【作者】 孔燕；

【导师】 何维； 崔莲仙；

【作者基本信息】 中国协和医科大学 ， 免疫学， 2008， 博士

【摘要】 UL16结合蛋白（UL16 Binding Proteins，ULBPs）是一个NK细胞活化型受体一NKG2D的配体家族，目前发现的同系物包括ULBP1～5。ULBPs可应激性表达在一些肿瘤细胞或病原体感染细胞的表面。ULBP1、2、和3之间存在55-60%同源性，而ULBP4序列则变化较大，且相关研究较少，仅限于ULBP4-NKG2D相互作用和活化NK细胞方面。γδT细胞表面表达NKG2D，可识别MICA、ULBPs分子等配体分子。研究发现，γδT细胞表面的T细胞受体（TCR）γ和δ链异质二聚体分子也识别MICA。这种NKG2D和TCRγδ对相同配体分子双重识别的生物学意义尚不完全明了，据推测，可能与确保固有免疫细胞能及时清除表达应激分子的受损细胞有关的免疫监视功能有关。本研究关注以下四个科学问题：第一，回答TCRγδ是否识别ULBP4分子?这一问题至今尚无答案。事实上，ULBPs家族分子是否可与TCRγδ结合，至今无结构生物学的证据。由于ULBP4具有一定的基因表达的多样性，我们推测机体可能会启动具有受体多样性的TCRγδ对其产生识别，故以ULBP4分子为靶分子，探讨TCRγδ对其识别的可能性。在这一问题框架下，我们要研究δ1链，或δ2链对其结合的特异性，并探究δ链中与配体结合起关键作用的部位一互补决定区（CDR）3的基因序列变化的特点。第二，澄清ULBP4反应性γδT细胞的特性，如主要亚群，细胞因子分泌谱系和肿瘤细胞毒等。第三，利用抗体阻断实验分析ULBP4-NKG2D和ULBP4-TCRγδ双重识别的生物学意义。第四，通过ULBP4在EB病毒感染B淋巴细胞和肿瘤细胞表面的表达和ULBP4介导的γδT细胞细胞毒分析，来探究ULBP4在免疫监视（尤其是在细胞恶变早期）中的作用。我们希望通过针对以上四个科学问题的科学研究可基本阐明γδ-T细胞对ULBP4识别的机制，并为系统阐释ULBP4的生物学意义提供有价值的资料。为解决上述科学问题，我们进行了以下研究：（1）利用293ET真核表达系统表达了人ULBP4分子胞外区（1～226氨基酸）的重组蛋白ULBP4_1-226；利用pcDNA真核表达载体系统在EL4细胞中稳定表达全长的ULBP4基因，这些系统的建立为后续的研究奠定了基础。（3）首次使用固相化ULBP4分子诱导OEC和CC中的肿瘤浸润γδT淋巴细胞的体外扩增，获得纯度为80%的Vδ2 T细胞，并开展ULBP4反应性γδT细胞TCR Vδ-CDR3区的结构生物学研究。（4）利用配体受体酶联免疫结合实验、流式细胞术和γδTCR基因转染的TCR-β链缺陷型的T细胞系J．RT3-T3．5细胞平台证实，ULBP4与TCRγ9δ2特异结合。（5）ELISA法检测ULBP4刺激γδT细胞产生细胞因子的谱系。MTT方法检测γδT细胞对表达ULBP4的肿瘤细胞的杀伤活性。（6）在抗体封闭实验中，分别分析了anti-NKG2D、anti-TCRγδ、anti-NKG2D+anti-TCRγδ单抗封闭前后，ULBP4活化的γδT细胞分泌IFN-γ水平及对ULBP4阳性的肿瘤细胞细胞毒的变化。实验结果显示，anti-NKG2D+anti-γδTCR单抗联合阻断的效果强于anti-γδTCR或anti-NKG2D单独阻断作用。（7）结合流式细胞术和组织化学方法，检测ULBP4在不同来源肿瘤细胞、组织和EB病毒转化前后B淋巴细胞表面的表达。分析ULBP4介导的γδT细胞细胞毒效应。综上，γδT细胞可凭借表面的NKG2D和TCRγδ结合并识别与肿瘤和感染相关的应激分子ULBP4，其自身发生细胞活化，对肿瘤细胞，恶变细胞，病毒感染细胞迅速产生细胞毒效应，体现了固有免疫系统在免疫监视中所发挥的直接与迅捷的特点。以ULBP4为靶的免疫诊断与治疗策略可能为肿瘤的早期诊断与干预提供新型手段。更多还原

【Abstract】 Ph.D.candidate:Yan KongSupervisor:Prof.Wei He,Prof.Lianxian CuiULBPs (UL16 binding proteins),is a ligand family for NKG2D activatingreceptor of human NK cells.Five members of ULBPs family have been identifiedto date and they are stressfully expressed in most epithelial tumours orpathogen-infected tissue cells.Amino acid sequence analyses indicate thatULBP1,ULBP2 and ULBP3 are 55～60% identical,while newly found ULBP4 ismore divergent,which only few reports related to,but focus on the mechanism ofNK cell activation via ULBP4 - NKG2D interaction.MostγδT cells express NKG2D on their surface,which could recognize ULBPs,MICA and other ligands.Furthmore,many results has demonstrated that theγδTcells homodimer receptor,TCRγδalso mediated the recognition of MICA by theVδ1 subset ofγδT cells.However,it is not clear about the biological significance ofrecognition of the same ligand mediated by TCRγδand NKG2D simultaneously,which preditly benefit ed the clear of cells by innate immune cells during immunedefenseThe present study focused on four scientific questions as follows:First,Demonstrate whether ULBP4 could recognized byγδT cells viaTCRγδ.It is still enigmatic.In fact,there is no evidence in structure biology so farabout the ULBP4-TCRγδbinding.We proposed that TCRγδrespond to a diversityof ULBP4 gene due to its extremely diversity,analyzed the specificity ofδ1 orδ2 bind to ULBP4 and explored the sequence features of TCRδ-CDR3.Second,clarify the immunological effects of ULBP4 -reactiveγδT cells,including analyzing the phentype of ULBP4 -reactiveγδT cells、measuringcytokine secreted by ULBP4-inducedγδT cells and identifing the target cells lysis.Third,analyze biological significance of recognition of the same ligandmediated by TCRγδand NKG2D simultaneously by antibodies blockade.Finaly,Study the roles of ULBP4 in immunosurveillance through analysis of theULBP4 expression on different tumor cells or tissue or EB virus- transformed Blymphocytes,and ULBP4-mediate target cells lysis.To solve the scientific problems as mentioned above,we performed theinvestigations as follows.(1) we successfully expressed and purified recombinantextracellular segments (1-226aa) of ULBP41-226 protein utilizing 293ET eukaryoticexpressing system and established stable transfection of ULBP4 into EL4 cells,which make foundation for further researches.(2) The immobilized ULBP4 couldinduce marked expansion of human OEC- or CC -derivedγδTILs of Vδ2phenotype mostly in vitro andδ-CDR3 region of ULBP-reactiveγδT cells wasanalyzed (3) ULBP4 could bind to TCRγ9δ2 identified by Enzyme-linkedimmunosorbent binding assay or flow cytometry and stimulate the activation ofJ.RT3-T3.5-TCRγ9δ2 via TCRγ9δ2.(4) Cytokine secreted byγδT cells afterstimulation with ULBP4 were analyzed by ELISA and the cytotoxicity of Vδ2 T cellsto EL4/ULBP4-EL4 tumor cell lines were compared.(5) We compared the level ofIFN-γsecreted by ULBP-reactiveγδT cells and target cells lysis before or afterblockade with anti-NKG2D、anti-TCRγδ、anti-NKG2D +anti-TCRγδmAb.Our dataindicate that the percentages of blocking by combination anti-NKG2D andanti-TCRγδantibodies is stronger than anti-NKG2D mAb alone or anti-γδTCR mAbalone.(6) ULBP4 expressed on different tumor cells or tissue or EB virus-transformed B lymphocytes were analyzed by flow cytometry or immunohistochemistry.Taken these data together,humanγδT cells could bind to and recognizeULBP4 stressfully expressed in tumours or pathogen-infected tissue cell via TCRand NKG2D signal,be actived,display strong cytotoxic toward tumors、transformed and virus infected cells,Which reflect the instant and direct responseof innate immune cells during immune defense.更多还原