肿瘤患者CD4^＋CD25^＋调节性T细胞流式细胞术分析

目的：分析比较肿瘤患者和健康人外周血CD4^＋CD25^＋调节性T细胞的特点,为肿瘤免疫学研究及治疗探索新方法。方法：收集并分离30例肿瘤患者和32例健康人的外周血单个核细胞（PBMCs）,用荧光标记的抗人CD4及抗人CD25单抗标记肿瘤患者和健康人PBMCs细胞,FCM检测CD4^＋CD25^＋Treg细胞,分析CD4^＋CD25^＋Treg细胞在肿瘤患者和健康人中的差别。结果：肿瘤患者的CD4^＋CD25^＋Treg细胞百分数明显高于健康人（年龄〈55者62．4vs40．4;年龄≥55者53．1vs31．0,P〈0．05）。结论：肿瘤患者的CD4^＋CD25^＋Treg细胞高于健康对照,为肿瘤免疫治疗提供新策略,通过删除CD4^＋CD25^＋Treg细胞,有可能增强抗瘤免疫反应。     

CD4^＋CD25^＋调节T细胞与肿瘤免疫治疗策略

随着分子生物技术的不断发展，肿瘤的生物治疗在临床应用中的地位日渐突出，其中CD4^＋CD25^＋Foxp^3＋调节T细胞（CD4^＋CD25^＋Foxp^3＋regulatory T cell，Treg）在抑制肿瘤免疫方面起着重要作用，文章就其近年来在肿瘤免疫领域的研究进展作一综述。     

CD4^＋CD25^＋调节T细胞与肿瘤免疫治疗策略

随着分子生物技术的不断发展，肿瘤的生物治疗在临床应用中的地位日渐突出，其中CD4^＋CD25^＋Foxp^3＋调节T细胞（CD4^＋CD25^＋Foxp^3＋regulatory T cell，Treg）在抑制肿瘤免疫方面起着重要作用，文章就其近年来在肿瘤免疫领域的研究进展作一综述。     

原发性肝细胞癌微环境中CD4^＋CD25^＋调节性T细胞分布与局部免疫的关系

目的通过检测原发性肝细胞癌（hepatocellular carcinoma,HCC）局部肝癌组织和癌旁组织中T细胞亚群的分布情况,探讨在肝癌发生、发展过程中CD4^＋CD25^＋调节性T细胞与局部免疫状态的关系。方法54例肝癌组织和癌旁组织及10例正常肝组织用免疫组织化学S—P法标记CD4^＋T细胞、CD8^＋T细胞及CD4^＋CD25^＋调节性T细胞（CD4^＋CD25^＋Tr细胞）,并对组织中CD4^＋CD25^＋Tr细胞与CD4^＋T、CD8^＋T及CD4^＋T／CD8^＋T值进行相关性分析。结果54例肝癌、癌旁组织中CD4^＋CD25^＋Tr细胞单个高倍视野平均数分别为5．6208±2．7235和3．8554±1．6018（P=0．001）;肝癌中CD4^＋CD25^＋Tr细胞数目与肿瘤大小、有无子灶有关,组间差异有显著性（P〈0．01）;肝癌中CD4^＋CD25^＋Tr数量与CD4^＋T细胞的数量以及CD4^＋T／CD8^＋T值呈显著负相关（r=-0．459,P=0．015;r=-0．563,P=0．011）,而与浸润性CD8^＋T细胞的数量分布无关（r=-0．485,P=0．072）。结论在肝癌微环境中CD4^＋T淋巴细胞表达降低,CD4^＋CD25^＋Tr细胞表达增高,后者可能通过抑制CD4^＋T淋巴细胞的增殖来抑制肝癌局部免疫,从而促进肿瘤的进展以及侵袭或转移。     

CD4^＋CD25^＋调节性T细胞在肿瘤免疫及化疗方面的研究进展

Treg（CD4^＋CD25^＋调节性T细胞）是具有独特免疫调节功能的T细胞亚群，抑制免疫反应，在机体免疫稳态维持、肿瘤免疫以及移植耐受等方面发挥着重要的作用影响其抑制作用的表面分子标记有Foxp3、CD127、GITR等越来越多的研究表明在实体肿瘤和血液恶性肿捕患者中Treg数目增多。在乳腺癌、卵巢癌、肺癌以及肝癌等多种实体肿瘤患者外周血和肿痛局部微环境中，Treg比例增高，且数目与患者肿瘸进展程度和预后、生存率呈负相关血液恶性肿瘤患者中Treg数目呈肿瘤发展阶段相关性增长。但是，Treg增高的机制尚不清楚。去除Treg或封闭其抑制功能，可以增强抗肿瘤免疫反应。化疗作为一种重要的治疗肿瘤的方法，对肿瘤患者外周血中的Treg数目可产生一定影响。化疗药物可能通过抑制肿瘤微环境中血管生成，促进Treg凋亡，从而起到有效控制肿瘤的作用，据报道环磷酰胺、多西紫杉醇和氟达拉滨等化疗药物均可使肿瘤明显退化，患者生存期延长但也有报道证明肿瘤患者接受化疗后，外周血中Treg数目增多。化疗究竞时Treg会产生怎样的影响，目前尚无定论，有待于进一步研究因此，Treg在肿瘤治疗方面的应用成为研究的热点，如何清除或逆转Treg的抑制作用是肿瘤免疫治疗的一个关键问题。     

结直肠癌患者外周血CD4＋ CD25high调节性T细胞的作用

目的探讨结直肠癌患者外周血CD4＋CD25high调节性T细胞（Treg）的作用。方法采用流式细胞仪测定36例结直肠癌患者及30例健康对照者外周血中CD4＋CD25highFoxP3 Treg、CD4＋CD25highTreg、CD4＋CTLA-4 Treg及CD4＋T细胞水平。结果与健康对照组比较,结直肠癌组患者外周血CD4＋CD25highFoxP3 Treg显著增多（P〈0.01）,CD4＋CD25high Treg和CD4＋CTLA-4 T细胞也增多（P〈0.05）。结论CD4＋CD25highFoxP3 Treg可能在结直肠癌发展过程中起着重要作用。     

食管癌患者CD4^＋ CD25^＋调节性T细胞的检测及意义

目的：探讨CD4^＋ CD25^＋调节性T细胞在食管癌局部及全身免疫中的作用。方法：流式细胞仪检测97例食管癌患者外周血和20例肿瘤组织的CD4^＋ CD25^＋调节性T细胞比例,比较不同病理类型、不同分期等食管癌患者外周血及肿瘤局部组织的CD4^＋ CD25^＋调节性T细胞的分布变化。结果：食管癌患者肿瘤组织CD4^＋ CD25^＋调节性T细胞比例为（18.97±2.38）%,高于患者外周血比例〔（17.57±3.99）%〕,差异无统计学意义,t=1.511,P〉0.05;食管癌患者肿瘤组织及外周血中CD4^＋ CD25^＋调节性T细胞占CD4＋ T淋巴细胞的比例,均高于同期健康对照组患者外周血的比例（9.35±1.41）%,差异有统计学意义,t值分别为12.111和8.332,P值均〈0.01。CD4^＋ CD25^＋调节性T细胞水平与临床分期（F=9.384）、有无淋巴结转移（t=2.326）有关,P值均〈0.05。结论：食管癌患者全身及肿瘤局部均存在免疫异常,推测CD4^＋ CD25^＋调节性T细胞可能参与了食管癌的发生与发展。     

CD4+CD25+调节性T细胞在肿瘤免疫领域的研究进展

CD4^ CD25^ 调节性T细胞(Regulatory T cell，Treg)在维持自身免疫耐受中起重要作用。本文就其在肿瘤免疫领域的研究进展进行综述。     

激发性CD28单抗对肿瘤患者外周血FOXP3^＋Treg细胞的下调作用及意义

目的探究肿瘤患者外周血单个核细胞（PBMC）中FOXP3^＋Treg经CD28单抗激发后的改变及意义。方法采用腹水诱生法及免疫亲和层析法进行单抗的制备和纯化,Ficoll密度梯度离心法获取外周血PBMC,经激发性CD28单抗10μg／mL共培养后经免疫荧光及流式细胞术分析其中FOXP3^＋Treg的表达以及FOXP3^＋Fas^＋Treg和FOXP3^＋FasL^＋Treg的表达。结果免疫荧光和流式细胞术显示肿瘤患者外周血中FOXP3^＋Treg表达比例为（6．208±2．754）％,而对照组为（2．653±0．638）％。FOXP3^＋FasL^＋Treg细胞经CD28单抗活化后FasL表达上调为（7．252±2．23）％,而健康对照组为（7．96±2．938）％,FOXP3^＋Fas^＋Treg活化前后的比例分别为（1．467±0．590）％和（3．692±1．822）％。结论肿瘤患者外周血中PBMC经CD28单抗激发后FOXP3^＋Treg的比例下调（P〈0．01）,提示该抗体能够使肿瘤患者的免疫功能得到一定程度的恢复与提高。     

CD4^＋T细胞与肿瘤免疫

CD4^＋T细胞不仅辅助激活CD8^＋T细胞,而且对记忆性细胞毒性T淋巴细胞（CTL）应答的产生和维持起重要作用,并具有直接的抗肿瘤功能。另外CD4^＋CD25^＋调节性T细胞（Tregs）具有免疫负调控功能。在肿瘤免疫抑制及免疫逃逸中发挥重要作用,是肿瘤免疫治疗失败的重要原因。近年肿瘤免疫治疗已获得很大进步,相关肿瘤疫苗的研究也备受关注.     

CD4~+ CD25~+调节性T细胞及其在肿瘤免疫治疗中的意义

CD4+CD25+调节性T细胞是具有独特免疫调节功能的T细胞亚群。近年来研究发现各种恶性肿瘤患者外周血及肿瘤环境中该细胞比例增加,去除CD4+CD25+调节性T细胞或封闭其抑制功能可以增强抗肿瘤免疫反应。CD4+CD25+调节性T细胞成为肿瘤免疫治疗的新靶点。     

CD4+CD25+调节性T细胞与大肠癌的关系

近来CD4 CD25 调节性T细胞对肿瘤免疫调节的抑制作用正受到越来越多的重视,许多恶性肿瘤患者外周血都存在CD4 CD25 调节性T细胞比例上调,机体抗肿瘤免疫力下降.大肠癌的发病率正逐年上升,其发病分子机制已为熟知,免疫机制研究不多,国内尚未见大肠癌与该类细胞关系的报道,现对CD4 CD25 调节性T细胞在大肠癌的发生、发展、治疗中的研究综述如下.     

CD4~+CD25~+调节性T细胞与抗肿瘤免疫

CD4~+CD25~+调节性T(CD4~+CD25~+Tr)细胞是一类维持机体自身耐受的T细胞亚群,分布广泛,但不同组织表型有所不同。它们可由胸腺自然产生,也可在外周血中诱导产生,其活化要依赖于特异性抗原的存在。CD4~+CD25~+Tr细胞发挥抑制效应是通过细胞接触依赖或分泌细胞因子这两种方式。去除CD4~+CD25~+Tr细胞或抑制其功能,重新募集效应性T细胞能够增强机体抗肿瘤作用,这将成为一种可行的肿瘤免疫治疗方法。     

Low molecular weight fraction secreted by SKOV3 cells expands peripheral CD4+CD25+ regulatory T cells and enhances their suppressive capacity

The increase of CD4+CD25+ regulatory T cells in patients with ovarian carcinoma has been verified. Here we investigated the effects of supernatant derived from ovarian carcinoma cell SKOV3 on peripheral regulatory T cells. Supernatant from SKOV3 was collected and fractionated into three different molecular weight fractions (MWFs). The proliferation of the CD4+CD25+ regulatory T cells cultured in complete RPMI 1640 medium with the different stimulators was detected. The phenotype (GITR and CTLA-4) of natural and expanded CD4+CD25+ T cells was detected by flow cytometry. Foxp3 mRNA expression of low MWF-expanded CD4+CD25+ T cells was detected by RT-PCR. Those expanded CD4+CD25+ regulatory T cells showed enhanced capacity to suppress CD4+CD25− T proliferation and increased expression of GITR and CTLA-4. In brief, low molecular weight fraction of supernatant secreted by SKOV3 could expand peripheral CD4+CD25+ regulatory T cells and enhance their suppressive function.     

胃癌患者调节性T细胞胞内外细胞因子的检测及其意义

背景与目的:目前认为CD4 CD25 调节性T细胞与胃癌患者的免疫功能抑制密切相关,但CD4 CD25 调节性T细胞发挥免疫抑制功能的作用机制并不十分清楚.本研究通过检测胃癌患者CD4 CD25 调节性T细胞产生具有不同生物活性的细胞因子干扰素-γ(interferon-γ,IFN-γ)、白介素4(interleukin-4,IL-4)、IL-10及肿瘤生长因子-β(tumor growth factor-β,TGF-β)的分泌情况,进一步探讨这些细胞因子在胃癌患者CD4 CD25 调节性T细胞发挥免疫抑制功能的作用.方法:按常规方法制备患者外周血单个核淋巴细胞,采用免疫磁珠分选方法分离CD4 CD25 T细胞及CD4 CD25-T细胞后,用细胞内细胞因子染色法及ELISA法分别研究CD4 CD25 T细胞在胞内及胞外产生具有不同生物活性的细胞因子IFN-γ、IL-4、IL-1O及TGF-β的水平.结果:(1)与健康对照组比较,胃癌患者分泌内细胞因子IFN-γ、IL-4及IL-10的CD4 CD25 T细胞占CD4 细胞的百分比均显著增高(P＜0.05).(2)培养96 h后,上清液的各种细胞因子水平,无论是胃癌患者还是健康对照组,CD4 CD25 T细胞分泌的IL-10及TGF-β均显著高于CD4 CD25-T细胞(P＜0.05).CD4 CD25 T细胞分泌的IFN-γ显著低于CD4 CD25-T细胞(P＜0.05).结论:CD4 CD25 调节性T细胞体外免疫抑制作用的发挥可能与一些抑制性细胞因子有关,特别是细胞因子TGF-β在胃癌CD4 CD25 调节性T细胞的免疫抑制功能中起着相当重要的作用.     

CD4+CD25+ regulatory T cells in patients with gastrointestinal malignancies: possible involvement of regulatory T cells in disease progression

BACKGROUND: Active suppression by CD4+CD25+ regulatory T cells plays an important role in the down-regulation of the response of T cells to foreign and self antigens. Experimental tumor models in mice revealed that regulatory T cells inhibit antitumor immune responses. The purpose of the current study was to demonstrate the possible involvement of CD4+CD25+ regulatory T cells in immune system impairment in patients with gastrointestinal malignancies. METHODS: The phenotypes of lymphocytes, particularly those of CD4+CD25+ T cells, were analyzed in peripheral blood in 149 patients with gastrointestinal malignancies and in ascites in 7 patients with peritoneal dissemination. In addition, cytokine production after in vitro stimulation was examined in CD4+CD25+ and CD4+CD25- T cells isolated from patients with malignant disease. RESULTS: Compared with healthy volunteers, patients with gastrointestinal malignancies had a higher proportion of CD4+CD25+ T cells in peripheral blood, due to the presence of a drastically smaller number of CD4+CD25- T cells. Among patients with gastric carcinoma, those with higher percentages of CD4+CD25+ T cells had a poorer prognosis than did those with lower percentages. CD4+CD25+ T cells also were present in greater proportions in ascites from patients who had advanced-stage disease with peritoneal dissemination. Isolated CD4+CD25+ T cells from patients with malignant disease produced interleukin (IL)-4 and IL-10 but not IL-2 or interferon-gamma; these cells also inhibited cytokine production by CD4+CD25- T cells after in vitro stimulation. CONCLUSIONS: The relative increase in CD4+CD25+ regulatory T cells may be related to immunosuppression and tumor progression in patients with gastrointestinal malignancies. This finding suggests that the use of immunomodulatory therapy to treat patients with gastrointestinal malignancies may be an effective strategy.     

CD+4CD+25Foxp+3调节性T细胞在T细胞非霍奇金淋巴瘤中的研究进展

 CD+4 CD+25 调节性T细胞（CD+4 CD+25 Treg）是一个具有独特免疫调节功能的T细胞亚群, 天然的CD+4 CD+25 Treg起源于胸腺,获得性CD+4 CD+25 Treg可在外周由CD+4 CD-25 T细胞诱导产生,其分子表面表达特异性的转录抑制因子Foxp3,又可表达CD4、CD25、CTLA-4 （CD152） 、GITR等膜分子,主要功能具有免疫抑制性和免疫无能性。近年来研究发现,其在非霍奇金淋巴瘤（NHL）中存在表达异常,某些NHL外周血中或瘤内均存在CD+4 CD+25 Treg表达升高,且有研究表明其表达量随肿瘤增长和分期而增加。增加的CD+4 CD+25 Treg可加速肿瘤生长及再发,且可抑制自身效应性T细胞（CD+4 T／CD+8 T）功能,在肿瘤免疫逃逸机制中发挥一定作用。文章就CD+4 CD+25 Foxp3+调节性T细胞在T细胞非霍奇金淋巴瘤（T-NHL）（主要为皮肤T细胞淋巴瘤及成年人T细胞淋巴瘤）中的研究进展进行综述。     

Human CD4+ CD25+ regulatory T cells suppress NKT cell functions

CD4+CD25+ regulatory T cells play an important role in peripheral tolerance. These cells have been reported to be capable of suppressing the response of CD4+CD25- T cells in vitro. The depletion of these cells evokes effective immune responses to tumor cells in vivo. In this study, we demonstrate that CD4+CD25+ T cells also suppress all subsets of Valpha24+NKT cells (Valpha24+CD4-CD8- double negative, Valpha24+CD4+, and Valpha24+CD8+) in both proliferation and cytokine production [IFN-gamma, interleukin-4 (IL-4), IL-13, and IL-10]. This suppression is mediated by cell-to-cell contact but not by a humoral factor or the inhibition of antigen-presenting cells. Moreover, the cytotoxic activity of Valpha24+NKT cells against some tumor cell lines is suppressed by CD4+CD25+ T cells. This finding is important in developing an effective immunotherapy for cancer.     

Correlation between CD4+CD25+Treg Cells and CCR4 in Nasopharyngeal Carcinoma

OBJECTIVE CD4+CD25+ T regulatory (Treg) cells are a population of T cells which suppress an overactive immune system. CCR4 is a chemokine receptor involved in the recruitment of lymphocytes. Nasopharyngeal carcinoma (NPC) is resistant to immunosurveillance, owing to the increased number of tumor-infiltrating Treg cells which are recruited to the tumor by CCR4.METHODS The percentage of CD4+CD25+Treg cells and CCR4+ cells in tissue or peripheral blood (PB) lymphocytes of patients with untreated NPC or normal subjects was analysed by flow cytometry. RESULTS In both tissue and PB lymphocytes, the percentage of CD4+CD25+ Treg cells and CCR4+ cells was significantly elevated in patients with NPC in comparison with that in the normal tissue of controls. Furthermore, in the patients with NPC, a higher percentage of CD4+CD25+ Treg cells was found in the tumor-infiltrating (T1) lymphocyte population than in the PB population. In the NPC patient group, a general trend towards an increased percentage of Tl Treg cells was found in the patients with advanced stage NPC. The number of CD4+CD25+ Treg cells was positively related to the number of CCR4+ cells in the tumor and in the PB of the patients with NPC, while the number of CD4+CD25+ Treg cells was negatively related to the number of CD4+CD25- T cells.CONCLUSION Immunosuppression was observed in NPC, especially at the tumor sites. CD4+CD25+ Treg cells may suppress CD4+CD25- T cells. CCR4 may have an important role in the recruitment of CD4+CD25+ Treg cells to tumor sites, thus causing resistance to immunosurveillance.     

非小细胞肺癌患者引流区淋巴结中CD4+CD25+调节性T细胞的检测及临床意义

目的分析非小细胞肺癌(NSCLC)患者引流区淋巴结中CD4 CD25 调节性T细胞在淋巴结局部免疫抑制状态的形成以及在肺癌发生发展中的作用。方法手术切除53例NSCLC患者引流区淋巴结,采用双标记的间接免疫荧光法检测CD4 CD25 调节性T细胞数量,实时荧光定量逆转录-聚合酶链反应(RT-PCR)法检测细胞因子TGF-β1、IL-10的表达水平,常规免疫组化方法检测CD8 T细胞的数量。结果NSCLC患者引流区转移淋巴结中,CD4 CD25 调节性T细胞(28.80%±8.06%)明显高于未转移淋巴结(15.48%±4.66%,P<0.01)。随肺癌的进展,引流区淋巴结中CD4 CD25 调节性T细胞数量增多。在转移的纵隔淋巴结(N2)和肺内淋巴结(N1)中,CD4 CD25 调节性T细胞数量分别为32.58%±7.52%和22.76%±4.67%(P<0.01)。在进展期(Ⅲ)和早期(Ⅰ Ⅱ)NSCLC患者引流区淋巴结中,CD4 CD25 调节性T细胞数量分别为30.42%±7.47%和16.22%±4.88%(P< 0.01)。NSCLC患者引流区淋巴结中的CD4 CD25 调节性T细胞数量与其自身的CD8 T细胞的数量呈负相关(r=-0.756,P<0.001)。在NSCLC患者引流区淋巴结中,CD4 CD25 调节性T细胞数量与抑制性细胞因子TGF-β1和IL-10的表达水平呈正相关(TGF-β1:r=0.645,P<0.001;IL-10:r=0.769,P<0.001)。结论NSGLC患者引流区淋巴结的CD4 CD25 调节性T细胞数量与肺癌的发展密切相关。一方面,检测肺癌患者引流区淋巴结的免疫状况为评价NSCLC患者疾病的进展程度和预后提供了一个新的免疫学指标;另一方面,在NSCLC的生物治疗中,控制CD4 CD25 调节性T细胞数量,阻断其发挥免疫抑制作用,具有广阔的临床应用前景。