肝癌患者外周血CD4~+CD25~+调节性T细胞的变化及其意义

目的 探讨应用流式细胞术检测肝癌患者外周血中CD4~+CD25~+调节性T细胞的变化及意义.方法 应用三色免疫荧光流式细胞仪测定37例肝癌患者及30例肝硬化患者外周血T细胞亚群CD4~+CD25~+/CD~+比值.采用酶联免疫吸附试验(ELISA)法检测外周血中转化生长因子β1(TGF-B1)的表达水平.结果 肝癌患者外周血CD4~+CD25~+/CD4~+比值较肝硬化患者显著增高,两者比较差异有统计学意义(P<0.05);肝癌患者外周血中CD4~+CD25~+T细胞水平与肝癌原发肿瘤的大小、TGF-βl呈正相关(P<0.05).结论 肝癌患者外周血中CD4~+CD25~+调节性T细胞增多,对肝癌患者具有免疫抑制作用.     

调节性CD4~+CD25~+T细胞的分离纯化及免疫功能研究

目的 制备调节性CD~+ CD25~+T细胞(Treg)分析其免疫功能,诱导局部免疫耐受防治同种异体复合组织移植(CTA)排斥反应.方法 采用免疫磁珠法(MACS)从雄性大鼠脾脏细胞分离CD4~+CD25~+Treg(1×10~6),2%锥虫蓝染色检测活性、流式细胞术分析其纯度,在5 mg/L抗CD3的刺激下观察其反应性、增殖及其与200 U/ml细胞介素(IL)-2的关系.结果 从8只雄性大鼠脾脏分选出的CD4~+CD25~+Treg活性平均为(97.90±0.36)%及纯度为(96.05±0.41)%,CD3刺激呈低反应,按比例培养抑制率为89%,IL-2可使CD4~+CD25~-抑制逆转.结论 MACS能快速分选出较高纯度的CD4~+CD25~+Treg,并且活性良好在体外具有免疫无能及免疫抑制作用,能满足动物CTA排斥反应研究的需要.     

不同分期前列腺癌患者外周血CD4~+ CD25~+ Foxp3~+调节性T细胞的变化及与胰岛素抵抗的相关性

目的 :观察不同分期前列腺癌患者外周血单个核细胞CD4+CD25+Foxp3+调节性T细胞的变化及与胰岛素抵抗的关系。方法:采用流式细胞术检测62例前列腺癌患者(患者组,临床TNM分期Ⅰ期5例、Ⅱ期16例、Ⅲ期21例、Ⅳ期20例)外周血单个核细胞(PBMC)中CD4+CD25+Foxp3+调节性T细胞数目,计算CD4+CD25+Foxp3+调节性T细胞占CD4+T淋巴细胞的百分率;并检测其空腹胰岛素及空腹血糖水平,计算胰岛素抵抗指数(HOMA-IR);采用ELISA法测定外周血胰岛素样生长因子1(IGF-1)水平,分析CD4+CD25+Foxp3+调节性T细胞与胰岛素抵抗的相关性,并与42例健康体检者进行对照。结果:与健康对照组相比,前列腺癌患者HOMAIR明显升高(6.68±1.66 vs 3.68±1.42),IGF-1水平明显下降[(96.39±21.21)ng/ml vs(164.56±30.58)ng/ml],PBMC CD4+CD25+Foxp3+Treg占CD4+T淋巴细胞的百分率[(13.88±0.96)%vs(5.33±0.65)%]及CD4+CD25+Foxp3+Treg绝对值[(3.55±0.29)×107vs(1.99±0.78)×107]明显升高(P0.05,P0.01)。患者PBMC CD4+CD25+Foxp3+Treg占CD4+T淋巴细胞的百分率及CD4+CD25+Foxp3+Treg绝对数﹑HOMA-IR均随TNM分期逐渐加重而增加,IGF-1逐渐下降;相关性分析表明:CD4+CD25+Foxp3+Treg/CD4+T及CD4+CD25+Foxp3+Treg绝对数均与HOMA-IR呈明显正相关(r分别为0.689、0.722,P0.01),与IGF-1呈明显负相关(r分别为-0.896、-0.747,P0.01)。结论:前列腺癌患者存在不同程度的胰岛素抵抗,且随着疾病程度的加重,外周血CD4+CD25+Foxp3+调节性T细胞数目和比例及胰岛素抵抗逐渐加重;CD4+CD25+Foxp3+调节性T细胞可能通过调节胰岛素抵抗参与其形成和发展。     

CD4+CD25+调节性T细胞与免疫耐受的研究进展

CD4 CD25 调节性T细胞的主要功能是抑制自身反应性T细胞,其在维持机体T细胞内环境稳定,调节和保持对自身抗原耐受之间的平衡以及移植免疫耐受方面具有重要作用。本文主要就CD4 CD25 调节性T细胞的功能、作用机制以及免疫抑制药物对调节性T细胞的影响等研究进展进行综述。     

CD4+CD25+调节性T细胞与免疫耐受的研究进展

CD4＋CD25＋调节性T细胞的主要功能是抑制自身反应性T细胞,对于维持机体T细胞内环境的稳定,调节和保持对自身抗原耐受之间的平衡以及移植免疫耐受具有重要作用。本文主要就CD4＋CD25＋调节性T细胞的功能和作用机制以及免疫抑制药物对其的影响等研究进展进行综述。     

CD4+CD25+调节性T细胞与移植免疫耐受

CD4^＋CD25^＋调节性T细胞是一类具有特殊免疫调节功能的T细胞亚群,它所介导的免疫抑制在移植免疫耐受的诱导和维持中起关键作用。本文对近年来CD4^＋CD25^＋调节性T细胞的作用机制及免疫耐受等方面的研究进行了综述。     

胃癌患者外周血调节性T细胞检测及初步分析

目的 检测胃癌患者外周血调节性T细胞(Treg)水平并对其进行初步分析.方法 四色流式细胞仪检测术前胃癌患者(n=25)及正常健康者(n=25)外周血CD4+ CD25+ FOXP3+Treg水平.结果 正常对照组CD4+ CD25+ FOXP3+ Treg/PBL为(1.229±0.656)%,胃癌组CD4+ CD25+ FOXP3+ Tres/PBL为(1.993±0.830)%.胃癌患者外周血Treg较正常对照组明显升高(P<0.01).结论 胃癌患者周血Treg水平升高,可因其免疫负调作用而导致免疫抑制.     

CD4~+CD25~+调节性T细胞与肿瘤免疫的研究进展

CD4+CD25+调节性T细胞(Treg)是一类具有免疫调节功能的T细胞亚群,可通过直接接触和细胞因子依赖等方式下调机体免疫反应,维持自身免疫耐受。近年来,Treg细胞已成为自身免疫、肿瘤免疫及移植免疫等领域的研究热点。本文就该类细胞的研究现状及其在肿瘤治疗方面的作用进行简要综述。     

CD4~+CD25~+调节性T细胞体内外培养扩增方法的研究进展

随着医学的进步及外科技术的提升,器官移植已成为治疗终末期疾病的有效手段。免疫抑制剂的出现对器官移植的发展具有划时代的意义,在移植术中及术后应用免疫抑制剂可避免、减轻或逆转排斥反应,从而大大提高了移植物的存活率。然而,应用免疫抑制剂的同时也带来了一系列问题,如感染几率增加、恶性肿瘤发生率升高、费用昂贵并且需要实时监测血药浓度等。诱导和维持移植物与受体之间产生免疫耐受,被认为是解决这一问题的有效办法,即在不使用免疫抑制剂的情况下使移植物获得与自身组织相同的地位而长期存活,同时又不损伤免疫系统对其他非己抗原的反应性。     

主动免疫治疗对反复胚胎植入失败患者CD4+CD25+调节性T细胞表达的影响

目的 探讨淋巴细胞主动免疫治疗对反复胚胎植入失败(RIF)患者外周血CD4+ CD25+调节性T淋巴细胞(CD4+CD25+Treg)表达的影响. 方法 选择在郑州大学第三附属医院生殖中心就诊的30例RIF患者(RIF组),采用荧光标记流式细胞分析技术,检测其淋巴主动免疫治疗前、后2周的外周血CD4+CD25+Treg表达,并选择同期正常未妊娠妇女20例作对照(对照组). 结果 (1)RIF组治疗前CD4+CD25+Treg占CD4+T细胞的比例显著低于对照组,差异有统计学意义(P＜0.05);(2)RIF组治疗后CD4+CD25+Treg的表达率明显高于治疗前,差异有统计学意义(P＜0.05). 结论 RIF的发生可能与CD4+CD25+Treg的表达下降有关;淋巴细胞主动免疫治疗可上调CD4+CD25+Treg的表达,调控母胎免疫耐受,有利于胚胎植入成功.     

Expression of CD39 by Human Peripheral Blood CD4+CD25+ T Cells Denotes a Regulatory Memory Phenotype

We have shown that CD39 and CD73 are coexpressed on the surface of murine CD4⁺Foxp3⁺ regulatory T cells (Treg) and generate extracellular adenosine, contributing to Treg immunosuppressive activity. We now describe that CD39, independently of CD73, is expressed by a subset of blood‐derived human CD4⁺CD25⁺CD127lo Treg, defined by robust expression of Foxp3. A further distinct population of CD4⁺CD39⁺ T lymphocytes can be identified, which do not express CD25 and FoxP3 and exhibit the memory effector cellular phenotype. Differential expression of CD25 and CD39 on circulating CD4⁺ T cells distinguishes between Treg and pathogenic cellular populations that secrete proinflammatory cytokines such as IFNγ and IL‐17. These latter cell populations are increased, with a concomitant decrease in the CD4⁺CD25⁺CD39⁺ Tregs, in the peripheral blood of patients with renal allograft rejection. We conclude that the ectonucleotidase CD39 is a useful and dynamic lymphocytes surface marker that can be used to identify different peripheral blood T cell‐populations to allow tracking of these in health and disease, as in renal allograft rejection.     

免疫磁珠两步法分离大鼠脾脏CD4+CD25+调节性T细胞

目的探索利用磁性细胞分离(MACS)系统高效、快速地分离大鼠脾脏CD4+CD25+调节性T细胞.方法采用免疫磁珠两步法分离大鼠脾脏内的CD4+CD25+调节性T细胞, 首先采用"鸡尾酒"抗体和抗IgG磁珠阴性分选CD4+ T细胞,再用抗CD25-PE抗体和抗PE磁珠阳性分选获得CD4+CD25+T 细胞.分离后的细胞经流式细胞仪检测分离纯度; 台盼蓝染色检测细胞存活率; 体外增殖实验检测其对CD4+CD25-T细胞的免疫抑制作用.结果阴性分选获得的CD4+T细胞纯度为(83.6±2.5)%(79%～87%), 阳性分选后获得的CD4+CD25+T细胞纯度为(90.2±1.8)%(86%～93%), 细胞存活率为(92.8±3.4)%(92%～95%), 体外增殖实验表明,CD4+CD25+T细胞能明显抑制CD4+CD25-T 细胞的增殖(P＜0.01). 结论采用MACS系统阴性加阳性分选可以高效、快速地获得理想纯度和有免疫抑制功能的大鼠CD4+CD25+调节性T细胞.     

Relative Resistance of Human CD4+ Memory T Cells to Suppression by CD4+CD25+ Regulatory T Cells

Successful expansion of functional CD4⁺CD25⁺ regulatory T cells (T_reg) ex vivo under good manufacturing practice conditions has made T_reg‐cell therapy in clinical transplant tolerance induction a feasible possibility. In animals, T_reg cells home to both transplanted tissues and local lymph nodes and are optimally suppressive if active at both sites. Therefore, they have the opportunity to suppress both naïve and memory CD4⁺CD25^? T cells (Tresp). Clinical transplantation commonly involves depleting therapy at induction (e.g. anti‐CD25), which favors homeostatic expansion of memory T cells. Animal models suggest that T_reg cells are less suppressive on memory, compared with naïve Tresp that mediate allograft rejection. As a result, in the context of human T_reg‐cell therapy, it is important to define the effectiveness of T_reg cells in regulating naïve and memory Tresp. Therefore, we compared suppression of peripheral blood naïve and memory Tresp by fresh and ex vivo expanded T_reg cells using proliferation, cytokine production and activation marker expression (CD154) as readouts. With all readouts, naïve human Tresp were more suppressible by approximately 30% than their memory counterparts. This suggests that T_reg cells may be more efficacious if administered before or at the time of transplantation and that depleting therapy should be avoided in clinical trials of T_reg cells.     

CD4+ CD25bright+ Regulatory T Cells Can Mediate Donor Nonreactivity in Long-Term Immunosuppressed Kidney Allograft Patients

CD4+ CD25bright+ FoxP3+ T cells are potent regulators of T-cell reactivity, but their possible involvement in donor-specific nonresponsiveness after clinical kidney transplantation remains to be elucidated. We assessed the proliferative donor-reactivity in 33 kidney allograft recipients who were maintained on a combination of proliferation inhibitors (mycophenolate mofetil (MMF) or Azathioprine (Aza)) and prednisone, long (> 5 years) after transplantation. Of the 33 patients, 8 still exhibited donor-reactivity, whereas 25 were classified as donor nonreactive patients. Within these 25 donor nonreactive patients, we assessed the involvement of CD4+ CD25bright+ regulatory T cells both by depleting them from the responder population as well as by reconstituting them to the CD25(-/dim) effector population. The absence of proliferation in these 25 patients, was abolished in 7 (28%) recipients upon depletion of the CD4+ CD25bright+ T cells. Reconstitution of these cells suppressed the donor-reactivity in a dose-dependent manner. Adding-back CD4+ CD25bright+ T cells inhibited the anti-third party response in all recipients, indicating that functional CD4+ CD25bright+ T cells circulate despite more then 5 years of immunosuppressive treatment. Altogether, we conclude that in long-term immunosuppressed kidney allograft patients functional regulatory CD4+ CD25bright+ T cells circulate but that these cells mediate donor non reactivity only in a subset of patients.     

Allergic Conjunctivitis Renders CD4+ T Cells Resistant to T Regulatory Cells and Exacerbates Corneal Allograft Rejection

Allergic diseases rob corneal allografts of immune privilege and increase immune rejection. Corneal allograft rejection in BALB/c allergic hosts was analyzed using a short ragweed (SWR) pollen model of allergic conjunctivitis. Allergic conjunctivitis did not induce exaggerated T‐cell responses to donor C57BL/6 (B6) alloantigens or stimulate cytotoxic T lymphocyte (CTL) responses. Allergic conjunctivitis did affect T regulatory cells (Tregs) that support graft survival. Exogenous IL‐4, but not IL‐5 or IL‐13, prevented Treg suppression of CD4⁺ effector T cells isolated from naïve mice. However, mice with allergic conjunctivitis developed Tregs that suppressed CD4⁺ effector T‐cell proliferation. In addition, IL‐4 did not inhibit Treg suppression of IL‐4Rα^?/? CD4⁺ T‐cell responses, suggesting that IL‐4 rendered effector T cells resistant to Tregs. SRW‐sensitized IL‐4Rα^?/? mice displayed the same 50% graft survival as nonallergic WT mice, that was significantly less than the 100% rejection that occurred in allergic WT hosts, supporting the role of IL‐4 in the abrogation of immune privilege. Moreover, exacerbation of corneal allograft rejection in allergic mice was reversed by administering anti‐IL‐4 antibody. Thus, allergy‐induced exacerbation of corneal graft rejection is due to the production of IL‐4, which renders effector T cells resistant to Treg suppression of alloimmune responses.     

大鼠CD4^＋CD25^＋调节性T细胞的分离及功能鉴定

目的：研究利用免疫磁珠分选法稳定分离正常大鼠脾脏CD4^＋CD25^＋调节性T细胞的方法。方法：采用免疫磁珠两步法分离大鼠脾组织CD4^＋CD25^＋T细胞。首先采用藻红蛋白（PE）标记的抗CD25抗体和抗PE多功能磁珠试剂盒阳性分选CD25^＋T细胞,再用抗异硫氰酸荧光素（FITC）标记抗体和抗IgG磁珠阳性分选获得CD4^＋CD25^＋T细胞。分离后的细胞经流式细胞仪检测分离纯度,台盼蓝染色检测细胞存活率,体外增殖实验检测其对CD4^＋CD25^-T细胞的免疫抑制作用。结果：两次阳性分选后获得的CD4^＋CD25^＋T细胞纯度为（90.4±1.6）%,细胞存活率为（92.6±2.4）%。体外增殖实验表明,CD4^＋CD25^＋T细胞能明显抑制CD4^＋CD25^-T细胞的增殖（P〈0.01）。结论：采用免疫磁珠法两次阳性分选,可稳定地获得纯度理想并有免疫抑制功能的大鼠CD4^＋CD25^＋T细胞。     

Human FOXP3+ Regulatory T Cells in Transplantation

CD4+CD25+FOXP3+ suppressive regulatory T cells (Treg) represent a subset of immune regulatory cells. Based on experimental results, Treg have recently been considered as a potential treatment option in several diseases. Compared with murine Treg, human CD4+CD25+FOXP3+ cells are less well characterized and understood, so a thorough understanding of their biology is vital before clinical applications can be initiated. This review summarizes knowledge on generation, phenotypic characteristics and function of human Treg. The possible role of these cells in organ transplantation, as well as interactions between immunosuppression and Treg are also discussed.