【作者】 崔永生；

【导师】 李一；

【作者基本信息】 吉林大学 ， 免疫学， 2007， 博士

【摘要】 免疫系统的功能之一免疫监视在清除肿瘤细胞方面发挥着重要作用。当机体内正常的细胞发生突变转化成肿瘤细胞时,免疫系统可对肿瘤细胞产生一系列免疫应答,消灭肿瘤细胞。然而临床上肿瘤发生的事实表明,肿瘤细胞可通过某些机制诱导肿瘤细胞的耐受,逃避免疫监视而在机体内生长、转移。多种机制可参与肿瘤细胞逃避免疫监视,包括肿瘤抗原的缺失、MHC I分子表达下降、共刺激分子缺乏及死亡受体信号缺陷等。近年来发现具有免疫调节功能的CD4+CD25+调节性T细胞(Regulatory T cells, Treg)在保持自身耐受和免疫稳定的同时,也可能以某种机制抑制着免疫系统对肿瘤细胞的免疫应答。这可能是肿瘤细胞难以诱导有效免疫应答的重要因素。本课题从临床肿瘤病人和小鼠动物模型两方面入手,对CD4+CD25+ Treg与肿瘤的相关性进行了以下三方面的研究:1、肺癌、乳腺癌患者外周血中CD4+CD25+Treg细胞数量和功能变化的研究。结果显示肺癌和乳腺癌患者外周血中CD4+CD25+Treg细胞数量增加,CD4+CD25+Treg细胞功能相关基因Foxp3 mRNA表达水平均明显高于非肿瘤患者。2、Lewis肺癌小鼠中CD4+CD25+Treg细胞与免疫监视功能的研究。结果表明荷瘤鼠体内及肿瘤局部引流淋巴结内CD4+CD25+Treg细胞比例增高,Foxp3 mRNA表达水平增强;荷瘤鼠脾脏及胸腺T淋巴细胞增殖功能及脾细胞中CTLs杀伤功能明显降低。3、抗肿瘤药物环磷酰胺(CTX)和卡介苗(BCG)疗法对Lewis肺癌小鼠CD4+CD25+Treg细胞与免疫监视功能影响的研究。结果显示单独应用CTX可降低CD4+CD25+Treg细胞的数量及Foxp3 mRNA表达,使T淋巴细胞增殖功能及脾细胞中CTLs杀伤功能增强。而单独应用BCG既可以促进效应细胞的活化,同时也可以活化肿瘤局部的CD4+CD25+Treg细胞。联合应用CTX和BCG显示既可以降低CD4+CD25+Treg细胞的数量及功能,又可以增强肿瘤免疫功能。研究结果提示在肿瘤的情况下,外周和肿瘤局部的CD4+CD25+Treg细胞的数量增多,并且功能增强。这种CD4+CD25+Treg细胞可干扰免疫系统对肿瘤的免疫应答,参与肿瘤的免疫逃逸、诱导肿瘤的免疫耐受,促进肿瘤的发生和发展。如果以CD4+CD25+Treg细胞为靶点,通过降低其数量、抑制其增殖或削弱其功能,可使机体免疫系统充分发挥对肿瘤细胞的杀伤效应。因此新的肿瘤治疗策略是在刺激肿瘤免疫应答的同时抑制或清除CD4+CD25+Treg细胞,使肿瘤免疫治疗更加有效和完善。更多还原

【Abstract】 Immunological surveillance of immune system is that immune system may recognize and destroy tumor cells. But tumor cells may continuously grow by various kinds of ways or mechanisms that escape from immune system recognition and elimination, such as down regulation MHC I molecule, deprivation of tumor antigens, deficiency of death receptor signal, deficiency of co-stimulation, production immune suppression factors and so on. At present, it had been confirmed that CD4+CD25+ regulatory T cells (Treg) could actively suppress or modulate the self reactive T cells in peripheral and play an important role in maintaining peripheral tolerance. Apart from in self-tolerance, CD4+CD25+Treg might be involved in tumor– tolerance by suppressing tumor immunity.After the detection of CD4+CD25+ regulatory T cells by Sakaguchi in 1995, they are in the focus of intense research in immunology. Naturally occurring CD4+CD25+ regulatory T cells are generated in thymus, and they represent 5-10% of total CD4+T lymphocytes. The TCR of CD4+CD25+ Treg may recognize self and foreign antigens, and have priority in self ones. Activated CD4+CD25+ Treg can inhibit the proliferation and cytokines secretion of CD4+ and CD8+ T cell, and also inhibit NK, B cell and DC. CD4+CD25+ Treg inhibit the proliferation of effector cells by cell-cell contact and/or secreting soluble cytokine such as TGF-βand IL-10. The transcription factor Foxp3, CTLA-4 and membrane-bound TGF-βmay have effect in the inhibition. We have discovered that CD4+CD25+ Treg hamper immune responses that immune system aim directly at tumor when they maintain immunologic homeostasis.So, CD4+CD25+Treg may play its function not only in promoting growth of tumor but also in causing troubles in tumor immunotherapy especially in tumor vaccine treatments.It may helpful for tumor immunotherapy to research the function of CD4+CD25+ Treg in tumor and the effect of antitumor drug and immunoenhancer on CD4+CD25+ Treg. So we should promote the immune response to tumor and downregulate the inhibition of CD4+CD25+ Treg simultaneously in the therapy.In this research, we have studied the CD4+CD25+Treg in tumor patients and Lewis lung cancer model from three parts:1. Changes of CD4+CD25+Treg in lung cancer and breast cancer patients.The results showed that the number of CD4+CD25+Treg was significantly enhanced in peripheral blood mononuclear cells from patients with lung cancer and breast cancer. The expressions of Foxp3 mRNA were increased. It means that tumor cells could induce the proliferation, amplification and activation of CD4+CD25+Treg by expressing tumor antigens or secreting immune inhibition factors, so it can impair effects of effector cells’on tumor, and improve the tumor survival and development.2. Changes of CD4+CD25+Treg and immunological surveillance in Lewis lung cancer model.The results showed that the number of CD4+CD25+Treg was markedly enhanced and expressions of Foxp3 mRNA were increased in thymus, spleen and tumor regions from mice with Lewis lung cancer. The mRNA expressions of CTLA-4 and IL-10 that are molecules related with CD4+CD25+Treg functions were increased also than normal mice. The T cell proliferation was decreased in spleen. The function of CTLs in spleen was decreased. It might be implicated that tumor cells not only stimulated peripheral and local CD4+CD25+Treg proliferation and activation, but also stimulated CD4+CD25+ Treg generation in thymus, interfered immune response to tumor cells. It may be one of the mechanisms of tumor cells evading from immune elimination.3. Influence of CTX and BCG on CD4+CD25+ Treg and immunological surveillance.The studies of this part, three kinds of therapy method were employed including CTX alone, BCG alone and combination of CTX and BCG. 1) Influence of CTX on CD4+CD25+ Treg and immunological surveillance.CTX could decrease the number and interfere the function of CD4+CD25+Treg in the thymus and spleen, and down-regulate the expression of Foxp3 mRNA in splenocyte and Foxp3, CTLA-4 and IL-10 mRNA in lymph node at the tumor local, and also enhance the proliferation of T lymphocyte and function of CTLs in the spleen. Our experiments proved that CTX might enhance the tumor immune response by inhibiting the function of CD4+CD25+Treg and delay tumor growth. It also demonstrated that CD4+CD25+Treg has the functions of inhibiting effector cells, and indirectly promote development of tumor.2) Influence of BCG on CD4+CD25+Treg and immunological surveillance.The result showed that BCG has two sides of functions: one side is that it can enhance the proliferation and cytotoxicity of immune cells, and inhibit tumor growth, and another side is it could also enhance the expression of Foxp3 mRNA in tumor region. So tumor effector cells and CD4+CD25+Treg can be both activated by BCG in tumor region simultaneously. This might be the reasons why the treatment of tumor with BCG can’t act as expected, because activation of CD4+CD25+Treg by BCG can inhibit the activity function of effector cells and hamper the killing effective of effector cells.3) Combination of CTX and BCG on CD4+CD25+ Treg and immunological surveillance.Combination of CTX and BCG could not only reduce the number of CD4+CD25+Treg in the thymus and spleen, but also down-regulate the expression of Foxp3 mRNA in splenocyte and lymph node in the tumor region, and then it can improve the proliferation and cytotoxic function of splenocyte obviously. It suggests that the inhibition of CD4+CD25+Treg by CTX and promotion of effector cells function by BCG simultaneously can make tumor immunotherapy more reasonable and effective.Our research suggests that in the body with tumor growth, the number and the function of CD4+CD25+Treg are promoted in peripheral and tumor region. This group of cells can hamper the immune response to tumor, help tumor to escape from immune system recognition and elimination, induce the immune tolerance to tumor, promote the generation and development of tumor cells. So it can be a new way if we cure the cancer patients by aiming at the CD4+CD25+Treg. So it may be a new treatment strategy to reinforce the immune response by enhance the effector cells and diminish the CD4+CD25+Treg meanwhile. To sum up, tumor can escape from immunologic surveillance in various kinds of mechanisms, and one of them is the function of CD4+CD25+Treg that could inhibit tumor immune response. Previous strategies of tumor immunotherapy mainly focused on how to enhance the tumor immune response, and ignore the negative effect of Treg on tumor immunotherapy. Our experiment suggested that development of tumor might be related with CD4+CD25+Treg, and CD4+CD25+Treg could be involved in immune evasion of tumor and induce tumor tolerance.Our experiment and other reports all discovered that by decreasing the number or function of CD4+CD25+Treg can delay the speed of tumor growth, and the effect will be better if eliminating CD4+CD25+ Treg completely, but it will unavoidably lead to autoimmune diseases. Therefore, it is a significant question that how to properly impair the function of CD4+CD25+Treg, so that it will make CD4+CD25+Treg that neither hamper tumor treatment, nor evoke autoimmune diseases. So far, the specific markers of CD4+CD25+Treg haven’t been identified. CD25, CTLA-4, GITR also express in effector cells , so interfering CD4+CD25+Treg by this molecules will also interfere the function of effector cells, and have disadvantagement for the whole immunotherapy. Therefore, we will gain preferably therapeutic efficacy in tumor immunotherapy by searching the specific marker, precisely controlling the number and function of CD4+CD25+Treg.So promotion of tumor immune response and inhibition of CD4+CD25+Treg at same time may make tumor immunotherapy more reasonable and effective.To sum up, tumor can escape from immunologic surveillance, recognition and elimination, and CD4+CD25+Treg are important in this program. They proliferate and activate in tumor development, and have protective effect on tumor cells. Previous tumor immunotherapy may focus on how to enhance the cytotoxic effect of effector cells, and ignore the harmful effect of Treg to tumor immunotherapy. The result of our research suggests that CD4+CD25+Treg are important in the generation and development of tumor.更多还原