1、論文實例：重組卡介苗誘導T細胞免疫應答的分子、細胞機理研究作者簡介：焦新安，男，1964年09月出生，1997年02月師從于揚州大學劉秀梵教授，于1999年12月獲博士學位。摘要牛結核分枝桿菌BCG是世界上廣泛使用的疫苗，亦被認為是表達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)與宿主免疫系統相互作用機理尚未闡明，而從免疫調節生物學角度探討BCG或rBCG誘導T細胞應答的規律亦未涉足。為此，本研究以表達大腸桿菌麥芽糖結合蛋白(MalE)的rBCG(rBCGMalE)為模型，以期闡明rBCG與抗原提呈細胞相互作用的規律、rBCG表達產物抗原表位多樣性、rBCG和BCG

2、誘導的T細胞應答規律與調控因素。1重組卡介苗表達的MalE蛋白T細胞表位的鑒定在體外抗原提呈試驗中，證實rBCGMalE成功表達MalE蛋白的四種T細胞抗原表位，即p68-82、p100-114、p151-165和p277-291。T細胞增殖試驗和ELIOT試驗結果均表明這種表達是功能性的，rBCGMalE可刺激小鼠產生MalE蛋白及其多肽特異的T細胞增殖和IFN-應答。表位作圖顯示，BCG表達的MalE未形成新的抗原表位或使隱蔽表位暴露出來，并進一步證實MalEp68-82多肽表位是MalEH-2d限制性主要T細胞表位，而p100-114、p151-165和p277-291多肽表位為次要表位

3、。本研究結果表明，作為表達和運送外源抗原的BCG載體，它功能性地表達了外源抗原MalE蛋白，并能向宿主免疫系統有效地傳遞，這進一步證明BCG是一種優良的疫苗載體。2重組卡介苗感染早期樹突細胞的作用具有抗原提呈細胞(APC)功能的吞噬性細胞，通過刺激T細胞應答可促進細菌清除，從而在抵抗細菌感染中起重要作用。巨噬細胞(M?)是胞內寄生菌的優選宿主細胞，并貯存大量的抗原物質，而樹突細胞(DC)卻是非常強勢的APC。然而在體內針對細菌的T細胞應答中，這兩種有吞噬活性的細胞之作用還未闡明。為此，本研究以rBCGMalE為材料，對M?和DC誘導抵抗細菌的T細胞應答中的相對作用進行了探討。在小鼠i.v.接種

4、rBCGMalE12小時后，脾臟M?和DC的感染率相當，但在來自體內的體外試驗(Exvivo)中，用針對MalE蛋白的特異T細胞雜交瘤僅測出DCS表面存在免疫原性的MalE多肽/MHC復合物，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)分子表達水平升高，并分泌IL-12p40，因而首次證實DC在激發針對分枝桿菌T細胞應答中的關鍵作用。進一步試驗還顯示，脾臟DCCD8和CD8亞群在體內是相同勢能的APC，但IL-12的產生主要來自CD8亞群。這表明在rBCG感染早期，DC在體內條件下不僅在針對分枝桿菌獲得性免疫中發揮主導作用，

5、而且還通過分泌IL-12在自然免疫中起重要作用。研究中還首次發現，rBCG在其感染早期能在DC中存活，但數量沒有增長。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胞。這些結果為BCG感染與免疫機理提供了新認識，對結核病控制有重要價值，同時，為以BCG作載體研制新型疫苗提供了重要理論依據。3表達MalE重組卡介苗誘導T細胞應答的動力學應用免疫磁性分離技術去除免疫小鼠脾臟中CD4和CD8T細胞后，在ELIOT試驗中證實，rBCGMalE誘導的T細胞應答是CD4T細胞依賴的。對MalE、D特異T細胞應答的動態分析結果表明，MalE、BCGwt誘導的

6、特異CD4T細胞應答存在Th1/Th2平衡轉換現象，即起始階段為Th1應答，一段時間后出現Th2應答，并逐步形成Th1/Th2混合應答。在此基礎上，進一步分析了rBCGMalE誘導針對MalE不同T細胞表位的應答規律，結果發現，隨著感染與免疫過程的發展，針對MalEp68-82和p277-291的特異T細胞應答從起初的Th1應答向Th1/Th2混合類型變遷，非常有趣的是，針對p100-114的特異T細胞應答呈現典型的Th1/Th2平衡轉換，而針對p151-165的T細胞應答僅是Th2類型，而且在免疫后較長時間才出現。這些結果不僅進一步驗證rB CGMalE誘導的T細胞應答規律，而且還揭示Mal

7、E蛋白不同T細胞表位的功能多樣性。此外，研究中證實rBCG不同表達構建產生的MalE及其表達量亦直接影響它們誘導免疫應答的質和量。這些結果亦為疫苗的分子設計提供了新思路。關鍵詞重組BCG，MalE，T細胞表位，樹突細胞，巨噬細胞，IL-12，Th1/Th2應答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