免疫组库测序技术介绍课件

1、The diversity of Invaders . . . BacteriaVirusesparasites such as fungi, protista, & wormsPhysical injuryThe diversity of Invaders . . The Immune System The Immune System http:/www.nInnate immune systema general response to anything other than recognized “self cells”macrophages- able to ingest small

2、foreign invadersMast cells /basophils release histamine that dilates blood vesselscauses redness Innate immune systema general Adaptive immune systema specific counter-assault against a “known foreign” invader previously recognizedAdaptive immune systema specifKey-lockEvery mature T cell (B cell) ex

3、presses a single TCR (BCR) with specificity for a limited number of antigens.T cell receptorKey-lockEvery mature T cell (BHow 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

4、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 n

5、umber of T cells in the human body to be about 5 x 1011.How many T cells in your body?B lymphocyte developmentHow antibody diversity is generatedJaneway C., Immunobiology, 2004Stem cellChromosome 14, heavy chainB lymphocyte developmentHow anB lymphocyte developmentHow antibody diversity is generated

6、Janeway C., Immunobiology, 2004Stem cellChromosome 14, heavy chainPro-B cellB lymphocyte developmentHow anB lymphocyte developmentHow antibody diversity is generatedJaneway C., Immunobiology, 2004Stem cellB cellChromosome 14, heavy chainPro-B cellB lymphocyte developmentHow anHumanHeavy chain V gene

7、 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 sequencesHumanHea

8、vy chain V gene segmenDiversity 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 sta

9、ble 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 autoimmun

10、ity in common variable immunodeficiency (CVID), in severe combined immunodeficiency (SC1D), Diversity and healthyIntrinsicTraditional methods to measure the diversity of TCR/BCRflow cytometryFluorescently labelled monoclonal antibodies against TCRV family gene segmentsResolution: gene segment levelL

11、imited to a few TCRV family genesPCR + Sanger sequencingResolution: gene sequenceLow throughputTraditional methods to measureHigh throughput sequencing B cells and T cellsStephen Quake labScience, 2009PNAS, 2011Sci. Transl. Med, 2013Fire labSci. Transl. Med2009Pamer labNat. Medicine2013Han labPNAS,

12、2010Warren labSci. Transl. Med2010Georgiou labNat. Biotechnology2010High throughput sequencing B cThe first use of high throughput sequencing 12003700 unique antibodies in a fishStephen QuakeThe first use of high throughpThe diversity of human TCR420,000 unique CDR3 nucleotide in a few milliliter bl

13、ood3*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 S

14、uper 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 r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

15、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

16、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 fr

17、om 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TImmune response to flu vaccineDay 0Day 7Day 28Flu Vaccine: TIV & LAIVPlasma blastsDay 7Collect 5ml bloodImmune response to flu vaccineExperiment flowBlood samplePatient TissuesSingle cell Suspen

18、sionPBMCTCD8Genomic DNARNAAmplify the library by Multiplex PCRIllumina Hiseq 2000Alignment and ClusteringExperiment flowBlood samplePatBroad VDJ gene segment usage patternVDJBroad VDJ gene segment usage pA snapshot of immune systemDay 7, after vaccination. Elderly subjectWe amplified antibody sequen

19、ces 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 systemDayImmune reperotire is a dynamics systemDay 0Day 7Day 28Vaccine specific antibody lineag

20、e13% of total sequencesVaccine specific antibody lineage0.01% of total sequencesElderly subjectPlasma blastsDay 7Immune reperotire is a dynamicReduced antibody diversity in elderlyAge 8-17Age 18-32Age 70-100MutationEach data point represent a lineageComparison of 3 subjectsSequences in a lineageReduced antibody diversity in Reduced antibody diversity in elderly70100 ye