细胞与分子免疫学课程课件

细胞与分子免疫学课程课件演示文稿目前一页\总数八十三页\编于十九点（优选）细胞与分子免疫学课程课件目前二页\总数八十三页\编于十九点3Chapter7LymphocyteMaturationandExpressionofAntigenReceptorGenes目前三页\总数八十三页\编于十九点4GeneralFeaturesofLymphocyteMaturation

ThematurationofBandTlymphocytesconsistsof:LineagecommitmentandproliferationExpressionofantigenreceptorgenesSelectionofthematurerepertories目前四页\总数八十三页\编于十九点5Earlymaturation:PluripotentstemcellsgiverisetoalllineagesofbloodcellsItisdifficulttopreciselydefinethemechanismsbywhichstemcellsbecometothelymphoidlineage.Bcell:boneborrowTcell:thymus3)Bythegenemutationsinexperimentalanimalsorhumanpatients.i.Transcriptionfactorsii.Growthfactorsiii.Growthfactorsreceptor目前五页\总数八十三页\编于十九点6Example1Bubbleboydisease1.MutatedinIL-7geneandIL-7receptorgenehaveprofounddeficienciesinmatureTandBcells.2.MutationsinachainoftheIL-2receptorgchain,leadstoanimmunodeficiencydisease:X-linkedseverecombinedimmunodeficiencydisease,bubbleboy.目前六页\总数八十三页\编于十九点7AntigenReceptorGeneRecombinationandExpressionTheremaybe107or

moredifferentTandBlymphocyteclonesinoneindividual,eachcloneproduceonespecificantigenreceptor.Bysomaticrecombination,eachindividualdoesnotneedtohavesuchenormouslygenes.Duringthepre-Bcellorpre-Tcellstage,animmatureformofantigenreceptorisformedwhichtotransducesignalstoinducefurthermaturationInmorematurelymphocytes,completeantigenreceptorexpressed,whichpromotecellmaturation.目前七页\总数八十三页\编于十九点8SelectionProcessThatShapetheBandTLymphocyteRepertoiresThepreservationofusefulspecificitiesiscalledpositiveselection:Tcellsinthymus.Bcellsmaturingarenotwellknown.2.Negativeselectionistheprocessthateliminatesdevelopinglymphocyteswithstrongbindingtoself-antigen.目前八页\总数八十三页\编于十九点9AcquisitionofFunctionalCompetenceBcellscansecreteantibodies.TcellscandifferentiateintodistinctsubsetsofTcells.Expressionofavarietyofcellsurfaceandintracellularmoleculesthatparticipateinlymphocyteactivationandeffectorfunctions.目前九页\总数八十三页\编于十九点10FormationofFunctionalAntigenReceptorGenesinBandTLymphocytesElucidationofthemechanismsofantigenreceptorgeneexpressionisoneofthelandmarkachievementsofmordernimmunology1胚系理论(GermlineTheory)在胚系基因组中包含了免疫基因库，可对外界的众多抗原发生应答，并能遗传。Whataretheproblemswiththistheory?2体细胞突变理论（SomaticMutationModel）基因组中的免疫球蛋白基因相对较少，抗体的多样性主要是由体细胞基因突变引起的。Whataretheproblemswiththistheory?目前十页\总数八十三页\编于十九点11胚系理论与体细胞突变理论所遇到的问题1胚系理论1）基因的种类少于抗体的多样性Somanyspecificities–sofewgenes2体细胞突变理论（SomaticMutationModel）

1）部分编码V区基因有众多的变化但部分编码C区的基因却相对稳定。2）不同类的同型的免疫球蛋白具有相同的V区。Samevariableregionsondifferentisotypes目前十一页\总数八十三页\编于十九点123Dreyer–Bennett假说：免疫球蛋白单一的肽链是由两个分隔的基因编码：每个免疫球蛋白类基因可能只有单个C区基因，在胚系基因组中与V区基因是分隔开的。在抗体产生细胞的发育过程中，其中一个独立的V区基因序列将与C区序列结合成为完整的V＋C基因，然后在细胞内表达。两个基因–一条多肽链：此模型可以解释如下问题：1）一个基因的部分片断是多变的，而另一部分相对不变。2）一个V区可以与不同类的C区结合，产生出不同类型的抗体。目前十二页\总数八十三页\编于十九点134ThegeneticfoundationofantibodydiversityTheNobelPrizeinPhysiologyorMedicine1987"forhisdiscoveryofthegeneticprincipleforgenerationofantibodydiversity“in1976.SusumuTonegawa

JapanMassachusettsInstituteofTechnology(MIT)

Cambridge,MA,USAb.1939目前十三页\总数八十三页\编于十九点14ProofoftheDreyer-BennetthypothesisVVVVVVVVVVVVVAmechanismtorearrangeVandCgenesinthegenomesothattheycanfusetoformacompleteImmunoglobulingene.CVCAsingleCregiongeneencodedinthegermlineandseparatefromthemultipleVregiongenesFindawaytoshowtheexistenceofmultipleVgenesandrearrangementtotheCgene目前十四页\总数八十三页\编于十九点15ProofoftheDreyer-BennetthypothesisVVVCVVVVVVSizefractionatebygelelectrophoresisCVVVVVVVVVCVVVVVVVVVCutgermlineDNAwithrestrictionenzymesVVVVVVVVVCArangeoffragmentsizesisgeneratedBlotwithaVregionprobeBlotwithaCregionprobeThefollowingexampledescribeseventsononlyONEofthechromosomes目前十五页\总数八十三页\编于十九点16CVVVVVCVVVVVSizefractionatebygelelectrophoresisVVVVCVBlotwithaVregionprobeBlotwithaCregionprobeCutmatureBcellDNAwithrestrictionenzymesVVVBlotwithaVregionprobeBlotwithaCregionprobeCVVVVVVSizefractionatebygelelectrophoresis-comparethepatternofbandswithgermlineDNAVandCprobesdetectthesamefragmentSomeVregionsaremissingTheCfragmenthasgotlargerVVVVCVEvidenceforgenerecombination目前十六页\总数八十三页\编于十九点17OrganizationofIgandTCRGenesintheGermline1.OrganizationofIgGeneLociThreeseparatelociencodetheIgchains.chromosome14:Hchainlocuschromosome2:kchainchromosome22:lchain2)Multiplecopiesofatleastthreedifferenttypesofgenesegments:V:300base,separatebynoncodingDNA,atthe5’endofeachVregionisanucleotidesequencewhichiscalledleaderpeptide.目前十七页\总数八十三页\编于十九点18C,eachIglocushasadistinctarrangementandnumberofCgene.Inhuman:Igklightchain:asingleCgeneIgllightchain:fourfunctionalCgenesIgHheavychain:ninedifferentIgisotypesandsubtypes.Jsegments,30-50basepairslong.Dsegments,inthehumanIgheavychainlocus.1.OrganizationofIgGeneLoci目前十八页\总数八十三页\编于十九点192.OrganizationofTCRGeneLociInthreeseparateloci:TCRa,dchain:chromosome14TCRbchain:chromosome7TCRgchain:chromosome7V:likeIgVgeneC:twoCgenesineachofthehumanTCRbandTCRgchain,oneCgeneineachofTCRa,dchain.J:inallTCRloci,betweenVandCgenes.D:inTCRbchainanddchain.目前十九页\总数八十三页\编于十九点20AntigenReceptorGeneRecombinationDiversityofantigenreceptorgenes目前二十页\总数八十三页\编于十九点21MechanismsofSomaticRecombinationofAnitgenReceptorGenes有限的DNA是怎样产生无限的专一性的?V区是怎样找到J区的？为什么V区不与V区相连？DNA是怎样断裂的？DNA是怎样重新组合的？目前二十一页\总数八十三页\编于十九点22RSS(重排信号序列)

，recombinase(重组酶)

，12-23rule(一个规则)。1、RecombinationSignalSequences(RSS),重排信号序列：基因片断两端存在两个特异的保守序列：7聚体与9聚体。

7聚体与9聚体之间间隔是23bp或者是12bp。RSS:7聚体---间隔---九聚体(Theheptamer–spacer–nonamer)

MechanismsofSomaticRecombinationofAnitgenReceptorGenes目前二十二页\总数八十三页\编于十九点23

RSS（重排信号序列）目前二十三页\总数八十三页\编于十九点242、重组酶：一组参与V、（D）、J基因片断重组的酶，包括以下几种：RAG-1与RAG-2(recombination-activatinggenes):

一种内切酶，只表达在T和B淋巴细胞不成熟阶段。末端脱氧核苷酸转移酶(terminaldeoxynucleotidyltransferase,TdT)：表达于T、B细胞前体，此酶可将数个核苷酸通过不需要模板的方式加到DNA的断段。其他切开发夹结构的内切酶，参与修复DNA双链断段的DNA外切酶、DNA合成酶等，如DNA连接酶IV，DNA依赖的蛋白激酶。目前二十四页\总数八十三页\编于十九点25重组酶所催化的重排步骤RAG蛋白复合物结合到RSS。蛋白复合物拉近将要相连的DNA片断。互补链切断，发夹结构形成。其他DNA修饰蛋白结合到发夹结构剪切RSS末端。发夹结构被随机剪切，碱基或者增加或者减少。DNA连接酶连接产生编码连接点和信号连接。目前二十五页\总数八十三页\编于十九点2623-mer=twoturns12-mer=oneturn3、Molecularexplanationofthe12-23ruleInterveningDNAofanylength23V9712DJ79目前二十六页\总数八十三页\编于十九点2723-mer12-merLoopofinterveningDNAisexcisedTheshapegeneratedbytheRSS’sactsasatargetforrecombinases7997V1V2V3V4V8V7V6V5V9DJV1DJV2V3V4V8V7V6V5V9Anappropriateshapecannotbeformediftwo23-merflankedelementsattemptedtojoin(i.e.the12-23rule)3.Molecularexplanationofthe12-23rule目前二十七页\总数八十三页\编于十九点28Thisrulecanexplainwhyinheavychain,theVcannotdirectlybindtoJ.目前二十八页\总数八十三页\编于十九点29V7239D7129JV723972397129D7129J72397129VDJRecombinationactivatinggeneproducts,(RAG1&RAG2)and‘highmobilitygroupproteins’bindtothe

RSSThetwoRAG1/RAG2complexesbindtoeachotherandbringtheVregionadjacenttotheDJ

region互补链断裂，发夹结构形成4.StepsofIggenerecombination目前二十九页\总数八十三页\编于十九点30VDJ72397129Anumberofotherproteins,(Ku70:Ku80,XRCC4andDNAdependentproteinkinases)bindtothehairpinsandtheheptamerends.VDJThehairpinsattheendoftheVandDregionsareopened,andexonucleasesandtransferasesremoveoraddrandomnucleotidestothegapbetweentheVandDregionVDJ72397129DNAligaseIVjoinstheendsoftheVandDregiontoformthecodingjointandthetwoheptamerstoformthesignaljoint.4.StepsofIggenerecombination目前三十页\总数八十三页\编于十九点31V1V2V3V4V9DJLoopingoutworksifallVgenesareinthesametranscriptionalorientationV1V2V3V9DJ缺失性重排DJ7129V47239V17239D7129JHowdoesrecombinationoccurwhenaVgeneisinoppositeorientationtotheDJregion?V45.重排方式：

以重链重排为例非缺失性重排LL目前三十一页\总数八十三页\编于十九点32DJ7129V47239V4andDJinoppositetranscriptionalorientationsDJ7129V472391.DJ7129V472393.DJ7129V472392.DJ7129V472394.Non-deletionalrecombination目前三十二页\总数八十三页\编于十九点33DJ7129V472391.DJV4712972393.VtoDJligation-codingjointformationDJ7129V472392.Heptamerligation-signaljointformationDJV471297239FullyrecombinedVDJregionsinsametranscriptionalorientationNoDNAisdeleted4.目前三十三页\总数八十三页\编于十九点347D129J6.GenerationofDiversityoftheBandTCellRepertoiresJunctionaldiversity:Pnucleotideadditions7V239D7129JV7239TCCACAGTGAGGTGTCACATGTGACACTACACTGTGTherecombinasecomplexmakessinglestrandednicksatrandomsitesclosetotheendsoftheVandDregionDNA.7D129J7V239CACAGTGGTGTCACGTGACACCACTGTGTCAGATTADJVTCAGATTAUUThe2ndstrandiscleavedandhairpinsformbetweenthecomplementarybasesatendsoftheVandDregion.目前三十四页\总数八十三页\编于十九点35V2V3V4V8V7V6V5V97239CACAGTGGTGTCAC7129GTGACACCACTGTGVTCAGUDJATTAUHeptamersareligatedbyDNAligaseIVVandDregionsjuxtaposedVTCAGUDJATTAU目前三十五页\总数八十三页\编于十九点36VTCAGUDJATTAUEndonucleasecleavessinglestrandatrandomsitesinVandDsegmentVTC~GAAGDJATTA~TAThenucleotidesthatflipout,becomepartofthecomplementaryDNAstrand6.1GenerationofthepalindromicsequenceIntermsofGtoCandTtoApairing,the‘new’nucleotidesarepalindromic.ThenucleotidesGAandTAwerenotinthegenomicsequenceandintroducediversityofsequenceattheVtoDjoin.VTCAGUDJATTAURegionstobejoinedarejuxtaposedThenickedstrand‘flips’out目前三十六页\总数八十三页\编于十九点376.2JunctionalDiversity

–

NnucleotideadditionsVTC~GAAGDJATTA~TATerminaldeoxynucleotidyltransferase(TdT)addsnucleotidesrandomlytothePnucleotideendsofthesingle-strandedVandDsegmentDNACACTCCTTATTCTTGCAAVTC~GAAGDJATTA~TACACACCTTATTCTTGCAAComplementarybasesannealVDJDNApolymerasesfillinthegapswithcomplementarynucleotidesandDNAligaseIVjoinsthestrandsTC~GAAGATTA~TACACACCTTATTCTTGCAADJTA~TAExonucleasesnibblebackfreeendsVTC~GACACACCTTATTCTTGCAAVTCDTAGTT

ATATAG

C目前三十七页\总数八十三页\编于十九点38VDJTCGACGTTATATAGCTGCAATATAJunctionalDiversityNNNNNNNNNNNNNNNGermline-encodednucleotidesPalindromic(P)nucleotides-notinthegermlineNon-template(N)encodednucleotides-notinthegermlineCreatesanessentiallyrandomsequencebetweentheVregion,DregionandJregioninheavychainsandtheVregionandJregioninlightchains.目前三十八页\总数八十三页\编于十九点39JunctionalDiversity目前三十九页\总数八十三页\编于十九点40MaturationofBLymphocytes1.Pro-Bcell2.Pre-Bcell3.ImmatureBcell4.MatureBcell目前四十页\总数八十三页\编于十九点41MaturationofBLymphocytes目前四十一页\总数八十三页\编于十九点42StagesofBLymphocyteMaturation:1.Pro-Bcell:1)notproduceIg2)Maker:CD19andCD103)RAGproteinsarefirstexpressed4)TdTenzymeisexpressedmostabundantly5)IgaandIgbof

BCRcomplexstartstoexpress.6)FirstrecombinationofIggenesoccursinheavychains目前四十二页\总数八十三页\编于十九点43RecombinationinPro-Bcell:目前四十三页\总数八十三页\编于十九点442.Pre-BcellAprimarytranscriptthatincludestherearrangedVDJcomplexandtheproximalCgenesisproduced;FunctionalmRNAforthemheavychainisproduced;mproteininpre-Bcellsaretranslated;membrane-boundantigenreceptorisnotexpressed;Pre-Bcellreceptorsareexpressedonthecellsurfaceatlowlevels.Onlyinhematopoietictissues;目前四十四页\总数八十三页\编于十九点45目前四十五页\总数八十三页\编于十九点468)Thepre-BCRregulatesfurthersomaticrecombinationofIggenesintwoways:8.1)Fortheproductiverearrangements:allelicexclusionexsits.Forthenonproductiveone,thesecondallelicchromosomescanrecombine.目前四十六页\总数八十三页\编于十九点47刘老师：您好！上节课讲到内切酶在V和D上随机切开增加了VD间连接的多样性。那么这个区域将来编码成蛋白质即抗体后，并不位于V片段的CDR区，如何能够增加抗体的多态性？还有一个问题是TdT随机增加几个碱基从而增加了基因的多态性，那么在重排机制中如何保证随机增加碱基后使得重排的基因的碱基数依旧是3的倍数而不发生编码错误？如果编码的不是3的倍数，是否是在转录水平上随机添加碱基U来使编码恢复正常。谢谢老师！来自崔雪晶目前四十七页\总数八十三页\编于十九点48C,eachIglocushasadistinctarrangementandnumberofCgene.Inhuman:Igklightchain:asingleCgeneIgllightchain:fourfunctionalCgenesIgHheavychain:ninedifferentIgisotypesandsubtypes.Jsegments,30-50basepairslong.Dsegments,inthehumanIgheavychainlocus.1.OrganizationofIgGeneLoci目前四十八页\总数八十三页\编于十九点492.OrganizationofTCRGeneLociInthreeseparateloci:TCRa,dchain:chromosome14TCRbchain:chromosome7TCRgchain:chromosome7V:likeIgVgeneC:twoCgenesineachofthehumanTCRbandTCRgchain,oneCgeneineachofTCRa,dchain.J:inallTCRloci,betweenVandCgenes.D:inTCRbchainanddchain.目前四十九页\总数八十三页\编于十九点508)Thepre-BCRregulatesfurthersomaticrecombinationofIggenesintwoways:8.1)Fortheproductiverearrangements:allelicexclusionexsits.Forthenonproductiveone,thesecondallelicchromosomescanrecombine.目前五十页\总数八十三页\编于十九点51上堂课回顾：1Ig与TCR基因在染色体基因座位上的排列特点2重排机制

1）一个信号序列

2）一个原则

3）一组酶其中P碱基与N碱基的加入是导致抗体出现多样性的主要原因：VDJTCGACGTTATATAGCTGCAATATAVTCAGUDJATTAU目前五十一页\总数八十三页\编于十九点524）缺失性重排5）非缺失性重排3、B细胞的成熟：祖B细胞，前B细胞，非成熟B细胞，成熟B细胞目前五十二页\总数八十三页\编于十九点534、B细胞表面受体的重排：重链：等位基因排斥轻链：同种型的排斥目前五十三页\总数八十三页\编于十九点54Pro-BcellPre-Bcell目前五十四页\总数八十三页\编于十九点558.2)thepre-BCRregulatessomaticrecombinationisbystimulatinglightchaingenerearrangement.3.ImmatureBcellkchainandlchaingenearerearranged.IgMisexpressed.InasimilarmannerasintheIgheavychainlocus.

kchainrearrangesafterheavychainrearrangementandbeforelchain.Lightchainisotypeexclusion.TheassembledIgMmoleculesareexpressedonthecellsurfaceinassociationwithIgaandIgb.ImmatureBcellsdonotproliferateanddifferentiateinresponsetoantigens.目前五十五页\总数八十三页\编于十九点56目前五十六页\总数八十三页\编于十九点574.MatureBcellCoexpressedmanddheavychainsinassociationwiththekandllight

chaintoproduceIgMandIgD.ThecoexpressionofIgMandIgDmaketheBcellacquirethefunctionalcompetence.ItsuggeststhatIgDistheessentialactivatingreceptorofmatureBcells.Canresponsetoantigens.Withoutantigens,theywilldieinafewdaysorweeks.ThemajorityofBcellsareIgD+IgM+.目前五十七页\总数八十三页\编于十九点58目前五十八页\总数八十三页\编于十九点59SelectionoftheMatureBcellRepertoireTherepertoireofmatureBcellsispositivelyselectedfromthepoolofimmaturecellsbeforeleavingtheboneborrow.isdifferentfromtheTcellsselection,perhapsservetopreserveallthecellswiththecapacitytorecognizeantigens,regardlessofspecificity.目前五十九页\总数八十三页\编于十九点602)Negativeselection:SelfantigensdeliverstrongsignalstoIgM–expressingimmatureBlymphocytesthathappentoexpressreceptorsspecificfortheseselfantigens.AntigenrecognitionleadstoapoptoticdeathofimmatureBcells.SomeimmatureBcellsthatrecognizeselfantigensmaybeinducedtochangetheirspecificitiesbyaprocesscalledreceptorediting.目前六十页\总数八十三页\编于十九点61B-1CellsBcellsdescribedto-date:B-2cellsAsecondpopulationofBcellsexists,calledB-1cellsAccountforapproximately5%totalBcellsinhumansFoundpredominantlyinperitonealandpleuralcavitiesRelationshiptoB-2cellsnotunderstoodUsealimitedsetofVgenesegmentsSynthesizelow-affinityIgMinresponsetomanybacteriaThoughttohaveanimportantroleasafirstlineofdefenseagainstmanypathogens;maybepartofinnateimmunity?ExpresstheCD5molecule.B-1cellsspontaneouslysecreteIgMantibodiesthatoftenreactwithmicrobialpolysaccharidesandlipids.目前六十一页\总数八十三页\编于十九点62B-1SubsetofBlymphocytes目前六十二页\总数八十三页\编于十九点63MaturationofTlymphocytesPro-TcellPre-TcellDoublepositiveSinglepositiveNaïvematureTcell目前六十三页\总数八十三页\编于十九点641.Pro-TcellRecentarrivalsfromtheboneborrow.ContainTCRgenesintheirgermlineconfiguration,withoutexpressionofTCR,CD3orzchainsorCD4orCD8RAG-1andRAG-2arefirstexpressed.4)Db-to-Jbrearrangementsoccurfirst目前六十四页\总数八十三页\编于十九点65目前六十五页\总数八十三页\编于十九点662.Pre-Tcellstage:Vb-to-DJb

rearrangementsoccurPrimaryRNAandmRNAThepromoterinthe5’flankingregionsofVbgenesfunctiontogetherwithapowerfulenhancerthatislocated3’oftheCb2

gene.Thepre-Tcellreceptorwasformed.Signalsfromthepre-TCRstimulateproliferationofthepre-Tcells,recombinationattheachainlocus.Fromthedouble-negativetothedouble-positivestage.目前六十六页\总数八十三页\编于十九点67目前六十七页\总数八十三页\编于十九点683.DoublepositiveThymocytesexpressbothCD4andCD8molecules.TherearrangementoftheTCRachaingenesandtheexpressionofTCRabheterodimersoccurinthedouble-positivepopulation.TCRagenerecombinationcommences.Pre-TCRhasnoallelicexclusionofachain.30%ofmatureperipheralTcellsdoexpresstwodifferentTCRs,withdifferentachainsbutthesamebchains.目前六十八页\总数八十三页\编于十九点69目前六十九页\总数八十三页\编于十九点704.SinglepositiveAfterselection,cellsmatureintoCD4+orCD8+Tcells.CD4+cellsacquiretheabilitytoproducecytokinesCD8+cellsbecomecapableofproducingmoleculesthatkillothercells.目前七十页\总数八十三页\编于十九点71RoleofthethymusinTcellMaturationThethymusisthemajorsiteofmaturationofTcells.2)Tlymphocytesoriginatefromprecursorsbutseedthethymus.SelectionprocessesinthematurationofMHC-restrictedabTCells目前七十一页\总数八十三页\编于十九点723)Thethymicenvironmentprovidesstimulitomakethymocytesproliferateandmaturate.Thestimulicomefromcellsincluding:Thymicepithelialcells;Bonemarrow–derivedmacrophages;Dendriticcells;ii)TwotypesofmoleculesproducedbythenonlymphoidMHCmolecules:epithelialcells,dendriticcellsandmacrophagesCytokines:thymicstromalcellsincludingepithelialcells,StimulatetheproliferationofimmatureTcells,especiallyIL-7.目前七十二页\总数八十三页\编于十九点73PositiveSelectionofThymocytes:DevelopmentoftheSelfMHC-RestrictedTcellRepertoire1.PositiveselectionDouble–positivethymocytesareproducedspontaneouslyInthethymiccortex,immaturecellsencounterMHCTCRrecognizetheMHCI-peptidescomplex,theCD8interactswiththemolecules,theTcellwilldeveloptoCD8+Tcell.ThymocytescannotrecognizeselfMHC-restrictedwilldiebyapoptosis.目前七十三页\总数八十三页\编于十九点74Inthepositiveselection,threemoleculesareessentialinmaturationofTcells:selfMHCmolecules;TCR;peptidesboundtoMHCTable7-2.Deve