肿瘤免疫治疗中CIK细胞、Treg细胞及其相关性研究
|
摘要
目的:用免疫活性细胞输注的过继性细胞免疫疗法(adoptive cell immunotherapy ACI)是肿瘤生物治疗的研究热点之一。此治疗方法不仅是常规手术、放疗、化疗等抗肿瘤治疗的补充,它对于促进患者免疫系统的重建、消除残留病变等都有良好的效果。更为晚期不适于手术治疗或无法承受放、化疗所带来的副作用的患者开辟了一个新的治疗途径,成为人类抗肿瘤治疗最有希望的措施之一。
细胞因子诱导的杀伤细胞CIK(cytokine induced killer,CIK),作为一种新型高效的免疫活性细胞因其具有增殖能力强、杀瘤谱广、杀瘤活性强等其它一些效应细胞无法比拟的优越特性,在过继性细胞免疫治疗中得到了广泛的应用。如何提高CIK细胞的数量、增强其抗肿瘤杀伤活性以及抗肿瘤特异性等方面是目前针对于CIK细胞的研究关键。树突状细胞(DC)是迄今为止发现的效力最强的抗原递呈细胞,具有高效摄取、加工、递呈肿瘤相关抗原,并激活初始免疫反应的独特功能。本实验利用DC细胞与CIK细胞共同培养,并且负载特异性抗原,以便获得数量更多、杀伤活性更高,更具有特异性的抗肿瘤效应细胞。
但是在实际应用过程中,抗肿瘤效应细胞在肿瘤微环境中通常被诱导进入"免疫无能"(immune anergy)状态,因此不能有效地识别和杀伤肿瘤细胞。去除体内的免疫抑制细胞,充分发挥效应细胞的功能,对于提高肿瘤的治疗效果具有重要意义。CD4~+CD25~+Treg细胞是新发现的具有免疫抑制作用的细胞群,目前较为常用的是根据产生的途径不同将CD4~+CD25~+ T细胞分为自然产生的调节性T细胞(natural regulatory T cells nTreg)和诱导产生的调节性T细胞(induced regulatory T cells iTreg)两类。大量研究发现在胃癌、肺癌、乳腺癌等癌症患者的外周血、肿瘤浸润淋巴结中都可以检测出CD4~+CD25~+T细胞,CD4~+CD25~+T细胞可能通过识别肿瘤抗原,活化后发挥其免疫抑制作用,抑制机体抗肿瘤免疫应答的产生,使机体处于对肿瘤低应答或无应答的一种状态。这些研究说明CD4~+CD25~+Treg细胞对于肿瘤的免疫治疗是一个重要的障碍。因此寻找既能增强免疫效应细胞的抗肿瘤作用又能抑制或清除CD4~+CD25~+T细胞作用的方法,将为提高和改善肿瘤免疫治疗提供一个崭新的途径。
本实验将特异性抗原致敏的成熟DC与CIK细胞共同培养,作为抗肿瘤免疫效应细胞,从形态学、对细胞增殖和凋亡的影响以及对肿瘤细胞杀伤活性等不同方面观察不同的阶段应用免疫磁珠分选去除CD4~+CD25~+Treg细胞成分的特异性抗原致敏的DC-CIK细胞对B16黑色素瘤动物模型的作用,来探讨和分析CIK细胞、Treg细胞在抗肿瘤免疫中所起的作用以及相互关系,来为抗肿瘤免疫治疗的临床应用提供更多的治疗思路和理论基础。
方法:
本实验以C57BL/6小鼠的黑色素瘤动物模型作为研究对象,制备黑色素瘤动物模型,并应用放射性钴60对小鼠黑色素瘤动物模型进行照射,使小鼠机体内形成非髓性淋巴细胞删除状态,使C57BL/6小鼠的黑色素瘤动物模型的免疫机制丧失。
取小鼠外周血单个核细胞(PBMC)制备树突状细胞和CIK细胞,将两者共同培养,并且负载肿瘤特异性抗原,并检测其免疫表型。以免疫磁珠分选方法分别在培养前及培养后去除CD4~+CD25~+Treg细胞成分。分别得到去除CD4~+CD25~+Treg细胞成分再经体外诱导产生的特异性抗原负载的DC-CIK效应细胞、经体外诱导产生肿瘤特异性抗原负载的DC-CIK效应细胞,再用磁珠分选方法去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK效应细胞和未去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK效应细胞共三组抗肿瘤免疫效应细胞。
然后再将制备好的达到非髓性淋巴细胞删除状态的C57BL/6小鼠的黑色素瘤动物模型随机分为4组。第一组:回输先通过磁珠分选去除CD4~+CD25~+Treg细胞成分再经体外诱导产生的特异性抗原负载的DC-CIK效应细胞,第二组:回输先经体外诱导产生肿瘤特异性抗原负载的DC-CIK效应细胞,再用磁珠分选方法去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK效应细胞。第三组:回输未去除CD4~+CD25~+Treg细胞成分的DC-CIK细胞组。第四组:对照组。
通过测量各组的肿瘤体积、重量、计算抑瘤率,比较其抑瘤作用,并应用透射电镜观察特异性抗原负载的DC-CIK细胞及杀伤肿瘤细胞的形态学表现。应用流氏细胞技术(FCM)检测各组肿瘤细胞的增殖周期中G0/G1期、S期、G2/M期细胞比率、细胞增殖指数(proliferation Index,PI)和细胞凋亡指数(apoptosis Index ,AI)的变化,观察特异性抗原负载的DC-CIK对肿瘤细胞增殖和凋亡的影响。
采用MTT染色法检测培养前去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组、培养后去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组、未去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组三组抗肿瘤免疫效应细胞对B16黑色素瘤肿瘤细胞的杀伤效应。
结果:
采用特异性肿瘤抗原致敏的DC细胞与CIK细胞共同培养,分析其免疫表型,透射电镜观察特异性抗原负载的DC-CIK细胞体积增大,核有切迹,细胞质内细胞器丰富,粗面内质网扩张,细胞表面有突起,与肿瘤细胞密切接触,大量肿瘤细胞凋亡、坏死。
培养前去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组肿瘤平均体积0.0377±0.0128cm3 ,培养后去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组肿瘤平均体积0.0359±0.0131cm3,未去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组肿瘤平均体积0.1278±0.0362 cm3,而对照组肿瘤平均体积0.4052±0.0429cm3。三组实验组肿瘤体积均明显小于对照组(P<0.05),不同阶段去除CD4~+CD25~+Treg细胞成分的两组之间比较,虽然培养前去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组较培养后去除CD4~+CD25~+Treg细胞成分组体积略小,但没有显著性差异(P >0.05)。但两组与未去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组比较肿瘤体积有统计学差异(P<0.05)。
三组特异性抗原致敏的DC-CIK效应细胞组抑瘤率明显高于对照组(P<0.05);去除CD4~+CD25~+Treg细胞成分两组抑瘤率明显高于未去除CD4~+CD25~+Treg细胞DC-CIK效应细胞组(P<0.05),但两组之间没有明显差异(P >0.05)。
瘤体重量对照组3.361±1.07g,培养前去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组0.958±0.32g,培养后去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组0.939±0.41g,未去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组1.651±0.57g。同样三实验组瘤体重量均明显小于对照组(P<0.05)。但不同阶段去除CD4~+CD25~+Treg细胞成分的两组肿瘤重量都小于未去除CD4~+CD25~+Treg细胞成分组,并且有统计学差异(P<0.05)。不同阶段去除CD4~+CD25~+Treg细胞成分的两组之间则没有显著性差异(P >0.05)。
透射电镜可以观察到特异性抗原负载的DC-CIK细胞能促进肿瘤细胞凋亡超微结构的改变。流式细胞(FCM)检测癌细胞增殖周期中的变化;实验组三组与对照组比较G0/G1期细胞比率、AI显著升高,S期无明显变化,G2/M期细胞比率、PI显著下降;去除CD4~+CD25~+Treg细胞成分两组G0/G1期细胞比率、AI明显高于未去除CD4~+CD25~+Treg组,G2/M期细胞比率、PI显著低于未去除CD4~+CD25~+Treg组,S期无明显变化。去除CD4~+CD25~+Treg细胞成分两组之间比较G0/G1期、S期、G2/M期细胞比率和AI、PI均没有明显差异(P >0.05)。
采用MTT法检测培养前去除CD4~+CD25~+Treg细胞DC-CIK细胞组,培养后去除CD4~+CD25~+Treg细胞DC-CIK细胞组和未去除CD4~+CD25~+Treg细胞DC-CIK细胞组三组效应细胞对于B16黑色素瘤细胞的杀伤作用,结果表明三组效应细胞对肿瘤细胞都有较强的杀伤作用。培养前去除CD4~+CD25~+Treg细胞DC-CIK细胞组和培养后去除CD4~+CD25~+Treg细胞DC-CIK细胞组对B16黑色素细胞的杀伤作用明显高于(P<0.05)未去除CD4~+CD25~+Treg细胞DC-CIK细胞组。且不同效靶比之间比较,效靶比越高杀伤作用越强。培养前去除CD4~+CD25~+Treg细胞DC-CIK细胞组和培养后去除CD4~+CD25~+Treg细胞DC-CIK细胞组两组之间比较对B16黑色素细胞的杀伤作用没有显著性差异(P >0.05)。
结论:
1、特异性抗原致敏的DC-CIK细胞对B16黑色素瘤具有明显的抑瘤作用,并且对B16黑色素瘤的肿瘤细胞有较强的杀伤作用。特异性抗原致敏的DC-CIK细胞可以影响肿瘤细胞的细胞周期,并且具有诱导肿瘤细胞凋亡的作用。说明CIK细胞是一种具有强大肿瘤杀伤能力的新一代过继免疫抗肿瘤细胞。
2、去除CD4~+CD25~+Treg细胞成分后的特异性抗原致敏的DC-CIK细胞抗肿瘤免疫的作用更加高效而且特异,说明CD4~+CD25~+Treg细胞能够对特异性抗原致敏的DC-CIK细胞杀伤肿瘤细胞的活性形成明显的抑制。去除CD4~+CD25~+Treg细胞,重新募集效应性T细胞能够增强机体的抗肿瘤作用,这将成为一种可行的肿瘤免疫治疗方法。
3、不同时段去除CD4~+CD25~+Treg细胞后,从抑瘤作用、对于肿瘤细胞的细胞周期的影响和对肿瘤细胞的杀伤作用上都有一定的差异,但没有统计学意义,说明CD4~+CD25~+Treg细胞各个亚群的来源和作用机制还需要进一步的研究。
Objective: Adoptive immunotherapy is one of the hotest subject in biotherapy of tumor. The treatment is not only the supplement to conventional therapy such as surgical operation、chemotherapy and radiotherapy ,but also apply hope to people who can not sustain the side effects of radiotherapy and chemotherapy any longer.Adoptive immunotherapy has good therapeutic effect to the tumor patients in immune reconstruction and eliminate remained pathological.
Cytokine induced killer (CIK)cells is one of the new type immunocompetence cells, they have high efficiency in anti-tumor immunity. Cytokine induced killer cells have widespread using in adoptive immune therapy ,because they showed highly effcient cytolytic effector, faster proliferation speed rate then other immunocompetence cells in anti-tumor immunity.The key of studies to cytokine induced killer cells are how to enhance their cytolytic effcient and anti-tumor specific effcient,obtain more cytokine induced killer cells. Dendritic cells(DCs) are the most potent antigen presenting cells(APC) with the ability to acquire,process,present,antigens and the unique capability of initating primary immune responses against tumor-associated antigens. Dendritic cells(DCs) and cytokine induced killer(CIK) cells were separated and culture respectively,then dendritic cells(DCs) were loaded with specific tumor antigen. By using this methods ,we can co-culture tumor Antigen-sensitized Dendritic cells (DCs) with cytokine induced killer( CIK)cells. The tumor antigen-sensitized Dendritic cells (DCs) with cytokine induced killer( CIK)cells has higher cytolytic effcient and more specific anti-tumor specific effcient.We can obtain more quantity immunocompetence cells than cytokine induced killer( CIK)cells which did not loaded with specific tumor antigen.
But in practice, at the tumor circumstances anti-tumor immunocompetence cells were be revulsanted into immune anergy state constantly . so the anti-tumor immunocompetence cells killing effect descend obviously. It has very importmant significance effect to increase the anti-tumor immunotherapy that delete immunodepression cells. CD4~+CD25~+ regulatory T cells is one of the new discovered cells,they are the main subsets of regulatory T cells that play an essential role in maintaining immunological self-tolerance. They have the function of down-regulates the tumor immunity mediated by T cells. CD4~+CD25~+ regulatory T cells also divided into two groups by produced channel.The two groups are natural regulatory T cells (nTreg) and induced regulatory T cells( iTreg). CD4~+CD25~+ regulatory T cells increases in the peripheral biood and tumor infiltration lymphoglandula in many tumor patients,such as breast cancer、lung cancer、gastric cancer. CD4~+CD25~+ regulatory T cells suppress immune responses mainly by cell contract-dependent interactions or secreting soluble cytikines.Depletion or attenuation of CD4~+CD25~+ regulatory T cells will evoke effective anti-tumor immunity,which may become a feasible immunotherapy for cancer.
With this experimental, we use the specific antigen-sensitized DC-CIK cells as the anti-tumor immunotherapy cells.We observe the effect on B16 melanoma tumor animal experimental modle by DCs loaded with tumor antigen through morphological observation、cells proliferation and apoptosis、killing effect. By this way, we may analyse and discuss the relationship between CIK cells and regulatory T cells in anti-tumor immunity. We want to find more treament methods and theory foundation to clinical therapy in anti-tumor immunotherapy through the experimental.
Methods:
The B16 melanoma tumor cells were incubated in the C57 BL/6 mouse to build the animal models.Then using the total body irradiation by 60Co-ray to the animal models to eliminate it’s immunol function. Then the animal models in a no-marrew lymphocyte deletion state.
Peripheral biood mononuclear cells(PBMC) were isolated from C57 mouse. DCs were induced from adherent cells by some cytokines and CIK cells were generated from non- adherent cells. Mature DCs co-cultured with CIK cells,then loaded with specific tumor antigen, analyze specific antigen-sensitized DC-CIK immune epitope. Before and after culture ntigen-sensitized DC-CIK MACS (magnetic cell sorting) separating system was used to delete CD4~+CD25~+ regulatory T cells. So we have three gpoups anti-tumor immunocompetence cells. The first group: after by using MACS separating system to delete CD4~+CD25~+ regulatory T cells, then culture and acquire the specific antigen-sensitized DC-CIK cells. The second group: culture and acquire the specific antigen-sensitized DC-CIK cells ,then use MACS separating system to delete CD4~+CD25~+ regulatory T cells in this specific antigen-sensitized DC-CIK cells. The third group: the specific antigen-sensitized DC-CIK cells which did not delete CD4~+CD25~+ regulatory T cells.
Then randomize divided the animal models into four groups. The first group: after by using MACS separating system to delete CD4~+CD25~+ regulatory T cells, then culture and acquire the specific antigen-sensitized DC-CIK cells were injectedinto the animal models. The second group: culture and acquire the specific antigen-sensitized DC-CIK cells ,then use MACS separating system to delete CD4~+CD25~+ regulatory T cells in this specific antigen-sensitized DC-CIK cells were injectedinto the animal models.The third group: the specific antigen-sensitized DC-CIK cells which did not delete CD4~+CD25~+ regulatory T cells were injectedinto the animal models.The fourth group:control group.
According to the change of tumor volume、tumor weight and the tumor inhibition rates, inhibitory effect of the specific antigen-sensitized DC-CIK cells on B16 melanoma cells were estimated. Morphological characteristics of immune cells were observed by electronic microscope.With flow cytometry equipment,the rate of tumor cells in G0/G1、S、G2/M stage、proliferation index and the apoptosis index were respectively detected. To study the effect of cytokine induced killer cells on B16 melanoma cells proliferation and apoptosis in the animal models.Determine the killing effect on tumors cells by MTT method.
Results:
Co-culture tumor antigen-sensitized Dendritic cells (DCs) with cytokine induced killer (CIK) cells,analyze the immune epitope. The specific antigen-sensitized DC-CIK cells were bigger than the normal lymphocytes and notch always appears on the cellar nucleus observed under TEM. Plenty of active-functioned organelles such as dilated endoplasmic reticulum were observed in the cytoplasm of the specific antigen-sensitized DC-CIK cells. Protrusions on the surface of the specific antigen-sensitized DC-CIK cells contacted closely with the tumor cells. Apoptosis and necrosis of the cells could be widely observed in tumor tissues.
The tumor volume of the first group is 0.0377±0.0128cm3 ;the second group is 0.0359±0.0131cm3;the third group is 0.1278±0.0362 cm3;control group is 0.4052±0.0429cm3. The tumor volume of the three experiment groups were significant smaller than the control group(P<0.05). The first and second groups were significant smaller than the third group(P<0.05).There was no significant difference between the first groups and the second groups in tumor volume (P >0.05).
Tumor inhibition rate of the third group(59.19%) was lower than that of either the first group(91.70% ,P<0.05) or the second group(92.64% ,P<0.05) . There was no significant difference between the first groups and the second groups in tumor inhibition rate (P >0.05). The tumor inhibition rate of the three experiment groups were significant higher than the control group(P<0.05).
The tumor weight of control group is 3.361±1.07g, the first group is 0.958±0.32g ;the second group is 0.939±0.41g;the third group is 1.651±0.57g. The tumor weight of the three experiment groups were significant lower than the control group(P<0.05). The first and second groups were significant lower than the third group(P<0.05). There was no significant difference between the first groups and the second groups in tumor weight (P >0.05).
By using flow cytometry equipment,compared with the three experiment groups and the control group, the rate of tumor cells in G0/G1 and apoptosis Index were significantly higher,on the other hand , the rate of tumor cells in G2/M stage and proliferation Index were significantly lower,but there was not greatly change in S stage. The first and second groups compared with the third group have the same circumstances. There was no significant difference between the first groups and the second groups in the rate of tumor cells in G0/G1、apoptosis index、G2/M stage and proliferation index(P >0.05).
The specific antigen-sensitized DC-CIK cells showed strong killing effect on B16 melanoma tumor cells. The killing effect on B16 melanoma tumor cells of the three experiment groups were significant lower than the control group(P<0.05). The first and second groups were significant higher than the third group(P<0.05). There was no significant difference between the first groups and the second groups in killing effect on B16 melanoma tumor cells (P >0.05).
Conclusion:
1. The specific antigen-sensitized DC-CIK cells have high efficiency in inhibition on B16 melanoma. The specific antigen-sensitized DC-CIK cells may have an effect on the tumor cells cycle and induce them apoptosis.CIK cells is a new generation of adoptive immunocell has high efficiency in killing tumors cells.
2. The specific antigen-sensitized DC-CIK cells have high efficiency and more specifical in inhibition effect on B16 melanoma tumor cells after delete CD4~+CD25~+ regulatory T cells. CD4~+CD25~+ regulatory T cells could exert inhibitory effect on The specific antigen-sensitized DC-CIK cells killing function. Deletion CD4~+CD25~+ regulatory T cells and recruitment of effector T cells will evoke effective anti-tumor immunity,which may have become a feasible immunotherapy for cancer.
3. There was no significant difference between the deletion CD4~+CD25~+ regulatory T cells in different stage through tumor inhibition rate、tumor cells apoptosis and proliferation、the killing effect. We must do more study to explain the possible mechansim and relationship between the main subsets of CD4+CD25+ regulatory T cells.
细胞因子诱导的杀伤细胞CIK(cytokine induced killer,CIK),作为一种新型高效的免疫活性细胞因其具有增殖能力强、杀瘤谱广、杀瘤活性强等其它一些效应细胞无法比拟的优越特性,在过继性细胞免疫治疗中得到了广泛的应用。如何提高CIK细胞的数量、增强其抗肿瘤杀伤活性以及抗肿瘤特异性等方面是目前针对于CIK细胞的研究关键。树突状细胞(DC)是迄今为止发现的效力最强的抗原递呈细胞,具有高效摄取、加工、递呈肿瘤相关抗原,并激活初始免疫反应的独特功能。本实验利用DC细胞与CIK细胞共同培养,并且负载特异性抗原,以便获得数量更多、杀伤活性更高,更具有特异性的抗肿瘤效应细胞。
但是在实际应用过程中,抗肿瘤效应细胞在肿瘤微环境中通常被诱导进入"免疫无能"(immune anergy)状态,因此不能有效地识别和杀伤肿瘤细胞。去除体内的免疫抑制细胞,充分发挥效应细胞的功能,对于提高肿瘤的治疗效果具有重要意义。CD4~+CD25~+Treg细胞是新发现的具有免疫抑制作用的细胞群,目前较为常用的是根据产生的途径不同将CD4~+CD25~+ T细胞分为自然产生的调节性T细胞(natural regulatory T cells nTreg)和诱导产生的调节性T细胞(induced regulatory T cells iTreg)两类。大量研究发现在胃癌、肺癌、乳腺癌等癌症患者的外周血、肿瘤浸润淋巴结中都可以检测出CD4~+CD25~+T细胞,CD4~+CD25~+T细胞可能通过识别肿瘤抗原,活化后发挥其免疫抑制作用,抑制机体抗肿瘤免疫应答的产生,使机体处于对肿瘤低应答或无应答的一种状态。这些研究说明CD4~+CD25~+Treg细胞对于肿瘤的免疫治疗是一个重要的障碍。因此寻找既能增强免疫效应细胞的抗肿瘤作用又能抑制或清除CD4~+CD25~+T细胞作用的方法,将为提高和改善肿瘤免疫治疗提供一个崭新的途径。
本实验将特异性抗原致敏的成熟DC与CIK细胞共同培养,作为抗肿瘤免疫效应细胞,从形态学、对细胞增殖和凋亡的影响以及对肿瘤细胞杀伤活性等不同方面观察不同的阶段应用免疫磁珠分选去除CD4~+CD25~+Treg细胞成分的特异性抗原致敏的DC-CIK细胞对B16黑色素瘤动物模型的作用,来探讨和分析CIK细胞、Treg细胞在抗肿瘤免疫中所起的作用以及相互关系,来为抗肿瘤免疫治疗的临床应用提供更多的治疗思路和理论基础。
方法:
本实验以C57BL/6小鼠的黑色素瘤动物模型作为研究对象,制备黑色素瘤动物模型,并应用放射性钴60对小鼠黑色素瘤动物模型进行照射,使小鼠机体内形成非髓性淋巴细胞删除状态,使C57BL/6小鼠的黑色素瘤动物模型的免疫机制丧失。
取小鼠外周血单个核细胞(PBMC)制备树突状细胞和CIK细胞,将两者共同培养,并且负载肿瘤特异性抗原,并检测其免疫表型。以免疫磁珠分选方法分别在培养前及培养后去除CD4~+CD25~+Treg细胞成分。分别得到去除CD4~+CD25~+Treg细胞成分再经体外诱导产生的特异性抗原负载的DC-CIK效应细胞、经体外诱导产生肿瘤特异性抗原负载的DC-CIK效应细胞,再用磁珠分选方法去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK效应细胞和未去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK效应细胞共三组抗肿瘤免疫效应细胞。
然后再将制备好的达到非髓性淋巴细胞删除状态的C57BL/6小鼠的黑色素瘤动物模型随机分为4组。第一组:回输先通过磁珠分选去除CD4~+CD25~+Treg细胞成分再经体外诱导产生的特异性抗原负载的DC-CIK效应细胞,第二组:回输先经体外诱导产生肿瘤特异性抗原负载的DC-CIK效应细胞,再用磁珠分选方法去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK效应细胞。第三组:回输未去除CD4~+CD25~+Treg细胞成分的DC-CIK细胞组。第四组:对照组。
通过测量各组的肿瘤体积、重量、计算抑瘤率,比较其抑瘤作用,并应用透射电镜观察特异性抗原负载的DC-CIK细胞及杀伤肿瘤细胞的形态学表现。应用流氏细胞技术(FCM)检测各组肿瘤细胞的增殖周期中G0/G1期、S期、G2/M期细胞比率、细胞增殖指数(proliferation Index,PI)和细胞凋亡指数(apoptosis Index ,AI)的变化,观察特异性抗原负载的DC-CIK对肿瘤细胞增殖和凋亡的影响。
采用MTT染色法检测培养前去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组、培养后去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组、未去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组三组抗肿瘤免疫效应细胞对B16黑色素瘤肿瘤细胞的杀伤效应。
结果:
采用特异性肿瘤抗原致敏的DC细胞与CIK细胞共同培养,分析其免疫表型,透射电镜观察特异性抗原负载的DC-CIK细胞体积增大,核有切迹,细胞质内细胞器丰富,粗面内质网扩张,细胞表面有突起,与肿瘤细胞密切接触,大量肿瘤细胞凋亡、坏死。
培养前去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组肿瘤平均体积0.0377±0.0128cm3 ,培养后去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组肿瘤平均体积0.0359±0.0131cm3,未去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组肿瘤平均体积0.1278±0.0362 cm3,而对照组肿瘤平均体积0.4052±0.0429cm3。三组实验组肿瘤体积均明显小于对照组(P<0.05),不同阶段去除CD4~+CD25~+Treg细胞成分的两组之间比较,虽然培养前去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组较培养后去除CD4~+CD25~+Treg细胞成分组体积略小,但没有显著性差异(P >0.05)。但两组与未去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组比较肿瘤体积有统计学差异(P<0.05)。
三组特异性抗原致敏的DC-CIK效应细胞组抑瘤率明显高于对照组(P<0.05);去除CD4~+CD25~+Treg细胞成分两组抑瘤率明显高于未去除CD4~+CD25~+Treg细胞DC-CIK效应细胞组(P<0.05),但两组之间没有明显差异(P >0.05)。
瘤体重量对照组3.361±1.07g,培养前去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组0.958±0.32g,培养后去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组0.939±0.41g,未去除CD4~+CD25~+Treg细胞成分的特异性抗原负载的DC-CIK细胞组1.651±0.57g。同样三实验组瘤体重量均明显小于对照组(P<0.05)。但不同阶段去除CD4~+CD25~+Treg细胞成分的两组肿瘤重量都小于未去除CD4~+CD25~+Treg细胞成分组,并且有统计学差异(P<0.05)。不同阶段去除CD4~+CD25~+Treg细胞成分的两组之间则没有显著性差异(P >0.05)。
透射电镜可以观察到特异性抗原负载的DC-CIK细胞能促进肿瘤细胞凋亡超微结构的改变。流式细胞(FCM)检测癌细胞增殖周期中的变化;实验组三组与对照组比较G0/G1期细胞比率、AI显著升高,S期无明显变化,G2/M期细胞比率、PI显著下降;去除CD4~+CD25~+Treg细胞成分两组G0/G1期细胞比率、AI明显高于未去除CD4~+CD25~+Treg组,G2/M期细胞比率、PI显著低于未去除CD4~+CD25~+Treg组,S期无明显变化。去除CD4~+CD25~+Treg细胞成分两组之间比较G0/G1期、S期、G2/M期细胞比率和AI、PI均没有明显差异(P >0.05)。
采用MTT法检测培养前去除CD4~+CD25~+Treg细胞DC-CIK细胞组,培养后去除CD4~+CD25~+Treg细胞DC-CIK细胞组和未去除CD4~+CD25~+Treg细胞DC-CIK细胞组三组效应细胞对于B16黑色素瘤细胞的杀伤作用,结果表明三组效应细胞对肿瘤细胞都有较强的杀伤作用。培养前去除CD4~+CD25~+Treg细胞DC-CIK细胞组和培养后去除CD4~+CD25~+Treg细胞DC-CIK细胞组对B16黑色素细胞的杀伤作用明显高于(P<0.05)未去除CD4~+CD25~+Treg细胞DC-CIK细胞组。且不同效靶比之间比较,效靶比越高杀伤作用越强。培养前去除CD4~+CD25~+Treg细胞DC-CIK细胞组和培养后去除CD4~+CD25~+Treg细胞DC-CIK细胞组两组之间比较对B16黑色素细胞的杀伤作用没有显著性差异(P >0.05)。
结论:
1、特异性抗原致敏的DC-CIK细胞对B16黑色素瘤具有明显的抑瘤作用,并且对B16黑色素瘤的肿瘤细胞有较强的杀伤作用。特异性抗原致敏的DC-CIK细胞可以影响肿瘤细胞的细胞周期,并且具有诱导肿瘤细胞凋亡的作用。说明CIK细胞是一种具有强大肿瘤杀伤能力的新一代过继免疫抗肿瘤细胞。
2、去除CD4~+CD25~+Treg细胞成分后的特异性抗原致敏的DC-CIK细胞抗肿瘤免疫的作用更加高效而且特异,说明CD4~+CD25~+Treg细胞能够对特异性抗原致敏的DC-CIK细胞杀伤肿瘤细胞的活性形成明显的抑制。去除CD4~+CD25~+Treg细胞,重新募集效应性T细胞能够增强机体的抗肿瘤作用,这将成为一种可行的肿瘤免疫治疗方法。
3、不同时段去除CD4~+CD25~+Treg细胞后,从抑瘤作用、对于肿瘤细胞的细胞周期的影响和对肿瘤细胞的杀伤作用上都有一定的差异,但没有统计学意义,说明CD4~+CD25~+Treg细胞各个亚群的来源和作用机制还需要进一步的研究。
Objective: Adoptive immunotherapy is one of the hotest subject in biotherapy of tumor. The treatment is not only the supplement to conventional therapy such as surgical operation、chemotherapy and radiotherapy ,but also apply hope to people who can not sustain the side effects of radiotherapy and chemotherapy any longer.Adoptive immunotherapy has good therapeutic effect to the tumor patients in immune reconstruction and eliminate remained pathological.
Cytokine induced killer (CIK)cells is one of the new type immunocompetence cells, they have high efficiency in anti-tumor immunity. Cytokine induced killer cells have widespread using in adoptive immune therapy ,because they showed highly effcient cytolytic effector, faster proliferation speed rate then other immunocompetence cells in anti-tumor immunity.The key of studies to cytokine induced killer cells are how to enhance their cytolytic effcient and anti-tumor specific effcient,obtain more cytokine induced killer cells. Dendritic cells(DCs) are the most potent antigen presenting cells(APC) with the ability to acquire,process,present,antigens and the unique capability of initating primary immune responses against tumor-associated antigens. Dendritic cells(DCs) and cytokine induced killer(CIK) cells were separated and culture respectively,then dendritic cells(DCs) were loaded with specific tumor antigen. By using this methods ,we can co-culture tumor Antigen-sensitized Dendritic cells (DCs) with cytokine induced killer( CIK)cells. The tumor antigen-sensitized Dendritic cells (DCs) with cytokine induced killer( CIK)cells has higher cytolytic effcient and more specific anti-tumor specific effcient.We can obtain more quantity immunocompetence cells than cytokine induced killer( CIK)cells which did not loaded with specific tumor antigen.
But in practice, at the tumor circumstances anti-tumor immunocompetence cells were be revulsanted into immune anergy state constantly . so the anti-tumor immunocompetence cells killing effect descend obviously. It has very importmant significance effect to increase the anti-tumor immunotherapy that delete immunodepression cells. CD4~+CD25~+ regulatory T cells is one of the new discovered cells,they are the main subsets of regulatory T cells that play an essential role in maintaining immunological self-tolerance. They have the function of down-regulates the tumor immunity mediated by T cells. CD4~+CD25~+ regulatory T cells also divided into two groups by produced channel.The two groups are natural regulatory T cells (nTreg) and induced regulatory T cells( iTreg). CD4~+CD25~+ regulatory T cells increases in the peripheral biood and tumor infiltration lymphoglandula in many tumor patients,such as breast cancer、lung cancer、gastric cancer. CD4~+CD25~+ regulatory T cells suppress immune responses mainly by cell contract-dependent interactions or secreting soluble cytikines.Depletion or attenuation of CD4~+CD25~+ regulatory T cells will evoke effective anti-tumor immunity,which may become a feasible immunotherapy for cancer.
With this experimental, we use the specific antigen-sensitized DC-CIK cells as the anti-tumor immunotherapy cells.We observe the effect on B16 melanoma tumor animal experimental modle by DCs loaded with tumor antigen through morphological observation、cells proliferation and apoptosis、killing effect. By this way, we may analyse and discuss the relationship between CIK cells and regulatory T cells in anti-tumor immunity. We want to find more treament methods and theory foundation to clinical therapy in anti-tumor immunotherapy through the experimental.
Methods:
The B16 melanoma tumor cells were incubated in the C57 BL/6 mouse to build the animal models.Then using the total body irradiation by 60Co-ray to the animal models to eliminate it’s immunol function. Then the animal models in a no-marrew lymphocyte deletion state.
Peripheral biood mononuclear cells(PBMC) were isolated from C57 mouse. DCs were induced from adherent cells by some cytokines and CIK cells were generated from non- adherent cells. Mature DCs co-cultured with CIK cells,then loaded with specific tumor antigen, analyze specific antigen-sensitized DC-CIK immune epitope. Before and after culture ntigen-sensitized DC-CIK MACS (magnetic cell sorting) separating system was used to delete CD4~+CD25~+ regulatory T cells. So we have three gpoups anti-tumor immunocompetence cells. The first group: after by using MACS separating system to delete CD4~+CD25~+ regulatory T cells, then culture and acquire the specific antigen-sensitized DC-CIK cells. The second group: culture and acquire the specific antigen-sensitized DC-CIK cells ,then use MACS separating system to delete CD4~+CD25~+ regulatory T cells in this specific antigen-sensitized DC-CIK cells. The third group: the specific antigen-sensitized DC-CIK cells which did not delete CD4~+CD25~+ regulatory T cells.
Then randomize divided the animal models into four groups. The first group: after by using MACS separating system to delete CD4~+CD25~+ regulatory T cells, then culture and acquire the specific antigen-sensitized DC-CIK cells were injectedinto the animal models. The second group: culture and acquire the specific antigen-sensitized DC-CIK cells ,then use MACS separating system to delete CD4~+CD25~+ regulatory T cells in this specific antigen-sensitized DC-CIK cells were injectedinto the animal models.The third group: the specific antigen-sensitized DC-CIK cells which did not delete CD4~+CD25~+ regulatory T cells were injectedinto the animal models.The fourth group:control group.
According to the change of tumor volume、tumor weight and the tumor inhibition rates, inhibitory effect of the specific antigen-sensitized DC-CIK cells on B16 melanoma cells were estimated. Morphological characteristics of immune cells were observed by electronic microscope.With flow cytometry equipment,the rate of tumor cells in G0/G1、S、G2/M stage、proliferation index and the apoptosis index were respectively detected. To study the effect of cytokine induced killer cells on B16 melanoma cells proliferation and apoptosis in the animal models.Determine the killing effect on tumors cells by MTT method.
Results:
Co-culture tumor antigen-sensitized Dendritic cells (DCs) with cytokine induced killer (CIK) cells,analyze the immune epitope. The specific antigen-sensitized DC-CIK cells were bigger than the normal lymphocytes and notch always appears on the cellar nucleus observed under TEM. Plenty of active-functioned organelles such as dilated endoplasmic reticulum were observed in the cytoplasm of the specific antigen-sensitized DC-CIK cells. Protrusions on the surface of the specific antigen-sensitized DC-CIK cells contacted closely with the tumor cells. Apoptosis and necrosis of the cells could be widely observed in tumor tissues.
The tumor volume of the first group is 0.0377±0.0128cm3 ;the second group is 0.0359±0.0131cm3;the third group is 0.1278±0.0362 cm3;control group is 0.4052±0.0429cm3. The tumor volume of the three experiment groups were significant smaller than the control group(P<0.05). The first and second groups were significant smaller than the third group(P<0.05).There was no significant difference between the first groups and the second groups in tumor volume (P >0.05).
Tumor inhibition rate of the third group(59.19%) was lower than that of either the first group(91.70% ,P<0.05) or the second group(92.64% ,P<0.05) . There was no significant difference between the first groups and the second groups in tumor inhibition rate (P >0.05). The tumor inhibition rate of the three experiment groups were significant higher than the control group(P<0.05).
The tumor weight of control group is 3.361±1.07g, the first group is 0.958±0.32g ;the second group is 0.939±0.41g;the third group is 1.651±0.57g. The tumor weight of the three experiment groups were significant lower than the control group(P<0.05). The first and second groups were significant lower than the third group(P<0.05). There was no significant difference between the first groups and the second groups in tumor weight (P >0.05).
By using flow cytometry equipment,compared with the three experiment groups and the control group, the rate of tumor cells in G0/G1 and apoptosis Index were significantly higher,on the other hand , the rate of tumor cells in G2/M stage and proliferation Index were significantly lower,but there was not greatly change in S stage. The first and second groups compared with the third group have the same circumstances. There was no significant difference between the first groups and the second groups in the rate of tumor cells in G0/G1、apoptosis index、G2/M stage and proliferation index(P >0.05).
The specific antigen-sensitized DC-CIK cells showed strong killing effect on B16 melanoma tumor cells. The killing effect on B16 melanoma tumor cells of the three experiment groups were significant lower than the control group(P<0.05). The first and second groups were significant higher than the third group(P<0.05). There was no significant difference between the first groups and the second groups in killing effect on B16 melanoma tumor cells (P >0.05).
Conclusion:
1. The specific antigen-sensitized DC-CIK cells have high efficiency in inhibition on B16 melanoma. The specific antigen-sensitized DC-CIK cells may have an effect on the tumor cells cycle and induce them apoptosis.CIK cells is a new generation of adoptive immunocell has high efficiency in killing tumors cells.
2. The specific antigen-sensitized DC-CIK cells have high efficiency and more specifical in inhibition effect on B16 melanoma tumor cells after delete CD4~+CD25~+ regulatory T cells. CD4~+CD25~+ regulatory T cells could exert inhibitory effect on The specific antigen-sensitized DC-CIK cells killing function. Deletion CD4~+CD25~+ regulatory T cells and recruitment of effector T cells will evoke effective anti-tumor immunity,which may have become a feasible immunotherapy for cancer.
3. There was no significant difference between the deletion CD4~+CD25~+ regulatory T cells in different stage through tumor inhibition rate、tumor cells apoptosis and proliferation、the killing effect. We must do more study to explain the possible mechansim and relationship between the main subsets of CD4+CD25+ regulatory T cells.
引文
1 Lu PH, Negrin RS. A novel population of expanded human CD3+CD56+ cells derived from T cells with potent in vivo antitutmor activity in micewith severe combined immunodeficiency [J]. J Immunol, 1994, 153(4):1687-1696
2 Sakaguchi S, Sakaguchi N, Asano M, et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptorα-chains(CD25). Breackdown of single mechanism of self- tolerance causes various autoimmune diseases[J]. J Immunol, 1935,160:1151-1164
3谢遵讲,曹立敏,贺业春,等。体外培养小鼠树突状细胞的扩增鉴定及形态学观察[J].解剖科学进展.2005,11(3):240-243
4 Hishii M, Kurnick JT, Ramiraz T,et al. Studies of zhe mechanism of cytolysis by tumor-infiltrating lymphocytes[J].Clin Exp Immunol,1999,116(3):388-394
5 William LG,Daniel AH, Kono K, et al. The EWS-WT1 gene fusion in desmoplastic small round cell tumor[J]. Seminars in Cancer Biology, 2005,15(3):197-205
6 Raghavan S, Hilmgren J, Angela MT, et al. CD4+CD25+ suppressor Tcells regulate pathogen induced inflammation and disease[J]. FEMS Immunol Med Microbiol, 2006,14(2):121-127
7 Nguyen S, Dhedin N, Vemant JP, et al. NK—cell reconstitution after haploydentical hem atopoietic stem cell transpiantantions inmaturity of NK cells and inhibitory effect of NKG2A override GvL effect[J]. Blood, 2005,105(9):4135-4142
8 Liebrich W, Schlag P, Manasterski M, et al. In vitro and clinical characterization of a Newcastle disease virus-modified autologous tumor cell vaccine for treatment of colorectal cancer patients[J]. Eur J Cancer,1991,27(6):708-710
9 Leemhuis T, Wells S, Scheffold C, et al. A phase I trial of autologous cytokine-induced killer cells for the treatment[J]. Biology of biood and marrow transplation,2005,11:181-187
10 Marten A, Renoth S, Toal M, et al. Enhanced lyticactivity of cytokine-induced killer cells against multiple myelom a cells after co-culture with idiotype-pulsed dendritic cells. HHaematologica,2001;86:1029-1037
11张嵩,王恩忠,白春学,等。共培养的树突状细胞与CIK细胞治疗结肠癌血源性肺转移的实验研究[J]。肿瘤,2003,23(6):448-451
12 Metha BA, Schmidt-Wolf , Weissman IL, et al. Two path ways of exocytosis of cytoplasmic granule contents and target cell killing by cytokine-induced CD3+CD56+ killer cells [J]. Blood, 1995, 86(9):3493-3499
13 Zoll B, Lefterova P, Csipai M, et al. Generation of cytokine-induced killer cells using exogenous interleukin-2,7 or 12[J]. Cancer Immunol Immunother, 1998, 47(3) :221-226
14 Shevach EM. Control of T cell activation by CD4+CD25+ T suppressor T cells[J]. Immunol Rev, 2001,182:59-67
15 Sakaguchi S. Immunologic tolerance maintained by CD4+CD25+ regulatory T cells: their common role in controlling autoimmunity,tumor immunity and transplantation tolerance[J]. Immunol Rev, 2001,182:18-32
16 Hishii M, Kurnick JT, Ramiraz T,et al. Studies of zhe mechanism of cytolysis by tumor-infiltrating lymphocytes[J].Clin Exp Immunol,1999,116(3):388-394
17 Cureil TJ, Coukos G, Zou L, et al.Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival[J]. Nat Med,2004,10(9):942-949
18 Woo EY, Chu CS, Goletz TJ, et al. Regulatory CD4+CD25+ T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer[J]. Cancer Res,2001,61(7):1766-1772
19 Biyanage UK, Moore TT, Joo HG, et al. Prevalence of regulatory T cells is increased in peripheral biood and tumor in roenvironment of patients with pancreas or breast adenocarcinoma[J]. J Immunol,2005,169(5):2756-2761
20 Taams L, Vukmanovic-Stejic M, Salmon M, et al. Immune regulation by CD4+CD25+ regulatory T cells: implications for transplantation tolerance[J]. Transpl Immunol, 2003, 11(3):277-285
21 Chattopadhyay S, Chakraborty NG, Mukherji B, et al. Regulatory T cells and tumor immunity. Cancer Immunol Immunother, 2005, 54(12):1153-1161
22 La Cava A, Fang CJ, Singh RP, et al. Manipulation of immune-regulation in systemic lupus erythermatosus[J]. Autoimmun Rev,2005,4(9):515-519
23 Oluwole SF, Oluwole OO, DePaz HA, et al. CD4+CD25+ regulatory T cells mediate acquired transplant tilerance[J]. Transpl Immunol, 2003,11(4):287-293
1 Wang FS, Liu MX, Zhang B, et al.Antitumor activities of human autologous cytoikn induced killer(CIK) cells against hepatocellular carcinoma cells in vitro and in vivo[J]. World Gastroenterol, 2002,8(3):464-468
2 Gorschluter M, Ziske C, Glasmacher A, et al. Current clinical and laboratory strategies to augment the efficacy of immunotherapy in multiple myeloma [J]. Clin Cancer Res, 2001, 7(8):2195-2204
3 Leemhuis T, Wells S, Scheffold C, et al. A phase I trial of autologous cytokine-induced killer cells for the treatment[J]. Biology of biood and marrow transplation,2005,11:181-187
4 Sakaguchi S, Sakaguchi N, Asano M, et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptorα-chains(CD25). Breackdown of single mechanism of self- tolerance causes various autoimmune diseases[J]. J Immunol, 1995,160:1151-1164
5 Shevach EM. Control of T cell activation by CD4+CD25+ T suppressor Tcells[J]. Immunol Rev, 2001,182:59-67
6 Schmidt-Wolf IG,Negrin RS, Kiem HP, et al.Use of a SCID mouse/human lymphom a model to avaluate cytokine-induced Killer cells with potent antitumor cell activity[J]. J Exp Med, 1991, 174(1):139-149
7 Chattopadhyay S, Chakraborty NG, Mukherji B. Regulator T cells and tumor immunity. Cancer Immunol Immunother[J], 2005, 54(12):1153-1161
8 Verneris MR, Komacker M, Mailander V, et al. Resistance of ex vivo expanded CD3+CD56+ T cells to Fas-mediated apoptosis[J]. Cancer Immunol Immunother, 2000, 49(6):335-345
9 Holy C,Bang CD, Nerrin RS, et al. Expansion of Philadelphia chromosome-negative CD3+56+ cytotoxic cells from chronic myeloid leukemia patients:in vitro and in vivo efficacy in severe combined immunodeficiency disease mice[J]. Blood, 1998, 92(9):3318-3327
10 Rudin CM, Thompson CB. Apoptosis and disease:regulation and clinical relevance of programmed cell death[J]. Annu Rev Med,1997,48(2):267-281
11 Uraushihara K, Takanon KT, Ko K, et al. Regulation of murine inflammatory bowel disease by CD25+ and CD4+CD25+glucocorticoid-Induced TNF receptor family-related gene regulatory T cells[J]. Proc Natl Acad Sci USA,2003,171(2):708-716
12 Taams L, Vukmanovic-Stejic M, Salmon M, et al. Immune regulation by CD4+CD25+ regulatory T cells: implications for transplantation tolerance[J]. Transpl Immunol, 2003, 11(3):277-285
13 Hsieh CS, Rudensky AY. The role of TCR specificity in naturally arising CD4+CD25+ regulatory T cell biology.Curr Top Microbiol Immunol, 2005,293:25-42
14 Bacchetta R, Gregori S, Roncarolo MG, et al. CD4+CD25+ regulatory T cell merchanisms of induction and effector function[J]. Autoimmun Rev, 2005,4(8):491-496
15 Orentas RJ, Kohler ME, Johnson BD, et al. Suppression of anti-cancerimmunity by regulatory T cells:back to future. Semin Cancer Biol,2006,16(2):137-149
16 Klein L, Khazaie K, Boehmer H, et al. In two dynamics of antigen-specific regulatory T cells not predicted from behavior in vitro[J]. Proc Natl Acad Sci USA,2003,100(15):8886-8891
17 Jonuleit H, Adema G, Schmitt, et al.Immune regulation by regulatory T cells imphcations for transplantation[J]. Transpl Immunol, 2004, 12(5):267-276
1 Cao JP, Jiang ZM, Zhang XC, et al. The proliferation phenotype change and anti-tumor activity of CIK cells[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2005,21(5):583-586
2 Fheger D, Kufer P, Eeier I, et al. A bispecific single-chain antibody directed against EpCAM/CD3 in combination with the cytokines interferon a and interleukin-2 efficiently retargets T and CD3+CD56+ natural-killer-like T lymphocytes to EpCAM-expressing tumor cells[J]. Cancer Immunol Immunother, 2000, 49(8):441-448
3 Schmidt-Wolf IG,Negrin RS, Kiem HP, et al.Use of a SCID mouse/human lymphom a model to avaluate cytokine-induced Killer cells with potent antitumor cell activity[J]. J Exp Med, 1991, 174(1):139-149
4 Sakaguchi S, Sakaguchi N, Asano M, et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptorα-chains(CD25). Breackdown of single mechanism of self- tolerance causes various autoimmune diseases[J]. J Immunol, 1995,160:1151-1164
5 Kawaida H, Kono K, Takahashi A, et al. Distribution of CD4+CD25high regulatory T-cells in tumor-drarning lymph nodes in patients with gastric tumor[J]. Surgical Research, 2005,124(1):151-157
6 Jonuleit H, Adema G, Schmitt, et al.Immune regulation by regulatory T cells imphcations for transplantation[J]. Transpl Immunol, 2004, 12(5):267-276
7 Seliger B, Maeurer MJ, Ferrone S, et al. Antigen-processing machinery breakdown and tumor growth[J].Immunol Today,2000,21(2):455-464
8 Geroea F, Bajdani B, Nisii C, et al. Reciprocal activating interaction between natural killer cells and dendritic cells.[J]Exp Med, 2002,195(3):327-333
9 Marten A, Ziske C, Schottker B, et al. Interactions between dendritic cells and cytokine-induced killer cells lead to an activation of both population[J]. J Immunother, 2001, 24(5):502-510
10 Marten A, Renoth S, Von LT, et al. Enhanced lytic activity of cytokine-induced killer cells against multiple myeloma cells after co-culture with idiotype-pulsed dendritic cells[J]. Haematologica, 2001, 86(7):1029-1037
11 Metha BA, Schmidt-Wolf , Weissman IL, et al. Two path ways of exocytosis of cytoplasmic granule contents and target cell killing by cytokine-induced CD3+CD56+ killer cells [J]. Blood, 1995, 86(9):3493-3499
12 Zoll B, Lefterova P, Csipai M, et al. Generation of cytokine-induced killer cells using exogenous interleukin-2,7 or 12[J]. Cancer Immunol Immunother, 1998, 47(3) :221-226
13 Thompson C, Powrie F. Regulatory T cells. Curr Opin Pharmacol, 2004,4(4):408-414
14 Hsieh CS, Rudensky AY. The role of TCR specificity in naturally arising CD4+CD25+ regulatory T cell biology.Curr Top Microbiol Immunol, 2005,293:25-42
15 Fontenot J,Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ [J].Nat Immunol, 2003,4(4):330-336
16 Taams L, Vukmanovic-Stejic M, Salmon M, et al. Immune regulatory by CD4+CD25+ regulatory T cells implications for transplantation tolerance[J]. Transpl Immunol, 2003,11(3):277-285
17 Blazar BR, Taylor PA. Regulatory T cells. Biol Blood Marrow Transplant,2005,11(2):46-49
18 Bacchetta R, Gregori S, Roncarolo MG, et al. CD4+CD25+ regulatory T cell merchanisms of induction and effector function[J]. Autoimmun Rev, 2005,4(8):491-496
19 Orentas RJ, Kohler ME, Johnson BD, et al. Suppression of anti-cancer immunity by regulatory T cells:back to future. Semin Cancer Biol,2006,16(2):137-149
20 Yamaguchi T, Sakaguchi S, Aderson DE, et al. Regulatory T cells in immune surveillance and treatment of cancer. Semin Cancer Biol,2006,16(2):115-123
21 Nomura T, Saksguchi S. Naturally arising CD4+CD25+ regulatory T cells in tumor immunity [J]. Curr Top Microbiol Immunol, 2005,293(12):2870-2892
1 Schmidt-Wolf IG,Negrin RS, Kiem HP, et al.Use of a SCID mouse/human lymphom a model to avaluate cytokine-induced Killer cells with potent antitumor cell activity[J]. J Exp Med, 1991, 174(1):139-149
2 Holy C, Bangs CD, Chang P, et al. Expansion of Philadelphia Chromosome-Negative CD3+CD56+ Cytotoxic Cells from Chronic Myeloid Leukemia Patients: In Vitro Efficacy in Severe Combined Immunodeficiency Disease Mice[J]. Blood, 1998,92(9):3318-3327
3 Alvamas JC, Linn YC, Hope EG, et al. Expansion of cytotoxic CD3+CD56+ cells from peripheral blood progenitor cells of patients undergoing autologous hematopoietic cell transplantation [J]. Biol Blood Marrow Transplant, 2001, 7(4):216-222
4 Fheger D, Kufer P, Eeier I, et al. A bispecific single-chain antibody directed against EpCAM/CD3 in combination with the cytokines interferon a and interleukin-2 efficiently retargets T and CD3+CD56+ natural-killer-like T lymphocytes to EpCAM-expressing tumor cells[J]. Cancer Immunol Immunother, 2000, 49(8):441-448
5 Gorschluter M, Ziske C, Glasmacher A, et al. Current clinical and laboratory strategies to augment the efficacy of immunotherapy in multiple myeloma [J]. Clin Cancer Res, 2001, 7(8):2195-2204
6任欢,邢淑贤,徐红薇,等。CIK体外增殖及体内外杀瘤活性的实验研究[J]。中国肿瘤生物治疗杂志,1999,6(1):17-21
7 Metha BA, Schmidt-Wolf , Weissman IL, et al. Two path ways ofexocytosis of cytoplasmic granule contents and target cell killing by cytokine-induced CD3+CD56+ killer cells [J]. Blood, 1995, 86(9):3493-3499
8 Verneris MR, Komacker M, Mailander V, et al. Resistance of ex vivo expanded CD3+CD56+ T cells to Fas-mediated apoptosis[J]. Cancer Immunol Immunother, 2000, 49(6):335-345
9 Holy C,Bang CD, Nerrin RS, et al. Expansion of Philadelphia chromosome-negative CD3+56+ cytotoxic cells from chronic myeloid leukemia patients:in vitro and in vivo efficacy in severe combined immunodeficiency disease mice[J]. Blood, 1998, 92(9):3318-3327
10 Finker S, Trojaneck B, Lefterova P, et al. Increase of proliferation rate and enhancement of antitumor cytotoxicity of expanded human CD3+CD56+ immunologic effector cells by receptor mediated transfetion with the interleukin-7 gene[J]. Gene Ther, 1998, 5 (1):31-39
11 Zoll B, Lefterova P, Csipai M, et al. Generation of cytokine-induced killer cells using exogenous interleukin-2,7 or 12[J]. Cancer Immunol Immunother, 1998, 47(3) :221-226
12 Marten A, Ziske C, Schottker B, et al. Interactions between dendritic cells and cytokine-induced killer cells lead to an activation of both population[J]. J Immunother, 2001, 24(5):502-510
13 Marten A, Renoth S, Von LT, et al. Enhanced lytic activity of cytokine-induced killer cells against multiple myeloma cells after co-culture with idiotype-pulsed dendritic cells[J]. Haematologica, 2001, 86(7):1029-1037
14 Wang ZH, Simimoto H, Tani K, et al. IL-12 synergizes with B7-1 enhance the antitumor immunity in C57BL/6 Micc[J]. International Journal of Modern Cancer Therapy, 2000, 3(3):45-49
15 Wang ZH, Zhang QY, Zhang CY, et al. Antitumor immnity and vaccine effect induced by IL-12 synergizes B7-1 gene transfected cells[J]. Chinese Journal of Cancer Research, 2003, 15(1):5-8
16 Zoll B, Lefterova P, Ebert O, et al. Modulation of cell surface markers onNK-like T lymphocytes by using IL-2,IL-7 or IL-12 in vitro stimulation[J]. Cytokine, 2000, 12(3):1385-1390
17 Ense N, Lu S, Alcid MG, et al. Pooled umbilical cord blood as a possible universal donor formarrow reconstitution and use in nuclear accidents[J]. Life Sci, 2001, 69:1531-1539
18童春容,耿彦彪,陆道培,等。自体细胞因子诱导的杀伤细胞治疗急性白血病的临床研究[J]。北京医科大学学报,2000, 32(5):473-477
19陈复兴,刘军权,张南征,等。自身细胞因子诱导的杀伤细胞过继性免疫治疗恶性肿瘤的临床观察[J]。癌症,2002,21(7):797-801
20张宏文,彭晓,张晓燕,等。脐带血DCS对CIK细胞杀伤活性的影响研究。临床血液学杂志,2002,15(1):26-28
21 Schmidt-wolf, Leiterova P, John V, et al. Sensitivity of multidrug resistant tumor cell lines to immunological effector cells[J]. Cell immunol, 2003,169(1):85-90
1 Sakaguchi S, Sakaguchi N, Asano M, et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptorα-chains(CD25). Breackdown of single mechanism of self- tolerance causes various autoimmune diseases[J]. J Immunol, 1995,160:1151-1164
2 Shevach EM. Control of T cell activation by CD4+CD25+ T suppressor T cells[J]. Immunol Rev, 2001,182:59-67
3 Sakaguchi S. Immunologic tolerance maintained by CD4+CD25+ regulatory T cells: their common role in controlling autoimmunity,tumor immunity and transplantation tolerance[J]. Immunol Rev, 2001,182:18-32
4 Chattopadhyay S, Chakraborty NG, Mukherji B. Regulator T cells and tumor immunity. Cancer Immunol Immunother[J], 2005, 54(12):1153-1161
5 Bacchetta R, Gregori S, Roncarolo MG, et al. CD4+ regulatory T cells Mechanisms of induction and effector function[J]. Autoimmun Rev, 2004, 4(8):491-496
6 Read S, Powrie F. CD4+ regulatory T cells[J]. Curr Opintion Immunol, 2001, 13(15):644-649
7 Green EA, Choi Y, Flavell RA, et al. Pancreatic lymph node-derived CD4+CD25+ regulatory T cells:highly potent regulators of diabetes that require TRANCE-RANK signals[J]. Immunity, 2002, 16(2):183-191
8 Fontenot J, Gavin MA, Rudensky AY, et al. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells[J]. Nat Immunol, 2003, 4(4):330-336
9 Taams L, Vukmanovic-Stejic M, Salmon M, et al. Immune regulation byCD4+CD25+ regulatory T cells:implications for transplantation tolerance[J]. Transpl Immunol, 2003, 11(3):277-285
10 Goleva E, Cardona ID, Ou LS, et al. Factors that regulate naturally occurring T regulatory cell-mediated suppression[J]. Allergy Clin Immunol. 2005, 116(7):1094-1100
11 Jonuleit H, Adema G, Schmitt, et al.Immune regulation by regulatory T cells imphcations for transplantation[J]. Transpl Immunol, 2004, 12(5):267-276
12 Roll HK, Yama S, Stemman RM, et al. The interactions of DC cells with antigen-specific regulatory T cells that suppress autoimmunity[J]. Semin Immunol, 2006, 18(1):93-102
13 Zoltan F, Huber S, Read S, et al. Development and function of CD4+CD25+ regulatory T cells[J]. Curr Opin Immunol, 2004, 16(3):203-208
14 Jonuleit H, Schmitt E. The regulatory T cells family:dietarent subsets and their interlations[J]. J Immunol, 2003, 171(12):6323-6327
15 Hori S, Nomura T, Sakaguchi S, et al. Control of regulatory T cells development by the transcription factor Foxp3[J]. Science, 2003, 299(5609):1057-1061
16 Belkaid Y, Piccirillo DA, Mendez S, et al. CD4+CD25+ regulatory T cells control leishmania major persistence and immunity[J]. Nature, 2002,420(6915):502-507
17 Shimizu J, Yamazaki S, Takahashi T, et al. Stimulation of CD4+CD25+ regulatory T cells through GITR breaks immunological self-tolerance[J]. Nat Immunol, 2002, 3(2):135-142
18 Kazuhiko N, Atsushi K, Warren S, et al. Cell contact-dependent immunosuppression by CD4+CD25+ regulatory T cells in mediated by cell surface-bound transforming growth factor–β[J]. J Exp Med,2001,194(5):629-644
19 Cureil TJ, Coukos G, Zou L, et al.Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival[J]. Nat Med,2004,10(9):942-949
20 Woo EY, Chu CS, Goletz TJ, et al. Regulatory CD4+CD25+ T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer[J]. Cancer Res,2001,61(7):1766-1772
21 Biyanage UK, Moore TT, Joo HG, et al. Prevalence of regulatory T cells is increased in peripheral biood and tumor in roenvironment of patients with pancreas or breast adenocarcinoma[J]. J Immunol,2005,169(5):2756-2761
2 Sakaguchi S, Sakaguchi N, Asano M, et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptorα-chains(CD25). Breackdown of single mechanism of self- tolerance causes various autoimmune diseases[J]. J Immunol, 1935,160:1151-1164
3谢遵讲,曹立敏,贺业春,等。体外培养小鼠树突状细胞的扩增鉴定及形态学观察[J].解剖科学进展.2005,11(3):240-243
4 Hishii M, Kurnick JT, Ramiraz T,et al. Studies of zhe mechanism of cytolysis by tumor-infiltrating lymphocytes[J].Clin Exp Immunol,1999,116(3):388-394
5 William LG,Daniel AH, Kono K, et al. The EWS-WT1 gene fusion in desmoplastic small round cell tumor[J]. Seminars in Cancer Biology, 2005,15(3):197-205
6 Raghavan S, Hilmgren J, Angela MT, et al. CD4+CD25+ suppressor Tcells regulate pathogen induced inflammation and disease[J]. FEMS Immunol Med Microbiol, 2006,14(2):121-127
7 Nguyen S, Dhedin N, Vemant JP, et al. NK—cell reconstitution after haploydentical hem atopoietic stem cell transpiantantions inmaturity of NK cells and inhibitory effect of NKG2A override GvL effect[J]. Blood, 2005,105(9):4135-4142
8 Liebrich W, Schlag P, Manasterski M, et al. In vitro and clinical characterization of a Newcastle disease virus-modified autologous tumor cell vaccine for treatment of colorectal cancer patients[J]. Eur J Cancer,1991,27(6):708-710
9 Leemhuis T, Wells S, Scheffold C, et al. A phase I trial of autologous cytokine-induced killer cells for the treatment[J]. Biology of biood and marrow transplation,2005,11:181-187
10 Marten A, Renoth S, Toal M, et al. Enhanced lyticactivity of cytokine-induced killer cells against multiple myelom a cells after co-culture with idiotype-pulsed dendritic cells. HHaematologica,2001;86:1029-1037
11张嵩,王恩忠,白春学,等。共培养的树突状细胞与CIK细胞治疗结肠癌血源性肺转移的实验研究[J]。肿瘤,2003,23(6):448-451
12 Metha BA, Schmidt-Wolf , Weissman IL, et al. Two path ways of exocytosis of cytoplasmic granule contents and target cell killing by cytokine-induced CD3+CD56+ killer cells [J]. Blood, 1995, 86(9):3493-3499
13 Zoll B, Lefterova P, Csipai M, et al. Generation of cytokine-induced killer cells using exogenous interleukin-2,7 or 12[J]. Cancer Immunol Immunother, 1998, 47(3) :221-226
14 Shevach EM. Control of T cell activation by CD4+CD25+ T suppressor T cells[J]. Immunol Rev, 2001,182:59-67
15 Sakaguchi S. Immunologic tolerance maintained by CD4+CD25+ regulatory T cells: their common role in controlling autoimmunity,tumor immunity and transplantation tolerance[J]. Immunol Rev, 2001,182:18-32
16 Hishii M, Kurnick JT, Ramiraz T,et al. Studies of zhe mechanism of cytolysis by tumor-infiltrating lymphocytes[J].Clin Exp Immunol,1999,116(3):388-394
17 Cureil TJ, Coukos G, Zou L, et al.Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival[J]. Nat Med,2004,10(9):942-949
18 Woo EY, Chu CS, Goletz TJ, et al. Regulatory CD4+CD25+ T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer[J]. Cancer Res,2001,61(7):1766-1772
19 Biyanage UK, Moore TT, Joo HG, et al. Prevalence of regulatory T cells is increased in peripheral biood and tumor in roenvironment of patients with pancreas or breast adenocarcinoma[J]. J Immunol,2005,169(5):2756-2761
20 Taams L, Vukmanovic-Stejic M, Salmon M, et al. Immune regulation by CD4+CD25+ regulatory T cells: implications for transplantation tolerance[J]. Transpl Immunol, 2003, 11(3):277-285
21 Chattopadhyay S, Chakraborty NG, Mukherji B, et al. Regulatory T cells and tumor immunity. Cancer Immunol Immunother, 2005, 54(12):1153-1161
22 La Cava A, Fang CJ, Singh RP, et al. Manipulation of immune-regulation in systemic lupus erythermatosus[J]. Autoimmun Rev,2005,4(9):515-519
23 Oluwole SF, Oluwole OO, DePaz HA, et al. CD4+CD25+ regulatory T cells mediate acquired transplant tilerance[J]. Transpl Immunol, 2003,11(4):287-293
1 Wang FS, Liu MX, Zhang B, et al.Antitumor activities of human autologous cytoikn induced killer(CIK) cells against hepatocellular carcinoma cells in vitro and in vivo[J]. World Gastroenterol, 2002,8(3):464-468
2 Gorschluter M, Ziske C, Glasmacher A, et al. Current clinical and laboratory strategies to augment the efficacy of immunotherapy in multiple myeloma [J]. Clin Cancer Res, 2001, 7(8):2195-2204
3 Leemhuis T, Wells S, Scheffold C, et al. A phase I trial of autologous cytokine-induced killer cells for the treatment[J]. Biology of biood and marrow transplation,2005,11:181-187
4 Sakaguchi S, Sakaguchi N, Asano M, et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptorα-chains(CD25). Breackdown of single mechanism of self- tolerance causes various autoimmune diseases[J]. J Immunol, 1995,160:1151-1164
5 Shevach EM. Control of T cell activation by CD4+CD25+ T suppressor Tcells[J]. Immunol Rev, 2001,182:59-67
6 Schmidt-Wolf IG,Negrin RS, Kiem HP, et al.Use of a SCID mouse/human lymphom a model to avaluate cytokine-induced Killer cells with potent antitumor cell activity[J]. J Exp Med, 1991, 174(1):139-149
7 Chattopadhyay S, Chakraborty NG, Mukherji B. Regulator T cells and tumor immunity. Cancer Immunol Immunother[J], 2005, 54(12):1153-1161
8 Verneris MR, Komacker M, Mailander V, et al. Resistance of ex vivo expanded CD3+CD56+ T cells to Fas-mediated apoptosis[J]. Cancer Immunol Immunother, 2000, 49(6):335-345
9 Holy C,Bang CD, Nerrin RS, et al. Expansion of Philadelphia chromosome-negative CD3+56+ cytotoxic cells from chronic myeloid leukemia patients:in vitro and in vivo efficacy in severe combined immunodeficiency disease mice[J]. Blood, 1998, 92(9):3318-3327
10 Rudin CM, Thompson CB. Apoptosis and disease:regulation and clinical relevance of programmed cell death[J]. Annu Rev Med,1997,48(2):267-281
11 Uraushihara K, Takanon KT, Ko K, et al. Regulation of murine inflammatory bowel disease by CD25+ and CD4+CD25+glucocorticoid-Induced TNF receptor family-related gene regulatory T cells[J]. Proc Natl Acad Sci USA,2003,171(2):708-716
12 Taams L, Vukmanovic-Stejic M, Salmon M, et al. Immune regulation by CD4+CD25+ regulatory T cells: implications for transplantation tolerance[J]. Transpl Immunol, 2003, 11(3):277-285
13 Hsieh CS, Rudensky AY. The role of TCR specificity in naturally arising CD4+CD25+ regulatory T cell biology.Curr Top Microbiol Immunol, 2005,293:25-42
14 Bacchetta R, Gregori S, Roncarolo MG, et al. CD4+CD25+ regulatory T cell merchanisms of induction and effector function[J]. Autoimmun Rev, 2005,4(8):491-496
15 Orentas RJ, Kohler ME, Johnson BD, et al. Suppression of anti-cancerimmunity by regulatory T cells:back to future. Semin Cancer Biol,2006,16(2):137-149
16 Klein L, Khazaie K, Boehmer H, et al. In two dynamics of antigen-specific regulatory T cells not predicted from behavior in vitro[J]. Proc Natl Acad Sci USA,2003,100(15):8886-8891
17 Jonuleit H, Adema G, Schmitt, et al.Immune regulation by regulatory T cells imphcations for transplantation[J]. Transpl Immunol, 2004, 12(5):267-276
1 Cao JP, Jiang ZM, Zhang XC, et al. The proliferation phenotype change and anti-tumor activity of CIK cells[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2005,21(5):583-586
2 Fheger D, Kufer P, Eeier I, et al. A bispecific single-chain antibody directed against EpCAM/CD3 in combination with the cytokines interferon a and interleukin-2 efficiently retargets T and CD3+CD56+ natural-killer-like T lymphocytes to EpCAM-expressing tumor cells[J]. Cancer Immunol Immunother, 2000, 49(8):441-448
3 Schmidt-Wolf IG,Negrin RS, Kiem HP, et al.Use of a SCID mouse/human lymphom a model to avaluate cytokine-induced Killer cells with potent antitumor cell activity[J]. J Exp Med, 1991, 174(1):139-149
4 Sakaguchi S, Sakaguchi N, Asano M, et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptorα-chains(CD25). Breackdown of single mechanism of self- tolerance causes various autoimmune diseases[J]. J Immunol, 1995,160:1151-1164
5 Kawaida H, Kono K, Takahashi A, et al. Distribution of CD4+CD25high regulatory T-cells in tumor-drarning lymph nodes in patients with gastric tumor[J]. Surgical Research, 2005,124(1):151-157
6 Jonuleit H, Adema G, Schmitt, et al.Immune regulation by regulatory T cells imphcations for transplantation[J]. Transpl Immunol, 2004, 12(5):267-276
7 Seliger B, Maeurer MJ, Ferrone S, et al. Antigen-processing machinery breakdown and tumor growth[J].Immunol Today,2000,21(2):455-464
8 Geroea F, Bajdani B, Nisii C, et al. Reciprocal activating interaction between natural killer cells and dendritic cells.[J]Exp Med, 2002,195(3):327-333
9 Marten A, Ziske C, Schottker B, et al. Interactions between dendritic cells and cytokine-induced killer cells lead to an activation of both population[J]. J Immunother, 2001, 24(5):502-510
10 Marten A, Renoth S, Von LT, et al. Enhanced lytic activity of cytokine-induced killer cells against multiple myeloma cells after co-culture with idiotype-pulsed dendritic cells[J]. Haematologica, 2001, 86(7):1029-1037
11 Metha BA, Schmidt-Wolf , Weissman IL, et al. Two path ways of exocytosis of cytoplasmic granule contents and target cell killing by cytokine-induced CD3+CD56+ killer cells [J]. Blood, 1995, 86(9):3493-3499
12 Zoll B, Lefterova P, Csipai M, et al. Generation of cytokine-induced killer cells using exogenous interleukin-2,7 or 12[J]. Cancer Immunol Immunother, 1998, 47(3) :221-226
13 Thompson C, Powrie F. Regulatory T cells. Curr Opin Pharmacol, 2004,4(4):408-414
14 Hsieh CS, Rudensky AY. The role of TCR specificity in naturally arising CD4+CD25+ regulatory T cell biology.Curr Top Microbiol Immunol, 2005,293:25-42
15 Fontenot J,Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ [J].Nat Immunol, 2003,4(4):330-336
16 Taams L, Vukmanovic-Stejic M, Salmon M, et al. Immune regulatory by CD4+CD25+ regulatory T cells implications for transplantation tolerance[J]. Transpl Immunol, 2003,11(3):277-285
17 Blazar BR, Taylor PA. Regulatory T cells. Biol Blood Marrow Transplant,2005,11(2):46-49
18 Bacchetta R, Gregori S, Roncarolo MG, et al. CD4+CD25+ regulatory T cell merchanisms of induction and effector function[J]. Autoimmun Rev, 2005,4(8):491-496
19 Orentas RJ, Kohler ME, Johnson BD, et al. Suppression of anti-cancer immunity by regulatory T cells:back to future. Semin Cancer Biol,2006,16(2):137-149
20 Yamaguchi T, Sakaguchi S, Aderson DE, et al. Regulatory T cells in immune surveillance and treatment of cancer. Semin Cancer Biol,2006,16(2):115-123
21 Nomura T, Saksguchi S. Naturally arising CD4+CD25+ regulatory T cells in tumor immunity [J]. Curr Top Microbiol Immunol, 2005,293(12):2870-2892
1 Schmidt-Wolf IG,Negrin RS, Kiem HP, et al.Use of a SCID mouse/human lymphom a model to avaluate cytokine-induced Killer cells with potent antitumor cell activity[J]. J Exp Med, 1991, 174(1):139-149
2 Holy C, Bangs CD, Chang P, et al. Expansion of Philadelphia Chromosome-Negative CD3+CD56+ Cytotoxic Cells from Chronic Myeloid Leukemia Patients: In Vitro Efficacy in Severe Combined Immunodeficiency Disease Mice[J]. Blood, 1998,92(9):3318-3327
3 Alvamas JC, Linn YC, Hope EG, et al. Expansion of cytotoxic CD3+CD56+ cells from peripheral blood progenitor cells of patients undergoing autologous hematopoietic cell transplantation [J]. Biol Blood Marrow Transplant, 2001, 7(4):216-222
4 Fheger D, Kufer P, Eeier I, et al. A bispecific single-chain antibody directed against EpCAM/CD3 in combination with the cytokines interferon a and interleukin-2 efficiently retargets T and CD3+CD56+ natural-killer-like T lymphocytes to EpCAM-expressing tumor cells[J]. Cancer Immunol Immunother, 2000, 49(8):441-448
5 Gorschluter M, Ziske C, Glasmacher A, et al. Current clinical and laboratory strategies to augment the efficacy of immunotherapy in multiple myeloma [J]. Clin Cancer Res, 2001, 7(8):2195-2204
6任欢,邢淑贤,徐红薇,等。CIK体外增殖及体内外杀瘤活性的实验研究[J]。中国肿瘤生物治疗杂志,1999,6(1):17-21
7 Metha BA, Schmidt-Wolf , Weissman IL, et al. Two path ways ofexocytosis of cytoplasmic granule contents and target cell killing by cytokine-induced CD3+CD56+ killer cells [J]. Blood, 1995, 86(9):3493-3499
8 Verneris MR, Komacker M, Mailander V, et al. Resistance of ex vivo expanded CD3+CD56+ T cells to Fas-mediated apoptosis[J]. Cancer Immunol Immunother, 2000, 49(6):335-345
9 Holy C,Bang CD, Nerrin RS, et al. Expansion of Philadelphia chromosome-negative CD3+56+ cytotoxic cells from chronic myeloid leukemia patients:in vitro and in vivo efficacy in severe combined immunodeficiency disease mice[J]. Blood, 1998, 92(9):3318-3327
10 Finker S, Trojaneck B, Lefterova P, et al. Increase of proliferation rate and enhancement of antitumor cytotoxicity of expanded human CD3+CD56+ immunologic effector cells by receptor mediated transfetion with the interleukin-7 gene[J]. Gene Ther, 1998, 5 (1):31-39
11 Zoll B, Lefterova P, Csipai M, et al. Generation of cytokine-induced killer cells using exogenous interleukin-2,7 or 12[J]. Cancer Immunol Immunother, 1998, 47(3) :221-226
12 Marten A, Ziske C, Schottker B, et al. Interactions between dendritic cells and cytokine-induced killer cells lead to an activation of both population[J]. J Immunother, 2001, 24(5):502-510
13 Marten A, Renoth S, Von LT, et al. Enhanced lytic activity of cytokine-induced killer cells against multiple myeloma cells after co-culture with idiotype-pulsed dendritic cells[J]. Haematologica, 2001, 86(7):1029-1037
14 Wang ZH, Simimoto H, Tani K, et al. IL-12 synergizes with B7-1 enhance the antitumor immunity in C57BL/6 Micc[J]. International Journal of Modern Cancer Therapy, 2000, 3(3):45-49
15 Wang ZH, Zhang QY, Zhang CY, et al. Antitumor immnity and vaccine effect induced by IL-12 synergizes B7-1 gene transfected cells[J]. Chinese Journal of Cancer Research, 2003, 15(1):5-8
16 Zoll B, Lefterova P, Ebert O, et al. Modulation of cell surface markers onNK-like T lymphocytes by using IL-2,IL-7 or IL-12 in vitro stimulation[J]. Cytokine, 2000, 12(3):1385-1390
17 Ense N, Lu S, Alcid MG, et al. Pooled umbilical cord blood as a possible universal donor formarrow reconstitution and use in nuclear accidents[J]. Life Sci, 2001, 69:1531-1539
18童春容,耿彦彪,陆道培,等。自体细胞因子诱导的杀伤细胞治疗急性白血病的临床研究[J]。北京医科大学学报,2000, 32(5):473-477
19陈复兴,刘军权,张南征,等。自身细胞因子诱导的杀伤细胞过继性免疫治疗恶性肿瘤的临床观察[J]。癌症,2002,21(7):797-801
20张宏文,彭晓,张晓燕,等。脐带血DCS对CIK细胞杀伤活性的影响研究。临床血液学杂志,2002,15(1):26-28
21 Schmidt-wolf, Leiterova P, John V, et al. Sensitivity of multidrug resistant tumor cell lines to immunological effector cells[J]. Cell immunol, 2003,169(1):85-90
1 Sakaguchi S, Sakaguchi N, Asano M, et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptorα-chains(CD25). Breackdown of single mechanism of self- tolerance causes various autoimmune diseases[J]. J Immunol, 1995,160:1151-1164
2 Shevach EM. Control of T cell activation by CD4+CD25+ T suppressor T cells[J]. Immunol Rev, 2001,182:59-67
3 Sakaguchi S. Immunologic tolerance maintained by CD4+CD25+ regulatory T cells: their common role in controlling autoimmunity,tumor immunity and transplantation tolerance[J]. Immunol Rev, 2001,182:18-32
4 Chattopadhyay S, Chakraborty NG, Mukherji B. Regulator T cells and tumor immunity. Cancer Immunol Immunother[J], 2005, 54(12):1153-1161
5 Bacchetta R, Gregori S, Roncarolo MG, et al. CD4+ regulatory T cells Mechanisms of induction and effector function[J]. Autoimmun Rev, 2004, 4(8):491-496
6 Read S, Powrie F. CD4+ regulatory T cells[J]. Curr Opintion Immunol, 2001, 13(15):644-649
7 Green EA, Choi Y, Flavell RA, et al. Pancreatic lymph node-derived CD4+CD25+ regulatory T cells:highly potent regulators of diabetes that require TRANCE-RANK signals[J]. Immunity, 2002, 16(2):183-191
8 Fontenot J, Gavin MA, Rudensky AY, et al. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells[J]. Nat Immunol, 2003, 4(4):330-336
9 Taams L, Vukmanovic-Stejic M, Salmon M, et al. Immune regulation byCD4+CD25+ regulatory T cells:implications for transplantation tolerance[J]. Transpl Immunol, 2003, 11(3):277-285
10 Goleva E, Cardona ID, Ou LS, et al. Factors that regulate naturally occurring T regulatory cell-mediated suppression[J]. Allergy Clin Immunol. 2005, 116(7):1094-1100
11 Jonuleit H, Adema G, Schmitt, et al.Immune regulation by regulatory T cells imphcations for transplantation[J]. Transpl Immunol, 2004, 12(5):267-276
12 Roll HK, Yama S, Stemman RM, et al. The interactions of DC cells with antigen-specific regulatory T cells that suppress autoimmunity[J]. Semin Immunol, 2006, 18(1):93-102
13 Zoltan F, Huber S, Read S, et al. Development and function of CD4+CD25+ regulatory T cells[J]. Curr Opin Immunol, 2004, 16(3):203-208
14 Jonuleit H, Schmitt E. The regulatory T cells family:dietarent subsets and their interlations[J]. J Immunol, 2003, 171(12):6323-6327
15 Hori S, Nomura T, Sakaguchi S, et al. Control of regulatory T cells development by the transcription factor Foxp3[J]. Science, 2003, 299(5609):1057-1061
16 Belkaid Y, Piccirillo DA, Mendez S, et al. CD4+CD25+ regulatory T cells control leishmania major persistence and immunity[J]. Nature, 2002,420(6915):502-507
17 Shimizu J, Yamazaki S, Takahashi T, et al. Stimulation of CD4+CD25+ regulatory T cells through GITR breaks immunological self-tolerance[J]. Nat Immunol, 2002, 3(2):135-142
18 Kazuhiko N, Atsushi K, Warren S, et al. Cell contact-dependent immunosuppression by CD4+CD25+ regulatory T cells in mediated by cell surface-bound transforming growth factor–β[J]. J Exp Med,2001,194(5):629-644
19 Cureil TJ, Coukos G, Zou L, et al.Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival[J]. Nat Med,2004,10(9):942-949
20 Woo EY, Chu CS, Goletz TJ, et al. Regulatory CD4+CD25+ T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer[J]. Cancer Res,2001,61(7):1766-1772
21 Biyanage UK, Moore TT, Joo HG, et al. Prevalence of regulatory T cells is increased in peripheral biood and tumor in roenvironment of patients with pancreas or breast adenocarcinoma[J]. J Immunol,2005,169(5):2756-2761
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |