Studies on naïve CD4+CD25+T cells inhibition of naïve CD4+CD25- T cells in mixed lymphocyte cultures

BackgroundNaïve CD4⁺CD25⁺T cells suppress immune responses in a non-antigen specific manner. The effects of naïve CD4⁺CD25⁺T cells in suppressing alloimmune responses as assayed in the mixed lymphocyte culture (MLC) is poorly understood.MethodThe alloreactivity of naïve CD4⁺CD25⁺, CD4⁺CD25⁻ and unfractionated CD4⁺T cells from DA rats was compared in MLC with MHC incompatible stimulator cells. The response of Lewis and PVG cells to semi-allogeneic (Lewis × PVG)F₁ cells and fully allogeneic stimulators were compared. Potential mechanisms of suppression were examined by blocking T cell cytokines, produced by activated CD4⁺CD25⁺T cells.ResultsProliferation of CD4⁺CD25⁻T cells was significantly greater than unfractionated CD4⁺T cells to both allogeneic and syngeneic stimulator cells. CD4⁺CD25⁺T cells had no response to syngeneic stimulators and very low proliferative responses to alloantigen due to the Foxp3⁻ cells. Admixing CD4⁺CD25⁺T cells with CD4⁺CD25⁻T cells at a ratio of 1:10 reduced the proliferation to that of unfractionated CD4⁺T cells. At a ratio of 1:1 proliferation was nearly totally suppressed, IL-2, IL-4 and IL-5 mRNA induction was reduced but IFN-γ, IL-10, TGF-β and inducible nitric oxide (iNOS) mRNA induction was spared. The inhibition by CD4⁺CD25⁺T cells was not due to their consumption of IL-2 nor to anti-CD25mAb that had been used to enrich the cells being releases and blocking the IL-2 receptor on CD4⁺CD25⁻T cells that had been activated by alloantigen and induced to express CD25. Blocking IFN-γ, IL-10, TGF-β, IL-5 or iNOS did not prevent CD4⁺CD25⁺T cell's inhibition of CD4⁺CD25⁻T cell proliferation. Blocking IFN-γ or iNOS enhanced CD4⁺CD25⁻T cell proliferation only in the absence of CD4⁺CD25⁺T cells. Depletion of CD4⁺CD25⁺T cells enhanced responses to syngeneic stimulator cells, but this anti-self suppression did not regulate the response to alloantigen on semi-allogeneic stimulators.ConclusionsTwo independent mechanisms that control proliferation of CD4⁺CD25⁻T cells in MLC were identified that naive CD4⁺CD25⁺T cells mediated by cell to cell contact and not release of cytokines produced in the cultures, and that CD4⁺CD25⁻T cells producing IFN-γ to induce iNOS.     

Analysis of circulating regulatory T cells (CD4+CD25+CD127?) after cryosurgery in prostate cancer

This study was performed to assess the response of regulatory T cells (Tregs) following cryosurgery in prostate cancer (PCa) patients by measuring their frequency and immune function. Blood was collected prior to and at 4 and 8 weeks after treatment in 30 patients with high-risk PCa who underwent cryosurgery and from 15 healthy volunteers. Circulating CD4⁺CD25⁺CD127⁻ Tregs were isolated. Their frequency was detected by flow cytometry, and immune suppressive function was evaluated by measuring the proliferation of CD4⁺CD25⁻ T cells cocultured with Tregs. The results showed that the percentage of circulating CD4⁺CD25⁺CD127⁻ Tregs was increased in PCa patients compared to healthy volunteers (7.6%±0.73% vs. 5.8%±0.54%, P<0.001). The frequency of circulating CD4⁺CD25⁺CD127⁻ Tregs was reduced 4 weeks after cryosurgery compared to before surgery (6.3%±0.58% vs. 7.6%±0.73%, P<0.001), and the decrease persisted for 8 weeks. However, the suppressive function of Tregs was increased in eight of 12 patients, which might contribute to cancer recurrence. Then the response of circulating Tregs is complicated after cryosurgery for PCa, and further studies are warranted.     

CD+4CD+25调节性T淋巴细胞的研究进展

CD+4CD+25 Treg细胞(调节性T淋巴细胞)是一类特殊的T淋巴细胞群体,其能够识别自身抗原并抑制自身反应性细胞的免疫反应,是自身免疫耐受不可缺失的一环,同时也可在炎症情况下抑制效应性细胞过度活化,防止免疫反应过强而对机体造成损伤.在CD+4CD+25 Treg 细胞缺失或功能障碍情况下,将导致机体发生自身性疾病,而在细胞过度活化情况下,则机体发生肿瘤的概率大为增加.故研究CD+4CD+25 Treg细胞对于临床疾病的治疗具有重大意义.我们就以下几个方面进行综述.(1)CD+4CD+25 Treg细胞表面特异性分子标志;(2)CD+4CD+25 Treg细胞理想的分离扩增方法;(3)分析其维持免疫耐受可能的分子细胞机制;(4)CD+4CD+25 Treg细胞在自身免疫疾病治疗、肿瘤免疫、以及移植排斥的防治等临床疾方面的作用.     

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。     

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细胞与移植物抗宿主病

造血干细胞移植是治疗造血系统疾病的有效方法,但是发生率及死亡率都很高的移植物抗宿主病(GVHD)限制了这一治疗方法的广泛应用.目前免疫抑制药物虽然是最有效的治疗GVHD的办法,但是患者长期服用容易导致严重感染和肿瘤.因此,人们在寻找能够诱导抗原特异性的移植耐受的方法,以减少或减轻GVHD发生.目前发现体内存在一类CD4+CD25+免疫调节性T细胞(regulatory T cells,Treg细胞)在减少或减轻GVHD发生方面显示了一定的应用前景,本文就CD4+CD25+Treg细胞在GVHD防治中的应用前景进行了综述.     

CD4+CD25+调节性T细胞与移植物抗宿主病

造血干细胞移植是治疗造血系统疾病的有效方法,但是发生率及死亡率都很高的移植物抗宿主病(GVHD)限制了这一治疗方法的广泛应用。目前免疫抑制药物虽然是最有效的治疗GVHD的办法,但是患者长期服用容易导致严重感染和肿瘤。因此,人们在寻找能够诱导抗原特异性的移植耐受的方法,以减少或减轻GVHD发生。目前发现体内存在一类CD4 CD25 免疫调节性T细胞(regulatoryTcells,Treg细胞)在减少或减轻GVHD发生方面显示了一定的应用前景,本文就CD4 CD25 Treg细胞在GVHD防治中的应用前景进行了综述。     

CD4+CD25+调控T细胞对小鼠胃癌的影响

目的在体研究CD4^＋ CD25^＋调控T（Treg）细胞对小鼠胃癌的影响。方法检测荷瘤小鼠不同时相点Treg细胞比例的变化。并应用免疫磁珠法（MACS法）分选、纯化小鼠Treg细胞：以不同剂量的Treg细胞及Treg细胞拈抗剂Anti-GITR注入荷瘤小鼠，3周后观察小鼠移植瘤的体积及胃癌细胞凋亡率的变化。结果正常小鼠脾脏Treg细胞为（3．86±0．07）％，在小鼠胃癌模型中Treg细胞比例逐渐升高，第3周时为（4．12±0．13）％，明显高于对照组（P=0．015）。在体应用Treg细胞达到2．0×10^5时，小鼠移植瘤的体积明显增大（P=0．013），胃癌细胞凋亡率明显降低（P=0．012）。当应用Treg细胞拮抗剂Anti-GITR剂量为100μg时，种植瘤的体积明显小于对照组（P=0．008），其凋亡比例明显高于对照组（P=0．021）。结论在胃癌小鼠模型中，随着胃癌的进展，Treg细胞的比例有增高的趋势。应用Treg细胞能促进小鼠胃癌生长。降低胃癌细胞的凋亡；相反、应用Treg细胞拈抗剂Anti-GITR可以提高小鼠机体的抗胃癌作用。     

CD4+CD25+调节性T细胞在肿瘤免疫中的作用研究

CD4+CD25+调节性T细胞(Treg)在自身免疫耐受、免疫自稳、肿瘤免疫中发挥着重要作用,它可以抑制自身抗原或者非自身抗原如肿瘤抗原引起的免疫反应.人们对Treg细胞的免疫抑制作用已进行了相关的研究和临床应用,最新研究表明其能够诱导肿瘤特异性抗原和局部的免疫反应.此综述将讨论Treg细胞的相关表面分子及其在肿瘤免疫...     

结肠癌患者外周血 CD4+ CD25+调节性 T细胞检测的临床意义

目的 探讨结肠癌患者外周血CD4^＋CD25^＋调节性T细胞检测的临床意义。方法采用流式细胞技术对51例结肠癌患者和30例健康体检者外周血淋巴细胞CD4及CD25进行了检测。结果（1）结肠癌组CD4^＋CD25^＋细胞明显高于正常对照组（P〈0．05），CD4^＋CD25^＋细胞则显著低于正常对照组（P〈0．05）；（2）结肠癌组手术后患者CD4^＋CD25^＋、CD4^-CD25^＋细胞与手术前相比显著下降（P〈0．05）；（3）结肠癌组淋巴结转移患者CD4^＋CD25^＋细胞明显高于未转移组（P〈0．001）。结论CD4^＋CD25^＋调节性T细胞数可以反映肿瘤患者免疫系统存在抑制状态，且可能与肿瘤的免疫耐受有关，并可反映肿瘤淋巴结转移状况。     

胃癌患者手术前后CD4+CD25+调节性T细胞的变化

目的 研究胃癌患者手术前、后调节性T细胞(Treg)及FoxP3表达的变化.方法 采用流式细胞术检测20例胃癌患者术前及其中15例接受了手术者术后1周(简称术后)以及15例因胃部不适接受胃镜检查的自愿者(正常对照组)外周血中Treg数量的变化,用RT-PCR法检测Treg的特异性分子标志物FoxP3的转录水平,同时用免疫组织化学法检测胃癌组织中FoxP3蛋白的表达情况.结果 胃癌患者术前外周血中CD4+T细胞中的CD4+ CD25+比例明显高于正常对照组[(19.39±5.58)%比(9.91±3.23)%,P＜0.01],而术后CD4+ CD25+比例较术前明显下降[(13.50±5.93)%比(19.39±5.58)%,P＜0.05].胃癌患者术前外周血中FoxP3转录水平明显高于正常对照组(0.86±0.03比0.64±0.02,P＜0.01),而术后较术前明显下降(0.73±0.04比0.86±0.03,P＜0.05),提示FoxP3转录水平与Treg变化一致.胃癌患者外周血中CD4+T细胞在单个核细胞中的比例与正常对照组相比明显下降(P＜0.01),而手术前、后变化不明显.20例胃癌患者中13例胃癌癌细胞的细胞浆中有不同程度的FoxP3蛋白表达(强阳性2例,中阳性6例,弱阳性5例),7例胃癌患者的胃癌细胞中不表达.结论 Treg可能通过免疫抑制作用在胃癌的发生、发展中发挥作用,肿瘤组织本身可能是引起Treg变化的重要始动因素.     

Impact of immunosuppressive drugs on CD4+CD25+FOXP3+ regulatory T cells: does in vitro evidence translate to the clinical setting?

Success of solid-organ transplantation requires the continuous administration of immunosuppressive drugs to prevent graft rejection. The currently prescribed immunosuppressive medication targets the immune system in a nonspecific fashion, leading to debilitating side effects that diminish patient survival and quality of life. Therefore, it is important to minimize immunosuppression, but this requires the development of alternative therapeutic strategies to induce and maintain transplant tolerance. One such strategy would be to allow and facilitate the induction of alloantigen-specific immune regulation by regulatory T cells (Treg). Recent experimental studies indicate that several commonly used immunosuppressive drugs have detrimental effects on the induction and function of Treg, whereas other drugs appear to spare these cells or may even be beneficial. These differential effects may be explained by differences in signaling pathways between Treg and effector T cells. In this review, we provide a comprehensive overview of the current literature on the effects of immunosuppressive drugs on CD4+CD25+FOXP3+ Treg and discuss whether these in vitro data are substantiated by in vivo evidence from the clinic. A greater understanding of the impact of immunosuppression on Treg may help to create future opportunities to manipulate the host allo-immune response and achieve operational tolerance in transplantation.     

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.     

CD4+CD25+调节性T细胞对同种胰岛移植免疫耐受的影响

目的 观察体外分离的CD4+CD25+调节性T细胞对同种胰岛移植免疫耐受的影响.方法 免疫磁珠法分离CD4+CD25+调节性T细胞,体外试验观察其对CD4+CD25-T细胞增殖的影响.将数量达1×106的CD4+CD25+调节性T细胞回输胰岛移植受体,对比其对移植物存活的影响.结果 分离的CD4+CD25+调节性T细胞体外试验可明显抑制CD4+CD25-T细胞的增殖.单纯胰岛移植组移植物物存活期为(5.57±0.79)d,回输受体CD4+CD25+调节性T细胞数量1×106、2×106时,胰岛移植物存活时间分别为(15.29±2.29)d和(25.43±2.30)d(P《0.01).CD4+CD25+调节性T细胞回输胰岛移植受体可显著延长移植物存活期,诱导免疫耐受的作用为剂量依赖性.结论 CD+CD25+调节性T细胞体外、体内试验可抑制效应性T细胞功能,诱导胰岛移植免疫耐受.     

原发性肝癌患者体内的CD4+CD25+ T细胞变化及其意义

目的:探讨原发性肝细胞癌(HCC)患者体内CD4+CD25+T细胞变化的及其意义。 方法:实验分HCC组（n=20）与正常人对照组(n=10)，采用免疫荧光标记法，流式细胞仪分离出外周血CD4+CD25+T细胞，分析两组CD4+CD25+T细胞所占T细胞的比例。计算并比较癌组织与癌旁组织中CD4+CD25+T细胞所占T细胞的比例。结果:HCC组机体的CD4+CD25+T 细胞比例为(19.3±3.0) %，明显高于对照组(5.2±1.6) % (P<0.05)。癌组织中CD4+CD25+T 细胞比例为(6.5±2.9) %，癌旁组织中CD4+CD25+T 细胞比例为(5.8 ±2.1) %， (P>0.05)。结论:原发性肝细胞癌患者机体免疫功能降低的原因之一可能是外周血CD4+CD25+T 细胞数明显增加。     

CD4+CD25+T细胞对同种异体小鼠心脏移植的免疫调节作用

目的 观察CD4+CD25+T细胞(Treg)对小鼠同种异体心脏移植的免疫调节作用.方法 流式细胞仪检测正常小鼠和胸腺切除+PC61小鼠淋巴结、脾脏和外周血的Treg的比例.将供体鼠BALB/C心脏移植到受体鼠B6腹腔内,观察对照组(n=6)、胸腺切除组(THY,n=8)、hCTLA4Ig组(n=8)、DST+hCTLA4Ig组(n=8)和THY+PC61+DST+hCTLA4Ig组(n=6)小鼠心脏移植后生存时间和移植心脏病理学检查.结果 正常B6小鼠淋巴结、脾脏和外周血的Treg的比例分别为5.1%、4.5%和1. 7%,明显高于胸腺切除+PC61处理组(1. 8%、1.7%、0.7%).移植心脏平均存活时间在对照组和胸腺切除组分别为(8.2±2.9)d和(7.6±3.0)d,两组间差异无统计学意义(P＞0.05);而在hCTLA4Ig组和DST+hCTLA4Ig组分别为(43.0±11.8)d和(135.0±29.7)d,均较对照组或胸腺切除组明显延长(P＜0.01);THY+PC61+DST+hCTLA4Ig组移植心脏平均存活时间(25.8±8.9)d,也明显较对照组明显延长,但短于hCTLA4Ig组和DST+hCTLA4Ig组(P＜0.01).DST+hCTLA4Ig组移植的心脏存活时间(135.0±29.7)d明显高于其他各组(P＜0.01),其病理组织学表现为间质内有较多的淋巴细胞浸润,伴毛细血管增生,管壁增厚,间质纤维化.结论 CD4+CD25+T细胞水平对同种异体心脏移植的免疫耐受具有免疫调节作用.

Abstract：

Objective To investigate the immunoregulation effects of CD4 + CD25 + T cells in mice heart allograft transplantation. Methods Flow cytometry was used to analyze the contents of CD4 + CD25 +T regulatory cells (Tregs) of the lymph nodes, spleen and blood in the normal mice group and the thymusectomy (THY) + PC61 group. BALB/C mice served as the donors and C57BL/6 (B6) mice as the recipients. Five groups were established, including control group ( n = 6 ), THY group ( n = 8 ), hCTLA4Ig group ( n = 8 ), DST ( donor-specific T-depleted spleen cells) + hCTLA4Ig group ( n = 8) and THY + PC61+ DST + hCTLA4Ig group (n = 6). The survival time after heart allograft transplantation was observed and pathological examination was done in different groups. Results In control group, the rate of Tregs in lymph nodes, spleen and blood was 5. 1%, 4. 5% and 1.7% respectively, which was significantly higher than in THY + PC61 group ( 1. 8% , 1. 7% and 0. 7% respectively). The average survival time of control and THY groups was 8. 2 ± 2.9 and 7.6 ± 3. 0 days respectively ( P ＞ 0. 05 ). The average survival time of hCTLA4Ig and DST + hCTLA4Ig groups was 43.0 ± 11.8 and 135.0 ± 29. 7 days respectively, which was significantly longer than in control group or THY group ( P ＜0. 01 ). The average survival time of THY +PC61 + DST + hCTLA4Ig group was 25.8 ± 8.9 days, which was significantly longer than in control group,but shorter than in hCTLA4Ig group or DST + hCTLA4Ig group ( P ＜ 0. 01 ). The survival time in DST +hCTLA4Ig group was 135.0 ± 29. 7 days, which was significantly longer than other groups ( P ＜ 0. 01 ).The pathological examination revealed that there were more lymphocytes infiltration and capillary vessel proliferation in the desmohemoblast in the transplanted heart of DST + hCTLA4Ig group. Conclusion CD4 +CD25 +T cells regulate the immune tolerance in the allograft transplantation.