論文實(shí)例：重組卡介苗誘導(dǎo)T細(xì)胞免疫應(yīng)答的分子、細(xì)胞機(jī)理研究

1、論文實(shí)例：重組卡介苗誘導(dǎo)T細(xì)胞免疫應(yīng)答的分子、細(xì)胞機(jī)理研究作者簡介：焦新安，男，1964年09月出生，1997年02月師從于揚(yáng)州大學(xué)劉秀梵教授，于1999年12月獲博士學(xué)位。摘要牛結(jié)核分枝桿菌BCG是世界上廣泛使用的疫苗，亦被認(rèn)為是表達(dá)與傳遞外源抗原的理想活載體之一，然而迄今對BCG或重組BCG(rBCG)與宿主免疫系統(tǒng)相互作用機(jī)理尚未闡明，而從免疫調(diào)節(jié)生物學(xué)角度探討B(tài)CG或rBCG誘導(dǎo)T細(xì)胞應(yīng)答的規(guī)律亦未涉足。為此，本研究以表達(dá)大腸桿菌麥芽糖結(jié)合蛋白(MalE)的rBCG(rBCGMalE)為模型，以期闡明rBCG與抗原提呈細(xì)胞相互作用的規(guī)律、rBCG表達(dá)產(chǎn)物抗原表位多樣性、rBCG和BCG

2、誘導(dǎo)的T細(xì)胞應(yīng)答規(guī)律與調(diào)控因素。1重組卡介苗表達(dá)的MalE蛋白T細(xì)胞表位的鑒定在體外抗原提呈試驗(yàn)中，證實(shí)rBCGMalE成功表達(dá)MalE蛋白的四種T細(xì)胞抗原表位，即p68-82、p100-114、p151-165和p277-291。T細(xì)胞增殖試驗(yàn)和ELIOT試驗(yàn)結(jié)果均表明這種表達(dá)是功能性的，rBCGMalE可刺激小鼠產(chǎn)生MalE蛋白及其多肽特異的T細(xì)胞增殖和IFN-應(yīng)答。表位作圖顯示，BCG表達(dá)的MalE未形成新的抗原表位或使隱蔽表位暴露出來，并進(jìn)一步證實(shí)MalEp68-82多肽表位是MalEH-2d限制性主要T細(xì)胞表位，而p100-114、p151-165和p277-291多肽表位為次要表位

3、。本研究結(jié)果表明，作為表達(dá)和運(yùn)送外源抗原的BCG載體，它功能性地表達(dá)了外源抗原MalE蛋白，并能向宿主免疫系統(tǒng)有效地傳遞，這進(jìn)一步證明BCG是一種優(yōu)良的疫苗載體。2重組卡介苗感染早期樹突細(xì)胞的作用具有抗原提呈細(xì)胞(APC)功能的吞噬性細(xì)胞，通過刺激T細(xì)胞應(yīng)答可促進(jìn)細(xì)菌清除，從而在抵抗細(xì)菌感染中起重要作用。巨噬細(xì)胞(M?)是胞內(nèi)寄生菌的優(yōu)選宿主細(xì)胞，并貯存大量的抗原物質(zhì)，而樹突細(xì)胞(DC)卻是非常強(qiáng)勢的APC。然而在體內(nèi)針對細(xì)菌的T細(xì)胞應(yīng)答中，這兩種有吞噬活性的細(xì)胞之作用還未闡明。為此，本研究以rBCGMalE為材料，對M?和DC誘導(dǎo)抵抗細(xì)菌的T細(xì)胞應(yīng)答中的相對作用進(jìn)行了探討。在小鼠i.v.接種

4、rBCGMalE12小時(shí)后，脾臟M?和DC的感染率相當(dāng)，但在來自體內(nèi)的體外試驗(yàn)(Exvivo)中，用針對MalE蛋白的特異T細(xì)胞雜交瘤僅測出DCS表面存在免疫原性的MalE多肽/MHC復(fù)合物，而M?卻沒有。同樣，在rBCGMalE感染后，僅在DCs觀察到CD40、B7.1(CD80)和B7.2(CD86)分子表達(dá)水平升高，并分泌IL-12p40，因而首次證實(shí)DC在激發(fā)針對分枝桿菌T細(xì)胞應(yīng)答中的關(guān)鍵作用。進(jìn)一步試驗(yàn)還顯示，脾臟DCCD8和CD8亞群在體內(nèi)是相同勢能的APC，但I(xiàn)L-12的產(chǎn)生主要來自CD8亞群。這表明在rBCG感染早期，DC在體內(nèi)條件下不僅在針對分枝桿菌獲得性免疫中發(fā)揮主導(dǎo)作用，

5、而且還通過分泌IL-12在自然免疫中起重要作用。研究中還首次發(fā)現(xiàn)，rBCG在其感染早期能在DC中存活，但數(shù)量沒有增長。鑒于DC提呈rBCG抗原能力的迅速喪失，表明除M?外DC亦是rBCG或BCG感染早期的貯存宿主細(xì)胞。這些結(jié)果為BCG感染與免疫機(jī)理提供了新認(rèn)識，對結(jié)核病控制有重要價(jià)值，同時(shí)，為以BCG作載體研制新型疫苗提供了重要理論依據(jù)。3表達(dá)MalE重組卡介苗誘導(dǎo)T細(xì)胞應(yīng)答的動(dòng)力學(xué)應(yīng)用免疫磁性分離技術(shù)去除免疫小鼠脾臟中CD4和CD8T細(xì)胞后，在ELIOT試驗(yàn)中證實(shí)，rBCGMalE誘導(dǎo)的T細(xì)胞應(yīng)答是CD4T細(xì)胞依賴的。對MalE、D特異T細(xì)胞應(yīng)答的動(dòng)態(tài)分析結(jié)果表明，MalE、BCGwt誘導(dǎo)的

6、特異CD4T細(xì)胞應(yīng)答存在Th1/Th2平衡轉(zhuǎn)換現(xiàn)象，即起始階段為Th1應(yīng)答，一段時(shí)間后出現(xiàn)Th2應(yīng)答，并逐步形成Th1/Th2混合應(yīng)答。在此基礎(chǔ)上，進(jìn)一步分析了rBCGMalE誘導(dǎo)針對MalE不同T細(xì)胞表位的應(yīng)答規(guī)律，結(jié)果發(fā)現(xiàn)，隨著感染與免疫過程的發(fā)展，針對MalEp68-82和p277-291的特異T細(xì)胞應(yīng)答從起初的Th1應(yīng)答向Th1/Th2混合類型變遷，非常有趣的是，針對p100-114的特異T細(xì)胞應(yīng)答呈現(xiàn)典型的Th1/Th2平衡轉(zhuǎn)換，而針對p151-165的T細(xì)胞應(yīng)答僅是Th2類型，而且在免疫后較長時(shí)間才出現(xiàn)。這些結(jié)果不僅進(jìn)一步驗(yàn)證rB CGMalE誘導(dǎo)的T細(xì)胞應(yīng)答規(guī)律，而且還揭示Mal

7、E蛋白不同T細(xì)胞表位的功能多樣性。此外，研究中證實(shí)rBCG不同表達(dá)構(gòu)建產(chǎn)生的MalE及其表達(dá)量亦直接影響它們誘導(dǎo)免疫應(yīng)答的質(zhì)和量。這些結(jié)果亦為疫苗的分子設(shè)計(jì)提供了新思路。關(guān)鍵詞重組BCG，MalE，T細(xì)胞表位，樹突細(xì)胞，巨噬細(xì)胞，IL-12，Th1/Th2應(yīng)答MolecularandCellularMechanismsofTCellImmune ReoesInducedbyRecombinantMycobacteriumbovisBCGAtractMycobacteriumbovisBCGisthemostwidelyusedvaccineallovertheworld,andrepresen

8、tsoneofthemostpromisinglivevectorstodeliverforeignantigetotheimmunesystem.However,untilnow,littleisknownaboutthemechanismsofinteractionbetweenBCGorrecombinantBCG(rBCG)andhostimmunesystem,andthedetailedcharacteristicsofcell-mediatedimmunityinducedbyrBCGorBCG.Toaddrethesequestio,therBCGexpreingMalEpro

9、teinofEscherichiacoli(rBCGMalE)wasusedasmodelstraintostudythemechanismsbywhichMalEproteinispresentedbymajorhistocompatibilitycomplexcla(MHC)moleculestoTcells,todefinethefunctionaldiversityofTcellepitopesofexpreedMalEfromrBCG,andtodemotratethecharacteristicsandregulationmechanismsofThreoesinitiatedby

10、rBCGMalE.1.IdentificationofTcellepitopesofMalEproteinexpreedbyrecombinantMycobacteriumbovisBCGTheepitoperepertoireofexpreedMalEfromrBCGwasanalyzedinvitrobyantigenpresentationaayusingdendriticcells(DCs)asantigenpresentingcell(APC)pulsedwithrBCGMalE、BCGwtorpurifiedMalEprotein.FourTcellepitopesofMalEpr

11、oteinpresentedonthesurfaceofthoseDCspulsedwithrBCGMalEandpurifiedMalEproteinwererecognizedbyCD4ThybridomasecificforMalEpeptidesp68-82,p100-114,p151-165andp277-291,reectively,whereasDCspulsedwithBCGwtfailedtostimulatetheseThybridomas.TheresultsalsoshowedthatrBCGMalEinducedinvivoTcellproliferationandI

12、FN-reoesagaitMalEproteinanditspeptidesorDantigenwhileBCGwtonlytriggeredreoesecificforD.rBCGMalEfunctionallyexpreedthefourTcellepitopesofMalEproteinandepitopemaingfrommiceimmunizedwithrBCGMalEshowedthatnonovelepitop esorcrypticepitopeswererevealedinrBCG,andthep68-82wasimmunodominantepitopeandtheother

13、sweresubdominantepitopes.ItwasfurthershownthatBCGisoneofthemostpromisinglivevectorstoexpreanddeliverforeignantigetotheimmunesystem.2.TheroleofdendriticcellsduringtheearlystagesofarecombinantMycobacteriumbovisBCGinfectionPhagocyticcellswithAPCfunctioplayanimportantroleinresistancetobacterialinfection

14、inturnthattheycanstimulateTcellreoesenhancingbacterialclearance.Macrophages(M?)areprivilegedhostcellsforintracellularbacteriaandthusrepresentalargereservoirofantigenicmaterial,butDCsaremuchmorepotentAPC.Therefore,inTcellimmunitytobacteriatheroleofthesetwophagocyticcellsisnotclearlyestablished.Here,w

15、einvestigatedivivotherelativecontributionofM?andDCsuetstotheantibacterialTcellreoetoMycobacteriumbovisBCG.Twelvehoursafteri.v.administrationofrBCGMalE,therateofinfectionintheleenwascomparableforM?andDC.However,thepresenceofimmunogenicMalEpeptides/MHCcomplexeswasdetectedexvivoonDC,butnotonM?,usingTce

16、llhybridomasecificfortheMalEprotein.Likewise,upregulationofCD40,B7.1andB7.2moleculesandproductioofIL-12p40followinginfectionwasonlyoervedforDC,confirmingtheexclusiveroleofDCinstimulatingTcellreoes.CD8andCD8leenDCwereequallypotentantigenpresentingcellsinvivo,buttheproductionofIL-12wasmainlyaociatedwi

17、ththeCD8suet.Altogether,thesedataindicatedthatinvivoDCplayedaprimaryrolenotonlyinacquiredimmunitytomycobacteriaintheearlytimesofinfection,butalsoiniateimmunitythroughIL-12secretion.Strikingly,BCGbacillussurvivebutremainstableinnumberintheDCduringtheearlystagesofinfection.AsantigenpresentationbyDCisr

18、apidlylost,thissuggeststhatDCmayrepresentahiddenreservoirformycobacteria.Theseresultswereveryusefulforthepreventionoftuberculosisandrelatedinfectio,andforthedevelopmentofnewvaccinesbasedonBCGvector. 3.KineticsofTcellimmunereoesinducedbyrecombinantMycobacteriumbovisBCGexpreingMalEproteinInordertodete

19、rminethecharacteristicsofTcellreoesinducedbyrBCGMalE,thelenocytesfromimmunizedmiceweretreatedtodepleteCD4orCD8 Tcellsbyanimmunomagneticselection,thenthesecellswereusedtomeasuretheIFN-orIL-2reoestoMalEproteinoritspeptidesinELIOTtest.NoIFN-orIL-2reoesagaitMalEanditspeptidesweredetectedafterdepletionof

20、CD4Tcells,incontrast,highlevelofIFN-orIL-2reoesagaitMalEanditspeptideswerestilldetectedafterremovingCD8Tcells.TheseresultsclearlyindicatedthatTcellreoesinducedbyrBCGMalEwereCD4Tcell-dependent.TheresultsofanalyzingTcellreoestoMalEproteinorDafterrBCGMalEimmunizationshowedthatinitialTh1reoestoBCGorrBCG

21、weremodulatedduringthecourseofinfectiontobecomeamixedTh1/Th2reoes.ToverifythisphenomenonofTh1/Th2shift,wefurtheranalyzedthekineticreoestoMalEpeptidesafterrBCGMalEimmunization.Tcellreoesagaitp68-82andp277-291wereshiftfromTh1typetoTh2type,thenbecomeamixedTh1/Th2reoe.Intriguingly,Tcellreoestop100-114weretypicalshiftofTh1/Th2reoes,whileonlyTh2reoestop151-165weredetectedatlatertimepoints.ThisrevealedthatMalEproteinhadfunctiona