【作者】 刘英姿；

【导师】 单保恩；

【作者基本信息】 河北医科大学 ， 免疫学， 2011， 博士

【摘要】 目的:脑胶质瘤,尤其是高级别胶质瘤是颅内最常见的恶性肿瘤,肿瘤呈浸润性生长,手术很难将其全部切除,尽管结合放化疗等综合治疗,但胶质瘤患者的预后仍十分不理想,复发率高,高级别胶质瘤患者的生存时间甚至不足1年。随着分子生物学研究的不断进展,胶质瘤的免疫治疗逐步得到人们的重视。胶质瘤细胞的侵袭性与其复杂的病理生理学特点有关,其逃逸免疫监视能力限制了机体产生有效抗肿瘤免疫反应,调节性T细胞(CD4~+CD25~+Foxp3~+ T细胞,Treg)在肿瘤细胞免疫逃逸过程中扮演重要角色。Treg细胞具有免疫抑制作用,对维持机体免疫稳态发挥着重要作用。Treg细胞在抑制效应性T细胞功能的同时,也降低机体对肿瘤细胞的免疫反应,间接促进了肿瘤细胞的生长。在多种恶性肿瘤组织中(如乳腺癌,卵巢癌,肺癌,肝癌,恶性淋巴瘤等)有大量Treg细胞浸润,其在患者外周血淋巴细胞中的比例也明显升高。并且,Treg细胞的数量与肿瘤的发展和预后呈负相关,清除Treg细胞或抑制其功能有助于机体抗肿瘤免疫功能的恢复。Treg细胞分为天然型和诱导型,前者由胸腺产生,后者可在外周由非调节性T细胞转化而来。在肿瘤微环境中,异常增多的Treg细胞也分为两类,一种是在肿瘤细胞趋化下,由外周迁徙而来,一种是在肿瘤局部特定环境中由非调节性T细胞转化而来。浸润在肿瘤中的Treg细胞在肿瘤细胞作用下分化成熟,具有肿瘤特异性,可阻止效应性T细胞(主要是CD8~+和CD4~+T细胞)的功能。在肿瘤局部,Treg细胞和效应性T细胞在数量上的不平衡是引起肿瘤细胞免疫逃逸的重要因素。因此打破这种不平衡成为肿瘤免疫治疗新的策略。白细胞介素18(IL-18)作为前炎因子,常高表达在多种自身免疫疾病组织及体液中,如类风湿性关节炎、结节病、红斑狼疮等。在这些患者体内,Treg细胞明显减少,而效应性CD8~+T细胞功能由于失去Treg细胞的抑制作用而明显上调,导致自身免疫性疾病发生。IL-18的高表达应与Treg细胞的减少存在某种关联。此外,IL-18本身也具有明显抗肿瘤功能,包括诱导T细胞和NK细胞产生IFN-γ,增强NK细胞和CTL细胞的细胞毒性,促进活化的CD4~+T细胞分化成为辅助性T细胞和效应性T细胞。在肿瘤动物模型研究及临床试验中,IL-18已被证实具有抗肿瘤功能,但在肿瘤微环境中是否能够逆转Treg细胞的免疫抑制作用,尚缺乏研究报道。本研究旨在探讨Treg细胞在脑胶质瘤组织中的数量与肿瘤发展的关系,探讨其免疫抑制性功能的机制,以及IL-18对Treg细胞的调节作用。方法:1收集2007年9月至2009年9月在河北医科大学第四医院神经外科治疗的脑胶质瘤患者的外周血及肿瘤组织标本,共76例。同期因脑疝行脑内减压术获得的正常脑组织标本及健康人群外周血标本作为阴性对照。分离肿瘤组织及正常脑组织中浸润淋巴细胞及患者、健康对照者外周血单个核细胞。采用流式细胞术检测组织中及外周血中Treg细胞、CD4~+T CD8~+T细胞的含量及比率,分析其与脑胶质瘤患者临床、病理学特征及预后的关系。采用ELISA法测定脑胶质瘤患者外周血中TGF-β1的含量。采用逆转录聚合酶链反应(RT-PCR)技术定量检测外周血单个核细胞中Foxp3mRNA的含量,分析两者表达的关系。2收集2008年9月至2009年9月间在河北医科大学第四医院神经外科治疗的5例脑胶质瘤患者手术切除的新鲜肿瘤组织标本。采集同期健康志愿者外周血标本及行脑内减压术获得正常脑组织为对照。分别培养正常人脑星形胶质原代细胞及原代胶质瘤细胞,采用免疫组织化学染色法进行鉴定。胶质瘤组织体外原代培养细胞及星形胶质细胞体外原代培养均获成功,并经免疫组化染色鉴定、证实。分离组织中浸润淋巴细胞及外周血单个核细胞后,采用Mini MACS免疫磁珠分离技术获得组织和外周血中CD4~+CD25~+、CD4~+CD25-及CD8~+T细胞。经免疫磁珠两次分选后,CD4~+CD25~+T细胞的纯度达89~92%,CD4~+CD25-T细胞纯度在90%以上,CD8~+T细胞纯度达87%~ 94%。按照不同比例将CD4~+CD25~+ T和CD4~+CD25-T细胞或CD8~+T细胞共同培养,采用[3H]-TdR掺入法检测CD4~+CD25~+ T对CD4~+CD25-T细胞及CD8~+T细胞增殖的影响。采用Western Blot法对不同来源的CD4~+CD25~+T细胞中Foxp3、CTLA-4、GITR、CCR4和CCR8蛋白进行定量检测。采用ELISA法对胶质瘤组织原代培养细胞及不同来源CD4~+CD25~+T细胞培养上清中TGF-β1、CCL12和CCL22进行定量检测。3应用逆转录病毒载体LXSN,将插入IL-18基因的质粒感染PA317包装细胞,经G418筛选后获得表达IL-18分子的PA317阳性细胞克隆,用IL-18/PA317培养上清转染9L细胞,筛选出高表达IL-18的IL-18/9L细胞克隆,传代培养。将雄性F344大鼠随机分为2组,每组15只,分别颅内接种9L细胞和IL-18/9L细胞,每组分别在颅内接种肿瘤细胞后7天、14天、21天时各处死3只大鼠,每组剩余的6只大鼠用于观察生存期。采用逆转录聚合酶链反应(RT-PCR)技术定量检测各组大鼠脑胶质瘤组织中IL-18mRNA的表达。在颅内接种肿瘤细胞21天时,采用[3H]-TdR掺入法检测各组大鼠脾细胞增殖反应情况,ELISA法检测各组大鼠脾细胞IFN-γ的产生情况。采用流式细胞术检测各组胶质瘤组织中Treg细胞、CD4~+T、CD8~+T细胞的含量及比率,分析其与大鼠胶质瘤生长及生存期的关系。取接种9L细胞21天时的大鼠肿瘤组织,分离肿瘤组织中浸润淋巴细胞,利用Mini MACS免疫磁珠分离系统,分选CD4~+CD25~+T细胞和CD4~+CD25-T细胞,将两者共同培养,培养基中加入IL-18,观察CD4~+CD25~+T细胞免疫抑制功能的变化。结果:1胶质瘤患者肿瘤组织和外周血单个核细胞中Treg细胞比例变化:流式细胞学检测结果显示,星形胶质瘤患者外周血单个核细胞中CD4~+CD25~+Foxp3~+T细胞的含量为10.41±2.13%,明显高于健康人外周血(4.35±1.07%)(p<0.01)。脑胶质瘤组织中Treg细胞比例为35.45±2.47%,明显高于健康对照脑组织(Treg细胞含量2.53±0.75%)。胶质瘤组织中浸润的Treg细胞含量与肿瘤的恶性程度呈正相关,而与患者年龄、性别、肿瘤大小、部位无关。不同病理学级别胶质瘤组织中CD8~+T细胞比例无明显差别(p>0.05)。胶质瘤患者TIL或PBMC中Treg细胞比例变化:将胶质瘤患者TIL中Treg细胞所占比例的均值(M)作为阈值,分为高比值组(≥M)和低比值组(<M),绘制两组的生存曲线,应用log-rank法进行统计学分析,结果显示,Treg细胞比例与患者预后无明显关联,高比值组患者的生存期与低比值组无统计学差异(p>0.05)。而CD8~+T细胞在肿瘤组织的浸润数量亦与患者预后无关(p>0.05)。而以肿瘤组织中CD8~+T/Treg细胞比值作为观测指标时,其比值与胶质瘤患者预后呈正相关,高比值组患者的生存期明显长于低比值组,两者具有显著差异(p<0.05)。胶质瘤患者外周血单个核细胞Foxp3mRNA表达及TGF-β1含量:胶质瘤患者外周血单个核细胞中均可见Foxp3mRNA表达,明显高于健康人,并随胶质瘤恶性级别的增加而升高(p<0.05)。胶质瘤患者外周血中均可检测到TGF-β1,并随着肿瘤恶性程度的升高而增加。直线回归分析显示,外周血TGF-β1含量与PBMC中Foxp3mRNA的表达呈正相关(r=0.940,p<0.05)。2不同来源Treg细胞F0xp3、CTLA-4、GITR蛋白表达:Western Blot分析结果显示,不同来源的CD4~+CD25~+T细胞均检测到Foxp3、CTLA-4及GITR蛋白表达,而CD4~+CD25-T细胞无相应蛋白表达。源自胶质瘤患者外周血及肿瘤组织中的Treg细胞,Foxp3表达明显高于健康人。而在不同来源CD4~+CD25~+ T细胞之间,CTLA-4和GITR的表达并无明显区别(p>0.05)。源自胶质母细胞瘤患者TIL中的CD4~+ CD25~+T细胞高表达CCR4(0.64±0.21),显著高于健康者外周血中CD4~+CD25~+T细胞的含量(0.27±0.13)(p<0.05)(Fig2-8),但CCR8在胶质母细胞瘤患者中和健康者外周血中均未见明显表达。CD4~+CD25~+T细胞对CD4~+CD25-T及CD8~+T细胞增殖的影响:在抗人CD3单克隆抗体作用下,将不同比例CD4~+CD25~+T细胞和CD4~+CD25-T细胞及CD8~+T细胞共同培养,CD4~+CD25~+T细胞可明显抑制CD4~+CD25-T细胞及CD8~+T细胞的增殖。随着CD4~+CD25~+T细胞数量的增加,对CD4~+CD25-T细胞及CD8~+T细胞的抑制率逐步升高。原代胶质瘤细胞、不同来源CD4~+CD25~+T细胞培养上清中TGF-β1的水平:ELISA检测结果显示,体外培养的原代胶质瘤细胞及不同来源CD4~+CD25~+T细胞均分泌TGFβ1,而在正常人脑星形胶质细胞培养上清中,未检测到TGF-β1。在来源于胶质母细胞瘤患者TIL及PBMC的CD4~+ CD25~+T细胞中, TGF-β1的分泌量分别为6.21±1.14μg/ml和5.47±1.05μg/ml,两者无明显差异(p>0.05),但均明显高于健康者外周血中的CD4~+CD25~+T细胞(2.32±0.61μg/ml)(p<0.05)。原代胶质瘤细胞大量分泌CCL22(12.17±1.23μg /ml),明显高于CCL12(4.68±0.83μg/ml)(p<0.05)。3接种亲代鼠脑胶质瘤细胞9L的F344大鼠肿瘤组织中,CD4~+CD25~+ Foxp3~+T细胞的浸润数量明显增多,并呈现出时间依赖性。接种IL-18/9L和9L细胞大鼠脑肿瘤组织中IL-18mRNA表达:分别取接种IL-18/9L细胞和9L细胞21d时的大鼠脑肿瘤组织,经RT-PCR分析,结果表明:IL-18/9L细胞组大鼠脑肿瘤组织中有IL-18mRNA表达,而接种9L细胞组大鼠脑肿瘤组织中无IL-18mRNA表达,说明IL-18/9L细胞在荷瘤大鼠体内仍可表达IL-18。IL-18对大鼠脾细胞IFN-γ产生的影响:接种IL-18/9L细胞组大鼠脾细胞可产生高水平IFN-γ(178.2±13.1pg/ml),明显高于接种9L细胞组(40.7±5.3pg/ml)(p<0.01)。IL-18对CD4~+CD25~+ T细胞免疫抑制功能的影响:在不同CD4~+CD25~+T:CD4~+CD25-T细胞比例下,IL-18均不能影响CD4~+CD25~+ T细胞对CD4~+CD25-T细胞的增殖抑制,与对照组比较无显著差异(p>0.05)。接种IL-18/9L组大鼠生存期为66.3±5.02d,明显长于接种9L细胞组大鼠(43.5±3.87d)(p<0.05)。结论:1胶质瘤患者外周血、肿瘤组织中Treg细胞含量明显增加。胶质瘤组织中CD8~+T/Treg细胞比值与患者的预后呈正相关。Treg细胞在胶质瘤组织中的比例尚不能单独作为患者预后的指标。胶质瘤患者外周血中Treg细胞的数量与血浆TGF-β1的水平呈正相关。2 Treg细胞在胶质瘤中聚集和发挥免疫抑制作用的可能机制是:胶质瘤细胞通过CCL22/CCR4趋化作用,驱使Treg细胞由外周向肿瘤部位迁移,并在肿瘤部位通过分泌TGF-β1诱导非Treg细胞向Treg细胞转化和成熟、Treg细胞通过细胞-细胞接触和分泌抑制性细胞因子TGF-β1发挥免疫抑制作用,肿瘤组织中Treg细胞数量与其免疫抑制功能密切相关。3在荷9L/F344细胞大鼠脑胶质瘤模型中,Treg细胞数量明显增多,并呈现时间依赖性。在鼠脑胶质瘤模型中,IL-18可通过刺激脾细胞增殖,促进IFN-γ分泌,促进CD8~+T细胞在肿瘤部位的增殖,提高CD8~+ T/ Treg细胞的比率,解除Treg细胞在局部对效应性细胞的抑制作用可抑制鼠脑胶质瘤的生长,而IL-18在体外对Treg细胞增殖和功能无明显作用。更多还原

【Abstract】 Objective: Gliomas, especially high-grade gliomas are the most common malignant brain tumor showed infiltrative growth. It’s very difficult to cut the tumor completely. The overall prognosis of glioma patients is still not ideal in spite of comprehensive treatment including radiotherapy and chemotherapy. The recurrence rate of glioma was high and the survival time for patients with high-grade glioma were even less than 1 year. With the continuous development of molecular biology, glioma immunotherapy is arousing people’s attention gradually. The aggressive nature of this neoplasia is closely related to its complex pathophysiology. In particular, evasion of the immune system by gliomas limits effective anti-tumoral responses. Current evidence suggests a major role in the evasion of immune rejection by CD4~+CD25~+ regulatory T cells (Tregs).Tregs are lymphocytes that have a physiological role in the modulation of the immune response. Specifically, these cells prevent autoimmunity by inhibiting autoreactive effector T lymphocytes, which downregulates the anti-tumoral response and promotes the growth of tumor. Many studies have found a large population of Tregs were infiltrated in the tissue and blood of many malignancies including breast, ovarian, lung, liver carcinoma and malignant lymphoma. It’s suggested that the population of Tregs was correlated negatively with the progression and prognosis of tumor. Depletion of Tregs or inhibition of its function has been associated with recovery of anti-tumoral response.Tregs are divided into thymus-derived Tregs or natural Tregs (nTregs) and Inducible Tregs (iTregs). nTreg development takes place in the thymus and iTregs are very similar in function to nTregs but derive from non-Treg cells in the periphery under specific stimulation. The abnormal increasing Tregs in tumor microenvironment are also divided into two groups. One group includes Tregs migrated from periphery under the influence of chemokines. The other group are induced from CD4~+CD25–T cells upon the exposure to the suppressive cytokine milieu at the tumor site. Tregs separated from tumor infiltrated lymphocytes usually represent tumor-specific antigen. The differentiation and maturation of Tregs take place under the action of tumor cells, which prevent the function of effector T cells (mainly CD8~+, CD4~+). Imbalance between Tregs and conventional T cells play a role in the immune escape of tumor cells and recovery of imbalance may be a new strategy for tumor treatment.As pro-inflammatory cytokine, interleukin 18(IL-18) has also been implicated in multiple autoimmune associated disease, such as rheumatoid arthritis, sarcoidosis, lupus erythematosus. Tregs have been reported to be decreased or to have decreased functional activity in a number of autoimmune disorders, and it is possible that an imbalance of Tregs and effector T cells due to differential IL-18 signaling contributes to the loss of tolerance. Several preclinical studies have suggested that IL-18 may have significant antitumor effects. IL-18 enhances the production of IFN-γby T cells and NK cells and can augment the cytolytic activity of NK cells and cytotoxic T lymphocytes (CTL). IL-18 promotes the differentiation of activated CD4 T cells into helper effector cells of Th1 or Th2 type. It’s not clear, however, that IL-18 can manipulate the function of Tregs in tumor microenvironment.Aim of our investigation is to discuss the expression of Tregs in cancer tissue from patients with glioma, the correlation between the population of Tregs and progression of tumor, investigate the inhibitory mechanism of Tregs for immune fanction, analyze the manipulation of IL-18 on Tregs.Methods:1Tumor tissue and blood of 76 patients with astrocytoma were collected, who were operated in the department of neurosurgery of the 4th hospital affiliated to Hebei Medical University from September, 2007 to September, 2009. Normal brain tissue and blood were used for control, which were from patients with decompression surgery for herniation and health volunteers. TILs and PBMCs were separated from tumor tissue and blood. The frequency of Tregs, CD4~+T cells and CD8~+T cells and ratio between Tregs and other T cells in TILs and PBMC were detected by flow cytometry. Then, the frequency and ratio of Tregs were analyzed and observe the relationship between Tregs and clinical pathological characters, prognosis of patients with astrocytoma.Expression of TGF-β1 in periphery blood were analyzed by ELISA. The Foxp3mRNA in PBMC were detected by RT-PCR, and analyze the relationship between the expression of Foxp3mRNA and TGF-β1.2 Tumor tissue and blood of 5 patients with glioblastoma were collected, who were operated in the department of neurosurgery of the 4th hospital affiliated to Hebei Medical University from September, 2008 to September, 2009. Normal brain tissue and blood were used for control, which were from patients with decompression surgery for herniation and health volunteers. Primary glioma cells and primary normal astrocyte were cultured in vitro and identified by immunohistochemistry, respectively. Primary glioma cells and primary normal astrocytes were cultured successfully in vitro, and identified by immunohistochemistry. CD4~+CD25~+T cells, CD4~+CD25-T cells and CD8~+T cells from TIL and PBMC were isolated by immunomagnetic separation system. After magnetic activated cell sorting, the purity of CD4~+CD25-, CD8~+T cells was 89~92%, higher than 90% and 87~94%, respectively.CD4~+CD25-T cells were then co-cultured with CD4~+CD25~+T cells in different population. [3H]-TdR incorporation were used to determine the proliferation of CD4~+CD25-T cells.Expression of Foxp3, CTLA-4, GITR, CCR4 and CCR8 protein were detected by West Blot. The secretion of TGF-β1, CCL22 and CCL12 in the medium supernatant of primary glioma cells, primary normal astrocyte and different CD4~+CD25~+T cells.3 30 male Fisher344 rats were divided randomly into 2 groups: 9L group and IL-18/9L group.9L cells and IL-18/9L cells were inoculated into the right cerebral hemisphere, respectively. Three mice of each group were killed on the 7th, 14th and 21st day after inoculation of tumor cells, the spleens and tumors were obtained from mice. 6 rats remained of every group were observed for over survival time.Expression of IL-18mRNA in rat tumor tissue were detected by RT-PCR. [3H]-TdR incorporation were used to determine the proliferation of CD4~+CD25-T cells. The frequency of Tregs, CD4~+T cells and CD8~+T cells and ratio between Tregs and other T cells in TILs and PBMC were detected by flow cytometry on 21st day after inoculation of tumor cells. IFN-γproducted by splenocytes were detected with ELISA.On 21st day after inoculation of tumor cells, CD4~+CD25~+T cells, CD4~+CD25-T cells and CD8~+T cells from TIL and PBMC of rats were isolated by immunomagnetic separation system. CD4~+CD25-T cells were then co-cultured with CD4~+CD25~+T cells in different population and IL-18. [3H]-TdR incorporation were used to determine the proliferation of CD4~+CD25-T cells. The frequency and ratio of Tregs in TIL of every group on specified time were analyzed and observe the relationship between Tregs and tumor progression by flow cytometry.Results:1 The result of flow cytometry instructed that the mean frequency of Tregs in PBMC of patients with astrocytoma(10.41±2.13%) was higher than that of volunteers(4.35±1.07%, p<0.05). The mean frequency of Tregs in TIL of patients (35.45±2.47%) was higher than that of control(2.53±0.75%, p<0.05). In comparison to autologous patient blood, the frequency of Tregs in tumor tissue were correlated significantly with the malignancy of glioma but age, sex, tumor size and tumor location. There was not a significant difference of the frequency of CD8~+T cells in TILs according to pathological grade.In all patients were divided into higher group(≥M) and lower group(<M) according to the mean frequency(M) of T cells in TILs. The result showed that the ratio of CD8~+/Treg is a significant prognostic factor for the patients but Treg or CD8~+T cell alone.Expression of Foxp3mRNA in blood of patients was significantly higher than in controls(p<0.05). Moreover, higher level of TGF-β1 in the blood of patients in comparison to the blood from control individuals(p<0.05). Expression of Foxp3mRNA in blood was correlated significantly with the level of TGF-β1(r=0.940, p<0.05).2 CD4~+CD25-, CD8~+T cells were respectively co-cultured with CD4~+CD25~+T cells in different population. With increasing population of CD4~+CD25~+T cells, the proliferation of CD4~+CD25-, CD8~+T cells was significantly suppressed by CD4~+CD25~+T cells.The result of ELISA showed that the level of TGF-β1 was observed in supernatant of primary glioma cells and different CD4~+CD25~+T cells but normal astrocytes. In comparison to CD4~+CD25~+T cells of health volunteers (2.32±0.61μg/ml), the higher secretion of TGF-β1 was observed in CD4~+CD25~+T cells from tumor tissue (6.21±1.14μg/ml) and autologous blood (5.47±1.05μg/ml). Moreover, the secretion of CCL22 was very higher than that ofCCL12.The result of West Blot instructed that higher level of CCR4, FOXP3 was measured in CD4~+CD25~+T cells from the TIL in comparison to that from control individuals (p<0.05). There was not a significant difference for CTLA-4, GITR between different CD4~+CD25~+T cells. Expression of CCR8 was not observed in any supernatant.3 For rats inoculated with 9L cells, a time dependent increase in the frequency of CD4~+CD25~+Foxp3~+Treg at the tumor bearing site was observed.Expression of IL-18mRNA was measured in IL-18/9L group but in 9L group, which indicated that IL-18/9L cell can represent IL-18 at site of tumor. The splenocytes from the rat inoculated with IL-18/9L cells can secret IFN-γ(178.2±13.1pg/ml) that were higher than with 9L cells (40.7±5.3pg/ml, p<0.05).In suppression assay, IL-18 failed to influence the inhibitory function of CD4~+CD25~+T cells in vitro.Compared with 9L group (43.5±3.87d), the mean overall survival time of IL-18/9L group (66.3±5.02d) was significant longer. Conclusion:1 Infiltration of Tregs is significantly higher in the tumor and blood of patients with astrocytoma. The ratio of CD8~+/Treg is a significant prognostic factor for the patients but Treg or CD8~+T cell alone. Expression of Foxp3mRNA in blood was correlated significantly with the level of TGF-β1 in patients with astrocytoma.2 Tregs infiltrated in glioma may migrated from periphere under the influence of CCR4/CCL22. Glioma cells can sectret high level TGF-β1 which may turn non-Tregs into Tregs. Cell-cell contact seems to be the important way by which Treg present inhibitory function in glioma. The population of Tregs play a important role in its function.3 For rats inoculated with 9L cells, a time dependent increase in the frequency of Treg at the tumor bearing site was observed. Although, IL-18 failed to influence the inhibitory function of CD4~+CD25~+T cells in vitro, it can increase the amount of CD8~+T cells, especially the ratio of CD8~+/Treg, promote the secretion of IFN-γand upregulate anti-tumor immunoresponse.更多还原