免疫组库测序技术介绍

Immuno-Seq: exploring the diversity of immune system,Jiankui HeSouth University of Science and Technology of China(SUSTC), Shenzhen, China,The diversity of Invaders . . .,BacteriaVirusesparasites such as fungi, protista, & wormsPhysical injury,The Immune System,,Innate immune system,a general response to anything other than recognized “self cells”,macrophages,- able to ingest small foreign invaders,Mast cells /basophils,release histamine that dilates blood vesselscauses redness,Adaptive immune system,a specific counter-assault against a “known foreign” invader previously recognized,Key-lock,Every mature T cell (B cell) expresses a single TCR (BCR) with specificity for a limited number of antigens.,T cell receptor,How 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.,B lymphocyte development,How antibody diversity is generated,Janeway C., Immunobiology, 2004,Stem cell,Chromosome 14, heavy chain,B lymphocyte development,How antibody diversity is generated,Janeway C., Immunobiology, 2004,Stem cell,Chromosome 14, heavy chain,Pro-B cell,B lymphocyte development,How antibody diversity is generated,Janeway C., Immunobiology, 2004,Stem cell,B cell,Chromosome 14, heavy chain,Pro-B cell,Antibody repertoire estimates,Joint diversity3-5bp random insertion/deletion,Theoretical calculation:Immune system only uses 83 geneSegment to code 1014 BCR sequences,Diversity and healthy,Intrinsic 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),Traditional methods to measure the diversity of TCR/BCR,flow cytometryFluorescently labelled monoclonal antibodies against TCRV family gene segmentsResolution: gene segment levelLimited to a few TCRV family genesPCR + Sanger sequencingResolution: gene sequenceLow throughput,High throughput sequencing B cells and T cells,Stephen Quake labScience, 2009PNAS, 2011Sci. Transl. Med, 2013,Fire labSci. Transl. Med2009,Pamer labNat. Medicine2013,Han labPNAS, 2010,Warren labSci. Transl. Med2010,Georgiou labNat. Biotechnology2010,The first use of high throughput sequencing,12003700 unique antibodies in a fish,Stephen Quake,The diversity of human TCR,420,000 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.,Super 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.,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, et al., Sci Transl Med. 2013 Feb 6;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”。,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,Immune response to flu vaccine,Day 0,Day 7,Day 28,Flu Vaccine: TIV & LAIV,Plasma blastsDay 7,Collect 5ml blood,Experiment flow,Broad VDJ gene segment usage pattern,V,D,J,A snapshot of immune system,Day 7, after vaccination. Elderly subject,We 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 vaccine,Immune reperotire is a dynamics system,Day 0,Day 7,Day 28,Vaccine specific antibody lineage13% of total sequences,Vaccine specific antibody lineage0.01% of total sequences,Elderly subject,Plasma blastsDay 7,Reduced antibody diversity in elderly,Age 8-17,Age 18-32,Age 70-100,Mutation,Each data point represent a lineage,Comparison of 3 subjects,Sequences in a lineage,Reduced antibody diversity in elderly,70100 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 offer,Lab Members,Postdoc: Zhoufang LiTechnician:Luwen NingMeng ZhangStudents:Jiajie Guo Yin TongYing Xu Peng LiYao Chen Ronghai Cheng,Coll