《细胞进展TCRMH》PPT课件.ppt

T细胞识别抗原的分子基础,一、TCR及其共受体 二、MHC分子及其与抗原肽的相互作用 三、MHC多聚体及其应用,T细胞对抗原的识别是 特异性免疫应答发生的先决条件,T细胞识别抗原的物质基础是其表面的TCR TCR识别的配体是与MHC分子结合的抗原肽 TCR识别需要CD4/CD8分子作为共受体,Antigen recognition by T cells requires peptide antigens and presenting cells that express MHC molecules,MHC分子提呈抗原肽供T细胞识别,一、T细胞抗原受体（TCR） 结构 基因及多样性形成的机制 与抗原肽/MHC复合物的相互作用,The structure of the T cell receptor,Structure of TCR is similar to that of Fab,图1,Three sets of key antigen-recognition receptors have similar overall anatomies,图2,Domain structure: Ig gene superfamily Monovalent No alternative constant regions Never secreted Heterodimeric, chains are disuphide-bonded Very short intracytoplasmic tail Positively charged amino acids in the TM region Antigen combining site made of juxtaposed Va and Vb regions 30,000 identical specificity TcR per cell,The T cell antigen receptor, TcR are highly variable in an individual Diversity focuses on small changes in the shape presented at the end of the T cell receptor (CDR). The mechanisms responsible for variability are closely related to the developmental stages of T cells The mechanisms of diversity generation are similar to those used in the immunoglobulin genes with respect to: Gene organisation, rearrangement and random joining error,T cell antigen receptor diversity,Generation of diversity in the TcR,COMBINATORIAL DIVERSITY Multiple germline segments In the human TcR Variable (V) segments: 70, 52 Diversity (D) segments: 0, 2 Joining (J) segments: 61, 13 The need to pair and chains to form a binding site doubles the potential for diversity JUNCTIONAL DIVERSITY Addition of non-template encoded (N) and palindromic (P) nucleotides at imprecise joints made between V-D-J elements SOMATIC MUTATION IS NOT USED TO GENERATE DIVERSITY IN TcR,Organisation of TcR genes,TcR genes segmented into V, (D), J & C elements (VARIABLE, DIVERSITY, JOINING & CONSTANT) Closely resemble Ig genes (aIgL and bIgH),This example shows the mouse TcR locus,TcR a gene rearrangement by SOMATIC RECOMBINATION,Spliced TcR mRNA,Rearrangement very similar to the IgL chains,Rearranged TcR 1 transcript,Spliced TcR mRNA,TcR b gene rearrangement SOMATIC RECOMBINATION,D-J Joining,V-DJ joining,C-VDJ joining,Generation of diversity in the TcR,COMBINATORIAL DIVERSITY Random recombination of multiple germline segments JUNCTIONAL DIVERSITY Reading D region in 3 frames Addition of P & N nucleotides NO SOMATIC MUTATION In TcR,If TcR did undergo somatic mutation:,TcR interacts with entire top surface of MHC-peptide antigen complex Somatic mutation in the TcR could mutate amino acids that interact with the MHC molecule causing a complete loss of peptide-MHC recognition,T cell receptor - and -chain gene rearrangement and expression,Element,Immunoglobulin, TcR,Variable segments,Diversity segments,D segments in all 3 frames,Joining segments,Joints with N & P nucleotides,No. of V gene pairs,Junctional diversity,Total diversity,H,51,30,Rare,5,2,3519,3640,1013,1013,1016*,1016,69,0,-,5,(1)*,52,70,2,0,Often,-,13,61,2,1,* Only half of human k chains have N & P regions *No of distinct receptors increased further by somatic mutation,Estimate of the number of human TcR and Ig,V(D)J rearrangements during T cell development -chain first, then -chain,V(D)J rearrangements during T cell development -chain first, then -chain,The outline structures of the CD4 and CD8 co-receptor molecules,Coreceptor interactions,CD4: monomer, 4 IgSF domains, binding MHC II. CD8: 2 chains with single IgSF domain, CD8: conventional MHC I CD8: nonclassical MHC I The CD8-MHC interaction has a Koff at least 2 orders of magnitude faster than TCR-pMHC interactions, and the CD4-MHC interaction seems to be much less stable. Coreceptors bind to the same MHC molecule engaged by the TCR, and associate with the TCR via the signaling molecules p56lck and ZAP-70.,The binding sites for CD4 and CD8 on MHC lie in the Ig-like domains,Antigen recognition leads to weak phosphorylation of the TCR The combined avidity of 2-site interactions involving coreceptor binding to the ectodomain of the MHC molecule and to intracellular components of the TCR (via CD4-p56lck SH2 domain interacts with phosphorylated ITAM of CD3 or ZAP-70 associated with TCR), is sufficient to recruit coreceptor and p56lck to the triggered TCR-pMHC complex. Stably recruited p56lck then is expected to amplify the initial phosphorylation signal. The signal amplification remains entirely subservient to agonistic TCR signaling.,A possible scenario for co-receptor interaction,Coreceptor interactions and structure,hierarchical affinity differences in TCR-pMHC and coreceptor-MHC complexes ensure these events occur sequentially.,The T cell receptor binds to the MHC:peptide complex,二、MHC分子,（一）MHC-I类和-II类分子 （二）MHC生物学功能,The genetic organization of the MHC in human and the mouse,（一）经典的HLA-I类和-II类抗原,1 HLA-I类分子结构 * 由链（重链）和链（轻链）组成的异源二聚体，其中重链由HLA-I类基因编码且为跨膜链，具有多态性。 * 可分为4个区 (1)肽结合区：结合抗原肽，多态性区域 (2)Ig样区：与CD8分子结合的部位 (3)跨膜区：将HLA分子锚定在胞膜上 (4)胞浆区：与信息转导有关,The structure of an MHC class I molecules (determined by X-ray crystallography),MHC-I类分子结构,链：43KDa,链：12KDa,2.HLA-II类分子的结构 * 由链和链组成的异源二聚体， 链和链均由HLA-II类基因编码且均为跨膜链，均具有多态性。 * 可分为4个区 (1)肽结合区：结合抗原肽，多态性区域 (2)Ig样区：与CD4分子结合的部位 (3)跨膜区：将HLA分子锚定在胞膜上 (4)胞浆区：与信息转导有关,The structure of an MHC class II molecules (resemble MHC class I molecules in structure),MHC-II类分子结构,链：34KDa,链：29KDa,（二）MHC生物学功能及其分子机制,1、MHC分子的生物学功能 抗原提呈 MHC分子的抗原结合凹槽选择性结合抗原肽 形成MHC分子-抗原肽复合物 以MHC限制性的方式供T细胞识别 启动特异性免疫应答。 * CD4+T细胞识别抗原肽/MHC-II复合物 * CD8+T细胞识别抗原肽/MHC-I复合物,MHC分子提呈抗原肽供T细胞识别,TCR识别抗原肽的MHC限制性,2、MHC分子与抗原肽的相互作用,（1）抗原肽和MHC分子相互作用的分子基础 MHC分子方面 * MHC的多态性集中在结合区 MHC分子与抗原肽结合的部位是肽结合区中的肽结合槽，是多态性集中的区域，即型别不同的MHC分子的抗原肽结合槽部位的氨基酸组成和顺序存在差异。,等位基因多态性集中于肽结合区,抗原肽方面 * 抗原肽借助锚定残基与MHC分子的抗原肽结合槽结合 锚定残基：抗原肽并不是整段地与抗原肽结合槽结合，而是以2个或数个氨基酸残基结合于MHC分子的抗原肽结合槽中，抗原肽上这些氨基酸残基为锚定残基。,抗原肽以锚着残基与MHC抗原肽结合槽结合,Peptide binds to MHC-I molecule through anchor residues,Peptides binding to MHC-II molecules are variable in length and residues lie at various distances from the ends of the peptide,（2）抗原肽与MHC分子相互作用 * 抗原肽借助锚定残基与MHC分子抗原结合槽结合 * 不同MHC等位基因产物，其抗原结合槽结构存在差异，从而决定其选择性结合不同的抗原肽 * 能够与同一型别MHC分子结合的抗原肽，其锚定位和锚定残基往往相同或相似,3、MHC型别影响个体对抗原应答性质和强度 * 不同MHC提呈同一抗原分子的不同抗原肽 应答针对的表位不同 * 同一抗原肽与不同MHC结合的亲合力不同 应答的强度不同,MHC alleles control the immune responsiveness,MHC分子提呈抗原肽供T细胞识别,（一）MHC-抗原肽四聚体 MHC-抗原肽四聚体是借助亲和素-生物素结合的原理，在体外将MHC分子（MHC-I类分子包括2微球蛋白）及其所提呈的抗原肽组装成的MHC/抗原肽四聚复合物，作为TCR结合的配体，用于检测和分离特异性T细胞,三、MHC-抗原肽四聚体技术 （MHC-peptide tetramer）,HLA-抗原肽四聚体结构示意图,通过生物素-亲和素的作用，将4个 MHC-I/抗原肽单体连接成四聚体，亲和素分子上还结合有荧光标记物质（PE），用于检测或分离抗原特异性T细胞,HLA-抗原肽四聚体用于检测或分离特异性T细胞,（二）HLA-I/抗原肽四聚体的应用,1、检测病毒抗原特异性CTL * CMV抗原肽/HLA-I四聚体 CMV特异性T细胞频率的检测可以作为肝脏移植后CMV感染或激活的早期诊断指标之一 * EBV抗原肽/HLA-I四聚体 移植患者在术后4周，EB病毒抗体为阴性的患者中，可检测到EB病毒特异性的T细胞扩增，可以作为术后EBV感染的检测和诊断指标。,* HCV抗原肽/HLA I四聚体 丙型病毒肝炎患者中，与康复的患者比较，慢性肝炎患者白细胞中的特异性CTL数量升高，但功能受损，表现为后者的特异性CTL的增殖能力下降，特异性杀伤能力下降和产生IFN-能力下降，而且IL-2不能恢复受损的特异性CTL功能。但是这些患者的流感病毒特异性CTL的功能是正常的。,2、研究自身免疫病发生机制,* GAD65/HLA-DR4四聚体 65 kDa glutamic acid decarboxylase (GAD65)被认为是IDDM的自身抗原，其中含有DR4结合的免疫显性表位（555-567），外周血标本中GAD65/HLA-DR4特异性T细胞识别相应的抗原提呈细胞提呈抗原肽后，用GAD65/HLA-DR4四聚体和流式细胞仪检测，所有IDDM患者和部分高危个体的标本能够检测到活化的GAD65/HLA-DR4特异性T细胞（CD4+/CD25+），而正常对照组却未能观察到。,3、评价肿瘤疫苗的效果 例如HLA-I/Melan-A用于免疫治疗方法的评估。 4、制备抗原特异性T细胞 * 流式细胞分选-体外培养法制备抗原特异性的T淋巴细胞：被特定HLA/抗原肽四聚体结合的T细胞克隆通过荧光激活的细胞分选方法分离出来，在体外用传统的方法进行刺激并扩增。 * MHC四聚体修饰APC-体外培养法制备抗原特异性的T淋巴细胞：用亲和素-抗B细胞抗体融合蛋白（anti-CD20 B9E9-scFvSA）将HLA-I/抗原肽复合物（HLA-I类分子经过生物素化）结合到自身B细胞表面，将经过处理的B细胞作为抗原提呈细胞，与PBMN在体外培养8-14天，仅需一次刺激便可产生特异性CTL。这种方法为体外扩增抗原特异性T细胞提供了有效手段，例如制备病毒抗原、肿瘤抗原特异性的CTL。,5、与其他技术联合应用研究抗原特异性T细胞 * 四聚体技术与细胞内染色技术联合运用 能够检测抗原特异性T细胞的细胞周期（活化与增殖）、死亡方式（衰老和凋亡）、效应功能（胞毒能力和细胞因子产生）。有利于研究在各种环境和疾病状态下抗原特异性T细胞的产生、活化和功能，从而揭示细胞免疫应答的机制。,T细胞识别配体的多聚化提高了与受体结合的亲和力,pMHC四聚体：应用于特异性同种T细胞的检测,pMHC二聚体：诱导或抑制特异性同种T细胞,图1. pMHC四聚体结构示意图,图2. pMHC二聚体结构示意图,可溶性pMHC二聚体特异性抑制同种T细胞应答,图7. 可溶性pMHC二聚体诱导特异性的同种T细胞耐受的示意图,Tyr/HLA-A2二聚体抑制增殖作用具有pMHC特异性,图9.增殖抑制反应 a, HLA-A2-ve PBLs b, HLA-A2-ve PBLs 与T2/Tyr混合培养 c, b+抗原肽Tyr368-376 d, b+HIV/HLA-A2二聚体 e, b+Tyr/HLA-A2二聚体,Tyr/HLA-A2二聚体抑制同种反应体系中 CD8T细胞的增殖,图8. 在T2/Tyr与HLA-A2-ve PBLs混合培养体系中，Tyr/HLA-A2二聚体对T细胞的抑制作用具有浓度依赖性，并可被HLA-A2特异性抗体BB7.2阻断。 Xiufang Weng, Maohua Zhong, Zhihui Liang, Shengjun Lu, Xueling Chen, Janan Li, Juan Hao, Feili