免疫组库测序技术介绍

Immuno-Seq:exploringthediversityofimmunesystemJiankuiHeSouthUniversityofScienceandTechnologyofChina(SUSTC),Shenzhen,China现在是1页\一共有28页\编辑于星期三ThediversityofInvaders...BacteriaVirusesparasitessuchasfungi,protista,&wormsPhysicalinjury现在是2页\一共有28页\编辑于星期三TheImmuneSystem现在是3页\一共有28页\编辑于星期三Innateimmunesystemageneralresponsetoanythingotherthanrecognized“selfcells”macrophages-abletoingestsmallforeigninvadersMastcells/basophilsreleasehistaminethatdilatesbloodvesselscausesredness现在是4页\一共有28页\编辑于星期三Adaptiveimmunesystemaspecificcounter-assaultagainsta“knownforeign”invader[previouslyrecognized]现在是5页\一共有28页\编辑于星期三Key-lockEverymatureTcell(Bcell)expressesasingleTCR(BCR)withspecificityforalimitednumberofantigens.Tcellreceptor现在是6页\一共有28页\编辑于星期三HowmanyTcellsinyourbody?1.HumanbloodhasaboutonemillionTcellsineverymilliliter(ml).2.Theaverageadulthumanhasabout10litersofblood.3.Thereare10,000mlin10L.4,So,thereareaboutonemillionx10,000Tcellsinthebloodstream.Thiscomesto1010.About1/50ofallTcellsarefoundintheblood.Mostoftherestareinthespleenandlymphnodes.Somearealsofoundinthebonemarrow,liver,andothertissues.5.So,IwouldestimatethenumberofTcellsinthehumanbodytobeabout5x1011.现在是7页\一共有28页\编辑于星期三BlymphocytedevelopmentHowantibodydiversityisgeneratedJanewayC.,Immunobiology,2004StemcellChromosome14,heavychain现在是8页\一共有28页\编辑于星期三BlymphocytedevelopmentHowantibodydiversityisgeneratedJanewayC.,Immunobiology,2004StemcellChromosome14,heavychainPro-Bcell现在是9页\一共有28页\编辑于星期三BlymphocytedevelopmentHowantibodydiversityisgeneratedJanewayC.,Immunobiology,2004StemcellBcellChromosome14,heavychainPro-Bcell现在是10页\一共有28页\编辑于星期三HumanHeavychainVgenesegments~50HeavychainDgenesegments27HeavychainJgenesegments6AntibodyrepertoireestimatesVDJcombination50*27*6=8,000Potentialantibodydiversity~1014Jointdiversity3-5bprandominsertion/deletionTheoreticalcalculation:Immunesystemonlyuses83geneSegmenttocode1014BCRsequences现在是11页\一共有28页\编辑于星期三DiversityandhealthyIntrinsicfactorsthatinfluencetheexpressionoftheTCRrepertoireinhumans:humanMHC(HLA)systemandTCRgenepolymorphisms,Extrinsicfactors:environmentalfactors,suchasinfectionandsuperantigens.

InnormalindividualstheTCRrepertoireisstableandpolyclonal.clonalpopulationsarethehallmarkofmalignancy,clonaloroligoclonalpopulationsofTandBcellsmayoccurinnon-malignantconditionsincluding:humanimmunodeficiencyvirus(HIV)andEpstein-Barrvirus(EBV)infectionswithspecificityforvirus,intheelderly

inautoimmunity

incommonvariableimmunodeficiency(CVID),inseverecombinedimmunodeficiency(SC1D),现在是12页\一共有28页\编辑于星期三TraditionalmethodstomeasurethediversityofTCR/BCRflowcytometryFluorescentlylabelledmonoclonalantibodiesagainstTCRVfamilygenesegmentsResolution:genesegmentlevelLimitedtoafewTCRVfamilygenesPCR+SangersequencingResolution:genesequenceLowthroughput现在是13页\一共有28页\编辑于星期三HighthroughputsequencingBcellsandTcellsStephenQuakelabScience,2009PNAS,2011Sci.Transl.Med,2013FirelabSci.Transl.Med2009PamerlabNat.Medicine2013HanlabPNAS,2010WarrenlabSci.Transl.Med2010GeorgioulabNat.Biotechnology2010现在是14页\一共有28页\编辑于星期三Thefirstuseofhighthroughputsequencing1200~3700uniqueantibodies

inafishStephenQuake现在是15页\一共有28页\编辑于星期三ThediversityofhumanTCR420,000uniqueCDR3nucleotideinafewmilliliterblood3*106uniqueCDR3isestimatedforanadultWearrivedatsurprisinglyhighminimalestimatesof100millionuniqueTCRBsequencesinnaïveCD4andCD8T-cellrepertoiresofyoungadults.Naïverepertoirerichnessmodestlydeclinedtwo-tofivefoldinhealthyelderly.现在是16页\一共有28页\编辑于星期三Supersensitivityinminimalresiduediseasemonitoring(Leukemia)HighthroughputsequencingTCRcandetect0.001%frequencyoftumorcellsFlowcytometercandetect0.1%frequency.现在是17页\一共有28页\编辑于星期三给免疫系统“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接受流感疫苗后现在是18页\一共有28页\编辑于星期三Immuno-SeqImmuno-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,NIHDirector

2013年2月13日现在是19页\一共有28页\编辑于星期三ImmuneresponsetofluvaccineDay0Day7Day28FluVaccine:TIV&LAIVPlasmablastsDay7Collect5mlblood现在是20页\一共有28页\编辑于星期三ExperimentflowBloodsamplePatientTissuesSinglecellSuspensionPBMCTCD8GenomicDNARNAAmplifythelibrarybyMultiplexPCRIlluminaHiseq2000AlignmentandClustering现在是21页\一共有28页\编辑于星期三BroadVDJgenesegmentusagepatternVDJ现在是22页\一共有28页\编辑于星期三AsnapshotofimmunesystemDay7,aftervaccination.ElderlysubjectWeamplifiedantibodysequencesfromsinglesortedplasmaBcell.Wethenexpressedmonoclonalantibodiesaccordingtothesesequencesandverifiedtheirbindingtoeachofthethreevirusstrainsinthevaccine现在是23页\一共有28页\编辑于星期三ImmunereperotireisadynamicssystemDay0Day7Day28Vaccinespecificantibodylineage13%oftotalsequencesVaccinespecificantibodylineage0.01%oftotalsequencesElderlysubjectPlasmablastsDay7现在是24页\一共有28页\编辑于星期三ReducedantibodydiversityinelderlyAge8-17Age18-32Age70-100MutationEachdatapointrepresentalineageComparisonof3subjectsSequencesinalineage现在是25页\一共有28页\编辑于星期三Reducedantibodydiv