慢性移植肾肾病所致功能丧失移植肾切除与否对CD4~+CD25~+Treg细胞的影响

目的 探讨因慢性移植肾肾病(CAN)所致的功能丧失移植肾切除与否对CD4~+ CD25~+调节性T淋巴细胞(Treg细胞)的影响.方法 30例移植肾功能丧失患者,经移植肾活检证实发生CAN,患者群体反应性抗体(PRA)均无增高.根据是否行移植肾切除分为切除组(n=17)和保留组(n=13).两组在原发病构成比、HLA错配及移植后用药等影响机体免疫因素诸方面的差异均无统计学意义.所有受者在移植肾功能丧失后除均服用泼尼松5 mg/d外,近3个月未再服用其他抗排斥反应药物,切除组的患者切除术后仍口服泼尼松5 mg/d,直到术后2个月停用.分别于移植肾切除术前、术后1个月和2个月检测患者外周血单个核细胞中CD4~+ CD25~(high)T淋巴细胞/CD4~+T淋巴细胞(CD4~+ CD25~(high)/CD4~+)比率、细胞毒性T淋巴细胞相关抗原(CTLA-4)表达比率和叉状头螺旋转录因子(Foxp3)表达率.结果 切除组术前CD4~+ CD25~+/CD4~+比率为(1.47±0.19)%,术后2个月为(1.08±0.16)%;保留组相应时间点CD4~+ CD25~(high)/CD4~+比率分别为(1.44±0.25)%和(1.77±0.24)%,切除组术后2个月时CD4~+ CD25~(high)/CD4~+比率明显低于保留组(P<0.01).切除组术前CTLA-4表达比率为(76.82±5.31)%,术后2个月为(72.56±4.99)%;保留组相应时间点CTLA-4表达比率分别为(76.20±4.22)%和(75.24±4.26)%,两组间各时间点的CTLA-4表达比率的差异均无统计学意义(P>0.05).切除组术前Foxp3表达率为(79.77±1.59)%,术后2个月时为(69.07±4.37)%;保留组相应时间点Foxp3表达率分别为(79.56±1.75)%和(79.09±2.05)%,切除组术后2个月时Foxp3表达率明显低于保留组,差异有统计学意义(P<0.01).结论 移植肾切除后CD4~+ CD25~(high)/CD4~+比率及其表面标志性因子Foxp3的表达明显下降,提示移植肾切除可明显抑制CD4~+ CD25~+ Treg的活性.     

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.     

胃癌患者CD4+CD25+Foxp3+调节性T细胞与TGF-β1检测的意义

目的:探讨胃癌患者外周血CD4+CD25+Foxp3+调节性T细胞(CD4+CD25+Foxp3+Tergs)以及血清TGF-β1水平的变化及意义。方法:检测并比较42例胃癌患者(胃癌组)与24例健康体检者(对照组)外周血CD4+CD25+Foxp3+Tergs与血清TGF-β1水平,分析两者水平与胃癌患者临床病理因素的关系。结果:胃癌组CD4+CD25+Foxp3+Tergs和TGF-β1水平均明显高于对照组(均P<0.05)。胃癌患者外周血CD4+CD25+Foxp3+Tergs水平与TNM分期、淋巴结转移有关(均P<0.05),而血清TGF-β1的表达水平与TNM分期、分化程度、淋巴结转移有关(均P<0.05);胃癌组患者外周血内CD4+CD25+Foxp3+Tergs的表达水平与血清内TGF-β1的表达水平呈明显正相关(r=0.801,P<0.05)。结论:胃癌患者外周血CD4+CD25+Foxp3+Tergs水平及血清TGF-β1水平升高,检测两者水平有助于患者病情与预后的判断。     

CD4+CD25+调节性T细胞在维持小鼠肝脏移植自发性免疫耐受状态中的作用

目的 探讨CD4+CD25+调节性T细胞在维持小鼠肝脏移植免疫耐受状态中的作用.方法 进行小鼠原位肝脏移植,诱导出移植免疫耐受后,向受体注射抗CD25抗体(PC61)以去除CD4+CD25+T细胞,检测受体内CD4+CD25+T细胞数量及叉状头/翅膀状螺旋转录因子(Foxp3)的表达以确定CD4+CD25+T细胞完全被清除,同时观察受体生存时间.结果 与同种同系小鼠肝脏移植结果 相似,同种异系肝脏移植小鼠的生存时间亦均超过70 d.移植免疫耐受诱导后,PC61不同注射方案均能完全去除受体小鼠肝脏、脾脏及血液中的CD4+CD25+T细胞,且移植肝脏中Foxp3 mRNA的表达也明显降低,表明完全去除了CD4+CD25+调节性T细胞,但肝脏移植动物生存时间并未受到影响.结论 CD4+CD25+调节性T细胞对于小鼠肝脏移植自发性免疫耐受的维持并非必需.     

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.     

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。     

不同免疫抑制剂对肝移植受者外周血中调节性T淋巴细胞比例的影响

目的 探讨西罗莫司(SRL)和钙调磷酸酶抑制剂(CNI)对肝移植受者外周血中CD4+CD25high T淋巴细胞水平的影响.方法 排除肝移植远期移植肝功能异常的受者,将移植肝功能长期(超过2年)稳定的受者47例纳入研究,其中免疫抑制方案使用SRL者15例(SRL组),使用CNI(均为他克莫司)者32例(CNI组).以同期38名健康成人志愿者作为正常对照.使用流式细胞仪检测各组受试者外周血中单个核细胞CD4、CD25及Foxp3的表达水平,比较各组间外周血中CD4+CD25high调节性T淋巴细胞(Treg细胞)的差异.结果 与正常对照组相比,CNI组外周血淋巴细胞中CD4+ CD25high T淋巴细胞的比例显著减少(P<0.05),SRL组CD4+ CD25high T淋巴细胞的比例显著升高(P<0.05).SRL组、正常对照组和CNI组受试者外周血中CD4+ CD25high Foxp3+ Treg 细胞占CD4+ T淋巴细胞的比例依次降低,分别为1.88%(1.56%～2.60%)、1.15%(0.57%～1.48%)和0.84%(0.46%～1.45%),3组间两两比较,差异均有统计学意义(P<0.01或P<0.05).CD4+ CD25 high T淋巴细胞表达Foxp3的阳性率超过95%,CD4+ CD25 low T淋巴细胞表达Foxp3的阳性率低于20%,CD4+ CD25-T淋巴细胞不表达Foxp3.结论 SRL可促进肝移植受者外周血中Treg细胞水平的升高,而CNI可降低Treg细胞的水平.     

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。     

树突状细胞与Foxp3+细胞在瘢痕疙瘩中的免疫调节作用

目的 探讨树突状细胞与Foxp3+细胞(T调节细胞,regulatory T cell,Treg)在瘢痕疙瘩发病机制中的相互关系和免疫调节作用.方法 应用磁珠分离、流式细胞术和ELISA法检测瘢痕疙瘩患者(K组,15例)和正常人(N组,15例)外周血中Foxp3+细胞和成熟树突状细胞表面分子MHCII、CD83的表达和功能,分离外周血中树突状细胞分别与CD44 CD25-细胞共培养,测定Foxp3+细胞生成和IL-10的表达.结果 ①K组Foxp3+细胞占CD4+CD25+细胞的(1.45±0.22)%,细胞培养上清中IL-10的浓度为(一),明显低于N组[(5.63±0.95)%,(137±12)ng/L],P<0.05;②K组MHCⅡ+CD83+细胞占(85.47±4.13)%,培养上清中IL-12 p70的浓度为(263±21)ng/L,明显高于N组[(12.79±6.84)%,(一)],P<0.05;③K组DC与CD4+CD25-细胞共培养,3 d后诱导的Foxp3+细胞占CD4+细胞的(0.27±0.18)%,分泌的IL-10浓度为(一),明显低于N组DC与CD4+CD25-细胞共培养诱导的Foxp3+细胞含量和IL-10浓度[(2.53±0.72)%,(79.6±3.24)ng/L],P<0.05.结论 ①外周血中Foxp3+细胞的表达减少,功能降低提示瘢痕疙瘩患者可能存在外周的主动免疫抑制功能减弱;②瘢痕疙瘩患者中树突状细胞与Foxp3+细胞之间存在免疫调节作用;③Foxp3+细胞与瘢痕疙瘩的发病关系密切.     

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。     

Gut microbiota intervention by pre and probiotics can induce regulatory T cells and reduce the risk of severe acute GVHD following allogeneic hematopoietic stem cell transplantation

BackgroundAcute graft-versus-host disease (aGVHD) is one of the leading causes of limitation and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Numerous studies have shown that changes in the gut microbiome diversity increased post-transplant problems, including the occurrence of aGVHD. Probiotics and prebiotics can reconstitute the gut microbiota and thus increase bacterial metabolites such as short-chain fatty acids (SCFAs) that have immunomodulatory effects preventing aGVHD in recipients of allo-HSCTs.Methods/Study DesignWe conducted a pilot randomized clinical trial to investigate whether oral synbiotics are associated with the prevention or reduction in occurrence/severity and mitigate complications of aGVHD following allo-HSCT. A commercially available synbiotic mixture containing high levels of 7 safe bacterial strains plus fructo-oligosaccharides as a prebiotic was administered to allo-HSCT recipients. Out of 40 allo-HSCT patients, 20 received daily a synbiotic 21 days prior to transplantation (days −21 to day 0). In contrast, in the control group 20 recipients of allo-HSCT did not receive a symbiotic therapy.ResultsWithin first 100 days of observation, the incidence of severe (grade III/IV) aGVHD in the a synbiotic-therapy group was 0% (0 out of 20 patients), whereas it was 25% (5 out of 20 patients) in the control group (P = 0.047). The median percentage of CD4 + CD25 + Foxp3+ regulatory T cells (Tregs) among CD4+ lymphocytes on day 28 after HSCT in the synbiotic group was higher (2.54%) than in control group (1.73%; P = 0.01). There was no difference in Treg cells on day 7 after HSCT between two groups. However, the median percentage and the absolute count of Tregs in patients who experience aGVHD was significantly lower on days 7 and 28 after HSCT (both P < 0.05). The overall 12-month survival (OS) rate was higher (90%) in the symbiotic-treated patients than in the control group (75%), but the difference was not statistically significant (P = 0.234).ConclusionOur preliminary findings suggest that synbiotic intake before and during the conditioning regimen of allo-HSCT patients may lead to a reduction in the incidence and severity of aGVHD through the induction of CD4 + CD25 + Foxp3+ regulatory T cells, thus contributing to the improvement of transplant outcomes. Much larger studies are needed to confirm our observations.     

Characterization of CD3+CD4-CD8- (double negative) T cells reconstitution in patients following hematopoietic stem-cell transplantation

Background

CD3+CD4−CD8−double negative (DN) T cells, as a distinct subset of regulatory T cells (Tregs), played a pivotal role in patients following hematopoietic stem-cell transplantation.

Methods

This study examines the behavior of CD3+CD4−CD8− double negative (DN) T cells in 73 patients at days 30, 60, 90 and 180 after allo-HSCT.

Results

There was no significant difference in neutrophil and platelet engraftment between the higher and lower absolute counts of 30 days DN Tregs (p = 0.674, 0.863, respectively). The reconstitution of DN Tregs was significantly slower than that of CD8+, CD4+, and CD3+CD8+CD28− T cells (p < 0.001), but significantly faster than that of CD19+ and CD4+CD25+ T cells (p < 0.001, p = 0.032, respectively). Importantly, in the HLA mismatched group, DN Tregs reconstitution had significant effect on aGVHD (p = 0.027) and there was significant correlation between aGVHD and DN Tregs reconstitution (p = 0.035). DN Tregs reconstitution was significantly faster in the patients who were devoid of aGVHD than that of patients who developed aGVHD. Furthermore, we compared the absolute value of DN Tregs at 30 days, 60 days, 90 days and 180 days after allo-HSCT with grade aGVHD and found an inverse linear relationship in the HLA mismatched group (n = 37, P < 0.001, r = − 0.573).

Conclusions

The successful expansion of DN Tregs at 60 days after allo-HCST may help avoid severe manifestations of aGVHD in the HLA mismatched group, suggesting that DN Tregs have potential protection effect against aGVHD.     

The influence of immuosuppressive therapy on the development of CD4+CD25+ T cells after renal transplantation

A growing number of studies suggest that CD4(+)CD25(+) T regulatory (Treg) cells play a significant role to downregulate the immune response to alloantigens. In this study, we investigated the possible influence of immunosuppressive therapy, including cyclosporine (CsA) or rapamycin (sirolimus), on the level of CD4(+)CD25(+), CD4(+)CD25(+)FOXP3(+), and CD4(+)CD25(+)CTLA-4(+) T cells in the peripheral blood of renal allograft recipients. The study was performed on renal allograft recipients who displayed uneventful stable courses (RAR-S; n = 15) versus biopsy-proven chronic rejection (RAR-CH; n = 12). The patients were divided based on the immunosuppressive protocol: group 1 (prednisone+CsA+Aza) and group II (prednisone+sirolimus). The control group consisted of 10 healthy blood donors. We examined the expression of CD4, CD25, CTLA-4, and Foxp3 in peripheral blood T cells. Flow cytometry was performed with a FACSCalibur (BD Biosciences) instrument with data analyzed using Cell Quest software. The percentage of CD4(+)CD25(+)Foxp3(+) T cells in rapamycin (sirolimus) treated patients did not differ from that observed in healthy individuals, but was significantly higher compared with CsA-treated patients. CsA therapy resulted in a reduction in the percentage of CD4(+)CD25(+)CTLA-4(+) and CD4(+)CD25(+)Foxp3(+) regulatory T cells after renal transplantation in both groups (RAR-S and RAR-CH) compared with patients treated with rapamycin or to healthy donors. The type of immunosuppressive therapy (with or without calcineurin inhibitors) may have an important role in tolerance induction and graft function.     

全身照射预处理供体对异基因大鼠肝移植的作用及对受体内CD4~+CD25~+调节性T细胞的影响

目的 探讨全身照射(TBI)预处理诱导大鼠肝移植术后急性排斥反应的发生机制,及CD4~+ CD25~+调节性T细胞的变化在诱导免疫耐受中的作用.方法 以雄性Lewis、DA大鼠为供、受体,随机分为正常对照组、同种肝移植组、自发免疫耐受组、急性排斥反应组.观察各组受体的生存时间及生存率,检测受体术后外周血中ALT、TB含量、Foxp3~+ CD4~+ CD25~+ 调节性T细胞和T细胞亚群上GITR的表达,检测受体术后第14天移植肝的病理变化和受体脾脏CTL杀伤活性.结果 自发免疫耐受组,术后经历短暂排斥反应最终获得免疫耐受并长期存活.急性排斥反应组,在术后第17～21天死亡,与其他组相比,外周血血清中ALT、TB含量明显升高,而Foxp3~+ CD4~+ CD25~+调节性T细胞比例明显降低.TBI预处理大鼠供肝致受体外周血中CD3~+ CD4~+ T细胞上GITR表达降低,CD3~+CD8~+T细胞上GITR表达增加,提高CTL的杀伤活性.结论 通过TBI清除供体大鼠肝移植物中携带的旁路淋巴细胞,致受体外周血中Foxp3~+ CD4~+ CD25~+调节性T细胞表达降低,而使CD3~+ CD8~+T细胞上GITR表达增加,共同诱导大鼠肝移植术后急性排斥反应发生和耐受障碍.     

脾切除对心脏移植大鼠外周血淋巴细胞凋亡及调节性T淋巴细胞的影响

目的 探讨脾切除对同种异体心脏移植大鼠外周血淋巴细胞凋亡及调节性T淋巴细胞的影响.方法 以Wistar大鼠为供者、SD大鼠为受者,进行腹部异位心脏移植,同时切除受者的脾脏(心脏移植切脾组),并以不切脾者为对照(心脏移植对照组),另设不行任何处理的对照组和单纯切脾的单纯切脾组.术后第1、3、5、7天.取各组受者的移植心脏和外周血,观察移植心脏的组织学变化和细胞超微结构改变情况,以流式细胞仪检测外周血淋巴细胞的凋亡率及CD4+ CD25+ T淋巴细胞的变化,逆转录聚合酶链反应检测CD4+ CD25+ T淋巴细胞上Foxp3 mRNA的表达情况,记录移植心脏的存活时间.结果 心脏移植对照组移植心脏存活时间为(7.47±2.24)d,心脏移植切脾组移植心脏存活时间为(17.63±4.54)d,二者间的差异有统计学意义(P<0.05).心脏移植对照组的移植心脏肿胀,质硬,色暗,间质水肿、出血,弥漫性炎症细胞浸润,大量心肌细胞坏死、溶解,横纹不清;心脏移植切脾组的移植心脏质软,色红,局部灰白,外膜下以及细胞间局灶性水肿,炎症细胞浸润,心肌细胞结构完整,横纹清晰;心脏移植切脾组的细胞超微结构改变轻于心脏移植对照组.心脏移植切脾组术后第5天和第7天的淋巴细胞凋亡率分别为(7.62±2.15)%和(9.41±3.82)%,明显高于心脏移植对照组(P<0.05,P<0.05).心脏移植切脾组术后第3、5、7天时的CD4+ CD25+ T淋巴细胞明显多于心脏移植对照组(P<0.01,P<0.01,P<0.01),其Foxp3 mRNA的表达也较心脏移植对照组明显上调.结论 脾切除使心脏移植大鼠外周血淋巴细胞凋亡率增加,调节性T淋巴细胞增多,其Foxp3 mRNA表达上调,这些变化与移植心脏病理改变呈负相关.     

Absence of CD4CD25 regulatory T cell expansion in renal transplanted patients treated in vivo with Belatacept mediated CD28-CD80/86 blockade

AIMS: Belatacept is a new recombinant molecule (CTLA4-Ig) that interferes with the second activation signal of T lymphocytes. CTLA4-Ig induced T cell allograft tolerance in rodents but not in primates. We examined the changes in peripheral lymphocyte subsets, including regulatory T cells, in renal transplant patients treated with Belatacept. METHODS: A cross-sectional immunological study was carried out 6 months after transplantation in 28 patients enrolled in the Belatacept phase II study. Eighteen patients received Belatacept, mycophenolate mofetil and steroids (Belatacept group), while the control group of 10 patients received cyclosporine, mycophenolate mofetil and steroids (CsA group). Lymphocyte subsets were examined by flow cytometry. Foxp3 mRNA expression was measured by quantitative PCR. RESULTS: The number of T lymphocytes and the percentage of CD3+ T cells were similar in both groups. However, the percentage of CD3+ CD4+ T cells was lower in the Belatacept group than in the control CsA group (B=42.5%+/-13.7 vs CsA=52.9%+/-9, p<0.005), and the percentage of CD3+ CD8+ cells was higher in the Belatacept group than in the control (B=32.9%+/-6.7 vs CsA=19.5%+/-8.2, p<0.0002). The percentage of CD19+ cells was similar in both groups. Among CD56+cells, only the percentage of CD16+ cells was significantly higher in the Belatacept group than in the control (B=82%+/-12 vs CsA=59.7%+/-25, p=0.01). Among CD4 and CD8 T cells the percentage of activated lymphocytes expressing CTLA4, HLA-DR or CD40L was similar in both groups. The percentage of CD4+CD25+ T cells was higher in the CsA group. The percentage of regulatory CD4+CD25+ cells with bright CD25 staining was similar in both groups (B=3.6+/-2.3% vs CsA=4.7+/-1.9%, ns) as was the expression of FoxP3. CONCLUSION: Our results indicated that Belatacept did not induce regulatory T cell expansion in vivo. We suggest that Belatacept treatment should be maintained after transplantation to allow graft acceptance.     

Extracorporeal photochemotherapy is accompanied by increasing levels of circulating CD4+CD25+GITR+Foxp3+CD62L+ functional regulatory T-cells in patients with graft-versus-host disease

BACKGROUND: Extracorporeal photochemotherapy (ECP) produces clinical improvements in refractory/resistant graft-versus-host disease (GvHD). Immunological mechanisms of ECP are still under investigation. METHODS: We have evaluated the changes in frequency and immunophenotype of circulating regulatory T cells (T-regs) in 10 patients undergoing allogeneic hematopoietic stem cell transplantation, receiving ECP for acute (n=4) or chronic (n=6) GvHD. T-regs were monitored for expression of surface CD4, CD25, GITR, CD45RO, CD62L and intracytoplasmic Foxp3. T-regs were sorted by fluorescence-activated cell sorting to perform functional assays by interferon (IFN)-gamma enzyme-linked immunospot and real-time quantitative polymerase chain reaction (RQ-PCR) to measure Foxp3, transforming growth factor (TGF)-beta, and interleukin (IL)-10 mRNA. RESULTS: ECP was accompanied by a significant increase of CD4+CD25+ T-regs after six procedures, increasing from 8.9% to 29.1% of total CD4 (P<0.05), with a simultaneous increase of glucocorticoid induced tumor necrosis factor receptor expression on CD4+CD25+ cells (from 15% to 40.8%, P<0.05). This increase was sustained after 12 procedures. T-regs expressed high levels of CD62L, CD45RO, and Foxp3. Sorted CD4+CD25+ T-regs were potently inhibitory toward the CD4+CD25- fraction, when matched with an allogeneic target (IFN-gamma secretion was reduced by 79%). Trans-well experiments showed that cell-to-cell contact was necessary to exert inhibitory activity. RQ-PCR revealed a significant expression of Foxp3 in CD4+CD25+ T-regs, but there was virtually no detection of TGF-beta and IL-10. GvHD improved in all patients, allowing tapering or discontinuation of immunosuppressive drugs. CONCLUSION: Our study shows a time correlation between ECP and increasing percentages of circulating functional T-regs. Albeit suggestive, our results need to be confirmed on larger series to determine the actual role of T-reg in mediating the clinical effect of ECP.