免疫组库测序技术介绍.ppt

Immuno Seq exploringthediversityofimmunesystem JiankuiHeSouthUniversityofScienceandTechnologyofChina SUSTC Shenzhen China ThediversityofInvaders BacteriaVirusesparasitessuchasfungi protista wormsPhysicalinjury TheImmuneSystem Innateimmunesystem ageneralresponsetoanythingotherthanrecognized selfcells macrophages abletoingestsmallforeigninvaders Mastcells basophils releasehistaminethatdilatesbloodvesselscausesredness Adaptiveimmunesystem aspecificcounter assaultagainsta knownforeign invader previouslyrecognized Key lock EverymatureTcell Bcell expressesasingleTCR BCR withspecificityforalimitednumberofantigens Tcellreceptor HowmanyTcellsinyourbody 1 HumanbloodhasaboutonemillionTcellsineverymilliliter ml 2 Theaverageadulthumanhasabout10litersofblood 3 Thereare10 000mlin10L 4 So thereareaboutonemillionx10 000Tcellsinthebloodstream Thiscomesto1010 About1 50ofallTcellsarefoundintheblood Mostoftherestareinthespleenandlymphnodes Somearealsofoundinthebonemarrow liver andothertissues 5 So IwouldestimatethenumberofTcellsinthehumanbodytobeabout5x1011 Blymphocytedevelopment Howantibodydiversityisgenerated JanewayC Immunobiology 2004 Stemcell Chromosome14 heavychain Blymphocytedevelopment Howantibodydiversityisgenerated JanewayC Immunobiology 2004 Stemcell Chromosome14 heavychain Pro Bcell Blymphocytedevelopment Howantibodydiversityisgenerated JanewayC Immunobiology 2004 Stemcell Bcell Chromosome14 heavychain Pro Bcell Antibodyrepertoireestimates Jointdiversity3 5bprandominsertion deletion Theoreticalcalculation Immunesystemonlyuses83geneSegmenttocode1014BCRsequences Diversityandhealthy IntrinsicfactorsthatinfluencetheexpressionoftheTCRrepertoireinhumans humanMHC HLA systemandTCRgenepolymorphisms Extrinsicfactors environmentalfactors suchasinfectionandsuperantigens InnormalindividualstheTCRrepertoireisstableandpolyclonal clonalpopulationsarethehallmarkofmalignancy clonaloroligoclonalpopulationsofTandBcellsmayoccurinnon malignantconditionsincluding humanimmunodeficiencyvirus HIV andEpstein Barrvirus EBV infectionswithspecificityforvirus intheelderlyinautoimmunityincommonvariableimmunodeficiency CVID inseverecombinedimmunodeficiency SC1D TraditionalmethodstomeasurethediversityofTCR BCR flowcytometryFluorescentlylabelledmonoclonalantibodiesagainstTCRVfamilygenesegmentsResolution genesegmentlevelLimitedtoafewTCRVfamilygenesPCR SangersequencingResolution genesequenceLowthroughput HighthroughputsequencingBcellsandTcells StephenQuakelabScience 2009PNAS 2011Sci Transl Med 2013 FirelabSci Transl Med2009 PamerlabNat Medicine2013 HanlabPNAS 2010 WarrenlabSci Transl Med2010 GeorgioulabNat Biotechnology2010 Thefirstuseofhighthroughputsequencing 1200 3700uniqueantibodiesinafish StephenQuake ThediversityofhumanTCR 420 000uniqueCDR3nucleotideinafewmilliliterblood3 106uniqueCDR3isestimatedforanadult Wearrivedatsurprisinglyhighminimalestimatesof100millionuniqueTCRBsequencesinna veCD4andCD8T cellrepertoiresofyoungadults Na verepertoirerichnessmodestlydeclinedtwo tofivefoldinhealthyelderly Supersensitivityinminimalresiduediseasemonitoring Leukemia HighthroughputsequencingTCRcandetect0 001 frequencyoftumorcellsFlowcytometercandetect0 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 V D J N 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 Immuneresponsetofluvaccine Day0 Day7 Day28 FluVaccine TIV LAIV Collect5mlblood Experimentflow BroadVDJgenesegmentusagepattern V D J Asnapshotofimmunesystem Day7 aftervaccination Elderlysubject WeamplifiedantibodysequencesfromsinglesortedplasmaBcell Wethenexpressedmonoclonalantibodiesaccordingtothesesequencesandverifiedtheirbindingtoeachofthethreevirusstrainsinthevaccine Immunereperotireisadynamicssystem Day0 Day7 Day28 Vaccinespecificantibodylineage13 oftotalsequences Vaccinespecificantibodylineage0 01 oftotalsequences Elderlysubject Reducedantibodydiversityinelderly Age8 17 Age18 32 Age70 100 Mutation Eachdatapointrepresentalineage Comparisonof3subjects Sequencesinalineage Reducedantibodydiversityinelderly 70 100yearoldvolunteershadasignificantlylessdiversecollectionofantibodiesthantheyoungervolunteers Thatis aspeopleaged theirimmunesystemhadamorelimitedrepertoireofantibodiestooffer LabMembers Postdoc ZhoufangLiTechnician LuwenNingMengZhangStudents JiajieGuoYinTongYingXuPengLiYaoChenRonghaiCheng Collabora