免疫组库测序技术介绍ppt课件

The diversity of Invaders.BacteriaVirusesparasites such as fungi,protista,&wormsPhysical injuryThe diversity of Invaders.1The Immune System http:/www.nobelprize.orgThe Immune System http:/www.n2Innate immune systema general response to anything other than recognized“self cells”macrophages-able to ingest small foreign invadersMast cells/basophils release histamine that dilates blood vesselscauses redness Innate immune systema general 3Adaptive immune systema specific counter-assault against a“known foreign”invader previously recognizedAdaptive immune systema specif4Key-lockEvery mature T cell(B cell)expresses a single TCR(BCR)with specificity for a limited number of antigens.T cell receptorKey-lockEvery mature T cell(B5How many T cells in your body?1.Human blood has about one million T cells in every milliliter(ml).2.The average adult human has about 10 liters of blood.3.There are 10,000 ml in 10 L.4,So,there are about one million x 10,000 T cells in the bloodstream.This comes to 1010.About 1/50 of all T cells are found in the blood.Most of the rest are in the spleen and lymph nodes.Some are also found in the bone marrow,liver,and other tissues.5.So,I would estimate the number of T cells in the human body to be about 5 x 1011.How many T cells in your body?6B lymphocyte developmentHow antibody diversity is generatedJaneway C.,Immunobiology,2004Stem cellChromosome 14,heavy chainB lymphocyte developmentHow an7B lymphocyte developmentHow antibody diversity is generatedJaneway C.,Immunobiology,2004Stem cellChromosome 14,heavy chainPro-B cellB lymphocyte developmentHow an8B lymphocyte developmentHow antibody diversity is generatedJaneway C.,Immunobiology,2004Stem cellB cellChromosome 14,heavy chainPro-B cellB lymphocyte developmentHow an9HumanHeavy chain V gene segments50Heavy chain D gene segments27Heavy chain J gene segments6Antibody repertoire estimatesVDJ combination50*27*6=8,000Potential antibody diversity1014Joint diversity3-5bp random insertion/deletionTheoretical calculation:Immune system only uses 83 geneSegment to code 1014 BCR sequencesHumanHeavy chain V gene segmen10Diversity and healthyIntrinsic factors that influence the expression of the TCR repertoire in humans:human MHC(HLA)system and TCR gene polymorphisms,Extrinsic factors:environmental factors,such as infection and superantigens.In normal individuals the TCR repertoire is stable and polyclonal.clonal populations are the hallmark of malignancy,clonal or oligoclonal populations of T and B cells may occur in non-malignant conditions including:human immunodeficiency virus(HIV)and Epstein-Barr virus(EBV)infections with specificity for virus,in the elderly in autoimmunity in common variable immunodeficiency(CVID),in severe combined immunodeficiency(SC1D),Diversity and healthyIntrinsic11Traditional methods to measure the diversity of TCR/BCRflow cytometryFluorescently labelled monoclonal antibodies against TCRV family gene segmentsResolution:gene segment levelLimited to a few TCRV family genesPCR+Sanger sequencingResolution:gene sequenceLow throughputTraditional methods to measure12High throughput sequencing B cells and T cellsStephen Quake labScience,2009PNAS,2011Sci.Transl.Med,2013Fire labSci.Transl.Med2009Pamer labNat.Medicine2013Han labPNAS,2010Warren labSci.Transl.Med2010Georgiou labNat.Biotechnology2010High throughput sequencing B c13The first use of high throughput sequencing 12003700 unique antibodies in a fishStephen QuakeThe first use of high throughp14The diversity of human TCR420,000 unique CDR3 nucleotide in a few milliliter blood3*106 unique CDR3 is estimated for an adult We arrived at surprisingly high minimal estimates of 100 million unique TCRB sequences in nave CD4 and CD8 T-cell repertoires of young adults.Nave repertoire richness modestly declined two-to fivefold in healthy elderly.The diversity of human TCR We 15Super sensitivity in minimal residue disease monitoring(Leukemia)High throughput sequencing TCR can detect 0.001%frequency of tumor cellsFlow cytometer can detect 0.1%frequency.Super sensitivity in minimal r16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,et al.,Sci Transl Med.2013 Feb 6;5(171):171接受流感疫苗后给免疫系统“拍照”“Immuno-Seq”技术使用新一代测序17Immuno-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”。There are numerous tests to gauge the health of your heart.But no such widely accepted test exists for the many parts of the immune system.How can we tell if the immune system is strong or weak?Or quantify how badly its malfunctioning when we suffer from asthma,allergies,or arthritis?-Francis Collins,NIH Director 2013年2月13日Immuno-SeqImmuno-Seq技术可以观察和分析T18Immune response to flu vaccineDay 0Day 7Day 28Flu Vaccine:TIV&LAIVPlasma blastsDay 7Collect 5ml bloodImmune response to flu vaccine19Experiment flowBlood samplePatient TissuesSingle cell SuspensionPBMCTCD8Genomic DNARNAAmplify the library by Multiplex PCRIllumina Hiseq 2000Alignment and ClusteringExperiment flowBlood samplePat20Broad VDJ gene segment usage patternVDJBroad VDJ gene segment usage p21A snapshot of immune systemDay 7,after vaccination.Elderly subjectWe amplified antibody sequences from single sorted plasma B cell.We then expressed monoclonal antibodies according to these sequences and verified their binding to each of the three virus strains in the vaccineA snapshot of immune systemDay22Immune reperotire is a dynamics systemDay 0Day 7Day 28Vaccine specific antibody lineage13%of total sequencesVaccine specific antibody lineage0.01%of total sequencesElderly subjectPlasma blastsDay 7Immune reperotire is a dynamic23Reduced antibody diversity in elderlyAge 8-17Age 18-32Age 70-100MutationEach data point represent a lineageComparison of 3 subjectsSequences in a lineageReduced antibody diversity in 24Reduced antibody diversity in elderly70100 year old volunteers had a significantly less diverse collection of antibodies than the younger volunteers.That is,as people aged,their immune system had a more limited repertoire of antibodies to offerReduced antibody diversity in 25Lab MembersPostdoc:Zhoufang LiTechnician:Luwen NingMeng ZhangStudents:Jiajie Guo Yin TongYing Xu Peng LiYao Chen Ronghai ChengCollaborators:Pinghua Liu(Boston U),Hengye Man(Boston U)Zhigang Liu(Shenzhen U),Zhanhui Wang(SMU),Zhiming Cai(2nd Hospital of Shenzhen),Xiao Liu(BGI)Xiao PingChen(CAS),Hua Zhang(SMU)Lab web:www.sustc-Lab MembersPostdoc:Collaborat26免疫组库测序服务提供商深圳市瀚海基因生物科技有限公司深圳市瀚海基因生物科技有限公司专注于提供人，小鼠，猴子等的TCR/BCR免疫组库测序网址：免疫组库测序服务提供商深圳市瀚海基因生物科技有限公司27