【作者】 张蘋；

【导师】 张利宁； 陈万军；

【作者基本信息】 山东大学 ， 免疫学， 2007， 博士

【摘要】 目的调节性细胞(regulatory T cell,Treg)是近几年来免疫学的研究热点之一。随着对其研究的不断深入,发现它在维持免疫耐受、控制自身免疫性疾病和移植排斥反应等方面发挥重要的保护作用;在肿瘤中,调节性T细胞发挥着抑制机体抗肿瘤效应的作用。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 cells)的特性研究的较多,已知CD4~+CD25~+Treg细胞有两大类:天然CD4~+CD25~+调节性T细胞(natural occurring regulatory T cell,nTreg)和诱导性CD4~+CD25~+调节性T细胞(inducible regulatory T cells,iTreg)。天然CD4~+CD25~+调节性T细胞在胸腺中产生,存在于正常机体内,占CD4~+T细胞的5-10%;主要标记有CD25、CTLA-4、CD62L、GITR和新近发现的转录因子Foxp3等;诱导而来的CD4~+CD25~+调节性T细胞是在某些条件下(如TGF-β、IL-10等)由CD4~+CD25~-T细胞转化而来,iTreg与nTreg具有相同的表面标志和抑制功能。最近CD4~+CD25~+Treg特异性标志转录因子Foxp3的发现及在体外用细胞因子TGF-β诱导CD4~+CD25~-T细胞转化成具有调节功能的CD4~+CD25~+Treg细胞的成功,大大推动了CD4~+CD25~+调节性T细胞的研究。对于调节性T细胞的深入研究为利用CD4~+CD25~+Treg细胞治疗自身免疫病,控制器官移植后排斥反应带来了新的希望。目前用CD4~+CD25~+调节性T细胞治疗疾病的主要瓶颈是调节性T细胞的数量不足。在小鼠中,CD4~+CD25~+Treg细胞占外周血CD4~+T细胞的5-10%,而人的CD4~+CD25~+Treg细胞仅占外周血CD4~+T细胞5%以下。由此看见,从单个供者分离足够数量的CD4~+CD25~+Treg细胞,用于各种疾病的治疗是不可能的。因此,获得足够数量CD4~+CD25~+Treg细胞是其应用的关键问题之一,探索有效的扩增方法是非常必要的。已知4-1BB协同刺激分子在普通T细胞的活化增殖中发挥重要的作用,但对CD4~+CD25~+Treg细胞的作用尚不清楚。本研究的目的之一是研究4-1BB协同刺激通路在CD4~+CD25~+Treg细胞活化、增殖中的作用;寻找获得足够数量调节性T细胞的途径和方法;推动4-1BB协同刺激通路在调节性T细胞中作用的研究。近几年的研究发现,多种肿瘤患者外周血、肿瘤浸润淋巴组织(TIL)、局部引流淋巴结、腹水中CD4~+CD25~+Treg细胞的数量明显增高,这些增加的CD4~+CD25~+Treg细胞可能参与了肿瘤的免疫逃逸,但对于这些细胞的来源还不清楚。明确CD4~+CD25~+Treg细胞的来源,对寻找清除CD4~+CD25~+Treg细胞的方法,建立有效的肿瘤免疫治疗方法有重要的价值。因此,研究的目的之二是以H22肝癌荷瘤小鼠为模型,研究CD4~+CD25~+Treg细胞数量的变化及其与肿瘤大小的关系,探讨肿瘤生长过程中CD4~+CD25~+Treg细胞数量升高的机制。方法一、4-1BB单抗对CD4~+CD25~+Treg细胞的扩增1.用免疫磁珠的方法从小鼠单个脾细胞中分离纯化CD4~+CD25~-T细胞和CD4~+CD25~+Treg细胞。2.在体外,在不同的培养条件下,用~3H-TdR掺入法检测4-1BB单抗对于CD4~+CD25~+Treg细胞增殖的影响。3.用ELISA方法检测4-1BB单抗扩增的CD4~+CD25~+Treg细胞的培养上清中IL-2和INF-γ细胞因子的表达。4.用RT-PCR和流式细胞术(FCM)的方法检测CD4~+CD25~+Treg细胞的各种表型及Foxp3的表达。5.在体外,用~3H-TdR掺入法测定CD4~+CD25~+Treg细胞的对CD4~+CD25~-T细胞的抑制作用。二、肝癌对CD4~+CD25~+Treg细胞的诱导1.制备H22肝癌荷瘤BALB/c小鼠。2.用FCM检测H22肝癌荷瘤小鼠淋巴结、脾中CD4~+CD25~+T细胞的数量。3.用RT-PCR和FCM的方法检测H22肝癌荷瘤BALB/c小鼠脾脏和淋巴结中Foxp3的表达。4.在体外,用~3H-TdR掺入法检测荷瘤小鼠来源的CD4~+CD25~+T细胞的抑制作用。5.通过转输荷瘤小鼠来源的CD4~+CD25~+T细胞,在裸鼠体内检测CD4~+CD25~+T细胞对CD4~+CD25~+T细胞抗肿瘤作用的抑制效应。6.用ELISA的方法测定H22肝癌荷瘤SCID小鼠血浆中细胞因子TGF-β的表达。结果一、4-1BB单抗对CD4~+CD25~+Treg细胞的扩增1.CD4~+CD25~-T细胞和CD4~+CD25~+T细胞的纯度FCM的方法检测的结果显示,本研究所用得CD4~+CD25~-T细胞的纯度均达到95%以上;CD4~+CD25~+T细胞的纯度可以达到90%。2.4-1BB单抗对CD4~+CD25~+Treg细胞的扩增效应及其对IL-2的依赖T细胞增殖实验的结果表明,在没有外源性IL-2或APCs细胞存在的情况下,只有CD3单抗或/和4-1BB单抗均不能扩增CD4~+CD25~+Treg细胞。但是,在低浓度的细胞因子IL-2存在的情况下,4-1BB单抗可以协同CD3抗体有效地扩增CD4~+CD25~+Treg细胞。在有APCs细胞而没有外源性IL-2存在时,这种扩增作用同样有效。进一步用抗IL-2抗体中和IL-2的作用后,尽管还有APCs细胞的存在,4-1BB单抗也不能扩增CD4~+CD25~+Treg细胞,从另一个方面证明4-1BB单抗对CD4~+CD25~+Treg细胞的扩增需要IL-2存在。3.4-1BB单抗扩增的CD4~+CD25~+T细胞的特征3.1 4-1BB单抗扩增的CD4~+CD25~+T细胞依然表达高水平Foxp3已知Foxp3是调节性T细胞的特异性标志,为了确定4-1BB单抗扩增后CD4~+CD25~+T细胞是否仍然是调节性T细胞,我们分别采用RT-PCR、荧光定量PCR和流式细胞术的方法从RNA和蛋白质水平分别检测了Foxp3的表达,RT-PCR的结果显示:4-1BB单抗扩增的CD4~+CD25~+T细胞仍然表达高水平的Foxp3。荧光定量PCR的结果分析显示,与对照组相比,4-1BB单抗扩增的CD4~+CD25~+T细胞的Foxp3的mRNA表达略有升高。流式细胞术的结果发现,4-1BB单抗扩增的CD4~+CD25~+T细胞的Foxp3的表达与对照组无明显的差异。以上的结果提示4-1BB单抗扩增的CD4~+CD25~+T细胞仍然具有调节性T细胞的特征。3.2 4-1BB单抗扩增的CD4~+CD25~+Treg细胞产生低水平的IL-2,高水平IFN-γELISA的方法检测显示,尽管4-1BB单抗扩增的普通CD4~+CD25~-T细胞产生高水平的IL-2,但是,4-1BB单抗扩增的CD4~+CD25~+Treg细胞的培养上清中仅检测到低水平的IL-2。不同的是,4-1BB单抗扩增的CD4~+CD25~+Treg细胞产生较高水平的IFN-γ,其意义尚不清楚。3.3 4-1BB单抗扩增的CD4~+CD25~+Treg细胞仍然保持其抑制作用从BALB/c小鼠新分离的CD4~+CD25~-T细胞用CD3抗体和APCs细胞刺激的同时,加入4-1BB单抗扩增的CD4~+CD25~+Treg细胞。培养3天后,用~3H-TdR掺入法检测细胞的增殖情况。结果显示,4-1BB单抗扩增的CD4~+CD25~+Treg细胞可以抑制CD4~+CD25~-T细胞的增殖。扩增后的CD4~+CD25~+Treg细胞的抑制率可以达到35.36%;与新鲜分离的CD4~+CD25~+Treg细胞的抑制率无明显的差别。二、肝癌对CD4~+CD25~+Treg细胞的诱导1.H22肝癌荷瘤鼠的脾脏及淋巴结中CD4~+CD25~+T细胞的比例升高H22肝癌细胞接种于BALB/c小鼠使之荷瘤,4-5周后处死小鼠,用流式细胞术分别检测脾脏、淋巴结、引流淋巴结细胞表面分子CD4、CD25的表达情况。结果显示,与未荷瘤BALB/c小鼠相比,H22肝癌荷瘤鼠脾脏、淋巴结和引流淋巴结中CD4~+CD25~+T/CD4~+T的比例明显增加,脾脏:17.28±0.06%vs11.08±0.04%P<0.05;引流淋巴结:18.8±0.06%vs9.5±0.03%,P<0.01:非引流淋巴结:16.28±0.02%vs9.5±0.03%,P<0.01。引流淋巴结中的比例略高于非引流淋巴结,但两者间的差异无统计学意义(P>0.05)。2.小鼠体内CD4~+CD25~+T细胞的数量与肿瘤的大小成正比用不同浓度的H22肝癌细胞制备荷瘤小鼠,隔日测量肿瘤的大小,3-4周后,处死小鼠,分离其脾脏、淋巴结和引流淋巴结,制备单细胞悬液,用FCM的方法检测细胞表面分子CD4、CD25的表达。结果显示,脾脏或淋巴结中CD4~+CD25~+T/CD4~+T细胞比例的增高与肿瘤大小呈正比,说明CD4~+CD25~+T细胞数量的升高有利于肿瘤的生长。3.H22肝癌诱导Treg特异性标志─转录因子Foxp3的表达RT-PC电泳图经过分析软件分析的结果显示,与未注射H22肝癌细胞的BALB/c小鼠相比,H22肝癌荷瘤小鼠的脾脏、淋巴结(包括引流淋巴结和非引流淋巴结)中转录因子Foxp3的表达增加。进一步用FCM的方法检测Foxp3蛋白的表达。结果发现,在H22肝癌荷瘤小鼠的脾脏中,转录因子Foxp3蛋白的表达明显高于对照组BALB/c小鼠。4.H22荷瘤鼠升高的CD4~+CD25~+T细胞在体外失去增殖能力,但具有抑制CD4~+CD25~-T细胞增殖的功能从H22肝癌荷瘤小鼠脾脏纯化的CD4~+CD25~+T细胞,用CD3单抗和APCs细胞刺激,~3H-TdR掺入增殖实验的结果显示,H22肝癌荷瘤小鼠来源的CD4~+CD25~+T细胞在体外不增殖,处于失能状态。T细胞增殖抑制实验可以看到,CD4~+CD25~+T细胞可明显抑制CD4~+CD25~-T细胞的增殖,抑制率达到34.59%。5.H22荷瘤鼠的CD4~+CD25~+T细胞转输荷瘤裸鼠后可抑制CD4~+CD25~-T细胞抗肿瘤效应在H22肝癌荷瘤的裸鼠体内中输入纯化的CD4~+CD25~-T细胞,同时按照CD4~+CD25~+T:CD4~+CD25~-T=2:1的比例输入CD4~+CD25~+T细胞。结果发现,荷瘤9天后,只注射CD4~+CD25~-T细胞的裸鼠的肿瘤体积明显小于其他两组,CD4~+CD25~+T和CD4~+CD25~-T细胞混合注射组的裸鼠肿瘤体积大于单独注射CD4~+CD25~-T细胞的H22肝癌裸鼠组,说明CD4~+CD25~+T在体内具有抑制CD4~+CD25~-T的抗肿瘤作用。6.H22肝癌在裸鼠和SCID鼠体内可诱导CD4~+CD25~-T细胞转变成为CD4~+CD25~+Foxp3~+Treg为了确定荷瘤小鼠升高的CD4~+CD25~+T细胞是否是从普通的CD4~+CD25~-T细胞诱导产生,我们将从正常BALB/c小鼠纯化的CD4~+CD25~-T转输给荷瘤的裸鼠或SCID小鼠。5-10天后,分离荷瘤裸鼠脾脏、淋巴结,用FCM的方法检测是否有CD4~+CD25~+Treg的产生。结果显示大部分转输的CD4~+CD25~-T细胞的在荷瘤的裸鼠或SCID鼠中变成了CD4~+CD25~+T细胞。为了进一步证明这些细胞是否为调节性T细胞,用RT-PCR的方法检测各组H22荷瘤裸鼠Foxp3基因的表达。与未注射CD4~+CD25~-T细胞的H22荷瘤裸鼠相比,注射CD4~+CD25~-T细胞的荷瘤裸鼠的脾脏和淋巴结中Foxp3表达增加。流式细胞术检测显示在荷瘤的SCID小鼠中这些转化的CD4~+CD25~+T细胞高表达Foxp3蛋白。分析流式细胞术的结果发现,CD4~+CD25~+Foxp3~+T细胞占CD4~+T细胞的80-85%。以上的结果表明H22肝癌可诱导CD4~+CD25~-T细胞转变CD4~+CD25~+Foxp3~+Treg细胞,这可能是荷瘤鼠体内调节性T细胞增高的原因之一。7.H22肝癌细胞在体内外产生的高水平TGF-β已知TGF-β在体外可诱导CD4~+CD25~-T细胞转变成CD4~+CD25~+Treg细胞。H22肝癌细胞可诱导CD4~+CD25~-T细胞转变CD4~+CD25~+Foxp3~+Treg细胞的原因是否是TGF-β的作用尚不清楚。ELISA检测结果显示,H22肝癌细胞培养的上清液中含有高水平的TGF-β细胞因子(1.2ug/ml)。另外,荷瘤SCID小鼠的血浆中含有大量的TGF-β,这提示H22肝癌细胞在体内外可以产生的高水平的TGF-β,这可能是肿瘤诱导CD4~+CD25~+Treg细胞产生的原因之一。8.用TGF-βRI基因敲除小鼠证明TGF-β参与肿瘤对CD4~+CD25~+Foxp3~+Treg的诱导将特异性T细胞敲除TGF-βRI小鼠的CD4~+CD25~-T输入荷瘤的Rag1~+小鼠体内。15天后用FCM的方法检测小鼠的脾脏、淋巴结及引流淋巴结中CD4、CD25、Foxp3分子的表达。结果显示,与输入野生型细胞的荷瘤小鼠相比,基因敲出小鼠来源的CD4~+CD25~-T转化为CD4~+CD25~+Foxp3~+Treg的数量明显的减少。证明TGF-β参与肿瘤对CD4~+CD25~+Foxp3~+Treg的诱导结论一、4-1BB单抗对CD4~+CD25~+Treg细胞的扩增1.4-1BB单抗能够扩增CD4~+CD25~+Treg细胞,其扩增作用需要细胞因子IL-2的参与。2.4-1BB单抗扩增的CD4~+CD25~+Treg细胞产生低水平的细胞因子IL-2,但产生高水平的IFN-γ。3.4-1BB单抗扩增的CD4~+CD25~+Treg细胞依然表达转录因子Foxp3,并仍然保持其抑制作用。二、肝癌对CD4~+CD25~+Treg细胞的诱导1.H22肝癌荷瘤小鼠的脾脏及淋巴结中CD4~+CD25~+T细胞的比例较正常BALB/c小鼠升高;升高的CD4~+CD25~+T细胞与H22肝癌小鼠的肿瘤体积的比例呈正相关;其脾脏和淋巴结转录因子Foxp3表达增加。2.在体外,H22肝癌小鼠体内升高的CD4~+CD25~+T细胞处于增殖失能状态,但具有抑制CD4~+CD25~-T细胞增殖的功能;在体内,H22肝癌小鼠的CD4~+CD25~+T细胞促进荷瘤裸鼠的肿瘤的生长。3.在H22肝癌裸鼠和SCID小鼠体内,CD4~+CD25~-T细胞可以诱导成为CD4~+CD25~+Foxp3~+T细胞。4.H22荷瘤SCID小鼠血浆中细胞因子TGF-β表达增加。5.在体内,细胞因子TGF-β参与了CD4~+CD25~+Foxp3~+Treg的转化创新性一、4-1BB单抗对CD4~+CD25~+Treg细胞的扩增1.在国内外率先证明4-1BB单抗活化的协同刺激通路能有效扩增CD4~+CD25~+Treg细胞,其扩增作用依赖于IL-2细胞因子的存在。2.发现4-1BB单抗扩增的CD4~+CD25~+Treg细胞产生高水平的IFN-γ。二、肝癌对CD4~+CD25~+Treg细胞的诱导1.首先在小鼠H22肝癌荷瘤模型中证实肝癌的生长上调CD4~+CD25~+T细胞的比例,且CD4~+CD25~+T细胞数量的升高与H22肝癌小鼠的肿瘤生长呈正相关。2.在国内外首次证明H22肝癌可诱导CD4~+CD25~-T细胞转变成为CD4~+CD25~+Foxp3~+Treg细胞。3.在体内证实,肿瘤释放的细胞因子TGF-β参与了CD4~+CD25~+Foxp3~+Treg的诱导转化。本研究的局限性1.4-1BB单抗刺激CD4~+CD25~+Treg细胞产生高水平IFN-γ的机制和意义尚有待进一步研究。2.TGF-β细胞因子在H22肝癌细胞诱导CD4~+CD25~+Treg细胞中的作用机制有待于进一步证明。目的PDCD4(Programmed Cell Death 4)是1995年Shibahara等在小鼠体内发现的与细胞凋亡有关的基因,可通过与真核细胞翻译起始因子eIF4A发生结合,从而抑制核糖体复合物的形成和蛋白质的合成,抑制细胞的生长。近年来,在PDCD4转基因小鼠和基因敲除小鼠的研究证明PDCD4对肿瘤具有明显抑制作用,是一种新的抑癌基因。关于PDCD4在人类肿瘤发生中的作用资料尚少。目前已知,PDCD4在来自肾脏、肺脏、结肠、乳腺以及神经胶质细胞的肿瘤细胞系中均有表达较低或缺失。在原发性肺癌中PDCD4缺失率高达83%,而且PDCD4缺失与肿瘤的分化程度降低以及预后效果不良密切相关;在胰腺癌中亦发现癌组织中的PDCD4表达与癌旁组织相比明显降低,这提示人PDCD4可能也是一种抑癌基因。但PDCD4在其他肿瘤的表达特点如何?有何临床意义?这些问题目前尚未见报道。胶质瘤是中枢神经系统中最常见的肿瘤,其中50%是恶性肿瘤。尽管有手术、放疗和化疗等多种治疗措施,但患者的预后仍然较差。已有的研究显示6种起源于中枢神经系统肿瘤细胞系中有5株PDCD4蛋白表达完全丢失,这些结果是否提示PDCD4可能在来源于神经系统的原发肿瘤中也存在高频缺失?这促使我们更关注PDCD4在神经系统肿瘤中的表达特点和临床价值。研究结果将对神经胶质细胞瘤的治疗提供新的策略。方法1.病例的收集:收集山东大学齐鲁医院神经外科2005-2006年神经胶质瘤患者的手术标本总共30例,以及其中3例患者的癌旁组织,所有患者术前未接受任何放、化疗治疗。2.用Trizol一步法提取肿瘤标本以及胶质瘤细胞系U251RNA和蛋白。3.分别用RT-PCR、Western blot和免疫组化方法检测肿瘤组织、神经胶质瘤细胞系U251中PDCD4 mRNA和蛋白的表达情况。4.统计学分析PDCD4在不同胶质瘤病例中的表达与胶质瘤临床病理特点间的关系。结果1.PDCD4 mRNA在人胶质瘤中的表达或缺失RT-PCR的检测结果显示:3例神经胶质瘤癌旁组织PDCD4 mRNA均表达,而且表达水平较高。然而,大部分的肿瘤组织标本都出现了不同程度的RNA水平的表达降低,甚至是PDCD4 mRNA表达缺失。在30例肿瘤组织标本中,14例标本PDCD4 mRNA表达缺失,缺失率为47%(14/30),而在剩余的16例肿瘤组织标本中,与癌旁组织表达相比较有12例标本(40%,12/30)出现了不同程度的表达降低。神经胶质瘤细胞系U251,PDCD4 mRNA表达也出现缺失。2.Western Blot发现PDCD4蛋白在胶质瘤中高频缺失Western blot结果显示,3例神经胶质瘤癌旁组织PDCD4蛋白均有表达,而且表达水平较高,PDCD4阳性表达条带亮度明显较强。然而,大部分的肿瘤组织标本都出现了不同程度PDCD4蛋白的表达降低,甚至缺失;而且缺失率与mRNA水平相比较有明显提高。在30例神经胶质瘤组织标本中,有23例肿瘤标本未见阳性条带出现(77%,23/30);有6例肿瘤标本阳性条带与癌旁组织相比较亮度出现明显降低。神经胶质瘤细胞系U251,PDCD4蛋白表达也出现缺失。3.免疫组化进一步证实PDCD4在胶质瘤病例中出现高频率表达缺失免疫组化的结果显示,3例神经胶质瘤癌旁组织PDCD4蛋白均有表达,而且表达水平较高,PDCD4棕黄色阳性染色明显。然而,大部分的肿瘤组织标本都出现了不同程度的蛋白水平的表达降低(棕黄色阳性染色较弱),甚至是出现了PDCD4蛋白表达的缺失,未见明显棕黄色阳性染色出现。在30例神经胶质瘤组织标本中,有23例肿瘤标本(77%)未见棕黄色阳性染色出现;有6例肿瘤标本棕黄色阳性染色与癌旁组织相比较出现明显降低。4.PDCD4表达缺失与胶质瘤临床病理类型以及病例分级之间的相关性为了进一步阐明和了解PDCD4在神经胶质瘤发生、发展中的作用,我们详细分析了PDCD4表达缺失与神经胶质瘤临床病理特点间的关系,结果显示PDCD4表达与神经胶质瘤的组织学分型、病理学分级均无明显相关性,有关PDCD4表达与神经胶质瘤病人预后的关系,正在追踪调查中。结论1.PDCD4 mRNA在人胶质瘤中出现低表达或缺失,PDCD4 mRNA的缺失率达47%。2.胶质瘤病例中PDCD4在蛋白表达水平出现高频率缺失,缺失率达77%。3.PDCD4表达缺失与胶质瘤临床病理类型以及病例分级之间无明显相关性。创新性和意义本研究在国内外首次发现PDCD4在胶质瘤中的表达出现高频率缺失,而且针对PDCD4与胶质瘤临床病理特点间的关系进行具体的分析。为进一步系统地探讨PDCD4对胶质瘤发生、发展的影响以及发现PDCD4对肿瘤发生的抑制机制提供了实验依据和理论条件。研究的局限性1.病例数尚少,正在扩大病例数量。2.PDCD4表达与神经胶质瘤病人预后的关系,正在追踪调查。更多还原

【Abstract】 ObjectiveRegulatory T cells（Tregs）are one of hot topics in immunology research for the past a few years.With the further research of regulatory T cells,it has been known that Tregs exert a very important protective role in maintaining immune tolerance, controlling autoimmune disease and transplantation rejection reaction.But in tumor, they suppress the anti-tumor effect of immune system.Among regulatory T cells, CD4⁺CD25⁺Treg cells are the most studied population.It has been known that CD4⁺CD25⁺Treg cells have two sub-sets:naturally occurring CD4⁺CD25⁺Treg cells （nTregs）and inducible CD4⁺CD25⁺Treg cells（iTregs）.The nTreg cells are from thymus and account for approximately 5-10%of CD4⁺T cells.The major markers for the Treg include CTLA-4,GITR and newly-discovered transcription factor Foxp3,etc. They could suppress self-reactive T cells through cell-cell contact.The iTreg cells ale converted from CD4⁺CD25^-T cells under certain conditions（e.g.TGF-β,IL-10,etc）. They have the same surface markers and suppressive function as nTregs have.But they differ from nTregs in that they exert suppressive function through cytokine.Recently,the discovery of CD4⁺CD25⁺Treg cells specific marker-Foxp3 and the TGF-βassisted in vitro conversion of CD4⁺CD25^-T cells into the CD4⁺CD25⁺Treg cells which have the suppressive function have greatly promoted development of CD4⁺CD25⁺Treg cells research.It sheds light into treating autoimmune diseases and controlling transplantation rejection reaction through using CD4⁺CD25⁺Treg cells. Several prominent immunologists have predicted that the most valuable clinical application of CD4⁺CD25⁺Treg cells is in managing Graft-versus-Host Disease （GVHD）and transplantation rejection reaction.The bottleneck of administering the CD4⁺CD25⁺Treg cells for disease treatment is the insufficiency of Treg cells.The CD4⁺CD25⁺Treg cells account for 5-10%of peripheral CD4⁺T cells in mice but less than 5%in human beings.In order to treat diseases,the amount of CD4⁺CD25⁺Treg cells from one donor is insufficient. Therefore,it becomes imperative to find ways obtaining enough CD4⁺CD25⁺Treg cells.We already knew that co-stimulatory molecule plays an important role in T cells activation and proliferation.In recent years,we found that 4-1BB also expresses on the surface of CD4⁺CD25⁺Treg cells.However,the function of 4-1BB on CD4⁺CD25⁺ Treg cells remains unknown.Hence,one of the purposes of this research is to identify 4-1BB’s role in the proliferation of CD4⁺CD25⁺Treg cells.The research not only explores new ways to provide sufficient amount of CD4⁺CD25⁺Treg cells,but offers theoretical basis for administering CD4⁺CD25⁺Treg cells in GVHD.CD4⁺CD25⁺Treg cells play an important protective role in autoimmune disease and transplantation rejection reaction.But in tumor,they depress anti-tumor capability of the body.In recent years,it has been known that lymphocytes in the vicinity of tumor have not only effector CD8⁺T cells or CD4⁺T cells,but also plenty of CD4⁺CD25⁺Treg cells.These CD4⁺CD25⁺Treg cells are involved in tumor immunity escape.Increased numbers of CD4⁺CD25⁺Treg cells are found in cancer patients’ peripheral blood,tumor infiltrating lymph tissue（TIL）,draining lymph nodes and ascites.However,origin of these cells is unknown.To identify the origin of CD4⁺CD25⁺Treg cells is useful for exploring to eliminate these cells and establish effective tumor immunology therapy modality.Therefore,the second purpose of the research into study the correlation between increased CD4⁺CD25⁺Treg cells and tumor size and explore the mechanism the CD4⁺CD25⁺Treg cells elevation in tumor growth using H22 hepatic carcinoma-bearing BALB/c mice as model.Methods1.The expansion effect of 4-1BB monoclonal antibody on naturally occurring CD4⁺CD25⁺Treg cells（1）To isolate and purify CD4⁺CD25^-T cells from mice spleen and CD4⁺CD25⁺Treg cells by using mice CD4⁺CD25⁺T cell isolation Kit.（2）In vitro,detect the effect of 4-1BB monoclonal antibody on proliferation of CD4⁺CD25⁺Treg cells was detected using 3H-TdR incorporation.（3）To detect the effect of 4-1BB monoclonal antibody on cytokine production of CD4⁺CD25⁺Treg cells using ELISA.（4）To detect CD4⁺CD25⁺Treg cells phenotypes and Foxp3 expression using RT-PCR and FCM.（5）In vitro,the inhibitive effect of 4-1BB mAb-expanded CD4+CD25+Treg cells was detected using 3H-TdR incorporation.2.Induction of hepatic carcinoma on CD4⁺CD25⁺Treg cells（1）To prepare H22 hepatic carcinoma BALB/c mice.（2）To detect the change of CD4⁺CD25⁺T cells numbers in tumor bearing mice using FCM.（3）To detect Foxp3 expression in H22 hepatic carcinoma bearing BALB/c mice spleen and lymph nodes using RT-PCR and FCM.（4）In vitro,detect the inhibitive effect of tumor bearing mice-originated CD4⁺CD25⁺T cells using 3H-TdR incorporation.（5）To infuse tumor bearing mice-originated CD4⁺CD25⁺T cells and CD4⁺CD25^-T cells to tumor bearing nude mice,then detect inhibitive function of CD4⁺CD25⁺T cells to CD4+CD25^-T cells anti-cancer effect.（6）To detect expression of cytokine TGF-βin plasma of tumor bearing SCID mice using ELISA.ResultsⅠ.4-1BB mAb and CD4⁺CD25⁺Treg cells expansion.1.Purification of CD4⁺CD25^-T cells and CD4⁺CD25⁺T cells.The results from FCM showed that purification of CD4⁺CD25^-T cells could reach 95%and purification of CD4⁺CD25⁺T cells could reach 90%.2.The expansion effect of 4-1BB mAb on CD4⁺CD25⁺Treg cells and its dependence on IL-2.The results from T cell proliferation showed that without exogenous IL-2 or APCs cells,CD3 TCR signal in presence or absence of 4-1BB co-stimulatory signal can’t expand CD4⁺CD25⁺ Treg cells.However,with co-existence of low concentration cytokine IL-2,4-1BB could synergistically expand CD4⁺CD25⁺ Treg cells with the presence of CD3 antibody.With the co-existence of APCs cells but no exogenous IL-2 cells,the same effect could also be observed.Furthermore,after the action of IL-2 was neutralized by ani-IL-2 mAb,4-1BB mAb can’t expand CD4⁺CD25⁺ Treg cells even with the presence of APCs cells.These results demonstrated that IL-2 was necessary for expansion of CD4⁺CD25⁺ Treg cells stimulated by 4-1BB Ab.3.The characteristics of 4-1BB mAb expanded CD4⁺CD25⁺ Treg cells.3.1.4-1BB mAb expanded CD4⁺CD25⁺ Treg cells still express Foxp3.The Foxp3 is a specific marker for regulatory T cells.In order to confirm whether 4-1BB mAb-expanded CD4⁺CD25⁺ T cells still have regulatory characteristics,we tested Foxp3 expression on RNA and protein level by using RT-PCR,real time PCR and FCM.The RT-PCR results showed that 4-1BB mAb-expanded CD4⁺CD25⁺Treg cell still express high level of Foxp3.Real time PCR results showed that 4-1BB mAb-expanded CD4⁺CD25⁺Treg cells have slightly elevated levels of Foxp3 mRNA, comparing with control group.FCM results indicated that Foxp3 expression had no significant difference between 4-1BB mAb expanded CD4⁺CD25⁺Treg cells and CD4⁺CD25⁺Treg cells in control group.The results suggested that 4-1BB mAb expanded-CD4⁺CD25⁺Treg cells still are regulatory T cells features. 3.2 4-1BB mAb expanded CD4⁺CD25⁺Treg cells produce low level of IL-2 and high level of IFN-γ.The detection of ELISA showed that although 4-1BB mAb expanded CD4⁺CD25⁺T cells produce high level of IL-2,4-1BB mAb expanded CD4⁺CD25⁺T reg cells only produce low level of IL-2.The difference is that 4-1BB mAb expanded CD4⁺CD25⁺Treg cells produce high level of IFN-γ.The significance remains unknown.3.3.4-1BB mAb expanded-CD4⁺CD25⁺Treg cells remain inhibitive function.Use CD3 antibody and APCs cells to stimulate newly isolated BALB/c CD4⁺CD25⁺T cells,then cultured with 4-1BB mAb expanded CD4⁺CD25⁺Treg cells. After 3 days,use ³H-TdR incorporation assay to detect cell proliferation.Inhibition rate of post expansion CD4⁺CD25⁺Treg cells could reach 35.36%,which has no significant difference from newly isolated CD4⁺CD25⁺Treg cells.Ⅱ.Induction of hepatic carcinoma on CD4⁺CD25⁺Treg cells.1.The number of CD4⁺CD25⁺T cells in the spleen and lymph nodes of H22 hepatic carcinoma mice is elevated.BALB/c mice were inoculated subcutaneously with H22 hepatic carcinoma cells. 4-5 weeks later,appeared with tumor.Use FCM to detect spleen,lymph nodes and draining lymph nodes cell surface marker CD4 and CD25 expression.The results show that comparing with no tumor bearing BALB/c mice,the percentage of CD4⁺CD25⁺T/CD4⁺T increases significantly in H22 hepatic carcinoma bearing mice spleen,lymph nodes and draining lymph nodes.Spleen:17.28+0.06%vs. 11.08±0.04%,P＜0.05;draining lymph nodes:18.8±0.06%vs.9.5±0.03%,P＜0.01; non-draining lymph nodes:16.28±0.02%vs.9.5±0.03%,P＜0.01.Draining lymph nodes have slightly higher percentage than non-draining lymph nodes,but there is no statistically significant difference（P＞0.05）.2.The CD4⁺CD25⁺T cells number is positively correlated with tumor size.Prepare tumor bearing mice from H22 hepatic carcinoma with different concentration and measure tumor size every other day.After 3-4 weeks,kill mice, isolate spleen,lymph nodes,draining lymph nodes and then isolate cells.Use FCM to detect CD4,CD25 expression on cell surface.The results show that with the increase of tumor sizes in H22 hepatic carcinoma bearing mice,the percentage of spleen CD4⁺CD25⁺T/CD4⁺T cells increases,which indicates CD4⁺CD25⁺T count will benefit tumor growth.3.H22 hepatic carcinoma induced Treg specific marker-Foxp3 expression.RT-PCR electrophoresis results show that comparing with no tumor bearing BALB/c mice,Foxp3 expression increases in the spleen and lymph nodes（including draining lymph nodes and non-draining lymph nodes）of H22 tumor beating mice. Further test of Foxp3 protein expression through FCM shows that transcript factor Foxp3 protein expression in H22 tumor beating mice is significantly higher than that of the control group BALB/c mice.4.In vitro,H22 tumor bearing mice CD4⁺CD25⁺T cells are anergy and can inhibit CD4⁺CD25⁺T cells proliferation.Isolated H22 tumor bearing mice spleen CD4⁺CD25⁺T cells,stimulate with CD3 mAb and APCs cells,culture for 3 days under 37℃,5%CO2;3H-TdR incorporation proliferation results show that CD4⁺CD25⁺T cells from H22 tumor bearing mice don’t proliferate.The suppressive assay shows that the CD4⁺CD25⁺T cells could inhibit the proliferation of the CD4⁺CD25^-T cells.The ration of inhibition is 34.59%.5.In vitro,the increased CD4⁺CD25⁺T cells from the tumor bearing BALB/c mice can inhibit CD4⁺CD25^-T cells anti-tumor effect.Infuse purified CD4⁺CD25^-T cells and CD4⁺CD25⁺T cells into H22 tumor bearing nude mice at a ratio of CD4⁺CD25⁺T:CD4⁺CD25^-T=2:1.After 9 days,tumor size in nude mice only received CD4⁺CD25^-T cells infusion is significantly smaller than the other two groups,tumor size of nude mice received mixture of CD4⁺CD25^-T cells CD4⁺CD25⁺T cells is smaller than H22 hepatic carcinoma nude mice not receiving any infusion.6.In H22 tumor-bearing mice and SCID mice,CD4⁺CD25^-T cells can convert into CD4⁺CD25⁺Foxp3⁺T cells.In order to identify whether elevated CD4⁺CD25⁺T cells from tumor bearing mice are converted from CD4⁺CD25^-T cells,we use the purified CD4⁺CD25^-T cells of regular BALB/c mice to inject to tumor bearing nude mice or SCID mice.After 5-10 days,isolate tumor bearing nude mice spleen,lymph nodes,and use FCM to detect presence of CD4⁺CD25⁺Treg.The results show that most infused CD4⁺CD25^-T cells in tumor bearing nude mice or SCID mice converted into CD4⁺CD25⁺T cells.In order to further test whether these cells are regulatory T cells,we adopted RT-PCR to detect H22 tumor bearing nude mice Foxp3 gene expression.Comparing with H22 tumor bearing nude mice not receiving CD4⁺CD25^-T cells,the ones injected with CD4⁺CD25^-T cells have elevated Foxp3 gene expression in spleen and lymph nodes. FCM shows that in tumor bearing SCID mice,these CD4⁺CD25⁺T cells have high expressed Foxp3 protein.FCM shows that CD4⁺CD25⁺Foxp3⁺T cells account for 80-85%of CD4⁺T cells.The aforementioned results demonstrate that H22 hepatic carcinoma can induce conversion of CD4⁺CD25^-T cells into CD4⁺CD25⁺Foxp3Treg cells.This is could be one of the reasons tumor bearing mice have elevated levels of regulatory T cells.7.The TGF-βcytokine expression increased in the plasma of the H22 hepatic carcinoma and this could be one of the reasons of tumor induced CD4⁺CD25⁺Treg cells production.We already knew that in vitro,TGF-βcan induce conversion of CD4⁺CD25^-T cells into CD4⁺CD25⁺Treg cells.Whether TGF-βplayed a role in CD4⁺CD25^-T conversion into CD4⁺CD25⁺Foxp3^Treg cells in H22 tumor bearing mice is unknown.ELISA test shows that H22 hepatic carcinoma supernatant has higher levels of TGF-βcytokines （1.2ug/ml）.Also,tumor bearing SCID mice plasma have large amounts of TGF-β,this indicates that H22 tumor bearing mice can produce high level of TGF-β.This could be one of the reasons tumor induced CD4⁺CD25⁺Treg cells,but more research needs to be done.8.In vivo,TGF-βcytokine is involved in CD4⁺CD25⁺Foxp3⁺Treg conversion.Tumor-bearing Rag1^-/-mice were injected with CD4⁺CD25^-T cells,which comes from T cell specific TGF-βRI knockout mice.After 15 days,CD4、CD25、Foxp3 molecules expression in mice spleen,LN and draining LN were measured by FCM. The results show that the conversion of CD4⁺CD25^-T cells into CD4⁺CD25⁺Foxp3⁺Treg Cells is much less in tumor-bearing Rag1^-/-mice injected knockout mice CD4⁺CD25^-T cells than in Tumor-bearing Rag1^-/-mice injected the wild type CD4⁺CD25^-T cells. ConclusionⅠ.The effect of 4-1BB mAb on expansion of CD4⁺CD25⁺Treg cells.1.In vitro,the expansion of natural CD4⁺CD25⁺Treg cells by agonistic anti-4-1BB antibody needs APC or exogenous IL-2.2.The expanded CD4⁺CD25⁺Treg by 4-1BB mAb produce a few IL-2,but large amounts of IFN-γ3.4-1BB mAb-expanded CD4⁺CD25⁺Treg cells still express Foxp3.4.The CD4⁺CD25⁺Treg cells expanded by anti-4-1BB antibody remain suppressive activity to CD4⁺CD25^-T cells.Ⅱ.Hepatic carcinoma induces CD4⁺CD25⁺Treg cells.1.The number of CD4⁺CD25⁺T cells in lymph nodes and spleen of H22 tumor-bearing mice is higher than that of tumor free mice.The number of CD4⁺CD25⁺T cells in draining lymph nodes is higher than that in non-draining lymph nodes.2.The number of CD4⁺CD25⁺T/CD4⁺T cells is positively related to tumor size in H22 tumor-bearing mice.3.Transcript factor Foxp3 expression increases in the spleen and lymph nodes of H22 hepatic carcinoma mice. 4.In vitro,the CD4⁺CD25⁺T cells from H22 tumor bearing-mice are anergy and can inhibit proliferation of conventional CD⁺CD25T cells.5.In vivo,the CD4⁺CD25⁺T cells can inhibit the anti-tumor effect of conventional CD4+^CD25^-T cells in tumor bearing-nude mice.6.The proliferation of H22 tumor cells can induce the conversion of CD4⁺CD25^-T into CD4⁺CD25⁺foxp3⁺T cells in SCID mice.7.The H22 cells can secret high level of TGF-βin condition of culture or in tumor-bearing SCID mice.8.In vivo,TGF-βcytokine is involved in CD4⁺CD25⁺Foxp3⁺Treg conversion.OriginalityⅠ.4-1BB mAb affect CD4⁺CD25⁺Treg cells proliferation.1.We discovered for the first time that 4-1BB mAb-activated costimulatory pathway can effectively expand CD4⁺CD25⁺Treg cells in dependent manner of IL-2.2.We have demonstrated that 4-1BB mAb-expanded CD4⁺CD25⁺Treg cells maintain regulatory T calls features,expression of Foxp3,production of low levels of IL-2 and immune suppression to conventional T cells.Ⅱ.Hepatic carcinoma can induce CD4⁺CD25⁺Treg cells.1.We have found that the proliferation of H22 hepatocellular carcinoma can increase the number of CD4⁺CD25⁺T cells in spleen and lymph nodes and the elevated number of CD4⁺CD25⁺T cells is positively correlated with size of tumor growth in mice.2.The proliferation of H22 hepaocellular carcinoma in SCID mice can induce conversion of CD4⁺CD25^-T cells into CD4⁺CD25⁺Foxp3⁺Treg cells,which is one reason for increase of CD4⁺CD25⁺Tregs cells in tumor-bearing mice.3.Be confirmed in vivo,TGF-βreleased by tumor is involved in CD4⁺CD25⁺Foxp3⁺Treg cells conversion.Limitations of this study1.Although it has been found in this research that 4-1BB mAb can stimulate CD4⁺CD25⁺Treg cells to produce high level of IFN-γ,its mechanism and significance have not been studied.2.The function of TGF-βin conversion of CD4⁺CD25⁺Treg cells by H22 tumor cells needs to be studied further. ObjectiveProgrammed cell death 4,PDCD4,was cloned Shibahara et al in from mouse cDNA.The expression of PDCD4 was up-regulated during apoptosis in all cell lines tested.Programmed cell death 4（PDCD4）is a translation factor that binds to eIF4A Recent evidence suggests that PDCD4 acts a tumor suppressor oncogene and might represent a promising target for future antitumor therapy.There is e few reports about the function of PDCD4 gene in human tumor progress.Recently,it is known that the tumor cells which derive from kidney,lung and colon express less PDCD4 or lack.In the original lung cancer,the frequency of PDCD4 lack reaches to 83%,and there is a relationship between PDCD4 lack and tumor differentiation or the patient’s prognosis.The same results had been found in the pancreatic tumor.These demonstrate that PDCD4 is a suppressor oncogene. However,It is unknown that the expression character of PDCD4 gene in other organ tumor and whether it is significant in clinical.Glioma is one of the common tumors in central nervous system,A half of the gliomas are histological malignant.Although the operation,radiotherapy and chemotherapy have been used for cancer treatments,the prognosis of patients with glioma is bad.It has been reported that five of six cell lines from central nervous system,lacked the PDCD4 expression.However,the situation of PDCD4 in primary nervous tumors is unknown.The objective of this research clarifies the character of PDCD4 expression in gliomes and clinical significance.The results maybe provide the new strategy for the gliomas therapy.Methods1.Glioma specimens were obtained from 30 patients with tumor aged between 40 and 60 years（mean 50 years）who underwent operations at the Department of Neurosurgery in QiLu Hospital of Shandong University from March 2005 to March 2006.No patient had received adjuvant immunosuppressive treatment including radiotherapy or chemotherapy prior to surgery.The specimens were immediately frozen in liquid nitrogen after surgical tissue removal,and then kept at -80℃.The three normal glial tissues were all acquired from tumor adjacent tissues.2.Total RNAs and protein were extracted from 30 glioma specimens,3 normal tissue specimens and glioma cell line（U251）cells using a modified TRIzol（?）one-step extraction method.3.The PDCD4 mRNA and protein expression in 30 cases of glioma specimens,3 normal tissue specimens and glioma cell line cells were detected by RT-PCR,Western blot and immunohistochemistry.4.The relationship between the PDCD4 expression and the pathological characters was analyzed by staticallyResults 1.Lacked or Decreased expression of PDCD4 mRNA in gliomas.To study role of PDCD4 in the gliomas,we firstly examined the PDCD4 expression at RNA level by semi-quantitative RT-PCR according to normalized OD value of PDCD4 mRNA expression toβ-actin:-,no detection;+,0.01 to 0.49;++,0.5 to 0.99;+++,1.0 to 1.99;++++,=2.00.As illustrated in Fig.1,all of three normal brain tissues adjacent to tumor lesion expressed high level of PDCD4 mRNA, whereas the most of gliomas expressed reduced or lacked PDCD4 mRNA.In 30 glioma specimens,14（47%,14/30）lost PDCD4 mRNA expression.In the remaining 16 glioma samples,12（40%,12/30）appeared differently reduction in PDCD4 mRNA expression compared with three normal tissue controls.For example,PDCD4s were lost in the glioma sample 2,3,7 and distinctly reduced in the glioma sample 4,5,6; while glioma sample 1 was almost identical with the normal tissue control.U251 is cell line originated from glioma.It has reported that the cell line almost loses expression of PDCD4 mRNA.Our result was identical with the previous report.2.High frequent loss of PDCD4 protein expression in gliomas.Based on above studies,we further examined the expression of PDCD4 protein by Western blot.The data from Western blot experiment showed that the loss of PDCD4 proteins was more than that of PDCD4 mRNA.In 30 cases of the gliomas,23 cases（77%）exhibited no relevant PDCD4 bands and other 6 glioma cases showed weak PDCD4 bands,compared with normal brain tissues adjacent to tumor,which showed strong PDCD4 expression.Only sample 1,4,6 had slight expression.While other samples,e.g.,sample 2,3,5,7 were lost in the protein level.Note worthily, some gliomas such as sample 5 expressed PDCD4 mRNA but not PDCD4 protein. These suggested that loss of PDCD4 expression in certain gliomas might occur after mRNA transcription.3.The high frequent loss of PDCD4 protein expression in gliomas was confirmed further by immunohistochemistry.Immunohistochemisry analysis showed that the PDCD4 protein expression in the tumor tissue sections were similar to those from Western blot analysis.The tumor adjacent tissue such as case 24 showed very strong PDCD4 expression,while its paired tumor tissue exhibited decreased PDCD4 staining.In certain gliomas,such as case 29,even there was almost no relevant PDCD4 staining.In addition,it showed that the positive PDCD4 staining localized in cytoplasm.4.The loss of PDCD4 in gliomas had no signification relationships with tumor grade or histological typeTo explore the significance of lacked PDCD4 protein in gliomas,we further analyzed the relationship between loss of PDCD4 proteins and clinic pathological parameters.As expected,there was no significant correlation of lacked PDCD4 expression with sex,age,and tumor size.Unexpectedly there was also no significant relationship between loss of PDCD4 expression and tumor grade or histological type. We are following up the relationship between loss of PDCD4 expression and the prognosis of the gliomas patientsConclusion 1.There are a lacked or decreased expression of PDCD4 mRNA in gliomas,the rate of the lack of PDCD4 expression to 47%.2.High frequent loss of PDCD4 protein expression is found in gliomas,the rate of the lack of PDCD4 expression to 77%.3.The loss of PDCD4 in gliomas had no signification relationships with tumor grade or histological type.OriginalityThis is the first time to discover that high frequent loss of PDCD4 protein expression in gliomas,and to research the relationship between PDCD4 expression and tumor grade or histological type.It provides theoretical basis for PDCD4 on glioma pathogenesis and its inhibitive mechanism on tumor.Limitation1.The more cases are need for next step study.2.With limited follow up time,the correlation between PDCD4 expression and prognosis of patients with glioma has not been finished.We are continuing this follow up study.更多还原