Short-term anti-CD25 monoclonal antibody treatment and neogenetic CD4(+)CD25(high) regulatory T cells in kidney transplantation

CD4(+)CD25(high) T cells named regulatory T (Treg) cells are generated and play a key role in the induction and maintenance of transplant tolerance in organ recipients. Interleukin-2 (IL-2) enhance the development of effector cells and is essential for generation of Treg cells. The effect of the anti-CD25 monoclonal antibody (anti-CD25mAb) induction therapy on the neogenetic CD4(+)CD25(high)Treg cells is important for therapeutic strategies in kidney transplant. To clarify the question, a prospective study was conducted in 21 living donor kidney transplant recipients who randomly divided into the anti-CD25mAb group (Daclizumab) with 11 patients and the control group with 10 patients. The frequency of CD4(+)CD25(high)Treg cells in total CD4(+) T cells was analyzed by flow cytometry and FoxP3 expression by RT-PCR in peripheral blood, and results were compared at day 0, 3, 13, 17, 27 posttransplantation. There was no significant difference in patient characteristics and allograft survival. The present study showed that in vivo antigen-specific Treg cells population were generated and expanded after transplant. Both groups showed a significant increase in the frequency of CD4(+)CD25(high)Treg cells and higher level of FoxP3 mRNA after transplantation while the serum creatinine declined. Compared with the control group, recipients with anti-CD25mAb injection had significantly lower percentage of CD4(+)CD25(high) in total CD4(+) cells (1.13%+/-0.13% vs 1.94%+/-0.22%, P=0.00; 3.75%+/-0.28% vs 7.11%+/-0.51%, P=0.00) on day 3, 17 after transplantation. While, the percentage was not significantly different on day 10, 27 (3.72%+/-0.19% vs 4.36%+/-0.28%, P=0.08; 7.84%+/-0.35% vs 8.56%+/-0.36%, P=0.16). However, there was not obvious difference in Foxp3 expression level associated with the source of the CD4(+)CD25(high)Treg cells at the different time point after transplant. Our data indicated that CD4(+)CD25(high)Treg cells were transiently affected by anti-CD25mAb, without depletion. In conclusion, the short-term treatment with anti-CD25mAb might not prevent the production, proliferation of neogenetic Treg cells in organ transplant.     

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.     

Effect of GK1.5 monoclonal antibody dosage on survival of pig proislet xenografts in CD4+ T cell-depleted mice

Treatment of CBA/H mice with 5 injections of anti-CD4 (GK1.5 mAb) terminating on day 10 posttransplant resulted in long-term survival (greater than or equal to 6 weeks) of fetal pig proislet (pancreatic islet precursor) xenografts. The GK1.5 mAb dose determined the duration of CD4+ T cell depletion and the extent to which the survival of pig proislet xenografts was prolonged. Sustained depletion of CD4+ T cells (0%, 1%, and 9% of total T cells in peripheral lymph nodes at 2, 4, and 6 weeks, respectively) and survival of proislet xenografts at 6 weeks posttransplant was observed when transplant recipients were treated with 5.4 mg GK1.5 mAb/injection. Treatment of transplanted mice with a suboptimal dose of GK1.5 mAb (0.2 mg/injection) resulted in the same level of depletion at 2 weeks posttransplant but a more rapid recovery of CD4+ T cells in the periphery (24% of total T cells at 4 weeks) and only temporary prolongation in xenograft survival (less than or equal to 4 weeks). Control xenografts showed evidence of graft destruction by as early as 6-7 days posttransplant and were completely rejected by 2 weeks. The rejection reaction consisted predominantly of CD4+ T cells, eosinophils and F4/80-positive macrophages. Only small numbers of CD8+ T cells were identified. CD4+ T cells therefore represented the major T cell component of the cellular infiltrate. In contrast, surviving xenografts in GK1.5 mAb-treated recipient mice showed essentially an absence of CD4+ T cells but presence of CD8+ T cells. This finding may be attributable to the increase (1.7-3.1-fold) in the absolute size of the population of CD8+ T cells in the periphery following GK1.5 mAb treatment in vivo. Compared with isolated fetal pig proislets, which contained only a small population of insulin-producing cells in addition to glucagon- and somatostatin-positive cells, surviving pig proislet xenografts contained mainly insulin-positive beta cells with smaller populations of glucagon- and somatostatin-positive cells. Fetal pig proislets therefore differentiate into insulin-producing islet tissue posttransplant and thus show evidence of normal development of endocrine function.     

OX40信号调节抑制小鼠CD4+CD25+调节性T淋巴细胞Foxp3的表达

目的 探讨共刺激信号OX40对体外诱导的小鼠CD4+ CD25+适应性调节性T淋巴细胞(iTreg)的Foxp3表达的影响.方法 制备C57BL/6小鼠淋巴细胞悬液,经免疫磁珠法分选,获得CD4+ CD25-静息T淋巴细胞,与抗CD3单克隆抗体、抗CD28单克隆抗体、转化生长因子β1、白细胞介素2共孵育,诱导产生Foxp3+ iTreg.在此基础上,于培养体系中加入OX40激动型抗体及其对照抗体,利用流式细胞仪分析研究OX40信号刺激对iTreg Foxp3表达的影响.结果 C57BL/6小鼠淋巴结中CD4+ CD25+天然调节性T淋巴细胞(Treg)比例为(5.0±0.4)％,体外诱导培养的CD4+CD25+ Treg比例为(71.8±13.4)％,其中Foxp3阳性表达占(74.9±1.9)％.OX40激动型抗体组CD4+ CD25+ Treg细胞比例为(80.0±1.6)％,其中Foxp3表达水平为(59.2±0.7)％;OX40激动型抗体对照抗体组CD4+ CD25+ Treg细胞比例为(86.0±1.4)％,其中Foxp3表达水平为(70.0±0.8)％,两组间差异有统计学意义(P＜0.05).结论 静息T淋巴细胞可以在体外诱导培养获得高纯度iTreg;OX40信号刺激可以显著抑制CD25+ iTreg细胞Foxp3的表达.     

CD4＋CD25＋调节性T细胞与Foxp3表达在白癜风发病中的作用

目的：研究CD4＋CD25＋调节性T细胞（CD4＋CD25＋Treg细胞）与Foxp3基因表达与白癜风发病的关系及可能机制。方法：以确诊为进展期白癜风且治疗显效的24例患者为研究对象,患者确诊符合白癜风诊断及疗效标准。以16例健康志愿者作为对照。应用流式细胞术检测正常对照、白癜风患者治疗前后各自外周血中CD4＋CD25＋调节性T细胞的比例。采用密度梯度离心法从治疗前后的外周血中提取单个核细胞,以逆转录聚合酶链扩增方法检测其中Foxp3 mRNA的表达水平。结果：进展期且治疗显效的24例白癜风患者外周血中CD4＋CD25＋调节性T细胞的比例以及CD4＋CD25＋调节性T细胞在总CD4＋T细胞中所占比例明显均低于正常对照组（P〈0．05）。治疗后显效的进展期白癜风患者CD4＋CD25＋调节性T细胞的比例与治疗前相比明显升高（P〈0．05）,CD4＋CD25＋调节性T细胞中Foxp3 mRNA的表达水平比治疗也前明显增加（P〈0．05）。结论：进展期白癜风患者体内存在CD4＋CD25＋调节性T细胞的异常,CD4＋CD25＋Treg细胞的数量的减少和Foxp3表达的降低所造成的CD4＋CD25＋Treg细胞免疫抑制功能爱损可能是白癜风发病的一个因素。     

Foxp3 is critical for human natural CD4+CD25+ regulatory T cells to suppress alloimmune response

Naturally occurring CD4+CD25+ regulatory T cells (nTregs) that express high level of Foxp3 actively suppress pathological and physiological immune responses, contributing to the maintenance of immunological self-tolerance and immune homeostasis. Although Foxp3 is required for nTreg development and appears to be necessary for mature murine Treg function, the precise role of Foxp3 in regulating natural human Treg function in alloimmune response is unclear. In this study, we used siRNA-mediated gene silencing to knockdown Foxp3 expression in natural human Tregs and investigated the importance of Foxp3 in maintaining human nTreg suppressive function. We showed that Foxp3 knockdown resulted in impaired phenotype and nonresponsiveness, downregulated expression of function molecules, and reduced production of suppressive cytokines in nTregs. These changes correlated with diminished nTreg activity in suppressing proliferation of effector CD4+CD25- T cells, their cytotoxicity against allogeneic target cells and production of effector cytokines in response to allogeneic stimulation. Thus, this study shows that ongoing Foxp3 expression is required for natural human Tregs to maintain their phenotype and suppressive function in the alloimmune response.     

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.     

转化生长因子-β1联合白细胞介素-2体外诱导非肥胖糖尿病小鼠调节性T细胞的产生

目的 体外诱导非肥胖糖尿病(NOD)小鼠CD4+ CD25+Foxp3+调节性T细胞(Treg)的产生并检测其免疫抑制功能.探讨外源性白细胞介素(IL)-2在诱导方案中的作用.方法 分选NOD小鼠童贞T细胞(Naive T),利用Anti-CD3、Anti-CD28刺激,同时给予转化生长因子-β1(TGF-β1)和白细胞介素-2(IL-2),共同培养5 d,收获诱导性Treg(iTreg),经流式细胞仪检测其表型.利用体外T细胞增殖体系,对比NOD小鼠天然Treg(nTreg),评价iTreg的免疫抑制能力.将诱导方案中的IL-2撤除以观察其作用.结果 TGF-B1联合IL-2能诱导NOD小鼠Naive T转化为iTreg,较对照组有统计学意义[(41.33±3.21)%比(8.00±3.00)%,P《0.05].iTreg可有效抑制T细胞增殖,其能力与nTreg的差异元统计学意义[(40.33±1.03)%比(38.33±3.06),P》0.05].外源性IL-2有利于iTreg的产生[(41.33±3.21)%比(15.00±1.00)%,P《0.05].结论 TGF-β1联合IL-2可在体外诱导Naive T转化为具有免疫抑制功能的Treg.     

A novel mechanism of action for anti-thymocyte globulin: induction of CD4+CD25+Foxp3+ regulatory T cells

T cell-depleting agents are being tested as part of clinical tolerance strategies in humans with autoimmunity and transplantation. The immunosuppressive activity of anti-thymocyte globulin (ATG) has been thought to result primarily from depletion of peripheral lymphocytes. Herein is reported for the first time that ATG but not anti-CD52 mAb (alemtuzumab) or the IL-2R antagonists causes rapid and sustained expansion of CD4+CD25+ T cells when cultured with human peripheral blood lymphocytes. These cells display enhanced expression of the regulatory markers glucocorticoid-induced TNF receptor, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and forkhead box P3 and efficiently suppress a direct alloimmune response of the original responder lymphocytes. It is interesting that the cells do not suppress memory responses to the recall antigen mumps. Ex vivo expansion of regulatory T cells is due mainly to conversion of CD4+CD25- into CD4+CD25+ T cells and to a lesser degree to proliferation of natural CD4+CD25+ T cells. The induction of regulatory T cells depends on production of Th2 cytokines in the generating cultures. These novel data suggest that ATG not only may promote expansion/generation of regulatory T cells but also may be useful in future ex vivo expansion of these cells for cellular therapy in autoimmunity and clinical transplantation.     

The significantly enhanced frequency of functional CD4CD25Foxp3 T regulatory cells in therapeutic dose aspirin-treated mice

CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells, produced in the thymus or periphery as a functionally mature T cell subpopulation, play pivotal roles in maintenance of self-tolerance and negative regulation of immune responses. Aspirin (ASA) is widely used to reduce pain, the risk of cardiovascular diseases and allo-graft rejection. However, the effect of ASA on CD4⁺CD25⁺Foxp3⁺ Treg cells has yet to be determined. The frequency, phenotype and immunosuppressive function of CD4⁺CD25⁺Foxp3⁺ Treg cells were detected in BALB/c mice treated with low or high doses of ASA for 4 weeks. ASA significantly decreased the percentage and number of CD4⁺ T cells in the periphery, while ASA remarkably increased the percentage of CD4⁺CD25⁺Foxp3⁺ Treg cells in CD4⁺T cells. The total cell numbers of thymocytes were significantly decreased in ASA-treated mice, but the number of CD4⁺ CD25⁺Fxop3⁺ cells and its ratio in CD4⁺CD8⁻ thymocytes were markedly enhanced in the thymi of ASA-treated mice. The phenotype of CD4⁺CD25⁺ Treg cells, including the expressions of CD44, CD45RB, CD62L, CD69, GITR and CTLA-4, did not show detectable changes in ASA-treated mice. CD4⁺CD25⁺ Treg cells in ASA-treated mice exhibited unimpaired immunosuppressive function on CD4⁺CD25⁻ T effector cells. ASA significantly enhanced the frequency of functional CD4⁺CD25⁺Foxp3⁺ Treg cells in mice in a therapeutic dose range. The different effects of ASA on CD4⁺CD25⁺Foxp3⁺ Treg cells and CD4⁺CD25⁻ T cells may potentially make hosts susceptible to tolerance induction which would be beneficial for tolerance induction in patients with autoimmune diseases or allo-grafts. This study may have potential impacts in the clinical application of ASA.     

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+ regulatory T cells mediate acquired transplant tolerance

The Holy Grail of clinical organ transplantation is the safe induction of allograft tolerance. Transplant tolerance has been successfully induced in animal models. Since T cells play a pivotal role in graft rejection, modulating T cell function has been the primary focus of studies aimed at inducing transplant tolerance. Rodent models of transplant tolerance induction include central deletion and peripheral mechanisms involving activation-induced cell death (AICD), anergy, immune deviation, and production of regulatory T cells. These mechanisms are not mutually exclusive. Although clonal deletion and anergy limit self-reactive T cells in the thymus, these mechanisms alone are not sufficient for controlling self-reactive T cells in the periphery. There is now evidence that the adult animal harbors two functionally distinct populations of CD4(+) T cells; one mediates autoimmune disease and the other dominantly inhibits it. The latter cells express CD4, CD25 and CTLA-4. These thymus-derived T cells have recently been shown to mediate the induction and maintenance of transplant tolerance. These CD4(+)CD25(+) T cells are similar in origin, phenotype, and function to those that maintain natural self-tolerance and T cell homeostasis in the periphery. Against this background, is it possible that alloantigen specific regulatory T cells might be generated and expanded ex vivo before organ transplantation and then infused to induce long-term tolerance, perhaps without the need for chronic immunosuppression?     

乳腺癌患者CD4+CD25+Foxp3+调节性T细胞的变化及意义

目的:探讨乳腺癌患者CD4+CD25+Foxp3+调节性T细胞(简称Foxp3+Treg)的变化及意义。方法:选择40例乳腺癌患者和32例乳腺良性肿瘤患者,采用流式细胞术检测外周血Foxp3+Treg、CD8+CD28+T细胞、NK细胞水平;用Western blot和RT-PCR病变乳腺组织Foxp3蛋白与m RNA表达。结果:乳腺癌患者外周血中Foxp3+Treg比例较乳腺良性肿瘤患者明显升高,而CD8+CD28+T细胞、NK细胞比例明显降低(均P0.05),且乳腺癌患者外周血Foxp3+Treg水平与CD8+CD28+T细胞和NK细胞水平呈负相关(r=-0.631,r=-0.578,均P0.05);乳腺癌患者术后外周血Foxp3+Treg水平较术前明显降低(P0.05)。乳腺癌组织中Foxp3蛋白与m RNA的表达均较乳腺良性肿瘤组织明显升高(均P0.05)。结论:Foxp3+Treg和其标记分子Foxp3在乳腺癌患者中的表达增加,且可能通过抑制CD8+CD28+T细胞和NK细胞而产生肿瘤免疫抑制。