IP10,ITAC及其雙功能分子誘導(dǎo)的抗腫瘤免疫及機(jī)制研究

IP10,ITAC及其雙功能分子誘導(dǎo)的

抗腫瘤免疫及機(jī)制研究Anti-tumorimmunityandthemechanismsinducedbyIP10,ITACandtheirdualfunctionalmoleculeResearchBackgroundAnti-tumorimmunotherapyAnti-angiogenesisAnti-tumorStrategiesSurgeryRadiation

therapychemotherapy?

moleculesBiologicTherapeuticsGoalsofanti-tumorimmunotherapy：effectorcellsactivationandrecruitment.Directedmovementoflymphocytesmediatedbychemokines.Chemokines,asuperfamilyofcytokines,actbywayofG-protein-coupledreceptors.ResearchBackgroundResearchBackgroundcancerAngiogenesisInflammationInfection-Co-receptorofHIVReproductiveHematopoiesisThymusdevelopmentImmunomodulatorgraftrejectionchemokinesChemokinesenhanceanti-tumorimmunityTh1

ImmatureAPCApoptosiscellsmaturationSLCCTLCTLTumorcellIP10,ITACMIP,CXCL16KillingMigrationIP10,ITACSDF-1AngiogenesisofchemokinesintumordevelopmentVECproliferationVECmigrationIL-8,IP10,ITACRecruitmentofeffectorcellsAnti-angiogenesisEnhancinganti-tumorefficacy?chemokines

BRAK

MIP-1α,MIP-2

IP10,ITAC

PF4

SDF-1

BCA-1

CXCsuperfamilynon-ELRchemokineSimilarstructureCommonreceptorCXCR3ChemotacticactivityforactivatedTcellsAnti-angiogenesiscandidatesRecruitmentofeffectorcellsAnti-angiogenesisBreastcancerOneofthecommonmalignanciesforwomenOneoftheleadingcausesofdeathforwomenismetastaticbreastcancerTwotherapeuticstrategies:enhancinganti-tumorimmunityandanti-angiogenesisTumorcellsderivedchemokinesControllymphocytesinfiltrationintumorsRegulateangiogenesisAsautocrineorparacrinegrowthfactorsControlactivityoftumorcellsitself

IP10,

ITACIP10,

ITACTheexpressionofchemokinesinhumanbreastcancerbiopsyandtumorcelllinesPartIIP10andITACexpressioninhumanbreastcancerbiopsies*P<0.05**GAPDHIP10ITACinnertumoroutertumorLane1,GAPDH;lane2,PF4;lane3,MIG;lane4,IP10;lane5,ITAC;lane6,IL-8;lane7,MCP-1,;lane8,MCP-2;lane9,MCP-3;lane10,Frak;lane11,MIP-2;lane12,MIP-1a;lane13,MIP-1b;lane14,BCA-1;lane15,LARC;lane16,BRAK;lane17,SDF-1.Chemokinesexpressioninbreastcancercelllines12345678910111213141516174T1MCF-712345678910111213141516174T1celllineBALB/cmicederivedmammarycarcinomacelllineGrowthcharacteristicsparallelinvasivehumanmammarycarcinomaSpontaneousmetastasistolungandlymphnodeResistanttohormone-therapy4T1mammarycarcinomamodelIP10-mediatedanti-tumoreffectsImmunomodulatorAnti-angiogenesisReferencesDorsey,etal.,CancerRes,2002.Huang,etal.,IntJcancer,2004.Pertle,etal.,JImmunol,2001.Arenberg,etal.,CancerImmunother,2001.

?

Anti-tumoreffectsofITACInductionofTh1-typeresponseThemostpotentagonistofCXCR3withthehighestreceptoraffinityInductionofCXCR3internalizationPromotingTcell-endothelialcellcontactInhibitionofCCR3-mediatedTh2-typeresponseBConstructionandidentificationofpcDNA3-IP10ACCCATGGGCGGCCGCCTGCAGACCAGGTCTATAAGAATCCGGCCGCCAAGGCATCCACTGTAACCACAGAAGGTAGCGTGGAGTGTGCACGTGACTTTTAGAGACAAATATTTTTCATAGCCAAGTAGAACAGTTTAGATGTATCGGACCTTCTAGAAAGTTCTGCAGCCTGGTAATACGTGGCTGCATGTTCCAAGACAGCAGAGGGTCAGGTTCCTGGCACAGAGTTCTTATTGGAGGGCTCACAGTCAGACGTTCCCAGGATGTCACATGTTTTGACGCCTTAAAAAATTCTTTTTTTCTATTGCCTGCATTATGAGGCGAGCTTGCTTGGATCTGGGGTCCAGGCACCTTTGTCGTTTATGAGCCTTCATAGTAACAATCACTTCAACTTTGTCGCAGCCGTTACTCGGGTAAATTACAGAAGCTTTCTCGATCTCTGCCATTTTGACGGCTTTCATCCCGGGGCCGATGCAAAGACAGCGCCCCTGTTTGAACATAAGGAAGCCTTGAGCAGCTGTGGCCCAAGCCGCTGCAGCTCTCTGCC340bpM12FigA.M.Marker;2.Undigested;3.DigestedwithEcoRI/XhoI.DNAsequencingGGCAGAGAGCTGCAGCGGCTGCTGAGACGAACAGGAAGGTCACAGCCATAGCCCTGGCTGCGATCATCTGGGCCACAGCTGCTCAAGGCTTCCTTATGTTCAAACAGGGGCGCTGTCTTTGCATCGGCCCCGGGATGAAAGCCGTCAAAATGGAGAGATCGAGAAGCTTGAAATCATCCCTGCGAGCCTATCCTGCCCACGTGTTGAGATCATTGCCACGATGAAAAAGAATGATGAGCAGAGATGTCTGAATCCGGAATCTAAGACCATCAAGAATTTAATGAAAGCGTTTAGCCAAAAAAGGTCTAAAAGGGCTCCTCTCGAGGATCCGGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTAB580bp12MFigA.1.DigestedwithEcoRI/XhoI;2.Undigested;M,Marker.ConstructionandidentificationofpcDNA3-ITACDNAsequencingIP10andITACexpressionintransfected4T1cellsA*B**P<0.054T1-IP10Cell4T1-ITACCellSchemeofchemotaxisassayusinga48-wellchemotaxischamberabcIL-2activatedlymphocytesSupernatantsoftransfectedcellsChemotacticactivityof4T1-IP10and4T1-ITACcellsB*A**P<0.05IP10andITACexpressionarelowinhumanbreastcancerbiopsyandtumorcelllinespcDNA3-IP10andpcDNA3-ITACcanexpressIP10andITAC4T1-IP10and4T1-ITACcellsexpressfunctionalIP10andITACwithchemotacticactivitySummariesAnti-tumoreffectsandmechanismsofIP10andITACinmurine4T1mammarycarcinomaPartIIInvivoTumorigenesisStudies

1x105cellspermouseParental4T1cells4T1-pcDNA3cells4T1-IP10cells4T1-ITACcellsTheeffectsofIP10andITACgeneintroductiononthegrowthof4T1cellsinBALB/cmice*P<0.05B*A**P<0.05*A*BTumorigenesisinimmunocompetentmiceonday304T14T1-pcDNA34T1-IP104T1-ITAC

Survivalrateofthetumor-bearingmiceABThegrowthkineticsof4T1-IP10and4T1-ITACcellsinvitro

ABResearchIP10andITAChaveanti-tumoreffectsIP10andITACgeneintroductiondonotalterthetumorigenicityof4T1cellsIP10andITACprolongthelifespanoftumor-bearingmiceIP10andITACexpressionintumortissues**P<0.01A**B*******ChemotacticactivityofrecombinantproteinsIP10andITAC*P<0.05Theamountsoftumorinfiltratedlymphocytes*P<0.05B*A**Anti-Thy1*P<0.05**LymphocytesinfiltrationintumorsH&E4T14T1-pcDNA34T1-IP104T1-ITACThesubsetsofthetumorinfiltratedlymphocytes*P<0.05AB**C4T14T1-pcDNA34T1-IP104T1-ITACCXCR3*P<0.05ProliferationoftumorinfiltratedlymphocytesA***B******Proliferationofsplenocytesintumor-bearingmice*P<0.05**A***B***Cytotoxicactivityoftumorinfiltratedlymphocytes*P<0.05B***A******Cytotoxicactivityofsplenocytesintumor-bearingmice*P<0.05B***A*****IFN-γproductionintumorinfiltratedlymphocytes*P<0.05*B*A*ExperimentallungmetastasismodelInoculationof2x106

parental4T1,4T1-pcDNA3,4T1-IP10and4T1-ITAC7daysInjectionof1x105wildtype4T1cells14daysRemovetumor28daysMetastaticnodeonthelungsLungremovedforcolonyassayNormal4T14T1-pcDNA34T1-IP104T1-ITAC

ADisseminatedmetastaticnodesandlungweight*P<0.05B**Theamountsofclonogenicmetastaticcells*P<0.05A*B*SectionsoflungtissuesstainedwithH&EA.4T1B.4T1-pcDNA3C.4T1-IP10D.4T1-ITACABCDIP10andITACAnti-tumorimmunityAnti-angiogenesis?TheeffectsofIP10andITAConthegrowthofvascularendothelialcellsBA*P<0.05**MigrationofvascularendothelialcellsinaninvitroscrapingassaybFGF+ITACbFGF+IP10bFGFRPMI1640*P<0.05

*bFGF20ng/mlIP10100ng/mlITAC100ng/mlNeovascularizationinmousecorneamicropocketmodel*P<0.05*

bFGF20ng/mlIP10100ng/mlITAC100ng/mlbFGF+ITACbFGF+IP10bFGFPBSGrowthkineticsofendothelialcellsintumorcellconditioned-mediumBA*P<0.05***A.4T1B.4T1-pcDNA3C.4T1-IP10D.4T1-ITACHistologicalassayofvascularityintumorsIP10andITAChaveanti-tumoreffectsRecruitmentoflymphocytesandanti-angiogenesisparticipateinthetumorgrowthinhibitionIP10andITACcaninduceprotectiveanti-tumorimmunityITACinducesrobustanti-tumorimmuneresponseIP10showsobviousangiostaticactivitySummariesReferencesSautyA,etal.JImmunol,2001,167:6576-82MeyerM,etal.EurJImmunol2001,31:2521-27Soejima,etal.JImmunol,2001,167:6576-82LusterAD,etal.FASEBJ,2000,14:2055-61ChemotaxisCa++mobilityTransendothelialmigrationAdhesionInhibitingendotheliummigrationIP10ITACCurrentfindingsBiologiceffectsof

IP10

and

ITACBiologiceffects4T1-IP104T1-ITAC腫瘤淋巴細(xì)胞浸潤程度++++++腫瘤浸潤淋巴細(xì)胞特異增殖應(yīng)答++++++腫瘤浸潤淋巴細(xì)胞特異性殺傷活性++++++腫瘤浸潤淋巴細(xì)胞IFN-γ分泌++++++脾細(xì)胞特異性增殖應(yīng)答++++++脾細(xì)胞特異性殺傷活性++++++Inhibitingmigrationofendothelium++++++Inhibitinggrowthofendothelium+++++Inhibitingneovascularization+++++Micro-vasculardensity+++++Infiltrationoflymphocyteintumors++++++SpecificproliferativeresponseofTIL++++++SpecificcytotoxicactivityofTIL++++++IFN-γproductionofTIL++++++Specificproliferativeresponseofsplenocytes++++++Specificcytotoxicactivityofsplenocytes++++++Designingandconstructionofadualfunctionalmolecule

PartIIIStructureofchemokines1.N-terminal（beforeconservedCys)2.N-LOOP(afterthe2rdconservedCys)3.?-sheet4.C-terminalα-helixTwo-stepbindingmodelN-LoopregionrecognizereceptorN-terminalactivatereceptorC-terminalECVDesigningaofdualfunctionalmoleculeITIPIP10ConfigurationofITIPandIP10MSIinsightII2000versionHomogenecitybetweenITIPandIP1066.6667%StructuresimilaritybetweenITIPandIP10

（RMS＝1.08358）FigA.1,N-terminalofITAC;2,C-terminalofIP10.FigB.M,marker;1,ITIP.A210bp12168bp380bpM1BAmplificationofITIPcodinggene168bp210bpConstructionandidentificationofpcDNA3-ITIPABATACGACTCACTATAGGGCGACATATGATCGATGATATCCCATGGGCGGCCGCCTGCAGACCAGGTCTCGGAATTCGAGAGATCTCCAAAGCCCAGGCAGAGAGCTGCAGCGGCTGCTGAGATGAACAGGAAGGTCACAGCCATAGCCCTGGCTGCGATCATCTGGGCCACAGCTGCTCAAGGCTTCCTTATGTTCAAACAGGGGCGCTGTCTTTGCATCGGCCCCGGGATGAAAGCCGTCAAAATGGCAGAGATCGAGAAGCTTGAAATCATCCCTGCGAGCCTATCCTGCCCACGTGTTGAGATCATTGCCACGATGAAAAAGAATGATGAGCAGAGATGTCTGAATCCGGAATCTAAGACCATCAAGAATTTAATGAAAGCGTTTAGCCAAAAAAGGTCTAAAAGGGCTCCTTAACTGGAGAGAAGCCACAAGACTGGAGATCTGGATCCCTCGAGTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCCGCTTTCCAGTCGGGAAAC380bpM12FigA.M.Marker;1.Undigested;2.DigestedwithEcoRI/XhoI.DNAsequencingITIPexpressionandchemotaxicactivity*P<0.05Fig.A1,4T1;2,4T1-pcDNA3;3,4T1-ITIP.123A***BITIPcanformchemokine-likeconfigurationITIPcodinggeneisamplifiedpcDNA3-ITIPexpressITIP4T1-ITIPcellsexpressfunctionalITIPwithchemotaxicactivitySummariesAnti-tumoreffectsandmechanismsofthedualfunctionalmoleculePartIVInvivoTumorigenesisStudies

5x104cellspermouseParental4T1cells4T1-pcDNA3cells4T1-IP10cells4T1-ITACcells4T1-ITIPcells4T1-ITAC+IP10Thegrowthof4T1cellsharboringITIPgeneinimmunocompetentmice*P<0.05A*BA*P<0.05Thegrowthof4T1-ITIPinnudemiceB**Thesubsetsofthetumorinfiltratedlymphocytes*P<0.05A**BTheproliferativeresponseoflymphocytesfrom4T1-ITIPtumor-bearingmice*P<0.05A**SplenocytesTIL**BCytotoxicactivityofthelymphocytesfrom4T1-ITIPtumor-bearingmice*P<0.05B***A***TILSplenocytesIFN-γproductionoftumorinfiltratedlymphocytes*P<0.05*InteractionbetweenAPCandTcellsCD40CD40LB7CD28ICAM-1LFA-1ChemokineChemokinereceptorReferences:Nanki,etal.,JImmunol,2000.Whiting,etal.,JImuunol,2004.MHC-AgAPCTCRTcellCo-stimulatorsTheeffectsofIP10andITACproteinontheproliferationof4T1-primedlymphocytes*P<0.054T1－

+++IP10

－－+－

ITAC

－

－

－

+*A4T1-++