免疫組庫測(cè)序技術(shù)介紹

Immuno-Seq:exploringthediversityofimmunesystemJiankuiHeSouthUniversityofScienceandTechnologyofChina(SUSTC),Shenzhen,China目前一頁\總數(shù)二十八頁\編于十三點(diǎn)ThediversityofInvaders...BacteriaVirusesparasitessuchasfungi,protista,&wormsPhysicalinjury目前二頁\總數(shù)二十八頁\編于十三點(diǎn)TheImmuneSystem目前三頁\總數(shù)二十八頁\編于十三點(diǎn)Innateimmunesystemageneralresponsetoanythingotherthanrecognized“selfcells”macrophages-abletoingestsmallforeigninvadersMastcells/basophilsreleasehistaminethatdilatesbloodvesselscausesredness目前四頁\總數(shù)二十八頁\編于十三點(diǎn)Adaptiveimmunesystemaspecificcounter-assaultagainsta“knownforeign”invader[previouslyrecognized]目前五頁\總數(shù)二十八頁\編于十三點(diǎn)Key-lockEverymatureTcell(Bcell)expressesasingleTCR(BCR)withspecificityforalimitednumberofantigens.Tcellreceptor目前六頁\總數(shù)二十八頁\編于十三點(diǎn)HowmanyTcellsinyourbody?1.HumanbloodhasaboutonemillionTcellsineverymilliliter(ml).2.Theaverageadulthumanhasabout10litersofblood.3.Thereare10,000mlin10L.4,So,thereareaboutonemillionx10,000Tcellsinthebloodstream.Thiscomesto1010.About1/50ofallTcellsarefoundintheblood.Mostoftherestareinthespleenandlymphnodes.Somearealsofoundinthebonemarrow,liver,andothertissues.5.So,IwouldestimatethenumberofTcellsinthehumanbodytobeabout5x1011.目前七頁\總數(shù)二十八頁\編于十三點(diǎn)BlymphocytedevelopmentHowantibodydiversityisgeneratedJanewayC.,Immunobiology,2004StemcellChromosome14,heavychain目前八頁\總數(shù)二十八頁\編于十三點(diǎn)BlymphocytedevelopmentHowantibodydiversityisgeneratedJanewayC.,Immunobiology,2004StemcellChromosome14,heavychainPro-Bcell目前九頁\總數(shù)二十八頁\編于十三點(diǎn)BlymphocytedevelopmentHowantibodydiversityisgeneratedJanewayC.,Immunobiology,2004StemcellBcellChromosome14,heavychainPro-Bcell目前十頁\總數(shù)二十八頁\編于十三點(diǎn)HumanHeavychainVgenesegments~50HeavychainDgenesegments27HeavychainJgenesegments6AntibodyrepertoireestimatesVDJcombination50*27*6=8,000Potentialantibodydiversity~1014Jointdiversity3-5bprandominsertion/deletionTheoreticalcalculation:Immunesystemonlyuses83geneSegmenttocode1014BCRsequences目前十一頁\總數(shù)二十八頁\編于十三點(diǎn)DiversityandhealthyIntrinsicfactorsthatinfluencetheexpressionoftheTCRrepertoireinhumans:humanMHC(HLA)systemandTCRgenepolymorphisms,Extrinsicfactors:environmentalfactors,suchasinfectionandsuperantigens.

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

inautoimmunity

incommonvariableimmunodeficiency(CVID),inseverecombinedimmunodeficiency(SC1D),目前十二頁\總數(shù)二十八頁\編于十三點(diǎn)TraditionalmethodstomeasurethediversityofTCR/BCRflowcytometryFluorescentlylabelledmonoclonalantibodiesagainstTCRVfamilygenesegmentsResolution:genesegmentlevelLimitedtoafewTCRVfamilygenesPCR+SangersequencingResolution:genesequenceLowthroughput目前十三頁\總數(shù)二十八頁\編于十三點(diǎn)HighthroughputsequencingBcellsandTcellsStephenQuakelabScience,2009PNAS,2011Sci.Transl.Med,2013FirelabSci.Transl.Med2009PamerlabNat.Medicine2013HanlabPNAS,2010WarrenlabSci.Transl.Med2010GeorgioulabNat.Biotechnology2010目前十四頁\總數(shù)二十八頁\編于十三點(diǎn)Thefirstuseofhighthroughputsequencing1200~3700uniqueantibodies

inafishStephenQuake目前十五頁\總數(shù)二十八頁\編于十三點(diǎn)ThediversityofhumanTCR420,000uniqueCDR3nucleotideinafewmilliliterblood3*106uniqueCDR3isestimatedforanadultWearrivedatsurprisinglyhighminimalestimatesof100millionuniqueTCRBsequencesinna?veCD4andCD8T-cellrepertoiresofyoungadults.Na?verepertoirerichnessmodestlydeclinedtwo-tofivefoldinhealthyelderly.目前十六頁\總數(shù)二十八頁\編于十三點(diǎn)Supersensitivityinminimalresiduediseasemonitoring(Leukemia)HighthroughputsequencingTCRcandetect0.001%frequencyoftumorcellsFlowcytometercandetect0.1%frequency.目前十七頁\總數(shù)二十八頁\編于十三點(diǎn)給免疫系統(tǒng)“拍照”“Immuno-Seq”技術(shù)使用新一代測(cè)序可以一次測(cè)出500萬個(gè)TCR受體或者BCR的RNA序列。通過生物信息學(xué)方法分TCR/BCR的多樣性和豐度，可以得知人體對(duì)感染，疫苗，和治療的免疫反應(yīng)狀況。VDJN.Jiang,etal.,SciTranslMed.2013Feb6;5(171):171接受流感疫苗后目前十八頁\總數(shù)二十八頁\編于十三點(diǎn)Immuno-SeqImmuno-Seq技術(shù)可以觀察和分析T細(xì)胞和B細(xì)胞，達(dá)到前所未有的深度和特異性。在疾病監(jiān)控，抗體生產(chǎn)，疫苗研究，健康體檢等領(lǐng)域有廣泛的應(yīng)用?，F(xiàn)在臨床上有許多種檢測(cè)可以監(jiān)控你的健康狀況，但是卻沒有一種被廣泛接受的方法可以檢測(cè)你免疫系統(tǒng)的各個(gè)方面。我們?cè)趺纯梢缘弥粋€(gè)人的免疫系統(tǒng)是很強(qiáng)健還是很虛弱？在人們受哮喘，過敏，關(guān)節(jié)炎折磨時(shí)，如何量化免疫系統(tǒng)的錯(cuò)誤？斯坦福大學(xué)的一個(gè)研究團(tuán)隊(duì)在開發(fā)這樣的檢測(cè)方面邁出了第一步，他們的方法是：給免疫系統(tǒng)“拍照”。Therearenumerousteststogaugethehealthofyourheart.Butnosuchwidelyacceptedtestexistsforthemanypartsoftheimmunesystem.Howcanwetelliftheimmunesystemisstrongorweak?Orquantifyhowbadlyit’smalfunctioningwhenwesufferfromasthma,allergies,orarthritis?------FrancisCollins,NIHDirector

2013年2月13日目前十九頁\總數(shù)二十八頁\編于十三點(diǎn)ImmuneresponsetofluvaccineDay0Day7Day28FluVaccine:TIV&LAIVPlasmablastsDay7Collect5mlblood目前二十頁\總數(shù)二十八頁\編于十三點(diǎn)ExperimentflowBloodsamplePatientTissuesSinglecellSuspensionPBMCTCD8GenomicDNARNAAmplifythelibrarybyMultiplexPCRIlluminaHiseq2000AlignmentandClustering目前二十一頁\總數(shù)二十八頁\編于十三點(diǎn)BroadVDJgenesegmentusagepatternVDJ目前二十二頁\總數(shù)二十八頁\編于十三點(diǎn)AsnapshotofimmunesystemDay7,aftervaccination.ElderlysubjectWeamplifiedantibodysequencesfromsinglesortedplasmaBcell.Wethenexpressedmonoclonalantibodiesaccordingtothesesequencesandverifiedtheirbindingtoeachofthethreevirusstrainsinthevaccine目前二十三頁\總數(shù)二十八頁\編于十三點(diǎn)ImmunereperotireisadynamicssystemDay0Day7Day28Vaccinespecificantibodylineage13%oftotalsequencesVaccinespecificantibodylineage0.01%oftotalsequencesElderlysubjectPlasmablastsDay7目前二十四頁\總數(shù)二十八頁\編于十三點(diǎn)ReducedantibodydiversityinelderlyAge8-17Age18-32Age70-100MutationEachdatapointrepresentalineageComparisonof3subjectsSequencesinalineage目前二十五頁\總數(shù)二十八頁\編于十三點(diǎn)Reducedantibodydiver