论文实例：重组卡介苗诱导T细胞免疫应答的分子、细胞机理研究

作者简介：焦新安，男，1964年09月出生，1997年02月师从于扬州大学刘秀梵教授，于1999年12月获博士学位。

摘要牛结核分枝杆菌BCG是世界上广泛使用的疫苗，亦被认为是表达与传递外源抗原的理想活载体之一，然而迄今对BCG或重组BCG(rBCG)与宿主免疫系统相互作用机理尚未阐明，而从免疫调节生物学角度探讨BCG或rBCG诱导T细胞应答的规律亦未涉足。为此，本研究以表达大肠杆菌麦芽糖结合蛋白(MalE)的rBCG(rBCG·MalE)为模型，以期阐明rBCG与抗原提呈细胞相互作用的规律、rBCG表达产物抗原表位多样性、rBCG和BCG诱导的T细胞应答规律与调控因素。

1．重组卡介苗表达的MalE蛋白T细胞表位的鉴定

在体外抗原提呈试验中，证实rBCG·MalE成功表达MalE蛋白的四种T细胞抗原表位，即p68-82、p100-114、p151-165和p277-291。T细胞增殖试验和ELIOT试验结果均表明这种表达是功能性的，rBCG·MalE可刺激小鼠产生MalE蛋白及其多肽特异的T细胞增殖和IFN-γ应答。表位作图显示，BCG表达的MalE未形成新的抗原表位或使隐蔽表位暴露出来，并进一步证实MalEp68-82多肽表位是MalEH-2d限制性主要T细胞表位，而p100-114、p151-165和p277-291多肽表位为次要表位。本研究结果表明，作为表达和运送外源抗原的BCG载体，它功能性地表达了外源抗原MalE蛋白，并能向宿主免疫系统有效地传递，这进一步证明BCG是一种优良的疫苗载体。

2．重组卡介苗感染早期树突细胞的作用

具有抗原提呈细胞(APC)功能的吞噬性细胞，通过刺激T细胞应答可促进细菌清除，从而在抵抗细菌感染中起重要作用。巨噬细胞(M?)是胞内寄生菌的优选宿主细胞，并贮存大量的抗原物质，而树突细胞(DC)却是非常强势的APC。然而在体内针对细菌的T细胞应答中，这两种有吞噬活性的细胞之作用还未阐明。为此，本研究以rBCG·MalE为材料，对M?和DC诱导抵抗细菌的T细胞应答中的相对作用进行了探讨。在小鼠i.v.接种rBCG·MalE12小时后，脾脏M?和DC的感染率相当，但在来自体内的体外试验(Exvivo)中，用针对MalE蛋白的特异T细胞杂交瘤仅测出DCS表面存在免疫原性的MalE多肽/MHCⅡ复合物，而M?却没有。同样，在rBCG·MalE感染后，仅在DCs观察到CD40、B7.1(CD80)和B7.2(CD86)分子表达水平升高，并分泌IL-12p40，因而首次证实DC在激发针对分枝杆菌T细胞应答中的关键作用。进一步试验还显示，脾脏DCCD8α＋和CD8α－亚群在体内是相同势能的APC，但IL-12的产生主要来自CD8α＋亚群。这表明在rBCG感染早期，DC在体内条件下不仅在针对分枝杆菌获得性免疫中发挥主导作用，而且还通过分泌IL-12在自然免疫中起重要作用。研究中还首次发现，rBCG在其感染早期能在DC中存活，但数量没有增长。鉴于DC提呈rBCG抗原能力的迅速丧失，表明除M?外DC亦是rBCG或BCG感染早期的贮存宿主细胞。这些结果为BCG感染与免疫机理提供了新认识，对结核病控制有重要价值，同时，为以BCG作载体研制新型疫苗提供了重要理论依据。

3．表达MalE重组卡介苗诱导T细胞应答的动力学

应用免疫磁性分离技术去除免疫小鼠脾脏中CD4＋和CD8＋T细胞后，在ELIOT试验中证实，rBCG·MalE诱导的T细胞应答是CD4＋T细胞依赖的。对MalE、D特异T细胞应答的动态分析结果表明，·MalE、BCG·wt诱导的特异CD4＋T细胞应答存在Th1/Th2平衡转换现象，即起始阶段为Th1应答，一段时间后出现Th2应答，并逐步形成Th1/Th2混合应答。在此基础上，进一步分析了rBCG·MalE诱导针对MalE不同T细胞表位的应答规律，结果发现，随着感染与免疫过程的发展，针对MalEp68-82和p277-291的特异T细胞应答从起初的Th1应答向Th1/Th2混合类型变迁，非常有趣的是，针对p100-114的特异T细胞应答呈现典型的Th1/Th2平衡转换，而针对p151-165的T细胞应答仅是Th2类型，而且在免疫后较长时间才出现。这些结果不仅进一步验证rBCG·MalE诱导的T细胞应答规律，而且还揭示MalE蛋白不同T细胞表位的功能多样性。此外，研究中证实rBCG不同表达构建产生的MalE及其表达量亦直接影响它们诱导免疫应答的质和量。这些结果亦为疫苗的分子设计提供了新思路。

关键词重组BCG，MalE，T细胞表位，树突细胞，巨噬细胞，IL-12，Th1/Th2应答

MolecularandCellularMechanismsofTCellImmune

ReoesInducedbyRecombinantMycobacteriumbovisBCG

Atract

MycobacteriumbovisBCGisthemostwidelyusedvaccineallovertheworld,andrepresentsoneofthemostpromisinglivevectorstodeliverforeignantigetotheimmunesystem.However,untilnow,littleisknownaboutthemechanismsofinteractionbetweenBCGorrecombinantBCG(rBCG)andhostimmunesystem,andthedetailedcharacteristicsofcell-mediatedimmunityinducedbyrBCGorBCG.Toaddrethesequestio,therBCGexpreingMalEproteinofEscherichiacoli(rBCG·MalE)wasusedasmodelstraintostudythemechanismsbywhichMalEproteinispresentedbymajorhistocompatibilitycomplexclaⅡ(MHCⅡ)moleculestoTcells,todefinethefunctionaldiversityofTcellepitopesofexpreedMalEfromrBCG,andtodemotratethecharacteristicsandregulationmechanismsofThreoesinitiatedbyrBCG·MalE.

1.IdentificationofTcellepitopesofMalEproteinexpreedbyrecombinantMycobacteriumbovisBCG

TheepitoperepertoireofexpreedMalEfromrBCGwasanalyzedinvitrobyantigenpresentationaayusingdendriticcells(DCs)asantigenpresentingcell(APC)pulsedwithrBCG·MalE、BCG·wtorpurifiedMalEprotein.FourTcellepitopesofMalEproteinpresentedonthesurfaceofthoseDCspulsedwithrBCG·MalEandpurifiedMalEproteinwererecognizedbyCD4＋ThybridomasecificforMalEpeptidesp68-82,p100-114,p151-165andp277-291,reectively,whereasD CspulsedwithBCG·wtfailedtostimulatetheseThybridomas.TheresultsalsoshowedthatrBCG·MalEinducedinvivoTcellproliferationandIFN-γreoesagaitMalEproteinanditspeptidesorDantigenwhileBCG·wtonlytriggeredreoesecificforD.rBCG·MalEfunctionallyexpreedthefourTcellepitopesofMalEproteinandepitopemaingfrommiceimmunizedwithrBCG·MalEshowedthatnonovelepitopesorcrypticepitopeswererevealedinrBCG,andthep68-82wasimmunodominantepitopeandtheothersweresubdominantepitopes.ItwasfurthershownthatBCGisoneofthemostpromisinglivevectorstoexpreanddeliverforeignantigetotheimmunesystem.

2.TheroleofdendriticcellsduringtheearlystagesofarecombinantMycobacteriumbovisBCGinfection

PhagocyticcellswithAPCfunctioplayanimportantroleinresistancetobacterialinfectioninturnthattheycanstimulateTcellreoesenhancingbacterialclearance.Macrophages(M?)areprivilegedhostcellsforintracellularbacteriaandthusrepresentalargereservoirofantigenicmaterial,butDCsaremuchmorepotentAPC.Therefore,inTcellimmunitytobacteriatheroleofthesetwophagocyticcellsisnotclearlyestablished.Here,weinvestigatedivivotherelativecontributionofM?andDCsuetstotheantibacterialTcellreoetoMycobacteriumbovisBCG.Twelvehoursafteri.v.administrationofrBCG·MalE,therateofinfectionintheleenwascomparableforM?andDC.However,thepresenceofimmunogenicMalEpeptides/MHCⅡcomplexeswasdetectedexvivoonDC,butnotonM?,usingTcellhybridomasecificfortheMalEprotein.Likewise,upregulationofCD40,B7.1andB7.2moleculesandproductioofIL-12p40followinginfectionwasonlyoervedforDC,confirmingtheexclusiveroleofDCinstimulatingTcellreoes.CD8α＋andCD8α－leenDCwereequallypotentantigenpresentingcellsinvivo,buttheproductionofIL-12wasmainlyaociatedwiththeCD8α＋suet.Altogether,thesedataindicatedthatinvivoDCplayedaprimaryrolenotonlyinacquiredimmunitytomycobacteriaintheearlytimesofinfection,butalsoiniateimmunitythroughIL-12secretion.Strikingly,BCGbacillussurvivebutremainstableinnumberintheDCduringtheearlystagesofinfection.AsantigenpresentationbyDCisrapidlylost,thissuggeststhatDCmayrepresentahiddenreservoirformycobacteria.Theseresultswereveryusefulforthepreventionoftuberculosisandrelatedinfectio,andforthedevelopmentofnewvaccinesbasedonBCGvector.

3.KineticsofTcellimmunereoesinducedbyrecombinantMycobacteriumbovisBCGexpreingMalEprotein

InordertodeterminethecharacteristicsofTcellreoesinducedbyrBCG·MalE,thelenocytesfromimmunizedmiceweretreatedtodepleteCD4＋orCD8 Tcellsbyanimmunomagneticselection,thenthesecellswereusedtomeasuretheIFN-γorIL-2reoestoMalEproteinoritspeptidesinELIOTtest.NoIFN-γorIL-2reoesagaitMalEanditspeptidesweredetectedafterdepletionofCD4＋Tcells,incontrast,highlevelofIFN-γorIL-2reoesagaitMalEanditspeptideswerestilldetectedafterremovingCD8＋Tcells.TheseresultsclearlyindicatedthatTcellreoesinducedbyrBCG·MalEwereCD4＋Tcell-dependent.TheresultsofanalyzingTcellreoestoMalEproteinorDafterrBCG·MalEimmunizationshowedthatinitialTh1reoestoBCGorrBCGweremodulatedduringthecourseofinfectiontobecomeamixedTh1/Th2reoes.ToverifythisphenomenonofTh1/Th2shift,wefurtheranalyzedthekineticreoestoMalEpeptidesafterrBCG·MalEimmunization.Tcellreoesagaitp68-82andp277-291wereshiftfromTh1typetoTh2type,thenbecomeamixedTh1/Th2reoe.Intriguingly,Tcellreoestop100-114weretypicalshiftofTh1/Th2reoes,whileonlyTh2reoestop151-165weredetectedatlatertimepoints.ThisrevealedthatMalEproteinhadfunctionaldiversityofTcellepitopes.AlltheresultsinthisstudysuggestedthatthecharacteristicsofCD4＋TcellreoesinducedbyrBCG·MalEhadinitialtendencytohighlevelofTh1reoes,andlateron,thereoesbecametoshiftfromTh1toTh2andgotmixtureofbothtypesofreoesundertheregulationofTh1/Th2balancemechanism.Thisiscorrelatedwellwiththeabilityof bothmyeloid(CD8α－)andlymphoid(CD8α＋)DCsuetstostimulateMalE-ecificTcellsfollowingrBCG·MalEinfection.Inaddition,preliminaryresultsfrommiceimmunizedwith3differentrBCG·MalEstraialsosuggestedthatthenatureofTcellreoesagaitMalEproteinoritspeptideswereinfluencedbydifferentlevelsandlocalizationofMalEexpreionaccordingtothedifferentexpreioncaettesforrBCG.Theseresultsalsoshowsomenewcluesfortherationaldesignofvaccines.