【作者】 刘春梅；

【导师】 张利宁；

【作者基本信息】 山东大学 ， 免疫学， 2010， 博士

【摘要】 目的调节性T细胞(regulatory T cell, Treg)是具有免疫负调控作用的T亚群,它在维持外周免疫耐受,控制自身免疫性疾病、过敏反应和诱导移植耐受等方面发挥重要的保护作用,成为近几年来免疫学的研究热点之一。目前,在调节性T细胞的研究中,对CD4+CD25+调节性T细胞(CD4+CD25+Treg)的特性研究得较多。已知CD4+CD25+Treg主要有两大类：一类是天然CD4+CD25+调节性T细胞(natural occurring regulatory T cell, nTreg),在胸腺中产生,存在于正常机体内,在小鼠脾脏和人外周血中占CD4+T细胞的5-10%；另一种是诱导性CD4+CD25+调节性T细胞(inducible regulatory T cell, iTreg),在外周合适的条件(如TCR刺激和细胞因子TGF-β或IL-10)下可以由CD4+CD25-T细胞转化而来。iTreg与nTreg具有相同的表面标记和抑制功能,主要标记有CTLA-4、GITR和Foxp3,其中转录因子Foxp3是目前广为应用的Treg最重要的特异性标志,不仅是后者分化发育的标志,更是其功能性标志。近年来,利用Treg的免疫抑制效应控制机体过度或不适的免疫应答进而防治疾病成为免疫治疗的新策略。然而,有限的Treg数量限制了Treg的临床应用,因此寻找扩增或诱导CD4+CD25+Treg的有效方法,并深入探索其机制,成为推动Treg临床应用的前提和基础。树突状细胞(dendritic cells, DCs)是机体重要的抗原递呈细胞。已有的研究显示DCs不仅在效应性T细胞的活化和增殖方面发挥重要作用,而且具有扩增和诱导Treg产生的作用。负载抗原的DCs在外源性IL-2存在的情况下可以扩增nTreg；在细胞因子如TGF-β、IL-10或Vitamin D等存在的情况下,DCs可以将CD4+CD25-T细胞诱导转化为Foxp3+Treg。然而,在这些诱导和扩增的实验方法中,CD4+CD25-T细胞和CD4+CD25+T细胞都需要从CD4+T细胞群体中分离纯化出来,然后在体外加入不同的外源性细胞因子进行诱导或扩增,使得整个实验过程变得复杂化而且成本也相对较高。既然CD4+T细胞群体不仅含有CD4+CD25+的nTreg,也含有CD4+CD25-的iTreg前体细胞,那么我们是否可以不加外源性细胞因子,不需分离纯化而利用DCs直接刺激CD4+T细胞群体产生Treg?故本研究的目的一是探索DCs是否可直接从CD4+群体中产生Treg,哪种类型DCs(同种同型与同种异型、未成熟与成熟)作用最强,机制如何?进而建立简便、价廉的产生Treg的方法,推动Treg的临床应用。DCs在体内分化、成熟和发挥作用时常常处于不同的微环境,氧压是微环境中的重要因素。生理或病理情况下组织细胞经常处于不同氧压的微环境,如在动脉栓塞、器官移植过程中,早期局部组织是处于一种远远低于大气氧压的低氧或乏氧状态。当外界因素撤除或手术致使原本处于低氧状态的组织血流通畅,氧供随之恢复,即发生再氧合。这种乏氧-再氧合的病理生理过程,经常见于缺血再灌注损伤。那么不同氧压对DCs的分化成熟及功能有何影响?不同氧压微环境中产生的DCs对T细胞分化特别是对Treg分化有何影响?目前都尚不清楚。我们在前期研究中发现乏氧可抑制DCs的表型成熟和对T细胞的刺激功能。故本课题的目的二是在前期研究的基础上,进一步研究乏氧-再氧合条件下小鼠DCs表型及功能变化,分析再氧合DCs对Th细胞及调节性T细胞的分化影响,并对其内在分子机制进行探讨。这为解释缺血再灌注损伤的发病机制,控制器官移植的排斥反应以及肿瘤免疫治疗奠定一定的基础。总之,本课题包括两部分内容：一、同种异型DCs在调节性T细胞产生中的作用及其机制研究；二、再氧合DCs在调节性T细胞产生中的作用及其机制研究。第一部分同种异型DCs在调节性T细胞产生中的作用及其机制研究方法一、小鼠骨髓来源DCs的诱导1.分离C57BL／6或BALB／c小鼠的骨髓细胞,利用细胞因子GM-CSF和IL-4诱导BM-DCs。2.隔2天半量换液,第6天时给予LPS(1μg／ml)刺激24h,以诱导成熟。二、自体DCs或同种异型DCs刺激CD4+T细胞产生Treg的能力1.用免疫磁珠分离纯化BALB／c小鼠脾脏CD4+T细胞,分别与自体(同基因型)DCs或同种异型DCs共培养。2.流式细胞术(FCM)检测培养体系中CD4+CD25+Treg的比例。三、人外周血单个核细胞来源的DCs刺激CD4+T细胞产生Treg的能力1.获取不同人外周血10-15m1,分离单个核细胞(PBMC)。2.免疫磁珠法分离CD4+T细胞。3.PBMC静置2h后去除未贴壁细胞,给予重组人GM-CSF和IL-4诱导成DCs。4.将不同人的CD4+T细胞与DCs做同种异型或自体的细胞共培养。5.流式细胞术(FCM)检测培养体系中CD4+CD25+Foxp3+Treg的比例。四、增殖抑制试验检测CD4+CD25+Treg的抑制功能1.将第一步中同种异型混合淋巴细胞培养产生的Treg (generated Treg)纯化后作为抑制细胞,同种异型DCs作为刺激细胞,标记了CFSE的CD4+CD25-T细胞作为反应细胞,反应细胞：抑制细胞按照不同比例共培养。2.新鲜分离的天然Treg作为实验阳性对照,流式检测反应细胞的增殖情况。五、细胞因子在CD4+CD25+Treg产生中的作用(一)探索IL-2在同种异型DCs刺激Treg产生中的作用1.分离小鼠脾脏CD4+CD25+T细胞,标记CFSE,与同种异型DCs共培养,并给予IL-2刺激,5天后FCM检测同种异型DCs对CD4+CD25+T细胞的增殖情况。2.分离CD4+CD25+T细胞,标记CFSE,然后与CD4+CD25-T细胞按照原来比例混合成CD4+T细胞整体,与同种异型DCs共培养,培养5天后收集上清,ELISA检测IL-2的水平。3.IL-2阻断试验：上述体系中加入抗IL-2的中和性抗体,对照组加入同型对照IgG,5天后流式检测CD4+CD25+T细胞的增殖情况。(二)探索TGF-p在同种异型DCs刺激Treg产生中的作用1.分离小鼠脾脏CD4+CD25-T细胞,标记CFSE,与同种异型DCs共培养,不同时间后流式检测Foxp3的表达和CFSE的情况。2.收集不同时间的上清,ELISA检测TGF-p水平。3.TGF-p阻断试验：上述体系中加入不同浓度的TGF-p受体Ⅰ激酶抑制剂Ⅱ(ALK-5抑制剂Ⅱ),培养5天后检测Foxp3的表达。结果一、在无外源性细胞因子存在的情况下,小鼠同种异型DCs可刺激CD4+T细胞高效产生Treg,但自体DCs无此作用新鲜纯化的CD4+T细胞纯度大于95%,其中CD4+CD25+Foxp3+T细胞的比例小于10%。自体BM-DCs与抗CD3抗体联合刺激CD4+T细胞3-5天后,T细胞大量活化,CD4+CD25+T细胞的比例明显增加(>90%),但其中Foxp3+T细胞的比例仍然小于10%。然而,用同种异型BM-DCs刺激CD4+T细胞后,尽管活化的CD4+CD25+T细胞的比例较自体DCs刺激体系低(50%-70%),然而其中Foxp3+T细胞的比例高达80%以上。随着培养时间的延长,体系中CD4+CD25+Foxp3+T细胞的比例增加5-7倍。值得注意的是,未成熟同种异型DCs和成熟同种异型DCs刺激CD4+T细胞产生Treg的能力无明显差异。二、人同种异型DCs在没有外源性细胞因子的情况下,同样可以增加CD4+T细胞中Treg的比例为了进一步证明同种异型DCs刺激Treg产生的能力,我们从人PBMC中用重组人GM-CSF和IL-4分别诱导同种异型或自体DCs,与CD4+T细胞共培养,流式检测体系中CD4+CD25+Foxp3+Treg的比例。结果显示,自体DCs活化的CD4+CD25+T细胞中Foxp3+细胞表达在10%左右,而同种异型DCs活化的CD4+CD25+T细胞中Foxp3+Treg的比例高达40%左右,其作用与小鼠的同种异型DCs相似。三、同种异型DCs产生的Treg在体外具有抑制功能为了进一步证明同种异型DCs刺激产生的Treg (generated Treg)在体外是否保持其特有的抑制功能,我们将小鼠同种异型DCs刺激产生的CD4+CD25+T细胞纯化后作为抑制细胞,按照不同比例与反应细胞CD4+CD25-T细胞共培养,一定时间后流式检测反应细胞的增殖情况,结果发现,同种异型DCs刺激产生的Treg在体外具有明显的抑制功能,与新鲜分离的nTreg一致。四、内源性IL-2和TGF-β在同种异型DCs刺激CD4+T细胞产生Treg中发挥重要作用为了探讨同种异型DCs刺激CD4+T细胞高效产生Treg的机制,我们进一步研究了内源性IL-2和TGF-β在同种异型DCs刺激CD4+T细胞产生Treg中的作用。1.IL-2在同种异型DCs刺激Treg扩增中发挥重要作用1)用CFSE标记和流式细胞术证明在外源性IL-2存在的情况下,DCs可以有效的扩增nTreg分离纯化CD4+CD25+T细胞后用CFSE标记,然后与同种异型BM-DCs共培养,并给予外源性IL-2刺激,5天后流式检测细胞增殖情况,发现同种异型DCs可以有效扩增CD4+T细胞中的nTreg。2)用抗体阻断的方法证明内源性的IL-2在同种异型DCs刺激Treg扩增中发挥重要作用分离纯化CD4+CD25+T细胞后用CFSE标记,然后与CD4+CD25-T细胞按照原有的比例混合,与同种异型BM-DCs共培养,ELISA检测上清中细胞因子IL-2的水平,发现同种异型DCs刺激组中IL-2的水平明显高于对照组。为了进一步验证体系中IL-2的作用,在如上体系中同时加入抗IL-2的中和抗体,5天后收集细胞,流式检测细胞CFSE增殖情况,发现与对照组IgG相比,IL-2阻断抗体可以明显抑制CD4+T细胞群体中CD4+CD25+T细胞的增殖。这说明同种异型DCs对CD4+CD25+Treg的有效扩增依赖于体系中的内源性IL-2。2.TGF-β在同种异型DCs诱导Treg产生中发挥重要作用1)用CFSE标记和流式术证明同种异型DCs可以诱导Treg的产生新鲜分离小鼠脾脏CD4+CD25-T细胞,用CFSE标记,然后与同种异型BM-DCs共培养不同时间后,收集细胞,流式检测Foxp3的表达情况。结果显示,第5天时,同种异型DCs刺激的CD4+CD25-T细胞与新鲜分离的CD4+CD25-T细胞相比,Foxp3+T细胞比例明显增加。随着培养时间的延长,Foxp3+T细胞比例逐渐增加。当培养到第13天时,Foxp3的表达达到第5天时的两倍。这表明在没有外源性TGF-p存在的情况下,同种异型DCs可以诱导CD4+CD25-T向CD4+CD25+Foxp3+T细胞转化。2)同种异型DCs刺激体系中存在内源性TGF-β的产生收集培养不同时间的上清,ELISA检测上清中TGF-p的水平,结果提示,培养到第5天时,总的TGF-p水平明显增加。并随着培养时间的延长,其水平逐渐升高。当培养第13天时,TGF-β水平达到第5天时的2-3倍。3)用抑制剂阻断的方法证明内源性TGF-β在同种异型DCs刺激Treg产生中发挥重要作用为进一步验证产生的TGF-p在CD4+CD25+Foxp3+T细胞诱导中的作用,我们在如上体系中加入不同浓度的TGF-p受体Ⅰ激酶抑制剂Ⅱ(ALK-5抑制剂Ⅱ),定时间后,流式检测Foxp3的表达。结果显示,体系中CD4+Foxp3high T细胞的百分比和绝对数随着ALK-5抑制剂Ⅱ浓度的增加而逐渐降低,且CD4+Foxp3dim T细胞的百分比和绝对数显著下降,与加入的ALK-5抑制剂Ⅱ浓度具有明显的剂量依赖关系。所以我们推测CD4+Foxp3dim T细胞可能是转化的Foxp3+T细胞的潜在群体,且TGF-p在转化过程中发挥着重要的作用。结论1.小鼠同种异型BM-DCs和人PBMC诱导的同种异型DCs,在无外源性细胞因子的情况下,都可以增加CD4+T细胞中Treg的比例,产生的Treg在体外对T细胞增殖具有抑制功能。2.同种异型BM-DCs刺激的体系中,内源性IL-2和TGF-p在Treg的产生中发挥着重要的作用。创新性和意义1.首次发现同种异型BM-DCs在没有外源性细胞因子的情况下可以刺激CD4+T细胞高效产生Treg,并探讨了其机制。2.首次将CD4+T细胞作为一个整体来寻找获得足够数量Treg的方法,为优化后续研究的实验方法、制定Treg的临床治疗策略提供了一定的理论依据。研究的局限性1.同种异型BM-DCs刺激CD4+T细胞产生Treg的具体内在分子调控机制有待于进一步深入研究。2.所获得Treg应用于临床,其条件仍需进一步优化。第二部分再氧合DCs在调节性T细胞产生中的作用及其机制研究方法一、再氧合对DCs的表型影响1.分离C57BL／6小鼠骨髓细胞,分别在常、乏氧条件下利用GM-CSF和IL-4诱导DCs,并给予LPS刺激。2.对乏氧条件下分化的DCs进行再氧合不同时间。3.流式检测再氧合DCs表面协同刺激分子及MHC-Ⅱ类分子的表达。二、再氧合对DCs的Th细胞增殖、分化的影响1.分离CD4+T细胞并标记CFSE,与再氧合的同种异型DCs或自体DCs共培养,后者给予抗CD3抗体刺激。2.流式检测再氧合DCs刺激CD4+T细胞增殖的能力。3.收集细胞,流式检测再氧合DCs刺激CD4+T细胞的分化情况。三、再氧合影响DCs表型及功能的分子机制研究1.将乏氧下分化的DCs进行再氧合,Real time PCR的方法检测其表达四种腺苷受体的mRNA水平。2.以不同腺苷受体激动剂处理再氧合的DCs,流式检测其表型的变化。3.流式检测不同浓度A2AR腺苷受体激动剂对再氧合的DCs表型的影响。4.ELISA检测A2AR激动剂对再氧合DCs的细胞因子表达的影响。结果一、再氧合可逆转乏氧对DCs成熟表型的抑制为了研究再氧合对DCs表型的影响,我们在乏氧条件下诱导DCs后对其进行再氧合不同时间,流式检测细胞表面分子的表达。结果显示乏氧抑制DCs协同刺激分子CD80、CD86以及MHC-Ⅱ类分子的表达。再氧合6小时后其表达百分比和荧光强度均明显上调,且荧光强度随再氧合时间的延长而逐渐增加,再氧合24h、48h可达到高峰。这提示再氧合可以逆转乏氧对DCs表型的抑制,促进DCs向成熟方向分化。二、再氧合DCs刺激CD4+T细胞增殖的能力较乏氧DCs明显增强无论是在同种异型混合淋巴细胞反应体系中,还是在CD3单抗刺激的体系中,乏氧DCs刺激CD4+T细胞增殖的能力与常氧DCs相比非常弱。再氧合后,其诱导CD4+T细胞增殖的能力明显提高。这提示无论针对同种异型抗原,还是自体抗原,再氧合DCs均具有很强的抗原递呈功能。三、再氧合DCs降低Treg的数量而上调Th17和Thl细胞应答进一步我们检测了同种异型混合淋巴细胞反应体系中,再氧合的DCs对CD4+T细胞分化的影响。结果显示,再氧合DCs诱导产生的CD4+CD25+Foxp3+Tregs,与常氧成熟和未成熟DCs相比,明显减少；相反,再氧合DCs可显著诱导CD4+T细胞向IL-17+Th17细胞分化,其比例明显高于未成熟DCs,甚至常氧成熟DCs刺激产生的IL-17+Th17细胞。同时,IFN-γ+Th1细胞的比例和上清中IFN-γ的水平也明显高于常氧成熟和未成熟DCs刺激组。我们进一步研究了再氧合DCs的细胞因子分泌情况。结果证实再氧合24h的DCs上清中IL-6水平显著升高,而TGF-p的水平则与乏氧组相比明显降低。这提示再氧合DCs分泌的细胞因子更有利于使CD4+T细胞向Th17方向分化。总之,这些结果表明再氧合DCs具有很强的驱动Th17和Thl细胞介导的炎性反应的能力。四、腺苷-A2AR通路在DCs乏氧-再氧合过程中发挥着重要的作用1.再氧合可上调腺苷受体A2AR在DCs的表达,而对A1R、A2BR及A3R的表达无明显影响为了研究腺苷通路是否参与乏氧-再氧合对DCs表型和功能的影响,我们首先采用Real time PCR方法检测了再氧合DCs的四种腺苷受体的mRNA表达水平,结果显示,与常氧及乏氧DCs相比,再氧合DCs表达的A2AR水平呈现明显升高趋势；而各组之间A1R、A2BR及A3R的表达则无明显变化。2.A2AR受体激动剂可抑制再氧合DCs成熟相关表型的上调为了进一步确认腺苷通路在乏氧-再氧合对DCs表型和功能的影响,我们在将乏氧下分化的DCs进行再氧合时,给予不同腺苷受体激动剂刺激,流式检测DCs的表型。筛选结果提示,与对照组(再氧合组)相比,A2AR受体选择性激动剂CGS21680可以抑制再氧合对乏氧DCs表型的上调作用,即抑制MHC-Ⅱ和协同刺激CD80、CD86的表达。而A1R、A2BR、A3R的激动剂则没有此功能。3.A2AR激动剂对再氧合DCs表型的抑制具有浓度依赖性体外进一步检测了不同浓度的A2AR受体选择性激动剂CGS21680对再氧合DCs表型的影响。流式结果显示,随着CGS21680浓度的增加,协同刺激分子CD80、CD86及MHC-Ⅱ表达的平均荧光强度逐渐降低,与对照组相比有显著性差异,这进一步提示A2AR通路确实在DCs的乏氧-再氧合过程中发挥着重要的作用。4.A2AR激动剂可明显抑制再氧合DCs促炎细胞因子产生,而上调抑炎细胞因子产生我们进一步检测了不同腺苷受体激动剂对再氧合DCs的细胞因子分泌情况,ELISA结果显示A2AR选择性激动剂CGS21680可以明显抑制再氧合DCs炎性细胞因子IL-1p、IL-6和TNF-a等的分泌,而上调其免疫抑制因子TGF-p的分泌。这说明腺苷受体通路参与再氧合逆转乏氧对DCs功能的影响,A2AR受体通路可能是再氧合上调DCs表型和功能的一个重要调节分子。结论一、再氧合可以逆转乏氧对DCs成熟表型及功能的抑制作用1.再氧合可明显促进乏氧下分化DCs的表型成熟。2.再氧合可明显促进DCs对同种异型及同种同型抗原的递呈功能,明显增强DCs对CD4+T细胞增殖的能力,可降低Treg的数量而促进CD4+T细胞向Th17和Th1方向分化。二、腺苷受体通路参与再氧合逆转乏氧对DCs表型及功能的影响1.再氧合DCs表达高水平腺苷受体A2AR。2.A2AR激动剂CGS21680抑制再氧合逆转乏氧对DCs表型的能力,并具有浓度依赖性。3.A2AR激动剂CGS21680抑制再氧合DCs炎性细胞因子的表达。创新性和意义1.首次发现再氧合可明显逆转乏氧对DCs的成熟表型及功能的影响,这为解释缺血再灌注损伤的发病机制提供有力的证据,证明乏氧-再氧合可能是导致缺血再灌注损伤的内在机制之一。2.首次发现A2AR腺苷受体通路抑制再氧合对乏氧DCs表型及功能的逆转作用,为寻找从免疫学角度治疗缺血再灌注损伤的新策略提供新思路。研究的局限性1.腺苷通路参与再氧合促进乏氧DCs表型及功能成熟的内在分子机制仍需进一步研究。2.腺苷受体激动剂应用于治疗IRI的体内实验有待于进一步补充。更多还原

【Abstract】 Object:Regulatory T cells (Tregs) have been described as the most potent immunosuppressive cells and play a critical role in maintaining peripheral immunologic tolerance and controlling alloimmune disorders, infections, allergy, and transplantation tolerance. Among the Tregs, the characteristics of CD4+CD25+ regulatory T cells are well illuminated. CD4+CD25+ Tregs have two major subtypes, naturally occurring Tregs (nTregs) and induced Tregs (iTregs). The nTregs arise from the thymus and represent 5-10% of CD4+ T cells in mice and in human blood. The iTregs can differentiate from the CD4+CD25-Foxp3- precursors in the periphery outside the thymus under some conditions, such as TCR stimulation plus IL-10, or TGF-β.The iTregs had the same suppressive function as nTregs, both expressed CTLA-4, GITR and Foxp3. The transcript factor Foxp3, as the specific marker of Tregs, plays a critical role in the differentiation, development and function of Tregs. The utilization of Tregs to prevent or cure diseases by controlling inappropriate immune response has been a new strategy for immunotherapy. The adoptive transfer of Tregs was found to control experimental alloimmune diseases, and prevent allograft rejection in rodents. But the limited number of Tregs was the obstacle for their clinical application. So it’s necessary to explore new methods to expand or induce CD4+CD25+ Tregs to facilitate the clinical application.Dendritic cells (DCs) are professional antigen presenting cells (APCs). The accumulating data showed that, DCs play the important role not only in the activation and proliferation of effector T cells, but also in both expansion of nTregs and induction of iTregs. On one hand, the antigen-loaded DCs together with IL-2 are able to expand nTregs. On the other hand, DCs with TGF-P, IL-10 or Vitamin D can induce naive CD4 CD25-T into Foxp3+Tregs. However, in previous protocols for the expansion or induction of Tregs, CD4+CD25-T or CD4+CD25+T cells needed to be purified from total CD4+T cell population and exogenous cytokines were required, which were costly and relatively complicated. Since total CD4+T cells contain both nTregs and CD4+CD25- precursors of iTregs, so whether is it feasible to generate Tregs directly from total CD4+ population stimulated by DCs without separation from each other and exogenous cytokines? Therefore, the first purpose of this study is to explore whether it is feasible or not to generate Tregs directly from total CD4+ population stimulated by DCs, which type of DCs (autogeneic or allogeneic, immature or mature DCs) work well, what is the mechanism? It is important to set up simpler and less-costly methods of generating Tregs to facilitate their clinical application.When DCs happen to differentiate, mature, and execute their responsibility, they are often in different microenvironment, in which oxygen tension is the important factor. While, under most physiological or pathological conditions, tissues and cells are always in different oxygenic microenvironment. For example, in the process of arterial embolism or organ transplantation, hypoxia was always found during the early phage. But after removing of the external factor or operation, the following return of blood flow and oxygen delivery in the hypoxic tissues lead to rapid change of oxygen tension. This reoxygenation (re-exposure to saturated oxygen) is a very important patho-physiological process, and it often exists ischemia-reperfusion injury (IRI). However, it is unclear whether the oxygenic microenvironment affects the differentiation and function of DCs, whether the DCs differentiated under different oxygenic microenvironment affect the T cell differentiation including the generation of Tregs. In the previous study, we found that hypoxia can inhibit the phenotypic maturation and function of DCs to stimulate T cell proliferation. Therefore, the second purpose of this study is to further explore the effect of hypoxia-reoxygenation (from hypoxia to normoxia) on the phenotype and function of mouse DCs and to analyze the effect of reoxygenated DCs on the T cell differentiation including the generation of Tregs on the basis of previous study. Also the internal molecular mechanism is researched. It is much helpful for explaining the pathogenesis of IRI, graft rejection in organ transplantation, and the immunotherapy for tumor.In a word, this project contains two parts, the first part is "The effect of allogeneic DCs on the generation of regulatory T cells and its mechanism"; the second part is "The effect of reoxygenated DCs on the generation of regulatory T cells and its mechanism".PART I THE EFFECT OF ALLOGENEIC DCS ON THE GENERATION OF REGULATORY T CELLS AND ITS MECHANISMMethods:1. To induce murine bone marrow derived DCs1) To separate murine bone marrow cells from C57BL/6 or BALB/c mice and stimulate with GM-CSF and IL-4.2) To stimulate BM-DCs maturation by 1μg/ml LPS or not at day 6 for 24h.2. To detect the ability of murine DCs to generate CD4+CD25+Tregs in different mixed lymphocyte reaction1) To purify CD4+T cells from the splenocytes of BALB/c mice, and cocultured with autogeneic (auto-DCs) or allogeneic DCs (allo-DCs).2) To detect the proportion of CD4+CD25+Tregs in the different cultural systems at the indicated time by Flow Cytometry (FCM).3. To detect the ability of human DCs to generate CD4+CD25+Tregs in allogeneic MLR1) To separate peripheral blood monouclear cells (PBMC) from different two human blood.2) To separate CD4+T cells by human CD4 immunomagnetic beads.3) To induce DCs by stimulating the adherent cells by recombinant human GM-CSF and IL-4.4) To coculture the induced human DCs with autogeneic or allogeneic human CD4+T cells for days.5) To detect the proportion of CD4+CD25+Foxp3+Treg in the cultures by FCM.4. To detect the suppressive function of the obtained CD4+CD25+Treg in allogeneic DCs stimulated system1) To separate the generated CD4+CD25+T cells from the above allogeneic MLR for using as suppressors. Freshly separated CD4+CD25-T cells were used as responders after labeling CFSE. Allogeneic DCs were stimulators.2) To detect the cell proliferation by FCM, freshly isolated CD4+CD25+T cells were used as positive suppressive control.5. To explore the mechanism of CD4+CD25+Tregs increasing5.1 To explore the effect of IL-2 on the generation of CD4+CD25+Tregs by allogeneic DCs1) To purify the CD4+CD25+T cells and label CFSE, to detect the effect of allo-DCs to stimulate the proliferation of Tregs in the presence of exogenous IL-2.2) To purify the CD4+CD25+T cells and label CFSE, remix with the CD4+CD25-T cells at the intrinsic ratio as the whole CD4+T cells, and to detect the effect of allo-DCs on the IL-2 production of the whole CD4+T cells.3) IL-2 blocking assay:to detect the inhibitive effect of anti-IL-2 neutralizing antibody on the proliferation of CFSE-labeled CD4+CD25+T cells abovementioned.5.2 To explore the effect of TGF-βon the generation of CD4+CD25+Tregs by allogeneic DCs1) To purify the CD4+CD25-T cells and label CFSE, to detect the ability of allo-DCs to induce Tregs without of exogenous cytokines.2) To detect the expression of cytokine TGF-P in the supernatant of above MLR using ELISA.3) TGF-βblocking assay: to detect the effect of different concentration of TGF-βRI Kinase InhibitorⅡ(ALK-5 inhibitorⅡ) on the expression of Foxp3. Results1. The murine allogeneic DCs effectively generated CD4+CD25+Foxp3+Tregs from total CD4+T cells population without exogenous cytokines in vitro, but the autogeneic DCs did notIn freshly purified total CD4+T cells (purity>95%), the percentage of CD4+CD25+Foxp3+Tregs was less than 10%. In cocultured CD4+T cells with auto-DCs, CD4+CD25+T cells were increased significantly (more than 90%), but only a small population (less than 10%) were Foxp3+Tregs among them. However, in cocultured CD4+T cells with allo-DCs, the percentage of Foxp3+Tregs in these CD4+CD25+T cells was more than 80%, which was closed to the freshly purified CD4+ T cells. As the cultural time extended, the proportion of CD4+CD25+Foxp3+T cells in total CD4+T cells was increased by 5-7 folds. Notably, the maturation of DCs did not significantly affect the generation of CD4+CD25+Foxp3+T cells, as both immature and mature allo-DCs were capable of increasing the percentage of CD4+CD25+Foxp3+ T cells.2. The human allogeneic DCs could generate CD4+CD25+Foxp3+ Tregs from total CD4+T cells population without exogenous cytokines in vitro as well as murine allogeneic DCsTo further demonstrate the ability of allo-DCs to generate Tregs in vitro, we repeated the abovementioned experiments in human systems. The results showed that, in freshly purified CD4+T cells (purity>95%), the percentage of CD4+CD25+ Foxp3+ T cells was about 5-6%. In auto-DCs stimulated system, although the CD4+CD25+T cells increased significantly, but only a small population (about 10%) were Foxp3+Tregs among them. However, in allo-DCs stimulated system, the percentage of Foxp3+ Tregs in these CD4+CD25+T cells was about 40%, which was much higher than that in the autogeneic DCs stimulated system. These data suggested that, human allo-DCs, as well as mouse DCs, can effectively generate CD4+CD25+Foxp3+Tregs from total CD4+T cells population without exogenous cytokines in vitro.3. Allogeneic DCs-generated Tregs possess the suppressive function in vitro To examine whether the allo-DCs generated Tregs possess suppressive function in vitro, we purified CD4+CD25+T cells (suppressor) from the abovementioned allo-DCs stimulated system, and added these cells to CFSE labeled CD4+ CD25-T cells (responder) at gradually increasing ratio of suppressor:responder, and stimulated with allo-DCs. The FCM result showed that the proliferation of CSFE-labeled CD4+CD25-T cells was effectively suppressed by the allo-DCs generated Tregs as well as the freshly purified nTregs.4. The endogenous IL-2 and TGF-βplay an important role in the generation of Tregs in allo-DCs stimulated systemsIn order to investigate the mechanism of generation of increased Tregs by allo-DCs, we further detect the role of endogenous IL-2 and TGF-P in the generation of Tregs in allo-DCs stimulated systems.4.1 IL-2 plays an important role in the expansion of CD4+CD25+Tregs in the allogeneic DCs stimulated system1) Tregs could proliferate when stimulated by allo-DCs in presence of exogenous IL-2CD4+CD25+ nTregs were purified and labeled with CFSE, and then co-cultured with allo-DCs plus IL-2 for indicated days. The FCM results showed that allo-DCs effectively expanded CD4+CD25+T cells in presence of exogenous IL-2.2) The endogenous IL-2 plays an important role in the generation of Tregs in allo-DCs stimulated systemCSFE labeled CD4+CD25+T cells were remixed with purified CD4+CD25-T cells according to their ratio in total CD4+ population and then coculured them with allo-DCs without cytokines. The ELISA result showed that the level of IL-2 in supernatant of coculture of CD4+T cells with allo-DCs was significant increased after 5 days of allostimulation compared with that in control group. To further clarify the role of IL-2 in proliferation of Tregs in this system, we administrated anti-IL-2 monoclonal antibody to block the role of IL-2 in MLR. The result showed that, blockage of IL-2 was able to markedly inhibit the proliferation of nTregs in CD4+ population. These results indicate that significant amounts of IL-2 in coculture plays an important role in the expansion of CD4+CD25+Tregs in CD4+ population by allo-DCs.4.2. TGF-βplays an important role in the induction of CD4+CD25+Tregs in the allogeneic DCs stimulated system1) Tregs could be induced by allo-DCsIn order to clarify whether the conversion of CD4+CD25+Tregs from CD4+CD25T cells was involved in allo-DCs generated Tregs in our system, the CFSE labeled CD4+CD25-T cells were cocultured with allo-DCs for different times and a dynamic analysis of Foxp3 expression was performed by FCM. The results showed that Foxp3+T cells increased significantly after 5 days coculture compared with freshly purified CD4+CD25-T cells. After 13 days of allostimulation, the expression of Foxp3 increased nearly two folds compared with that at day 5 of allostimulation. This indicates that CD4+CD25+Foxp3+ T cells can be induced by allo-imDCs without exogenous cytokines in vitro.2) Endogenous TGF-βexisted in allo-DCs stimulated systemThen the level of TGF-βin the supernatant of coculture was detected by ELISA. The results indicated that the total TGF-βsignificantly increased at day 5 and then elevated gradually along with the time of allostimulation. At day 13, the concentration of TGF-βwas about two times more than that at day 5 culture.3) The endogenous TGF-βplays an important role in the generation of Tregs in allo-DCs stimulated systemTo further confirm the role of endogenous TGF-βin the differentiation of Tregs by allo-DCs, we used the TGF-βRI Kinase InhibitorⅡ(ALK-5 inhibitorⅡ) to block the TGF-βsignal and then measured the change of percentage and total number of Foxp3+Tregs. We found that the percentage and absolute number of CD4+Foxp3high cells were gradually decreased when the concentration of ALK-5 inhibitorⅡincreased. Additionally, in this culture, CD4+Foxp3dim cells markedly decreased after administration of TGF-βRI Kinase InhibitorⅡin a dose-dependent manner. These results suggested that CD4+Foxp3dim cells may be the main population derived from the conversion of Foxp3+ Tregs, and TGF-β, to some extent, contributes to the conversion of CD4+CD25+T cells.Conclusions 1. The murine allogeneic DCs effectively generated CD4+CD25+Foxp3+Tregs from total CD4+T cells population without exogenous cytokines, and the generated Tregs also possessed the suppressive function in vitro.2. The human allogeneic DCs from PBMC could also generate CD4+CD25+Foxp3+ Tregs from total CD4+T cells population effectively.3. The mechanism of the increased CD4+CD25+Foxp3+Tregs:The endogenous IL-2 and TGF-β. play the important role in the generation of CD4+CD25+Foxp3+Tregs stimulated by allogeneic DCs.Originality1. It’s the first time to provide evidence that the murine allogeneic but not autogeneic DCs effectively generated CD4+CD25+Foxp3+Tregs from total CD4+T cells population without exogenous cytokines in vitro and to explore the mechanism.2. It’s the first time to look the CD4+T cells as a whole population to investigate the expansion and induction of Tregs. It is important to provide theoretical foundation for optimizing continuous empirical methods and application of Treg in the clinical therapy.Limitation of this study1. The detailed molecular mechanism for the allogeneic DCs to increase the proportion of Tregs in CD4+T population need to be further explored.2. The empirical methods need to be further optimized before the application of generated Treg in the clinical therapy. Methods1. Effect of reoxygenation on the phenotype of DCs1) To induce murine BM-DCs using GM-CSF and IL-4 under normoxic or hypoxic conditions, and stimulate maturation by LPS.2) To transform DCs differentiated under hypoxic condition to normoxia for various periods of continuous culture.3) To detect the phenotype (CD80, CD86, and MHC classⅡmolecules) of reoxygenated DCs by FCM.2. Effect of reoxygenation on the function of DCs to drive Th cells to proliferate and differentiate1) To separate CD4+T cells and label with CFSE and then coculture them with reoxygenated allogeneic or autogeneic DCs.2) To assay the CD4+T cells proliferation by FCM.3) To assay the CD4+T cells differentiation stimulated by reoxygenated DCs via FCM. 3. To explore the mechanism of the effect of reoxygenation on the phenotype and function of DCs1) To detect the expression of four adenosine receptors on reoxygenated DCs by real time PCR.2) To detect the effect of four adenosine receptor agonists on the phenotype of reoxygenated DCs by FCM.3) To detect the effect of different concentration of A2AR agonist on the phenotype of reoxygenated DCs by FCM.4) To detect the effect of A2AR agonist on the cytokines secreted by reoxygenated DCs by ELISA.Results 1. Reoxygenation reversed the effect of hypoxia on the phenotypic maturation of DCsTo determine whether oxygen delivery could influence the phenotypic maturation of hypoxic DCs, reoxygenation was performed on hypoxic DCs for various periods of time, related surface markers were examined by FCM. The results showed that reoxygenation obviously upregulated the expression of CD80, CD86 and MHC classⅡmolecules on DCs which were inhibited by hypoxia. And the percentage and mean fluorecence intensity (MFI) was gradually increased with the time of reoxygenation, and peaked after 24-48h of reoxygenation. This indicated that the reoxygenated DCs displayed a full mature phenotype.2. Reoxygenation enhanced the ability of hypoxic DCs to stimulate CD4+T cell proliferationSince reoxygenation promoted the phenotypic maturation of hypoxic DCs, we further analyzed the effects of reoxygenated DCs on the proliferation of CD4+T cells. The results showed that hypoxic DCs could not effectively drive T cells proliferation, but DCs reoxygenated for 6 h stimulated CD4+T cells to proliferate vigorously, not only in allogeneic DCs but also in anti-CD3 stimulated system. These data suggested that reoxygenation markedly enhances the ability of hypoxic DCs to stimulate CD4+T cells proliferation.3. Reoxygenated DCs decreased Foxp3+Treg cells, and induced Th17, Thl cell immune responsesIn order to investigate the CD4+T cells differentiation driven by reoxygenated DCs in the allogeneic MLR, the intracellular staining was performed. The FCM results showed that, the proportion of CD4+CD25+Foxp3+Tregs in the reoxygenated DCs stimulated system was much lower than that in the normoxic mature and immature DCs stimulated systems. However, reoxygenated DCs induced significantly higher number of IL-17+ CD4+T (Th17) cells than DCs differentiated under hypoxic or normoxic conditions did. And also the proportion of IFN-y+Thl cells in the allogeneic MLR and the level of IFN-y in the supernatant were also higher. In addition, we analyzed the cytokines profile of reoxygenated DCs and found that, hypoxic DCs exposed to oxygen for 24h produced significantly higher levels of IL-6 and slightly lower levels of TGF-β, .compared with that in hypoxia-differentiated DCs, suggesting the elevated IL-6 secretion by reoxygenated DCs along with significant amounts of TGF-β. abrogates Treg generation but drives CD4+T cells toward Th17 cells. Taken together, these results suggested that reoxygenated DCs had strong capacity to drive immune response toward a proinflammatory direction.4. The adenosine-A2AR pathway plays an important role in the process of hypoxia-reoxygenation of DCs4.1 Reoxygenation upregulated the mRNA level of A2AR on the DCs, but had no effect on the expression of A1R, A2BR and A3RTo investigate that did the adenosine receptor pathway play the important role in the process of reoxygenation, the expression of A1R, A2AR, A2BR and A3R on the mRNA level were detected by real time PCR. The results demonstrated that the A1, A2B and A3 receptors expression levels remained unaltered in response to reoxygenation compared with their levels in response to normoxia and hypoxia. In contrast, the expression of A2AR was predominantly increased in reoxygenated DCs.4.2 The effect of adenosine receptor agonists on the phenotype of reoxygenated DCsTo further clarify the effect of adenosine receptor pathway on the phenotype of reoxygenated DCs, different agonists for A1R, A2AR, A2BR and A3R were administrated when reoxygenation was performed on the hypoxic DCs. Twenty four hours later, the phenotype was detected by FCM. The results showed that, A2AR specific agonist CGS21680 could downregulate the expression of MHC-II, CD80 and CD86 of reoxygenated DCs, while the other agonists couldn’t. It’s that CGS21680 could restrain the upregulated function of reoxygenation on the expression of MHC-II, CD80 and CD86 which were inhibited by hypoxia.4.3 The A2AR specific agonist affects the phenotype of reoxygenated DCs in a concentration-dependent mannerFurthermore, we also studied the effect of different concentration of A2AR specific agonist CGS21680 on the phenotype of reoxygenated DCs, and the data demonstrated that MFI of MHC-II, CD80 and CD86 on the reoxygenated DCs were gradually decreased with the increased concentration of CGS21680, which was coincident with that in normoxic DCs. This indicated that A2AR signaling pathway did play an important role in the reoxygenated DCs.4.4 The A2AR agonist can obviously inhibit the proinflammatory cytokines secreted by reoxygenated DCs, but promote the anti-inflammatory cytokines’ secretionWe further detected the effect of A2AR agonist on the cytokines secreted by reoxygenated DCs by ELISA. The results showed that A2AR specific agonist CGS21680 could inhibit the proinflammatory cytokines IL-1β, IL-6 and TNF-α, which were enhanced by reoxygenation. Well, the level of TGF-βwas significantly increased after the CGS21680 treatment. All these data suggested that A2AR signaling pathway may be a mediator for reoxygenation up-regulating the phenotype and function of hypoxic DCs.Conclusions1. Reoxygenation reversed the inhibition of maturation and function of DCs by hypoxia1) Reoxygenation upregulated the mature phenotype of DCs.2) Reoxygenation enhanced the ability of DCs to stimulate CD4+T cell proliferation, and induced Th1 and Th17 cell immune responses with decreased Foxp3+Treg cells.2. Adenosine receptor pathway may be a mediator for reoxygenation to upregulate the phenotype and function of DCs1) Reoxygenated DCs predominantly expressed higher level of A2AR, compared to that in normoxic and hypoxic DCs.2) A2AR specific agonist CGS21680 downregulated the phenotype of reoxygenated DCs in a concentration dependent manner.3) A2AR specific agonist CGS21680 inhibited the secretion of proinflammatory cytokines of reoxygenated DCs.Originality1. It’s the first time to provide evidence that reoxygenation can reverse the maturation and function of hypoxic DCs. It is helpful for explaining the pathogenesis mechanism of IRI, and it also demonstrated that hypoxia-reoxygenation maybe one of internal mechanisms of IRI.2. It is the first time to provide evidence that, A2AR signaling pathway can inhibit the ability of reoxygenation to reverse the maturation and function of DCs. This is important for exploring new strategies to prevent or treat IRI at the immunologic point of view.Limitation of this study1. A2AR signaling pathway inhibits the ability of reoxygenation to reverse the maturation and function DCs, but the internal detailed molecular mechanism need to be further studied.2. Experiments of adenosine receptor agonist to treat IRI in animal model need to be further completed.更多还原