Potent antitumor effect elicited by gp96-peptide complexes pulsed by dendritic
cell on mice of H22 liver cancer
obiliaryDepartment,FirstHospital‟
;MicrobiologyDepartment,Medicalcollege‟
;Xi‟anJiaotongUniversity,Xi‟an710061,China
;Xi‟anJiaotongUniversity.Xi‟an710061,China
;lAbstract]Objective:ToimproveDC—basedtumorvaccination,westudied
whetherdendriticceils
;(DCs)whichcoculturedwithH22livercancercells—derivedheatshockprot
ein(HSP)glycoprotein96
;(gP96)affecttheTcell—activatingpotentialinvitroandtheinductionoftumorimmunityinvivo?Meth?
;ods:Maturationofmurinebonemarrow—derivedDCwasinducedbyGM—
CSFplusIL一4,whichmimiced
;theimmunostimulatoryeffectofDC.CocuhuredDCandgp96一
peptidecomplexeswereusedtovaccineH22
;livercancerce11sofmice.Usingmurinemodelswecomparedtheimmunogenecityof19t2modifiedbygp96?
;peptidescomplexesderivedfrommurinelivercancercellsaloneorinactivetu
morcells.Toverifythespeci—
;ficitvofthevaccine,iitroassayswereexecuted.Serumcytokinelevelswerequantifiedtoexplorethe
;supposedpathwayofDCmodifiedbygp96peptidecomplexesanditseffectonantitumorimmuneresponse?
;ResuIts:DCmodifiedbygp96一
peptidecomplexescanactivatespleenlymphocyteandthelattercanspecifi—
;callvkillH22cellsbutnotEhrilichascitescarcinomacells.ModifiedDCcanin
antigen— ducepotenttumor—
;speeifieimmuneresponse,augmenttheproliferationofThlcells,andinhibittumorgrowth.Conclusion:
;Inthisstudy,wehavedevelopedanovelDC—mediatedtumorvaccinebycom
bingthegp96antigenicpep—
;tidescomplexesandinducingimmuneresponseagainstspecifictumorcells.gp96canbeidentifiedasapo—
;tentDCactivator.
;[Keywords]dendriticcell;gp96一peptidecomplexes;1ivercancer;vaccine
;Increasingevidencehasestablishedthatimmu—
;nizationwithheatshockprotein(HSP)isolated
;fromcancercellsorvirus—infectedcellscanelicit
;protectiveimmuneresponse.Thisparadigmhas
;alsobeensubstantiatedbyHSPglycoprotein96
;(gp96)whichcanimmunizeandinduceimmunere—
;sponseagainstspecificantigens[„.Dendriticcells
;(DCs)arehighlyspecializedantigenpresentingcells ;(APC)thatareuniqueintheirpotencytoinduceT ;cell—dependentAg—specificimmuneresponses【4].A
;numberofinvestigatorshavedemonstratedthatDC—
;basedvaccinescangeneratespecificantitumorim—
;munityinmurinetumormodelsc一川.Thesevaccines
;werebasedonfusionorcocuhureofDCandtumor ;cellsontumorpeptide—ortumorlysate—pulsed
;DCc.,.BecauseHSPhasthepromiscuousabilityto ;„
;:SupportedbytheNationalNaturalScienceFoundationof ;China(No.30200369)
;，
;:Correspondingauthor.E—mail:yangwei@21cn.com
;chaperoneandpresentabroadrepertoireoftumor ;antigenstoAPC[..wereasonedthattheymayim—
;provethetherapeuticpotentialofDC——basedvac——
;cines.Towardthisgoal,westudiedwhethermodi—
;fiedDCswhichcocuhuredwithH221ivercancer ;cellsderivedgp96affectstheTcell—activatingpo-
;tentialinvitroandtheinductionoftumorimmunity ;invivo.
;MATERIALSANDMETHoDS
;MiceBALB/cmice,aged6-8weeksand
20gwereprovidedbyLaboratorialAn—— ;weighted18——
;imalsCenterofFourthMilitaryMedicalUniversity ;andmaintainedunderpathogen—freeconditions.An—
;imalcarewasprovidedinaccordancewiththeproce—
;duresoutlinedinthe”ModernMedicalLaboratory
;Animals”(People‟sMilitaryMedicalPress,1999).
;CelllinesH22livercancercellsandEhrilich ;ascitescarcinomacellsweregiftsofTeachingand ;ResearchingRoomofImmunologyandPathology, ;
;980r口ofMedicalCollegesofPLA2006;21(2) ;Xi‟anJiaotongUniversityandmaintainedinRP—
;MI1640supplementedwith10%fetalbovine ;SerUii1-
;Isolationofgp96一peptidecomplexesgp96was
;purifiedfromH22livercancercellofBALB/c ;mouseasdescribedc?.Inbrief,carcinomatous ;asciteswaschallengedwithasecondheattreatment
;at42Cfor12hinwater,homogenizedin30
;mmol/LNaHC03,O.5mmol/LPMSF(pH7.1),
;byrepeatedtwiceoffreezingandthawingcycles.A ;100000gsupernatantwasobtainedandappliedto ;ConAsepharoseaffinitychromatographycolumn ;(AmershamPharmaciaBiotech)whichequilibrated ;with30mmol/LNaHC03.Theglycoproteinswere ;elutedby1Oa—methylmannoside.Theeluatewas
;appliedtoMonoQFPLCcolumn(AmershamPhar—
;maciaBiotech),equilibratedwith20mmol/LTris ;?
;HCl(DH8.5),0.5mol/LNaClandelutedovera ;20—600mmol/LNaC1gradient.Thesamplebuffer ;ateverypeakswerecollectedandfolloweddialyzing ;andconcentrating.PreparationsanalyzedbySDS—
;PAGEsilverstainingandWesternblotwithanti—
;Grp94antibody(StressGensCo).
;PreparationofDCinvitroBALB/cmicebone ;marrowwasflushedfromfemursandtibiaswith ;physiologicalsaline.Afterlysedredbloodcellsby ;hyposmsis,theremainingswerewashed3times ;withHank‟ssolution.Themurinebonemarrow—de—
;rivedprecursercellswereplated10./Lto6-wellcul—
;tureplatesandmaintainedinRPMI1640culture ;mediumwith10FCSinthepresenceof100ng/ml
CSFand10ng/mlrecombi— ;recombinantmouseGM—
;nantmouseIL一4(AmershamPharmaciaBiotech). ;1ooselyadherentcellswereharvestedonday3and ;day7toverifybyS100immunohistochemistryand ;electronmicroscopicphotograph.Finally,the10./L ;DCscoculturedwith250ngpurifiedgp96-peptide ;complexesat37C,5%Co2for2h.
;ImmunizationFortyBALB/cmicewereallo—
;catedto4groups(一10).Forconstructingmurine
;livercancermodels,10./LH22livercancercells ;weregivensubcutaneouslyattherootofrightback ;leg.After3doftumorchallenge,fortherapeutic ;immunization,2×10./LmodifiedDC,6ug/ml ;gp96proteininactivatedH22livercancercells,for ;hlankcontrol,thephysiologicalsaline(blankcon—
;trolgroup)wereinjectedtomiceevery3d,totally ;4timesaltogether.
;ELISAandC,releaseassayInoculatedmice ;werekilledafter21d.Centrifugateofmouseserum
;werecollectedandthelevelsofIL一10andIFN-7
;weredeterminedbyELISAkit(EndogenCo.).The ;cytotoxicitywasevaluatedbyastandardC(pro—
;videdbyPathologicalTeachingandResearching ;RoomofXi‟anJiaotongUniversity)releaseassay.
;H22cellcollectedfromfreshhydropertoneumwere ;usedasatarget(at1000cellsperwel1)ina6-hour ;assay.Toassesstumorspecific,theEhrilichascites ;carcinomacellsfromfreshhydropertoneumwereal—
;sousedasatarget.Theproportionofeffectspleen ;cellsandtargetcellswas25:l-TheCI(specific ;killrate)wasevaluatedbyfollowedformula:CI ;一(experimentaltremacpm—naturallyreleased
;tremacpm)/(maxreleasedtremacpm—naturally
;releasedtremacpm)×100.
;StatisticalanalysisTheKruskal—WallisHtest
;wasusedforstatisticalanalysis.
;RESULTS
;DCculturePlentyo{non—adhereroundcells
;wereobservedattheinitialstageofculture.Great ;changeappearedafter3d.Theburringdendritic ;couldbeseenonthecell‟sbodyattheday4(Fig
;1),andthecellsweretypicallysuspended,gradual—
;ly,thecellsgrewinclusters.After7d,theloosely ;adherentcellswerespreadaroundthecultureflask ;equably.Whenweobservedthroughscanningelec—
;tronmicroscope(SEM),thebiggercells‟body
;couldbeseen.Plentyofbiforkeddendriticplasma ;tubergrewonthemembraneofburringcells.Some ;oftuberpresentedsliceshapeaccordingwiththe ;topicalshapecharacterofDC(Fig2).DCwas ;stainedbyimmunohistochemicalmethodpresenting ;S一100proteinstrongpositiveterra—cotta(Fig3).
;ConcentratiOnchangeofIFN-Y.IL-IOin ;serumTheconcentrationofIFN—rwasthehighest
;inDCimmunitygroup,butthelowestincontrol ;group,andgreatdifferenceexistedamongevery ;group(H一26.542,P一0.0000).Theconcentra—
;tionofIL一10wasthelowestinDCimmunitygroup, ;
;
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;1O0J0rnalofMedicalCollegesofPLA2006;21(2) ;livercancerspecificantigeninaddtotheexistedin—
;dividualvarience.HSP—basedvaccinesworkacross ;tumortypesandbypasstheneedforidentifyingthe