【作者】 王群；

【导师】 张利宁；

【作者基本信息】 山东大学 ， 医学免疫学， 2009， 博士

【摘要】 第一篇乏氧及CD137通路对DC功能的影响目的乏氧是指局部组织器官处于一种远远低于大气氧压的极低氧压状态,主要见于许多生理及病理条件下,如远离毛细血管末端的组织,炎症或肿瘤组织,以及器官移植、低血容量性休克、肝脏手术、心血管疾病等缺血情况。乏氧可促进肿瘤细胞对放疗和化疗的抵抗,并可诱发其表型改变,从而促进其转移扩散,因此治疗后其预后往往较差。尽管乏氧对肿瘤细胞生物学特性和行为的影响已有大量研究报道,但是在肿瘤、炎症等乏氧微环境中,免疫细胞及免疫功能如何发生变化,尚不完全清楚。目前有限的研究主要集中于乏氧对巨噬细胞的影响,散在的体外实验显示乏氧可抑制T细胞功能,如细胞活化增殖明显减少,细胞因子表达显著降低等,但具体的内在机制并不清楚。树突状细胞(Dendritic cells,DC)作为连接固有免疫和适应性免疫应答的抗原递呈细胞,其成熟状态直接影响其抗原递呈功能及刺激T细胞增殖的能力。未成熟DC主要位于外周非免疫器官或组织,摄取抗原之后向引流淋巴结迁移并逐渐分化为成熟DC,成熟DC高表达MHC-Ⅱ类分子及协同刺激分子CD80、CD86等,具有很强的抗原递呈能力,从而诱导适应性免疫应答的产生。DC的归巢及迁徙往往需要经过炎症、肿瘤等病理组织,其中乏氧微环境对DC的状态和功能影响如何?目前尚不明确。已有研究显示乏氧条件下分化的人单核细胞来源的DC,其基质金属蛋白酶9(matrix metalloproteinase 9,MMP9)的表达明显降低,迁移能力明显下降,但是乏氧对小鼠DC的成熟及功能的影响,则鲜有报到。此外,缺血部位血液的再灌注可导致乏氧组织氧压的迅速提升,而这种氧压的急剧变化对DC及其所介导的适应性免疫的影响如何?目前尚无研究涉及。因此,本课题在研究乏氧条件下小鼠骨髓来源DC(bone marrow derived-DC,BM-DC)表型及功能变化的基础上,进一步探索了再氧合对乏氧DC表型及功能的影响,这对于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等许多方面都具有重要的指导意义。CD137-CD137L是继CD28-B7之外的另一对重要的协同刺激分子。CD137属于肿瘤坏死因子受体(tumor necrosis factor receptor,TNFR)超家族,主要表达于活化的T细胞,是一种可诱导的T细胞膜表面受体。CD137-CD137L通路可依赖或不依赖CD28-B7途径提供协同刺激信号,导致CD4~+、CD8~+T细胞的活化、增殖及分化,在适应性免疫应答中,尤其是初次应答晚期以及再次应答中发挥着重要作用。近年研究显示CD137亦可表达于小鼠脾脏和骨髓来源的DC表面,刺激性抗CD137单抗可增强DC分泌IL-6和IL-12等细胞因子及其刺激T细胞增殖的能力,表明CD137通路在调节和增强DC功能方面具有不容忽视的作用。因此,本课题在研究乏氧、再氧合对小鼠DC功能影响的基础上,进一步探讨了CD137通路对乏氧DC功能的影响,以期通过CD137通路增强乏氧DC的功能,进而增强固有免疫应答或适应性免疫应答,从而为肿瘤以及移植排斥等的免疫治疗提供新的思路和方法。方法一、常、乏氧条件下DC的表型及功能变化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诱导未成熟DC,LPS刺激DC成熟。2.流式细胞术检测常、乏氧条件下分化DC表面的协同刺激分子CD80,CD86及MHC-Ⅱ类分子的表达差异。3.RT-PCR和ELISA法分别检测常、乏氧条件下分化DC所分泌细胞因子IL-1β,IL-6,TNF-α,TGF-β以及基质金属蛋白酶MMP9的表达差异。4.CFSE法检测常、乏氧条件下分化DC刺激CD4~+T细胞增殖能力的差异。二、再氧合对乏氧条件下DC表型及功能的影响1.对乏氧条件下分化的DC进行不同时间的再氧合。2.流式细胞术检测再氧合DC的表型变化(CD80,CD86及MHC-Ⅱ类分子)。3.CFSE方法检测再氧合DC刺激CD4~+T细胞增殖能力的变化。5.流式细胞术检测再氧合DC刺激CD4~+T细胞向IFN-γ~+Th1,IL-4~+Th2细胞的分化。5.ELISA方法检测再氧合DC刺激CD4~+T细胞增殖体系中细胞因子IFN-γ和IL-4的表达水平。三、CD137通路对乏氧条件下分化DC表型及功能的影响1.以抗CD137单克隆抗体刺激乏氧条件下分化的DC。2.流式细胞术检测抗CD137单抗刺激DC的表型变化(CD80,CD86及MHC-Ⅱ类分子)。3.RT-PCR法检测抗CD137单抗刺激DC所分泌细胞因子以及基质金属蛋白酶MMP9的表达变化。4.CFSE法检测抗CD137单抗刺激DC刺激CD4~+T细胞增殖能力的变化。结果一、乏氧对DC表型及功能的影响1.骨髓来源DC的诱导和纯度自C57BL/6小鼠分离骨髓细胞,在GM-CSF和IL-4存在的情况下,分别于常、乏氧条件下诱导分化未成熟DC,并于第6天用LPS刺激18-24小时,以诱导DC的成熟。形态学结果显示,无论在常、乏氧条件下,至诱导第5-7天,光镜下均可见具有典型树突状形态的DC。流式细胞术检测结果显示,所收获BM-DC表面CD11c的表达均在80%以上,达到所要求的DC纯度。提示乏氧条件并不影响DC的诱导分化。2.乏氧抑制DC的表型成熟为了研究乏氧微环境是否影响DC的表型成熟,我们分别收集常、乏氧条件下以LPS刺激成熟的小鼠BM-DC,流式细胞术检测其成熟相关表型。结果显示,乏氧条件下诱导分化并以LPS刺激的DC仍保持未成熟或半成熟的表型特征,其表面CD80,CD86以及MHC-Ⅱ类分子的表达,无论是百分比,还是平均荧光强度,均显著低于LPS刺激的常氧成熟DC,其中MHC-Ⅱ类分子的平均荧光强度(MFI)降低尤为明显(P＜0.01),表明LPS不能诱导乏氧条件下分化DC表面协同刺激分子以及MHC-Ⅱ类分子的表达上调,提示乏氧微环境可抑制DC的表型成熟。3.乏氧抑制DC炎性细胞因子IL-1,IL-6和TNF-α的分泌以及MMP9的产生,上调免疫抑制因子TGF-β的分泌为了研究乏氧微环境是否影响DC的细胞因子分泌及迁移等功能,我们分别收集常、乏氧条件下以LPS刺激成熟的小鼠BM-DC,采用RT-PCR和ELISA方法检测评价相关细胞因子和基质金属蛋白酶MMP9的表达水平。结果显示,乏氧条件下诱导分化的DC,其IL-1β,IL-6和TNF-α的mRNA和蛋白表达水平均明显低于常氧成熟DC;而其TGF-β的mRNA和蛋白表达水平却显著高于常氧成熟DC,表明乏氧可抑制DC分泌炎性细胞因子IL-1,IL-6和TNF-α,但却上调免疫抑制因子TGF-β的分泌。此外,我们发现,较常氧成熟DC相比,乏氧条件分化并以LPS刺激的DC,其MMP9的mRNA水平明显降低,提示乏氧可能通过抑制MMP9的表达而抑制DC的迁移功能。4.乏氧抑制DC刺激同种异型CD4~+T细胞增殖的能力为了探索乏氧微环境对DC致敏T细胞能力的影响,我们分别以常、乏氧条件下诱导产生的C57BL/6小鼠的DC刺激来自BALB/c小鼠的CD4~+T细胞,用流式细胞术检测CD4~+T细胞增殖率的不同。结果显示,以LPS刺激成熟的常氧DC,可诱导约40-50%左右的CD4~+T细胞的增殖;然而乏氧条件分化并以LPS刺激的DC,其诱导同种异型CD4~+T细胞增殖的能力很弱,只能诱导大约10%左右的CD4~+T细胞的增殖,甚至显著低于常氧未成熟DC刺激CD4~+T细胞增殖的能力(30%左右),可见LPS诱导的乏氧DC其刺激CD4~+T细胞增殖的能力与常氧DC相比非常弱,说明乏氧微环境抑制LPS诱导的DC的功能成熟,尤其是其介导适应性免疫应答的能力。二、再氧合对乏氧DC表型及功能的影响1.再氧合促进乏氧DC的表型成熟为了研究氧气再给予对乏氧DC表型的影响,我们对乏氧条件下分化的DC进行不同时间的再氧合,流式细胞术检测DC表面成熟相关表型的变化。结果显示再氧合可已明显上调乏氧DC表面协同刺激分子CD80,CD86以及MHC-Ⅱ类分子的表达,且随时间延长其表达强度逐渐增强,再氧合6小时的DC其表面CD80,CD86以及MHC-Ⅱ类分子的表达已明显高于乏氧DC,至再氧合24到48小时达到高峰,甚至远高于LPS刺激的常氧成熟DC,呈现显著成熟的表型。这些结果提示,当给以充足的氧供,LPS刺激的乏氧DC具有完全甚至更强的向成熟DC分化的潜能。2.再氧合增强DC刺激CD4~+T细胞增殖的能力由于再氧合可导致乏氧DC的表型成熟,我们进一步研究了这些再氧合DC刺激CD4~+T活化增殖的能力。在同种异型增殖体系中,再氧合6小时的乏氧DC可强烈地刺激诱导CD4~+T细胞的增殖,其刺激CD4~+T细胞增殖的能力较乏氧DC显著提高,可刺激约71.1%的CD4~+T细胞增殖,远远超过常氧成熟DC对CD4~+T细胞的刺激增殖(增殖率约为40.5%)(P＜0.01)。在抗CD3抗体存在的同种同型增殖体系中,同样可以观察到再氧合DC可强烈刺激CD4~+T细胞的增殖(增殖率92.0%),显著高于常氧成熟DC对CD4~+T细胞的刺激增殖(增殖率约为71.7%)(P＜0.05)。提示再氧合可显著增强乏氧DC刺激CD4~+T细胞增殖的能力。3.再氧合DC诱导CD4~+T细胞向Th1细胞分化为了搞清再氧合DC对效应性T细胞分化的影响,我们用流式细胞术检测了混合淋巴细胞反应体系中CD4~+效应T细胞的亚群。结果发现,与乏氧或常氧条件下分化的DC相比,再氧合DC可显著诱导CD4~+T细胞向IFN-γ~+Th1细胞分化(约为9.1%),明显高于未成熟DC刺激产生的IFN-γ~+Th1细胞,甚至高于常氧成熟DC刺激产生的IFN-γ~+Th1细胞(约为6.8%);进一步的ELISA结果证实再氧合DC刺激体系中存在高水平IFN-γ,显著高于乏氧DC及常氧未成熟DC刺激体系(P＜0.05)。同时,再氧合DC可刺激CD4~+T细胞向IL-4~+Th2细胞分化,但其比例较少(约为2.1%);ELISA结果亦显示再氧合DC刺激体系中存在较高水平的IL-4。提示再氧合DC可诱导CD4~+T细胞向Th1和Th2细胞分化,但更倾向于使其向Th1细胞分化。三、CD137通路对乏氧DC表型及功能的影响1.CD137单抗上调乏氧DC的成熟表型为了研究CD137通路对乏氧DC的作用,我们分别检测了常、乏氧条件下诱导分化并以LPS刺激的DC表面CD137分子的表达情况,结果发现两者表面均有CD137分子的表达,但总体表达强度并不太高;乏氧具有轻微下调DC表面CD137表达的趋势,但未达到显著统计学差异。提示乏氧微环境对DC表面CD137表达的影响并不大。为了进一步探讨CD137分子的存在对乏氧DC的表型成熟的影响,我们用流式细胞术检测了CD137单抗刺激的乏氧DC表面协同刺激分子CD80,CD86以及MHC-Ⅱ类分子的表达。结果发现,乏氧条件下,刺激性CD137单抗可明显上调DC表面CD80,CD86以及MHC-Ⅱ类分子的表达,但无论是表达百分率还是表达强度均未达到常氧成熟DC表面的表达程度。这些结果表明CD137单抗可通过CD137分子促进乏氧DC向接近成熟的表型方向转变,但并不能促进其表型的完全成熟。2.CD137单抗可促进乏氧DC炎性细胞因子及MMP9的产生为了研究CD137通路对乏氧条件下分化DC迁移及炎性细胞因子分泌功能的影响,我们用RT-PCR法检测了DC中MMP9以及细胞因子IL-1β,IL-6,IL-12p40和TNF-α等的mRNA表达水平。结果发现,CD137单抗刺激可上调乏氧DC中炎性细胞因子IL-1β,IL-6,IL-12p40和TNF-α的mRNA水平,但未达到常氧成熟DC中的水平;此外,CD137单抗刺激亦可在一定程度上增加乏氧DC中MMP9的表达。提示CD137单抗不仅可促进乏氧DC炎性细胞因子的分泌;还可能通过MMP9的表达增加提高其迁移能力。3.CD137单抗对乏氧DC刺激CD4~+T细胞增殖能力的影响由于CD137单抗可在一定程度上刺激乏氧DC的表型成熟,我们进一步研究了CD137单抗对乏氧DC刺激CD4~+T细胞增殖能力的影响。我们用来自C57BL/6小鼠的CD137单抗刺激的乏氧DC作为刺激细胞,用来自BALB/c小鼠的CD4~+T细胞作为反应细胞,进行T细胞增殖实验,结果显示CD137单抗刺激的乏氧DC其诱导CD4~+T细胞增殖率略高于无CD137单抗作用的乏氧DC,但未达到统计学意义。提示CD137通路对乏氧DC诱导CD4~+T细胞增殖的能力无明显影响。结论一、乏氧抑制LPS诱导的DC成熟及功能1.乏氧可抑制LPS诱导的DC的成熟表型。2.乏氧可降低DC分泌炎性细胞因子IL-1,IL-6和TNF-α,增高免疫抑制因子TGF-β的表达。3.乏氧可抑制DC刺激CD4~+T细胞增殖的能力。二、再氧合促进乏氧DC的成熟及功能1.再氧合显著促进乏氧DC的表型成熟。2.再氧合显著增强乏氧DC刺激CD4~+T细胞增殖的能力。3.再氧合DC诱导CD4~+T细胞向Th1细胞分化。三、CD137通路可促进乏氧DC的成熟及其功能1.CD137单抗可上调乏氧DC的成熟表型。2.CD137单抗可促进乏氧DC分泌炎性细胞因子IL-1,IL-6,IL-12和TNF-α。3.CD137单抗对乏氧DC诱导CD4~+T细胞增殖的能力无明显影响。创新性一、在国内外率先证明乏氧可抑制小鼠LPS诱导的DC表型成熟,抑制其分泌炎性细胞因子及刺激CD4~+T增殖的能力,为研究乏氧微环境对免疫功能的影响提供了新的实验依据。二、首先发现再氧合可明显促进DC的表型成熟,显著增强DC刺激CD4~+T细胞增殖的能力,说明再氧合对乏氧抑制DC成熟及功能具有逆转效应,这对于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的免疫治疗等许多方面都具有重要的指导意义。三、首先发现CD137单抗可促进DC的表型成熟及炎性细胞因子的分泌。对于解释CD137抗体体内抗肿瘤的作用机制有一定价值。局限性一、再氧合促进乏氧DC成熟及功能的作用机制尚需深入研究。二、CD137单抗对乏氧DC的表型成熟及细胞因子分泌影响的机制和意义,以及CD137单抗刺激的乏氧DC刺激CD8~+T增殖的能力有待进一步研究。第二篇CD137和CD137L在人原发肿瘤组织中的表达及意义目的CD137作为肿瘤坏死因子超家族的一员,主要表达于活化的T淋巴细胞和NK细胞表面;其配体CD137L主要表达于抗原递呈细胞表面,如成熟的树突状细胞(dendritic cells,DC),活化的B细胞和巨噬细胞。CD137-CD137L介导的共刺激信号可增强T细胞活化,促进心脏同种异型移植物的排斥反应,清除实验诱导的小鼠肿瘤。然而,人类CD137和CD137L的表达并不局限于免疫细胞,人CD137-CD137L通路的功能也远比小鼠复杂。CD137蛋白已被证明可表达于原发恶性肿瘤的血管壁;CD137L亦被发现可表达于几种不同的人类肿瘤细胞系,体外实验证明其是有功能的。迄今为止,关于CD137L在人类原发肿瘤中的表达尚无研究报道。因此,本研究的目的主要是分析CD137和CD137L在人类原发肿瘤中的表达,并深入研究它们在肿瘤免疫中的潜在作用。方法一、病例收集:63例组织标本来自山东大学齐鲁医院的手术病人,其中包括12例正常组织,15例上皮及间叶组织来源的良性肿瘤组织(腺瘤和平滑肌瘤),36例上皮来源的恶性肿瘤组织(鳞状细胞癌和腺癌)。二、免疫组织化学法分析CD137和CD137L在上述冰冻切片中的表达。三、RT-PCR法分析CD137L在9株人类肿瘤细胞系中的mRNA表达,其中包括3株肝癌细胞系,2株肺癌细胞系,2株结肠癌细胞系,1株淋巴瘤和1株白血病细胞系。四、为了分析肿瘤细胞表面CD137L的作用,将表达CD137L的肿瘤细胞与表达CD137的活化T淋巴细胞或CHO细胞共培养,ELISA法检测细胞因子(IL-8,IFN-γ)的表达水平。结果一、人类原发肿瘤组织可表达CD137和CD137L,但其表达部位不同CD137和CD137L仅表达于人类良性(2/15,3/15)或恶性(15/36,21/36)肿瘤组织中,但不表达于正常组织中(0/12,0/12)。CD137表达于肿瘤组织血管壁的上皮细胞和平滑肌细胞,而CD137L表达于肿瘤细胞。尽管样本来自于不同的组织类型,CD137和CD137L的表达更常见于恶性肿瘤组织,尤其是中度或低度分化的恶性肿瘤组织。三、人类肿瘤细胞系表达高水平的CD137L上述实验结果显示人类原发肿瘤组织中的肿瘤细胞可表达CD137L,我们进一步用RT-PCR法检测了来源于不同原发组织的肿瘤细胞细胞系表面CD137L的表达。结果显示,正常未刺激的外周血单个核细胞不表达CD137L mRNA,但所有检测的肿瘤细胞系(HepG2.2.15,BEL7402,HLE,HT29,L78,A2,U937,HL60,H6)均显示高水平的CD137L表达。出乎意料的是非肿瘤细胞系,人脐静脉内皮细胞系ECV304也表达CD137L。这些结果显示CD137L不仅表达于人类的原发肿瘤组织,也表达于肿瘤细胞系和一些非肿瘤细胞系,提示CD137L的表达可能与细胞的快速生长有关。三、肿瘤细胞表达的CD137L是有功能的为了研究肿瘤细胞表面CD137L的功能,我们将表达CD137L的肿瘤细胞与表达CD137的活化T淋巴细胞共培养,ELISA法检测IFN-γ的分泌。结果显示,与不CD137的静息T细胞相比,表达CD137的活化T淋巴细胞与L78细胞共培养可产生高水平的IFN-γ(P＜0.001),用抗CD137L单抗封闭CD137-CD137L通路,IFN-γ的分泌水平则明显降低(P＜0.001)。为了验证表达于肿瘤细胞表面的CD137L能否向肿瘤细胞传递信号,我们将不同的肿瘤细胞与表达CD137的CD137-CHO细胞共培养,ELISA法检测IL-8的表达。结果显示HepG2.2.15表面的CD137L与CD137-CHO表面的CD137交联可促进肿瘤细胞IL-8的分泌(P=0.038)。结论一、CD137和CD137L可表达于不同的人类原发肿瘤组织,但其表达部位不同。CD137表达于肿瘤组织血管壁的上皮细胞和平滑肌细胞,而CD137L表达于肿瘤细胞。二、肿瘤细胞表达的CD137L是有功能的,肿瘤细胞L78表面的CD137L与T细胞表面的CD137交联可诱导T细胞产生高水平的IFN-γ,HepG2.2.15表面的CD137L与CD137的交联可促进肿瘤细胞分泌IL-8,从而提示肿瘤组织中CD137和CD137L的表达可影响肿瘤的发展。创新性首次证明CD137和CD137L可表达于不同的人类原发肿瘤组织,肿瘤细胞表达的CD137L是有功能的,从而提示肿瘤组织中CD137和CD137L的表达可影响肿瘤的发展。局限性病例数有限,需扩大病例数以进行深入研究;对CD137和CD137L的表达分析,需进一步收集相同组织来源的标本进行研究。更多还原

【Abstract】 PARTⅠEffects of Hypoxia and CD137 Pathway on the Function of DCsObjectiveHypoxia,a status of very low oxygen tension in local tissues or organs,is always found in various physiological and pathophysiological tissues,such as microenvironments that are located far from the ends of capillaries,cancerous or inflamed tissues,as well as organ transplantation,hypovolemic shock,liver surgery and cardiovascular diseases.It was reported that hypoxia could promote tumor cells progression and resistance to chemotherapy and radiotherapy,leading to poor prognosis. Though many researches reported the influence of hypoxia on tumor growth and metastasis,it’s unclear that the change of immune cells and immune responses in such hypoxic microenvironments.Though limited data studied the effects of hypoxia on innate immunity such as macrophage,little reports referred to the effects on adaptive immunity,sporadic experiments showed that hypoxia could inhibit the fimction of T cells,the cell activation,proliferation,and the cytokine secretion decreased markedly, but detailed mechanism is largely unknown so far.Dendritic cells(DCs) are the critical antigen presenting cells linking innate and adaptive immunity,the status of DCs maturation can directly influence the ability of antigen presenting and stimulating T cell proliferation.Immature DCs mainly reside in non-lymphoid tissues,after uptake of antigen,these immature DCs migrate to the regional lymph nodes and differentiate into mature DCs,which express highly MHC classⅡmolecules,co-stimulatory molecules CD80,CD86,and have a potent capacity for antigen presentation,thereby initiate the specific immune response.The homing and migration of DCs need to go through the pathological tissues such as tumor and inflamed tissues,while the effects of hypoxic microenvironment in these tissues on DCs is unclear.Two reports suggested that human monocyte-derived DCs differentiated under hypoxia expressed decreased matrix metalloproteinase(MMP)-9 and have a reduced migratory capacity compared with those differentiated under normoxia,but further evidence for the effects of hypoxia on the function of DCs, especially on that of mouse DCs is lacking to date.In addition,the return of blood into ischemia tissues can result in the elevation of oxygen tension in hypoxic tissues,it is unclear whether the dramatic change of oxygen tension affects DCs as well as the T cell immune response mediated by these DCs.Therefore,in the current study,we focused on the effects of hypoxia on the phenotype and function of mouse DCs,further studied the effects of reoxygenation(from hypoxia to normoxia) on hypoxic DCs.It is much helpful for the control of ischemia reperfusion injury,graft rejection in organ transplantation,and the immunotherapy for tumor.CD137-CD137L is another pair of important co-stimulatory molecules besides CD28-B7 for T cells.As a member of tumor necrosis factor receptor superfamily, CD137 is an inducible T cell surface receptor and mainly expresses on activated T cells. CD137-CD137L pathway provides co-stimulatory signal for the activation, proliferation,differentiation of CD4~+ and CD8~+ T cells,and plays an important role in adaptive immunity,especially in the late phase of primary immune response and the secondary immune response.Recent studies showed that CD137 can be expressed on mouse splenic DCs and bone marrow-derived DCs(BM-DCs),agonistic mAb to CD137 increased the secretion of IL-6,IL-12 from DCs,importantly enhanced the ability of DCs to stimulate T cell proliferation,suggesting the important role of CD137 pathway on the modulation and enhancement of DCs function.Based on the above studies,we further explored the effects of CD137 pathway on hypoxic DCs,which aim to increase the function of hypoxic DCs,further enhance the adaptive immune response.It may provide a novel strategy for the immunotherapy for tumor and graft rejection.Methods1.Phenotye and function of DCs under normoxic or hypoxic conditions1) To isolate bone marrow cells from C57BL/6 mice,induce DCs using GM-CSF and IL-4,and stimulate maturation by LPS under normoxic or hypoxic conditions.2) To detect the expression of co-stimulatory molecules CD80,CD86,and MHC classⅡmolecules on DCs differentiated under normoxic or hypoxic conditions through Flow Cytometry.3) To detect the secretion of cytokines(IL-1β,IL-6,TNF-α,TGF-β) and expression of matrix metal proteinases MMP9 by DCs differentiated under normoxic or hypoxic conditions through RT-PCR and ELISA.4) To assay the CD4~+T cell proliferation stimulated by DCs differentiated under normoxic or hypoxic conditions through CFSE labeling.2.Effects of reoxygenation on the phenotype and function of DCs differentiated under hypoxic conditions1) To reoxygenate DCs differentiated under hypoxic conditions for various periods of time.2) To detect the change of phenotype(CD80,CD86,and MHC classⅡmolecules) on reoxygenated DCs by Flow Cytometry.3) To assay the CD4~+T cell proliferation stimulated by reoxygenated DCs via CFSE labeling.4) To assay the CD4~+T cell differentiation stimulated by reoxygenated DCs via Flow Cytometry.5) To detect the cytokine expression in CD4~+T cell proliferation system stimulated by reoxygenated DCs through ELISA.3.Effects of CD137 pathway on the phenotype and function of DCs differentiated under hypoxic conditions1) To treat DCs differentiated under hypoxic conditions using agonistic anti-CD137 Ab.2) To detect the change of phenotype(CD80,CD86,and MHC classⅡmolecules) on hypoxic DCs after anti-CD137 Ab stimulation by Flow Cytometry.3) To detect the change of cytokine secretion and MMP9 expression by hypoxic DCs after anti-CD137 Ab stimulation through RT-PCR.4) To assay the CD4~+T cell proliferation stimulated by anti-CD137 Ab treated-hypoxic DCs via CFSE labelling.Results1.Phenotype and function of DCs differentiated under normoxic or hypoxic conditions 1) Induction and purity of DCs derived from bone marrow cells of C57BL/6 miceBone marrow cellls were isolated from C57BL/6 mice and were induced into immature DCs under normoxic or hypoxic conditions in the presence of GM-CSF and IL-4.To induce maturation,immature DCs were treated with LPS for 18-24h.After 5-7 days of induction,cells with typical dendritic morphology were observed under microscope;results from Flow Cytometry showed that the purity of DCs differentiated under normoxic or hypoxic conditions as examined by CD11c expression were both above 80%,suggesting hypoxia has no influence on the induction and differentiation of DCs.2) Hypoxia inhibits the phenotypic maturation of DCsTo examine the effect of hypoxia on the phenotypic maturation of DCs, murine DCs derived from bone marrow(BM-DC) differentiated under normoxic or hypoxic conditions were stimulated with LPS,related surface markers were detected by Flow Cytometry.The results showed that DCs differentiated under hypoxic conditions displayed immature or semi-mature phenotype though stimulated with LPS,the expression of CD80,CD86 and MHC classⅡmolecules on these DCs was markedly lower than that of normoxic mature DCs, especially the mean fluorescence intensity(MFI) of MHC classⅡmolecules (P＜0.01),thus LPS fails to induce the up-regulation of co-stimulatory and MHC classⅡmolecules on hypoxia-differentiated DCs,suggesting hypoxia inhibits the phenotypic maturation of DCs.3) Hypoxia inhibits the production of proinflammatory cytokines IL-1,IL-6, TNF-α,and MMP9,but up-regulate the secretion of immune suppressive cytokine TGF-βby DCsTo determine the effects of hypoxia on the cytokine production and migration of DCs,BM-DCs differentiated under normoxic or hypoxic conditions were stimulated with LPS,expression of cytokines and MMP9 on these DCs were assayed by RT-PCR and ELISA.The results showed that DCs differentiated under hypoxic conditions secreted much lower levels of proinflammatory cytokines IL-1β,IL-6 and TNF-α,but higher levels of immune suppressive cytokine TGF-βas compared to DCs differentiated under normoxic conditions,suggesting hypoxia inhibits the production of proinflammatory cytokines IL-1,IL-6 and TNF-α,but up-regulate the secretion of immune suppressive cytokine TGF-βby DCs.In addition,hypoxia can inhibit the expression of MMP9,indicating hypoxia may decrease the migration of DCs through down-regulating the expression of MMP9.4) Hypoxia inhibits the ability of DCs to stimulate CD4~+T cell proliferationTo study the effects of hypoxia on the ability of DCs to prime CD4~+T cells, normoxic or hypoxic DCs from C57BL/6 mice were used to stimulate CFSE-labeled CD4~+T cells from BALB/c mice,and CD4~+T cell proliferation was examined by Flow Cytometry.The results showed that LPS-treated hypoxia-differentiated DCs had poor ability to promote CD4~+T cell proliferation compared with LPS-treated normoxia-differentiated DCs(about 10%vs.about 40-50%),suggesting hypoxia inhibits functional maturation of DCs,especially the ability to mediate adaptive immune response.2.Effects of reoxygenation on the phenotype and function of hypoxia-differentiated DCs1) Reoxygenation promotes the phenotypic maturation of DCsTo determine whether oxygen delivery can influence the phenotypic maturation of hypoxic DCs,reoxygenation was performed on hypoxic DCs for various periods of time,related surface markers were examined by Flow Cytometry.The results showed that reoxygenation of hypoxic DCs obviously up-regulated the expression of CD80,CD86 and MHC classⅡmolecules on hypoxic DCs with increased time.Compared with normoxic mature DCs, these DCs expressed higher levels of CD80,CD86 and MHC classⅡmolecules after 6h of reoxygenation,and peaked after 24-48 h of reoxygenation,displaying a full mature phenotype.These results indicate that hypoxia-differentiated DCs possess fully or even more potential to become mature DCs when sufficient oxygen is available.2) Reoxygenation enhances 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 allogeneic or syngeneic CD4~+T cells.In the proliferation assay, mitomycin-treated DCs from C57BL/6 mice were co-cultured with CD4~+T cells from BALB/c(allogeneic) or C57BL/6 mice plus stimulation of CD3-specific antibody(syngeneic),respectively.DCs reoxygenated for 6 h stimulated vigorous CD4~+T cell proliferation,which was even stronger than normoxic mature DCs(for allogeneic CD4~+T cells,P＜0.01;for syngeneic CD4~+T cells,P＜0.05),suggesting reoxygenation markedly enhances the ability of hypoxic DCs to stimulate CD4~+T cell.3) Reoxygenated DCs induced Th1 cell differentiationWe then investigated CD4~+T cell differentiation driven by reoxygenated DCs in the MLR culture systems.Reoxygenated DCs induced significantly higher number of IFN-γ~+CD4~+T(Th1) cells than DCs differentiated under hypoxic or normoxic conditions did.The levels of IFN-γin the supernatants of MLR systems was also higher in reoxygenated DCs-mediated stimulation system than those in immature DCs(P＜0.05) and normoxia-differentiated mature DCs-mediated stimulation system.IL-4~+CD4~+T(Th2) cells induced by reoxygenated DCs were also elevated.These data indicate that reoxygenated DCs induce Th1 cell differentiation.Taken together,these results suggest that reoxygenated DCs have strong capacity to drive immune response toward a proinflammatory direction.3.Effects of CD137 pathway on phenotype and function of hypoxic DCs1) Anti-CD137 mAb partly promoted the phenotypic maturation of hypoxic DCsTo study the role of CD137 pathway on hypoxic DCs,CD137 expression on DCs differentiated under normoxic or hypoxic conditions were detected via Flow Cytometry.The results showed that CD137 expression was detected on both normoxic and hypoxic DCs,though hypoxia had a trend to down-regulate CD137 expression on DCs,no statistical difference was observed,indicating hypoxia has no obvious influence on the CD137 expression on DCs.To further explore the effects of CD137 on the phenotypic maturation of hypoxic DCs,anti-CD137 mAb was used to stimulate hypoxic DCs,result from Flow Cytometry showed that anti-CD137 mAb treatment markedly increased the expression of co-stimulatory molecules CD80,CD86,and MHC classⅡmolecules on hypoxic DCs,but the levels were still lower than that on normoxic mature DCs,suggesting anti-CD137 mAb may promote the switch of hypoxic DCs toward a more mature direction,but has a limited ability to promote the full phenotypic maturation of hypoxic DCs.2) Anti-CD137 mAb promoted the production of proinflammatory cytokines and MMP9 by hypoxic DCs To clarify the effects of CD137 pathway on the cytokine secretion and migration of hypoxic DCs,mRNA levels for IL-1β,IL-6,IL-12p40,TNF-αand MMP9 in hypoxic DCs stimulated with anti-CD137 mAb were detected by RT-PCR.The results showed that anti-CD137 mAb treatment increased mRNA levels for IL-1β,IL-6,IL-12p40,TNF-αand MMP9,suggesting anti-CD137 mAb may not only promotes the production of proinflammatory cytokines,but also increase the migration of hypoxic DCs through up-regulation of MMP9 expression.3) Effects ofanti-CD137 mAb on the ability of hypoxic DCs to stimulate CD4~+T cell proliferationSince anti-CD137 mAb partly promoted the phenotypic maturation of hypoxic DCs,we further studied the effects of anti-CD137 mAb on the ability of hypoxic DCs to stimulate CD4~+T cell proliferation.Hypoxic DCs from C57BL/6 were treated with anti-CD137 mAb,and then were used to stimulate CD4~+T cells from BALB/c mice,CD4~+T cell proliferation were assayed by Flow Cytometry,no obviously increased CD4~+T cell proliferation was observed when hypoxic DCs were stimulated with anti-CD137 mAb, suggesting anti-CD137 mAb has little influence on the ability of hypoxic DCs to stimulate CD4~+T cell proliferation.Conclusion1.Hypoxia inhibits the maturation and function of DCs induced by LPS1) Hypoxia inhibits the phenotypic maturation of DCs.2) Hypoxia inhibits the secretion of proinflammatory cytokines IL-1,IL-6 and TNF-α,but increases the production of immune suppressive cytokine TGF-βby DCs.3) Hypoxia impairs the ability of DCs to stimulate CD4~+T cell proliferation.2.Reoxygenation promotes the maturation and function of hypoxic DCs1) Reoxygenation promotes the phenotypic maturation of hypoxic DCs.2) Reoxygenation enhances the ability of DCs to stimulate CD4~+T cell proliferation.3) Reoxygenated DCs induced Th1 cell differentiation.3.CD137 pathway partly promotes the maturation and function of hypoxie DCs1) Anti-CD137 mAb partly promotes the phenotypic maturation of DCs.2) Anti-CD137 mAb promotes the secretion of proinflammatory cytokines IL-1, IL-6,IL-12 and TNF-αby DCs3) Anti-CD137 mAb has little influence on the ability of hypoxic DCs to stimulate CD4~+T cell proliferationOriginality1.Hypoxia can inhibit the maturation and function of DCsIt’s the first time to provide evidence that hypoxia can inhibit the phenotypic maturation,proinflammatory cytokines secretion,and the ability to stimulate CD4~+T cell proliferation of DCs.It provides novel experimental basis for studying the effects of hypoxic microenvironments on immune function.2.Reoxygenation can promote the maturation and function of hyoxic DCsIt’s the first time to provide evidence that reoxygenation can promote the phenotypic maturation and the ability to stimulate CD4~+T cells proliferation and Th1 cell differentiation of DCs,suggesting reoxygenation can reverse the inhibitory effects of hypoxic DCs.It is much helpful for the control of ischemia reperfusion injury,graft rejection in organ transplantation,and the immunotherapy for tumor.3.CD137 pathway partly promote the maturation and function of hypoxic DCsWe demonstrate that anti-CD137 mAb can partly promote the phenotypic maturation and proinflammatory cytokine of secretion of DCs.It’s helpful to interpret the mechanisms of in vivo anti-tumor immunity mediated by anti-CD137 mAb.Limitation1.Reoxygenation promotes the maturation and function of hypoxic DCs,but detailed mechanism need to be further studied.2.Mechanism for the effect of anti-CD137 mAb on the maturation and function of hypoxic DCs need to be further explored. PARTⅡAnalysis of CD137 and CD137L Expression in Human Primary Tumor TissuesObjectiveCD137,a member of the tumor necrosis factor receptor family,is expressed primarily on activated T lymphocytes and natural killer cells.In contrast,its ligand CD 137L is mainly expressed on antigen-presenting cells,such as mature dendritic cells, activated B cells,and macrophages.The co-stimulation through CD137-CD137L enhances T cell activation,promotes the rejection of cardiac allografts and skin transplants,and eradicates experimentally induced tumors in mice.However,expression of human CD137 and CD137L is not restricted to immune cells,and the functions of human CD137-CD137L pathway are more complex than that of mice.Expression of CD137 protein has been verified on blood vessel walls in primary malignant tumors.Expression of CD137L has also been found on several human carcinoma cell lines and its function has been analyzed in vitro.Up to now, however,no studies have reported the expression of CD137L in human primary tumor tissues.Therefore,the aim of this study was to assess the expression of CD137 and CD137L in human primary tumor tissues and their potential role in tumor immunity.Methods1.Surgical specimens:A total of 63 tissue specimens were obtained from patients who underwent operations at Qilu Hospital,Shandong University,including 12 human normal tissues,15 benign tumors of epithelial or mesenchymal origin (adenoma and leiomyoma),and 36 malignant tumors of epithelial origin (squamous cell carcinoma and adenocarcinoma).2.Expression of CD137 and CD137L was assessed by immunohistochemistry in the above frozen sections.3.The expression of CD137L on 9 human tumor cell lines(3 hepatocarcinoma,2 lung carcinoma,2 colon carcinoma,1 lymphoma,and 1 leukemia) was detected by RT-PCR.4.To analyze the role of CD137L expressed on tumor cells,tumor cells expressing CD137L were co-cultured with activated T lymphocytes expressing CD137 or with Chinese hamster ovary cells expressing CD137 and then detected by ELISA the levels of cytokines(IL-8,IFN-γ) secreted by tumor cells or activated T cells.Results1.Human primary tumor tissues express CD137 and CD137L at different locationsThe expression of CD137 and CD137L was observed only in human benign (2/15,3/15) or malignant tumors(15/36,21/36),but not in normal tissues(0/12, 0/12).CD137 was expressed on the endothelial cells and smooth muscle cells of the vessel walls within tumor tissues,whereas CD137L was expressed on tumor cells.Though the samples were derived from many different tissue types,the expression of CD137 and CD137L was more common in malignant tumor tissues, especially in moderate or low-differentiated tumor tissues.2.Human tumor cell lines express high levels of CD137LOur results showed that tumor cells in human primary tumor tissues expressed CD137L.We also detected CD137L expression in human tumor cell lines derived from different primary tissues by RT-PCR.Normal un-stimulated peripheral blood mononuclear cells did not express CD137L mRNA but all investigated tumor cell lines(HepG2.2.15,BEL7402,HLE,HT29,L78,A2, U937,HL60,and H6) showed high levels of CD137L expression.Unexpectedly, non-tumor cell line,embryonic venous endotheliocytes ECV304,also expressed CD137L.These results indicated that CD137L was not only expressed in primary tumors but also in tumor cell lines and some non-tumor cell lines,suggesting CD137L expression may be related to rapid cell growth.3.CD137L expressed on tumor cells is functionalTo investigate the role of CD137L on tumor cells,we co-cultured tumor cells expressing CD137L with activated human T cells expressing CD137 in the presence of anti-CD3 mAb,and detected IFN-γsecretion by ELISA.The results showed that CD137-expressing T cells co-cultured with L78 cells produced higher levels of IFN-γthan CD137-1acking T cells(P＜0.001).After blocking CD137-CD137L pathway using anti-CD137L mAb,the levels of IFN-γsignificantly decreased(P＜0.001).This suggests that CD137L on L78 cells could conduct a co-stimulatory signal into T cells and this action could be specifically blocked by anti-CD 137L mAb.To confirm whether CD137L expressed on tumor cells could also signal back into tumor cells,different tumor cells were co-cultured with fixed CD137-CHO cells expressing CD137,and IL-8 was detected by ELISA.The results showed ligation of CD137L on tumor cells by CD137 expressed on CD137-CHO cells elevated the levels of IL-8 produced by HepG2.2.15 cells(P=0.038).Conclusion1.CD137 and CD137L are expressed in different human primary tumor tissues, but with different locations.CD137 was expressed on the endothelial cells and smooth muscle cells of the vessel walls within tumor tissues,whereas CD 137L was expressed on tumor cells.2.CD 137L expression found on tumor cell lines was functional because the ligation of CD137L on lung squamous carcinoma cells L78 with CD137 on T cells induced IFN-γproduction by T cells,and ligation of CD137L on hepatocarcinoma cells HepG2.2.15 with CD137 triggered tumor cells to produce IL-8,Suggesting the expression of CD137 and CD137L in tumor tissues may influence the progression of tumors.OriginalityIt is the first time to prove that CD137 and CD137L can be expressed by different human primary tumor tissues,and the expression of CD137L on tumor cells is functional,suggesting the expression of CD137 and CD137L in tumor tissues may influence the progression of tumors.LimitationMore cases need to be expanded for further study,and specimens derived from the same tissues should be collected for the CD 137 and CD 137L expression assay.更多还原