免疫组库测序技术介绍

Immuno-Seq:exploringthediversityofimmunesystemJiankuiHeSouthUniversityofScienceandTechnologyofChina(SUSTC),Shenzhen,ChinaThediversityofInvaders...•Bacteria•Viruses•parasitessuchasfungi,protista,&worms•Physicalinjurywormtrichura.jpg://://://•ageneralresponsetoanythingotherthanrecognized“selfcells”•aspecificcounter-assaultagainsta“knownforeign”invader[previouslyrecognized]Key-lock•EverymatureTcell(Bcell)expressesasingleTCR(BCR)withspecificityforalimitednumberofantigens.TcellreceptorHowmanyTcellsinyourbody?•1.HumanbloodhasaboutonemillionTcellsineverymilliliter(ml).•2.Theaverageadulthumanhasabout10litersofblood.•3.Thereare10,000mlin10L.•4,So,thereareaboutonemillionx10,000Tcellsinthebloodstream.Thiscomesto1010.•About1/50ofallTcellsarefoundintheblood.Mostoftherestareinthespleenandlymphnodes.Somearealsofoundinthebonemarrow,liver,andothertissues.•5.So,IwouldestimatethenumberofTcellsinthehumanbodytobeabout5x1011.BlymphocytedevelopmentHowantibodydiversityisgeneratedJanewayC.,Immunobiology,2004StemcellChromosome14,heavychainBlymphocytedevelopmentHowantibodydiversityisgeneratedJanewayC.,Immunobiology,2004StemcellChromosome14,heavychainPro-BcellBlymphocytedevelopmentHowantibodydiversityisgeneratedJanewayC.,Immunobiology,2004StemcellBcellChromosome14,heavychainPro-BcellHumanHeavychainVgenesegments~50HeavychainDgenesegments27HeavychainJgenesegments6AntibodyrepertoireestimatesVDJcombination50*27*6=8,000Potentialantibodydiversity~1014Jointdiversity3-5bprandominsertion/deletionTheoreticalcalculation:Immunesystemonlyuses83geneSegmenttocode1014BCRsequencesDiversityandhealthy•IntrinsicfactorsthatinfluencetheexpressionoftheTCRrepertoireinhumans:humanMHC(HLA)systemandTCRgenepolymorphisms,•Extrinsicfactors:environmentalfactors,suchasinfectionandsuperantigens.•InnormalindividualstheTCRrepertoireisstableandpolyclonal.•clonalpopulationsarethehallmarkofmalignancy,clonaloroligoclonalpopulationsofTandBcellsmayoccurinnon-malignantconditionsincluding:–humanimmunodeficiencyvirus(HIV)andEpstein-Barrvirus(EBV)infectionswithspecificityforvirus,–intheelderly–inautoimmunity–incommonvariableimmunodeficiency(CVID),–inseverecombinedimmunodeficiency(SC1D),TraditionalmethodstomeasurethediversityofTCR/BCR•flowcytometry–FluorescentlylabelledmonoclonalantibodiesagainstTCRVfamilygenesegments–Resolution:genesegmentlevel–LimitedtoafewTCRVfamilygenes•PCR+Sangersequencing–Resolution:genesequence–LowthroughputHighthroughputsequencingBcellsandTcellsStephenQuakelabScience,2009PNAS,2011Sci.Transl.Med,2013FirelabSci.Transl.Med2009PamerlabNat.Medicine2013HanlabPNAS,2010WarrenlabSci.Transl.Med2010GeorgioulabNat.Biotechnology2010Thefirstuseofhighthroughputsequencing•1200~3700uniqueantibodiesinafishStephenQuakeThediversityofhumanTCR•420,000uniqueCDR3nucleotideinafewmilliliterblood•3*106uniqueCDR3isestimatedforanadultWearrivedatsurprisinglyhighminimalestimatesof100millionuniqueTCRBsequencesinnaïveCD4andCD8T-cellrepertoiresofyoungadults.Naïverepertoirerichnessmodestlydeclinedtwo-tofivefoldinhealthyelderly.Supersensitivityinminimalresiduediseasemonitoring(Leukemia)•HighthroughputsequencingTCRcandetect0.001%frequencyoftumorcells•Flowcytometercandetect0.1%frequency.给免疫系统“拍照”•“Immuno-Seq”技术使用新一代测序可以一次测出500万个TCR受体或者BCR的RNA序列。•通过生物信息学方法分TCR/BCR的多样性和丰度，可以得知人体对感染，疫苗，和治疗的免疫反应状况。VDJN.Jiang,etal.,SciTranslMed.2013Feb6;5(171):171接受流感疫苗后Immuno-Seq•Immuno-Seq技术可以观察和分析T细胞和B细胞，达到前所未有的深度和特异性。在疾病监控，抗体生产，疫苗研究，健康体检等领域有广泛的应用。现在临床上有许多种检测可以监控你的健康状况，但是却没有一种被广泛接受的方法可以检测你免疫系统的各个方面。我们怎么可以得知一个人的免疫系统是很强健还是很虚弱？在人们受哮喘，过敏，关节炎折磨时，如何量化免疫系统的错误？斯坦福大学的一个研究团队在开发这样的检测方面迈出了第一步，他们的方法是：给免疫系统“拍照”。Therearenumerousteststogaugethehealthofyourheart.Butnosuchwidelyacceptedtestexistsforthemanypartsoftheimmunesystem.Howcanwetelliftheimmunesystemisstrongorweak?Orquantifyhowbadlyit’smalfunctioningwhenwesufferfromasthma,allergies,orarthritis?------FrancisCollins,NIHDirector2013年2月13日ImmuneresponsetofluvaccineDay0Day7Day28FluVaccine:TIV&LAIVPlasmablastsDay7Collect5mlbloodExperimentflowBloodsamplePatientTissuesSinglecellSuspensionPBMCTCD8GenomicDNARNAAmplifythelibrarybyMultiplexPCRIlluminaHiseq2000AlignmentandClusteringBroadVDJgenesegmentusagepatternVDJAsnapshotofimmunesystemDay7,aftervaccination.ElderlysubjectWeamplifiedantibodysequencesfromsinglesortedplasmaBcell.WethenexpressedmonoclonalantibodiesaccordingtothesesequencesandverifiedtheirbindingtoeachofthethreevirusstrainsinthevaccineImmunereperotireisadynamicssystemDay0Day7Day28Vaccinespecificantibodylineage13%oftotalsequencesVaccinespecificantibodylineage0.01%oftotalsequencesElderlysubjectPlasmablastsDay7Reducedantibodyd