【作者】 孙磊；

【导师】 陈丽；

【作者基本信息】 山东大学 ， 内科学， 2010， 博士

【摘要】 调节性T淋巴细胞(regulatory T cells, Tregs)是机体免疫系统的重要组成部分,在防止自身免疫,保持机体的自身耐受性中发挥着决定性作用。到目前为止已经发现了多种Tregs存在,其中CD4+ Treg研究最为广泛,包括CD4 +CD25+ Tregs, Tr1细胞,Th3细胞,CD4+ CD103+ T细胞和CD4+ CD25- Tregs. CD4+CD25+Treg细胞是目前研究最多的一类调节性T细胞。天然存在的CD4 +CD25+Treg (nTreg)约占CD4+细胞的5%～10%,通过细胞-细胞接触,分泌产生IL-10、TGF-β1及CTLA-4等细胞因子及共刺激分子,进而抑制T细胞、B细胞的分化及功能,是诱导和维持外周免疫耐受的重要细胞。该群细胞表达转录因子FoxP3 (forkhead box p3)、CD45RBlow、CD62L、CD103、细胞毒性T淋巴细胞抗原4 (CTLA-4)和糖皮质激素诱导的肿瘤坏死因子受体(GITR)等。CD4+CD25+Tregs作为一类调节性细胞,在抑制自身反应性细胞增殖,维持免疫环境的稳定方面起着重要作用。CD4+CD25+Treg细胞数量的减少、抑制功能的受损和(或)表面分子表达的缺陷,都可能导致自身免疫病的发生。在大量糖尿病的动物模型和1型糖尿病患者的研究中均发现CD4+CD25+Treg细胞的缺陷。另外,肿瘤患者外周血CD4+CD25+Treg细胞数量明显增多,并且在肿瘤浸润淋巴细胞或肿瘤相关淋巴细胞中,CD4+CD25+Treg细胞数量也明显增多。实验研究中也多将CD4+、CD25+、FoxP3+调节性T淋巴细胞数量的变化作为免疫激活剂和抗肿瘤治疗作用评价的观察指标之一。在同种／异种器官和／或细胞移植免疫中,CD4+CD25+ Treg细胞在免疫耐受中发挥重要的作用。Cohen等实验证明去除移植物中CD4+CD25+ T细胞加重移植物抗宿主病(GVHD),移植物中同时加入来自供鼠的新鲜分离的CD4+CD25+ T细胞减轻GVHD。转录因子Foxp3特异地高表达于CD4+CD25+ Treg细胞内,是叉头／翼状2螺旋转录因子家族的一个新成员。其最初的发现源于对免疫缺陷病Scurfy小鼠的研究。Scurfy小鼠患有一种X-连锁隐性遗传病,这种疾病由异常活化的CD4+T细胞介导,产生大量的细胞因子,造成对自身组织器官的损害。Brunkow于2001年在scurfy小鼠体内发现了Foxp3基因的突变,突变基因的蛋白质产物scurfin缺失功能性forkhead结构域,从而不能够发挥转录抑制因子的功能。类似的突变异常也出现在人类免疫失调-多发性内分泌腺病-肠病-X连锁综合征(Immunodysregulation, Polyendocrinopathy, Enteropathy, X-linked syndrom, IPEX),从而提示Foxp3基因在免疫系统稳态中的重要作用。对啮齿类动物的研究中,Foxp3对的关键作用已经得到公认,Foxp3特异的表达于调节性T细胞,并控制着调节性T细胞的发展和功能。但是在人类,Foxp3对CD4+CD25+ Treg细胞的调节作用尚存在许多争议,有研究证明在CD4+CD25-Foxp3- T细胞中诱导Foxp3表达并不能使这些细胞具备调节性T细胞的抑制功能,而且活化的非抑制性T细胞表面发现有Foxp3的表达,这就使得人们对Foxp3在Tregs中的作用产生了质疑。目前,体外扩增Tregs已经成为可能,进一步扩增的Tregs将被用于临床治疗一些自身免疫性疾病或者预防移植排斥,这样更加要求我们明确是哪个因子调控着Treg细胞的表面标志和抑制功能。目前对CD4+CD25+ T细胞介导的免疫抑制作用的效应机制还不明确,实验结果也存在着矛盾。有些实验支持CD4+CD25+ Treg细胞是通过与细胞接触发挥抑制作用；另一些研究表明CD4+CD25+ T细胞也可通过分泌细胞因子(IL-10,TGF-β)而介导抑制效应。我们以前的研究证实,在异种免疫反应中,扩增Tregs增强的抑制功能伴随着细胞因子IL-10的增加,表明了IL-10在异种免疫反应中有重要作用,但具体的作用机制仍不明确。但目前关于IL-10在Treg介导的抑制作用中究竟有无作用仍不明确。在本研究利用RNA干扰技术分别抑制Foxp3和IL-10基因表达,观察Foxp3和IL-10是否有助于维持体外扩增的人天然CD4+CD25+调节性T细胞的抑制性表型标志、功能基因及Treg介导的对异种／同种异体刺激的效应性T细胞的增殖反映的抑制作用。第一部分Foxp3在人天然CD4+CD25+调节性T细胞中的作用研究目的：通过抑制Foxp3基因表达,观察其对CD4+CD25+调节性T细胞表型及抑制功能的影响。研究方法：1.调节性T细胞的分离和体外培养。根据人CD4+CD25+淋巴细胞分离试剂盒分离人Tregs。并按照实验室成熟的扩增方法培养细胞2-3周后用于下一步试验。2. SiRNA转染：公司合成的Foxp3 siRNA与脂质体转染复合体转染至扩增的Tregs。非特异性siRNA转染的Tregs和未传染的Tregs作为对照组。3.采用实时定量PCR(realtime-PCR)和Western blotting、免疫荧光法分别检测Foxp3基因和蛋白的表达。实时定量PCR检测Tregs功能相关基因表达。4.流式细胞仪观察Tregs表型的改变。5. ELISA测定Tregs及活化的效应性CD4+CD25- T细胞分泌的细胞因子水平。6.增殖抑制试验：各组Tregs与同一个体的效应性CD4+CD25- T细胞,及其异种(猪)或同种异体(人)的外周血单个核细胞(PBMC)共培养5天,观察Tregs对效应性CD4+CD25- T细胞增殖的抑制功能变化。7.细胞毒性试验：分离以上共培养体系中的效应性CD4+CD25- T细胞,再与异种或同种异体PBMC共培养后,观察目标PBMC的调亡情况。7.统计学分析。结果：1.体外培养人Tregs 2-3周后可达新鲜Tregs数目的3500倍,流式细胞仪检测其CD4+CD25+双阳性细胞纯度达96%。2.用最适合的转染条件,本试验达到60-70%转染效率。3. Foxp3基因表达下调61%; Western blotting、免疫荧光结果提示了蛋白的表达下降；Foxp3基因抑制导致Tregs功能性相关基因表达下降。4. Foxp3基因抑制引起Tregs表型标志表达改变。5. Foxp3基因抑制导致Tregs分泌抑制性细胞因子TGF-β、IL-10减少,Tregs对效应性CD4+CD25- T细胞分泌的IFN-γ、IL-2的抑制作用减弱。6. Foxp3基因抑制导致不论是在异体或同种异体刺激实验中,Tregs对效应性CD4+CD25- T细胞增殖的抑制作用显著降低。结论：Foxp3 siRNA成功转染到人天然CD4+CD25+调节性T细胞内,并引起了基因抑制,蛋白表达减少。Foxp3基因抑制导致了Tregs表型标志表达改变,相关功能基因表达下调,对抑制效应性T细胞增殖的功能下降,抑制性细胞因子分泌下降,以及对抑制效应性T细胞产生的效应性细胞因子的作用减弱。通过以上结果证实不论在异种免疫反应还是同种免疫反应中,Foxp3是人天然CD4+CD25+调节性T细胞的关键调节因子。第二部分IL-10在人天然CD4+CD25+调节性T细胞中的作用研究目的：通过抑制IL-10基因表达,观察其对CD4+CD25+调节性T细胞表型及抑制功能的影响。研究方法：1.调节性T细胞的分离和体外培养。2. siRNA转染：公司合成的IL-10 siRNA与脂质体转染复合体转染至扩增的Tregs;非特异性siRNA转染的Tregs和未传染的Tregs作为对照组。3.采用实时定量PCR(realtime-PCR)和Western blotting分别检测IL-10基因和蛋白的表达。实时定量PCR检测Tregs功能相关基因的表达。4.流式细胞技术观察Tregs表型的改变。5.ELISA测定Tresg及活化的效应性CD4+CD25- T细胞分泌的细胞因子。6.增殖抑制试验：各组Treg与同一个体的效应性CD4+CD25- T细胞,及其异种(猪)或同种异体(人)的单个核细胞共培养5天,观察Treg对效应性CD4+CD25- T细胞增殖的抑制功能变化。利用transwell小室非接触式共培养的方法将以上细胞共培养后同样观察效应性CD4+CD25- T细胞增殖变化。在以上共培养体系中加入重组人白细胞介素10(rhIL-10),观察其对效应性CD4+CD25- T细胞增殖的作用及对减低的Treg抑制功能的逆转作用。7.统计学分析。结果：1.体外培养人Tregs 2-3周后可达新鲜Treg数目的3500倍,流式细胞仪检测其CD4+CD25+双阳性细胞纯度达96%。2.用最佳的转染条件,本试验可达到60-70%转染效率。3.IL-10基因表达下调68.9%; Western blotting结果提示了蛋白的表达下降；IL-10基因抑制没有影响Tregs功能性相关基因表达。4.IL-10基因抑制未引起Treg表型标志表达改变。5.IL-10基因抑制导致Treg分泌细胞因子IL-10减少,对TGF-β分泌没有影响。异体或同种异体刺激的效应性CD4+CD25- T细胞分泌的效应因子IFN-γ、IL-2的浓度未见改变。6.IL-10基因抑制导致异种抗原刺激实验中,Tregs对效应性CD4+CD25- T细胞增殖的抑制作用显著降低,但这种改变未见于同种异体抗原刺激试验中。7.RhIL-10使异种抗原刺激试验中降低的Tregs抑制功能部分恢复,对同种异体抗原刺激试验中Tregs功能没有影响。结论：IL-10 siRNA成功转染到人天然CD4+CD25+调节性T细胞内,并引起了基因抑制,蛋白表达减少。IL-10基因抑制没有影响Tregs表型标志表达,相关功能基因表达,Tregs对效应性T细胞分泌效应因子的抑制作用以及Tregs自身分泌抑制性细胞因子TGF-β。功能实验表明IL-10基因抑制降低了Tregs对异种抗原刺激的效应性CD4+CD25-T细胞增殖的抑制作用,而对同种异体抗原刺激的效应性CD4+CD25-T细胞的增殖抑制功能没有改变。通过以上结果证实IL-10在异种免疫而不是同种免疫中,影响人天然CD4+CD25+调节性T细胞的功能,表明在异种免疫反应中,Tregs的抑制作用除了通过细胞接触,细胞因子IL-10的分泌也起着重要的作用。而在同种异体免疫反应中,细胞接触才是主要的作用机制。更多还原

【Abstract】 Naturally occurring CD4+CD25+ regulatory T cells (nTregs), represent 5-10% of total CD4+ T cells in the peripheral circulation. They play an important role in controlling immune responses, silencing self-reactive T cells and mediating immunological self-tolerance and immune homeostasis. Tregs have been shown to be capable of suppressing CD4+CD25-effector T cell-mediated cellular responses through two possible ways:contact dependent and cytokine mediated mechanisms.In addition to high level expression of CD25, Tregs constitutively express a distinct set of cell surface and intracellular molecules. Predominant amongst is the the transcription factor Foxp3 (forkhead box P3) which is required for Treg development and function, and is sufficient to induce a Treg phenotype in murine CD4+CD25- T cells. Mutations in Foxp3 cause severe, multi-organ autoimmunity in both humans and mice. In mice Foxp3 has been shown to be both necessary and sufficient for the generation of suppressive CD4+CD25+ Tregs. It is a definitive marker of regulatory function because forced expression of Foxp3 in Foxp3- T cells leads to the acquisition of regulatory function and non-regulatory cells do not express Foxp3. Similarly in mice, Foxp3 expression was not upregulated in CD4+CD25-Foxp3- effector T cells following TcR stimulation. This combination of findings had led to the proposal that Foxp3 is a lineage specific marker of Tregs in mice. In humans T cells the situation is not so clear cut. Foxp3 expression is clearly important for the maintenance of self-tolerance as mutations in Foxp3 transcription leads to a rare X-linked/immune/polyendcrinopathy/enteropathy syndrome (IPEX) which manifests as a fatal disease with multiple autoimmune syndromes. However in human T cell biology, expression of Foxp3 is not always associated with suppressor function. Induction of Foxp3 in CD4+CD25- T cells with TCR stimulation and TGF-p did not result in anergic or suppressive activity. Unlike in their murine counterparts ectopic expression of Foxp3 in human CD4+ T cells was not an effective method for generating suppressive Treg in vitro. Furthermore activated non suppressive T cell clones express significant levels of Foxp3 suggesting that Foxp3 is not sufficient for Treg function and may also be implicated in T cell activation. These studies question the relevance of ongoing Foxp3 expression once Treg reach the peripheral. Naturally occurring CD4+CD25+ Treg are derived in the thymus and it has been shown that Foxp3 is an important element that identifies natural Treg during thymus selection. Once in the periphery these cells take on a regulatory role. However it is not clear from the published data that Foxp3 is an essential element in the maintenance of their regulatory phenotype. Substantial expansion of naturally occurring CD4+CD25+ Treg in culture is now possible and expanded Treg has been proposed as a therapeutic strategy for the treatment of autoimmune diseases and prevention of allograft rejection. Essential to this strategy is the development of cell markers whose expression and level of expression would be predictive of suppressive function. Hence it is important to determine whether Foxp3 is a transcription factor whose expression is essential for human Treg function and whether the level of Foxp3 suppression correlates with suppressive activity.CD4+CD25+ Tregs mediated suppression through cell contact dependent and /or cytokine mediated mechanisms. Our previous study showed the enhanced suppression shown by expanded Tregs was associated with the enhanced production of the suppressive cytokine IL-10, suggesting that IL-10 may be involved mechanistically in the regulation of xenogeneic response. However, the precise molecular mechanism by which Tregs suppress the activation and proliferation of other T cells is currently controversial. Some studies support the role of cytokines such as IL-10 and others emphasize the contribution of cell-to-cell cognate interactions with effector T cells on APC. Although many studies have reported that the in vitro suppression of alloimmune responses by natural Tregs was IL-10 independent and cell-cell contact dependent, the importance of IL-10 in expanded human Treg-mediated suppression of xenoresponse remains to be investigated.Gene targeting methods represent an important approach to study the function of individual single genes. RNA interference (RNAi) by small interfering RNA (siRNA) has been shown to specifically knock down a gene of interest in numerous cell types. Since siRNA can be introduced into mammalian cells by various methods, siRNA-mediated gene silencing has become a powerful tool to investigate gene function both in vitro and in vivo. In this study, a lipid-based siRNA delivery system was used to target and silence Foxp3 and IL-10 of in vitro expanded human Tregs. The aim was to determine the importance of Foxp3 and IL-10 expression for maintaining human Treg phenotype and their suppressive function in the human xeno and alloimmune response. Aims:Foxp3 is required for CD4+CD25+ T cells (Tregs) development and murine Treg function. The precise role of Foxp3 in regulating natural human Treg function remains to be defined by direct gene targeting approach.Methods:In vitro expanded human Tregs were transfected with Foxp3 siRNA by lipofectamine 2000. Foxp3 knockdown and Treg phenotype were evaluated by FACS, real-time PCR, Western-blotting and immunofluorescence. Treg suppressive function assay was performed by culturing human CD4+CD25- T cells with xeno- and allo PBMC in the presence or absence of Tregs in a coculture or transwell system for 5 days prior to assessment of proliferation and xeno and allo-cytotoxicity of CD4+CD25- T cells. The production of effector cytokines by xeno- and allo-reactive CD4+CD25- T cells as well as suppressive cytokines by Tregs in their cocultures was also examined.Results:Foxp3 knockdown resulted in impaired Treg phenotype and nonresponsiveness, downregulated expression of Treg function molecules, and reduced production of suppressive cytokines. These changes were correlated with diminished Treg-mediated suppression of CD4+CD25- T cells proliferation, xeno-and allo-cytotoxicity and effector cytokine production in xeno- and allogeneic response.Conclusions:Foxp3 expression is absolutely required for maintaining natural human CD4+CD25+ T cells phenotype and suppressive function in xeno- and alloimmune response in vitro. Aims:Cellular rejection of xenografts is predominantly mediated by CD4+ T cells. CD4+CD25+ T regulatory (Tregs) have been shown to be able to suppress CD4+ T cell-mediated anti-pig xenogeneic response in vitro. However, the precise mechanism(s) involved remain to be identified. In this study, we investigated whether IL-10 is required for human Tregs to suppress xenogeneic response in vitro.Methods:In vitro expanded human natural Tregs were transfected with IL-10 or non-specific siRNA by Lipofectamine 2000. Transfected Tregs were then analyzed for IL-10 gene and protein expression as well as their phenotypic characteristics. Human CD4+CD25- T cells were stimulated with allo or pig PBMC in the presence or absence of Tregs in a coculture or transwell system for evaluation of Treg suppressive activity. The production of effector cytokines by xeno-reactive CD4+CD25- T cells as well as suppressive cytokines by Tregs in their cocultures was examined by ELISA.Results. SiRNA mediated IL-10 knockdown resulted in substantially reduced production of IL-10 by human Tregs, leading to impaired Treg-mediated suppression of xeno-but not allo-reactive CD4+CD25- T cell proliferation. IL-10 knockdown had no impact on Treg phenotype and their production of suppressive cytokines.Conclusions:This study demonstrates that IL-10 is required for expanded human Tregs to suppress xeno-but not allo-immune response in vitro.更多还原