From www.bloodjournal.org by guest on January 5, 2019. For personal use only. Regular Article LYMPHOID NEOPLASIA The antigenic landscape of multiple myeloma: mass spectrometry fi – (re)de nes targets for T-cell based immunotherapy SimonWalz,1,2JulianeS. Stickel,1DanielJohannes Kowalewski,2 HeikoSchuster,2Katja Weisel,1LinusBackert,2,3 StefanKahn,2Annika Nelde,2TatjanaStroh,2Martin Handel,4Oliver Kohlbacher,3LotharKanz,1Helmut RainerSalih,1,5 Hans-Georg Rammensee,2,6 andStefanStevanovic´2,6 1DepartmentofHematologyandOncology,2InstituteforCellBiology,DepartmentofImmunology,and3AppliedBioinformatics,CenterforBioinformatics andDepartmentofComputerScience,UniversityofTu¨bingen,Tu¨bingen,Germany;4DepartmentofOrthopedics,HospitalGroupSouth-West,Calw, Germany;and5ClinicalCooperationUnitTranslationalImmunologyand6CancerImmunotherapyUnit,GermanCancerConsortium(DKTK),German CancerResearchCenter(DKFZ)PartnerSiteTu¨bingen,Tu¨bingen,Germany Key Points DirectanalysisofHLA-presentedantigensbymassspectrometryprovidesacomprehen- siveviewontheantigeniclandscapeofdifferenttissues/malignanciesandenablesthe (cid:129) Direct analysis of the HLA- identificationofnovel,pathophysiologicallyrelevantT-cellepitopes.Here,wepresentasys- presented peptidome tematicandcomparativestudyoftheHLAclassIandIIpresented,nonmutantantigenome identifies a distinct antigenic ofmultiplemyeloma(MM).QuantificationofHLAsurfaceexpressionrevealedelevatedHLA signature in MM. moleculecountsonmalignantplasmacellscomparedwithnormalBcells,excludingrelevant (cid:129) T-cell responses for these HLAdownregulationinMM.Analyzingthepresentationofestablishedmyeloma-associated T-cellantigensontheHLAligandomelevel,wefoundasubstantialproportionofantigenstobe antigens are detectable onlyinfrequentlypresentedonprimarymyelomasortodisplaysuboptimaldegreesofmye- exclusively in MM patients lomaspecificity.However,unsupervisedanalysisofourextensiveHLAliganddatasetdelin- and can be induced in vitro eatedapanelof58highlyspecificmyeloma-associatedantigens(includingmultiplemyeloma in response-naive patients. SETdomaincontainingprotein)whicharecharacterizedbyfrequentandexclusivepresen- tationonmyelomasamples.Functionalcharacterizationofthesetargetantigensrevealed peptide-specific,preexistingCD81T-cellresponsesexclusivelyinmyelomapatients,whichisindicativeofpathophysiologicalrelevance. Furthermore,invitroprimingexperimentsrevealedthat peptide-specificT-cellresponsescanbeinducedinresponse-naive myeloma patients.Together,ourresultsservetoguideantigenselectionforT-cell–basedimmunotherapyofMM.(Blood.2015;126(10):1203-1213) Introduction Antigen-specific immunotherapy holds the potential to induce malignantplasmacellsinthebonemarrow.14Despiterecentadvances clinically effective anticancer T-cell responses1,2 and might be intreatment,includinghigh-dosechemotherapyfollowedbyautolo- harnessedtoguideandincreasethespecificityofcancerimmuno- gousstemcelltransplantation,novelimmunomodulatorydrugs,and therapy in future combination trials.3 To this end, the exact proteasomeinhibitors,MMremainslargelyincurable.15,16Thisismostly knowledge of tumor-associated/specific T-cell epitopes is crucial. duetothepersistenceofminimalresidualdisease(MRD),whichleads After decades of research into overexpressed tumor antigens, more tohighrelapserates.17,18Sofar,theonlyestablishedimmunotherapeutic recentlythefocushasshiftedtothepatient-individualizedidentification approachforMMisallogenicstemcelltransplantation,whichisasso- ofmutation-derivedneoantigens.4,5Theencouragingfindingsofthese ciatedwithahighmorbidityandmortalityandremainsanoptionfor newstudies6-8haveledtoneoepitopesbeingviewedasthedominant only a fraction of patients.19-21 Antigen-specific T-cell–based targetsofanticancerimmuneresponses.9-11 immunotherapy,22,23especiallyintheconstellationofMRDcharac- However,analyzingtheantigenomeofhematologicmalignancies, terized by favorable effector-to-target ratios, might present an ef- wehaverecentlydemonstratedthatnonmutatedantigensarerelevant fective,lowside-effectoption.24 targetsofspontaneousantileukemiaT-cellresponses.12,13Thestrategy Anarrayofmyeloma-associatedT-cellantigenshasbeendescribed implementedinthesestudiesdifferentiallymapsthenaturallypresented inpreviousstudies.25-35Mostoftheseantigenswereidentifiedbased HLAligandomesofhematologiccellsinhealthanddiseasebymass ongeneexpressionanalysisandreverseimmunology.Someofthese spectrometryandwasfoundtoefficientlyidentifyrelevanttumor- antigens(WT1,36,37RHAMM,38,39hTERT,40andSurvivin40,41)have associatedantigens(TAAs). alreadyfoundtheirwayintoclinicaltrials,showingpromisingresults Here,wetranslatedthisapproachtomultiplemyeloma(MM),a intermsofinductionofspecificT-cellresponsesaswellasclinical low-gradeB-celllymphoma,characterizedbytheproliferationof responsesinsinglepatients.However,broadclinicaleffectivenesshas SubmittedApril 17,2015; accepted June 22,2015.Prepublished online as The publication costs of this article were defrayed in part by page charge BloodFirstEditionpaper,July2,2015;DOI10.1182/blood-2015-04-640532. payment. Therefore, and solely to indicate this fact, this article is hereby marked“advertisement”inaccordancewith18USCsection1734. S.W.andJ.S.S.contributedequallytothiswork. Theonlineversionofthisarticlecontainsadatasupplement. ThereisanInsideBloodCommentaryonthisarticleinthisissue. ©2015byTheAmericanSocietyofHematology BLOOD,3SEPTEMBER2015xVOLUME126,NUMBER10 1203 From www.bloodjournal.org by guest on January 5, 2019. For personal use only. 1204 WALZetal BLOOD,3SEPTEMBER2015xVOLUME126,NUMBER10 Table1.Patientcharacteristics Durieand Lightchain Previous UPN Sex Age(y) ISSStage Salmon restriction Cytogeneticrisk therapy HLAtyping Experiment 1 M 62 3 3B l High Yes A*26,A*30,B*15,B*42 Q;L 2 F 55 2 1A Non Standard No A*01,A*24,B*18,B*08 L 3 F 69 2 3B k Standard No A*02,A*01,B*08,B*37 L 4 M 72 3 3A k Standard No A*02,A*33,B*15,B*18 L 5 M 54 2 3A k Low No A*03,A*26,B*40,B*55 L 6 M 60 3 3B l High No A*02,A*24,B*07,B*27 L 7 F 39 3 3A k Standard No A*02,A*03,B*07,B*35 L 8 F 73 3 3B l Unknown No A*24,A*25,B*39,B*40 L 9 M 52 3 1B k Unknown No A*02,B*07,B*44 L 10 M 47 1 3A k Standard No A*03,A*33,B*07 L 11 F 72 2 3A k High Yes A*02,B*27,B*44 Q 12 F 68 2 3A k Standard No n.d. Q 13 M 74 1 3A k Standard No n.d. Q 14 F 50 1 3A l Standard No A*01,A*68,B*08,B*35 Q 15 M 62 1 3A k Standard Yes n.d. Q 16 M 74 3 2B k Standard Yes n.d. Q 17 M 68 1 3A k Unknown Yes A*03,A*33,B*14,B*18 Q 18 M 73 1 1A k Standard Yes A*02,A*32,B*40,B*44 Q 19 F 70 1 1A k Standard No n.d. Q 20 F 53 3 3A k Standard No n.d. Q 21 M 59 2 3A l Standard Yes n.d. Q 22 M 57 1 1A k Standard Yes n.d. Q 23 F 65 3 1A l Standard Yes n.d. Q 24 M 76 2 3A l Standard Yes n.d. Q 25 F 74 2 2A k Standard Yes A*01,A*02,B*08,B*44 Q 26 F 67 1 3A l Standard Yes n.d. Q 27 M 50 1 1A l Unkwnon Yes A*01,A*03,B*35,B*40 Q 28 M 70 3 3A l Unknown No n.d. Q 29 M 75 1 3A k Standard Yes n.d. Q CytogeneticriskaccordingtotheIMWGriskstratification.44 F,female;IMWG,InternationalMyelomaWorkingGroup;ISS,InternationalStagingSystem;L,HLAligandisolation;M,male;n.d.,notdetermined;Q,HLAquantification; UPN,uniformpatientnumber;y,years. notyetbeenachieved.Thesepreviousstudieswererestrictedtovery Myelomacelllines fewHLAallotypesandsingleantigens/epitopes,42limitingboththe ForHLAligandomeanalysis,myelomacelllines(MCLs;U266,RPMI8226, populationofpatientseligibleforthistherapeuticapproachandthe JJN3,LP-1,MM.1S)wereculturedintherecommendedcellmedia(RPMI1640, spectrumofinducibletumor-specificT-cellresponses.Ofnote,recent Gibco;IscovemodifiedDulbeccomedium[IMDM],Lonza)supplementedwith studiesdemonstratedlackingdegreesoftumorassociationforseveral 10%/20%fetalcalfserum,100IU/Lpenicillin,100mg/Lstreptomycin,and of these tumor antigens, both on the transcriptome level43 and im- 2 mmol/L glutamine at 37°Cand 5% CO. The MCLs RPMI 8226,JJN3, 2 portantlyalsoonthelevelofHLA-restrictedpresentation.12,13 MM.1S and LP-1 were obtained from the Department of Hematology and ByanalyzingtheantigeniclandscapeofMMdirectlyontheHLA Oncology,UniversityofTu¨bingen,Tu¨bingen,Germany. ligandlevel,wehereprovidenewinsightsonantigenicdistribution/ specificityandidentifyapanelofnovelmyeloma-associatedepitopes QuantificationofHLAsurfaceexpression suitedforantigen-specificimmunotherapy. HLAsurfaceexpressiononMMpatientandHVbonemarrowcellsincluding CD381CD1381myeloma cells/plasma cells, CD191CD201B cells, CD31 Tcells,andCD341CD382hematopoieticprogenitorcells(HPCs)wereana- lyzedusingtheQIFIKITbead–basedquantitativeflowcytometricassay(Dako) accordingtothemanufacturer’sinstructionsasdescribedpreviously.12Inbrief, Materials and methods sampleswerestainedwiththepan-HLAclassI–specificmonoclonalantibody (mAb)W6/32,HLA-DR–specificmAbL243(producedin-house)orimmu- Patients,blood,andbonemarrowsamples noglobulinGisotypecontrol(BioLegend),respectively.Surfacemarkerstaining wascarriedoutwithdirectlylabeledCD138,anti-k,anti-l,CD19,CD20 Bonemarrowmononuclearcells(BMNCs)andperipheralbloodmononuclear (BioLegend)andCD38,CD3,andCD34(BD)antibodies.7-aminoactinomycin cells(PBMCs)fromMMpatientsatthetimeofdiagnosisoratrelapsebefore D(BioLegend)wasaddedasviabilitymarkerimmediatelybeforeflowcytom- therapy,aswellasPBMCs,BMNCsandgranulocytesofhealthyvolunteers etricanalysisonaLSRFortessa(BDBiosciences). (HVs),wereisolatedbydensitygradientcentrifugation(Biocoll;Biochrom GmbH)anderythrocytelysis(ELbuffer;Qiagen).Informedconsentwasob- IsolationofHLAligandsfromprimarysamplesandMCLs tainedinaccordancewiththeDeclarationofHelsinkiprotocol.Thestudywas performedaccordingtotheguidelinesofthelocalethicscommittee(142/ HLA class I and II molecules were isolated using standard immunoaffinity 2013BO2).PatientcharacteristicsareprovidedinTable1.44HLAtyping purificationasdescribed45usingthepan-HLAclassI–specificmAbW6/32,the wascarriedoutbytheDepartmentofHematologyandOncology,University pan-HLAclassII–specificmAbTu3̈ 9,andtheHLA-DR–specificmAbL243 ofTu¨bingen,Tu¨bingen,Germany. (producedin-house). From www.bloodjournal.org by guest on January 5, 2019. For personal use only. BLOOD,3SEPTEMBER2015xVOLUME126,NUMBER10 T-CELLEPITOPESFORTHEIMMUNOTHERAPYOFMYELOMA 1205 AnalysisofHLAligandsbyliquidchromatography–tandem meannumberofspotsinthenegativecontrolwells(accordingtothecancer massspectrometry immunoguidingprogramguidelines51). HLAligandextractswereanalyzedin5technicalreplicatesasdescribed aAPCprimingofpeptide-specificTcells previously.13Inbrief,peptidesampleswereseparatedbynanoflowhigh- performance liquid chromatography (RSLCnano; Thermo Fisher Scien- Forthegenerationofartificialantigen-presentingcells(aAPCs),43106 tific)usinga50mm325cmPepMaprapidseparationliquidchromatography streptavidin-coatedpolystyreneparticles(BangsLaboratories)permilliliterwere column (Thermo Fisher Scientific) and a gradient ranging from 2.4% to resuspendedinPBE(phosphate-bufferedsaline/bovineserumalbumin/EDTA; 32.0%acetonitrileoverthecourseof90minutes.Elutingpeptideswere Gibco/Sigma Aldrich/Lonza) containing 200 pM biotinylated MHC-peptide analyzed in an online-coupled LTQ Orbitrap XL mass spectrometer monomerand20nMantihumanbiotinylatedCD28antibodyandincubatedat (Thermo Fisher Scientific) using a top 5 collision-induced dissociation roomtemperaturefor30minutes.Afterwashing,theaAPCswerestoredat4°C fragmentationmethod. priortouse.52CD81TcellsfromMMpatientsandHVwereenrichedbypositive selectionusingmagneticcellsorting(MiltenyiBiotec).Stimulationswerein- Databasesearchandspectralannotation itiatedin96-wellplateswith13106Tcellsplus23105aAPCsin200mLof T-cellmediumcomplementedwith5ng/mLhumanIL-12(PromoKine).IL-2 Dataprocessingwasperformedasdescribedpreviously.13Inbrief,theMascot (65U/mL;R&DSystems)wasaddedonday5.aAPCstimulationwasrepeated searchengine(Mascot2.2.04;MatrixScience)wasimplementedtosearchthe onday10,foratotalof3cycles. humanproteomeascomprisedintheSwiss-Protdatabase(20279reviewed proteinsequences,September2013)withoutenzymaticrestriction.Potential Tetramerstaining mutatedHLAligandsweresearchedimplementingadatabasecontainingthe humanproteomeconcatenatedwithproteinscontainingsingleaminoacid Thefrequencyofpeptide-specificCD81TcellswasdeterminedonaFACSCanto variantslistedintheCOSMICdatabase(http://cancer.sanger.ac.uk/cosmic/). IIcytometer(BDBioscience)bystainingwithanti-CD8(Biolegend)andHLA: Onlyrecurrentsingleaminoacidvariantsdescribedin2ormoresamples peptide-tetramer-phycoerythrinasdescribedpreviously.52Stainingwith ofhematologicoriginwereincluded.Oxidizedmethioninewasallowedasa tetramers containing the cytomegalovirus (CMV) pp65 A*02 peptide dynamicmodification.Thefalsediscoveryrate(FDR)wasestimatedusingthe NLVPMVATVservedaspositivecontrol,tetramerscontainingirrelevant;non- Percolatoralgorithm46andsetto5%.Peptidelengthswerelimitedto8to12aa primedA*02-restrictedcontrolpeptidesservedasnegativecontrols.Successful forHLAclassIand12to25aaforHLAclassII.Proteininferencewasdisabled, priming was considered if frequency of peptide-specific CD81 T cells was allowingformultipleproteinannotationsofpeptides.HLAannotationwas .0.1%ofviablecellsandatleastthreefoldhigherthanthefrequencyofpeptide- performedusingSYFPEITHI47oranextendedin-housedatabase.Experimen- specificCD81Tcellsinthenegativecontrol. talvalidationofpeptideidentificationsandHLAannotationswasperformedby massspectrometricandfunctionalcharacterizationofsyntheticpeptidesfor Softwareandstatisticalanalysis asubsetofpeptides. FlowcytometricdataanalysiswasperformedusingFlowJo7.2(TreeStar). In-house R and Python scripts were used for the generation of virtual PeptideandHLApeptidemonomersynthesis ligandomesanddefinitionofvirtualTAAsintheanalysisofTAAFDRs andfortheTAA-plateauregressionanalysis.ThestandardRheatmap.2 TheautomatedpeptidesynthesizerEPS221(Abimed)wasusedtosynthesize peptides using the 9-fluorenylmethyl-oxycarbonyl/tert-butyl (Fmoc/tBu) scriptwasusedfortheunsupervisedclusteranalysisofHLAligandsource strategy.48Syntheticpeptideswereusedforvalidationofliquidchromatography– proteins.GraphPadPrism6.0(GraphPadSoftware)wasusedforstatistical tandemmassspectrometry(MS)identificationsaswellasforfunctionalexper- analysis. Statistical analysis of HLA surface expression was based on iments.BiotinylatedrecombinantHLAmoleculesandfluorescentHLA- unpairedttests. peptidetetramerswereproducedasdescribedpreviously.49 Amplificationofpeptide-specificTcells Results PBMCsfromMMpatientsandHVswereculturedasdescribedpreviously.12,13 Inbrief,forCD81T-cellstimulation,PBMCswerepulsedwith1mg/mLper HLAclassIsurfaceexpressioniselevatedonmyelomacells peptideandculturedfor12daysaddinginterleukins4and7(IL-4andIL-7)on days0and1aswellasIL-2ondays3,5,7,and9.HLA-A*02(KLFEKVKEV)- AslossordownregulationofHLAexpressionontargetcellsmight andB*07(KPSEKIQVL)-restrictedcontrolpeptidesderivedfrombenigntissues severelyhampertheeffectivenessofT-cell–basedimmunotherapy,we (HV-exclusiveHLAligands)servedasnegativecontrol.Peptide-stimulated quantifiedHLAclassIandIIsurfacemoleculecountsonprimary PBMCswereanalyzedbyenzyme-linkedimmunospot(ELISPOT)assayson myelomacellscomparedwithautologoushematopoieticcellsand day12.ForCD41T-cellstimulation,culturewasperformedasdescribedfor plasmacellsderivedfromthebonemarrowofHVs.InMMpatients CD81Tcellsexceptfor2modifications:pulsingwascarriedoutwith10mg/mL (n520),HLAclassIexpressionwasfoundtobeheterogeneouswith HLAclassIIpeptideandnoIL-4orIL-7wasadded. meanexpressionlevelsonCD381CD1381myelomacellsof4160006 54500,whichwasfoundtobesignificantlyhigherascomparedwith IFNgELISPOTassay autologous normal CD191CD201 B cells (1985000 6 20500, Interferong(IFNg)ELISPOTassayswerecarriedoutasdescribedpreviously.50 P5.001),CD31Tcells(167500615500,P5.0002),andCD341 Inbrief,96-wellnitrocelluloseplates(Millipore)werecoatedwith1mg/mL CD382HPCs(204000632500,P5.002,Figure1A).Inaddition, IFNg mAb(Mabtech)andincubatedovernightat4°C.Plateswereblocked HLAclassIexpressiononprimaryMMcellswasalsofoundtobe with10%humanserumfor2hoursat37°C.PrestimulatedPBMCs(2.53105 significantlyhigherthanthatonCD381CD1381plasmacellsofHVs cellsperwell)werepulsedwith1mg/mL(HLAclassI)or2.5mg/mL(HLAclassII) (n 5 15, 291500 6 25500, P , .05; Figure 1C). No significant peptideandincubatedfor24to26hours.Readoutwasperformedaccordingto differencesinHLAclassIexpressionwereobservedwhencomparing themanufacturer’sinstructions.Phytohemagglutininwasusedaspositivecon- normalBcells,Tcells,andHPCsofMMpatientstothecorresponding trol.HLA-A*02(KLFEKVKEV)-andB*07(KPSEKIQVL)-restrictedcontrol cellpopulationsofHVs(supplementalFigure1,seesupplementalData peptidesderivedfrombenigntissues(HV-exclusiveHLAligands)servedas negativecontrol.SpotswerecountedusinganImmunoSpotS5analyzer(CTL). available on the Blood Web site). HLA-DR expression levels on T-cellresponseswereconsideredtobepositivewhen.10spotsperwellwere myelomacellsweregenerallyfoundtobemuchlowerthanHLAclassI countedandthemeanspotcountperwellwasatleastthreefoldhigherthanthe levels.MeanHLA-DRsurfacemoleculecountsonmyelomacells From www.bloodjournal.org by guest on January 5, 2019. For personal use only. 1206 WALZetal BLOOD,3SEPTEMBER2015xVOLUME126,NUMBER10 Figure1.HLAclassIandIIsurfaceexpressionon myelomapatientandHVbonemarrowcells.Quan- tification of HLA surface expression was performed using a bead-based flow cytometric assay. (A) HLA classIand(B)HLA-DRexpressiononCD381CD1381 primarymyelomacellscomparedwithautologous CD341CD382HPCs,CD191CD201Bcells,andCD31 Tcells.(C)HLAclassIand(D)HLA-DRexpression onprimaryMMcellscomparedwithbonemarrow–derived plasmacellsofHVs.n.s.,notsignificant;*P,.05; **P,.01;***P,.001. (2700067000)showednosignificantdifferencecomparedwith (13PBMC,5BMNC,5granulocytespecimens)yielded2899different autologousHPCs(3500065000)andTcells(18000613000)or peptidesrepresenting889sourceproteins(supplementalTable2). plasma cells of HVs (39500 6 5000) (Figure 1B,D). HLA-DR expressionofMMpatientCD191CD201Bcells(10400067000) HLAclassIligandomeprofilingidentifiesanovelpanelof wassignificantlyhighercomparedwithmyelomacells(P,.0001). myeloma-associatedantigens NocorrelationofHLAsurfaceexpressiononmyelomacellswith patientcharacteristicssuchassex,age,diseasestage,riskclassifica- Toidentifymyeloma-associatedantigens,wecomparativelyanalyzed theHLAligandomesoftheMMsampleandHVcohortsatthesource tion,orpriortherapywasobserved(supplementalTable1). proteinlevel.OverlapanalysisofHLAligandsourceproteinsidenti- fied2412proteins(correspondingto31.8%ofthemappedMMHLA MSidentifiesnaturallypresentedHLAligandsofprimaryMM sourceproteome)tobeexclusivelyrepresentedintheHLAligandomes samplesandMCLs ofMMsamples.OftheseMM-exclusivesourceproteins,68.3%were Mapping the HLA class I ligandomes of 10 myeloma patients and solelyidentifiedonMCLsamples,whereas13.2%ofproteinswere 5MCLs(Table1),weidentifiedatotalof17583differentpeptides foundtobepresentedbothonMCLsandprimaryMMsamples.A representing7574sourceproteins,attaining.80%ofthemaximum fractionof18.5%ofmyeloma-exclusivesourceproteinswasfoundto attainablecoverage(Figure2A).Themeannumberofuniquepeptide berestrictedtoprimaryMMsamples(Figure2B).Toidentifybroadly identifications (IDs) was 1059 IDs for primary myeloma sam- presented TAAs, myeloma-exclusivesource proteins were ranked ples and 2243 IDs for MCLs (supplemental Table 2). Overall, accordingtotheirfrequenciesofrepresentationintheMMsample peptidesrestrictedby20differentHLA-Aand-Ballotypeswere cohort(Figure2C).Tostatisticallyassessandoptimizethestringency identified in this study, covering 99.3% of the German popu- ofantigenidentification,wesimulatedrandomizedvirtualligandomes lation(calculatedaccordingtoBuietal53).Nomutatedpeptideiden- insilicoandcalculatedtheresultantnumberoffalse-positiveTAAs tificationswereobtainedwhenprocessingthedatasetagainsta atdifferentfrequenciesofrepresentation(Figure2D).Wesetthe referenceproteomecontainingrecurrentmutationsinhematologic frequencythresholdforHLAclassITAAdefinitionto.25%of malignancies. myeloma-exclusiveantigen presentation, yielding 58 TAAs with an Ascontrols,weanalyzedtheHLAclassIligandomesof45HV- estimatedFDRof4.1%(supplementalTable4).Thisnovelpaneloffre- derivedsamples(30PBMC,10BMNC,and5granulocytespecimens) quently presented myeloma-associated antigens was represented by identifyingatotalof20171differentpeptidesrepresenting7729source 197uniqueHLAclassIligandsandconstitutes0.8%ofthemapped proteins(supplementalTable3).TheHLAallotypedistributioninthe myelomaHLAligandsourceproteome.KyotoEncyclopediaofGenes HVcohortcovered.80%ofHLA-Aand-BallelesintheMMsample andGenomes(KEGG)pathwayanalysis55andfunctionalannotation cohort.54 Analysis of HLA class II ligandomes was performed for clusteringoftheseantigenswithrespecttotheirbiologicalfunc- 7MMpatientsand5MCLs.Atotalof6076uniquepeptidesrepresent- tion(GOTermBPFAT56)didnotidentifyanystatisticallysignifi- ing1743sourceproteinswereidentified.TheHLAclassIIHVcohort cantoverrepresentedpathwaysorfunctionalclusters.Notably,the From www.bloodjournal.org by guest on January 5, 2019. For personal use only. BLOOD,3SEPTEMBER2015xVOLUME126,NUMBER10 T-CELLEPITOPESFORTHEIMMUNOTHERAPYOFMYELOMA 1207 Figure2.ComparativeHLAligandomeprofilingandidentificationofmyeloma-associatedantigens.(A)SaturationanalysisofHLAclassIligandsourceprotein identificationsinMMpatients.NumberofuniqueHLAligandsourceproteinidentificationsasafunctionofcumulativeHLAligandsourceproteinidentificationsin10MMpatients. Exponentialregressionallowedfortherobustcalculation(R250.99)ofthemaximumattainablenumberofdifferentsourceproteinidentifications(dashedline).Thedottedline depictsthesourceproteomecoverageachievedinourMMpatientcohort.(B)OverlapanalysisofHLAclassIligandsourceproteinsofprimaryMMsamples(n510),MCLs(n55), andHVsamples(totaln545:PBMC[n530],BMNC[n510],granulocytes[n55]).(C)ComparativeprofilingofHLAligandsourceproteinsbasedonthefrequencyofHLA- restrictedpresentationinMMandHVligandomes.FrequenciesofMMs/HVspositiveforHLA-restrictedpresentationoftherespectivesourceprotein(x-axis)areindicatedonthe y-axis.Theboxonthelefthighlightsthesubsetofmyeloma-associatedantigensshowingMM-exclusivepresentationin.25%ofmyelomasamples.(D)Statisticalassessmentof false-positivemyeloma-antigenIDsatdifferentthresholdvalues.ThenumbersoforiginalTAAsidentifiedbasedontheanalysisoftheMMandHVcohortswerecomparedwith randomvirtualTAAs.VirtualMMandHVsamplesweregeneratedinsilicobasedonrandomweightedsamplingfromtheentiretyofproteinidentificationsinbothoriginalcohorts. Theserandomizedvirtualligandomesofdefinedsize(n5957proteins,whichisthemeannumberofproteinidentificationsinallanalyzedsamples)wereusedtodefineTAAsbased onsimulatedcohortsof15MMvs45HVsamples.Theprocessofproteinrandomization,cohortassembly,andTAAidentificationwasrepeated1000timesandthemeanvalueof resultantvirtualTAAswascalculatedandplottedforthedifferentthresholdvalues.ThecorrespondingFDRsforanychosenTAAthresholdarelistedbelowthex-axis.sum,summary. proto-oncogenemultiplemyelomaSETdomaincontainingpro- KAMEAASSL(A*02:01),SLLEQGLVEA(A*02:01)).Moreover, tein(MMSET)wasidentifiedasaTAAshowingrepresentationin MMSETwasdetectedonbothMMpatientswiththeoncogenic 33%ofMMpatientligandomesandwasfoundtoberepresented translocationt(4;14),butonlyon1of6patients(17%)withoutthis by3differentHLAligands(ASNPSNPRPSK(HLA-A*30:01), aberration. From www.bloodjournal.org by guest on January 5, 2019. For personal use only. 1208 WALZetal BLOOD,3SEPTEMBER2015xVOLUME126,NUMBER10 Figure3.Representationofestablishedmyeloma- associatedantigensintheHLAligandomesofMM and HV. (A) Representation of previously described MM-associatedantigensinHLAclassIligandomes. Barsindicaterelativerepresentation(%)ofrespective antigensbyHLAclassIligandsonprimaryMMsam- ples,MCLs,andHVsamples.Dashedlinesdividethe antigensinto4groupsaccordingtotheirdegreeofMM association(MMandMCL-exclusive,MCL-exclusive, mixedpresentation,HV-exclusive).(B-C)Distribution ofmyeloma-exclusiveantigenpresentationfor(B)pre- viouslydescribedantigensand(C)ligandome-defined TAAsonMCLs(white)andMMplusMCLs(shaded). Representationofestablishedmyeloma-associatedantigensin analysisofthe58HLAclassIantigenswiththe1135HLAclassII theHLAclassIligandome presentedMM-exclusiveproteinsidentifiedapanelof6class-spanning antigensrepresentedby31peptides(Figure4D-E;supplemental BasedonourextensiveHLAligandomedataset,weinvestigatedthe Table5). Functional characterization of synergisticHLA class II li- presentation of established myeloma-associated antigens within the gandsrevealedpeptide-specificT-cellresponsesinmyelomapatients differentsamplecohorts.Weidentified73differentHLAligandsre- for3of5testedpeptides(Figure4E,supplementalFigure3B). presenting22of25(88%)previouslydescribedmyelomaantigens.42 TheoverallcomparisonoftheHLAclassIandIIligandomesof We found 9 of the 22 detectable antigens (41%) to be exclusively MMsamplesrevealed80%(1622)ofHLAclassII–presentedproteins presentedonMMsamples,10of22antigens(45.5%)toberepresented tobealsopresentedonHLAclassI(Figure4F).Functionalannotation bothonMMandHVsamples,and3of22(13.6%)exclusivelypre- clustering(GOTermCCclusteringusingDAVID56)wasperformedon sented on HV-derived samples (Figure 3A). Of note, 7 of 9 MM- thetop500mostfrequentlypresentedproteinsineachHLAclassto exclusiveantigens(77.8%)wereonlydetectableonMCLs.Only2of9 identifythecellularcompartmentsfromwhichtheseproteinsderive. (22.2%)oftheseMM-exclusiveantigensHLAligandsweredetected Antigenspresented onclassIdisplayedhighlyenrichedclustersfor onprimaryMMpatientsamples(Figure3B).Forreference,only7of58 nuclear proteins as well as for ribosomal, cytoskeletal, and vesicle- (12.1%)ofthenewlydefinedmyelomaantigensshowedpresentation derivedproteins.Notably,thispatternwasrecapitulatedin theclus- exclusively on MCLs, whereas the majority (51 of 58 [87.9%]) of teringofproteinspresentedonbothHLAclasses,albeitwithahigher antigenswerealsopresentedonprimaryMMpatientsamplesaswell, rankingandanalmostthreefoldhigherenrichmentforvesicle-derived whichunderlinestheirpotentialasclinicaltargetantigens(Figure3C). proteins.HLAclassII–presentedantigensshowedintermediateenrich- Moreover,unsupervisedclusteringofsourceproteinpresentationin mentforplasmamembrane,vesicle-derived,andlysosomalproteins theHLAligandomesrevealedtheclusterofMCLstobehighlydistinct (supplementalTable6). fromprimaryMMsamples(supplementalFigure2). AnalysisofHLAclassIIligandomesidentifiespotentially HLAclassITAAsaretargetedbyspontaneousT-cellresponses synergisticvaccinecandidates inmyelomapatients As the direct involvement of CD41 T cells in tumor control is es- Functional characterization of the novel myeloma antigens was tablished,57wefurtheraimedtoidentifyHLAclassIIantigens.Overlap performedinpanelsof11HLA-A*02–and2HLA-B*07–restricted analysisofHLAclassIIligandsourceproteinsidentified1135my- peptides, including 2 HLA-A*02 ligands derived from MMSET elomaexclusiveantigens(Figure4A).ComparativeprofilingofHLA (Figure5A).Myeloma-associatedpeptideswereevaluatedin12-day class II ligandomes identified a single antigen (TFRC) represented recallIFNgELISPOTassaysusingPBMCobtainedfromMMpatients by 67 HLA class II ligands showing MM-exclusive presentation at andHVs.WeobservedIFNgsecretionfor5of11A*02ligandsand FDR,5%(Figure4B-C).Functionalcharacterizationofthemost 1 of 2 B*07 ligands inmyeloma patients, as shown exemplarily in abundantTFRCpeptide(NSVIIVDKNGRLV)byIFNgELISPOT Figure5C.Bothpeptides(P andP )derivedfromMMSETshowed 1 2 revealed memory T-cell responses in 2 of 5 MM patients (sup- specificT-cellrecognition in 2 of 16 (13%) and1 of8 (13%) MM plementalFigure3A). patients,respectively.Importantly,nomyelomapeptide-specific AsCD41Tcellsplaypivotalrolesintheinductionandmaintenance IFNg secretion was observed in 10 HLA-matched healthy controls ofantigen-specificCD81T-cellresponses,58-60weimplemented (Figure5B).Notably,T-cellresponseswereonlyobservedfor asecondapproachtoidentifypotentiallysynergisticHLAclassII– myeloma-associatedpeptidesidentifiedonprimarymyelomasamples restricted peptides derived from HLA class I TAAs. Overlap (10of13),andneverforthe3of13peptidesidentifiedonMCLsonly. From www.bloodjournal.org by guest on January 5, 2019. For personal use only. BLOOD,3SEPTEMBER2015xVOLUME126,NUMBER10 T-CELLEPITOPESFORTHEIMMUNOTHERAPYOFMYELOMA 1209 Figure4.IdentificationofsynergisticHLAclassII–restrictedmyeloma-associatedantigens.(A)OverlapanalysisofHLAclassIIligandsourceproteinsofprimaryMM samples(n57),MCLs(n55),andHVsamples(total,n523:PBMC[n513],BMNC[n55],granulocytes[n55]).(B)Statisticalanalysisoffalse-positivemyeloma-antigen identificationsatdifferentthresholdvalues,asdescribedinFigure2.Randomizedvirtualligandomesizesweresetto226proteinsandTAAsweredefinedbasedonsimulated cohortsof12MMvs23HVsamples.(C)ComparativeprofilingofHLAclassIIligandsourceproteinsbasedonthefrequencyofHLA-restrictedpresentationinMMandHV ligandomes.FrequenciesofMMs/HVspositiveforHLA-restrictedpresentationoftherespectivesourceprotein(x-axis)areindicatedonthey-axis.(D)Overlapanalysisof HLAclassITAAs(n558)andHLAclassIIMM-exclusiveantigens(n51135).(E)HLAclassITAAs,whichalsoyieldpotentiallysynergisticHLAclassIIligands.(F)Overlap analysiscomprisingtheentireHLAclassIandIIligandsourceproteomesofMMsamples.sum,summary;rep.,representation. Thefrequenciesofpeptide-specificT-cellresponsesdetectedinMM matchedligandomesofMMpatients(Figure5A).Duetolimitationsin patients by ELISPOT were generally in the same range as the thenumbersofcellsavailableforanalysis,furthercontrolswithtarget frequencies of presentation of the respective peptide in allotype- cellsexpressingthecorrespondingantigenscouldnotbeperformed. From www.bloodjournal.org by guest on January 5, 2019. For personal use only. 1210 WALZetal BLOOD,3SEPTEMBER2015xVOLUME126,NUMBER10 Figure5.Functionalcharacterizationofmyeloma-associatedantigens.(A)Myeloma-associatedT-cellepitopeswiththeircorrespondingHLArestrictionsandfrequenciesof immunerecognitionbymyelomapatient-derivedTcellsinIFNg-ELISPOTassays.(B)Exampleofmyeloma-associatedT-cellepitopesevaluatedinanIFNg-ELISPOTusingHV PBMCs.AnEBVepitopemixcontainingthefrequentlyrecognizedpeptidesBRLF109-117YVLDHLIVV(A*02)andEBNA3247-255RPPIFIRRL(B*07servedaspositivecontrol). Benign-tissue–derivedpeptidesKLFEKVKEV(HLA-A*02)andKPSEKIQVL(B*07)servedasnegativecontrol.(C)Examplesofmyeloma-associatedT-cellepitopesevaluatedin IFNg-ELISPOTsusingMMpatientPBMCs(n53).Resultsareshownonlyforimmunoreactivepeptides.AnEBVepitopemixcontaining5frequentlyrecognizedpeptides(BRLF109- 117YVLDHLIVV[A*02],EBNA3471-479RLRAEAQVK[A*03],EBNA3247-255RPPIFIRRL[B*07],BZLF1190-197RAKFKQLL[B*08],EBNA6162-171AEGGVGWRHW[B*44]) wasusedaspositivecontrol.Benign-tissue–derivedpeptidesKLFEKVKEV(HLA-A*02)andKPSEKIQVL(B*07)servedasnegativecontrol.(D-E)TetramerstainingofCD81Tcells after3cyclesofaAPC-basedinvitroprimingsusingTcellsderivedfrom(D)ahealthyindividualand(E)amyelomapatient:Leftmostpanels,P2-tetramerstainingofCD81Tcells primedwithP2-aAPCs(SLLEQGLVEA,A*02);leftmiddlepanels,exvivoP2-tetramerstainingofCD81Tcells;rightmiddlepanels,controlstainingwithA*02-tetramercontaining anonrelevantA*02-restrictedcontrolpeptide(KAMEAASSL,A*02)onCD81TcellsderivedfromthesamepopulationasTcellsdepictedintheleftpanels.Rightmostpanels,Positive control:tetramerstainingofCD81TcellsprimedwithCMV-aAPCs(NLVPMVATV,A*02).EBV,Epstein-Barrvirus;neg.,negative;pos.,positive;UPN,uniformpatientnumber. WethereforecannotexcludethatT-cellreactivityisdirectedagainst modified with a protecting group, and that these impurities are im- impuritiescontainedinthesyntheticpeptidebatch.Indeed,itiswell munogenic.However,HLA-A*02–and-B*07–restrictedcontrolpep- knownthatsyntheticpeptidescontainimpurities,forexample,peptides tidesderivedfrombenigntissues(HV-exclusiveHLAligands)usedin From www.bloodjournal.org by guest on January 5, 2019. For personal use only. BLOOD,3SEPTEMBER2015xVOLUME126,NUMBER10 T-CELLEPITOPESFORTHEIMMUNOTHERAPYOFMYELOMA 1211 allELISPOTsinthestudyathandneverresultedinsignificantIFNg cell lines but not in primary samples, indicating that selection of release(Figure5C). pathophysiologicallyrelevantantigensshouldbebasedonanalysis ofprimarytumorsamples. Antigen-specificTcellscanbeinducedinvitrofromnaive A notable exception was the established myeloma-associated TcellsofMMpatientsorHVs proteinMMSET,whichiscurrentlybeinginvestigatedasatargetfor thetherapyofpoor-prognosist(4;14)myelomapatients.74-77Although Toassesswhethermyelomaantigen-specificT-cellresponsescanbe MMSET-derived peptides werefrequently identified on t(4;14) my- inducedfromnaiveTcellsinvitro,weisolatedCD81Tcellsfrom1 eloma samples, we also detected MMSET peptides in the HLA li- healthyindividualand1MMpatient.WeperformedaAPCprimings gandomesof at(4;14)-negativepatientand 1 t(4;14)-negativeMCL using the MMSET-derived peptide SLLEQGLVEA (P2). Using (U266).Strikingly,functionalcharacterizationbyELISPOTrevealed HV-derived CD81 T cells, a population of 0.403% P2-tetramer memoryT-cellresponsestargetingtheseMMSET-derivedepitopes positiveCD81 Tcellswasdetectedafterinvitropriming.No exclusively in myeloma patients and not in HV. This suggests tetramer-positiveT-cellpopulations.0.1%weredetectableexvivo. myeloma-dependentprimingofanti-MMSETT-cellresponsesin AfterprimingofTcellsfromanMMpatientwithoutpreviousT-cell vivo in MM patients, which underscores thepathophysiological reactivityforP2(asdetectedby12-day-recallIFNg-ELISPOTand relevanceofthisantigen.InconcordancewiththeHLAligandomics exvivotetramerstaining),wedetectedtheinductionofasmallpop- data,wefoundtheseT-cellresponsesnottoberestrictedtot(4;14) ulationof0.236%P2-tetramerpositiveCD81Tcells(Figure5E). myelomapatients.ResultsofinvitroprimingssuggestthatMMSET- Importantly,controlstainingsperformedwithanA*02-tetramer specificCD81T-cellresponsescanbeinducedfromnaiveTcells, containinganonrelevantA*02controlpeptidewereperformedin bothinhealthyindividualsand,importantly,alsoinmyelomapa- paralleloncellsderivedfromthesamewellsasusedfortherelevant tients,albeitwithlimitedmagnitudes.Withthecurrentstrategies staininganddidnotyieldanyspecifictetramer-positiveT-cellpopu- focusingoninhibitionofMMSETbysmallmoleculesorsmall lations(Figure5D). interferingRNAs,78,79ouridentificationofmyeloma-exclusive MMSET-derivedT-cellepitopesprovidesnewoptionsfortarget- ingMMSETbyT-cell–basedimmunotherapy.Notably,thisther- apeuticstrategymaynotnecessarilyhavetoberestrictedtot(4;14) Discussion myelomas,asweobservedMMSET-presentationandimmunerec- ognitionirrespectiveofthemutationalstatus.Thismightbeexplained Withtheadventofimmunecheckpointmodulation,T-cell–basedim- bythe distortedcorrelation of gene expression andHLA-restricted munotherapyhasopenedupnewavenuesforthetreatmentofarangeof antigenpresentationaswellasbythesubclonaldistributionoft(4;14) solidtumors61-68andisundergoingclinicalevaluationinhematologic inmyelomacellsandgenomicplasticityoccurringoverthecourse malignancies.69,70Thesenoveltherapeuticoptionsmayfurtherbeim- ofdisease.73,80 proved in combination with antigen-specific immunotherapy, which Together,ourfindingsillustratehowantigenidentificationguided mayhelpinduceandguidespecificanticancerimmuneresponses.To byHLAligandomicscanpinpointnovelMM-associatedT-cellepi- thisend,theexactknowledgeoftumor-associatedepitopeswhichcan topesandallowfordirectassessmentofantigendistributionpatterns actasrejectionantigensisindispensable.4,8Inmyeloma,amultitudeof inpatientcohorts.InparalleltoourfindingswithMMSET,ourstudy studieshaveinvestigatedMM-associatedantigens,yieldingahandful featuresanextensivepanelofnovelantigenspreviouslynotassociated ofpromisingtargets.25-35However,thesestudieswerelimitedinscopeof withmyelomaorcanceringeneral.AnalogouslytoMMSET,wede- antigensandHLAallotypes.42Inourpreviousstudiesinacutemyeloid tectedpreexistingT-cellresponsesagainstasubstantialproportionof leukemiaandchroniclymphocyticleukemia,wedemonstratedthatthe thesetargetsinmyelomapatients,indicatingahighenrichmentforre- comprehensiveandcomparativeanalysisoftheHLA-presentedanti- levantMM-associatedantigens.Inconclusion,ourligandome-centric genomecanidentifyextensivepanelsofbroadlypresentedT-cellepi- studymayguidethedesignoffutureantigen-specificT-cellimmuno- topescoveringamultitudeofHLAallotypes.12,13Here,wetranslated therapyinMM. thissystematicapproachtoMM. Quantification of HLA surface expression on different cell pop- ulations in the bone marrow of myeloma patients and healthy vol- Acknowledgments unteersdemonstratedthatHLAlossordownregulationonmalignant plasma cells is of no concern, even in patients who received The authors thank Claudia Falkenburger, Patricia Hrstic, Nicole prior therapy. Comparative analysis of the HLA ligandomes of Zuschke,KatharinaGraf,andBeatePo¨mmerlforexcellenttechnical these cell populations revealed distinct antigenic signatures and identifiedapanelofmyeloma-associatedantigens.Theseantigens support. This work was supported by the German Cancer Consortium didnotcluster intoanyof themajortumor-associatedpathways (DKTK),theDeutscheForschungsgemeinschaft(DFG;SFB685), anddidnotshowenrichmentforhighlyprioritizedtargetschar- andtheEuropeanUnion(EU;ERCAdG339842MUTAEDITING). acterized by tumor-associated overexpression. This indicates a morecomplexinterplayofunderlyingmechanismssuchasprotein turnover71 and antigen processing as the source of altered antigen presentationontumorcellsandfurtherunderscorestheisolatedchar- Authorship acteroftheHLAligandomeandthedistortedcorrelationwithitsup- streamsources.72,73 Contribution:S.W.,J.S.S.,D.J.K.,S.S.,andH.-G.R.designedthe Importantly,asubstantialproportionofestablishedmyelomaanti- study;S.W.,J.S.S.,D.J.K.,H.-G.R.,L.K.,K.W.,H.R.S.,andS.S. genswasfoundtobeonlyinfrequentlypresentedonprimarymyelomas draftedthemanuscript;S.W.,J.S.S.,andH.S.performedflowcyto- ortoshowsuboptimaldegreesofmyelomaspecificity.Ofnote,the metric analysis and HLA surface expression quantitation; S.W., majority of theseantigens was selectively detected on myeloma D.J.K., J.S.S., and S.K. conducted MHC peptide immunoaffinity From www.bloodjournal.org by guest on January 5, 2019. For personal use only. 1212 WALZetal BLOOD,3SEPTEMBER2015xVOLUME126,NUMBER10 purification; S.W. and D.J.K. performed MS-based analyses and Conflict-of-interestdisclosure:Theauthorsdeclarenocompeting quantitation;S.W.,A.N.,T.S.,J.S.S.,andH.S.conductedinvitroT- financialinterests. cell experiments; J.S.S., L.K., K.W., M.H., and H.R.S. conducted Correspondence:JulianeS.Stickel,DepartmentofHematology patient data collection and medical evaluation; S.W., J.S.S., D.J.K., andOncology,UniversityofTu¨bingen,Otfried-Mu¨llerStrasse10, O.K.,andL.B.performedstatisticalanalyses;andS.S.,H.-G.R.,and 72076 Tu¨bingen, Germany; e-mail: [email protected] J.S.S.supervisedthestudy. tuebingen.de. References 1. SmallEJ,SchellhammerPF,HiganoCS,etal. 17. FerreroS,LadettoM,DrandiD,etal.Long-term 32. SchuberthPC,JakkaG,JensenSM,etal. 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