaro E, Sivori S, Della Chiesa M, Vitale M, Moretta
L. Early liaisons between cells of the innate immune system in inflamed
peripheral tissues. Trends Immunol. 26, 668-675 (2005).
40.
Bellora F, Castriconi R, Dondero A, et al. TLR activation of tumorassociated macrophages from ovarian cancer patients triggers cytolytic
activity of NK cells. Eur J Immunol. 44, 1814-1822 (2014).
41.
Whiteside T. The tumor microenvironment and its role in tumor
growth. Oncogene. 27, 5904-5912 (2008).
42.
Hoechst B, Voigtlaender T, Ormandy L, et al. Myeloid derived
suppressor cells inhibit natural killer cells in patients with hepatocellular
carcinoma via the NKp30 receptor. Hepatology. 50, 799-807 (2009).
43.
Sprinzl MF, Reisinger F, Puschnik A, et al. Sorafenib perpetuates
cellular anticancer effector functions by modulating the crosstalk
between macrophages and natural killer cells. Hepatology. 57, 23582368 (2013).
44.
Kalinski P. Regulation of immune responses by prostaglandin E2. J.
Immunol. 188, 21-28 (2012).
45.
Castriconi R, Cantoni C, Della Chiesa M, et al. Transforming growth
factor beta 1 inhibits expression of NKp30 and NKG2D receptors:
consequences for the NK-mediated killing of dendritic cells. Proc. Natl.
Acad. Sci. U S A. 100, 4120-4125 (2003).
46.
Marcenaro E, Della Chiesa M, Bellora F et al. IL-12 or IL-4 prime
human NK cells to mediate functionally divergent interactions with
dendritic cells or tumors. J. Immunol. 17, 3992-3998 (2005).
47.
Della Chiesa M, Carlomagno S, Frumento G, et al. The tryptophan
catabolite L-kynurenine inhibits the surface expression of NKp46- and
NKG2D-activating receptors and regulates NK-cell function. Blood. 108,
4118-4125 (2006).
48.
Ghiringhelli F, Menard C, Terme M, et al. CD4+CD25+ regulatory T
cells inhibit natural killer cell functions in a transforming growth factorbeta-dependent manner. J Exp Med. 202, 1075-1085 (2005).
49.
Sitrin J, Ring A, Garcia KC, Benoist C, Mathis, D. Regulatory T cells
control NK cells in an insulitic lesion by depriving them of IL-2. J. Exp.
Med. 210, 1153-1165 (2013).
50.
Li T, Yang Y, Hua X. Hepatocellular carcinoma-associated fibroblasts
trigger NK cell dysfunction via PGE2 and IDO. Cancer Lett. 318, 154-161
(2012).

51.
Li T, Yi S, Liu W, et al. Colorectal carcinoma-derived fibroblasts
modulate natural killer cell phenotype and antitumor cytotoxicity. Med.
Oncol. 30, 663 (2013).
52.
Pietra G, Manzini C, Rivara S, et al. Melanoma cells inhibit natural
killer cell function by modulating the expression of activating receptors
and cytolytic activity. Cancer Res. 72, 1407-1415 (2012).
53.
Raulet DH, Gasser S, Gowen BG, Deng W, Jung H. Regulation of
ligands for the activating NKG2D receptor. Annu. Rev. Immunol. 31, 413441 (2013).
54.
Krockenberger M, Dombrowski Y, Weidler C, et al. Macrophage
migration inhibitory factor contributes to the immune escape of ovarian
cancer by downregulating NKG2D. J. Immunol. 180, 7338-7348 (2008).
55.
Gubbels JA, Felder M, Horibata S, et al. MUC16 provides
immune protection by inhibiting synapse formation between NK and
ovarian tumor cells. Mol. Cancer. 9, 11 (2010).
56.
Castriconi R, Dondero A, Bellora F, et al. Neuroblastoma-derived
TGF-b1 modulates the chemokine receptor repertoire of human resting
NK cells. J. Immunol. 190, 5321-5328 (2013).
57.
Schlecker E, Fiegler N, Arnold A, et al. Metalloprotease-mediated
tumor cell shedding of B7.H6, the ligand of the natural killer activating
receptor NKp30. Cancer Res. 74, 3429-3440 (2014).
58.
Balsamo M, Manzini C, Pietra G, et al. Hypoxia downregulates the
expression of activating receptors involved in NK-cell-mediated target cell
killing without affecting ADCC. Eur. J. Immunol. 43, 2756-2764 (2013).
59.
Elboim M, Gazit R, Gur C, Ghadially H, Betser-Cohen G, Mandelboim,
O. Tumor immunoediting by NKp46. J. Immunol. 184, 5637-5644 (2010).
60.
Rosental B, Brusilovsky M, Hadad U, et al. Proliferating cell nuclear
antigen is a novel inhibitory ligand for the natural cytotoxicity receptor
NKp44. J. Immunol. 187, 5693-5702 (2011).
61.
Lankry D, Rovis TL, Jonjic S, Mandelboim, O. The interaction
between CD300a and phosphatidylserine inhibits tumor cell killing by NK
cells. Eur. J. Immunol. 43, 2151-2161 (2013).
62.
Ames E, Murphy WJ. Advantages and clinical applications of natural
killer cells in cancer immunotherapy. Cancer Immunol Immunother. 63,
21-28 (2014).
63.
Terme M, Ullrich E, Delahaye NF, Chaput N, Zitvogel L. Natural killer
cell-directed therapies: moving from unexpected results to successful
strategies. Nat Immunol. 9, 486-494 (2008).
64.
Atkins MB, Lotze MT, Dutcher JP, et al. High-dose recombinant
interleukin 2 therapy for patients with metastatic melanoma: analysis of
270 patients treated between 1985 and 1993. J Clin Oncol. 17, 2105-2116
(1999).
65.
*Ni J, Miller M, Stojanovic A, Garbi N, Cerwenka A. Sustained
effector function of IL-12/15/18-preactivated NK cells against established
tumors. J Exp Med. 209, 2351-2365 (2012).
*An important study in mice showing that IL-12, IL-15 and
IL-18 cytokine combination boost NK cell anti-tumor activity.
66.
Vacchelli E, Galluzzi L, Eggermont A, et al. Trial Watch:
Immunostimulatory cytokines. Oncoimmunology. 1, 493-506 (2012).

67.
Tarhini AA, Millward M, Mainwaring P, et al. A phase 2, randomized
study of SB-485232, rhIL-18, in patients with previously untreated
metastatic melanoma. Cancer. 115, 859-868 (2009).
68.
Conlon KC, Lugli E, Welles HC, et al. Redistribution,
Hyperproliferation, Activation of Natural Killer Cells and CD8 T Cells, and
Cytokine Production During First-in-Human Clinical Trial of Recombinant
Human Interleukin-15 in Patients With Cancer. J Clin Oncol. 33, 74-82
(2015).
69.
*Ardolino M, Azimi CS, Iannello A, et al. Cytokine therapy reverses
NK cell anergy in MHC-deficient tumors. J Clin Invest. 124, 4781-4794
(2014).
*For the first time the authors showed that IL-12 and IL-18
treatment restores responsiveness of anergic NK cells present
within MHC class I deficient solid tumors.
70.
Romagn F, Andr P, Spee P, et al. Preclinical characterization of 17F9, a novel human anti-KIR receptor therapeutic antibody that
augments natural killer-mediated killing of tumor cells. Blood. 114, 26672677 (2009).
71.
*Kohrt HE, Thielens A, Marabelle A, et al. Anti-KIR antibody
enhancement of anti-lymphoma activity of natural killer cells as
monotherapy and in combination with anti-CD20 antibodies. Blood. 123,
678-686 (2014).
* this paper provids a rationale for a treatment regimen
that includes mab-mediated KIR blockade and therapeutic mAbs
72.
Pahl JH, Rusian EN, Buddingh EP et al. Anti-EGFR antibody
Cetuximab enhances the cytolytic activity of natural killer cells towards
osteosarcoma. Clin. Cancer Res. 18, 432-441 (2012).
73.
Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat Rev
Cancer. 12, 278-287 (2012).
74.
Krieg S, Ullrich E. Novel immune modulators in hematology: impact
on NK cells. Front. Immunol. 3, 388 (2013).
75.
Benson DM Jr, Bakan CE, Zhang S, et al. IPH2101, a novel antiinhibitory KIR antibody, and lenalidomide combine to enhance the natural
killer cell versus multiple myeloma effect. Blood. 118, 6387-6391 (2011).
76.
Law TM, Motzer RJ, Mazumdar M, et al. Phase III randomized trial of
interleukin-2with or without lymphokine-activated killer cells in the
treatment of patients with advanced renal cell carcinoma. Cancer. 76,
824-832 (1995).
77.
Parkhurst MR, Riley JP, Dudley ME, Rosenberg SA. Adoptive transfer
of autologous natural killer cells leads to high levels of circulating natural
killer cells but does not mediate tumor regression. Clin Cancer Res. 17,
6287-6297 (2011).
78.
Miller JS, Soignier Y, Panoskaltsis-Mortari A, et al. Successful
adoptive transfer and in vivo expansion of human haploidentical NK cells
in patients with cancer. Blood. 105, 3051-3057 (2005).
79.
Geller MA, Cooley S, Judson PL, et al. A phase II study of allogeneic
natural killer cell therapy to treat patients with recurrent ovarian and
breast cancer. Cytotherapy. 13, 98-107 (2011).

80.
Iliopoulou EG, Kountourakis P, Karamouzis MV, et al. A phase I trial
of adoptive transfer of allogeneic natural killer cells in patients with
advanced non-small cell lung cancer. Cancer Immunol Immunother. 59,
1781-1789 (2010).
81.
Wennerberg E, Kremer V, Childs R, Lundqvist A. CXCL10-induced
migration of adoptively transferred human natural killer cells toward solid
tumors causes regression of tumor growth in vivo. Cancer Immunol
Immunother. 64, 225-235 (2015).
82.
Oberoi P, Wels WS. Arming NK cells with enhanced antitumor
activity: CARs and beyond. Oncoimmunology. (8), e25220 (2013).
83.
Pol J, Bloy N, Obrist F, et al. Trial Watch:: Oncolytic viruses for
cancer therapy. Oncoimmunology. 3, e28694 (2014).
84.
Miest TS, Cattaneo R. New viruses for cancer therapy: meeting
clinical needs. Nat Rev Microbiol. 12, 23-34 (2014).
85.
Yu W, Fang H. Clinical trials with oncolytic adenovirus in China. Curr.
Cancer Drug Targets. 7(2), 141148 (2007).
86.
Woller N, Grlevik E, Ureche C-I, Schumacher A, Khnel F. Oncolytic
viruses as anticancer vaccines. Front. Oncol. 4, 188 (2014).
87.
Chiocca EA, Rabkin SD. Oncolytic viruses and their application to
cancer immunotherapy. Cancer Immunol. Res. 2(4), 295300 (2014).
88.
Lopez C, Kirkpatrick D, Read SE, et al. Correlation Between Low
Natural Killing of Fibroblasts Infected with Herpes Simplex Virus Type 1
and Susceptibility to Herpesvirus Infections. J. Infect. Dis. 147, 1030-1035
(1983).
89.
Orange JS. Natural killer cell deficiency. J.
Allergy and Clin.
Immunol. 132, 515-525 (2013).
90.
Petitdemange C, Becquart P, Wauquier N et al. Unconventional
Repertoire Profile Is Imprinted during Acute Chikungunya Infection for
Natural Killer Cells Polarization toward Cytotoxicity. PLoS Pathog. 7,
e1002268 (2011).
91.
Marras F, Bozzano F, Ascierto ML, De Maria A. Baseline and dynamic
expression of activating NK cell receptors in the control of chronic viral
infections: the paradigm of HIV-1 and HCV. Front. Immunol. 5 (2014).
92.
Mandelboim O, Lieberman N, Lev M, et al. Recognition of
hemagglutinins on virus-infected cells by NKp46 activates lysis by human
NK cells. Nature. 409, 1055-1060 (2001).
93.
Arnon TI, Lev M, Katz G, Chernobrov Y, Porgador A, Mandelboim O.
Recognition of viral hemagglutinins by NKp44 but not by NKp30. Eur. J.
Immunol. 31, 2680-2689 (2001).
94.
Hershkovitz O, Rosental B, Rosenberg LA, et al. NKp44 receptor
mediates interaction of the envelope glycoproteins from the West Nile
and dengue viruses with NK cells. J. Immunol. 183, 2610-2621 (2009).
95.
Chisholm SE, Reyburn HT. Recognition of vaccinia virus-infected
cells by human natural killer cells depends on natural cytotoxicity
receptors. J. Virol. 80, 2225-2233 (2006).
96.
Ferrari de Andrade L, Smyth MJ, Martinet L. DNAM-1 control of
natural killer cells functions through nectin and nectin-like proteins.
Immunol. and Cell Biol. 92, 237-244 (2014).
97.
Cerboni C, Fionda C, Soriani A, et al. The DNA damage response: a
common pathway in the regulation of NKG2D and DNAM-1 ligand

expression in normal, infected and cancer cells. Front. Immunol. 4, 508


(2014)8
98.
Fogli M, Costa P, Murdaca G, et al. Significant NK cell activation
associated with decreased cytolytic function in peripheral blood of HIV-1infected patients. Eur. J. Immunol. 34, 2313-2321 (2004).
99.
De Maria A, Fogli M, Costa P, et al. The impaired NK cell cytolytic
function in viremic HIV-1 infection is associated with a reduced surface
expression of natural cytotoxicity receptors (NKp46, NKp30 and NKp44).
Eur. J. Immunol. 33, 2410-2418 (2003).
100.
Mavilio D, Lombardo G, Kinter A, et al. Characterization of the
defective interaction between a subset of natural killer cells and dendritic
cells in HIV-1 infection. J. Exp. Med. 203, 2339-2350 (2006).
101.
De Maria A, Mavilio D, Costa P, Dignetti P, Fogli M, Mingari MC.
Multiple HLA-class I-specific inhibitory NK receptor expression and IL-4/IL5 production by CD8+ T-cell clones in HIV-1 infection. Immunol. Lett. 72,
179-182 (2000).
102.
Ward J, Bonaparte M, Sacks J, et al. HIV modulates the expression of
ligands important in triggering natural killer cell cytotoxic responses on
infected primary T-cell blasts. 110, 1207-1214 (2007).
103.
Matusali G, Tchidjou HK, Pontrelli G, et al. Soluble ligands for the
NKG2D receptor are released during HIV-1 infection and impair NKG2D
expression and cytotoxicity of NK cells. FASEB J. 27, 2440-2450 (2013).
104.
Cerboni C, Neri F, Casartelli N, et al. Human immunodeficiency virus
1 Nef protein downmodulates the ligands of the activating receptor
NKG2D and inhibits natural killer cell-mediated cytotoxicity. J. Gen. Virol.
88, 242-250 (2007).
105.
Lisnic VJ, Krmpotic A, Jonjic S. Modulation of natural killer cell
activity by viruses. Curr. Opin. Microbiol. 13, 530-539 (2010).
106.
Jost S, Altfeld M. Evasion from NK cell-mediated immune responses
by HIV-1. Microbes Infect. 14, 904-915 (2012).
107.
Grauwet K, Cantoni C, Parodi M, et al. Modulation of CD112 by the
alphaherpesvirus gD protein suppresses DNAM-1-dependent NK cellmediated lysis of infected cells. Proc. Natl. Acad. Sci. U S A. 111, 1611816123 (2014).
108.
Crotta S, Brazzoli M, Piccioli D, Valiante NM, Wack A. Hepatitis C
virions subvert natural killer cell activation to generate a cytokine
environment permissive for infection. J. Hepatology 52, 183-190 (2010).
109.
Crotta S, Stilla A, Wack A, et al. Inhibition of Natural Killer Cells
through Engagement of CD81 by the Major Hepatitis C Virus Envelope
Protein. J. Exp.Med. 195:35-42 (2002).
110.
De Maria A, Fogli M, Mazza S, et al. Increased natural cytotoxicity
receptor expression and relevant IL-10 production in NK cells from
chronically infected viremic patients. Eur. J. Immunol. 37, 445-455 (2007).
111.
Amadei B, Urbani S, Cazaly A, et al. Activation of Natural Killer Cells
During Acute Infection With Hepatitis C Virus. Gastroenterology 138,
1536-1545 (2010).
112.
Golden-Mason L, Cox AL, Randall JA, Cheng L, Rosen HR. Increased
natural killer cell cytotoxicity and NKp30 expression protects against
hepatitis C virus infection in high-risk individuals and inhibits replication
in vitro. Hepatology 52, 1581-1589 (2010).

113.
Werner JM, Heller T, Gordon AM, et al. Innate immune responses in
hepatitis C virus-exposed healthcare workers who do not develop acute
infection. Hepatology 58, 1621-1631 (2013).
114.
Bozzano F, Picciotto A, Costa P, et al. Activating NK cell receptor
expression/function (NKp30,NKp46,DNAM-1) during chronic viraemic HCV
infection is associated with the outcome of combined treatment. Eur. J.
Immunol. 41, 2905-2914 (2011).
115.
Martin MP, Gao X, Lee J-H, et al. Epistatic interaction between
KIR3DS1 and HLA-B delays the progression to AIDS. Nat Genet 31, 429434 (2002).
116.
Marras F, Nicco E, Bozzano F, et al. Natural killer cells in HIV
controller patients express an activated effector phenotype and do not
up-regulate NKp44 on IL-2 stimulation. Proc. Natl. Acad. Sci. U S A. 10,
11970-11975 (2013).
117.
Rintoul JL, Lemay CG, Tai L-H, et al. ORFV: a novel oncolytic and
immune stimulating parapoxvirus therapeutic. Mol. Ther. J. Am. Soc.
Gene Ther. 20(6), 11481157 (2012).
118.
Granot T, Venticinque L, Tseng J-C, Meruelo D. Activation of
cytotoxic and regulatory functions of NK cells by Sindbis viral vectors.
PloS One. 6(6), e20598 (2011).
119.
Ogbomo H, Zemp FJ, Lun X, et al. Myxoma virus infection promotes
NK lysis of malignant gliomas in vitro and in vivo. PloS One. 8(6), e66825
(2013).
120.
Zhang J, Tai L-H, Ilkow CS, et al. Maraba MG1 virus enhances
natural killer cell function via conventional dendritic cells to reduce
postoperative metastatic disease. Mol. Ther. J. Am. Soc. Gene Ther. 22(7),
13201332 (2014).
121.
Dempe S, Lavie M, Struyf S, et al. Antitumoral activity of
parvovirus-mediated IL-2 and MCP-3/CCL7 delivery into human pancreatic
cancer: implication of leucocyte recruitment. Cancer Immunol.
Immunother. CII. 61(11), 21132123 (2012).
122.
Kottke T, Diaz RM, Kaluza K, et al. Use of biological therapy to
enhance both virotherapy and adoptive T-cell therapy for cancer. Mol.
Ther. J. Am. Soc. Gene Ther. 16(12), 19101918 (2008).
123.
Kottke T, Galivo F, Wongthida P, et al. Treg depletion-enhanced IL-2
treatment facilitates therapy of established tumors using systemically
delivered oncolytic virus. Mol. Ther. J. Am. Soc. Gene Ther. 16(7), 1217
1226 (2008).
124.
Freytag SO, Barton KN, Zhang Y. Efficacy of oncolytic adenovirus
expressing suicide genes and interleukin-12 in preclinical model of
prostate cancer. Gene Ther. 20(12), 11311139 (2013).
125.
Choi I-K, Lee J-S, Zhang S-N, et al. Oncolytic adenovirus coexpressing IL-12 and IL-18 improves tumor-specific immunity via
differentiation of T cells expressing IL-12R2 or IL-18R. Gene Ther.
18(9), 898909 (2011).
126.
Cerullo V, Diaconu I, Romano V, et al. An oncolytic adenovirus
enhanced for toll-like receptor 9 stimulation increases antitumor immune
responses and tumor clearance. Mol. Ther. J. Am. Soc. Gene Ther. 20(11),
20762086 (2012).

127.
John LB, Howland LJ, Flynn JK, et al. Oncolytic virus and anti-4-1BB
combination therapy elicits strong antitumor immunity against
established cancer. Cancer Res. 72(7), 16511660 (2012).
128.
Lemay CG, Rintoul JL, Kus A, et al. Harnessing oncolytic virusmediated antitumor immunity in an infected cell vaccine. Mol. Ther. J.
Am. Soc. Gene Ther. 20(9), 17911799 (2012).
129.
Prestwich RJ, Errington F, Steele LP, et al. Reciprocal human
dendritic cell-natural killer cell interactions induce antitumor activity
following tumor cell infection by oncolytic reovirus. J. Immunol. Baltim.
Md 1950. 183(7), 43124321 (2009).
130.
Komaru A, Ueda Y, Furuya A, et al. Sustained and NK/CD4+ T celldependent efficient prevention of lung metastasis induced by dendritic
cells harboring recombinant Sendai virus. J. Immunol. Baltim. Md 1950.
183(7), 42114219 (2009).
131.
Kaufmann JK, Chiocca EA. Glioma virus therapies between bench
and bedside. Neuro-Oncol. 16(3), 334351 (2014).
132.
Altomonte J, Wu L, Chen L, et al. Exponential enhancement of
oncolytic vesicular stomatitis virus potency by vector-mediated
suppression of inflammatory responses in vivo. Mol. Ther. J. Am. Soc.
Gene Ther. 16(1), 146153 (2008).
133.
Tomasec P, Wang ECY, Davison AJ, et al. Downregulation of natural
killer cell-activating ligand CD155 by human cytomegalovirus UL141.
Nat. Immunol. 6(2), 181188 (2005).
134.
Prodhomme V, Sugrue DM, Stanton RJ, et al. Human
cytomegalovirus UL141 promotes efficient downregulation of the natural
killer cell activating ligand CD112. J. Gen. Virol. 91(Pt 8), 20342039
(2010).
135.
Altomonte J, Wu L, Meseck M, et al. Enhanced oncolytic potency of
vesicular stomatitis virus through vector-mediated inhibition of NK and
NKT cells. Cancer Gene Ther. 16(3), 266278 (2009).
136.
Alvarez-Breckenridge CA, Yu J, Price R, et al. The histone
deacetylase inhibitor valproic acid lessens NK cell action against
oncolytic virus-infected glioblastoma cells by inhibition of STAT5/T-BET
signaling and generation of gamma interferon. J. Virol. 86(8), 45664577
(2012).
137.
Granot T, Meruelo D. The role of natural killer cells in combinatorial
anti-cancer therapy using Sindbis viral vectors and irinotecan. Cancer
Gene Ther. 19(8), 588591 (2012).
138.
Willmon C, Diaz RM, Wongthida P, et al. Vesicular stomatitis virusinduced immune suppressor cells generate antagonism between
intratumoral oncolytic virus and cyclophosphamide. Mol. Ther. J. Am. Soc.
Gene Ther. 19(1), 140149 (2011).
139.
Koch J, Steinle A, Watzl C, Mandelboim O. Activating natural
cytotoxicity receptors of natural killer cells in cancer and infection.
Trends Immunol. 34, 182-191 (2013).
140.
Hecht ML, Rosental B, Horlacher T, et al. Natural cytotoxicity
receptors NKp30, NKp44 and NKp46 bind to different heparan
sulfate/heparin sequences. J. Proteom Res. 8, 712-720 (2009).
141.
Pogge von Strandmann E, Simhadri VR, von Tresckow B, et al. A.
Human leukocyte antigen-B-associated transcript 3 is released from

tumor cells and engages the NKp30 receptor on natural killer cells.
Immunity. 27, 965-974 (2007).
142.
Reiners KS, Topolar D, Henke A, et al. Soluble ligands for NK cell
receptors promote evasion of chronic lymphocytic leukemia cells from NK
cell anti-tumor activity. Blood. 121, 3658-3665 (2013).
143.
Brandt CS, Baratin M, Yi EC, et al. The B7 family member B7-H6 is a
tumor cell ligand for the activating natural killer receptor NKp30 in
humans. J. Exp. Med. 206, 1495-1503 (2009).
144.
Schlecker E, Fiegler N, Arnold A, et al. Metalloprotease-mediated
tumor cell shedding of B7.H6, the ligand of the natural killer activating
receptor NKp30. Cancer Res. 74, 3429-3440 (2014).
145.
Baychelier F, Sennepin A, Ermonval M, Dorgham K, Debre P,
Vieillard. Identification of a cellular ligand for NKp44. Blood. 122, 29352942 (2013).
146.
Bottino C, Castriconi R, Pende D, et al. Identification of PVR
(CD155) and Nectin-2 (CD112) as cell surface ligands for the human
DNAM-1 (CD226) activating molecule. J. Exp. Med. 198, 557-567 (2003).
147.
Arnon TI, Achdout H, Lieberman N, et al. The mechanosms
controlling the recognition of tumor- and virus-infected cells by NKp46.
Blood. 103, 664-672 (2004).
148.
Jarahian M, Watzl C, Fournier P, et al. Activation of natural killer
cells by Newcastle disease virus hemagglutinin-neuraminidase. J. Virol.
83, 8108-8121 (2009).
149.
Jarahian M, Fiedler M, Cohnen A, et al. Modulation of NKp30- and
NKp46-mediated natural killer cell responses by poxviral hemagglutinin.
PLoS Pathog. 7, e1002195 (2011).
150.
Chisholm SE, Howard K, Gomez MV, Reyburn HT. Expression of ICP0
is sufficient totrigger natural killer cell recognition of herpes simplex
virus-infected cells by natural cytotoxicity receptors. J. Infect. Dis. 195,
1160-1168 (2007).
151.
Schepis D, DAmato M, Studah M, Bergstrom T,Karre K, Berg L.
Herpes simplex virus infection downmodulates NKG2D ligand expression.
Scand. J. Immunol. 69, 429-436 (2009).
152.
Costa P, Sivori S, Bozzano F et al IFN-alpha-mediated increase in
cytolytic activity of maturing NK cell upon exposure to HSV-infected
myelomonocytes. Eur. J. Immunol. 39, 147-158 (2009).
153.
Arnon TI, Achdout H, Levi O, et al. Inhibition of the NKp30 activating
receptor by pp65 of human cytomegalovirus. Nat. Immunol. 6, 515-523
(2005).
154.
Magri G, Muntasell A, Romo N, et al. NKp46 and DNAM-1 NK-cell
receptors drive the response to human cytomegalovirus-infected myeloid
dendritic cells overcoming viral immune evasion strategies. Blood. 117,
848-856 (2011).
155.
Madrid AS, Ganem D. Kaposis sarcoma-associated herpesvirus
ORF54/dUTPase downregulates a ligand for the NK activating receptor
NKp44. J. Virol. 86, 8693-8701 (2012)
156.
Dupuy S, Lambert M, Zucman D, et al. Human herpesvirus 8 (HVV8)
sequentially shapes the NK cell repertoire during the course of
asymptomatic infection and Kaposi sarcoma. PLoS Pathog. 8, e1002486
(2012).

157.
Holder KA, Stapleton SN, Gallant ME, Russell RS, Grant MD.
Hepatitis C virus-infected cells downregualte NKp30 and inhibit ex vivo
NK cell functions. J. Immunol. 191, 3308-3318 (2013).
158.
Bhat R, Rommelaere J. NK-cell-dependent killing of colon carcinoma
cells is mediated by natural cytotoxicity receptors (NCRs) and stimulated
by parvovirus infection of target cells. BMC Cancer. 13, 367- (2013).
159.
Vieillard V, Strominger K, Debr P. NK cytotxicity against CD4+ T
cells during HIV-1 infection: a gp41 peptide induces the expression of an
NKp44 ligand. Proc. Natl. Acad. Sci. U S A. 102, 10981-10986 (2005).
160.
Fausther-Bovendo H, Sol-Foulon N, Candotti D, et al. HIV escape
from natural killer cytotoxicity: nef inhibits NKp44L expression on CD4 + T
cells. AIDS. 23, 1077-1087 (2009).
161.
Richard J, Sindhu S, Pham TNQ, Belzile J-P, Cohen A. HIV-1 Vpr upregulates expression of ligands for the activating NKG2D receptor and
promotes NK cell-mediated killing. Blood. 115, 1354-1363 (2010).
162.
A. Muntasell, C. Vilches, A. Angulo, M. Lpez-Botet, Adaptive
reconfiguration of the human NK-cell compartment in response to
cytomegalovirus: A different perspective of the host-pathogen
interaction. European Journal of Immunology 43, 1133 (05/01, 2013).
163.
Bjorkstrom NK, Lindgren T, Stoltz M, Fauriat C, Braun M, Evander M,
et al. Rapid expansion and long-term persistence of elevated NK cell
numbers in humans infected with hantavirus. J. Exp.Med. 208, 1321
(2011).

FIGURELEGEND.
Figure1.RoleofNKcellsintumortherapy:potentialsynergies.NKcellscanplayapivotal
role in different therapeutic strategies. In NK cellbased immunotherapies NK cells are
directlyexploitedtokilltumorcells.IntumortargetedtherapiesNKcellscanparticipateto
the antitumor therapeutic effects through different mechanisms: mAbinduced ADCC or
drugincreasedNKcellcytotoxicity.Invirotherapy,NKcellsmayexerteitherapositiveora
negativeeffectontheefficacyoftherapy.Dependingon severalfactorssuchas virus type,
virusdoseandvirusreplicationrate,NKcellscaneitherparticipateinclearingthetumorby
killinginfectedtumorcells,or limit virus spreadwithinthetumor byprematurelykilling
infectedtumorcells. Thesearchforpossiblesynergisticeffectsamongdiversetherapeutic

approaches(yellowarrows)mayrepresentavaluablestrategytothedesignoffutureeffective
therapies.

Table 1: Major activating NK receptors and their ligand(s) on tumor cells


Activating
NK
receptor

Ligand(s) on
tumor cells

Ref.

NKp46
(CD335)

Unknown

4, 6, 139

HSPGa
BAT3b/BAG6c

140
139, 141,
142

NKp30
(CD337)

B7-H6
HSPGa
NKp44
(CD336)

NKG2D
DNAM-1
(CD226)

143, 144

PCNA *

140
60

MLL5e isoform

145

HSPGa
MICA/B,
ULBP1-6
PVR (CD155)
Nectin2
(CD112)

140
53

96, 146

* The interaction with NKp44 results in a paradoxical inhibitory signal.


a
HSPG: heparan sulfate proteoglycans; bBAT3: HLA-B-associated Transcript 3; cBAG6:
BCL-2-associated athanogene 6; dPCNA: proliferating cell nuclear antigen; eMLL5:
Mixed-lineage leukemia-5.

Table 2: Recently launched oncolytic clinical virotherapy trials in


cancer patients (based on www.clinicaltrials.gov).
Virus
CG0070

DNX-2401

VCN-01

ColoAd1

viral vector
human
adenovirus
expressing
GMCSF
oncolytic
adenovirus
human
adenovirus
expressing PH20
hyaluronidase
chimeric
Ad11p/Ad3
adenovirus

Indication(s)

Phase

Status

Route

Notes

Ref

Non-muscle
invasive bladder
cancer

II/III

Recruitin
g

i.ves.

as a single agent

NCT01438112

glioblastoma

glioblastoma or
gliosarcoma

solid tumors

pancreatic
cancer
Epithilial solid
tumors, bladder
and colorectal
cancer
overian
carcinoma
malignant
melanoma

Recruitin
g
Recruitin
g
Recruitin
g
Recruitin
g

i.b.p.
i.t.
i.v.
i.t.

combined with
temozolomide
combined with
IFN-
combined with
gemcitabine
combined with
gemcitabine

NCT01956734
NCT02197169
NCT02045602
NCT02045589

I/II

Recruitin
g

i.v.

as a single agent

NCT02028442

I/II

Recruitin
g

i.per.

as a single agent

NCT02028117

recruiting

e.v.

as a single agent

NCT01864759

NCT02285816

ICOVIR-5

oncolytic
adenovirus

MG1
Maraba
/MAGE-A3

rhabdo virus
expressing
tumorassociated
antigen MAGEA3

solid tumors

I/II

Recruitin
g

i.v.

Combined with
AdMa3,
adenovirus
expressing
MAGE-A3, as
priming
component

HF10

attentuated HSV1 virus

unresectable or
metastatic
melanoma

II

Recruitin
g

i.t

combined with
Ipilimumab

NCT02272855

glioma

i.t.c.

combined with
dexamethasone

NCT02031965

i.v. or i.t.

as a single agent

NCT00931931

i.pl.

as a single agent

NCT01721018

i.v.

as a single agent

NCT02192775

i.pl.

as a single agent

NCT01503177

HSV1716
Seprehvir

ICP34.5 lacking
HSV-1 mutant

non-CNS solid
tumors
malignant pleural
mesothelioma
Multiple myeloma

MV-NIS

GL-ONC1

malignant
mesothelioma
measles virus
peritoneal,
expressing
fallopian ovarian
thyroidal sodiumcarcinoma
iodide symporter
plasma cell
myeolma

attenuated
vaccinia virus
with fluorescent
reporter genes

I
I/II
II
I

Recruitin
g
Recruitin
g
Recruitin
g
Recruitin
g
Recruitin
g

II

Recruitin
g

i.per.

as a single agent

NCT02364713

I/II

Recruitin
g

i.v.

combined with
cyclophosphamid
e

NCT00450814

head and neck


cancer

Recruitin
g

i.t.

as a single agent

NCT01846091

head and neck


cancer

I/II

Not yet
recruiting

i.v.

combined with
cisplatin and
radiation therapy

NCT01584284

advanced solid
tumors

Recruitin
g

i.v.

as a single agent

NCT00794131

REOLYSIN

oncolytic
reovirus

combined with
FOLFIRI and
bevacizumab
combined with
paclitaxel and
carboplatin

metastatic
colorectal cancer

Recruitin
g

i.v.

non-small cell
lung cancer

II

Not yet
recruiting

i.v.

II

Recruitin
g

i.t.

as a single agent

NCT01227551

Recruitin
g

i.t.

as a single agent

NCT01628640

malignant
melanoma
primary
hepatocellular
carcinoma

NCT01274624
NTC00861627

CVA

coxsackievirus
A21

VSV-IFN

VSV virus
expressing IFN

ParvOryx

parvovirus H-1

glioblastoma

I/II

Not yet
recruiting

i.v. or i.t.
and i.t.c.

combined with
surgery

NCT01301430

NDV

new castle
disease virus

glioblastoma,
sarcoma and
neuroblastoma

I/II

Not yet
recruiting

i.v.

as a single agent

NCT01174537

Abbreviations: i.t., intratumoral; i.v., intravenous; i.per.: intraperitioneal; i.pl.: intrapleural; i.t.c.: into tumor
resection cavity; i.b.p.: in brain parychema; e.v., endvenous; i.ves.: intravesical

Table 3: Natural Cytotoxicity Receptors and their viral ligands on infected


cells
Virus

Influenza
virus
(IV)
Sendai virus
(SV)
Newcastle
disease virus
(NDV)
Vaccinia virus
(VV)
Dengue virus
(DV)
West Nile
virus
(WNV)
a

Activati
ng
NK
receptor
NKp46

Viral ligand(s)
on
infected cells

Ref.

HAa
(activation)

92

NKp44

93

NKp46

HA (activation)
HNb (activation)

147

NKp44
NKp46

HN (activation)
HN (activation)

93
148

NKp44
NKp30

HN (activation)
HA (inhibition)

149

NKp46
NKp44

HA (activation)
E-protein
(activation)
E-protein
(activation)

NKp44

94
94

HA: hemagglutinin; bHN: hemagglutinin-neuraminidase.

Table 4: Effect of viral infection on activating NK receptors and their ligands

Virus

Effect on activating
NK receptors

Herpes simplex
virus 1 (HSV-1)

Effect on ligand(s)
on infected cells

Ref.

Up-regulation of
NCR-L
(mediated by ICP0
protein)

150

24
Down-regulation of
NKp30-L and NKp46L
Up-regulation of
NKp46, NKp30, NKp44
on maturing NK cells
Herpes simplex
virus 2 (HSV-2),
Pseudorabies
virus
(PRV)
Cytomegalovir
us
(CMV)

151

Down-regulation of
NKG2D-L

152

Degradation of
Nectin2 (mediated
by gD)

107

Modulation of NKp30
function
(mediated by
pp65/UL83)

153

Down-regulation of
NKp46-L and NKp30L

154
105

Kaposis
sarcomaassociated
herpesvirus
(KHSV)
Human
herpesvirus 8
(HHV8)
Vaccinia virus
Hepatitis C
virus
(HCV)

Modulation of
NKG2D-L (mediated
by UL16, UL141,
UL142, miR-UL112)
Down-regulation of
NKp44-L (mediated
by ORF54/dUTPase)
Down-regulation of
NKp46, NKp30,
NKG2D

Up-regulation of
NKp46 and NKp30

155

Up-regulation of
NKG2D-L

156

Up-regulation of
NCR-L

95,
149
110

Down-regulation of
NKp30

91,,
157

Transient induction of
NKp30 and NKG2D
(acute infection and

111,
112

exposed-uninfected
pts)
Parvovirus
HIV-1

Up-regulation of
NCR-L
Down-regulation of
NCR

158
99

Up-regulation of
NKp44-L (mediated
by gp41S3peptide)

159

Intracellular
retention of NKp44-L
(mediated by nef)

160

Down-regulation of
NCR-L

102

Up-regulation of
NKG2D-L (mediated
by vpr)

102,
161
103

Shedding of NKG2DL
Expansion of CD56CD16+NCR- subset
Myxoma Virus
(MYXV)
HCMV
Chikungunya
virus
Hantavirus

Expansion of NKG2C+
subset
Expansion of NKG2C+
subset
Expansion of NKG2C+
subset

Down-regulation of
NKG2D-L (mediated
by nef)
Down-regulation of
Nectin-2

104
100
119
162
90
163