【作者】 危华锋；

【导师】 郭亚军； 李博华； 王皓； 戴建新； 赵健； 寇庚；

【作者基本信息】 第二军医大学 ， 肿瘤学， 2008， 博士

【摘要】 Treg（regulatory T cells,Treg）是一群独特的、具有免疫调节功能的、在调控免疫应答、促进和维持自身免疫耐受中发挥重要作用的T细胞。目前,越来越多的证据表明,Treg在肿瘤免疫逃避中也扮演重要角色。研究发现,包括乳腺癌、卵巢癌、胃癌、肺癌、淋巴瘤、黑色素瘤及肝癌等各种肿瘤病人的外周血或肿瘤组织中Treg的数量增加;卵巢癌中存在的高比例Treg与肿瘤病人不良预后密切相关。目前在各种实验性肿瘤模型中获得的证据表明,Treg在肿瘤免疫逃避中发挥显著的作用,在这些肿瘤模型中直接清除Treg或抑制其功能有效增强了治疗性肿瘤疫苗的效果。肿瘤细胞能够利用一套精细的直接或间接的主动机制逃避免疫系统的攻击,这些机制包括:抗原递呈途径的丢失或改变、死亡受体信号通路的变化以及产生大量具有免疫抑制功能的细胞因子在肿瘤周围形成免疫抑制性微环境。重要的是,肿瘤细胞还能直接招募Treg至肿瘤局部或诱导不成熟的髓性DC分泌TGF-β和IL-10,在上述细胞因子的作用下使CD4⁺CD25^-初始T细胞转化成Treg。基于Treg在肿瘤免疫逃避中的重要性,靶向Treg以提高抗肿瘤免疫应答已获得大家的共识。目前,各种靶向Treg的策略已在临床和临床前模型中进行验证,按其作用机制,归纳起来主要有以下两种:一、利用抗CD25抗体或IL-2免疫毒素直接清除Treg;二、通过靶向Treg表面抑制性受体抑制Treg的免疫调节功能或降低效应性T细胞对Treg的易感性。然后,通过白介素2受体（IL-2R,即CD25）清除Treg所带来的益处因为同时清除了激活的效应淋巴细胞以及潜在的诱导新的Treg的产生而丧失。理论上,功能性失活Treg不仅可以去除其免疫抑制活性,同时因维持其生理上的存在而避免源自外周淋巴细胞池的Treg再生成,这样将有效的增强效应淋巴细胞介导的抗肿瘤免疫应答。Foxp3是Treg发育、功能及维持外周生存最主要的调控因子。Foxp3基因突变（Scurfy小鼠）或敲除的小鼠,其体内Treg功能受损,早期就发展全身多器官自身免疫性疾病。同样在人类中,与小鼠Foxp3同源的FOXP3基因突变可引起人患IPEX综合征、Ｉ型糖尿病、克隆氏病等自身免疫性疾病,与Scurfy小鼠和Foxp3基因敲除小鼠的表型相似。Treg稳定特异地表达Foxp3转录因子,因此,在人和啮齿类动物中,Foxp3联合CD4和CD25分子能够用来鉴定Treg。Treg的功能需要Foxp3的持续表达,理论上,沉默Foxp3的表达能够逆转Treg的免疫抑制活性。然后,Foxp3主要在核内表达,因而传统的靶向策略鞭长莫及,而最近蓬勃发展的RNA干扰（RNA interfering,RNAi）技术为实现这一设想提供了实际可行的线路。RNAi的某些特性非常适合用于体内干扰Treg活性:首先,小干扰RNA（small interfering RNA,siRNA）沉默基因表达是暂时性的,允许灵活的掌握抑制时间的长短;其次,沉默Foxp3表达仅仅只抑制了Treg的活性,而并不干扰效应T细胞的功能;这两点对于临床应用很有价值。然后,目前应用siRNA面临的主要难点是体内把siRNA递送至细胞胞质内进而启动RNAi通路降解mRNA。虽然使用转染试剂能够局部递送siRNA,但是是非特异的,容易导致严重毒性。而通过细胞表面特异性受体介导siRNA递送将能使siRNA进入特殊细胞,达到最大的治疗效果,同时减少药物用量,避免非特异的基因沉默和细胞毒性。最近Song EW等利用人鱼精蛋白的核酸结合活性,构建了抗p24（一种HIV胞膜蛋白）Fab片段和鱼精蛋白的融合蛋白,这种蛋白能够把siRNA特异递送至HIV感染的淋巴细胞,进而沉默HIV胞膜糖蛋白gp160的表达。本研究中我们构建了小鼠CD3特异性单链抗体-鱼精蛋白融合蛋白（2C11scκtp）,利用2C11scκtp中鱼精蛋白段的核酸结合活性以及CD3单链抗体的导向功能,递送Foxp3特异性siRNA至CD3⁺T细胞中沉默Foxp3基因表达。体内外实验表明,2C11scκtp能够与siRNA结合并把siRNA特异递送至CD3⁺小鼠淋巴瘤细胞或小鼠淋巴细胞;2C11scκtp递送的Foxp3特异性siRNA能够特异沉默Treg中Foxp3蛋白的表达;Foxp3表达下调的Treg“无能”表型和免疫抑制功能丧失,刺激TCR后分泌Th1型细胞因子,不再能够抑制初始T细胞的增殖应答;此外,单链抗体递送的siRNA体内没有诱发干扰素免疫应答。具体实验内容如下:第一部分:克隆2C11sCκ和2C11sCκtp基因1.克隆2C11sCκ和2C11sCκtp基因:我们采用一步法逆转录PCR直接从产生仓鼠抗小鼠CD3抗体2C11-145的杂交瘤中扩增出VH和VL,测序正确后通过重叠PCR构建2C11sCκ和2C11sCκtp,两个PCR产物装入pGEM-Teasy载体扩增。2.构建2C11sCκ和2C11sCκtp表达载体:我们用HindⅢ和EcoRⅠ双酶切上述pGEM-Teasy载体获得2C11sCκ(和2C11sCκtp片段,然后装入经相同酶切的pcDNA3.1（+）中成功构建出含上述两个基因的表达载体。第二部分:表达和纯化2C11sCκ和2C11sCκtp蛋白1.表达2C11sCκ和2C11sCκtp蛋白:抽提上述构建的表达载体质粒,瞬时转染CHO,72h后收获上清,通过流式检测与表达小鼠CD3的YAC-1淋巴瘤细胞的结合表明2C11sCκ和2C11sCκtp蛋白构象正确,结合活性良好,在此基础上我们进行了稳定转染,筛选出了两株稳定高表达2C11sCκ和2C11sCκtp蛋白的CHO细胞克隆。2.纯化2C11sCκ和2C11sCκtp蛋白:大量扩增CHO细胞克隆,收集细胞上清,采用Ni⁺⁺离子金属鳌合层析纯化目的蛋白。SDS-PAGE和WB实验显示纯化的蛋白为单一条带,纯度90%以上,分子量与预期相符。纯化的2C11sCκ和2C11sCκtp蛋白体外显示良好的小鼠CD3结合活性。第三部分:鉴定小鼠Foxp3基因特异高效的siRNA1.构建稳定高表达鼠Foxp3的细胞系:因为转染小鼠T细胞很困难,因此我们需要构建表达鼠Foxp3的易于转染的细胞系。我们利用表达鼠Foxp3的MIGR1-MFoxp3逆转录病毒感染HELA细胞成功构建了高表达鼠Foxp3的细胞系（HELA-MF）,半定量、实时定量PCR和WB证实其高表达鼠Foxp3。2.筛选鼠Foxp3特异高效的siRNA:利用HELA-MF细胞,我们针对QIAGEN公司合成的3个鼠Foxp3特异的siRNA进行了筛选,发现siRNA A沉默效果最佳,因此,后续研究中我们主要使用siRNA A验证单链抗体介导的siRNA递送抑制Foxp3的效果。第四部分:2C11sCκtp蛋白递送siRNA1.2C11sCκtp蛋白把FAM荧光表达的siRNA特异递送至CD3⁺细胞:这部分结果显示2C11sCκtp蛋白能够与CD3分子和FAM-siRNA结合,把后者特异递送至CD3⁺YAC-1、EL-4鼠T淋巴瘤细胞和新鲜分离的小鼠T细胞中,而不是表达人CD3的Jurkat T细胞中。此外,注射2C11sCκtp蛋白与FAM-siRNA形成的复合物至小鼠体内,6h后流式检测小鼠脾细胞不同亚群的荧光摄取,发现只有CD3⁺ T细胞检测到FAM荧光的存在,而CD14⁺单核细胞、CD19⁺B细胞及CD11^c+DC胞内无FAM-siRNA存在。2.2C11sCκtp蛋白递送的Foxp3特异的siRNA沉默了自然Treg和TGF-β诱导的Treg中Foxp3的表达:研究表面,Foxp3特异的siRNA能有效被2C11sCκtp蛋白而不是对照2C11sCκ蛋白递送至上述两种Treg中,实时定量PCR、WB和胞内Foxp3直接染色都证实Foxp3的表达下调。第五部分:Foxp3表达沉默后Treg的表型和功能变化1.Foxp3表达沉默后Treg的表型:结果表明,随着Foxp3表达下调,Foxp3调控的相关基因表达也发生改变,CD25、CTLA4和GITR等表面标记分子表达下降,而IL-2、IL-7Rα和PDE3B等受Foxp3抑制的基因表达上调。2.Foxp3表达沉默后Treg的功能:结果显示,低表达Foxp3的Treg功能发生改变,体外受CD3和CD28双抗刺激后发生强力增殖,并分泌IL-2,IFN-γ和TNF-α等Th1细胞因子。这部分的结果提示沉默Foxp3表达后Treg的表型和功能向Th1逆转。第六部分:应用siRNA相关的干扰素免疫应答体内外研究都表明利用2C11sCκtp蛋白递送siRNA不会促发干扰素等非特异炎症免疫应答。结论:综上所述,我们的研究表明2C11sCκtp蛋白能够通过3′羧基端的鱼精蛋白结合siRNA,利用5′氨基端的单链抗体导向作用,把siRNA特异递送至CD3⁺T细胞内,递送的Foxp3特异的siRNA能够特异沉默Treg中Foxp3的表达,逆转Treg的表型和功能,使其向Th1细胞方向转变。我们的研究为逆转Treg免疫抑制功能进而增强肿瘤疫苗效果提供了新的策略。更多还原

【Abstract】 It is now evident that CD4⁺CD25⁺Foxp3⁺ regulatory T cells（Treg） represent a distinct lineage of T lymphocytes that plays a central role in controlling immune responses,and in promoting and maintaining self tolerance.In addition,Treg cells are.implicated in various immune evasion mechanisms used by cancers.Several studies have reported increased numbers of Treg cells in tumor tissue and/or peripheral blood of patients with various cancers,including breast,ovarian,gastric and lung cancers,lymphoma and melanoma associated with poor prognosis and survival of patients with ovarian cancer.Direct evident for the dominant role of Treg cells in immune evasion mechanisms has recently been provided in various experimental tumor models,where physical depletion of these cells or modulation of their function was the cause of therapeutic efficacy.Tumors employ an intricate set of direct and indirect active mechanisms to evade the immune system.These mechanisms include the loss or alterations of the antigen-presenting pathway,alterations in death receptor signaling,and production of an immunosuppressive cytokine milieu.Importantly,tumor cells can either directly recruit Treg cells into the tumor and/or induce immature myeloid DCs to secret TGF-βand/or IL-10 for conversion of CD4⁺CD25^-na（?）ve T-cells into Treg cells within the tumor microenvironment. Tumor-induced expansion of Treg cells is an obstacle to successful cancer immunotherapy. Because of their dominant role in tumor immune evasion mechanisms,Treg cells have become the target of intense studies for therapeutic purpose.Disrupting Treg suppression in combination with cancer vaccines presents an important therapeutic approach with a great likelihood of success in the clinic.At present,various strategies were brought about for targeting Treg cells,mainly including two categories:physical depletion of Treg cells using anti-CD25 mAbs and IL-2 toxins,and inhibiting Treg cell suppressive function and reducing effector T cell susceptibility to suppression by targeting cell-surface suppressive molecules on Treg cells.However,the potential benefit of Treg cells depletion through the interleukin- 2 receptor is lost by the concurrent elimination of activated effector lymphocytes and possibly by the de novo induction of Treg cells replenishment.In theory, the functional inactivation of Treg cells will maintain them at high numbers in tumors and avoid their replenishment from the peripheral lymphocyte pool,which has the capacity to further suppress the effector lymphocyte anti-tumor response.Foxp3 represents a lineage-specific marker for Treg,and its detection is widely used to identify these cells in vivo.The importance of Foxp3 in the development and function of Treg is demonstrated by experiments of nature in which spontaneous mutations of the Foxp3 gene cause severe X-linked autoimmune lymphoproliferative disorders.The scurfy mutation in mice,which is associated with a null mutation of the Foxp3 gene,results in complete loss of Treg,autoimmunity,and premature death.Similarly,mutations of FOXP3 in humans are responsible for a disease-called immuno dysfunction polyendocrinopathy enteropathy X-linked syndrome（IPEX）-in which the impairment of Treg function is associated with several autoimmune disorders such as enteropathy and type 1 diabetes. That Foxp3 is not only a marker of murine Treg but that it is also necessary for their function is a broadly accepted concept.In theory,silencing Foxp3 expression in Treg cells could lead to functional reversion of Treg cells.Owing to the intracellular localization of Foxp3,however,it is not readily accessible using traditional methods of in vivo targeting.The recent emergence of RNAi offers a novel opportunity for post-transcriptional suppression of foxp3 production in vivo. Several traits of RNAi technologies,in their current state,bode well for the potential applicability of this tool toward in vivo suppression of Treg cells activity.First,the transient nature of knockdowns mediated by pre-synthesized small interfering RNAs, would be advantageous in a clinical setting,allowing for the suppression of regulatory cells, only as long as therapeutically necessary.Second,the greatest advantage of this technique lies in its ability to repress regulatory pathways without depleting effector lymphocytes. However,the main obstacle to developing siRNA for Foxp3 silence is delivering it in vivo to Treg cells.Although transfection can deliver siRNAs locally,a systemic method to deliver siRNA to specific cells via cell-surface receptors would provide a means to introduce siRNA into desired cells to achieve maximal therapeutic benefit,decrease the amount of drug required and avoid nonspecific silencing and toxicity in bystander cells. Recently,song et al designed a protamine-antibody fusion protein to deliver siRNA to HIV-infected or envelope-transfected cells.The fusion protein was designed with the protamine coding sequence linked to the C terminus of the heavy chain Fab fragment of an HIV-1 envelope antibody,siRNA bound to fusion protein induced silencing only in cells expressing HIV-1 envelope.In this paper,we designed a mouse CD3-specific single-chain antibody（scFv） fragment-protamine fusion protein（2C11scκtp）for delivering Foxp3-specific siRNA to Treg cells.It was demonstrated that 2C11scκtp could specifically deliver siRNA to CD3⁺T lymphoma cells and mouse primary T cells in vivo and in vitro.Moreover,Foxp3-specific siRNA delivered by 2C11scκtp efficiently silenced Foxp3 expression in Treg cells. Importantly,Treg cells with down-regulated Foxp3 expression proliferated and secreted Th1-biased cytokines in response to TCR stimulation,which could not more inhibit na（?）ve T cell proliferation.Finally,siRNA delivered by 2C11scκtp do not trigger potentially toxic interferon responses.Part One:Cloning and construction of 2C11sCκand 2C11sCκtp fusion gene1.Cloning of 2C11sCκand 2C11sCκtp fusion gene.Antibody variable segments V_H and V_L were directly amplified from total mRNA of 2C11-145 hybridoma cells producing hamster anti-mouse CD3 antibody by one-step RT-PCR method.The sequences of VH and VL were verified by amplicon sequceing.After that,overlapping PCR was performed to generate 2C11 scFv.The 2C11 scFv was fused to human Cκchain and/or protamine gene to form 2C11sCκand 2C11sCκtp by second overlapping PCR.The PCR products of 2C11sCκand 2C11sCκtp were cloned into pGEM-Teasy vector to generate pGEM-2C11sCκand pGEM-2C11sCκp,and sequence verified.2.Construction of expression vector containing 2C11sCκand 2C11sCκtp gene. 2C11sCκand 2C11sCκtp gene removed from pGEM-2C11sCκand pGEM-2C11sCκtp were doublely digested by HindⅢand EcoRⅠendonucleases and cloned into pcDNA3.1（+） expression vector previously digested with the same enzyme to creat pcDNA3.1-2C11sCκand pcDNA3.1-2C11sCκtp,and sequence verified.Part Two:Expression and purification of 2C11sCκand 2C11sCκtp fusion protein1.Expression of 2C11sCκand 2C11sCκtp fusion protein.The pcDNA3.1-2C11sCκand pcDNA3.1-2C11sCκtp plasmids were transfected into CHO cells by the use of Lipofactamine 2000 according to the manufacturer’s protocol.After 72 hours,the supernatants were collected and assayed for fusion expression by flow cytometry.After one round screening,we identified two CHO cell clones which highly expressed 2C11sCκand 2C11sCκtp protein respectively.2.Purification of 2C11sCκand 2C11sCκtp protein.Supernatants were collected, and 2C11sCκand 2C11sCκtp protein were purified by Ni⁺⁺ chromatography.SDS-PAGE and western-blotting assays confirmed that purified 2C11sCκand 2C11sCκtp proteins were homogeneous,of which molecular mass were right.In vitro bind assays showed that 2C11sCκand 2C11sCκtp proteins were capable of effectively binding to CD3 molecule on lymphoma cell line YAC-1 or freshly isolated mouse lymph node cells.Part Three:Identification of potent and specific siRNAs for mouse Foxp3 gene1.Construction of cell lines stably expressing mouse Foxp3 gene.Because mouse T cells can not be easily transfected by conventional transfection methods such as liposome transfection,we need to construct a cell line stably expressing mouse Foxp3 gene and easily transfected.HELA cells were infected with MIGR1-MFoxp3 retrovirus, which expressed mouse Foxp3 and GFP gene.As a control,Hela cells were infected with MIGR1 retrovirus,which only expressed GFP gene.As shown in semi-quantitative PCR, real-time PCR and western-blotting assays,MIGR1-MFoxp3 retrovirus-infected HELA cells（HELA-MF cell） expressed high level of mouse Foxp3 mRNA and protein, compared with MIGR1 retrovirus-infected HELA cells（HELA-MR cell）.Thus, HELA-MF cells can act as an effective platform for screening mouse Foxp3 gene-specific siRNA.2.Identification of potent and specific siRNAs for mouse Foxp3 gene.We synthesized 3 siRNA from QIAGEN Corporation.After introduced into HELA-MF cells, siRNA A was shown to most potently and specifically silence mouse Foxp3 expression at the same concentration as siRNA B and siRNA C.Part Four:Delivering siRNA by 2C11sCκtp protein1.2C11sCκtp protein specifically delivered FAM-labeled siRNA to CD3⁺T lymphoma cells in vitro,and mouse T lymphocytes in vivo or in vitro.The results showed that 2C11sCκp protein could bind to mouse CD3 molecule and FAM-labeled siRNA,and specifically delivered siRNA to CD3⁺ YAC-1 and EL-4 lymphoma cell or freshly isolated mouse lymph node T cells,but not human CD3-expressing Jurkat lymphoma cells in vitro.Also,when complexes of 2C1sCκtp protein and FAM-labeled siRNA injected to C57BL/6 mice,FAM fluorescence could be easily detected in CD3⁺ T cells,but not CD14⁺ mononuclear cells,CD19⁺ B cells and CD11c⁺ DC by flow cytometry.2.Foxp3-specific siRNA delivered by 2C11sCκtp protein silenced Foxp3 expression in TGF-β-induced or naturally occurring Treg cells.Real-time PCR and Foxp3 staining by Foxp3-specific antibody showed that Foxp3-specific siRNA A delivered by 2C11sCκtp protein,but not 2C11sCκprotein significantly inhibited Foxp3 expression in TGF-β-induced or naturally occurring Treg cells at mRNA and protein level in vitro.Part Five:Phenotype and function alteration of Foxp3-silenced Treg cells1.Phenotype of Foxp3-silenced Treg cells.The results showed that Treg cells with low Foxp3 expression down-regulated CD25,CLTA4 and GITR expression and up-regulated IL-2,IL-7Rαand PDE3B expression,which genes were modulated by Foxp3 positively or negatively respectively.1.Function of Foxp3-silenced Treg cells.When stimulated by plate-coated anti-CD3 and CD28 mAbs,Foxp3-silenced Treg cells vigorously proliferated and secreted large quantities of IL-2,IFN-γand TNF-αcytokines,indicating biased toward Th1 immune response.Part Six:Interferon responses associated with application of siRNAThe in vitro and in vivo assays demonstrated that siRNA delivered by 2C11sCκtp protein did not trigger IFN-αand inflammatory cytokine TNF-αproduction,indicating no induction of nonspecific immune responses.Conclusion2C11sCκtp fusion protein with mouse CD3 targeting capacity and nucleic acid-binding activity can effective deliver Foxp3-specific siRNA to mouse CD3⁺ T cells, which resulted in efficient Foxp3 gene silence.Treg cells with down-regulated Foxp3 expression were reversed phenotypically and functionally,which have a tendency toward Th1 fate.In summary,our study may provide a promising approach for inhibiting Treg cells function and improving tumor vaccine efficacy.更多还原