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.     

高迁移率族蛋白B1对小鼠调节性T细胞与CD4+CD25-T细胞相互作用的影响

目的 观察高迁移率族蛋白B1(HMGB1)对调节性T细胞(Treg)与CD4+CD25-T细胞相互作用的影响,并初步探讨其影响Treg抑制功能的机制.方法 免疫磁珠法分离正常BALB/c小鼠脾脏CD4+CD25+Treg及CD4+CD25-T细胞.采用固相包被抗CD3/可溶性抗CD28进行辅助活化,以不同时间及浓度HMGB1刺激Treg,ELISA法分析HMGB1刺激对Treg分泌IL-10的影响.将HMGB1(1000μg/L)刺激后的Treg与CD4+CD25-T细胞共培养,MTT法观察其对Treg抑制CD4+CD25-T细胞反应的作用,并分析HMGB1刺激后的Treg对CD4+CD25-T细胞IL-2生成及细胞功能极化的影响.结果 经抗CD3/CD28辅助活化的CD4+CD25+Treg在HMGB1作用下IL-2生成无显著差异(P＞0.05),但随时间延长及剂量增加,IL-10生成明显减少(P＜0.05).经HMGB1刺激的Treg对CD4+CD25-T细胞增殖的抑制反应减弱,同时诱导CD4+CD25-T细胞IL-2产生及细胞功能极化的能力均下降(P＜0.05).结论 HMGB1可通过诱导Treg抑制功能的下调,从而影响CD4+CD25-T细胞功能,进而调节炎症反应过程.     

CD4^＋CD25^＋Treg细胞在移植免疫耐受的研究进展

CD4+CD25+Treg细胞是一类特殊的调节性T细胞,它具有免疫无能性和免疫抑制性两大功能特性,能通过多种机制诱导和维持免疫耐受,经过免疫磁珠分选后,能够在体外大量扩增,在器官移植领域有潜在的应用价值.     

抗原特异性CD4+CD25+T细胞对同种异体胰岛移植影响及作用机制研究

目的:探讨抗原特异性CD4+CD25+Treg细胞免疫对同种异体胰岛急性移植排斥反应的影响和机制。方法:用MACS分选供体抗原特异性CD4+CD25+Treg细胞免疫糖尿病BALB/cByJ受体小鼠，以ICR小鼠胰岛为供体行同种异体胰岛移植。观察移植后小鼠的存活时间、移植前后外周血CD4+和CD8+T细胞亚群的变化和移植物中Th1/Th2细胞因子mRNA表达水平的变化。结果:抗原特异性Treg细胞联合胰岛移植组(C组)胰岛移植物平均生存期为(34.57±17.15)d，显著长于单纯胰岛移植组(B组)的(10.6±1.82) d (P<0.01)；移植后第3天，C组外周血CD4+/CD8+的值显著低于B组(P<0.01)；C组移植物中IL-10,TGF-β mRNA表达比B组显著增强。B组移植物中IL-1β，IL-2及IFN-γ mRNA表达明显强于C组。结论:抗原特异性CD4+CD25+ Treg细胞可通过调节Th2/Th1之间的反应平衡而延长同种异体胰岛移植物的存活时间。     

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细胞对小鼠肝脏移植自发性免疫耐受的影响

目的 研究CD4+CD25+调节性T细胞在诱导自发性肝脏免疫耐受中的作用.方法 向受体和供体注射抗CD25抗体(PC61)后进行小鼠原位肝脏移植,观测其生存时间.术后20～30 d切取移植肝脏行HE染色,同时观察CD4+CD25+T细胞对CD4+T细胞和CD8+T细胞功能的影响.结果 去除受体而不是供体小鼠的CD4+CD25+T细胞可以导致肝移植排斥反应.而且,去除CD4+CD25+T细胞使移植物的白细胞浸润明显增多,组织损伤加重.同时,去除CD4+CD25+T细胞导致CD4+T细胞的增殖活性和CD8+T细胞的细胞毒活性明显增强.结论 受体来源的CD4+CD25+调节性T细胞在小鼠肝脏移植免疫耐受诱导中起重要作用.

Abstract：

Objective To examine the contribution of CD4+ CD25+ regulatory T cells to liver transplant tolerance. Methods After injection of anti-CD25 monoclonal antibody (mAb, PC61), mouse orthotopic liver transplantation was performed and survivals were determined. The paraffin-embedded sections of hepatic allografts were cut and stained with hematoxylin and eosin (HE). Furthermore, the effect of CD4+ CD25+ regulatory T cells on proliferative response of CD4+ T cells and cytotoxicity of CD8+ T cells was examined by depleting these regulatory T cells. Results Depletion of these cells in the recipients but not in the donors before liver transplantation caused rejection. Histological analyses of hepatic allografts with PC61 treatment showed extensive leukocyte infiltration and tissue destruction, whereas those in the control group showed minimal changes. Moreover, elimination of CD4+CD25+ T cells resulted in the enhancement of both proliferative response of CD4+ T cells and cytotoxicity of CD8+ T cells against donor-type alloantigen. Conclusions These results suggest that CD4+CD25+ regulatory T cells were important for tolerance induction to hepatic allografts.     

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。     

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.     

大黄素对大鼠肝移植后CD4~+CD25~+调节性T细胞的影响

目的研究大黄素对大鼠肝移植后CD4+CD25+调节性T细胞的比例以及对其免疫抑制功能的影响。方法双袖套法建立大鼠原位肝移植模型,以大黄素和环孢素A(CsA)分别在术后腹腔给药,并以给予PBS作为模型对照组,观察不同药物移植后大鼠存活时间的影响,以及对移植术后大鼠外周血中CD4+CD25+调节性T细胞(Tregulatory cells,Treg)亚群的变化以及免疫功能的影响。结果大黄素能显著延长大鼠原位肝移植的术后成活时间,大黄素组大鼠的平均存活时间(17.4±2.5)d与肝移植模型对照组(8.8±1.9)d相比差异具有统计学意义;大鼠肝移植后大黄素给药可显著上调受体大鼠外周血以及肝内CD4+CD25+Treg的比例,并显著增强CD4+CD25+Treg抑制效应性T细胞的增殖能力(P0.05)。结论大黄素能够延长大鼠原位肝移植的术后成活时间,并可能通过上调外周血以及肝内CD4+CD25+Treg的数量及免疫抑制功能来实现移植后免疫排斥反应的抑制。     

树突状细胞和CD4+CD25+调节性T细胞在免疫耐受中的作用及关系

诱导免疫耐受具有高效、低毒、经济等优点,在器官移植中有着极其重要的意义.在建立免疫耐受中,树突状细胞(DC)起到克隆清除、克隆无能、表达T细胞抑制因子、选择性激活辅助T细胞和诱导调节性T细胞尤其是CD4+CD25+调节性T细胞的产生等作用.而CD4+CD25+调节性T细胞主要以DC为作用靶点,影响其分化成熟,抑制DC向...     

GW5074对不成熟树突状细胞诱导初始性CD4+T淋巴细胞分化为Treg的影响

目的 建立一种稳定且高效的不成熟树突状细胞(imDC)体外培养方法,探讨细胞外信号调节激酶(ERK)1/2信号传导通路抑制剂GW5074对imDC体外诱导同种初始性CD4+T淋巴细胞分化为调节性T淋巴细胞(Treg)的影响.方法 从健康成人外周血单个核细胞(PBMC)中分离、培养成熟树突状细胞(mDC)和imDC,并对mDC和imDC的免疫表型和功能进行鉴定.取新生儿脐静脉血分离初始性CD4+T淋巴细胞.实验分为5组:(1)空白对照组为单纯培养的初始性CD4+T淋巴细胞,不做任何处理;(2)阳性对照组将imDC与初始性CD4+T淋巴细胞以1∶10的细胞比例混合培养;(3)低浓度GW5074组;(4)中浓度GW5074组;(5)高浓度GW5074组.后3组在阳性对照组基础上,分别加入终浓度为8、24和40μmol/L的GW5074.培养5 d后,用流式细胞仪检测初始性CD4+T淋巴细胞转化为Treg细胞的转化率.结果 imDC呈CD1a高表达,CD80和CD83低表达;mDC呈CD1a低表达,CD80和CD83高表达.imDC和mDC的刺激指数分别为1.12±0.03、2.85±0.07.空白对照组,阳性对照组,低、中及高浓度GW5074组的CD4+CD25+Treg转化率分别为(5.81±1.36)%、(35.73±2.07)%、(22.53±2.11)%、(11.55±1.73)%和(4.97±1.83)%,除空白对照组与高浓度GW5074组间的差异无统计学意义(P＞0.05)外,其余各组之间两两比较,差异均有统计学意义(P＜0.01).结论 通过应用重组人粒细胞-巨噬细胞集落刺激因子和重组人白细胞介素4联合诱导人外周血PBMC可获得高纯度imDC,ERK1/2信号传导通路在诱导免疫耐受中发挥作用,GW5074可抑制初始性CD4+T淋巴细胞向Treg转化.

Abstract：

Objective To establish a stable and efficient method of culturing imDCs in vitro,and to explore the effect of GW5074, which blocks ERK1/2 signal pathway in the process of imnature dentritic cells (imDCs) on inducing differentiation of the na(i)ve allogeneic CD4+ T cells into Treg cells in vitro. Methods The imDCs and mature DCs (mDCs) were isolated and cultured from the peripheral blood mononuclear cells (PBMC) derived from a healthy adult male volunteer, and they were identified by cell morphology, cell surface marker and cell functions respectively. Na(i)ve CD4+ T cells were isolated from newborn umbilical vein blood and were divided into 5 groups to be cultured: (1) Blank control group: Na(i)ve CD4+ T cells were cultured alone;(2) Positive control group: The irrDCs were Middle-concentration GW5074 group;(5) High-concentration GW5074 group. In the last three groups, imDCs and na(i)ve CD4+ T cells were co-cultured, the same as the positive control group, but these groups were added by GW5074 dilution at the concentrations of 8, 24, and 40μmol/Lrespectively. After co-culture for 5 days, the transformation ratio from naive CD4+T cells to Treg T cells was detected by flow cytometry. Results On the surface of imDCs, there was stronger pression of CD1a, but weaker expression of CD80 and CD83. On the contrary, on the surface of mDCs, there was weaker expression of CD1a, but stronger expression of CD80 and CD83. The stimulation index in imDCs group and mDCs group was 1.12±0.03 and 2.85±0. 07 respectively. The transformation ratio of Treg T cells in blank control group, positive control group, low-concentration GW5074 group, middle-concentration GW5074 group and high-concentration GW5074 group was (5. 81±1.36)%, (35.73±2.07)%, (22.53±2.11)%, (11.55±1.73)%, and (4.97±1.83)%respectively. One-way ANOVA analysis revealed that there was no significant difference between high-concentration GW5074 group and blank control group, P＞0. 05, but significant difference between the remaining groups, P＜0.01. Conclusion High purity of imDCs can be obtained from PBMC by induction with rhGM-CSF and rhIL-4. ERK1/2 signal pathway plays a role in inducing the immune tolerance. GW5074 can inhibit differentiation of na(i)ve CD4+ T cells into Treg T cells.     

CD4+CD25+T细胞联合CD154单抗抑制大鼠肝移植急性排斥反应的研究

目的探讨CD4 CD25 T细胞联合应用CD154单抗在抑制大鼠肝移植急性排斥反应中的作用。方法分离Lewis大鼠脾脏CD4 _CD25 T细胞后与DA大鼠脾细胞单向混合淋巴细胞反应行体外激活。用"二袖套法"行DA到Lewis的原位肝移植48例。A组为对照组;B、C组单独术前回输体外激活的CD4 CD25 T细胞或术后腹腔注射抗CD154单抗;D组联合应用CD4 CD25 T细胞和CD154单抗。每组大鼠12对。术后7 d各组处死6只受体,检测移植肝内T细胞亚群和细胞因子水平。余大鼠观察生存情况,死亡大鼠观察移植肝病理变化。结果D组受体生存期(52.00±10.64)d明显长于B、C组(P<0.01);移植肝内CD4 CD25 T细胞比例(16.43±4.28)%明显高于B、C组(P<0.05、P<0.01),而淋巴细胞浸润数量[(3.47±1.21)%×106]和(CD8 T细胞百分比(14.19±3.02)%明显低于B、C组(P<0.05、P<0.01);移植肝内白细胞介素- 2(IL-2)水平(6.44±1.83)ng/L低于B、C组(P<0.05),IL-10(43.72±7.55)ng/L和转化生长因子-β1(TGF-β1)(270.06±46.91)ng/L明显高于B、C组(P<0.05、P<0.01)。结论联合应用CD154单抗能明显增强CD4 CD25 调节性T细胞对大鼠肝移植急性排斥反应的抑制作用。     

CD4+CD25+ Regulatory T Cells in Transplantation: Progress, Challenges and Prospects

The involvement of CD4(+)CD25(+) regulatory T cells (Treg) in general immune homeostasis and protection from autoimmune syndromes is now well established. Similarly, there has been increasing evidence for Treg involvement in allograft rejection and current immunotherapies. However, despite significant advances in understanding the development, function, and therapeutic efficacy of Treg in certain well-defined rodent models, the relevance of Treg to clinical transplantation remains unclear. In this review, we summarize our current understanding of the role of Treg in immunity and organ transplantation in experimental and clinical settings. In addition, we review advances in using Treg as a form of immune therapy. The goal is to highlight the complexities and opportunities in the field and to provide evidence to support the use of antigen-specific Tregs in the context of transplantation to facilitate a robust and selective state of immune tolerance.     

门静脉输注供者脾细胞诱导的供者特异性免疫低反应性

目的 探讨经门静脉输注供者脾细胞能否诱导皮肤移植小鼠产生供者特异性的免疫低反应性及其可能机制.方法 取Balb/c小鼠,随机分为空白对照组(经小鼠门静脉输注RPMI 1640培养液)、受者脾细胞组(经小鼠门静脉输注Balb/c小鼠脾细胞)、供者脾细胞组(经小鼠门静脉输注C57BL/6小鼠脾细胞)、空白移植对照组(经小鼠门静脉输注RPMI 1640培养液,7 d后移植C57BL/6小鼠的皮肤)、实验对照组(经小鼠门静脉输注Balb/c小鼠脾细胞,7 d后移植C57BL/6小鼠的皮肤)、实验组(经小鼠门静脉输注C57BL/6小鼠脾细胞,7 d后移植C57BL/6小鼠的皮肤)以及第三方移植组(经小鼠门静脉输注C57BL/6小鼠脾细胞,7 d后移植C3H小鼠的皮肤).记录空白移植对照组、实验对照组、实验组和第三方移植组移植皮肤的存活时间,并观察移植皮肤的病理学变化;脾细胞输注后7 d,分别获取空白对照组、受者脾细胞组和供者脾细胞组小鼠的外周血、脾脏和肝脏,用流式细胞仪测定样本中CD4+CD25+Foxp3+调节性T淋巴细胞(CD4+CD25+Foxp3+Treg细胞)的比例.结果 实验组移植皮肤的存活时间为(19.8±4.6)d,明显长于空白移植对照组、实验对照组和第三方移植组,但仍未达到长期存活.皮肤移植后7 d,空白移植对照组和实验对照组的移植皮肤呈现重度急性排斥反应的病理学改变,而实验组移植皮肤呈现中度急性排斥反应的病理学改变.供者脾细胞组外周血、肝脏和脾脏中CD4+CD25+Foxp3+Treg细胞比例明显高于空白对照组和受者脾细胞组.结论 门静脉输注供者脾细胞可特异性地延长供者皮肤移植物的存活时间,减轻移植物的排斥反应,该效应可能与受者体内的CD4+CD25+Foxp3+Treg细胞增加有关.     

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.     

慢性乙型肝炎患者外周血调节性T细胞对树突状细胞功能的抑制作用

目的研究慢性乙型肝炎（CHB）患者外周血CD4＋CD25＋调节性T细胞（Treg）对树突状细胞（DCs）免疫功能的抑制作用,探讨治疗CHB的新方法。方法采用密度梯度离心法获得CHB患者和健康对照组（NC组）外周血单个核细胞（PBMC）;部分PBMC体外诱导培养获得DCs,部分PBMCs用特异性免疫磁珠分选获得CD4＋CD25＋Treg和CD4＋CD25-T细胞;不同来源的DCs和正常对照组CD4＋CD25-T细胞混合为反应细胞,将不同来源和不同比例的Treg分别加入到反应细胞中培养3 d,MTT法检测Treg抑制DCs的抑制指数（SI）,并在培养DCs的不同时间加入Treg,应用流式细胞术检测DCs表面共刺激分子CD80和HLA-DR的表达。结果来源于CHB患者及NC组的Treg均可抑制DCs的免疫作用,来源于CHB患者Treg抑制DCs能力显著高于NC,差异具有统计学意义（P ＜0.01）。不同比例的Treg均可抑制DCs的免疫功能,随着Treg比例的增高抑制作用也越明显,抑制指数亦越高。在DCs培养的不同时间加入相同比例的Treg,发现Treg对DCs表面分子CD80、HLA-DR的表达均有抑制作用,与对照组相比,差异具有统计学意义（P＜0.01）,同时发现加入Treg的时间越早,DCs表面分子表达降低越明显。结论 CHB患者Treg可显著抑制DCs免疫功能且呈时间和量的依赖,抑制DCs表面分子CD80和HLA-DR表达,可能是Treg抑制DC免疫功能的机制之一。