肿瘤免疫治疗中CTL细胞、Treg细胞及其相关性研究
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摘要
目的:恶性肿瘤发病率增高,严重危害人类健康。目前针对肿瘤手术、放疗、化疗三大常规治疗方法却远远不能改变肿瘤治疗的现状。近年来随着分子生物学技术的发展、免疫学理论不断丰富,现代免疫学技术也在不断地推陈出新,人们从细胞和分子水平上对肿瘤与免疫系统的关系有了更深刻的认识,为肿瘤免疫疗法在临床上的应用奠定了坚实的基础。过继性细胞免疫疗法(adoptive cell immunotherapy ACI)已经成为继手术、放疗和化疗后的第四种治疗模式。过继性细胞免疫疗法是指向肿瘤患者转输具有抗肿瘤活性的免疫细胞,直接杀伤肿瘤或激发机体免疫反应来杀伤肿瘤细胞,达到治疗肿瘤的目的。目前以抗体疗法、T细胞疗法和肿瘤疫苗为代表的肿瘤被动免疫和主动免疫治疗,己经取得显著的进展,作为肿瘤治疗的辅助手段在临床上显示出光明的应用前景。
过继免疫治疗的关键在于筛选具有肿瘤特异性杀伤作用的效应细胞,对靶细胞即肿瘤细胞产生致命的杀伤,以实现过继免疫治疗高效低毒的特点。具有肿瘤特异性的细胞毒T细胞(cytotoxic T lymphocyte CTL)具有杀瘤谱广、杀瘤活性高、增殖能力强等特点成为过继免疫治疗中常用效应细胞而倍受关注。如何提高CTL细胞数量及活性、增强其抗肿瘤特异性是提高过继免疫治疗效果的关键。
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C57BL/6小鼠DC细胞与T细胞共同培养,并且负载B16黑色素瘤特异性抗原,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DC-CTL。
但是在体内试验中发现,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,出现效应细胞与肿瘤细胞大量共存的尴尬局面。消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CD4~+细胞的新成员,目前较为常用的是根据产生的途径不同将CD4~+CD25~+Treg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~+Treg细胞。CD4~+CD25~+Treg细胞可能通过识别肿瘤抗原后活化发挥其免疫抑制作用。CD4~+CD25~+Treg通过细胞间相互接触或分泌细胞因子等机制,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~+Treg细胞阻抑作用的方法,将是提高和改善肿瘤免疫治疗的一个崭新途径。
本研究将B16黑色素瘤特异性抗原致敏的成熟DC与T细胞共同培养,诱导生成具有肿瘤特异性免疫效应细胞DC-CTL。而且应用免疫磁珠分选的方法(magnetic cell sorting,MACS)去除CD4~+CD25~+Treg细胞成分,从体外杀伤试验、体内的抑瘤效果、细胞形态学变化、对细胞增殖和凋亡的影响等多方面观察DC-CTL细胞的杀伤效果。同时比较于T细胞分化形成CTL的不同时期删除CD4~+CD25~+Treg细胞成分对DC-CTL特异性杀伤效果的影响。从而探究DC-CTL细胞、Treg细胞在抗肿瘤免疫中所起的作用以及相互关系,为肿瘤的免疫治疗提供理论基础及实验室资料。
方法:本实验以C57BL/6小鼠的B16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60Coγ对小鼠黑色素瘤动物模型进行照射,使小鼠机体达到非髓性淋巴细胞删除状态,则C57BL/6小鼠的黑色素瘤动物模型的免疫细胞丧失。便于效应细胞回输完成免疫重建。
取小鼠脾脏制备DC和T细胞,B16黑色素瘤细胞采用冻融法制备肿瘤全抗原。将B16黑色素瘤抗原致敏DC细胞完成肿瘤全抗原的负载。将DC与T共同培养,诱导T细胞分化产生效应细胞CTL。并通过流氏细胞术检测DC细胞的免疫表型,确定DC细胞成熟活性。用MACS法分别于DC细胞与T细胞共同培养前后删除CD4~+CD25~+Treg细胞成分,便得到培养前删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞与培养后删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞2组。另设未删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞组及对照组2组。
然后再将NMLD状态的B16黑色素瘤C57BL/6小鼠80只,按随机数字表法分为4组并经鼠尾静脉进行效应细胞DC-CTL回输。第1组:培养前删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞组。第2组:培养后删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞组。第3组:未删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞组。第四组:对照组。
采用MTT染色法检测4组的体外杀伤实验,观察效应细胞DC-CTL对B16黑色素瘤的杀伤效果。同时观察体内回输DC-CTL效应细胞后小鼠的生活习性、肿瘤的临床特征、DC-CTL细胞及靶细胞的超微结构与细胞增殖与凋亡特征等形态学表现。证实特异性抗原负载的DC-CTL细胞对肿瘤的杀伤效能。
最后,运用流氏细胞术(FACS)及酶联免疫吸附实验(ELISA)检测人体内Treg细胞、TNF-α及IL-12含量变化,证实化疗药物的抗肿瘤机制及对机体免疫系统的影响。
结果:DC细胞诱导负载B16黑色素瘤肿瘤抗原的DC-CTL细胞活化时可见细胞增大、胞质丰富、活力良好。与肿瘤细胞接触后则诱导肿瘤细胞出现凋亡及胀亡表现,高效杀伤肿瘤细胞。培养前删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞组、培养后删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞组、未删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞组MTT法体外杀伤试验:三组效应细胞对肿瘤细胞都有较强的杀伤作用。培养前删除CD4~+CD25~+Treg细胞DC-CTL细胞组和培养后删除CD4~+CD25~+Treg细胞DC-CTL细胞组对B16黑色素细胞的杀伤作用明显高于(P<0.05)未删除CD4~+CD25~+Treg细胞DC-CTL细胞组。且随效靶比升高杀伤作用增强。培养前后删除CD4~+CD25~+Treg细胞的DC-CTL组间比较对B16黑色素细胞的杀伤作用没有显著性差异(P >0.05)。
B16黑色素瘤的体内抑瘤试验三组实验组DC-CTL细胞均可使瘤体缩小。抑瘤率明显高于对照组(P<0.05);2组删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞组抑瘤率明显高于未删除CD4~+CD25~+Treg细胞DC-CTL效应细胞组(P<0.05),培养前后删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞组间比较没有明显差异(P >0.05)。
流氏细胞术检测肿瘤细胞增殖周期的变化;3组实验组与对照组比较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(P<0.05);删除CD4~+CD25~+Treg细胞成分两组G0/G1期细胞比率、AI明显高于未删除CD4~+CD25~+Treg组,G2/M期细胞比率、PI显著低于未删除CD4~+CD25~+Treg组,S期无明显变化(P<0.05)。删除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)。
化疗药物改变肿瘤患者体内Treg细胞、TNF-α及IL-12含量,三种指标不仅说明化疗药物对机体免疫机能有显著影响,而且还可以做为评估化疗效果的指标。
结论:
1、特异性抗原致敏的DC-CTL细胞对B16黑色素瘤细胞有较强的杀伤作用。特异性抗原致敏的DC-CTL细胞可以作用于肿瘤细胞的细胞周期,影响肿瘤细胞的增殖与凋亡。充分说明DC-CTL细胞是一种具有强大肿瘤杀伤能力且具有肿瘤特异性的过继免疫治疗的效应细胞。
2、删除CD4~+CD25~+Treg细胞成分后的特异性抗原致敏的DC-CTL细胞抗肿瘤免疫效应更具特异性与高效。说明CD4~+CD25~+Treg能够抑制具有肿瘤特异性的效应细胞DC-CTL。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,必将成为一种可行的肿瘤免疫治疗方法。
3、不同时间删除CD4~+CD25~+Treg细胞后,从抑瘤作用、对肿瘤细胞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。
4、化疗药物改变机体内CD4~+CD25~+Treg、TNF-α及IL-12含量,对机体免疫系统有显著影响。同时也是评估化疗效果的指标。联合使用中药可以减轻其毒副作用。
Objective: Adoptive immunotherapy is one of the hottest subjects in biotherapy of tumor. The treatment is not only the supplement to conventional therapy such as surgical operation、chemotherapy and radiotherapy ,but also giving the found base for the clinical treatment to the tumor now. With effector cells being input back into the patient, they can kill the target cell at once with little side effects and toxicant effects during the treatment. Adoptive immunotherapy has good therapeutic effect on the tumor, because it can minimize the tumor and kill the cells and block their transfer and recidivism.
Cytotoxic T lymphocyte (CTL)cells, as one of the new type immunocompetence cells, have high efficiency in anti-tumor immunity. CTL cells have widespread usage in tumor adoptive immune therapy, because they show highly effcient cytolytic effect, faster proliferation speed rate than other immunocompetence cells in anti-tumor immunity. The key of studies is to find how to enhance their cytolytic and anti-tumor specificity activity so that it will lead us to a bright future in tumor treatment.
Dendritic cells(DC) are the most potent antigen presenting cells(APC) with the ability to acquire, process, present antigens and the unique capability of initiating primary immune responses against tumor-associated antigens. DCs can turn the T cells into the active cells with high ability to anti-tumor. The killer cells are well known as the CTLs. We can separate DCs, T cells from the spleen and culture respectively, then DCs are loaded with specific tumor antigen-B16 melanoma. We can co-culture tumor Antigen-sensitized DCs with T cells and then the DCs can turn the T cells into the CTLs. The CTLs induced by the tumor antigen-sensitized DCs have higher activity, cytolytic effect and more anti-tumor specificity. We can obtain the effector cells DC-CTLs with the tumor specific antigen to B16 melanoma.
But in practice, the effector cells and tumor cells can be seen alive together peacefully in the tumor microenvironment. The anti-tumor immunocompetence cells are reduced into immune anergy and immunological tolerance constantly. So the effect of the CTLs to kill the tumor cells descends obviously. It is very important for us to delete the immunodepression cells and increase the anti-tumor effect. That is the new way to tumor immunotherapy.
CD4~+CD25~+ regulatory T cells are a new member of the CD4~+ T cells. They are the more important subset of regulatory T cells(Treg) that play an essential role in maintaining immunological self-tolerance. They have the function of down-regulating the tumor immunity mediated by T cells. CD4~+CD25~+ Tregs also are divided into two groups by produced channel. The two groups are natural regulatory T cells (nTreg) and induced regulatory T cells( iTreg). CD4~+CD25~+ Tregs increase in the peripheral blood and tumor infiltration lymphoglandula in many tumor patients, such as breast cancer, lung cancer, gastric cancer and so on. CD4~+CD25~+ Tregs suppress immune responses mainly by cell contract-dependent interactions or secreting soluble cytokines. Therefore, to delete or attenuate the effect of CD4~+CD25~+ Tregs will evoke effective anti-tumor immunity, which may become a feasible immunotherapy for cancer.
With this experiment, we use the specific antigen-sensitized DC-CTL cells as the anti-tumor immunotherapy cells to cure the B16 melanoma tumor animal model. We can observe not only the morphological difference, cell cycle,cell proliferation and cell apoptosis in vivo, but also the killing effect in vitro. Moreover, the deletion of the CD4~+CD25~+ Tregs by magnetic cell sorting (MACS) and observation the influence on the kill effection in the tumor treatment shows the relationship CD4~+CD25~+ Tregs and the adoptive immunotherapy in B16 melanoma. In this way, we may analyse and discuss the relationship between DC-CTL cells and regulatory T cells in anti-tumor immunity in order to find more treatment methods and theory foundation to clinical therapy in anti-tumor immunotherapy through the experiment.
Methods: The B16 melanoma tumor cells are cultured in vitro and injected into the C57BL/6 mouse body to make the animal models. Then the animal models are irradiated by 60Co-ray on the total body in order to eliminate its immunol function. Then the animal models in a no-marrow lymphocyte deletion(NMLD) state. It is a special period for us to restitut the immune system with new immunocells.
DCs and T cells are isolated from C57BL/6 mouse’spleen. By the analysis of the DC’s immune epitope to show the mature stage, the DCs are cultured and loaded with the B16 melanoma tumor special antigen. DCs can induce the T cells into the DC-CTLs with tumor specificity. Before and after culture antigen-sensitized DC-CTLs were gotten, the method of MACS system was used to delete CD4~+CD25~+ regulatory T cells. So we have two groups anti-tumor immunocompetence cells: DC-CTL without CD4~+CD25~+ Tregs. They are after and before the culture of DC-CTL by using MACS system to delete CD4~+CD25~+ Tregs respectively. We also have the third group: the specific antigen-sensitized DC-CTL cells without deletion CD4~+CD25~+ regulatory T cells, the forth group the control group.
80 cases of C57BL/6 mouse animal models have been randomly divided into four groups. The first group: the tumor specificity DC-CTL that gets before the culture to delete CD4~+CD25~+ Tregs; the second group :the tumor specificity DC-CTL that gets after the culture to delete CD4~+CD25~+ Tregs; the third group: the tumor specificity DC-CTL that gets without deletion CD4~+CD25~+ Tregs; the forth group: the control group that is to use normal sodium.
To inspect DC-CTLs’killing effect to B16 melanoma in vitro by the method of mono-nuclear cell direc cytotoxicity assay(MTT) and the effect of the inhibitory to the B16 melanoma in vivo. Such as the change of tumor volume、tumor weight and the tumor inhibition rates, inhibitory effect of the specific antigen-sensitized DC-CTL cells on B16 melanoma cells are estimated. Morphological characteristics of immune cells are observed by electronic microscope. Moreover, the rate of tumor cells in G0/G1、S、G2/M stage, proliferation index(PI) and apoptosis index(AI)are respectively detected with florescene-actevated cell sorter(FACS). To study the effect of DC-CTL cells on B16 melanoma cells proliferation and apoptosis in the animal models, is to show the significant effect to kill the tumor cells.
To check the CD4~+CD25~+ Tregs by FACS and the tumor necrosis factor-α(TNF-α), Interleukin-12(IL-12) by enzyme linked immunosorbent assay (ELISA) in patient with colorectal cancer at first. To find the change in the CD4~+CD25~+ Tregs, TNF-αand IL-12 and show the effect that the chemotherapy have done to the colorectal patients’immune system.
Results: CTL cells with the tumor specificity of B16 melanoma induced by DCs are analyzed in the morphology. The specific antigen-sensitized DC-CTL cells are bigger than the normal lymphocytes and notch always appears on the cell nucleus observed under transmission electron microscope(TEM). Plenty of active-functioned organelles such as dilated endoplasmic reticulum,cytochondriomes were observed in the cytoplasm of the specific antigen-sensitized DC-CTL cells. Protrusions on the surface of the specific antigen-sensitized DC-CTL cells contacted closely with the tumor cells. Apoptosis and necrosis of the cells can be widely observed in tumor tissues.
The kill effect in vitro by the method MTT, the tumor inhibition rate of the three experiment groups is significantly higher than the control group to B16 melanoma(P<0.05), and the inhibition rate increase according to the rate of the effect-target. The two groups that delete the CD4~+CD25~+ Tregs have higher tumor inhibition rate than the group that is without deletion. But there is no significant difference in tumor inhibition rate between the two groups that delete CD4~+CD25~+ Tregs before or after the culture (P >0.05).
The kill effect in vivo to the B16 melanoma in the three experimental groups has an obvious inhibition to the tumor compared with the control group. The volume, weight of the tumor and the inhibition rate is higher in the groups with deletion the CD4~+CD25~+ Tregs than the groups without deletion CD4~+CD25~+ Treg(sP<0.05). But there is no significant difference between the groups that delete the CD4~+CD25~+ Tregs before or after the culture (P >0.05).
By the method of FACS, compared with the three experimental groups and the control group, the rate of tumor cells in G0/G1 and AI are significantly higher; on the other hand, the rate of tumor cells in G2/M stage and PI is significantly lower. But there is no change in S stage. Comparing the two groups of deletion the CD4~+CD25~+ Tregs with the group without deletion, the difference in the cell cycle, AI and PI is significant in fact. But the difference between the two groups with deletion CD4~+CD25~+ Tregs befor and after the culture is not significant (P >0.05).
The medicine of the chemotherapy can change the CD4~+CD25~+ Tregs, TNF-αand IL-12 in the colorectal cancer and have effect to the patients’immune system. But on the other hand CD4~+CD25~+ Tregs, TNF-αand IL-12 are the index to estimate the effect of the chemotherapy.
Conclusion:
1 The specific antigen-sensitized DC-CTL cells have high efficiency in inhibition on B16 melanoma. The specific antigen-sensitized DC-CTL cells may have an effect on the tumor cells cycle and induce them apoptosis. DC-CTL cells are a new generation of adoptive immunocell with high efficiency in killing tumors cells.
2 The specific antigen-sensitized DC-CTL cells have high efficiency and more special in inhibition effect on B16 melanoma tumor cells after deleting CD4~+CD25~+Tregs. CD4~+CD25~+Tregs can exert inhibitory effect on The specific antigen-sensitized DC-CTL cells killing function. Deleting CD4~+CD25~+Tregs and recruitment of effector T cells will evoke effective anti-tumor immunity, which may have become a feasible immunotherapy for cancer.
3 There is no significant difference between the two groups in different stage with the deleting CD4~+CD25~+ Tregs by observing the tumor inhibition rate, tumor cells apoptosis and proliferation, the killing effect. We must do more study to explain the possible mechanism and relationship among the main subsets of regulatory T cells.
4 chemotherapy can change not only the CD4~+CD25~+Treg, TNF-αand IL-12 but also the immune system of patients. Combining with chinese medicine can reduce the side effects. It is a good way to cure the tumor.
过继免疫治疗的关键在于筛选具有肿瘤特异性杀伤作用的效应细胞,对靶细胞即肿瘤细胞产生致命的杀伤,以实现过继免疫治疗高效低毒的特点。具有肿瘤特异性的细胞毒T细胞(cytotoxic T lymphocyte CTL)具有杀瘤谱广、杀瘤活性高、增殖能力强等特点成为过继免疫治疗中常用效应细胞而倍受关注。如何提高CTL细胞数量及活性、增强其抗肿瘤特异性是提高过继免疫治疗效果的关键。
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C57BL/6小鼠DC细胞与T细胞共同培养,并且负载B16黑色素瘤特异性抗原,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DC-CTL。
但是在体内试验中发现,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,出现效应细胞与肿瘤细胞大量共存的尴尬局面。消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CD4~+细胞的新成员,目前较为常用的是根据产生的途径不同将CD4~+CD25~+Treg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~+Treg细胞。CD4~+CD25~+Treg细胞可能通过识别肿瘤抗原后活化发挥其免疫抑制作用。CD4~+CD25~+Treg通过细胞间相互接触或分泌细胞因子等机制,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~+Treg细胞阻抑作用的方法,将是提高和改善肿瘤免疫治疗的一个崭新途径。
本研究将B16黑色素瘤特异性抗原致敏的成熟DC与T细胞共同培养,诱导生成具有肿瘤特异性免疫效应细胞DC-CTL。而且应用免疫磁珠分选的方法(magnetic cell sorting,MACS)去除CD4~+CD25~+Treg细胞成分,从体外杀伤试验、体内的抑瘤效果、细胞形态学变化、对细胞增殖和凋亡的影响等多方面观察DC-CTL细胞的杀伤效果。同时比较于T细胞分化形成CTL的不同时期删除CD4~+CD25~+Treg细胞成分对DC-CTL特异性杀伤效果的影响。从而探究DC-CTL细胞、Treg细胞在抗肿瘤免疫中所起的作用以及相互关系,为肿瘤的免疫治疗提供理论基础及实验室资料。
方法:本实验以C57BL/6小鼠的B16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60Coγ对小鼠黑色素瘤动物模型进行照射,使小鼠机体达到非髓性淋巴细胞删除状态,则C57BL/6小鼠的黑色素瘤动物模型的免疫细胞丧失。便于效应细胞回输完成免疫重建。
取小鼠脾脏制备DC和T细胞,B16黑色素瘤细胞采用冻融法制备肿瘤全抗原。将B16黑色素瘤抗原致敏DC细胞完成肿瘤全抗原的负载。将DC与T共同培养,诱导T细胞分化产生效应细胞CTL。并通过流氏细胞术检测DC细胞的免疫表型,确定DC细胞成熟活性。用MACS法分别于DC细胞与T细胞共同培养前后删除CD4~+CD25~+Treg细胞成分,便得到培养前删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞与培养后删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞2组。另设未删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞组及对照组2组。
然后再将NMLD状态的B16黑色素瘤C57BL/6小鼠80只,按随机数字表法分为4组并经鼠尾静脉进行效应细胞DC-CTL回输。第1组:培养前删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞组。第2组:培养后删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞组。第3组:未删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞组。第四组:对照组。
采用MTT染色法检测4组的体外杀伤实验,观察效应细胞DC-CTL对B16黑色素瘤的杀伤效果。同时观察体内回输DC-CTL效应细胞后小鼠的生活习性、肿瘤的临床特征、DC-CTL细胞及靶细胞的超微结构与细胞增殖与凋亡特征等形态学表现。证实特异性抗原负载的DC-CTL细胞对肿瘤的杀伤效能。
最后,运用流氏细胞术(FACS)及酶联免疫吸附实验(ELISA)检测人体内Treg细胞、TNF-α及IL-12含量变化,证实化疗药物的抗肿瘤机制及对机体免疫系统的影响。
结果:DC细胞诱导负载B16黑色素瘤肿瘤抗原的DC-CTL细胞活化时可见细胞增大、胞质丰富、活力良好。与肿瘤细胞接触后则诱导肿瘤细胞出现凋亡及胀亡表现,高效杀伤肿瘤细胞。培养前删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞组、培养后删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞组、未删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞组MTT法体外杀伤试验:三组效应细胞对肿瘤细胞都有较强的杀伤作用。培养前删除CD4~+CD25~+Treg细胞DC-CTL细胞组和培养后删除CD4~+CD25~+Treg细胞DC-CTL细胞组对B16黑色素细胞的杀伤作用明显高于(P<0.05)未删除CD4~+CD25~+Treg细胞DC-CTL细胞组。且随效靶比升高杀伤作用增强。培养前后删除CD4~+CD25~+Treg细胞的DC-CTL组间比较对B16黑色素细胞的杀伤作用没有显著性差异(P >0.05)。
B16黑色素瘤的体内抑瘤试验三组实验组DC-CTL细胞均可使瘤体缩小。抑瘤率明显高于对照组(P<0.05);2组删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞组抑瘤率明显高于未删除CD4~+CD25~+Treg细胞DC-CTL效应细胞组(P<0.05),培养前后删除CD4~+CD25~+Treg细胞的具有肿瘤特异性的DC-CTL细胞组间比较没有明显差异(P >0.05)。
流氏细胞术检测肿瘤细胞增殖周期的变化;3组实验组与对照组比较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(P<0.05);删除CD4~+CD25~+Treg细胞成分两组G0/G1期细胞比率、AI明显高于未删除CD4~+CD25~+Treg组,G2/M期细胞比率、PI显著低于未删除CD4~+CD25~+Treg组,S期无明显变化(P<0.05)。删除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)。
化疗药物改变肿瘤患者体内Treg细胞、TNF-α及IL-12含量,三种指标不仅说明化疗药物对机体免疫机能有显著影响,而且还可以做为评估化疗效果的指标。
结论:
1、特异性抗原致敏的DC-CTL细胞对B16黑色素瘤细胞有较强的杀伤作用。特异性抗原致敏的DC-CTL细胞可以作用于肿瘤细胞的细胞周期,影响肿瘤细胞的增殖与凋亡。充分说明DC-CTL细胞是一种具有强大肿瘤杀伤能力且具有肿瘤特异性的过继免疫治疗的效应细胞。
2、删除CD4~+CD25~+Treg细胞成分后的特异性抗原致敏的DC-CTL细胞抗肿瘤免疫效应更具特异性与高效。说明CD4~+CD25~+Treg能够抑制具有肿瘤特异性的效应细胞DC-CTL。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,必将成为一种可行的肿瘤免疫治疗方法。
3、不同时间删除CD4~+CD25~+Treg细胞后,从抑瘤作用、对肿瘤细胞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。
4、化疗药物改变机体内CD4~+CD25~+Treg、TNF-α及IL-12含量,对机体免疫系统有显著影响。同时也是评估化疗效果的指标。联合使用中药可以减轻其毒副作用。
Objective: Adoptive immunotherapy is one of the hottest subjects in biotherapy of tumor. The treatment is not only the supplement to conventional therapy such as surgical operation、chemotherapy and radiotherapy ,but also giving the found base for the clinical treatment to the tumor now. With effector cells being input back into the patient, they can kill the target cell at once with little side effects and toxicant effects during the treatment. Adoptive immunotherapy has good therapeutic effect on the tumor, because it can minimize the tumor and kill the cells and block their transfer and recidivism.
Cytotoxic T lymphocyte (CTL)cells, as one of the new type immunocompetence cells, have high efficiency in anti-tumor immunity. CTL cells have widespread usage in tumor adoptive immune therapy, because they show highly effcient cytolytic effect, faster proliferation speed rate than other immunocompetence cells in anti-tumor immunity. The key of studies is to find how to enhance their cytolytic and anti-tumor specificity activity so that it will lead us to a bright future in tumor treatment.
Dendritic cells(DC) are the most potent antigen presenting cells(APC) with the ability to acquire, process, present antigens and the unique capability of initiating primary immune responses against tumor-associated antigens. DCs can turn the T cells into the active cells with high ability to anti-tumor. The killer cells are well known as the CTLs. We can separate DCs, T cells from the spleen and culture respectively, then DCs are loaded with specific tumor antigen-B16 melanoma. We can co-culture tumor Antigen-sensitized DCs with T cells and then the DCs can turn the T cells into the CTLs. The CTLs induced by the tumor antigen-sensitized DCs have higher activity, cytolytic effect and more anti-tumor specificity. We can obtain the effector cells DC-CTLs with the tumor specific antigen to B16 melanoma.
But in practice, the effector cells and tumor cells can be seen alive together peacefully in the tumor microenvironment. The anti-tumor immunocompetence cells are reduced into immune anergy and immunological tolerance constantly. So the effect of the CTLs to kill the tumor cells descends obviously. It is very important for us to delete the immunodepression cells and increase the anti-tumor effect. That is the new way to tumor immunotherapy.
CD4~+CD25~+ regulatory T cells are a new member of the CD4~+ T cells. They are the more important subset of regulatory T cells(Treg) that play an essential role in maintaining immunological self-tolerance. They have the function of down-regulating the tumor immunity mediated by T cells. CD4~+CD25~+ Tregs also are divided into two groups by produced channel. The two groups are natural regulatory T cells (nTreg) and induced regulatory T cells( iTreg). CD4~+CD25~+ Tregs increase in the peripheral blood and tumor infiltration lymphoglandula in many tumor patients, such as breast cancer, lung cancer, gastric cancer and so on. CD4~+CD25~+ Tregs suppress immune responses mainly by cell contract-dependent interactions or secreting soluble cytokines. Therefore, to delete or attenuate the effect of CD4~+CD25~+ Tregs will evoke effective anti-tumor immunity, which may become a feasible immunotherapy for cancer.
With this experiment, we use the specific antigen-sensitized DC-CTL cells as the anti-tumor immunotherapy cells to cure the B16 melanoma tumor animal model. We can observe not only the morphological difference, cell cycle,cell proliferation and cell apoptosis in vivo, but also the killing effect in vitro. Moreover, the deletion of the CD4~+CD25~+ Tregs by magnetic cell sorting (MACS) and observation the influence on the kill effection in the tumor treatment shows the relationship CD4~+CD25~+ Tregs and the adoptive immunotherapy in B16 melanoma. In this way, we may analyse and discuss the relationship between DC-CTL cells and regulatory T cells in anti-tumor immunity in order to find more treatment methods and theory foundation to clinical therapy in anti-tumor immunotherapy through the experiment.
Methods: The B16 melanoma tumor cells are cultured in vitro and injected into the C57BL/6 mouse body to make the animal models. Then the animal models are irradiated by 60Co-ray on the total body in order to eliminate its immunol function. Then the animal models in a no-marrow lymphocyte deletion(NMLD) state. It is a special period for us to restitut the immune system with new immunocells.
DCs and T cells are isolated from C57BL/6 mouse’spleen. By the analysis of the DC’s immune epitope to show the mature stage, the DCs are cultured and loaded with the B16 melanoma tumor special antigen. DCs can induce the T cells into the DC-CTLs with tumor specificity. Before and after culture antigen-sensitized DC-CTLs were gotten, the method of MACS system was used to delete CD4~+CD25~+ regulatory T cells. So we have two groups anti-tumor immunocompetence cells: DC-CTL without CD4~+CD25~+ Tregs. They are after and before the culture of DC-CTL by using MACS system to delete CD4~+CD25~+ Tregs respectively. We also have the third group: the specific antigen-sensitized DC-CTL cells without deletion CD4~+CD25~+ regulatory T cells, the forth group the control group.
80 cases of C57BL/6 mouse animal models have been randomly divided into four groups. The first group: the tumor specificity DC-CTL that gets before the culture to delete CD4~+CD25~+ Tregs; the second group :the tumor specificity DC-CTL that gets after the culture to delete CD4~+CD25~+ Tregs; the third group: the tumor specificity DC-CTL that gets without deletion CD4~+CD25~+ Tregs; the forth group: the control group that is to use normal sodium.
To inspect DC-CTLs’killing effect to B16 melanoma in vitro by the method of mono-nuclear cell direc cytotoxicity assay(MTT) and the effect of the inhibitory to the B16 melanoma in vivo. Such as the change of tumor volume、tumor weight and the tumor inhibition rates, inhibitory effect of the specific antigen-sensitized DC-CTL cells on B16 melanoma cells are estimated. Morphological characteristics of immune cells are observed by electronic microscope. Moreover, the rate of tumor cells in G0/G1、S、G2/M stage, proliferation index(PI) and apoptosis index(AI)are respectively detected with florescene-actevated cell sorter(FACS). To study the effect of DC-CTL cells on B16 melanoma cells proliferation and apoptosis in the animal models, is to show the significant effect to kill the tumor cells.
To check the CD4~+CD25~+ Tregs by FACS and the tumor necrosis factor-α(TNF-α), Interleukin-12(IL-12) by enzyme linked immunosorbent assay (ELISA) in patient with colorectal cancer at first. To find the change in the CD4~+CD25~+ Tregs, TNF-αand IL-12 and show the effect that the chemotherapy have done to the colorectal patients’immune system.
Results: CTL cells with the tumor specificity of B16 melanoma induced by DCs are analyzed in the morphology. The specific antigen-sensitized DC-CTL cells are bigger than the normal lymphocytes and notch always appears on the cell nucleus observed under transmission electron microscope(TEM). Plenty of active-functioned organelles such as dilated endoplasmic reticulum,cytochondriomes were observed in the cytoplasm of the specific antigen-sensitized DC-CTL cells. Protrusions on the surface of the specific antigen-sensitized DC-CTL cells contacted closely with the tumor cells. Apoptosis and necrosis of the cells can be widely observed in tumor tissues.
The kill effect in vitro by the method MTT, the tumor inhibition rate of the three experiment groups is significantly higher than the control group to B16 melanoma(P<0.05), and the inhibition rate increase according to the rate of the effect-target. The two groups that delete the CD4~+CD25~+ Tregs have higher tumor inhibition rate than the group that is without deletion. But there is no significant difference in tumor inhibition rate between the two groups that delete CD4~+CD25~+ Tregs before or after the culture (P >0.05).
The kill effect in vivo to the B16 melanoma in the three experimental groups has an obvious inhibition to the tumor compared with the control group. The volume, weight of the tumor and the inhibition rate is higher in the groups with deletion the CD4~+CD25~+ Tregs than the groups without deletion CD4~+CD25~+ Treg(sP<0.05). But there is no significant difference between the groups that delete the CD4~+CD25~+ Tregs before or after the culture (P >0.05).
By the method of FACS, compared with the three experimental groups and the control group, the rate of tumor cells in G0/G1 and AI are significantly higher; on the other hand, the rate of tumor cells in G2/M stage and PI is significantly lower. But there is no change in S stage. Comparing the two groups of deletion the CD4~+CD25~+ Tregs with the group without deletion, the difference in the cell cycle, AI and PI is significant in fact. But the difference between the two groups with deletion CD4~+CD25~+ Tregs befor and after the culture is not significant (P >0.05).
The medicine of the chemotherapy can change the CD4~+CD25~+ Tregs, TNF-αand IL-12 in the colorectal cancer and have effect to the patients’immune system. But on the other hand CD4~+CD25~+ Tregs, TNF-αand IL-12 are the index to estimate the effect of the chemotherapy.
Conclusion:
1 The specific antigen-sensitized DC-CTL cells have high efficiency in inhibition on B16 melanoma. The specific antigen-sensitized DC-CTL cells may have an effect on the tumor cells cycle and induce them apoptosis. DC-CTL cells are a new generation of adoptive immunocell with high efficiency in killing tumors cells.
2 The specific antigen-sensitized DC-CTL cells have high efficiency and more special in inhibition effect on B16 melanoma tumor cells after deleting CD4~+CD25~+Tregs. CD4~+CD25~+Tregs can exert inhibitory effect on The specific antigen-sensitized DC-CTL cells killing function. Deleting CD4~+CD25~+Tregs and recruitment of effector T cells will evoke effective anti-tumor immunity, which may have become a feasible immunotherapy for cancer.
3 There is no significant difference between the two groups in different stage with the deleting CD4~+CD25~+ Tregs by observing the tumor inhibition rate, tumor cells apoptosis and proliferation, the killing effect. We must do more study to explain the possible mechanism and relationship among the main subsets of regulatory T cells.
4 chemotherapy can change not only the CD4~+CD25~+Treg, TNF-αand IL-12 but also the immune system of patients. Combining with chinese medicine can reduce the side effects. It is a good way to cure the tumor.
引文
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8 Azria D, Ozsahin M, Rosenstein B. Radiation-induced sequelae measured by means of lymphocyte apoptosis: importance of certain single nucleotide polymorphisms[J]. Radiat Oncol Biol Phys, 2009,75(4):1275-1276
9 Frias M, Iglesias-Serret D, Cosialls AM, Akt inhibitors induce apoptosis in chronic lymphocytic leukemia cells[J]. Haematologica,2009,94(12): 1698-1707
10 Liu QL, Wang YS, Wang JX. Effect of insulin on functional status of cord blood-derived dendritic cells and on dendritic cell-induced CTL cytotoxicity against pancreatic cancer cell lines[J]. Hepatobiliary Pancreat Dis Int, 2009,8(5):529-534
11 Frumento G, Piazza T, Di Carlo E, et al. Targeting tumor-related immunosuppression for cancer immunotherapy[J]. Endocr Metab Immune Disord Drug Targets, 2006,6(3):233-237
12 Valzasina B, Piconese S, Guiducci C, et al. Tumor-induced expansion of regulatory T cells by conversion of CD4+CD25-lymphocytes is thymus and proliferation independent[J]. Cancer Res, 2006,66(8):4488-4495
13 Udagawa M, Kudo-Saito C, Hasegawa G, et al. Enhancement of immunologic tumor regression by intratumoral administration of dendritic cells in combination with cryoablative tumor pretreatment and Bacillus Calmette-Guerin cell wall skeleton stimulation[J]. Clin Cancer Res, 2006, 12(24):7465-7475
14 Lindley S, Dayan CM, Bishop A, et al. Defective suppressor function in CD4+CD25+T cells from patients with type 1 diabetes[J]. Diabetes, 2005, 54 (1):92-99
15 O' Reilly LA, Tai L, Lee L, et al. Membrane-bound Fas ligand only is essential for Fas-induced apoptosis[J]. Nature, 2009,461(7264):659-663
16 Deng YJ, Zhang LJ, Su XD, et al. Dendritic cell-tumor cell fusion vaccine prevents growth of subcutaneous transplanted esophageal carcinomas[J]. Chin J Cancer, 2009 ,28(10):1067-1071
17 Xu T, Sun BC, Li Q, Role of cytokines in promoting immune escape of FasL-expressing human colon cancer cells[J]. World J Gastroenterol, 2005,11(25):3915-3919
18 Morishima N, Owaki T, Asakawa M, et al. Augmentation of effector CD8+ T cell generation with enhanced granzyme Bexpression by IL-27[J]. Immunol, 2005,175(3):1686-1693
1 Yang I, Han S, Parsa AT. Heat-shock protein vaccines as active immunotherapy against human gliomas[J]. Expert Rev Anticancer Ther, 2009,9(11): 1577-1582
2 Kloverpris HN, Karlsson I, Thorn M, et al. Immune hierarchy among HIV-1 CD8+ T cell epitopes delivered by dendritic cells depends on MHC-I binding irrespective of mode of loading and immunization in HLA-A*0201 mice[J].APMIS, 2009, 117(11):849-855
3 MacConmara MP, Maung AA, Fujimi S, et al. increased CD4+ CD25+ T regulatory cell activity in trauma patients depresses protective Th1 immunity[J]. Ann Surg, 2006,244(4):514-523
4 Holtl L, Zeller Rieser C, Gander H, et al. Immunotherapy of metastatic renal cell carcinoma with tumor lysate-pulsed autologous dendritic cells [J]. Clin Cancer Ras, 2002,8(11):3369-3376
5 Tang ZH,Qiu WH, Wu GS, et al. The immunotherapeutic effect of dendritic cells vaccine modified with interleukin-18 gene and tumor cell lysate on mice with pancreatic carcinoma[J]. Word J Gastroenterol, 2002,8(5):908- 912
6 Terasawa H, Tsang K Y,Identification and characterization of a human agonist cytotoxic T-lymphocyte epitope of human prostate-specific antigen[J].Clin Cancer Ras, 2002,8(1):41-53
7 Dhodapar MV, Steinm an RM, Krasovsky J, et al. Amtigen-specific inhibition of effector T cell function in human after injection of immature dendritic cells [J]. Exp Med, 2001,7(4):761-765
8 Wang K, Zhou Q, Guo AL, et al. An autologous therapeutic dendritic cell vaccine transfected with total lung carcinoma RNA stimulates cytotoxic Tlymphocyte responses against non-small cell lung cancer [J]. Immunol Invest, 2009,38(7):665-680
9 Frumento G, Piazza T, Di Carlo E, Targeting tumor-related immunosupp- ression for cancer immunotherapy[J]. Endocr Metab Immune Disord Drug Targets, 2006,6(3):233-237
10 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
11 Chattopadhyay S, Chakraborty NG, Mukherji B, et al. Regulatory T cells and tumor immunity [J]. Cancer Immunol Immunother, 2005,54(12): 1153-1161.
12 Curiel TJ. Regulatory T cells and treatment of cancer[J]. Curr Opin Immunol, 2008,20(5):241-246.
13 uriel TJ. Tregs and ret hinking cancer immunotherapy [J]. Clin Invest,2007,11(7):1167-1174.
14 Valzasina B, Piconese S, Guiducci C, et al. Tumor induced expansion of regulatory T cells by conversion of CD4+CD25- lymphocytes is thymus and proliferation independent [J]. Cancer Res, 2006,6(6):4488-4495
15 Dudley ME, Wunderlich JR, Yang JC, et al. Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma [J]. J Clin Oncol,2005,23(10):2346-2357
16 Xie SJ, Fu ZX, Li DB, Experimental study of individualized cancer immunotherapy based on dendritic cells against gastric cancer [J]. Chinese Journal of Surgery, 2006, 44(7):476-480
17 Kozako T, Fukada K, Hirata S, et al. Efficient induction of human T-cell leukemia virus-1-specific CTL by chimeric particle without adjuvant as a prophylactic for adult T-cell leukemia[J]. Mol Immunol,2009,47(2-3): 606-613
18 Nguyen S, Dhedin N, Vemant JP, et al. NK—cell reconstitution after haploydentical hem atopoietic stem cell transpiantantions inmaturity of NKcells and inhibitory effect of NKG2A override GvL effect[J]. Blood, 2005,105(9):4135-4142
19 Raghavan S, Hilmgren J, Angela MT, et al. CD4+CD25+ suppressor T cells regulate pathogen induced inflammation and disease[J]. FEMS Immunol Med Microbiol, 2006,14(2):121-127
20 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
21 Xiong G, Husseiny MI, Song L, et al. Novel cancer vaccine based on genes of Salmonella pathogenicity island 2 [J]. Int J Cancer, 2009,126(11):2622 -2634
22 Yang JY, Cao DY, Xue Y, et al. Improvement of dendritic-based vaccine efficacy against hepatitis B virus-related hepatocellular carcinoma by two tumor-associated antigen gene-infected dendritic cells[J]. Hum Immunol, 2010,71(3):255-262
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9 Lundqvist A, Su S, Rao S, et al. Cutting edge: bortezomib-treated tumors sensitized to NK cell apoptosis paradoxically acquire resistance to antigen specific T cells[J]. Immunol, 2010,184(3):1139-1142
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12 Terasawa H, Tsang K Y,Identification and characterization of a human agonist cytotoxic T lymphocyte epitope of human prostate-specific antigen[J].Clin Cancer Ras, 2002,8(1):41-53
13 Dhodapar MV, Steinm an RM, Krasovsky J, et al. Amtigen-specific inhibition of effector T cell function in human after injection of immature dendritic cells[J]. Exp Med, 2001,7(4):761-765
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24 Deng YJ, Zhang LJ, Su XD, et al. Dendritic cell-tumor cell fusion vaccine prevents growth of subcutaneous transplanted esophageal carcinomas[J]. Chin J Cancer, 2009,28(10):1067-1071
25 Xiong G, Husseiny MI, Song L, et al. Novel cancer vaccine based on genes of Salmonella pathogenicity island 2 [J]. Int J Cancer, 2009,126(11): 2622- 2634
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7 Kilinc MO, Rowswell-Turner RB, Gu T, et al. Activated CD8+ T-effector/memory cells eliminate CD4+ CD25+ Foxp3+ T-suppressor cells from tumors via FasL mediated apoptosis[J]. Immunol, 2009,183 (12): 7656-7660
8 Azria D, Ozsahin M, Rosenstein B. Radiation-induced sequelae measured by means of lymphocyte apoptosis: importance of certain single nucleotide polymorphisms[J]. Radiat Oncol Biol Phys, 2009,75(4):1275-1276
9 Frias M, Iglesias-Serret D, Cosialls AM, Akt inhibitors induce apoptosis in chronic lymphocytic leukemia cells[J]. Haematologica,2009,94(12): 1698-1707
10 Liu QL, Wang YS, Wang JX. Effect of insulin on functional status of cord blood-derived dendritic cells and on dendritic cell-induced CTL cytotoxicity against pancreatic cancer cell lines[J]. Hepatobiliary Pancreat Dis Int, 2009,8(5):529-534
11 Frumento G, Piazza T, Di Carlo E, et al. Targeting tumor-related immunosuppression for cancer immunotherapy[J]. Endocr Metab Immune Disord Drug Targets, 2006,6(3):233-237
12 Valzasina B, Piconese S, Guiducci C, et al. Tumor-induced expansion of regulatory T cells by conversion of CD4+CD25-lymphocytes is thymus and proliferation independent[J]. Cancer Res, 2006,66(8):4488-4495
13 Udagawa M, Kudo-Saito C, Hasegawa G, et al. Enhancement of immunologic tumor regression by intratumoral administration of dendritic cells in combination with cryoablative tumor pretreatment and Bacillus Calmette-Guerin cell wall skeleton stimulation[J]. Clin Cancer Res, 2006, 12(24):7465-7475
14 Lindley S, Dayan CM, Bishop A, et al. Defective suppressor function in CD4+CD25+T cells from patients with type 1 diabetes[J]. Diabetes, 2005, 54 (1):92-99
15 O' Reilly LA, Tai L, Lee L, et al. Membrane-bound Fas ligand only is essential for Fas-induced apoptosis[J]. Nature, 2009,461(7264):659-663
16 Deng YJ, Zhang LJ, Su XD, et al. Dendritic cell-tumor cell fusion vaccine prevents growth of subcutaneous transplanted esophageal carcinomas[J]. Chin J Cancer, 2009 ,28(10):1067-1071
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18 Morishima N, Owaki T, Asakawa M, et al. Augmentation of effector CD8+ T cell generation with enhanced granzyme Bexpression by IL-27[J]. Immunol, 2005,175(3):1686-1693
1 Yang I, Han S, Parsa AT. Heat-shock protein vaccines as active immunotherapy against human gliomas[J]. Expert Rev Anticancer Ther, 2009,9(11): 1577-1582
2 Kloverpris HN, Karlsson I, Thorn M, et al. Immune hierarchy among HIV-1 CD8+ T cell epitopes delivered by dendritic cells depends on MHC-I binding irrespective of mode of loading and immunization in HLA-A*0201 mice[J].APMIS, 2009, 117(11):849-855
3 MacConmara MP, Maung AA, Fujimi S, et al. increased CD4+ CD25+ T regulatory cell activity in trauma patients depresses protective Th1 immunity[J]. Ann Surg, 2006,244(4):514-523
4 Holtl L, Zeller Rieser C, Gander H, et al. Immunotherapy of metastatic renal cell carcinoma with tumor lysate-pulsed autologous dendritic cells [J]. Clin Cancer Ras, 2002,8(11):3369-3376
5 Tang ZH,Qiu WH, Wu GS, et al. The immunotherapeutic effect of dendritic cells vaccine modified with interleukin-18 gene and tumor cell lysate on mice with pancreatic carcinoma[J]. Word J Gastroenterol, 2002,8(5):908- 912
6 Terasawa H, Tsang K Y,Identification and characterization of a human agonist cytotoxic T-lymphocyte epitope of human prostate-specific antigen[J].Clin Cancer Ras, 2002,8(1):41-53
7 Dhodapar MV, Steinm an RM, Krasovsky J, et al. Amtigen-specific inhibition of effector T cell function in human after injection of immature dendritic cells [J]. Exp Med, 2001,7(4):761-765
8 Wang K, Zhou Q, Guo AL, et al. An autologous therapeutic dendritic cell vaccine transfected with total lung carcinoma RNA stimulates cytotoxic Tlymphocyte responses against non-small cell lung cancer [J]. Immunol Invest, 2009,38(7):665-680
9 Frumento G, Piazza T, Di Carlo E, Targeting tumor-related immunosupp- ression for cancer immunotherapy[J]. Endocr Metab Immune Disord Drug Targets, 2006,6(3):233-237
10 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
11 Chattopadhyay S, Chakraborty NG, Mukherji B, et al. Regulatory T cells and tumor immunity [J]. Cancer Immunol Immunother, 2005,54(12): 1153-1161.
12 Curiel TJ. Regulatory T cells and treatment of cancer[J]. Curr Opin Immunol, 2008,20(5):241-246.
13 uriel TJ. Tregs and ret hinking cancer immunotherapy [J]. Clin Invest,2007,11(7):1167-1174.
14 Valzasina B, Piconese S, Guiducci C, et al. Tumor induced expansion of regulatory T cells by conversion of CD4+CD25- lymphocytes is thymus and proliferation independent [J]. Cancer Res, 2006,6(6):4488-4495
15 Dudley ME, Wunderlich JR, Yang JC, et al. Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma [J]. J Clin Oncol,2005,23(10):2346-2357
16 Xie SJ, Fu ZX, Li DB, Experimental study of individualized cancer immunotherapy based on dendritic cells against gastric cancer [J]. Chinese Journal of Surgery, 2006, 44(7):476-480
17 Kozako T, Fukada K, Hirata S, et al. Efficient induction of human T-cell leukemia virus-1-specific CTL by chimeric particle without adjuvant as a prophylactic for adult T-cell leukemia[J]. Mol Immunol,2009,47(2-3): 606-613
18 Nguyen S, Dhedin N, Vemant JP, et al. NK—cell reconstitution after haploydentical hem atopoietic stem cell transpiantantions inmaturity of NKcells and inhibitory effect of NKG2A override GvL effect[J]. Blood, 2005,105(9):4135-4142
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