【作者】 何小鹃；

【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】 尽管γδT细胞在机体抗感染、抗肿瘤的固有免疫应答中发挥重要的作用,但是目前所发现的配体抗原仅限于十余种,根本无法与承担特异性细胞免疫应答的αβT细胞可识别数以亿万计的抗原表位肽相比。此外,有关γδT细胞的表位蛋白多肽则尚无报道。因此,有必要进行γδT细胞蛋白抗原表位肽的研究,以澄清活化γδT细胞的结构基础。另一方面,活化的γδT细胞是机体免疫系统内杀伤肿瘤细胞的重要效应细胞,一旦获得具有活化γδT细胞的抗原表位多肽,可为体内外激活γδT细胞提供一个有效的活化手段。为此,我们所关注的第一个科学问题是是否存在人类γδT细胞的抗原表位?如果存在,多个串联抗原表位多肽是否比单个抗原表位肽更具刺激γδT细胞的能力?围绕上述两个科学问题我们开展了人类γδT细胞的抗原表位肽的鉴定研究。我们实验室先前根据三个来源于卵巢癌组织浸润的γδT细胞的TCRδ链互补决定区（CDR）3的（CDR3δ）基因序列,合成了三条CDR3δ多肽OT1、OT2、OT3,并且以这三条多肽为探针,在噬菌体随机十二肽库中筛选与CDR3δ多肽结合的十二肽,作为γδT细胞的候选抗原表位肽。我们获得了7个能与探针特异性结合的候选抗原表位肽。体外实验表明,这些多肽能在一定程度上增强γδT细胞杀伤肿瘤细胞的能力,提示这些多肽具有抗原表位活性。但是,可能由于这些多肽分子量小,因此,它们对γδT细胞的活化能力较弱。为了增强这些潜在抗原表位肽激活γδT细胞的能力,我们试图通过制备串联抗原多肽的方式来提高其刺激活性。具体的实验思路为:通过分子克隆技术,将表位肽串联,并与无关载体蛋白GST连接构建GST-表位融合蛋白表达载体,表达并鉴定了这些GST-表位融合蛋白的功能,以进一步阐明该项策略的可行性。这一研究的主要工作及其结果如下:首先,我们通过流式细胞仪检测、³H掺入法、MTT掺入法证实筛选到的十二肽能在一定程度上刺激γδT细胞增殖并增强其杀伤肿瘤的作用,提示这些十二肽可能是γδTCR特异性识别的抗原表位肽。这为进一步澄清活化γδT细胞的结构基础提供了依据。其次,采用同尾酶技术构建了含不同表位组合方式的GST-表位融合蛋白的表达载体,通过ELISA和流式细胞术检测,从分子水平和细胞水平证实这些原核表达的重组GST-表位融合蛋白能与探针OT3、OT3（V）-Fc分子及γδT细胞的TCR特异性结合。并且,利用CFSE染色和MTT掺入的方法,验证GST-表位融合蛋白可刺激γδT细胞群体的增殖作用。此外,GST-表位融合蛋白与γδT细胞孵育后,发现它们可以促进γδT细胞的杀伤作用,且GST-串联表位融合蛋白的作用要强于GST-单表位融合蛋白。通过半定量RT-PCR和进一步的ELISA定量分析,发现GST-表位融合蛋白能够促进γδT细胞分泌细胞因子IL-2、IFN-γ,和TNF-α;此外,Fas配体和颗粒酶B的表达也有所增加。最后,我们观察了GST-串联表位融合蛋白刺激的人γδT细胞对荷瘤裸鼠的抑瘤作用。结果显示,GST-串联表位融合蛋白刺激的人γδT细胞治疗组小鼠肿瘤的生长较未刺激γδT细胞治疗组相比明显受到抑制,小鼠的生存期也有所延长。综上,我们首次系统地验证了γδT细胞表位肽的存在。这些表位肽能在一定程度上刺激γδT细胞增殖,并增强其杀伤肿瘤的作用。其次,通过构建GST-表位融合蛋白并进行功能实验证实,串联表位肽的γδT细胞活化效果要优于单表位肽。这说明将表位串联起来提高表位的γδT细胞活化能力的新策略是完全可行的。这将为基于γδT细胞的过继免疫细胞治疗肿瘤提供新型活化手段。γδT细胞是一群特殊的T细胞,是连接固有免疫和特异性免疫之间不可或缺的桥梁。由于γδT细胞在外周血中只占很少的比例,分离纯化相对较繁琐,这为深入研究γδT细胞的结构和功能带来了一定的困难。因此,若能建立在体外可长期培养的γδT细胞克隆,尤其是建立单个的γδT细胞克隆,将极大地促进其抗原识别、细胞活化及功能等研究。为此,本文的第二方面工作为健康人γδT细胞克隆的建立及鉴定。在本实验中,我们首先探索了固相法扩增的γδT细胞的冻存与复苏条件。其次,以⁶⁰Co照射的同种异体PBMC作为饲养细胞,以有限稀释法对经过阳性分选的γδT细胞进行克隆化。用流式细胞术及分子生物学检测鉴定所获克隆,以MTT掺入法对所获克隆进行细胞毒检测。结果共获得五株γδT细胞克隆,这五株克隆均为Vγ9Vδ2亚型,CD4分子和CD8分子表达均为阴性,对肿瘤细胞SKOV3均具有一定的杀伤作用。在这5株γδT细胞克隆中,有两株为单克隆,且这两株单克隆经证实为相同克隆,其VδCDR3区序列为ACDTIPGDLVRADKLIFGKG,VγCDR3区序列为ALWEVGKLGKKIKVFGPG。另三株克隆为寡克隆,其中有两株为双克隆,另一株为三克隆。这五株γδT细胞克隆的相关功能实验正在进行之中。综上,本实验第二部分建立了正常人外周血γδT细胞克隆,为深入研究γδT细胞结构、表面标志、亚群及功能奠定了坚实的基础。更多还原

【Abstract】 γδT cells play an important role in anti-infection and anti-tumor immune responses, but only a few antigens recognized byγδT cells have been identified and antigenic peptide epitopes recognized byγδT cells were no reports so far.Therefore,it is necessary to study the antigenic epitope peptides recognized byγδT cells and thus clarify the structural basis of activation ofγδT cells.On the other hand,activatedγδT cells exert effector functions on tumor cells directly via cytotoxicity or indirectly via producing powerful cytokines.Once antigenic epitope peptides which could activateγδT cells were obtained,it can provide an effective means of activatingγδT cells in vitro and in vivo.To this end,the first scientific question we concerned is whether there exist antigenic epitope recognized by humanγδT cells? If there exist,whether the ability of stimulatingγδT cells of tandem epitope peptides was superior to that of single epitope peptide? Therefore,in the first part of my thesis,we identified the epitope peptides recognized byγδT cells.In previously study,we synthesized three CDR3-peptide OT1,OT2 and OT3 according to the gene sequence ofγδTCR CDR3 derived from tumor infiltrating lymphocyte（TIL） s in ovarian epithelial carcinoma（OEC）,and then we used the OT1, OT2 and OT3 as probe in an attempt to identify possible epitopes forγδT cells by screening a Ph.D.-12^TM Phage Display Peptide Library.We obtained seven peptides （EP1～EP7） recognized by probes as the candidates of epitope peptides,and these peptides could trigger the cytotoxicity ofγδT cells to tumor cells.However,perhaps because of their small molecular weight,the ability of activatingγδT cells of these peptides is weak. In order to enhance the functions of candidate epitope peptides,we tried to construct tandem peptides to improve their stimulation activity.The specific strategy for the experiment is to borrow a GST carrier protein and adopt a tandem-connection strategy to construct expression vectors of GST-epitope fusion protein,and the function of recombinant GST-epitope fusion protein would be verified to further clarify the strategy’s feasibility.The results of the present study were summarized as follows:Firstly,we synthesized these seven candidate epitope peptides and investigated their biological functions in vitro.The results confirmed that these peptides were able to induce significant proliferation response of humanγδT cells and could selectively trigger the growth ofγδT cells in vitro.Furthermore,we performed a MTT experiment to validate that cytotoxicity ofγδT cells against SKOV3 cells was promoted after pre-incubated with these peptides.All the results indicate that these peptides have the feature of antigen epitopes recognized byγδT cells,which provided a basis to further clarify the activation structure ofγδT cells.Secondly,we constructed four types of GST-epitope fusion proteins expression vectors containing single epitope or tandem epitopes by using isocaudamer technique.The results confirmed that the GST-epitope fusion proteins could bind to probe OT3, OT3（V）-Fc andγδTCR specially as shown by the analysis of ELISA and flow cytometry. Subsequently,we did CFSE,MTT assay,semi-quantity RT-PCR and ELISA to investigate the potential effect of GST- epitope fusion proteins to.γδT cells.The results of CFSE and MTT indicated that these GST-epitope fusion proteins could promote the proliferation ofγδT cells.Moreover,after pre-incubated with GST-epitope fusion proteins,significant cytotoxicity of.γδT cells to tumor cell lines was observed in a dose-dependant manner.In addition,the expression level of IL-2,IFN-γ,TNF-α,FasL and gramB were also found to be increased.Meanwhile,there were significant difference between GST-tandem epitope fusion protein groups and GST-single epitope fusion protein group,demonstrating that the function of epitopes were enhanced by tandem connected.Finally,the function of GST-epitope fusion proteins was further analyzed through experimental immunotherapy in subcutaneous tumor bearing nude mice models.Three intraperitoneal injections of.γδT cells pre-incubated with GST-EPt7 greatly decreased the sizes of SKOV3 tumors and prolonged the survival of tumor-bearing nude mice, significantly different from PBS control.In conclusion,for the first time,we systematically verified the existence of epitope peptides for.γδT cells;confirmed that the activation of tandem epitope peptides were superior to that of single epitope through the construction of four types of GST-epitope fusion proteins and functional tests,which clarify the strategy’s feasibility of enhancing the activation of epitope peptides by tandem connected.These results provide a new means for activatingγδT cells in tumor immunotherapy.γδT cells,a special group of T cells,are an indispensable bridge linking innate immunity and specific immunity.AsγδT cells account for only small proportion in the peripheral blood,it is relatively cumbersome to separate and purify,and hinder the in-depth study of the structure and function ofγδT cells.Therefore,establishment ofγδT cell clones in vitro,especiallyγδT cell single clones,will help us in-depth study of its antigen recognition,cell activation and functions.Therefore,in the second part of my thesis,we established healthy donor’sγδT cell clones.In this study,we firstly explored the freezing and thawing condition ofγδT cells. Secondly,γδT cells were cloned by limiting dilution after positive choice,with ⁶⁰Co irradiated allogeneic PBMC as feeder cells.Flow cytometry analysis and molecular biology technique were then used to identifyγδT cells clones,and MTT assay was used to verify their cytotoxicity.Results demonstrated that fiveγδT clones have been established. The subtype of these five clones is all Vγ9 Vδ2 and the expression of CD4 and CD8 molecule were negative.Among these five clones,two clones were monoclonal,and these two clones are identical,the amino acid sequence of VδCDR3 is ACDTIPGDLVRADKLIFGKG and the amino acid sequence of VγCDR3 is ALWEVGKLGKKIKVFGPG;other threeγδT clones are oligoclonal.All of these five clones could kill tumor cell line SKOV3.Functional study of these fiveγδT cell clones is now in process.In conclusion,in the second part of the thesis,we successfully established five normal peripheral bloodγδT cell clones,which laid a solid foundation for further study onγδT cells.更多还原