【作者】 张伟；

【导师】 蔡建辉；

【作者基本信息】 河北医科大学 ， 外科学（专业学位）， 2020， 博士

【摘要】 恶性肿瘤仍是全球范围内最主要的死亡原因之一。几十年来,恶性肿瘤的治疗因为缺乏可靠有效的治疗手段使得肿瘤患者的生存受到了挑战。术后复发转移或不可切除的进展期肿瘤,必须依靠放化疗等传统手段进行治疗,但临床疗效往往有限,并伴随着严重的不良反应。肿瘤免疫治疗的进步和发展,使得利用免疫疗法治疗癌症变得越来越重要。研究人员开发了多种激活免疫反应的策略,其中IL-2是最早的免疫增强药物之一,因其能够促进T细胞的增殖和分化,增强T细胞功能,而获批用于肾癌、黑色素瘤和非何杰金淋巴瘤的治疗,成为最早的肿瘤免疫治疗方法之一。但是,第一代免疫疗法的局限性在于其低应答率和高不良事件发生率。寻找可靠的免疫治疗策略及方法,通过调节T细胞活化和增殖来增强免疫治疗的疗效成为肿瘤免疫治疗的主要研究方向。近年研究发现,免疫检测点在T细胞活化及杀伤过程中,发挥着重要的调节功能,其中细胞毒性T淋巴细胞抗原4(Cytotoxic T-Lymphocyte Antigen 4,CTLA-4)、程序性死亡受体-1(programmed cell death-1,PD-1)、IDO-1、Tim3等免疫抑制分子对T细胞的免疫调节功能越来越受到重视。有研究证实,T细胞活化的早期出现CTLA-4的表达升高,与其配体B7结合而发挥竞争性抑制,使B7-CD28共刺激信号通路减弱,从而导致“免疫刹车”效应,阻碍了T细胞的活化。T细胞活化的同时,INF-γ大量释放,导致T细胞表面的PD-1大幅升高,与其配体PD-L1结合造成“T耗竭”,从而导致T细胞活化、增殖及杀伤功能的降低。因而,近年来对程序性死亡因子1(PD-1)和细胞毒性T淋巴细胞抗原4(CTLA-4)的研究进展迅速。针对免疫检查点的研究及治疗是目前免疫治疗研究的热点[1]。两种T细胞共抑制分子CTLA-4和PD-1的发现使得癌症免疫疗法和靶向疗法在如何治疗癌症,尤其是在晚期疾病患者中带来了革命性的变化[2]。因而大量临床前和临床研究围绕着PD-1和CTLA-4开展,其中以PD-1为靶点的免疫治疗在抗肿瘤、抗感染、抗自身免疫性疾病及器官移植等方面均有较深入的研究。以CTLA-4为靶点的免疫治疗,在抗肿瘤免疫等方面也表现出显著的治疗作用。随着CTLA-4和PD-1阻滞剂的深入研究及其在癌症治疗中的成功,靶向PD-1及CTLA-4的抗体类药物迅速出现并获得FDA批准上市,逐渐应用于多种恶性实体肿瘤的临床治疗[3]。CTLA-4是成功在临床上应用靶向的免疫检查点之一。针对CTLA-4的单克隆抗体靶向药物(ipilimumab)已获批用于治疗黑色素瘤等疾病,后续的研究表明在肺癌等多种肿瘤治疗中同样有效。迄今为止,已批准7种靶向CTLA-4/PD-1的药物用于治疗各种类型的癌症。众所周知T淋巴细胞是人体主要的免疫细胞,其中CD4~+T细胞主要表达于辅助T细胞(Th),被称为辅助性T细胞(helper T cell),而CD8~+T细胞被称为细胞毒性T细胞(cytotoxic T cell,CTL)是通过细胞裂解和细胞凋亡的方式特异性杀伤靶细胞而发挥着细胞毒性T细胞的作用,与细胞免疫以及抗肿瘤效应有关。CD4~+T细胞的主要功能是辅助CD8~+T细胞消灭体内的病毒、细菌以及异常发生的肿瘤细胞。大量研究表明PD-1阻断剂在可增强CD4+和CD8~+T细胞反应。本研究的目的在于,采用抗体阻断CTLA-4免疫抑制分子是否能有效的增强细胞毒性CD8~+T淋巴细胞的抗肿瘤效应。为此我们通过研究膀胱癌患者的外周血中T细胞PD-1和CTLA-4表达特点,检测PD-1和CTLA-4是否在活化的T细胞上存在高表达。随后,采用抗体阻断外周血T细胞表面的免疫性负调节分子CTLA-4,测试抗体封闭后能否增强细胞免疫反应和细胞杀伤毒性,验证阻断细胞毒T淋巴细胞表面CTLA-4分子的表达,(cytotoxic T cell,CTL)是否能增强其在体外的杀伤效率和皮下移植瘤模型的抗肿瘤效果。第一部分免疫抑制分子PD-1和CTLA-4表达特征的实验研究目的:检测PD-1和CTLA-4两种共抑制分子在膀胱癌患者和健康志愿者的外周血单核细胞的表达特征。方法:1.分别采集膀胱癌患者(n=62)及健康志愿者(n=32)外周血50ml,肝素抗凝并PBS稀释1倍;采用Ficoll法离心获得PBMC;吸出白膜层(PBMC)置于50ml离心管中,加入PBS,800×g、10min离心洗2次。在洗出血小板后获得PBMC。经分离培养,贴壁细胞培育DC细胞,检测DC表型,悬浮细胞培育CIK细胞。2.CD3抗体刺激T细胞活化。检测外周血中T淋巴细胞分泌IFN-γ和TNF-α的能力。T淋巴细胞特异性的表达CD3,并又进一步分为CD4~+T细胞、CD8~+T细胞亚群;3.流式检测膀胱癌患者及健康志愿者两组的PD-1和CTLA-4在CD4+和CD8~+T细胞的表达特点,4.进行PD-1和CTLA-4的表达与膀胱癌患者的年龄、性别、组织学分级、肿瘤大小等临床参数的相关性分析。结果:1.膀胱癌患者中CD4~+T细胞中PD-1的表达与健康的志愿者相比较无统计学差异(平均14.7%±3.5%比13.6%±4.6%,Fig.1A)。相比之下,在CD8~+T细胞中PD-1的表达明显高于健康的志愿者(平均频率12.8%±4.5%比8.1%±2.9%,Fig.1B)。2.在CD4~+T细胞中,CTLA-4的表达频率可与膀胱癌患者和健康献血者(Fig.1C)相比较表达无统计学差异。与此同时,在膀胱癌患者中,CD8~+T细胞中的CTLA-4的表达比健康对照组的表达高(平均10.2%±3.2%比7.5%±2.8%)(Fig.1D);3.在CD8~+T细胞上的PD-1表达与任何临床参数之间没有显著的相关性。然而CTLA-4的表达与肿瘤大小和TNM分期之间有密切的相关性(表1)。结论:PD-1和CTLA-4在膀胱癌患者的外周血中CD8~+T细胞的表达较健康志愿者明显升高。第二部分阻断CTLA-4的CD8~+T淋巴细胞体外抗肿瘤效应的实验研究目的:检测抗CTLA-4抗体封闭后的DC-CTL对肿瘤细胞的体外杀伤效应。方法:1.采集肿瘤病人外周血,提取血中的单状核细胞,Focill方法分离,然后贴壁法分离出DC细胞和T细胞;DC细胞扩增并负载细胞株抗原,制备出DC疫苗,2.检测UM-UC-3、TCCSUP、J82、T24,5637五种膀胱癌细胞CTLA-4配体CD80/86的表达水平,选择CD80/86高表达的细胞株作为靶细胞,3.T细胞培养扩增至第7天,应用抗CTLA-4抗体封闭CTLA-4后加入DC疫苗制备CTL;CTL继续扩增2天,应用流式细胞术检测第7、14和21天CTLA-4的表达,4.制备的肿瘤特异性细胞毒T细胞(CTL)分组:第一组:未干预组CTLs(特异性细胞毒性T淋巴细胞)、第二组:生理盐水空白对照组,第三组:抗CTLA-4抗体治疗组,检测CTL的细胞增殖,每7天进行一次检测,持续时间为21天;流式检测三组细胞的CTLA-4的表达水平,5.ELISA测定法测定实验组CTL和对照组CTL的培养基中的IFN-γ和TNF-α分泌量;6.实验组及对照组行96孔板细胞体外杀伤实验;结果:1.抗CTLA-4抗体封闭后,应用流式细胞术检测第7、14和21天CTLA-4的表达,抗CTLA-4治疗组CTL较对照组CTLA-4的表达明显受到抑制,二者相比结果具有统计学上的意义(P<0.05)(图.1A)。对照组CTLA-4在CTL上的表达从9.1%增加到17.6%;在CTLA-4阻断组中,CTLA-4表达从4.1%增加到9.2%。2.对未抗体干预组CTL、空白对照组和抗CTLA-4治疗组CTL的细胞增殖,每7天进行一次检测,持续时间为21天。在培养期间,我们发现CTL的增殖不受CTLA-4阻断的影响。随着时间延长,细胞的增殖逐渐增多,不同组的细胞增殖变化无明显差异,无统计学意义(图.1B)。3.抗CTLA-4治疗组和对照组CTL的上清液中,ELISA法检测细胞因子IFN-γ和TNF-α的分泌能力的变化。CTLA-4阻断后的CTL产生的IFN-γ和TNF-α水平更高(图.1C)。4.在5种膀胱癌细胞UM-UC-3、TCCSUP、J82、T24、5637中检测CTLA-4的配体CD80/86表达水平,其中T24表达最高,UM-UC-3表达水平最低,不同肿瘤细胞CD80/86表达水平相比具有统计学意义(P>0.05)(图.1D)。5.抗CTLA-4抗体阻断的CTLs对T24细胞的细胞毒性显著提高,且呈剂量依赖性,CTL上的CTLA-4的抗体阻断增强细胞的抗肿瘤活性。结论:CTLA-4诱导的T细胞失活被认为是免疫抑制的一种机制,阻断CTLA-4/B7通路后增强CTL对膀胱癌细胞的抗肿瘤活性。第三部分阻断CTLA-4的CD8~+T淋巴细胞体内抗肿瘤效应的实验研究目的:进一步验证抗CTLA-4封闭后的CTL抗肿瘤免疫杀伤效果,评估封闭CTLA-4后特异性CTL体内抗肿瘤效应。方法:1.动物模型建立:采用人源单个核细胞建立具有人免疫功能的SCID模型小鼠,并建立了T24细胞的异种移植模型,流式检测重建效率。免疫重建的小鼠,将5周龄雄性SCID小鼠(中国科学院)尾静脉注射4×107外周血淋巴细胞,肿瘤直径为6-9mm,建立SCID小鼠模型。2.预防试验抗-CTLA-4抗体阻断后的1×107CTLs和对照组通过尾静脉注射,然后SCID小鼠皮下移植T24细胞,剂量为1×106个细胞,悬浮于100μl PBS中(每组n=5)(图1A)。自开始接种后测量抗-CTLA-4抗体封闭组或对照CTL后小鼠的肿瘤体积(n=5)(图1B)。采用抗-CTLA-4抗体阻断或对照组CTL对肿瘤组织进行PCNA免疫组化染色(图1C)。3.治疗试验首先将T24细胞接种在SCID小鼠中。当肿瘤生长至接近100mm 3时,将小鼠随机分组,并用抗CTLA-4抗体处理的1×107个实验组CTL和对照组分别行尾静脉注射(每组n=5)。饲养条件遵循动物中心的指导方针:温度20-22℃;湿度50-70%;正常饮食,无特定病原体。每3天用游标卡尺测量肿瘤体积。27天后处死小鼠。通过计算肿瘤体积来监测肿瘤负荷。肿瘤体积计算如下:体积=(长度)×(宽度)2/2。自开始接种后测量抗-CTLA-4抗体封闭组或对照组CTL的小鼠的肿瘤体积(n=5)(图1E)。采用抗-CTLA-4抗体阻断或对照组CTL对肿瘤组织进行PCNA免疫组化染色(图1F)。通过CTLA-4抗体封闭后制备CTL进行杀伤实验。4.分组1)抗CTLA-4抗体封闭的CTL实验组;2)对照组CTL;3)(生理盐水)空白对照组。5.统计学分析数据报告为来自至少三次独立实验的平均值±标准差。使用SPSS(V13.0,Chicago,USA)进行统计学分析。通过χ2检验比较CTLA-4表达与临床参数之间的相关性。通过Student’s t-检验评估体外和体内测定中的组间差异。方差分析用于在多个组之间进行比较。P<0.05有统计学意义。结果:1.尾静脉注射单核细胞方法构建人源化SCID小鼠模型,将人源化移植瘤模型小鼠分为:CTLA-4抗体封闭组和CTL对照组,与对照组相比,抗CTLA-4抗体封闭组的肿瘤生长明显减慢,这表明被抗CTLA-4抗体封闭后肿瘤的生长出现延缓。与此同时,CTLA-4抗体实验组的PCNA阳性细胞也显著减少。2.预防性试验:接种1×107剂量的对照组CTL和抗-CTLA-4抗体封闭的CTL治疗组细胞,然后以1×106细胞的剂量皮下移植T24细胞(图.1A),每组(n=5)。与对照组相比,抗-CTLA-4实验组肿瘤生长明显减慢,说明CTLA-4抗体延缓了T24移植瘤的潜伏期(图1B),同时,抗-CTLA-4组中PCNA阳性细胞也明显减少(图.1C)。3.治疗性实验:与对照组相比,抗-CTLA-4治疗组的肿瘤体积明显减小,表明CTLA-4抗体封闭后在体内的抗肿瘤活性增强(每组n=5)(图1E)。同样,CTLA-4抗体封闭组CTL增殖指数PCNA及免疫反应活性也降低(图.1F)。4.CTLA-4抗体封闭组较CTL对照组对T24细胞有更强的抗肿瘤杀伤效应;CTLA-4抗体封闭组较生理盐水组对T24细胞有更强的抗肿瘤杀伤效应,二者相比具有统计学上的意义。DC-CTL组较生理盐水组有更强的抗肿瘤杀伤效应。5.ELISA法检测上清液中细胞因子IFN-γ和TNF-α分泌量:CTLA-4抗体阻断后的CTL产生更高水平的IFN-γ和TNF-α(表1)。结论:1.CTLA-4抗体封闭后增强了其对膀胱癌细胞免疫反应和细胞毒性,2.CTLA-4抗体封闭后CTL在皮下异种移植的SCID小鼠模型中显示出具有更好的抗肿瘤活性。3.CTLA-4抗体封闭后的CTL产生更高水平的IFN-γ和TNF-α。4.CTLA-4抗体封闭组肿瘤生长明显减慢,说明CTLA-4抗体延缓了肿瘤的生长。更多还原

【Abstract】 Malignant neoplasm remains one of the leading causes of death worldwide.For decades,the treatment of malignant tumors has challenged the survival of cancer patients because of the lack of reliable and effective treatment methods.Postoperative recurrent metastatic or unresectable advanced tumors must be treated by traditional means such as radiotherapy and chemotherapy,but the clinical efficacy is often limited and accompanied by severe adverse reactions.The progress and development of tumor immunotherapy make the use of immunotherapy to treat cancer more and more important.The researchers have developed a variety of strategies to activate the immune response.Among them,IL-2 is one of the earliest immune-enhancing drugs,because it can promote the proliferation and differentiation of T cells and enhance the function of T cells,and has been approved for the treatment of renal cell carcinoma,melanoma and non-hodgkin lymphoma,and has become one of the earliest methods of tumor immunotherapy.However,the limitations of first-generation immunotherapy lie in its low response rate and high incidence of adverse events.Find reliable immunotherapeutic strategies and methods to enhance T by regulating cell activation and proliferation.The therapeutic effect becomes the main research direction of tumor immunotherapy.Recent studies have found that immune detection sites play an important regulatory function in the process of T cell activation and killing.Among them,immunosuppressive molecules such as cytotoxicity T lymphocyte antigen 4(Cytotoxic T-Lymphocyte Antigen 4,CTLA-4),programmed death receptor-1(programmed cell death-1,PD-1),IDO-1,Tim3 have been paid more and more attention to the immune regulation function of T cells.Studies have shown that the expression of CTLA-4 in the early stages of T cell activation increases,the ligand exerts competitive inhibition B7 binding,weakening the B7-CD28 costimulatory signaling pathway,which leads to the "immune brake" effect and hinders the activation of T cells.T cell activation is accompanied by a large amount of INF-γ release,resulting in a large increase in the PD-1 on the surface of T cells,and a "T depletion" caused by the binding of its ligand,which leads to the decrease of cell activation,proliferation and killing function.thus,recent studies on programmed death factor 1(PD-1)and cytotoxic T lymphocyte antigen 4(CTLA-4)have progressed rapidly.The research and treatment of immune checkpoints is the focus of current immunotherapy research[1].the discovery of two T cell co-inhibiting molecular CTLA-4 and PD-1 makes cancer immunotherapy and targeted therapies revolutionize how to treat cancer,especially in patients with advanced disease [2].Therefore,a large number of preclinical and clinical studies are carried out around PD-1 and CTLA-4,in which immunotherapy targeting PD-1 has more in-depth research in anti-tumor,anti-infection,anti-autoimmune diseases and organ transplantation.Immunotherapy targeting CTLA-4,anti-tumor immunity,etc.aspects also showed significant therapeutic effects.With the in-depth study of CTLA-4 and PD-1 blockers and their success in cancer therapy,targeted PD-1 and CTLA-4 antibody drugs are rapidly emerging and approved for marketing,and are gradually applied to clinical treatment of multiple malignant solid tumors[3].CTLA-4 is one of the immune checkpoints successfully applied to the clinic.Monoclonal antibody targeting drugs(ipilimumab)targeting CTLA-4 have been approved for the treatment of diseases such as melanoma,and follow-up studies have shown to be equally effective in the treatment of multiple tumors such as lung cancer.7 targeted CTLA have been approved to CTLA-4/PD-1 drugs for the treatment of various types of cancer.T lymphocytes are known to be the main immune cells in the human body.Among them,CD4~+T cells are mainly expressed in helper T cells(Th),which are called helper T cells(helper T cell),while CD8 T cells are called cytotoxic cells(cytotoxic T cell,CTL)that specifically kill target cells by means of cell lysis and apoptosis,which are associated with cellular immunity and antitumor effects.The primary function of CD4~+T cells is to assist CD8~+Tcells in eliminating viruses,bacteria,and abnormalities in the body raw tumor cells.Numerous studies have shown that PD-1 blockers enhance CD4~+T and CD8~+T cellular responses.The aim of this study was to determine whether blocking CTLA-4 immunosuppressive molecules with antibodies could effectively enhance cytotoxicity CD8~+T the antitumor effect of lymphocytes.To this end,we examined whether PD-1 and CTLA-4 were highly expressed on activated T cells by studying the characteristics of cell PD-1 and CTLA-4 expression in peripheral blood of bladder cancer patients.Subsequently,the immune negative regulatory molecules on the surface of peripheral blood T cells were blocked by antibody CTLA-4,and whether the antibody could enhance the cellular immune response and cytotoxicity was tested to verify the blocking of cytotoxic T lymphocyte surface expression of CTLA-4 molecules,and(cytotoxic T cell,CTL)whether it can enhance its killing efficiency in vitro and the anti-tumor effect of subcutaneous transplanted tumor model.Part one Experimental Study on PD-1 and CTLA-4 Expression Characteristics of Immunosuppressive MoleculesObjective: To detect the expression characteristics of PD-1 and CTLA-4 co-inhibitory molecules in peripheral blood monocytes of bladder cancer patients and healthy volunteers.Method: 1.Patients with bladder cancer(n=62)and healthy volunteers(n=32)received 50 ml,heparin anticoagulant and diluted 1 times PBS.absorbent white film layer(PBMC)was obtained by centrifugation by Ficoll method and placed in a 50 ml centrifuge tube.the PBS,800×g、10min was centrifuged and washed twice.Get PBMC.after washing platelets Through isolation culture,adherent cells cultivate DC cells,detect DC phenotypes,and suspension cells cultivate CIK cells.2.CD3 antibodies stimulate T cell activation.to detect the ability of T lymphocytes to secrete IFN-γ 和 TNF-α in peripheral blood.T lymphocyte specific expression CD3,and further divided into CD4~+T cells,CD8~+T cell subsets;3.The expression characteristics of PD-1 and CTLA-4 in CD8~+T and CD4~+T cells of bladder cancer patients and healthy volunteers were measured by flow cytometry;4.The correlation between PD-1 and CTLA-4 expression and the age,sex,histological grade,tumor size and other clinical parameters of bladder cancer patients was analyzed.Results: 1.There was no statistical difference in the expression of PD-1 in CD4 T cells in bladder cancer patients compared with healthy volunteers(mean 14.7%±3.5% vs 13.6%,4.6%,Fig.1A).in contrast,PD-1 expression in CD8 T cells was significantly higher than in healthy volunteers(mean frequency 12.8%±4.5% vs 8.1%,2.9%,Fig.1B).2.The expression frequency of CTLA-4 in CD4 T cells was not statistically different compared with bladder cancer patients and healthy blood donors(Fig.1C).Meanwhile,in patients with bladder cancer,the expression of CTLA-4 in CD8~+T cells was higher than that in healthy controls(mean 10.2%±3.2% vs 7.5%±2.8%)(Fig.1D).3.There was no significant correlation between PD-1 expression on CD8 T cells and any clinical parameters(table 1).Nevertheless,there was a close correlation between CTLA-4 expression and tumor size and TNM staging(table 1).The comparison is statistically significant.Conclusion: The expression of PD-1 and CTLA-4 in peripheral blood of bladder cancer patients was significantly higher than that of healthy volunteers.Part Two Study on Anti-tumor Effect of Blocked CTLA-4 CD8~+T Lymphocytes in VitroObjective: The in vitro killing effect of DC-CTL after blocking antiCTLA-4 antibody on tumor cells was detected.Method: 1.The peripheral blood of tumor patients was collected,the mononuclear cells were extracted Focill the method,then the DC cells and T cells were isolated by adherent method.2.The expression level of CTLA-4 ligand CD80/86 in five bladder cancer cells was detected,and CD80/86 highly expressed cell lines were selected as target cells.3.T cell culture was expanded to 7 days,the CTLA-4 was sealed with anti CTLA-4 antibody and then prepared with DC vaccine.The expression of CTLA-4 was detected by flow cytometry.4.The as-prepared tumor-specific cytotoxic T cells(CTL)were grouped: group 1: non-intervention group CTLs(specific cytotoxicity T lymphocytes);group 2: saline blank control group;group 3: anti-antibody treatment group,the cell proliferation of the CTL was detected every 7 days for a duration of 21 days;and the expression level of the CTLA-4 in the three groups was detected by flow cytometry.5.ELISA assay for the amount of IFN-γ and TNF-α secreted in the medium of the experimental group CTL and the control group CTL;6.The experimental group and the control group were tested with 96-well plate cells in vitro.Results: 1.The expression of CTLA-4 CTLA-4 was detected by flow cytometry after the closure of anti CTLA-4 antibody.the expression of CTLA-4 in the anti CTLA-4 treatment group was significantly inhibited compared with that in the control group(P<0.05)(figure).1A).the expression of CTLA-4 on CTL in the control group increased from 9.1% to 17.6%;in the CTLA-4 blocking group,the expression of CTLA-4 increased from 4.1% to 9.2%.2.The cell proliferation CTL the unantibody intervention group CTL、the blank control group and the anti CTLA-4 treatment group was tested every 7 days for 21 days.we found that CTL proliferation was not affected by CTLA-4 blockade during culture.With the prolongation of time,the proliferation of cells gradually increased,and there was no significant difference in cell proliferation in different groups(Fig.1B).3.The change of cytokine IFN-γ and TNF-α secretion ability was detected by ELISA assay in the supernatant CTL the anti-drug treatment and control groups.The IFN-γ and TNF-α levels produced by the CTL after CTLA-4 blockade were higher(Fig.1C).4.CTLA-4 ligand CD80/86 expression levels were detected in 5 bladder cancer cell UM-UC-3、TCCSUP、J82、T24、5637,with the highest T24 expression and the lowest UM-UC-3 expression levels,which were statistically significant compared with different tumor cells(P>0.05)(fig.1D).5.The cytotoxicity of anti-CTLA-4 antibody-blocking CTLs to T24 cells was significantly increased and dose-dependent.CTLA-4 antibody blocking on the CTL enhanced the anti-tumor activity of the cells.Conclusion: CTLA-4 induced T cell inactivation is considered to be a mechanism of immunosuppression CTL antitumor activity against bladder cancer cells after blocking the CTLA-4/B7 pathway.Part three The Experimental Study anti-tumor effect of blocking CTLA-4 CD8~+T lymphocytes in vivoObjective: To further verify the CTL anti-tumor immune killing effect after anti-CTLA-4 closure,and to evaluate the specific in vivo anti-tumor effect after CTL closure.Method: 1.Animal model building: human-derived mononuclear cells were used to establish SCID model mice with human immune function,and xen model of T24 cells was established,and the efficiency of flow detection and reconstruction was established.immune reconstituted mice 5-week-old male SCID mice(chinese academy of sciences)were injected intravenously with 4×107 peripheral blood lymphocytes with a tumor diameter of 6-9 mm,to establish a SCID mouse model.2.Preventive testing The 1×107 CTLs after anti-CTLA-4 antibody blockade and the control group were injected through the caudal vein and then subcutaneously transplanted T24 cells in mice at a dose of 1×106 cells suspended in 100μl PBS(n 5 per group)(figure 1 A).tumor volume(n=5)was measured in mice after the anti-CTLA-4 antibody blocking group or control CTL from the beginning of inoculation(figure 1 B).PCNA immunohistochemical staining of tumor tissues was performed using anti-CTLA-4 antibody blockade or control group CTL(figure 1 C).3.For therapeutic assay,T24 cells were first inoculated in SCID mice.when the tumor grew to close to 100 mm 3,mice were randomly divided into groups,and 1×107 experimental groups control groups treated with antiCTLA-4 antibodies were injected intravenously(n=5,each group).Feeding conditions follow animal center guidelines: temperature 20-22℃;humidity 50-70%;normal diet,free of specific pathogens.tumor volume was measured with vernier caliper every 3 days.The mice were killed 27 days later.monitoring tumor load by calculating tumor volume.tumor volume is calculated as follows: volume =(length)×(width)2/2.tumor volume(n =5)was measured in mice CTL the anti-CTLA-4 antibody blocking group or control group since the beginning of inoculation(fig1E).PCNA immunohistochemical staining of tumor tissues was performed using anti-CTLA-4 antibody blockade or control group CTL(fig 1 F).killing experiments of CTL cells prepared after CTLA-4 antibody closure.4.Group 1)CTLA-4 antibody blocked CTLs experimental group;2)control group;3)saline control group(NS group)5.Statistical analysis Data were reported as mean ± standard deviation from at least three independent experiments.Statistical analysis was performed using SPSS(v13.0,chicago,il,usa).The correlation between CTLA-4 expression and clinical parameters was compared by χ2 test.Student’s t-tests were used to assess intergroup differences in in vitro and in vivo assays.ANOVA was used to compare between multiple groups.P<0.05 was statistically significant.Results: 1.The humanized SCID mouse model was constructed by the tail vein injection monocyte method.the humanized transplanted tumor model mice were divided into: CTLA-4 antibody blocking group and CTL control group.compared with the control group,the tumor growth in the anti CTLA-4 antibody blocking group was significantly slowed down,which indicated that the tumor growth was delayed after being blocked by anti-antibody.Meanwhile,the PCNA positive cells in the CTLA-4 antibody test group also decreased significantly.The cell proliferation CTL the unantibody intervention group CTL、the blank control group and the anti CTLA-4 treatment group was tested every 7 days for 21 days.we found that CTL proliferation was not affected by CTLA-4 blockade during culture.With the prolongation of time,the proliferation of cells gradually increased,and there was no significant difference in cell proliferation in different groups(Fig.1B).2.Prophylactic test: 1×107 doses of control CTL and anti-CTLA-4 antibody-blocked CTL-treated cells,then subcutaneously transplanted T24 cells at a dose of 1×106 cells(Fig.1A,each group(n = 5)Compared with the control group,the tumor growth of the anti-CTLA-4 CTLs experimental group was significantly slower,indicating that the anti-CTLA-4 CTLs delayed the latency of T24 xenografts(as shown in Figure 1B),and at the same time,anti-CTLA-4 PCNA positive cells in the CTL group were also significantly reduced(Fig.1C).3.Therapeutic experiments: Compared with the control group,the tumor volume of the anti-CTLA-4 CTLs treatment group was significantly reduced,indicating that the anti-tumor activity of anti-CTLA-4 CTLs was enhanced in vivo(n=5 per group)(Fig.1E).Similarly,the anti-CTLA-4 antibody-blocked CTLs also showed a decrease in PCNA and immunoreactivity in the CTLs(Fig.1F).4.The anti-CTLA-4 antibody-blocked DC-CTL treatment group had stronger anti-tumor killing effect on T24 cells than the DC-CTL treatment group;the anti-CTLA-4 antibody-blocked DC-CTL treatment group was more physiologically controlled than the saline control group(The NS group had a stronger anti-tumor effect on T24 cells,which was statistically significant.The DC-CTL treatment group had stronger anti-tumor killing effect than the saline control group(NS group),and the two were statistically significant.5.ELISA assay was performed to detect cytokine IFN-γ and TNF-α secretion in the supernatant: after antibody blockade produced higher levels of IFN-γ and TNF-α(table 1).Conclusion: 1.CTL enhances its immune response and cytotoxicity to bladder cancer cells after anti-CTLA-4 antibody is blocked.2.CTL showed better anti-tumor activity in a subcutaneous xenografted SCID mouse model after anti-CTLA-4 antibody blocking.3.CTLs after anti-CTLA-4 antibody blockade produce higher levels of IFN-γ and TNF-α.4.Tumor growth was significantly slowed in the anti-CTLA-4 CTL group,indicating that anti-CTLA-4 CTL delayed tumor growth.更多还原