共刺激分子B7-H1在膀胱癌免疫逃逸和免疫治疗中的作用研究
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摘要
在肿瘤发生发展的过程中，肿瘤细胞和机体的免疫系统相互影响，相互制约。肿瘤细胞可以通过多种途径逃避机体的免疫监视作用而使自身得以繁衍和发展，即发生肿瘤的免疫逃逸现象。对肿瘤免疫逃逸机制的研究一直是攻克肿瘤道路上的一个热点和难点。
T细胞介导的细胞免疫是机体抗肿瘤免疫的主要形式之一，肿瘤免疫应答的产生需要免疫系统有效识别和提呈肿瘤抗原，并活化特异性T细胞从而产生免疫杀伤作用。而肿瘤细胞往往可以通过异常表达和(或)缺如某些免疫分子，使特异性T细胞处于无反应或免疫耐受状态，甚至导致T细胞凋亡，从而逃避机体的免疫杀伤作用。B7-H1是一种最近新发现的抑制性共刺激分子，其与受体结合后可以提供抑制性信号从而诱导T细胞凋亡，抑制T细胞的活化和增殖，是近年来发现与肿瘤免疫逃逸有关的重要分子之一。目前研究已发现B7-H1在人肺癌、卵巢癌、结肠直肠癌、黑色素瘤、乳腺癌、胃癌、肾细胞癌等多种肿瘤组织表面都有大量表达，并且与肿瘤的发生发展、浸润转移等生物学行为密切相关。
膀胱癌是我国泌尿外科最常见的恶性肿瘤，其治疗后的高复发率及其恶性进展的生物学行为均提示在膀胱癌中还存在着某些机制使膀胱癌细胞能够逃避机体免疫细胞的杀伤作用，逃避机体的抗肿瘤免疫而继续生长。B7-H1因其在T细胞活化和肿瘤免疫逃逸中的重要作用，已成为目前国内外肿瘤学研究领域的焦点之一。但是B7-H1在膀胱癌中的表达情况如何，特别是B7-H1是否在膀胱癌免疫逃逸机制中发挥作用，以及干预B7-H1信号能否成为膀胱癌免疫治疗的新策略，目前国内外研究较少，鲜有报道。
因此，本研究旨在通过检测B7-H1在膀胱癌中的表达情况，并在体外检测B7-H1过表达对肿瘤特异性细胞毒性T淋巴细胞(cytotoxicity lymphocyte，CTL)抗膀胱癌免疫效应的影响，进而探讨其在膀胱癌免疫逃逸中的作用。在此基础上，通过B7-H1阻断型抗体阻断B7-H1的表达，检测B7-H1阻断对肿瘤特异性CTL及两种新型过继免疫细胞(CD3AK细胞和CD3／CD28共刺激活化T细胞)抗膀胱癌免疫效应的影响，进而探讨B7-H1阻断在膀胱癌免疫治疗中的作用，以期为膀胱癌的免疫治疗提供新的策略。
第一部分膀胱癌组织中B7-H1的表达及其临床意义
目的：检测B7-H1 mRNA及蛋白在膀胱癌组织中的表达情况，并探讨B7-H1的表达与膀胱癌发生发展的关系及其临床意义。
方法：采用RT-PCR方法检测B7-H1基因mRNA在膀胱癌组织中的表达情况，采用免疫组化法检测B7-H1蛋白及FasL蛋白在膀胱癌组织中的表达情况，并分析B7-H1的表达与膀胱癌各生物学指标及FasL表达的相关性，同时随访膀胱癌患者术后生存情况，将B7-H1的表达与患者术后生存时间进行生存分析。
结果：B7-H1 mRNA和蛋白在正常膀胱组织中均无表达，而在膀胱癌组织中表达明显增加，并且B7-H1的表达水平与膀胱癌的病理分级、临床分期等生物学指标密切相关。高分级组及浸润型组B7-H1 mRNA和蛋白的表达水平分别明显高于低分级组和浅表型组，复发组B7-H1蛋白的表达水平明显高于初发组。同时，膀胱癌组织中B7-H1蛋白的表达与FasL蛋白的表达显著相关。另外，B7-H1蛋白的表达与膀胱癌患者的预后具有明显相关性，B7-H1阳性表达的患者其术后生存率明显低于B7-H1阴性表达的患者，相关因素多变量分析发现B7-H1蛋白的表达可以作为膀胱癌的独立预后指标之一。
结论：B7-H1在膀胱癌中存在着过表达，并且其表达与膀胱癌的发生发展及预后密切相关，可以作为评价膀胱癌生物学行为的参考指标。B7-H1与其受体结合后可能通过上调FasL的表达，触发特异性T细胞发生凋亡，从而介导膀胱癌细胞逃避机体的免疫监视作用。
第二部分膀胱癌细胞系BIU-87中B7-H1的表达及LPS对其表达的诱导作用
目的：检测B7-H1在膀胱癌细胞系BIU-87中的表达情况以及LPS对其表达的诱导作用，同时通过LPS诱导形成高表达B7-H1的BIU-87细胞，从而为下一步实验研究做准备。
方法：采用RT-PCR方法和Western blot方法分别检测B7-H1 mRNA和蛋白在BIU-87细胞中的表达情况，随即给予不同浓度的LPS进行诱导，进而采用RT-PCR方法和Western blot方法分别检测诱导后B7-H1 mRNA和蛋白的表达情况。
结果：体外常规培养未经诱导的BIU-87细胞上有B7-H1 mRNA和蛋白的组成性表达，但表达量均较低。经不同浓度LPS(0．5-2μg／mL)体外诱导后，BIU-87细胞上B7-H1 mRNA和蛋白的表达量显著增加，并且随着LPS浓度的增加，B7-H1mRNA和蛋白的表达量亦呈升高趋势，其中当LPS浓度为1μg／mL时，B7-H1mRNA和蛋白的表达量到达高峰。
结论：膀胱癌BIU-87细胞上有B7-H1 mRNA和蛋白的组成性表达，但表达量较低，经LPS诱导后B7-H1 mRNA和蛋白的表达均显著增加。这表明可以通过LPS诱导形成高表达B7-H1的BIU-87细胞，进而模拟体内B7-H1高表达的膀胱癌局部微环境。
第三部分B7-H1参与膀胱癌细胞免疫逃逸的体外实验研究及其干预性治疗
目的：探讨B7-H1在膀胱癌细胞免疫逃逸中的作用以及干预B7-H1信号对肿瘤抗原特异性CTL抗膀胱癌免疫效应的影响。
方法：利用LPS体外诱导形成高表达B7-H1的膀胱癌BIU-87细胞，以模拟膀胱癌患者体内高表达B7-H1的肿瘤微环境。分离健康人外周血T细胞，以丝裂霉素灭活的BIU-87细胞为刺激细胞，激活形成肿瘤抗原特异性CTL。将高表达B7-H1的BIU-87细胞及对照组BIU-87细胞与肿瘤抗原特异性CTL混合培养，MTT法检测肿瘤抗原特异性CTL对BIU-87细胞的杀伤活性，PI染色流式细胞术检测肿瘤抗原特异性CTL自身的凋亡率。然后利用B7-H1单克隆抗体阻断BIU-87细胞表面B7-H1的表达，MTT法和流式细胞术分别检测其对肿瘤抗原特异性CTL抗膀胱癌活性和自身凋亡率的影响。
结果：肿瘤抗原特异性CTL对高表达B7-H1的BIU-87细胞的杀伤活性与对照组相比明显降低，而其自身的凋亡率却明显增加，体外存活时间亦明显缩短。B7-H1表达被阻断后，肿瘤抗原特异性CTL对BIU-87细胞的杀伤活性明显增强，其自身凋亡率亦明显降低，并且呈剂量依赖性。
结论：膀胱癌细胞可以通过表达B7-H1诱导肿瘤特异性CTL凋亡从而逃避肿瘤特异性CTL的杀伤作用，B7-H1可能在膀胱癌的发生发展及免疫逃逸机制中发挥重要的作用，干预B7-H1信号将有望成为膀胱癌免疫治疗的新策略。
第四部分B7-H1阻断对CD3AK细胞及CD3／CD28共刺激活化T细胞体外抗膀胱癌作用的影响
目的：探讨B7-H1阻断对CD3AK细胞及CD3／CD28共刺激活化T细胞体外抗膀胱癌作用的影响。
方法：分别利用CD3 mAb和CD3联合CD28 mAb刺激健康人外周血淋巴细胞诱导产生CD3AK细胞及CD3／CD28共刺激活化T细胞。然后于培养体系中加入B7-H1阻断型抗体阻断B7-H1通路，~3H-TdR渗入法检测两种免疫效应细胞的体外增殖能力，ELISA法检测其分泌IFN-γ、TNF-α和IL-10的水平。最后，将两种免疫效应细胞分别作用于膀胱癌BIU-87细胞，MTT法检测其对BIU-87细胞的体外杀伤活性。
结果：B7-H1被阻断后，CD3AK细胞及CD3／CD28共刺激活化T细胞的体外增殖能力明显增强，存活时间明显延长；并且其分泌IFN-γ、TNF-α的水平明显提高，而分泌IL-10的水平明显下降；同时B7-H1被阻断后，CD3AK细胞及CD3／CD28共刺激活化T细胞对BIU-87细胞的体外杀伤活性亦明显升高。
结论：阻断B7-H1通路可以促进和维持CD3AK细胞及CD3／CD28共刺激活化T细胞的体外增殖和活化，并增强其杀伤膀胱癌细胞的免疫效应。B7-H1阻断有可能成为辅助增强膀胱癌过继细胞免疫治疗疗效的新策略。
Interactions between the immune system and malignant cells play an important role intumorigenesis.Failure of the immune system to detect and reject transformed cells maylead to tumor development and tumors can use multiple mechanisms to escape fromimmune-mediated rejection.The study of tumor immuoescape mechanisms is always a hotspot and nodus on the way of tumor treatment.
T-cell mediated cytoimmunity is a main form of host antitumor immunity.Recognitionand presentation of tumor antigen and activation of tumor specific T-cell is necessary forthe start of tumor immune response.Tumor cells can promote the anergy or tolerance andeven apoptosis of T cells in order to escape host immune destruction by overexpressionor/and absence some immune molecules.B7-H1 is a recently discovered T-cell suppressivecostimulatory molecule that has been implicated as an important molecule in tumorimmunoescape.It can inhibit immune responses by inducing T-cell apoptosis,impairingcytokine production,and diminishing the cytotoxicity of activated T cells.A lot of humancancers,including lung,ovarian,colon,breast,kidney,lymphoma,and melanoma,havenow been reported to aberrantly express B7-H1 and the expression of B7-H1 was stronglyassociated with the genesis,development and aggressive biological behavior of tumors.
Bladder cancer is the most common malignant tumor of urology in China.The highrecurrence rate and aggressive biological behavior after operation indicate that there are still some mechanisms which can promote bladder cancer cells escaping the lethal effect ofimmune cells.B7-H1 has been a hot spot in the field of tumor research for its importantrole in tumor immunoescape.However,it is not clear that whether B7-H1 is aberrantlyexpressed in human bladder cancer,whether and how B7-H1 plays an important role inimmunoescape of bladder cancer and whether the manipulation of B7-H1 could become abeneficial target for immunotherapy in human bladder cancer.
Therefore,this research investigated the expression of B7-H1 in bladder cancer tissuesand the effects of B7-H1 overexpression on immunoescape of bladder cancer in vitro.Moreover,we further investigated the effects of B7-H1 blockade on the antitumorimmunity mediated by tumor antigen specific CTL and two immune effector cells (CD3AKcells and CD3/CD28-costimulated T cells) in order to explore a new strategy ofimmunotherapy in bladder cancer.
PartⅠExpression of B7-H1 in bladder cancer tissue and itsclinical significance
Objective:To investigate B7-H1 mRNA and protein expression in bladder cancer tissueand its clinical significance.
Methods:RT-PCR method was used to detect B7-H1 mRNA expression in 20 cases ofbladder cancer and immunohistochemical method was used to detect B7-H1protein and FasL protein expression in 50 cases of bladder cancer.Therelationship among B7-H1 expression,biological characters of bladder cancerand FasL expression was also analyzed.Meanwhile,the relationship betweenB7-H1 expression and survival time of bladder cancer patients was alsodetected using survival analysis.
Results:There was no B7-H1 expression in nomal bladder tissue.However,B7-H1 expression increased significantly in bladder cancer.B7-H 1 mRNA and proteinexpression were strongly associated with pathological grade and clinical stageof bladder cancer.Meanwhile B7-H1 protein expression was associated withrecurrence of bladder cancer and had a positive correlation with FasL proteinexpression.The survival rate was significantly worse in patients with B7-H1protein positive expression than in those with B7-H1 protein negativeexpression and multivariable analysis revealed that B7-H1 protein expressioncould be regarded as an independent factor in evaluating the prognosis ofbladder cancer.
Conclusion:B7-H1 expression is strongly associated with neoplastic progression andpoor prognosis of bladder cancer.B7-H1 may become one of parameters forunderstanding the biological behavior of bladder cancer.The basis for theseassociations may relate to the recognized ability of B7-H1 to involve in tumorimmune escape by upregulation of FasL and then inducing T-cell apoptosis.
PartⅡExpression of B7-H1 on BIU-87 cell line and the inductionby LPS in vitro
Objective:To investigate B7-H1 mRNA and protein expression on BIU-87 cell line and theinduction by LPS in vitro.To simulate bladder cancer microenvironment withLPS-induced high expression of B7-H1 on BIU-87 cell line and makepreparation for the next research.
Methods:B7-H1 mRNA and protein on BIU-87 cells were detected by RT-PCR andWestern blot methods respectively.Then various concentrations of LPS wereadded into the culture system to induce B7-H1 expression on BIU-87 cells. After that B7-H 1 mRNA and protein expression were also detected by RT-PCRand Western blot method respectively.
Results:B7-H1 mRNA and protein were constitutively expressed on BIU-87 cells at alow level.But after induced by LPS of various concentrations,B7-H1 mRNAand protein were further up-regulated in a concentration-dependent manner,with the most pronounced effect observed at 1μg/ml.
Conclusion:B7-H1 was constitutively expressed on BIU-87 cells at a low level and wasfurther up-regulated after induced by LPS.We could get B7-H1 high expressedBIU-87 cells by LPS induction and then simulate bladder cancermicroenvironment in vitro.
PartⅢEffect of B7-H1 on immunoescape of bladder cancer invitro and tumor immunotherapy by B7-H1 blockade
Objective:To investigate the effect of B7-H1 on immunoescape of bladder cancer and theeffect of B7-H1 blockade on the antitumor immunity mediated by tumorantigen specific CTL in vitro.
Methods:T cells were isolated from peripheral blood and induced to tumor antigenspecific CTL by stimulating with mitomycin inactivated BIU-87 ceils.Andthen tumor antigen specific CTL were co-cultured with BIU-87 cells whichhighly expressed B7-H1 by LPS induction.The cytotoxicity of tumor antigenspecific CTL against BIU-87 cells was measured by MTT method andapoptotic rate of tumor antigen specific CTL was measured by flow cytometrywith PI labeling technique.After that B7-H1 mAbs was used to block B7-H1pathway.The cytotoxicity and apoptotic rate of tumor antigen specific CTL were measured by MTT method and flow cytometry respectively.
Results:After co-cultured with BIU-87 cells which highly expressed B7-H1,thecytotoxicity of tumor antigen specific CTL against BIU-87 cells was depressedobviously.But its apoptotic rate increased greatly and its survival time in vitrowas significantly shortened.After B7-H1 blockade,the cytotoxicity of tumorantigen specific CTL returned to a high level and its apoptotic rate decreasedobviously in a concentration-dependent manner.
Conclusion:BIU-87 cells can escape the killing effect of tumor antigen specific CTL byinducing CTL apoptosis through B7-H1 pathway.B7-H1 may play animportant role in immunoescape of bladder cancer and the manipulation ofB7-H 1 may become a beneficial target for immunotherapy in bladder cancer.
PartⅣEffects of B7-H1 blockade on the cytotoxicity of CD3AK cellsand CD3/CD28-costimulated T cells againstbladder cancer cells in vitro
Objective:To investigate the effects of B7-H1 blockade on the cytotoxicity of CD3AKcells and CD3/CD28-costimulated T cells against bladder cancer cells in vitro.
Methods:Two immune effector cells,CD3AK cells and CD3/CD28-costimulated T cellswere produced by normal human peripheral blood lymphocytes stimulated withCD3 mAbs and CD3 plus CD28 mAbs respectively.Then B7-H1 mAbs wasadded to block B7-H1 pathway.The proliferation of these two immune effectorcells were measured by ~3H-thymidine incorporation assay and IFN-γ,TNF-αand IL-10 secretion were measured by ELISA method.Meanwhile,thecytotoxicity of these two immune effector cells against BIU-87 cells was measured by MTT method.
Results:Blockade of B7-H1 greatly promoted the proliferation of CD3AK cells andCD3/CD28-costimulated T cells and extended their survival time in vitro.Italso significantly enhanced their IFN-γand TNF-αsecretion but suppressedIL-10 secretion.Meanwhile,the cytotoxicity of CD3AK cells andCD3/CD28-costimulated T cells against BIU-87 cells was significantlyenhanced.
Conclusion:Blockade of B7-H1 can promote the proliferation and activation of CD3AKcells and CD3/CD28-costimulated T cells.It can also improve the antitumorimmunity mediated by them against bladder cancer.The manipulation ofB7-H1 may become a new strategy to improve the therapeutic effect ofadoptive cellular immunotherapy against bladder cancer.
T细胞介导的细胞免疫是机体抗肿瘤免疫的主要形式之一，肿瘤免疫应答的产生需要免疫系统有效识别和提呈肿瘤抗原，并活化特异性T细胞从而产生免疫杀伤作用。而肿瘤细胞往往可以通过异常表达和(或)缺如某些免疫分子，使特异性T细胞处于无反应或免疫耐受状态，甚至导致T细胞凋亡，从而逃避机体的免疫杀伤作用。B7-H1是一种最近新发现的抑制性共刺激分子，其与受体结合后可以提供抑制性信号从而诱导T细胞凋亡，抑制T细胞的活化和增殖，是近年来发现与肿瘤免疫逃逸有关的重要分子之一。目前研究已发现B7-H1在人肺癌、卵巢癌、结肠直肠癌、黑色素瘤、乳腺癌、胃癌、肾细胞癌等多种肿瘤组织表面都有大量表达，并且与肿瘤的发生发展、浸润转移等生物学行为密切相关。
膀胱癌是我国泌尿外科最常见的恶性肿瘤，其治疗后的高复发率及其恶性进展的生物学行为均提示在膀胱癌中还存在着某些机制使膀胱癌细胞能够逃避机体免疫细胞的杀伤作用，逃避机体的抗肿瘤免疫而继续生长。B7-H1因其在T细胞活化和肿瘤免疫逃逸中的重要作用，已成为目前国内外肿瘤学研究领域的焦点之一。但是B7-H1在膀胱癌中的表达情况如何，特别是B7-H1是否在膀胱癌免疫逃逸机制中发挥作用，以及干预B7-H1信号能否成为膀胱癌免疫治疗的新策略，目前国内外研究较少，鲜有报道。
因此，本研究旨在通过检测B7-H1在膀胱癌中的表达情况，并在体外检测B7-H1过表达对肿瘤特异性细胞毒性T淋巴细胞(cytotoxicity lymphocyte，CTL)抗膀胱癌免疫效应的影响，进而探讨其在膀胱癌免疫逃逸中的作用。在此基础上，通过B7-H1阻断型抗体阻断B7-H1的表达，检测B7-H1阻断对肿瘤特异性CTL及两种新型过继免疫细胞(CD3AK细胞和CD3／CD28共刺激活化T细胞)抗膀胱癌免疫效应的影响，进而探讨B7-H1阻断在膀胱癌免疫治疗中的作用，以期为膀胱癌的免疫治疗提供新的策略。
第一部分膀胱癌组织中B7-H1的表达及其临床意义
目的：检测B7-H1 mRNA及蛋白在膀胱癌组织中的表达情况，并探讨B7-H1的表达与膀胱癌发生发展的关系及其临床意义。
方法：采用RT-PCR方法检测B7-H1基因mRNA在膀胱癌组织中的表达情况，采用免疫组化法检测B7-H1蛋白及FasL蛋白在膀胱癌组织中的表达情况，并分析B7-H1的表达与膀胱癌各生物学指标及FasL表达的相关性，同时随访膀胱癌患者术后生存情况，将B7-H1的表达与患者术后生存时间进行生存分析。
结果：B7-H1 mRNA和蛋白在正常膀胱组织中均无表达，而在膀胱癌组织中表达明显增加，并且B7-H1的表达水平与膀胱癌的病理分级、临床分期等生物学指标密切相关。高分级组及浸润型组B7-H1 mRNA和蛋白的表达水平分别明显高于低分级组和浅表型组，复发组B7-H1蛋白的表达水平明显高于初发组。同时，膀胱癌组织中B7-H1蛋白的表达与FasL蛋白的表达显著相关。另外，B7-H1蛋白的表达与膀胱癌患者的预后具有明显相关性，B7-H1阳性表达的患者其术后生存率明显低于B7-H1阴性表达的患者，相关因素多变量分析发现B7-H1蛋白的表达可以作为膀胱癌的独立预后指标之一。
结论：B7-H1在膀胱癌中存在着过表达，并且其表达与膀胱癌的发生发展及预后密切相关，可以作为评价膀胱癌生物学行为的参考指标。B7-H1与其受体结合后可能通过上调FasL的表达，触发特异性T细胞发生凋亡，从而介导膀胱癌细胞逃避机体的免疫监视作用。
第二部分膀胱癌细胞系BIU-87中B7-H1的表达及LPS对其表达的诱导作用
目的：检测B7-H1在膀胱癌细胞系BIU-87中的表达情况以及LPS对其表达的诱导作用，同时通过LPS诱导形成高表达B7-H1的BIU-87细胞，从而为下一步实验研究做准备。
方法：采用RT-PCR方法和Western blot方法分别检测B7-H1 mRNA和蛋白在BIU-87细胞中的表达情况，随即给予不同浓度的LPS进行诱导，进而采用RT-PCR方法和Western blot方法分别检测诱导后B7-H1 mRNA和蛋白的表达情况。
结果：体外常规培养未经诱导的BIU-87细胞上有B7-H1 mRNA和蛋白的组成性表达，但表达量均较低。经不同浓度LPS(0．5-2μg／mL)体外诱导后，BIU-87细胞上B7-H1 mRNA和蛋白的表达量显著增加，并且随着LPS浓度的增加，B7-H1mRNA和蛋白的表达量亦呈升高趋势，其中当LPS浓度为1μg／mL时，B7-H1mRNA和蛋白的表达量到达高峰。
结论：膀胱癌BIU-87细胞上有B7-H1 mRNA和蛋白的组成性表达，但表达量较低，经LPS诱导后B7-H1 mRNA和蛋白的表达均显著增加。这表明可以通过LPS诱导形成高表达B7-H1的BIU-87细胞，进而模拟体内B7-H1高表达的膀胱癌局部微环境。
第三部分B7-H1参与膀胱癌细胞免疫逃逸的体外实验研究及其干预性治疗
目的：探讨B7-H1在膀胱癌细胞免疫逃逸中的作用以及干预B7-H1信号对肿瘤抗原特异性CTL抗膀胱癌免疫效应的影响。
方法：利用LPS体外诱导形成高表达B7-H1的膀胱癌BIU-87细胞，以模拟膀胱癌患者体内高表达B7-H1的肿瘤微环境。分离健康人外周血T细胞，以丝裂霉素灭活的BIU-87细胞为刺激细胞，激活形成肿瘤抗原特异性CTL。将高表达B7-H1的BIU-87细胞及对照组BIU-87细胞与肿瘤抗原特异性CTL混合培养，MTT法检测肿瘤抗原特异性CTL对BIU-87细胞的杀伤活性，PI染色流式细胞术检测肿瘤抗原特异性CTL自身的凋亡率。然后利用B7-H1单克隆抗体阻断BIU-87细胞表面B7-H1的表达，MTT法和流式细胞术分别检测其对肿瘤抗原特异性CTL抗膀胱癌活性和自身凋亡率的影响。
结果：肿瘤抗原特异性CTL对高表达B7-H1的BIU-87细胞的杀伤活性与对照组相比明显降低，而其自身的凋亡率却明显增加，体外存活时间亦明显缩短。B7-H1表达被阻断后，肿瘤抗原特异性CTL对BIU-87细胞的杀伤活性明显增强，其自身凋亡率亦明显降低，并且呈剂量依赖性。
结论：膀胱癌细胞可以通过表达B7-H1诱导肿瘤特异性CTL凋亡从而逃避肿瘤特异性CTL的杀伤作用，B7-H1可能在膀胱癌的发生发展及免疫逃逸机制中发挥重要的作用，干预B7-H1信号将有望成为膀胱癌免疫治疗的新策略。
第四部分B7-H1阻断对CD3AK细胞及CD3／CD28共刺激活化T细胞体外抗膀胱癌作用的影响
目的：探讨B7-H1阻断对CD3AK细胞及CD3／CD28共刺激活化T细胞体外抗膀胱癌作用的影响。
方法：分别利用CD3 mAb和CD3联合CD28 mAb刺激健康人外周血淋巴细胞诱导产生CD3AK细胞及CD3／CD28共刺激活化T细胞。然后于培养体系中加入B7-H1阻断型抗体阻断B7-H1通路，~3H-TdR渗入法检测两种免疫效应细胞的体外增殖能力，ELISA法检测其分泌IFN-γ、TNF-α和IL-10的水平。最后，将两种免疫效应细胞分别作用于膀胱癌BIU-87细胞，MTT法检测其对BIU-87细胞的体外杀伤活性。
结果：B7-H1被阻断后，CD3AK细胞及CD3／CD28共刺激活化T细胞的体外增殖能力明显增强，存活时间明显延长；并且其分泌IFN-γ、TNF-α的水平明显提高，而分泌IL-10的水平明显下降；同时B7-H1被阻断后，CD3AK细胞及CD3／CD28共刺激活化T细胞对BIU-87细胞的体外杀伤活性亦明显升高。
结论：阻断B7-H1通路可以促进和维持CD3AK细胞及CD3／CD28共刺激活化T细胞的体外增殖和活化，并增强其杀伤膀胱癌细胞的免疫效应。B7-H1阻断有可能成为辅助增强膀胱癌过继细胞免疫治疗疗效的新策略。
Interactions between the immune system and malignant cells play an important role intumorigenesis.Failure of the immune system to detect and reject transformed cells maylead to tumor development and tumors can use multiple mechanisms to escape fromimmune-mediated rejection.The study of tumor immuoescape mechanisms is always a hotspot and nodus on the way of tumor treatment.
T-cell mediated cytoimmunity is a main form of host antitumor immunity.Recognitionand presentation of tumor antigen and activation of tumor specific T-cell is necessary forthe start of tumor immune response.Tumor cells can promote the anergy or tolerance andeven apoptosis of T cells in order to escape host immune destruction by overexpressionor/and absence some immune molecules.B7-H1 is a recently discovered T-cell suppressivecostimulatory molecule that has been implicated as an important molecule in tumorimmunoescape.It can inhibit immune responses by inducing T-cell apoptosis,impairingcytokine production,and diminishing the cytotoxicity of activated T cells.A lot of humancancers,including lung,ovarian,colon,breast,kidney,lymphoma,and melanoma,havenow been reported to aberrantly express B7-H1 and the expression of B7-H1 was stronglyassociated with the genesis,development and aggressive biological behavior of tumors.
Bladder cancer is the most common malignant tumor of urology in China.The highrecurrence rate and aggressive biological behavior after operation indicate that there are still some mechanisms which can promote bladder cancer cells escaping the lethal effect ofimmune cells.B7-H1 has been a hot spot in the field of tumor research for its importantrole in tumor immunoescape.However,it is not clear that whether B7-H1 is aberrantlyexpressed in human bladder cancer,whether and how B7-H1 plays an important role inimmunoescape of bladder cancer and whether the manipulation of B7-H1 could become abeneficial target for immunotherapy in human bladder cancer.
Therefore,this research investigated the expression of B7-H1 in bladder cancer tissuesand the effects of B7-H1 overexpression on immunoescape of bladder cancer in vitro.Moreover,we further investigated the effects of B7-H1 blockade on the antitumorimmunity mediated by tumor antigen specific CTL and two immune effector cells (CD3AKcells and CD3/CD28-costimulated T cells) in order to explore a new strategy ofimmunotherapy in bladder cancer.
PartⅠExpression of B7-H1 in bladder cancer tissue and itsclinical significance
Objective:To investigate B7-H1 mRNA and protein expression in bladder cancer tissueand its clinical significance.
Methods:RT-PCR method was used to detect B7-H1 mRNA expression in 20 cases ofbladder cancer and immunohistochemical method was used to detect B7-H1protein and FasL protein expression in 50 cases of bladder cancer.Therelationship among B7-H1 expression,biological characters of bladder cancerand FasL expression was also analyzed.Meanwhile,the relationship betweenB7-H1 expression and survival time of bladder cancer patients was alsodetected using survival analysis.
Results:There was no B7-H1 expression in nomal bladder tissue.However,B7-H1 expression increased significantly in bladder cancer.B7-H 1 mRNA and proteinexpression were strongly associated with pathological grade and clinical stageof bladder cancer.Meanwhile B7-H1 protein expression was associated withrecurrence of bladder cancer and had a positive correlation with FasL proteinexpression.The survival rate was significantly worse in patients with B7-H1protein positive expression than in those with B7-H1 protein negativeexpression and multivariable analysis revealed that B7-H1 protein expressioncould be regarded as an independent factor in evaluating the prognosis ofbladder cancer.
Conclusion:B7-H1 expression is strongly associated with neoplastic progression andpoor prognosis of bladder cancer.B7-H1 may become one of parameters forunderstanding the biological behavior of bladder cancer.The basis for theseassociations may relate to the recognized ability of B7-H1 to involve in tumorimmune escape by upregulation of FasL and then inducing T-cell apoptosis.
PartⅡExpression of B7-H1 on BIU-87 cell line and the inductionby LPS in vitro
Objective:To investigate B7-H1 mRNA and protein expression on BIU-87 cell line and theinduction by LPS in vitro.To simulate bladder cancer microenvironment withLPS-induced high expression of B7-H1 on BIU-87 cell line and makepreparation for the next research.
Methods:B7-H1 mRNA and protein on BIU-87 cells were detected by RT-PCR andWestern blot methods respectively.Then various concentrations of LPS wereadded into the culture system to induce B7-H1 expression on BIU-87 cells. After that B7-H 1 mRNA and protein expression were also detected by RT-PCRand Western blot method respectively.
Results:B7-H1 mRNA and protein were constitutively expressed on BIU-87 cells at alow level.But after induced by LPS of various concentrations,B7-H1 mRNAand protein were further up-regulated in a concentration-dependent manner,with the most pronounced effect observed at 1μg/ml.
Conclusion:B7-H1 was constitutively expressed on BIU-87 cells at a low level and wasfurther up-regulated after induced by LPS.We could get B7-H1 high expressedBIU-87 cells by LPS induction and then simulate bladder cancermicroenvironment in vitro.
PartⅢEffect of B7-H1 on immunoescape of bladder cancer invitro and tumor immunotherapy by B7-H1 blockade
Objective:To investigate the effect of B7-H1 on immunoescape of bladder cancer and theeffect of B7-H1 blockade on the antitumor immunity mediated by tumorantigen specific CTL in vitro.
Methods:T cells were isolated from peripheral blood and induced to tumor antigenspecific CTL by stimulating with mitomycin inactivated BIU-87 ceils.Andthen tumor antigen specific CTL were co-cultured with BIU-87 cells whichhighly expressed B7-H1 by LPS induction.The cytotoxicity of tumor antigenspecific CTL against BIU-87 cells was measured by MTT method andapoptotic rate of tumor antigen specific CTL was measured by flow cytometrywith PI labeling technique.After that B7-H1 mAbs was used to block B7-H1pathway.The cytotoxicity and apoptotic rate of tumor antigen specific CTL were measured by MTT method and flow cytometry respectively.
Results:After co-cultured with BIU-87 cells which highly expressed B7-H1,thecytotoxicity of tumor antigen specific CTL against BIU-87 cells was depressedobviously.But its apoptotic rate increased greatly and its survival time in vitrowas significantly shortened.After B7-H1 blockade,the cytotoxicity of tumorantigen specific CTL returned to a high level and its apoptotic rate decreasedobviously in a concentration-dependent manner.
Conclusion:BIU-87 cells can escape the killing effect of tumor antigen specific CTL byinducing CTL apoptosis through B7-H1 pathway.B7-H1 may play animportant role in immunoescape of bladder cancer and the manipulation ofB7-H 1 may become a beneficial target for immunotherapy in bladder cancer.
PartⅣEffects of B7-H1 blockade on the cytotoxicity of CD3AK cellsand CD3/CD28-costimulated T cells againstbladder cancer cells in vitro
Objective:To investigate the effects of B7-H1 blockade on the cytotoxicity of CD3AKcells and CD3/CD28-costimulated T cells against bladder cancer cells in vitro.
Methods:Two immune effector cells,CD3AK cells and CD3/CD28-costimulated T cellswere produced by normal human peripheral blood lymphocytes stimulated withCD3 mAbs and CD3 plus CD28 mAbs respectively.Then B7-H1 mAbs wasadded to block B7-H1 pathway.The proliferation of these two immune effectorcells were measured by ~3H-thymidine incorporation assay and IFN-γ,TNF-αand IL-10 secretion were measured by ELISA method.Meanwhile,thecytotoxicity of these two immune effector cells against BIU-87 cells was measured by MTT method.
Results:Blockade of B7-H1 greatly promoted the proliferation of CD3AK cells andCD3/CD28-costimulated T cells and extended their survival time in vitro.Italso significantly enhanced their IFN-γand TNF-αsecretion but suppressedIL-10 secretion.Meanwhile,the cytotoxicity of CD3AK cells andCD3/CD28-costimulated T cells against BIU-87 cells was significantlyenhanced.
Conclusion:Blockade of B7-H1 can promote the proliferation and activation of CD3AKcells and CD3/CD28-costimulated T cells.It can also improve the antitumorimmunity mediated by them against bladder cancer.The manipulation ofB7-H1 may become a new strategy to improve the therapeutic effect ofadoptive cellular immunotherapy against bladder cancer.
引文
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2、 Pardoll DM. Spinning molecular immunology into successful Immunotherapy [J]. Nat Rev Immunol, 2002, 2(4): 227-238.
3、 Dong H, Zhu G, Tamada K, et al. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion [J]. Nat Med, 1999, 5(12): 1365-1369.
4、 Mazanet MM, Hughes CC. B7-H1 is expressed by human endothelial cells and suppresses T cell cytokine synthesis [J]. J Immunol, 2002, 169(7): 3581-3588.
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11、 Okazaki T, Maeda A, Nishimura H, et al. PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-dominain-containting tyrosine phosphatase 2 to phosphotyrosine [J]. Proc Natl Acad Sci USA, 2001, 98 (24): 13866-13871.
12、 Nishimura H, Agata Y, Kawasaki A, et al. Developmentally regulated expression of the PD-1 protein on the surface of double-negative (CD4~-CD8~-) thymocytes [J]. Int Immunol, 1996, 8(5): 773-780.
13、 Nishimura H, Honjo T. PD-1: an inhibitory immunoreceptor involved in peripheral tolerance [J]. Trends Immunol, 2001, 22(5): 265-268.
14、 Rietz C, Chen L. New B7 family members with positive and negative costimulatory function [J]. Am J Transplant, 2004, 4(1): 8-14.
15、 Dong H, Chen L. B7-H1 pathway and its role in the evasion of tumor immunity [J]. J Mol Med, 2003, 81(5): 281-287.
16、 Tamura H, Dong H, Zhu G, et al. B7-H1 costimulation preferentially enhances CD28-independent T-helper cell function [J]. Blood, 2001, 97(6): 1809-1816.
17、 Nishimura H, Minato N, Nakano T, et al. Immunological studies on PD-1-deficient mice: implication of PD-1 as a negative regulator for B cell responses [J]. Int Immunol, 1998, 10(10): 1563-1572.
18、 Selenko-Gebauer N, Majdic OA, Szekeres A, et al. B7-H1 (programmed death-1 ligand) on dendirtic cells is involved in the induction and maintenance of T cell anergy [J]. J Immunol, 2003, 170(7): 3637-3644.
19、 Ishida M, Iwai Y, Tanaka Y, et al. Differential expression of PD-L1 and PD-L2, ligands for an inhibitory receptor PD-1, in the cells of lymphohematopoietic tissues [J]. Immunol Lett, 2002, 84(1): 57-62.
20、 Iwai Y, Ishida M, Tanaka Y, et al. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade [J]. Proc Natl Acad Sci USA, 2002, 99 (19): 12293-12297.
21、 Hirano F, Kaneko K, Tamura H, et al. Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity [J]. Cancer Res, 2005, 65(3): 1089-1096.
22、 Blank C, Brown I, Peterson AC, et al. PD-L1/B7H-1 inhibits the effector phase of tumor rejection by T cell receptor (TCR) transgenic CD8~+ cells [J]. Cancer Res, 2004, 64(3): 1140-1145.
23、 Brown JA, Dorfman DM, Ma FR, et al. Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production [J]. J Immunol, 2003, 170(3): 1257-1266.
24、 Curiel TJ, Wei S, Dong H, et al. Blockade of B7-H1 improves myeloid dendritic cell-mediated antitumor immunity [J]. Nat Med, 2003, 9(5): 562-567.
1 Dery MA, Michaud MD, Richard DE. Hypoxia-inducible factor 1: regulation by hypoxic and non-hypoxic activators [J]. Int J Biochem Cell Biol. 2005, 37(3): 535-540.
2 Lee JW, Bae SH, Jeong JW, et al. Hypoxia-inducible factor (HIF-1) alpha: its protein stability and biological functions [J]. Exp Mol Med, 2004, 36(1): 1-12.
3 Semenza GL. Targeting HIF-1 for cancer therapy [J]. Nat Rev Cancer, 2003, 3(10): 721-732.
4 Talks KL, Turley H, Gatter KC, et al. The expression and distribution of the hypoxia-inducible factors HIF-1 alpha and HIF-2 alpha in normal human tissues, cancers, and tumor-associated macrophages [J]. Am J Pathol, 2000: 157(2): 411-421.
5 Brune B, von Knethen A, Sandau KB. Transcription factors p53 and HIF-1α as targets of nitric oxide [J]. Cell Signal, 2001, 13(8): 525-533.
6 Kimbro KS, Simons JW. Hypoxia-inducible factor-1 in human breast and prostate cancer [J]. Endocr Relat Cancer. 2006, 13(3): 739-749.
7 Enatsu S, Iwasaki A, Shirakusa T, et al. Expression of hypoxia-inducible factor-1 alpha and its prognostic significance in small-sized adenocarcinomas of the lung [J]. Eur J Cardiothorac Surg. 2006, 29(6): 891-895.
8 Kuwai T, Kitadai Y, Tanaka S. et al. Expression of NOS and HIF-1 alpha in human colorectal carcinoma and implication in tumor angiogenesis [J]. World J Gastroenterol. 2006, 12(29): 4660-4664.
9 Rajani R, Bijoyesh M, Zaver M, et al. Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor la [J]. Genes Dev, 2000, 14(1): 34-44.
10 Turner KJ, Moore JW, Jones A, et al. Expression of hypoxia-inducible factors in human renal cancer: relationship to angiogenesis and to the von Hippel-Lindau gene mutation [J]. Cancer Res, 2002, 62(10): 2957-2961.
11 Maynard MA, Ohh M. Von Hippel-Lindau tumor suppressor protein and hypoxia-inducible factor in kidney cancer [J]. Am J Nephrol. 2004, 24(1): 1-13.
12 Wiesener MS, Munchenhagen PM, Berger I, et al.Constitutive activation of hypoxia-inducible genes related to overexpression of hypoxia-inducible factor-1 alpha in clear cell renal carcinomas [J]. Cancer Res, 2001, 61(13): 5215-5222.
13 Kondo Y, Hamada J, Kobayashi C, et al. Over expression of hypoxia-inducible factor-1 alpha in renal and bladder cancer cells increases tumorigenic potency [J]. J Urol, 2005, 173(5): 1762-1766.
14 Lidgren A, Hedberg Y, Grankvist K, et al. The expression of hypoxia inducible factor 1 alpha is a favorable independent prognostic factor in renal cell carcinoma [J]. Clin Cancer Res, 2005, 11(3): 1129-1135.
15 Jones A, Fujiyama C, Blanche C, et al. Relation of vascular endothelial growth factor production to expression and regulation of hypoxia-inducible factor-1 alpha and hypoxia inducible factor-2 alpha in human bladder tumors and cell lines [J]. Clin Cancer Res, 2001, 7(5): 1263-1272.
16 Theodoropoulos VE. Lazaris Ach, Sofras F, et al. Hypoxia-inducible factor 1 alpha expression correlates with angiogenesis and unfavorable prognosis in bladder cancer [J]. Eur Urol, 2004, 46(2): 200-208.
17 Theodoropoulos VE, Lazaris AC, Kastriotis I, et al. Evaluation of hypoxia- inducible factor 1 alpha overexpression as a predictor of tumor recurrence and progression in superficial urothelial bladder carcinoma [J]. BJU Int, 2005, 95(3): 425-431.
18 Streeter EH, Harris AL. Angiogenesis in bladder cancer-prognostic marker and target for future therapy [J]. Surg Oncol, 2002, 11(1-2): 85-100.
19 Zhong H, De Marzo AM, Laughner E, et al. Overexpression of hypoxia- inducible factor 1 alpha in common human cancers and their metastases [J]. Cancer Res, 1999, 59(22): 5830-5835.
20 Zhong H, Semenza GL, Simons JW, et al. Up-regulation of hypoxia-inducible factor 1 alpha is an early event in prostate carcinogenesis [J]. Cancer Detect Prev. 2004, 28(2): 88-93.
21 Mabjeesh NJ, Willard MT, Frederickson CE, et al. Androgens stimulate Hypoxion-inducible Factor 1 Activation Via Autocrine Loop of Tyrosine Kinase Receptor/posphatidylinositol 3' -Ki-nase/ Protein Kinase B in Prostate Cancer cells [J]. Clin Cancer Res, 2003, 9 (7): 2416-2425.
22 Anastasiadis AG, Ghafar MA, Salomon L, et al. Human hormone-refractory prostate cancers can harbor mutations in the O(2)-dependent degradation domain of hypoxia inducible factor-1 alpha (HIF-1 alpha) [J]. J Cancer Res Clin Oncol, 2002, 128(7): 358-62.
23 Mabjeesh NJ, Post DE, Willard MT, et al. Geldanamycin induces degradation of hypoxia-inducible factor 1 alpha protein via the proteosome pathway in prostate cancer cells [J]. Cancer Res, 2002, 62(9): 2478-2482.
24 Alqawi O, Moghaddas M, Singh G. Effects of geldanamycin on HIF-1 alpha mediated angiogenesis and invasion in prostate cancer cells [J]. Prostate Cancer Prostatic Dis. 2006, 9(2): 126-35.
25 Wiedmann MW, Caca K. Molecularly targeted therapy for gastrointestinal cancer. Current Cancer Drug Targets [J]. 2005, 5(3): 171-193.
26 Mabjeesh Nj, Escuin D, Lavallee TM, et al. 2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF [J]. Cancer Cell, 2003, 3(4): 363-375.
27 Sun X, Kanwar JR, Leung E, et al. Gene transfer of antisense hypoxia inducible factor-la enhances the therapeutic efficacy of cancer immunotherapy [J]. Gene Ther, 2001, 8: 638-645.
2、 Pardoll DM. Spinning molecular immunology into successful Immunotherapy [J]. Nat Rev Immunol, 2002,2(4): 227-238.
3、 Dong H, Strome SE, Salomao DR, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion [J]. Nat Med, 2002, 8(8): 793-800.
4、 Strome SE, Dong H, Tamura H, et al. B7-H1 blockade augments adoptive T-cell immunotherapy for squamous cell carcinoma [J]. Cancer Res, 2003, 63(19): 6501-6505.
5、 Konishi J, Yamazaki K, Azuma M, et al. B7-H1 expression on non-small cell lung cancer cells and its relationship with tumor-infiltrating lymphocytes and their PD-1 expression [J]. Clin Cancer Res, 2004,10(15): 5094-5100.
6、 Thompson RH, Gillett MD, Cheville JC, et al. Costimulatory B7-H1 in renal cell carcinoma patients: Indicator of tumor aggressiveness and potential therapeutic target [J]. Proc Natl Acad Sci USA, 2004, 101(49): 17174-17179.
7、 Dong H, Zhu G, Tamada K, et al. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion [J]. Nat Med, 1999, 5(12): 1365-1369.
8、 Schreiner B, Mitsdoerffer M, Kieseier BC, et al. Interferon-beta enhances monocyte and dendritic cell expression of B7-H1 (PD-L1), a strong inhibitor of autologous T-cell activation: relevance for the immune modulatory effect in multiple sclerosis [J]. J Neuroimmunol, 2004, 155(1-2): 172-182.
9、 Dong H, Chen J. B7-H1 pathway and its role in the evasion of tumor immunity [J]. J Mol Med, 2003, 81(5): 281-287.
10、 Iwai Y, Ishida M. Tanaka Y, et al. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade [J]. Proc Natl Acad Sci USA, 2002, 99 (19): 12293-12297.
11、 HiranoF, Kaneko K, TamuraH, et al. Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity [J]. Cancer Res, 2005, 65(3): 1089-1096.
12、 Suda T, Takahashi T, Golstein P, et al. Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family [J]. Cell, 1993, 75(6):1169-1178.
13、 Hahne M, Rimold D, Schroter M, et al. Melanoma cells expression of Fas (AP0-1/CD95) ligand: implications for tumor immune escape [J]. Science, 1996, 274(5291): 1363-1366.
1、 Dong H, Strome SE, Salomao DR, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion [J]. Nat Med, 2002, 8(8): 793-800.
2、 Loke P, Allison JP. PD-L1 and PD-L2 are differentially regulated by Th1 and Th2 cells [J]. Proc Natl Acad Sci USA, 2003,100(9): 5336-5341.
3、 Qian Y, Deng J, Geng L, et al. TLR4 signaling induces B7-H1 expression through MAPK pathways in bladder cancer cells [J]. Cancer Invest, 2008, 26(8): 816-821.
4、 Igney FH, Kramrner PH. Immune escape of tumors: apoptosis resistance and tumor counterattack [J]. J Leukocyte Biology, 2002, 71(6): 907-920.
5、 Drake CG, Jaffee E, Pardoll DM. Mechanisms of immune evasion by tumors [J]. Adv Immunol, 2006, 90:51-81.
6、 Thompson RH, Gillett MD, Cheville JC, et al. Costimulatory B7-H1 in renal cell carcinoma patients: Indicator of tumor aggressiveness and potential therapeutic target [J]. Proc Natl Acad Sci USA, 2004,101(49): 17174-17179.
7、 Mazanet MM, Hughes CC. B7-H1 is expressed by human endothelial cells and suppresses T cell cytokine synthesis [J]. J Immunol, 2002, 169(7):3581-3588.
8、 Ishida M, Iwai Y, Tanaka Y, et al. Differential expression of PD-L1 and PD-L2, ligands for an inhibitory receptor PD-1, in the cells of lymphohematopoietic tissues [J]. Immunol Left, 2002, 84 (1): 57-62.
1、 Dong H, Strome SE, Salomao DR. et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion [J]. Nat Med, 2002, 8(8): 793-800.
2、 Strome SE, Dong H, Tamura H, et al. B7-H1 blockade augments adoptive T-cell immunotherapy for squamous cell carcinoma [J]. Cancer Res, 2003, 63(19): 6501-6505.
3、 Konishi J, Yamazaki K, Azuma M, et al. B7-H1 expression on non-small cell lung cancer cells and its relationship with tumor-infiltrating lymphocytes and their PD-1 expression [J]. Clin Cancer Res, 2004, 10(15): 5094-5100.
4、 Thompson RH, Gillett MD, Cheville JC, et al. Costimulatory B7-H1 in renal cell carcinoma patients: Indicator of tumor aggressiveness and potential therapeutic target [J]. Proc Natl Acad Sci USA, 2004, 101(49): 17174-17179.
5、 Dong H, Zhu G, Tamada K, et al. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion [J]. Nat Med, 1999, 5(12): 1365-1369.
6、 Dong H, Chen J. B7-H1 pathway and its role in the evasion of tumor immunity [J]. J Mol Med, 2003, 81(5): 281-287.
7、 Iwai Y, Ishida M, Tanaka Y, et al. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade [J]. Proc Natl Acad Sci USA, 2002, 99 (19): 12293-12297.
8、 Hirano F, Kaneko K, Tamura H, et al. Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity [J]. Cancer Res, 2005, 65(3): 1089-1096.
9、 Blank C, Brown I, Peterson AC, et al. PD-L1/B7H-1 inhibits the effector phase of tumor rejection by T cell receptor (TCR) transgenic CD8~+ cells [J]. Cancer Res, 2004, 64(3): 1140-1145.
10、 Brown JA, Dorfman DM, Ma FR, et al. Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production [J]. J Immunol, 2003, 170(3): 1257-1266.
11、 Curiel TJ, Wei S, Dong H, et al. Blockade of B7-H1 improves myeloid dendritic cell-mediated antitumor immunity [J]. Nat Med, 2003, 9(5): 562-567.
1、Yoshsiko I,Masayoshi I,Yoshimasa T,et al.Involvement of PD-LI on tumor cells in the escape from host immune system and tumor immunotherapy by PD-LI blockade [J].Proc Natl Acad Sci USA,2003,99(19):12293-12297.
2、Curti BD,Longo DL,Ochoa AC,et al.Treatment of cancer patients with ex vivo anti-CD3-activated killer cells and interleukin-2 [J].J Clin Oncol,1993,11 (4):652-660.
3、Garlic NK,LeFever AV,Siebenlist RE,et al.T cells coactivated with immobilized anti-CD3 and anti-CD28 as potential immunotherapy for cancer [J].J Immunother,1999,22(4):336-345.
4、Vasir B,Wu Z,Crawford K,et al.Fusions of dendritic cells with breast carcinoma stimulate the expansion of regulatory T cells while concomitant exposure to IL-12,CpG oligodeoxynucleotides,and anti-CD3/CD28 promotes the expansion of activated tumor reactive cells [J].J Immunol,2008,181(1):808-821.
5、秦建国，韩本立，韩民等，CD3AK细胞过继治疗对肝癌患者T淋巴细胞亚群及功能活性的影响[J]．中国普外基础与临床杂志，2002，9(3)：183-185．
6、罗志刚，谢江波．CD3单抗、CD28／CpGODN共刺激活化的PBMC对膀胱癌细胞杀伤作用的研究[J]．南华大学学报(医学版)，2004，32(1)：31-34．
7、Strome SE,Dong H,Tamura H,et al.B7-H1 blockade augments adoptive T-cell immunotherapy for squamous cell carcinoma [J].Cancer Res,2003,63(19):6501-6505.
8、De Vita F,Orditura M,Galizia G,et al.Serum Interleukin-10 levels as a prognostic factor in advanced non-small cell lung cancer patients [J].Chest,2000,117(2):365-373.
1、 Baxter AG, Hodgkin PD. Activation rules: the two signal theories of immune activation [J]. Nat Rev Immunol, 2002, 2(6): 439-446.
2、 Pardoll DM. Spinning molecular immunology into successful Immunotherapy [J]. Nat Rev Immunol, 2002, 2(4): 227-238.
3、 Dong H, Zhu G, Tamada K, et al. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion [J]. Nat Med, 1999, 5(12): 1365-1369.
4、 Mazanet MM, Hughes CC. B7-H1 is expressed by human endothelial cells and suppresses T cell cytokine synthesis [J]. J Immunol, 2002, 169(7): 3581-3588.
5、 Schreiner B, Mitsdoerffer M, Kieseier BC, et al. Interferon-beta enhances tnonocyte and dendritic cell expression of B7-H1 (PD-L1), a strong inhibitor of autologous T-cell activation: relevance for the immune modulatory effect in multiple sclerosis [J]. J Neuroimmunol, 2004, 155(1-2): 172-182.
6、 Dong H, Strome SE, Salomao DR, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion [J]. Nat Med, 2002, 8(8): 793-800.
7、 Strome SE, Dong H, Tamura H. et al. B7-H1 blockade augments adoptive T-cell immunotherapy for squamous cell carcinoma [J]. Cancer Res, 2003, 63(19): 6501-6505.
8、 Thompson RH, Gillett MD, Cheville JC, et al. Costimulatory B7-H1 in renal cell carcinoma patients: Indicator of tumor aggressiveness and potential therapeutic target [J]. Proc Natl Acad Sci USA, 2004, 101(49): 17174-17179.
9、 Finger LR, Pu J, Wasserman R, et al. The human PD-1 gene: complete cDNA, genomic organization, and developmentally regulated expression in B cell progenitors [J]. Gene, 1997, 197(1-2): 177-187.
10、 Prokunina L, Castillejo LC, Oberg F, et al. A regulatory polymorphism in PD-1 is associated with susceptibility to systemic lupus erythematosus in humans [J]. Nat Genet, 2002, 32(4): 666-669.
11、 Okazaki T, Maeda A, Nishimura H, et al. PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-dominain-containting tyrosine phosphatase 2 to phosphotyrosine [J]. Proc Natl Acad Sci USA, 2001, 98 (24): 13866-13871.
12、 Nishimura H, Agata Y, Kawasaki A, et al. Developmentally regulated expression of the PD-1 protein on the surface of double-negative (CD4~-CD8~-) thymocytes [J]. Int Immunol, 1996, 8(5): 773-780.
13、 Nishimura H, Honjo T. PD-1: an inhibitory immunoreceptor involved in peripheral tolerance [J]. Trends Immunol, 2001, 22(5): 265-268.
14、 Rietz C, Chen L. New B7 family members with positive and negative costimulatory function [J]. Am J Transplant, 2004, 4(1): 8-14.
15、 Dong H, Chen L. B7-H1 pathway and its role in the evasion of tumor immunity [J]. J Mol Med, 2003, 81(5): 281-287.
16、 Tamura H, Dong H, Zhu G, et al. B7-H1 costimulation preferentially enhances CD28-independent T-helper cell function [J]. Blood, 2001, 97(6): 1809-1816.
17、 Nishimura H, Minato N, Nakano T, et al. Immunological studies on PD-1-deficient mice: implication of PD-1 as a negative regulator for B cell responses [J]. Int Immunol, 1998, 10(10): 1563-1572.
18、 Selenko-Gebauer N, Majdic OA, Szekeres A, et al. B7-H1 (programmed death-1 ligand) on dendirtic cells is involved in the induction and maintenance of T cell anergy [J]. J Immunol, 2003, 170(7): 3637-3644.
19、 Ishida M, Iwai Y, Tanaka Y, et al. Differential expression of PD-L1 and PD-L2, ligands for an inhibitory receptor PD-1, in the cells of lymphohematopoietic tissues [J]. Immunol Lett, 2002, 84(1): 57-62.
20、 Iwai Y, Ishida M, Tanaka Y, et al. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade [J]. Proc Natl Acad Sci USA, 2002, 99 (19): 12293-12297.
21、 Hirano F, Kaneko K, Tamura H, et al. Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity [J]. Cancer Res, 2005, 65(3): 1089-1096.
22、 Blank C, Brown I, Peterson AC, et al. PD-L1/B7H-1 inhibits the effector phase of tumor rejection by T cell receptor (TCR) transgenic CD8~+ cells [J]. Cancer Res, 2004, 64(3): 1140-1145.
23、 Brown JA, Dorfman DM, Ma FR, et al. Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production [J]. J Immunol, 2003, 170(3): 1257-1266.
24、 Curiel TJ, Wei S, Dong H, et al. Blockade of B7-H1 improves myeloid dendritic cell-mediated antitumor immunity [J]. Nat Med, 2003, 9(5): 562-567.
1 Dery MA, Michaud MD, Richard DE. Hypoxia-inducible factor 1: regulation by hypoxic and non-hypoxic activators [J]. Int J Biochem Cell Biol. 2005, 37(3): 535-540.
2 Lee JW, Bae SH, Jeong JW, et al. Hypoxia-inducible factor (HIF-1) alpha: its protein stability and biological functions [J]. Exp Mol Med, 2004, 36(1): 1-12.
3 Semenza GL. Targeting HIF-1 for cancer therapy [J]. Nat Rev Cancer, 2003, 3(10): 721-732.
4 Talks KL, Turley H, Gatter KC, et al. The expression and distribution of the hypoxia-inducible factors HIF-1 alpha and HIF-2 alpha in normal human tissues, cancers, and tumor-associated macrophages [J]. Am J Pathol, 2000: 157(2): 411-421.
5 Brune B, von Knethen A, Sandau KB. Transcription factors p53 and HIF-1α as targets of nitric oxide [J]. Cell Signal, 2001, 13(8): 525-533.
6 Kimbro KS, Simons JW. Hypoxia-inducible factor-1 in human breast and prostate cancer [J]. Endocr Relat Cancer. 2006, 13(3): 739-749.
7 Enatsu S, Iwasaki A, Shirakusa T, et al. Expression of hypoxia-inducible factor-1 alpha and its prognostic significance in small-sized adenocarcinomas of the lung [J]. Eur J Cardiothorac Surg. 2006, 29(6): 891-895.
8 Kuwai T, Kitadai Y, Tanaka S. et al. Expression of NOS and HIF-1 alpha in human colorectal carcinoma and implication in tumor angiogenesis [J]. World J Gastroenterol. 2006, 12(29): 4660-4664.
9 Rajani R, Bijoyesh M, Zaver M, et al. Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor la [J]. Genes Dev, 2000, 14(1): 34-44.
10 Turner KJ, Moore JW, Jones A, et al. Expression of hypoxia-inducible factors in human renal cancer: relationship to angiogenesis and to the von Hippel-Lindau gene mutation [J]. Cancer Res, 2002, 62(10): 2957-2961.
11 Maynard MA, Ohh M. Von Hippel-Lindau tumor suppressor protein and hypoxia-inducible factor in kidney cancer [J]. Am J Nephrol. 2004, 24(1): 1-13.
12 Wiesener MS, Munchenhagen PM, Berger I, et al.Constitutive activation of hypoxia-inducible genes related to overexpression of hypoxia-inducible factor-1 alpha in clear cell renal carcinomas [J]. Cancer Res, 2001, 61(13): 5215-5222.
13 Kondo Y, Hamada J, Kobayashi C, et al. Over expression of hypoxia-inducible factor-1 alpha in renal and bladder cancer cells increases tumorigenic potency [J]. J Urol, 2005, 173(5): 1762-1766.
14 Lidgren A, Hedberg Y, Grankvist K, et al. The expression of hypoxia inducible factor 1 alpha is a favorable independent prognostic factor in renal cell carcinoma [J]. Clin Cancer Res, 2005, 11(3): 1129-1135.
15 Jones A, Fujiyama C, Blanche C, et al. Relation of vascular endothelial growth factor production to expression and regulation of hypoxia-inducible factor-1 alpha and hypoxia inducible factor-2 alpha in human bladder tumors and cell lines [J]. Clin Cancer Res, 2001, 7(5): 1263-1272.
16 Theodoropoulos VE. Lazaris Ach, Sofras F, et al. Hypoxia-inducible factor 1 alpha expression correlates with angiogenesis and unfavorable prognosis in bladder cancer [J]. Eur Urol, 2004, 46(2): 200-208.
17 Theodoropoulos VE, Lazaris AC, Kastriotis I, et al. Evaluation of hypoxia- inducible factor 1 alpha overexpression as a predictor of tumor recurrence and progression in superficial urothelial bladder carcinoma [J]. BJU Int, 2005, 95(3): 425-431.
18 Streeter EH, Harris AL. Angiogenesis in bladder cancer-prognostic marker and target for future therapy [J]. Surg Oncol, 2002, 11(1-2): 85-100.
19 Zhong H, De Marzo AM, Laughner E, et al. Overexpression of hypoxia- inducible factor 1 alpha in common human cancers and their metastases [J]. Cancer Res, 1999, 59(22): 5830-5835.
20 Zhong H, Semenza GL, Simons JW, et al. Up-regulation of hypoxia-inducible factor 1 alpha is an early event in prostate carcinogenesis [J]. Cancer Detect Prev. 2004, 28(2): 88-93.
21 Mabjeesh NJ, Willard MT, Frederickson CE, et al. Androgens stimulate Hypoxion-inducible Factor 1 Activation Via Autocrine Loop of Tyrosine Kinase Receptor/posphatidylinositol 3' -Ki-nase/ Protein Kinase B in Prostate Cancer cells [J]. Clin Cancer Res, 2003, 9 (7): 2416-2425.
22 Anastasiadis AG, Ghafar MA, Salomon L, et al. Human hormone-refractory prostate cancers can harbor mutations in the O(2)-dependent degradation domain of hypoxia inducible factor-1 alpha (HIF-1 alpha) [J]. J Cancer Res Clin Oncol, 2002, 128(7): 358-62.
23 Mabjeesh NJ, Post DE, Willard MT, et al. Geldanamycin induces degradation of hypoxia-inducible factor 1 alpha protein via the proteosome pathway in prostate cancer cells [J]. Cancer Res, 2002, 62(9): 2478-2482.
24 Alqawi O, Moghaddas M, Singh G. Effects of geldanamycin on HIF-1 alpha mediated angiogenesis and invasion in prostate cancer cells [J]. Prostate Cancer Prostatic Dis. 2006, 9(2): 126-35.
25 Wiedmann MW, Caca K. Molecularly targeted therapy for gastrointestinal cancer. Current Cancer Drug Targets [J]. 2005, 5(3): 171-193.
26 Mabjeesh Nj, Escuin D, Lavallee TM, et al. 2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF [J]. Cancer Cell, 2003, 3(4): 363-375.
27 Sun X, Kanwar JR, Leung E, et al. Gene transfer of antisense hypoxia inducible factor-la enhances the therapeutic efficacy of cancer immunotherapy [J]. Gene Ther, 2001, 8: 638-645.