The urgent need to recover MHC class I in cancers for effective immunotherapy.

The

urgent

need

to

recover

MHC

class

I

in

cancers

for

effective

immunotherapy

Federico

Garrido

,

Natalia

Aptsiauri

,

Elien

M

Doorduijn

,

Angel

M

Garcia

Lora

and

Thorbald

van

Hall

ImmuneescapestrategiesaimedtoavoidT-cellrecognition,

includingthelossoftumorMHCclassIexpression,are

commonlyfoundinmalignantcells.Tumorimmuneescapehas

proventohaveanegativeeffectontheclinicaloutcomeof

cancerimmunotherapy,includingtreatmentwithantibodies

blockingimmunecheckpointmolecules.Hence,thereisan

urgentneedtodevelopnovelapproachestoovercometumor

immuneevasion.MHCclassIantigenpresentationisoften

affectedinhumancancersandthecapacitytoinduce

upregulationofMHCclassIcellsurfaceexpressionisacritical

stepintheinductionoftumorrejection.Thisreviewfocuseson

characterizationofrejection,escape,anddormantprofilesof

tumorsanditsmicroenvironmentwithaspecialemphasison

thetumorMHCclassIexpression.Wealsodiscusspossible

approachestorecoverMHCclassIexpressionontumorcells

harboringreversible/‘soft’orirreversible/‘hard’geneticlesions.

SuchMHCclassIrecoveryapproachesmightwellsynergize

withcomplementaryformsofimmunotherapy.

Addresses 1

DepartamentodeBioquimica,BiologiaMolecularIIIeInmunologia, FacultaddeMedicina,UniversidaddeGranada,Granada,Spain 2_{Servicio de Ana´lisis Clı´nicos, UGC de Laboratorio Clı´nico, Hospital} Universitario Virgen de las Nieves, Granada, Spain

3

InstitutodeInvestigacionBiosanitariadeGranada(IBS.Granada), Granada,Spain

4_{ClinicalOncology,LeidenUniversityMedicalCenter,Albinusdreef2,} 2333ZALeiden,TheNetherlands

Corresponding authors: Garrido, Federico

([email protected])andvanHall,Thorbald ([email protected])

Current Opinion in Immunology 2016, 39:44–51

This review comes from a themed issue on Tumour immunology

Edited by Sjoerd H van der Burg and Francesco Marincola

ForacompleteoverviewseetheIssueandtheEditorial

Availableonline18thJanuary2016

http://dx.doi.org/10.1016/j.coi.2015.12.007

0952-7915/#2016TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBY-NC-NDlicense (http://creative-commons.org/licenses/by-nc-nd/4.0/).

Introduction

Cancerimmunotherapyinhumanshashistoricallyuseda variety of productsthat boostT lymphocyteresponses, such as IL-2 and IFN-a in melanoma and renal cell carcinoma and bacterial products as BCG in bladder

cancer therapy [1–3]. More recently, antigenic tumor peptidesor dendritic cells loaded with shared peptides havebeenintroducedtotheclinic[4,5].Thesetherapies createdgreatexpectationsamongclinicaloncologist be-causetheycould activatespecific anti-tumor T-cell im-munity.However, theobserved tumorregressionswere belowexpectations[6].Theabsenceor downregulation oftumorMHCclassI(MHC-I)moleculescouldbeoneof possible explanations for these disappointing results, since MHC-I expression oncancer cells is required for detectionanddestructionbyT-cells[7,8].MHC-Ilossor dowregulationisamajortumorescapemechanismfromT lymphocytes described in human tumors of different origin [9–12]. The HLA evaluation in human tumor tissues needs a complex approach since HLA class I (HLA-I) heavy chains are highly polymorphic and requires analysis of theexpression of six HLA-I alleles ontumorcellsurfacewhichdifferamongcancerpatients [13].ItisobviousthattheinformationabouttumorHLA expressionmostlycomesfromtheanalysisofprogressing tumors,whichhavealreadydevelopedescapestrategies. Incontrast,thetumorrejectionprofileisdifficulttostudy sincesuch regressinglesions either disappearinashort periodoftimeorprogresswhileacquiringthe immunoe-dited escape phenotype [14]. There are also evidences thatsometumorcellscansurviveinthehostina‘dormant state’ for long periods of time without being detected. Thesedormanttumorcells‘awake’in immune-compro-mised environments, especially whenCD4+ and CD8+ lymphocytesarenotpresentortheirnumbersareheavily reduced[15,16].

The

intimate

interaction

of

MHC

class

I

expression

by

tumors

and

the

T-cell

immune

pressure

process,buttheinteractionwiththehostimmunesystem determines the capacity of a given tumor cell clone to survive and disseminate. Therefore, a processof ‘selec-tion’,especiallyduetoT-cellimmunepressureonMHC-I deficienttumorvariants,mightrepresentanaturalprocess.

Weandothergroupshaveevidencethatthisstrong selec-tion processmediated bythe interaction ofMHC-I and

CD8+T-cellsinprimarytumorsistakingplaceduringthe earlystagesoftumordevelopmentleadingtoeithertumor rejection or immune escape via immunoediting [19,20]. TumorsarepredominantlyMHC-Ipositiveatearlystages. ThespecificantitumorCD8+T-cellsattackis progressive-lykillingMHC-IpositivecellsandselectingMHC nega-tive ones(Figure 1). The MHC-Iheterogeneity canbe observedinmanytumorsattheseearlystages.Finally,the

Figure1

MHC-I Intra-Tumor Heterogeneity and Patterns of T-Cell Immunoselection

Tumor Rejection Phenotype

Tumor Escape Phenotype

Dormant Tumor Phenotype

NO TUMOR (a)

(b)

(c)

CD8+ T-cell depletion

Dormant micrometastases

Tumor cell MHC class I +++

Dormant or awaken metastasis MHC class I +/++

Escaped tumor cell

MHC class I – T cellsCD8+

Awaken progressing macrometastasis

Current Opinion in Immunology

Tcellimmunoeditingleavestumorshomogeneously defi-cient or completely negative for MHC-I expression [20,21].Aclinicalexampleof T-cellmediated immuno-selectionofMHC-I negativetumorcellscame fromthe studyof melanomalesionsderivedfromasinglepatient duringthecourseofcancerprogression.Apointmutation presentinthecodon67ofthebeta2-microglobulingenein HLA-Inegativemelanomacellsfromtheheterogeneous primarytumorwasfound10monthslaterinanuniformly HLA-I-negativemetastaticlesion,stronglysuggestingan activeT-cellimmunoselectionofMHC-Inegative mela-nomacells[21].

Inamousecancermodel,averyclearexampleofTcell mediatedimmunoselectioncamefromtheassessmentof H-2expressioninmetastaticlungcoloniesobtainedfrom an H-2 negative tumor cell clone growing in immuno-competent and in immunodeficient mice[22,23].Lung metastaticcoloniesgrowinginimmunocompetent synge-neicmicewereH-2negative.Incontrast,colonies grow-ing in immunodeficient mice lacking T-cells wereH-2 positive.ThemechanismresponsiblefortheH-2 down-regulationinthistumorclonewasreversible(‘soft’)since the expression of H-2 antigens could be recovered by IFN-g [22,23]. We also observed that MHC-I positive tumor clones are highly immunogenic, while MHC-I negative variants have low immunogenicity. Neverthe-less,whenthenumberoflocallyinjectedtumorcellsfrom MHC-Ipositivecloneisconsiderablylarge,theygenerate localtumors anddevelopahigh numberof distant me-tastases,becauseare abletoovercomeT-cell responses andinduceimmunosuppression[22].Importantly, MHC-I positive lung metastases in this model could be completely eradicated byimmunotherapy [24]. In con-trast, MHC-I negative tumor clones have none or low metastaticcapacity.Theygeneratedormant micrometas-taseswithincreasedMHC-Iexpression,whichare capa-bleofinducingT-cellimmuneresponse(Figure1)[15].

Tumor

rejection

profile

There are only few reports describing the MHC-I ex-pression patterns in tumors undergoing rejection in humans.Thisrejectioncansometimesbeinducedusing different protocols of immunotherapy, including treat-mentwithIL-2,BCG,IFN-a,autologoustumorvaccine, peptidevaccinationortransfer ofautologous anti-tumor CD8+ T-cells, and is associated with theexpression of highlevelsofHLA-Imoleculesin tumorcells[20].Itis difficult to obtain regressing malignant lesions, since there are no clinical indications for surgical removal or biopsy.Inthissituation,werelyonlyontheevaluationof systemic CD8+ T-cell responses and intratumoral infil-tration [25]. Nevertheless, we had an opportunity to carefully analyze regressing andprogressing subcutane-ousmelanomalesionsintwo‘mixedresponder’patients afterautologousvaccination[14,20].Weobserveda mas-sive intratumoral infiltration of CD4+ and CD8+

lymphocytes (TILs) within the regressing melanoma lesionswithapositivecorrelationofhighMHC-Ilevels. In contrast, the lack of tumor HLA-I expression in progressinglesionscorrelatedwithabsenceofTILswith mostly peritumoral infiltration patterns [14,20]. These two opposite patterns in distinct lesions of the same patientreflectdifferentphenotypesoftumor microenvi-ronment, namely tumor rejection and tumor escape (Figure 1). Tumor regression was also associated with increasedtranscriptionalupregulationofHLAand inter-feronstimulationpathwaygenespointingtoanenhanced antigenpresentationcapabilityoftumorcells[14].This histological and molecular signature of tumor rejection mediated by CD8+ T-cells seems to be similarto that foundinallograftrejectionandgraftversushostdisease suggestinganexistenceofanimmunologicalcircuitryof rejection[26].

HighdegreeoftumorinfiltrationwithT-cellsis consid-ered to be a good prognostic factor [27] and has been includedintoanewtumorimmunologicalgradingsystem called‘immunoscore’[28].Wehavepreviouslyobserved in various types of cancers that the HLA-I negative tumors lack TILs. In contrast, HLA-I positive tumors arecharacterizedbyhighdegreeofintratumoral infiltra-tionwithCD8+Tcells[14,20].Thestatusofintratumoral infiltration,perhaps,reflectsthestageofcancerimmune escapeduringnaturalcancerprogression.Atearlystages therearemoreHLA-positivetumorcellsandmanyTILs, while at more advanced stages tumor contains more HLA-negativeescapevariantsandT-cellsarerestrained intheperitumoralarea(Figure1).

TumorHLA-Iexpressionpatternshavebeendiscussedat the12thInternationalHistocompatibilityWorkshop(Paris, France,June9–12,1996).Tumorssectionswereclassified asHLA-Inegative(<25%tumorcellsstained), heteroge-neous(between 25% and 75% tumorcells labeled) and positive(>75% tumorcellsstained)[13,29].Itwouldbe interestingtodeterminewhetherthelackofHLA-Iisthe causeofpoorT-cellrecruitmentintothetumoror,theother way around, the local infiltrating T cells mediate high HLA-I expression byproducing IFN-g. Arecent report indicatedthatthelatteroptioncould bevalid,atleastin sarcomas[30].Inthiscontext,thebroadened melanoma-reactiveCD8+Tcellresponsesreportedafteranti-CTLA-4 therapyinmelanoma[31,32,33]couldbeassociatedwith theupregulationofMHCexpressionandcouldleadtothe presentationofavarietyofpreviouslyhiddentumor spe-cificpeptides,whichsubsequentlyactivateapre-existing T-cell pool. Similar events were previously reported in clinical trials using peptide-based immunotherapy and weredefinedas‘epitopespreading’[25,34].

Tumor

escape

profile

human andexperimentaltumorsand reviewedin many previousreports[8,10,12,35].Tumorswiththisprofilecan bederivedfromestablishedprogressedtumorsafterthey hadescapedT-cellmediatedimmunosurveillance[11].A tumor derived from an HLA-I positive epithelium can losetotallyorpartiallytheexpressionofclassImolecules [9].ThetotalpercentageofvarioustypesofHLA-Iloss, includingtotalloss,haplotypeloss,orallelicloss,ranges from 65 to 90%, depending on the type of cancer [9,35,36].

AnotherevidenceoftumorescapeandtheresistancetoT cell immunity caused by MHC-I down-regulation has been corroborated bya cancer in a small mammal, the TasmanianDevil.AfacialtumorintheTasmanianDevil silencedthegenesforantigenpresentationatthe epige-neticlevelandtherebycreatedaninfectiouscancerthatis transmissible to histo-incompatible companions [37,38,39]. This curious case of a transmissible tumor clearly emphasizes the relevance of MHC loss for im-muneescapeoftumors.Theclearimpressioninthefield isthatmostMHC-Idefectsinhumancancersbelongto the categoryof ‘soft’-wired lesions [40,41–43]. Conse-quently,thistypeofimmuno-editingcanbecounteracted byclevertherapeutictargeting,suchas activationofthe interferon signaling pathway in cancers or intervention with HDACinhibitors[44].

Tumor

dormancy

profile

Clinical and experimental evidence indicate that the immunesystemcanmaintaincancercellsinadormant state [15,16]. Metastatic tumor cells can remainin a state ofequilibrium withthe immune systemfor long periodsoftimeduringwhichmetastaticcoloniesdonot progress and the cellular immune response does not reject the tumor. It resembles the symbiotic co-exis-tence we see in different species when each partner benefits from another inaparticular ‘statusquo’ ofno aggression.Despiteclinical reportssuggestingthat im-munosuppression is associated inhumans withclinical appearanceofmetastaticcolonies[45],theprofileofthe dormantmicroenvironment isnotpreciselyknown.We have developedamousetumormodel (GR9)inwhich several metastatic tumor nodules were kept in a per-manent state of immune-mediated dormancy in an immunocompetent host [15]. Interestingly, when the mice were depleted of CD8-T lymphocytes the coloniesstartedtogrowthresultinginovertmetastases. Moreover, the same tumor clone produced overt pul-monarymetastasesinnudemice.Tumorcellscapableof generating these dormant metastatic coloniesare very exceptional;theywerecompletelynegativeforMHC-I, but the dormant micrometastases recovered MHC-I surface expression (Figure 1) [15]. These results suggested that MHC-I surface expression and CD8+ T lymphocytes play an important role in immune-mediated dormancy [46] (Figure 1).

Antitumor strategies could be directed to harness the immuneresponsetomaintaincancercellsinapermanent dormantstateortofavoracompletetumorrejection.In theGR9mouse tumormodel,immunotherapy turneda highlymetastatictumorcloneintodormant micrometas-tases ([24] and GarciaLora et al., unpublished observa-tions).UpregulatingMHC-Iexpressionontumorcellsby cytokines, byincreasingFHITgeneexpression[47],by blockade of the immune-checkpointinhibitors, by sup-pressionofTregulatorycellsormyeloidsuppressorcells could lead to activation of anti-tumor T lymphocyte responses [44].An attractivestrategyfor therestoration of MHC-I expression is by epigenetic modifiers, like inhibitorsofDNAmethyltransferase(DNMT)orhistone deacetylase(HDAC).Inseveralrecentpapers,such reg-ulation attheepigeneticlevel was shown to beableto synergize with immunotherapy for the eradication of mouse tumor models [48,49,50]. Interestingly, IFN-g -inducedrestorationof‘soft’lesionsofMHC-I,oneofthe mostpowerfulinducersofthisgene,wasshowntopartly mediateitseffectbyinducingdemethylationof antigen-processing machineryrelatedgenes, includingtheTAP genes andLMP-2[51].

How

to

deal

with

‘hard’

lesions?

Gene

therapy

and

alternative

lymphocytes

Targetingthetumorescapephenotypeisoneofthemajor tasks ofthepresentandfuturecancertherapies[44].In theexamplesreferredtoabove,themolecularmechanism responsiblefortheHLA-Idownregulationisreversibleor ‘soft’[40,52].Incontrast,whenthegenesofHLAorbeta 2-microglobulinarecorrupteddueto mutationsor dele-tions resultingin loss of heterozygosity(LOH) at chro-mosomes 6 or 15, theHLA-I loss isirreversibledue to these ‘hard’ lesions [53–55]. In this case, tumor cells cannot recover the antigen presentation capacity and thetumor microenvironment retainstumor escape phe-notypefavoring cancerprogression.‘Hard’HLA-I aber-rationsintumors(LOHinchromosomes6or15andbeta 2-microglobulinmutations)areintherangeof30–40%of humancancers[55,56].InordertorestoreHLA-I expres-sioninhumantumorswith‘hard’lesionswehavemadea recombinant adenovirus carrying beta 2-microglobulin geneanddemonstratedarecoveryofHLA-Iexpression on tumor cells deficient in beta 2-microglobulin. This HLAreconstitutionalsorecoveredtumorcelldestruction bypeptidespecificCD8+T-cellsinHLA-restricted man-ner[57–59].

Thisanergicstate wasreversed withIL-12/IL-18 treat-ment and was even further enhanced by an improved form of IL-2, leading to NK-dependent tumor control. Another group showed thattransfer of in vitro pre-acti-vatedNKcells,incombinationwithbodyirradiation,was effective in eradication of tumors with ‘hard’ genetic lesionintheMHC-Ipathway[61].Interestingly,addition of HDAC inhibitors can increase the susceptibility of cancercellsforNKcellsbyupregulationofthe NKG2D-activatingligandMICA[62,63].Inadditionto upregula-tionof its ligands,theNKG2D receptor wasalso upre-gulated,whichledtoafurtherenhancedcytotoxicity of tumor cells [62].However,caution isneeded, as tumor cells treated with HDAC inhibitors might reduce their levels of other activating ligands, as shown for B7-H6, whichstimulatesNKcellsviatheNKp30receptor[64].

Some years ago, our group identified a novel group of CD8+Tcellswhichspecificallyrecognizecellswithlow MHC-Iexpressionduetoadefectinthepeptide trans-porterTAP [65,66]. TheseTcellsrecognizean alter-nativepeptiderepertoireonimmunoescapedtumorcells. Wenamed these peptides TEIPP, for ‘T cellepitopes associated with impaired peptide processing’ and they emerge in theresidual MHC-Imolecules as aresult of alternative antigen processing pathways [67,68]. We showedthattheprototypicTEIPPepitope,derivedfrom the housekeeping protein Trh4, is processed by signal peptidepeptidase andis,therefore,processed indepen-dentlyofTAP ortheproteasome [69].InanovelTCR transgenicmousemodelbasedontheTrh4-specificCD8+ Tcellclone,weobservedanefficientthymicselectionof theseTcells,indicatingthattheTEIPPTcellrepertoire isnotaffectedbycentraltolerance[70].Inaddition,the TEIPP T cells were effective in tumor control of the TAP-deficient RMA-S tumor. We anticipate that this CD8+ T cell subset can be exploited for treatment of immune-escapedtumors.

Conclusions

Wehavedefinedthefollowingthreemajortumor phe-notypes relevant for tumor-host interactions and anti-tumor immunity: rejection, escape and dormancy. We highlightedthekeyroleoftumorMHCexpressionthat influences the degree and composition the immune cellular infiltration. This type of cellular immune re-sponsemarkedlydeterminestheprognosisand clinical outcome in different types of malignancies. There is accumulating evidence suggesting that the efficacy of traditional(IL2,BCG,peptides,etc.)andnewly devel-oped immunotherapy (‘immune checkpoint’ blocking antibodies)dependsontheexpressionlevels of MHC-Iontumorscells[49].‘Soft’MHC-Imolecularlesionscan berecoveredby avariety ofinterventionsthat modify tumormicroenvironment insuch a way that Th1type cytokinesare released. ‘Hard’MHC molecularlesions canonlybecorrectedbytransferringtheappropriatewild

type MHC-I or beta 2-microglobulin gene, or by the applicationofnaturalkillercellsandTEIPP-specificT cells.Hence,identificationofmolecularaberrations re-sponsibleforalteredtumorMHC-Iexpression,aswellas monitoringthe evolution ofthisexpressionduring the course of treatment becomes essential for the success ofT-cell mediatedcancer immunotherapyand forthe development of novel complementary approaches forMHC-Iupregulation.Weareundoubtedly oversim-plifyingtheenormouscomplexityofthetumor microen-vironmentbutfuture findingsof keymoleculesand/or cells capable of overriding the immune escape routes usedbytumorcellswillcertainlyhelpininducing dura-bletumorrejection.Amongthem,MHCre-expressionis amajortargetforfuturestudies.

Acknowledgements

WewouldliketothankDrMBernalwhohashelpedusinpreparingthe figureforthemanuscript.Thisworkwassupportedbygrantsco-financedby FEDERfunds(EU)fromtheInstitutodeSaludCarlosIII(CP03/0111, PI12/02031,PI08/1265,PI11/01022,PI11/01386,PI14/01978,PI15/00528, RETICRD06/020,RD09/0076/00165,PT13/0010/0039),Juntade Andalucı´ainSpain(GroupCTS-143,andCTS-695,CTS-3952,CVI-4740 andPI09/0382grant),WorldwideCancerResearch15-1166grant,andby DutchCancerSociety(UL2010-4785,TvH).

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