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。     

重症急性胰腺炎患者外周血CD4+CD25high调节性T细胞的动态变化研究

目的 动态观察重症急性胰腺炎(severe acute pancreatitis,SAP)患者外周血CD4+CD25 high调节性T细胞(Treg)和Foxp3及细胞因子IL-10、IFN-γ及IL-4的变化,探讨其临床意义.方法 将2008年8月-2009年6月收治的42例SAP患者根据病程的变化分为3期:Ⅰ期(全身炎症反应综合征期即SIRS期);Ⅱ期(SIRS下调期);Ⅲ期(感染相关并发症期).对照组为同期住院的38例急性轻型胰腺炎(MAP)患者.分别抽取外周血,以流式细胞仪检测Treg及Foxp3百分率,酶联免疫吸附法(ELISA)检测IFN-γ、IL-4及IL-10的水平,并对以上指标进行相关性分析.结果 Treg百分率和IL-4水平在SAP Ⅰ期开始升高(Treg:1.37%±1.12%;IL-4:22.92±4.17),Ⅱ期高于Ⅰ期(Treg:3.12%±1.21%;IL-4:35.42±12.50),Ⅲ期升至最高(Treg:4.47%±1.04%;IL-4:76.04±14.58);IFN-γ水平在SAP Ⅰ期最高(978.57±213.29),Ⅱ期降低(571.43±157.14),在Ⅲ期降至最低(357.14±150.00);各期相比差异有统计学意义(P＜0.05),且均明显高于对照组(P＜0.05).Treg比例与IFN-γ水平负相关(r=-0.895,P＜0.01),与IL-4水平正相关(r=0.813,P＜0.01).结论 Treg可能通过抑制IFN-γ的分泌及促进IL-4的产生,对抗过度炎症反应对机体造成的损害.     

肝移植急性排斥反应者外周血CD4+CD25+Foxp3+调节性T淋巴细胞的变化

目的 探讨良性终末期肝病患者肝移植术后外周血CD4+CD25+叉状头螺旋转录因子(Foxp3)+调节性T淋巴细胞在急性排斥反应期的变化及意义.方法 2004年12月至2008年1月间,符合入选条件的良性终末期肝病患者共55例,按照术后是否发生急性排斥反应分为排斥组(14例)和无排斥组(41例).肝移植术前用流式细胞仪检测患者外周血CD4+CD25+Foxp3+T淋巴细胞占CD4+T淋巴细胞的百分率(简称CD4+CD25+Foxp3+T细胞百分率),出院后1年内每隔3～6个月复查;发生急性排斥反应时,于治疗前和治疗缓解后(3～6个月)复查.比较两组患者外周血CD4+CD25+Foxp3+T细胞百分率的变化,对排斥组发生急性排斥反应时外周血CD4+CD25+Foxr3+T细胞百分率与排斥反应活动指数(RAI的相关性进行统计学分析.结果 肝移植术前,排斥组与无排斥组外周血CD4+CD25+Foxp3+T细胞百分率的差异无统计学意义(P＞0.05).排斥组患者发生急性排斥反应时外周血CD4+CD25+Foxp3+T细胞百分率为(2.23±0.54)%,低于无排斥组的(2.99±0.86)%,差异有统计学意义(P＜0.01).排斥组中,患者发生急性排斥反应时外周血CD4+CD25+Foxp3+T细胞百分率低于未发生急性排斥反应时的(3.67±0.70)%,差异有统计学意义(P＜0.01).排斥组患者发生急性排斥反应时外周血CD4+CD25+Foxp3+T细胞百分率与RAI呈负相关(r=-0.80,P＜0.01).结论 监测肝移植受者外周血CD4+CD25+Foxp3+调节性T淋巴细胞的变化,可辅助诊断急性排斥反应及判断其严重程度.

Abstract：

Objective To investigate the expression of peripheral blood (PB) CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) in patients with benign end-stage liver disease after liver transplantation and the relationship between levels of PB Tregs and acute rejection. Methods A prospective analysis was performed on 55 consecutive patients who underwent liver transplantation.Fourteen out of 55 cases suffered from acute rejection after liver transplantation were defined as rejection group,while the rest patients were classified into no acute rejection group. PB was obtained from liver transplant patients at different time points longitudinally: pre-transplant, post-transplant within one year and acute rejection. The circulating CD4+ CD25+ Foxp3+ Tregs in PB were measured by flow cytometry. Blood samples were drawn during acute rejection, at the same time, liver biopsies were performed. The circulating CD4+ CD25+ Foxp3+ Tregs were compared between two groups.Results There was no difference between two groups in levels of circulating CD4+ CD25+ Foxp3 + Tregs cells pre-transplant. However, the levels of circulating CD4+ CD25+ Foxp3+ Tregs in rejection group were decreased significantly as compared with no-rejection group (2. 23 % ± 0. 54 % vs. 2. 99 % ±0. 86 %,P＜0.01). The frequency of CD4+ CD25+ Foxp3+ T cells was negatively correlated with rejection activity index (RAI) (r = - 0. 80, P＜0. 01 ). Conclusion Monitoring PB CD4+ CD25+ Foxp3+ Tregs levels may be helpful in evaluating the immune state and act as a more sensitive marker for acute rejection diagnosis in the patients following liver transplantation.     

The role of Foxp3+ regulatory T cells in liver transplant tolerance

The liver has long been considered a tolerogenic organ that favors the induction of peripheral tolerance. The mechanisms underlying liver tolerogenicity remain largely undefined. In this study, we characterized Foxp3-expressing CD4+ CD25+ regulatory T cells (Treg) in liver allograft recipients and examined the role of Treg in inherent liver tolerogenicity by employing the mouse spontaneous liver transplant tolerance model. Orthotopic liver transplantation was performed from C57BL/10 (H2b) to C3H/HeJ (H2k) mice. The percentage of CD4+ CD25+ Treg was expanded in the liver grafts and recipient spleens from day 5 up to day 100 posttransplantation, associated with high intracellular Foxp3 and CTLA4 expression. Immunohistochemistry further demonstrated significant numbers of Foxp3+ cells in the liver grafts and recipient spleens and increased transforming growth factor beta expression in the recipient spleens throughout the time courses. Adoptive transfer of spleen cells from the long-term liver allograft survivors significantly prolonged donor heart graft survival. Depletion of recipient CD4+ CD25+ Treg using anti-CD25 monoclonal antibody (250 microg/d) induced acute liver allograft rejection, associated with elevated anti-donor T-cell proliferative responses, CTL and natural killer activities, enhanced interleukin (IL)-2, interferon-gamma, IL-10, and decreased IL-4 production, and decreased T-cell apoptotic activity in anti-CD25-treated recipients. Moreover, CTLA4 blockade by anti-CTLA4 monoclonal antibody administration exacerbated liver graft rejection when combined with anti-CD25 monoclonal antibody. Thus, Foxp3+ CD4+ CD25+ Treg appear to underpin spontaneous acceptance of major histocompatability complex- mismatched liver allografts in mice. CTLA4, IL-4, and apoptosis of alloreactive T cells appear to contribute to the function of Treg and regulation of graft outcome.     

TGF-β Induces Foxp3 + T-Regulatory Cells from CD4 + CD25 − Precursors

CD4 + CD25 + regulatory T cells (Tregs) are potent suppressors, playing important roles in autoimmunity and transplantation tolerance. Understanding the signals necessary for the generation and expansion of Tregs is important for clinical cellular therapy, but only limited progress has been made. Recent reports suggest a role for TGF-beta in the generation of Tregs from CD4 + CD25 - precursors, but the mechanism remains unknown. Here, we demonstrate that TGF-beta2 triggers Foxp3 expression in CD4 + CD25 - precursors, and these Foxp3 + cells act like conventional Tregs. The generation of Foxp3 + Tregs requires stimulation of the T-cell receptor, the IL-2R and the TGF-beta receptor. More importantly, strong costimulation through CD28 prevents Foxp3 expression and suppressive function in an IL-4-dependent manner. Furthermore, TGF-beta-driven Tregs inhibit innate inflammatory responses to syngeneic transplanted pancreatic islets and enhance islet transplant survival. Thus, TGF-beta is a key regulator of the signaling pathways that initiate and maintain Foxp3 expression and suppressive function in CD4 + CD25 - precursors. TGF-beta and signaling through TGF-beta receptor, CD28 costimulation and IL-4 may be key components for the manipulation of Treg. The de novo generation of Foxp3 + cells from CD4 + cells has the potential to be used for treatment of autoimmune diseases and induction of transplant tolerance.     

CD4+CD25+Treg细胞对肿瘤特异性CTL杀伤效果的影响

目的 探讨CD4+CD25+Treg细胞对肿瘤特异性细胞毒T细胞(CTL)杀伤效果的影响及机制.方法 将C57BL/6小鼠80只随机分为4组,每组20只.A组:树突状细胞(DC)与T细胞共同培养前删除CD4+CD25+Treg;B组:DC与T细胞共同培养后删除CD4+CD25+Treg;C组:DC与T细胞共同培养时不删除CD4+CD25+Treg;对照组:无DC诱导的T细胞.应用脾脏来源DC细胞诱导T细胞制备CTL,在CTL形成的不同时期采用MACS法删除CD4+CD25+Treg.应用噻唑蓝(MTY)比色法检测不同组别CTL对B16黑色素瘤细胞的杀伤效果.同时应用酶联免疫吸附试验(ELISA)法检测细胞培养液中白细胞介素(IL)-2、干扰素(IFN)-γ含量变化.结果 3组实验组CTL杀伤率明显高于对照组(P<0.05).删除CD4+CD25+Treg的A组、B组CTL杀伤率明显高于未删除的C组(P<0.05).但CTL形成的不同时期删除CD4+CD25+Treg对CTL杀伤率的影响无统计学意义(P>0.05).IL-2、IFN-γ含量变化与杀伤率呈现相同的变化趋势.结论 删除CD4+CD25+Treg细胞可明显提高CTL的杀伤效果,是消除肿瘤免疫耐受机制的新途径.     

乳腺癌患者外周血CD4+CD25+Foxp3+调节性T细胞水平的检测及意义

目的：探讨乳腺癌患者外周血CD4+CD25+Foxp3+调节性T细胞（Treg）水平检测的意义。 方法：流式细胞术检测74例乳腺癌患者与30例健康对照者外周血CD4+CD25+Foxp3+Treg占CD4+T细胞百分比,分析CD4+CD25+Foxp3+Treg细胞水平与乳腺癌患者临床病理特征及相关免疫组化指标的关系。 结果：乳腺癌患者外周血CD4+CD25+Foxp3+Treg占CD4+T细胞的百分比高于健康对照者[（9.15± 2.24）% vs.（2.29±1.36）%],差异有统计学意义（P<0.05）。统计分析显示,乳腺癌患者外周血CD4+CD25+Foxp3+Treg细胞水平与肿瘤组织学分级、淋巴结转移、pTNM分期以及HER-2、pS2、nm23的表达有关（均P<0.05）,而与肿瘤大小、病理类型以及雌激素受体（ER）、孕激素受体（PR）、p53、Ki-67表达无关（均P>0.05）。进一步相关性分析显示,CD4+CD25+Foxp3+Treg细胞水平与肿瘤组织学分级、淋巴结转移数、pTNM分期、HER-2的表达呈正相关（r=0.583,r=0.333,r=0.919,r=0.604,均P<0.05）而与pS2、nm23表达呈负相关（r=-0.229,r=-0.401,均P<0.05）。 结论：乳腺癌患者外周血CD4+CD25+Foxp3+Treg细胞水平升高,并与与乳腺癌的进展、转移密切相关,对其检测可能有助于患者预后及治疗效果的评估。

     

CD4+CD25+调节性T细胞、Foxp3 mRNA及可溶性白细胞介素2受体检测在肾移植中的意义

目的 观察肾移植患者外周血中CD4+CD25+调节性T细胞水平及其表面特异性标志物Foxp3和可溶性白细胞介素2受体（sIL-2R）的变化，探讨其在诊断移植肾急性排斥反应中的作用和价值。 方法 选取42例维持性血液透析接受同种异体肾移植治疗的患者及30例健康体检对照者。在患者移植前、移植后1、2、4、8周或发生排斥反应时，以流式细胞仪检测外周血中CD4+CD25+调节性T细胞水平；荧光定量PCR检测Foxp3 mRNA表达；双抗体夹心酶联免疫吸附法（ELISB）检测血浆中sIL-2R水平。 结果 (1)移植后第1、2、4、8周急性排斥反应组CD4+CD25+调节性T细胞、Foxp3 mRNA水平明显低于同期未发生排斥的肾功能稳定组，而sIL-2R水平却显著高于肾功能稳定组。(2)血液透析患者外周血CD4+CD25+调节性T细胞[(9.22±3.53)%]、Foxp3 mRNA[（0.82±0.36）×10-3]及sIL-2R[（856.30±108.24） U/ml]水平与健康对照组[分别为(6.09±1.99)%、(0.50±0.28)×10-3、(247.35±11.24) U/ml]比较，差异均有统计学意义(P < 0.01)。(3)肾移植后随着肾功能的恢复，外周血CD4+CD25+调节性T细胞[(16.53±4.14)%]、Foxp3 mRNA[(4.97±1.94)×10-3]显著升高(P < 0.01)，而sIL-2R[（463.72±31.23）U/ml]水平明显降低(P < 0.01)。(4)当发生急性排斥反应时，CD4+CD25+调节性T细胞[(12.18±2.86)%]、Foxp3 mRNA[(3.15±1.22)×10-3]显著降低(P < 0.01)，而sIL-2R[(748.36±115.41) U/ml]水平明显升高(P < 0.01)，并且这些变化早于Scr的变化。(5)患者移植前后外周血CD4+CD25+调节性T细胞百分率与Foxp3 mRNA水平均呈正相关（分别为r ＝ 0.904、0.932，P < 0.01），但与sIL-2R水平无相关。 结论 外周血CD4+CD25+调节性T细胞、Foxp3 mRNA及sIL-2R水平的测定均可以作为肾移植患者移植后发生急性排斥反应的早期预测指标，并可判断预后。     

Reduction of Foxp3-expressing regulatory T cell infiltrates during the progression of renal allograft rejection in a mouse model

BACKGROUND: Regulatory T (Treg) cells are the immune suppressors in the maintenance of immune homeostasis and tolerance to self and non-self antigens, and may have therapeutic potential in the treatment of transplant rejection in patients. However, Treg cell development and action are poorly understood in transplantation. In this study, the association of CD4(+)Foxp3(+) infiltrates within renal allograft tissue with graft survival was investigated in a mouse model. METHODS: Kidney donors from C57BL/6J mice (H-2(b)) were transplanted to bilaterally nephrectomized Balb/c recipient mice (H-2(d)). Treg cells were examined with FACS and immunohistochemical staining. RESULTS: Here we showed that without any immunosuppressive regimen, kidney allografts were mostly rejected from 20 to 60 days after transplantation. During the progression of allograft rejection Foxp3(+) Treg phenotype infiltrates were significantly diminished, while intragraft expression of TGF-beta1, IL-6, IL-17 and IL-23 was up-regulated. The regulatory function of CD4(+)CD25(+) infiltrates was confirmed by their suppressive activity in mixed lymphocyte reaction. Further in vitro studies indicated that primary renal tubular epithelial cell (TEC) cultures produced high levels of IL-6 in response to allogeneic lymphocyte or IL-17 stimulation, and neutralization of IL-6 increased CD4(+)CD25(+)Foxp3(+) cells in co-cultures with TEC. CONCLUSION: Diminution of Foxp3(+) Treg infiltrates associates with renal allograft rejection, and neutralization of IL-6 activity enhances Foxp3(+) Treg cell differentiation. Our findings suggest that increase in intragraft IL-6 may down-regulate infiltrating Foxp3(+) Treg cells.     

The effect of immunosuppressive drug rapamycin on regulatory CD4+CD25+Foxp3+T cells in mice

CD4(+)CD25(+)Regulatory T (Treg) cells are crucial for negatively regulating immune responses. Rapamycin (rapa) is an immunosuppressive agent which is widely used for preventing acute graft rejection in patients and has been used to induce operational tolerance in mouse models. The aim of the present study was to determine the effect of rapa on CD4(+)CD25(+)Foxp3(+)Treg cells in a mouse model. After C57BL/6 mice were intraperitoneally given 1.5 mg/kg/day of rapa for 14 days, the percentages, cell numbers, phenotype and function of CD4(+)CD25(+)Treg cells were determined by flow cytometry as well as the in vitro and in vivo functional assays. The cell numbers of CD4(+) and CD4(+)CD25(+)Treg cell subsets were markedly decreased in rapa-treated mice as reported. However, rapa significantly enhanced the ratios of CD4(+)CD25(+)Treg cells or CD4(+)CD25(+)Foxp3(+)Treg cells to CD4(+)T cells in spleens and thymi of mice (P<0.01) respectively. Furthermore, splenic CD4(+)CD25(+)Treg cells in rapa-treated mice showed immunosuppressive ability on the immune response of T effector cells to alloantigens or mitogen as efficiently as the control CD4(+)CD25(+)Treg cells in vitro and in vivo. Thus, rapa could significantly enhance the percentages of CD4(+)CD25(+)Foxp3(+)Treg cells in the thymus and the periphery while keeping these cells functional, indicating that CD4(+)CD25(+)Treg cells are more resistant to rapa than other CD4(+)T cells. The different effects of rapa on CD4(+)CD25(+)Treg and T effector cells make rapa to be a favorable choice for inducing immune tolerance to self-, allo-, or xeno-antigens.     

Reduced CD4+ CD25++ CD45RA− Foxp3hi activated regulatory T cells and its association with acute rejection in patients with kidney transplantation

BackgroundIt was found that regulatory T cells (Tregs) importantly affect the maintenance of the kidney graft. However, Tregs are a heterogeneous population with less to more suppressive activity. The aim of this study was to determine the effects of different subsets of Tregs, as well as their ratio to effector T cells (Teff), on kidney transplantation outcomes.MethodsA total of 58 participants were enrolled in this study and divided into four groups: (i) first kidney transplant recipients (stable 1); (ii) second kidney transplant recipients (stable 2); (iii) transplant recipients with acute rejection (AR); and (iv) healthy control subjects. By using flow cytometer, the frequencies of CD4⁺ CD25⁺⁺ CD45RA⁻ Foxp3^hi activated Tregs (aTregs), CD4⁺ CD25⁺ CD45RA⁺ Foxp3^lo resting Tregs (rTregs), CD4⁺ CD25⁺ CD45RA⁻ Foxp3^lo non-suppressive T cells, CD4⁺ CD25⁺ Foxp3⁻ cells Teff, and total Tregs were analyzed in all subjects.ResultsThe frequency of aTregs (as well as the ratio of aTregs/Tregs) was significantly lower in the AR patients than the other three groups. In contrast to AR patients, stables 1 and 2 had a higher aTreg/Treg ratio than those in the control group. Although patients with AR had a significantly lower total Tregs than the other three groups, the balance of total Tregs and Teff was similar between patients with and without AR.ConclusionPatients with AR had poorer immunoregulatory properties than those with normal graft functioning, as well as those in the control group. These reduced immunoregulatory properties in patients with AR could lead to graft rejection.     

Relationship between T-helper cell 17/regulatory T cell imbalance and atherosclerosis cardiovascular disease in maintenance hemodialysis patients

Objective To investigate whether the T-helper cell 17 (Th17)/regulatory T cell (Treg) of patients on maintenance hemodialysis (MHD) present imbalance in terms of proportion and function, and if so, the relationship between such imbalance and atherosclerosis cardiovascular disease (ASCVD). Methods Fifty-seven MHD patients, included 25 with ASCVD and 32 without ASCVD, and 24 healthy volunteers were enrolled. The common carotid artery intima media thickness (CCA-IMT), carotid plaque and plaque area were determined with high-resolution B-mode ultrasonography. Treg (CD4+CD25+Foxp3+) and Th17 (CD4+IL-17+) were measured by flow cytometry. The Foxp3 and RAR-related orphan receptor γt (RORγt) mRNA expressions were measured by real-time PCR. TGF-β1, IL-10, IL-17 and IL-6 in serum were detected by ELISA. Results There were decreased Treg proportion, Foxp3 mRNA, TGF-β1 and IL-10, and increased Th17 proportion, RORγt mRNA, IL-17, IL-6 and Th17/Treg in the ASCVD group compared with that in the non-ASCVD group and healthy group (all P＜0.01). No correlation was observed between Treg and CCA-IMT, but IL-10 were negatively correlated with CCA-IMT (P＜0.05). Th17, IL-17 and ratio of Th17/Treg were positively correlated with CCA-IMT (all P＜0.05). MHD patients with carotid plaques had lower Treg, TGF-β1 and IL-10, higher Th17, IL-17 and ratio of Th17/Treg than those without carotid plaques (all P＜0.05). Moreover, Treg proportion was negatively correlated with carotid plaque area in MHD patients with carotid plaques (P＜0.01). Conclusions The Th17/Treg numerical and functional imbalance exists in MHD patients, especially in patients with ASCVD. This might act synergistically with micro-inflammation on immune-mediated atherosclerosis and contribute to the high incidence of ASCVD.     

不同免疫抑制方案对肾移植术受者CD4~+ Foxp3~+ 调节性T细胞的影响

目的 探讨不同免疫抑制剂方案对肾移植术受者外周血CD4~+ Foxp3~+调节性T细胞(regulatory T cells,Treg)表达水平的影响.方法 定群研究了2006年1月至2008年1月在本移植中心接受初次移植50例随访满1年肾移植受者,分为钙调神经蛋白抑制组(钙调神经蛋白抑制剂+吗替麦考酚酯+强的松)19例,其中环孢素组10例,他克莫司组9例;雷帕霉素组(雷帕霉素+吗替麦考酚酯+强的松)31例.另取20例行规律血液透析终末期肾病患者为对照组.采用流式细胞仪的方法检测3组外周血CD4~+ Foxp3~+ Treg占CD4~+ T细胞的比例,比较各组间表达水平与不同免疫抑制方案的关系.结果 钙调神经蛋白抑制剂组、雷帕霉素组和终末期肾病组3组年龄、性别比无统计学差异(P>0.05).钙调神经蛋白抑制剂组、雷帕霉素组2组冷缺血时间、HLA错配率、群体反应性抗体(PRA)和急性排斥反应发生率无统计学差异(P>0.05).雷帕霉素组和终末期肾病组CD4~+ Foxp3~+ T细胞占CD4~+ T细胞的比例均明显高于钙调神经蛋白抑制组,差异有统计学意义(P<0.01).使用环孢素患者和他克莫司患者外周血中CD4~+ Foxp3~+ T细胞占CD4~+ T细胞的比例之间无显著性差异(P>0.05).结论 肾移植术后服用雷帕霉素组患者外周血CD4~+ Foxp3~+ Treg占CD4~+ T细胞的比例显著高于服用钙调神经蛋白抑制组患者,提示雷帕霉素有助于诱导宿主对移植肾免疫耐受.     

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.     

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.     

Targeting acute allograft rejection by immunotherapy with ex vivo-expanded natural CD4+ CD25+ regulatory T cells

BACKGROUND: Natural CD4CD25 regulatory T (Treg) cells have been implicated in suppressing alloreactivity in vitro and in vivo. We hypothesized that immunotherapy using ex vivo-expanded natural Treg could prevent acute allograft rejection in mice. METHODS: Natural CD4+ CD25+ Treg were freshly purified from naive mice via automated magnetic cell sorter and expanded ex vivo by anti-CD3/CD28 monoclonal antibody (mAb)-coated Dynabeads. Suppression was assayed in vitro by mixed lymphocyte reaction and in vivo by targeting cardiac allograft rejection. Survival of Treg or effector T (Teff) cells after adoptive transfer in vivo was tracked by flow cytometry and all allografts were examined by histology and immunohistochemistry. RESULTS: By day nine in culture, 26.6+/-5.3-fold of expansion was achieved by co-culture of fresh natural Treg with anti-CD3/CD28 mAb-coated Dynabeads and interleukin-2. Ex vivo-expanded Treg exerted stronger suppression than fresh ones towards alloantigens in vitro and prevented CD4 Teff-mediated but only delayed CD4+/CD8+ Teff-mediated heart allograft rejection in Rag-/- mice. Long-term surviving allografts showed no signs of acute or chronic rejection with graft-infiltrating Treg expressing CD25 and FoxP3. Infused Treg persisted and expanded long-term in vivo and trafficked through the peripheral lymphoid tissues. CD25 expression was dynamic in vivo: maintained CD25 expression on Treg was indicative for the preservation of allosuppression, while significantly enhanced CD25 expression on CD4+ effector T cells was most likely associated with T-cell expansion and graft rejection. CONCLUSIONS: Therapeutic use of ex vivo-expanded natural CD4+ CD25+ Treg may be a feasible and nontoxic modality for controlling allograft rejection or perhaps inducing allograft tolerance.     

CD4+CD25+ regulatory T cells protect against injury in an innate murine model of chronic kidney disease

Studies of mechanisms of disease regulation by CD4+CD25+ regulatory T cells (Treg) have been focused on their interaction with effector T cells; however, the possibility that regulation might involve noncognate cells has not been explored in detail. This study investigated the effect of CD4+CD25+ Treg on macrophage proinflammatory properties and phenotype in vitro and found that they modulate macrophages by inhibiting their activation, leading to reduced proinflammatory cytokine production and a downregulated effector phenotype. For testing the in vivo significance of this effect, CD4+CD25+ T cells that expressed high levels of Foxp3 were reconstituted into SCID mice after induction of Adriamycin nephropathy, a noncognate model of chronic renal disease. CD4+CD25+ T cells significantly reduced glomerular and interstitial injury. In addition, there was a significant fall in the number of macrophages in both the glomeruli and interstitium of SCID mice that were reconstituted with Treg as compared with the Adriamycin alone group. Blockade of TGF-beta using neutralizing antibodies significantly impaired the protective effect of Treg. These findings delineate a TGF-beta-dependent Treg-macrophage inhibitory interaction that can explain cognate-independent protection by Treg.     

第三方骨髓间充质干细胞诱导同种异体移植受体免疫耐受机制的研究

目的 初步研究第三方骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)诱导同种异体移植受体免疫耐受的作用机制.方法 40只雌性C57BL/6小鼠作为供体,40只雄性BALB/C小鼠作为受体,建立稳定的同种异体皮肤移植模型,BMSCs取自SD大鼠骨髓.将40只BALB/C小鼠随机分为4组,每组10只.①空白对照组:只进行皮肤移植,未给予其他治疗;②环磷酰胺组(CP组):大剂量环磷酰胺(cyclophosphamide,CP)腹腔注射,200 mg/kg,连用2 d(q.d.);③单纯给予SD-BMSCs移植组(SD-BMSCs组):移植当天自受体小鼠尾静脉输注2×106个SD-BMSCs;④细胞药物联合应用组(CP+SD-BMSCs组):大剂量CP腹腔注射,200 mg/kg,连用2 d(q.d.),并于移植当天自受体小鼠尾静脉输注2×106个SD-BMSCs.检测指标包括:移植皮片存活情况;SD大鼠BMSCs表面抗原CD29、CD34、CD45和CD90鉴定;流式细胞仪检测受体脾脏调节性T细胞(CD4+、CD25+、Foxp3+、Treg细胞)的比例;ELISA检测受体外周血TGF-β、IL-10、IFN-γ的含量;异基因T淋巴细胞与经60Co照射的不同来源BMSCs共培养后,MTT法测定异基因T淋巴细胞增殖的情况.结果 CP+SD-BMSCs组皮肤移植物存活时间为(15.7 ±1.4)d,空白对照组为(6.1±1.1)d,CP组为(12.3±1.5)d,SD-BMSCs组为(12.6±1.8)d,CP+SD-BMSCs组皮肤移植物存活时间明显比后3组延长(P＜0.05).全骨髓贴壁培养的BMSCs表面抗原鉴定:CD29+、CD44+分别为99.7%和96.7%,CD34-、CD45-分别为1.6%和1.3%.流式细胞仪检测Treg含量SD-BMSCs组和CP+SD-BMSCs组明显高于空白对照组和CP组(P＜0.05);ELISA检测受体外局血SD-BMSCs组和CP组TGF-β和IL-10明显高于空白对照组,SD-BMSCs和CP组IFN-γ则明显低于空白对照组(P＜0.05);共培养结果显示:来源于C57小鼠和SD大鼠的BMSCs可以明显抑制T淋巴细胞的增殖反应(P＜0.05),而上述两组组间比较差异则无统计学意义(P＞0.05).结论 第三方BMSCs诱导同种异体移植免疫耐受作用可能与诱导受体Treg细胞增殖和促进免疫耐受因子的表达,抑制免疫排斥因子的表达有关.

Abstract：

Objective To study the immuno-tolerance mechanism of the third-party bone marrowderived mesenchymal stem cells ( BMSCs) in the allogeneic transplantation. Methods Forty female C57BL/ 6 mice and forty male BALB/C mice were respectively used as donors and recipients in skin allogenic graft model. Forty male BALB/C mice were divided randomly into 4 groups: blank control group, CP group, BMSCs group , CP + BMSCs group , with 10 mice in each group. Before skin graft, high-dose abdominal injection of cyclophosphamide ( 200 mg/kg,2 d,q. d. ) was performed in recipient mice in CP and CP + BMSCs groups. On the transplantation day, a bonus of 2 x 106 BMSCs from the SD rat (SD-BMSCs) were injected through the tail vein in the BMSCs and CP + BMSCs groups. The observation and HE staining of skin grafts were used. The expressions of CD29, CD34, CD45 and CD90 of cells were analyzed by using flow cytometry in order to identify BMSCs. The CD4+ , CD25+ , Foxp3+ and Treg cells of spleen were detected by flow cytometry. Cytokine in peripheral blood of recipient mice were measured by ELISA,including TGF-β, IL-10 and IFN-γ. T cells were co-cultured with 60 Co-irradiated bone marrow MSCs from different individuals. The proliferative activity of T cells were evaluated with MTT assay. Results The skin graft survival time was significantly prolonged in the CP + BMSCs group, as compared with that in the blank control group, the CP group, the BMSCs group, respectively. Cells cultured by whole bone marrow adherent cultivation showed CD29+ (99.7% ) ,CD44+ (96.7% ) ,CD34 (1.6% ) ,CD45( 1. 3% ). Compared with the control group and CP group, the ratio of the CD4+ ,CD25+ ,Foxp3+ and Treg cells significantly increased in the SD-BMSCs group and CP + BMSCs group (P ＜ 0. 05). Analysis of peripheral blood by ELISA showed significant high level of TGF-β, IL-10 and low level of IFN-γ in BMSCs group and CP group, compared with that in control group. When co-cultured with BMSCs from different individuals, T- lymphocytes proliferation decreased apparently in SD-BMSCs group and C57-BMSCs group (P ＜ 0. 05) , but there was no significant difference between SD-BMSCs group and C57-BMSCs group ( P ＞ 0. 05 ). Conclusions The immunotolerance mechanism of the third-party bone marrow-derived mesenchymal stem cells in the allogeneic transplantation might be associated with its effect on the proliferation of Treg cells and increasing expression of TGF-β and IL-10, decreasing expression of IFN-γ.     

New Insights into Mechanisms of Spontaneous Liver Transplant Tolerance: The Role of Foxp3-Expressing CD25+CD4+ Regulatory T Cells

Liver allografts in mice are accepted across MHC barriers without requirement for immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we investigated the role of Foxp3-expressing CD25⁺CD4⁺ regulatory T cells (Treg) in the induction of murine liver transplant tolerance. Foxp3⁺CD25⁺CD4⁺ T cells were increased in liver grafts and recipient spleens from day 5 to day 100 posttransplantation, associated with enhanced CTLA4 and TGF-β expression and IL-4 production. Depletion of recipient CD25⁺CD4⁺ T cells using anti-CD25 mAb (250 μg/day) induced acute liver allograft rejection. This was associated with a decreased ratio of Foxp3⁺ Treg: T effector cells, decreased IL-4 and elevated IL-10 and IL-2 production by graft-infiltrating T cells, and reduced apoptotic activity of graft-infiltrating CD4⁺ and CD8⁺ T cells in anti-CD25-mAb-treated recipients. Thus, the data suggest that Foxp3⁺CD25⁺CD4⁺Treg are involved in spontaneous acceptance of liver allografts in mice. The ratio of Treg to T effector cells appears to determine liver transplant outcome. CTLA4, IL-4, TGF-β and apoptosis of graft-infiltrating T cells are also associated with liver transplant tolerance and may contribute, at least in part, to the mechanisms of Treg-mediated immune regulation in this model.     

小鼠CD4+CD25+T调节细胞的分离、纯化及其功能检测

目的 探讨小鼠CD4+CD25+T调节细胞(Treg)的分离培养、纯化及其部分功能检测.方法 采用免疫磁珠分离法(MACS)对分离小鼠的脾淋巴细胞进行分选CD4+CD25+Treg细胞,锥虫蓝细胞染色检测其活性,流式细胞仪检测分选所得活性细胞的纯度,酶联免疫吸附试验(ELISA)检测培养上清液中白细胞介素(IL)-2、IL-10水平的浓度.结果 MACS分离的CD4+CD25+Treg细胞的纯度达83%～96%.体外培养中Treg组、T组和混合组IL-2和IL-10的平均水平分别为:(10.25±2.31)、(40.32±8.05)ng/L;(5 8.21±13.05)、(11.52±3.01)ng/L;(39.54±12.82)、(31.25±4.36)ng/L,数据差异有统计学意义(P＜0.05,P＜0.01).结论 采用MACS系统两步法,可获得高纯度、具有免疫抑制功能的Treg细胞,该细胞对CD4+CD25-T细胞的免疫抑制作用可能是通过IL-10对IL-2的调节作用实现的.