外周血CD4^＋CD25^high调节性T细胞比例变化在肾移植受者移植后免疫监测中的应用

背景：相关实验表明调节性T细胞在移植物免疫耐受中起重要作用。 目的：观察外周血CD4^＋CD25^high调节性T细胞比例变化与肾移植受者移植后免疫变化的相关性。 设计、时间及地点：回顾性病例分析,于2007-09／2008-07在广东省第二人民医院器官移植中心及其实验室完成。 参试者：52例病情稳定的维持性血液透析患者行同种异体肾移植治疗。 方法：肾移植后患者均服用三联免疫抑制剂。所有患者移植后发生急性移植肾排斥反应以及感染均按照相应指南诊断及治疗。分别于移植前和移植后1,2,4,8,12周以及发生排斥反应和感染时抽血检测外周血CD4^＋CD25^＋调节性T细胞。按其免疫力恢复情况分为正常组26例,排斥反应组17例,感染组9例。 主要观察指标：用流式细胞仪检测外周血CD4^＋CD25^high调节性T细胞的比例,所得结果进行相关分析。 结果：与正常组比较：排斥反应组CD4^＋CD25^highFoxP3／CD4^＋的比值降低（P〈0．05）,而感染组显著增高（P〈0．01）。与感染组比较：排斥反应组CD4^＋CD25^highFoxP3／CD4^＋的比值显著降低（P〈0．01）。 结论：肾移植后受者外周血CD4^＋CD25^high调节性T细胞比例与受者免疫状态密切相关。CD4^＋CD25^high调节性T细胞比例的变化可以反应机体的免疫状态的变化,其升高或降低可以作为预测肾移植受者移植后发生感染或排斥反应的指标之一。     

肾移植患者外周血中的CD4+ CD25+ CD127low/-调节性T细胞

背景:越来越多的实验在分析免疫耐受标志,以期能够更好地辅助患者进行移植后免疫抑制治疗。目的:分析肾移植后患者外周血中CD4+CD25+CD127low/-调节性T细胞在肾移植免疫耐受中的作用。方法:采集62例肾移植后患者(急性排斥反应组22例,移植稳定组40例)及20例健康对照者的外周抗凝血,经免疫染色,应用流式细胞仪分析CD4+CD25+CD127low/-调节性T细胞所占CD4+T细胞百分含量,同时采用ELISA方法检测患者血清中白细胞介素2和白细胞介素10的质量浓度。结果与结论:移植稳定组中CD4+CD25+CD127low/-调节性T细胞所占CD4+T细胞百分含量显著高于健康对照组和急性反应排斥组(P<0.01);CD4+CD25+CD127low/-调节性T细胞百分含量与白细胞介素2呈显著负相关(P<0.05),与白细胞介素10呈显著正相关(P<0.01)。提示CD4+CD25+CD127low/-调节性T细胞在肾移植后免疫耐受的机制中发挥了一定作用。     

肾移植后外周血CD4~+及CD8~+T细胞亚群计数的临床意义

背景:临床上常以流式细胞检测受者外周血CD4、CD8细胞比值来揭示与排斥或感染相关的关系。目的:探讨肾移植后排斥或感染时外周血CD4+及CD8+T细胞(简称CD4和CD8细胞)亚群计数的变化和意义。方法:应用流式细胞仪检测肾移植121例受者CD4、CD8细胞数进行检测。根据入院病情将患者分为移植后正常组、急性排斥组、肺部感染组进行观察。结果与结论:移植后正常患者和急性排斥患者相比,CD4、CD8细胞数差异均无显著性意义(P>0.05)。肾移植后肺部感染患者CD4、CD8细胞数则均显著低于移植后正常组(P<0.001)。当感染控制、症状改善时,CD4、CD8细胞数显著升高(P<0.001)。说明肾移植后CD4和CD8细胞计数可以作为免疫状态的参考,其对于感染的参考价值大于排斥,动态观察分析有助于指导治疗。     

肾移植患者外周血中的CD4~＋ CD25~＋ CD127~（low/-）调节性T细胞

背景：越来越多的实验在分析免疫耐受标志,以期能够更好地辅助患者进行移植后免疫抑制治疗。目的：分析肾移植后患者外周血中CD4＋CD25＋CD127low/-调节性T细胞在肾移植免疫耐受中的作用。方法：采集62例肾移植后患者（急性排斥反应组22例,移植稳定组40例）及20例健康对照者的外周抗凝血,经免疫染色,应用流式细胞仪分析CD4＋CD25＋CD127low/-调节性T细胞所占CD4＋T细胞百分含量,同时采用ELISA方法检测患者血清中白细胞介素2和白细胞介素10的质量浓度。结果与结论：移植稳定组中CD4＋CD25＋CD127low/-调节性T细胞所占CD4＋T细胞百分含量显著高于健康对照组和急性反应排斥组（P〈0.01）;CD4＋CD25＋CD127low/-调节性T细胞百分含量与白细胞介素2呈显著负相关（P〈0.05）,与白细胞介素10呈显著正相关（P〈0.01）。提示CD4＋CD25＋CD127low/-调节性T细胞在肾移植后免疫耐受的机制中发挥了一定作用。     

静脉输注过氧化氢溶液上调小鼠脾脏和外周血CD4+Fox03+调节性T细胞

背景:现已证实,天然生成的调节性T细胞在维护机体免疫稳态、抑制炎性反应、抗移植排斥、抑制抗肿瘤免疫应答以及防治自身免疫病中都有重要的功能和作用.目的:探讨静脉输注过氧化氢对BALB/c小鼠CD4+CD25+Foxo3+调节性T细胞的影响.方法:取BALB/c小鼠12只,随机分为两组.H2O2处理组小鼠尾静脉注射含有H2O2的PBS;PBS组小鼠静脉注射无菌PBS.分别在注射2周后处死小鼠,采集小鼠外周血、脾脏和胸腺,采用流式细胞仪检测其中CD4+CD25+Foxp3+T细胞比例.结果与结论:H2O2处理组小鼠外周血和脾脏CD4+Foxp3+T细胞比例、CD4+Foxp3+/CD4+细胞比例均高于对照组小鼠(P<0.05).提示静脉注射H2O2能上调小鼠外周血和脾脏内CD4+Foxp3+调节性T细胞的比例.     

肺癌患者外周血CD4+CD25+调节性T细胞的变化及意义

目的:探讨肺癌患者外周血中CD4+CD25+调节性T细胞的变化及意义.方法:采用流式细胞术检测肺癌患者60例、良性肺部疾病者20例、健康体检者20例的外周血CD4+CD25+调节性T细胞水平,同时用电化学发光法检测癌胚抗原、细胞角蛋白19片断-1和神经元特异性烯醇化酶水平.结果:肺癌组外周血CD4+CD25+调节性T细胞占CD4+ T细胞比例(20.93±4.47)%,明显高于良性肺病组(15.14±4.79)%和健康对照组(15.42±3.44)%,差异有统计学意义(P<0.01).外周血CD4+CD25+调节性T细胞水平与肺癌病理类型、临床分期无明显相关性,但外周血CD4+CD25+调节性T细胞水平较低者比增高者预后好.外周血CD4+CD25+调节性T细胞占CD4+ T细胞比例对肺癌的预测价值与癌胚抗原、细胞角蛋白19片断-1相似.结论:肺癌患者外周血中CD4+CD25+调节性T细胞水平明显增高,有可能成为肺癌预测及免疫治疗的靶点.     

脐血和成人外周血中CD4+CD25+调节性T细胞的表型及频率比较

目的:比较脐血和成人外周血中CD4+CD25+调节性T细胞的表型及频率.方法:密度梯度离心法分离15例新生儿脐血和12例正常健康成人外周血.应用流式细胞术分析CD4+CD25+调节性T细胞中CD45RA、CD45RO及胞内抗原Fouxp3的表达情况:检测CD4+CD25brught/CD4+T和CD4+CD25dim/CD4+T的百分比.结果:与成人外周血相比,脐血中CD4+CD25+调节性T细胞显示为独立的细胞群;CD4+T细胞、CD4+CD25dim/CD4+T比例增高:而CD4+CD25+/CD4+T、CD4+CD25brught/CD4+T的比例降低.在表型方面,脐血CD4+CD25+调节性T细胞大部分是CD45RA+的细胞,同时表达Foxp3,但含量较成人外周血低.结论:与成人外周血相比,脐血中存在数量较多的CD4+CD25+调节性T细胞,但功能尚未成熟,可能更适合作为调节性T细胞体外培养的标本来源.     

消化道恶性肿瘤患者外周血CD4＋CD25^high调节性T细胞分析——附117例检测报告

目的:探讨消化道恶性肿瘤患者外周血CD4+CD25high 调节性T细胞(regulatory T cell,Treg)占CD4+T细胞的比例及其临床意义.方法:采用流式细胞术检测117例消化道恶性肿瘤患者(消化道恶性肿瘤组)与15名健康体检者(对照组)的外周血CD4+CD25highTreg水平,并进行比较.结果:消化道恶性肿瘤组外周血CD4+CD25highTreg占CD4+T细胞的百分率为(4.4±1.6)%,明显高于对照组的(2.0±1.0)%(P＜0.05);Ⅳ期消化道恶性肿瘤外周血CD4+CD25hgh Treg占CD4+T细胞的百分率为(5.5±1.4)%,明显高于Ⅰ～Ⅲ期消化道恶性肿瘤的(3.8±1.4)%(P＜0.05).结论:消化道恶性肿瘤患者外周血CD4+CD25high曲Treg细胞比例明显升高,可作为检测这类患者免疫状态的指标.     

恶性肿瘤患者腹水及外周血中CD4+CD25+CD127low调节性T细胞表达及其意义

目的:探讨恶性肿瘤患者腹水及外周血中CD4+CD25+CD127low调节性T细胞水平及其临床意义.方法:以36名恶性肿瘤伴腹水患者、38名恶性肿瘤患者以及15名健康人为研究对象,以三色流式分析法对CD4+CD25+CD127low调节性T细胞进行分析.结果:恶性肿瘤伴腹水组患者腹腔积液及外周血CD4+CD25+CD127low调节性T细胞占CD4+T细胞比例为(8.328±1.012)%、(8.317±1.041)%,明显高于健康对照组(6.027±0.854)%和恶性肿瘤组(7.182±1.501)%,差异有显著性(P<0.01);恶性肿瘤组外周血CD4+CD25+CD127low调节性T细胞占CD4+T细胞比例明显高于健康对照组,差异有显著性(P<0.01);恶性肿瘤伴腹水患者腹腔积液与外周血CD4+CD25+CD127low调节性T细胞占CD4+T细胞比例无统计学差异(P>0.05).结论:恶性肿瘤患者腹水及外周血CD4+CD25+CD127low调节性T细胞升高,可促进肿瘤的进一步恶化,其机制可能是抑制肿瘤的免疫.     

单剂量巴利昔单抗对肾移植受者外周血Foxp3mRNA、CD4+CD25+调节性T细胞、白细胞介素2及其受体的影响

目的观察单剂量巴利昔单抗对肾移植受者外周血Foxp3mRNA、CD4+CD25+调节性T细胞、白细胞介素2及可溶性白细胞介素2受体的影响,分析其预防移植肾急性排斥反应的作用途径.方法选择2005-10,2006-12在上海解放军第四五五医院行同种异体肾移植的患者66例,移植前均行维持性血液透析治疗.按随机数字表法分为2组,常规组接受常规免疫抑制剂治疗,巴利昔单抗组在常规免疫抑制剂治疗的基础上加用巴利昔单抗(商品名舒莱,诺华制药公司,批号S20040059,20 mg/瓶),每组33例.另外选择年龄性别相配的健康体检者30例作为对照组.以上所有观察对象均知情同意,且得到医院伦理道德委员会批准.两组肾移植患者分别于移植前(手术当天)和移植后1,2,4,8周清晨采静脉血肝素抗凝,以流式细胞仪检测外周血中CD4+CD25+调节性T细胞含量,应用荧光定量PCR检测Foxp3mRNA的表达,应用双抗体夹心酶联免疫吸附法检测血浆中自细胞介素2及其受体,采用生化仪检测血肌酐及尿素氮水平,必要时行移植肾多普勒超声检查和病理活检,观察其急性排斥反应发生情况.结果66患者全部进入结果分析,无脱落.①常规组患者急性排斥反应的发生率21.12%(7/33)显著高于巴利昔单抗组6.1%(2/33)(p＜0.05).②移植后第1,2,4和8周巴利昔单抗组Foxp3、CD4+CD25+调节性T细胞和白细胞介素2水平均高于常规组(P＜0.05～0.01),可溶性白细胞介素2受体水平均低于常规组[(306.31±98.43,327.60±109.74,316.71±104.54,388.33±13242;410.14+112.04,442.82±118.94,450.80±123.63,445.61±115.24)U/mL(p＜0.05～0.01)].③两组肾移植患者无论移植前还是移植后,其血浆CD4+CD25+调节性T细胞百分率与Foxp3mRNA表达水平均呈明显正相关(r=0.892～0.932,P＜0.01).结论单剂量巴利昔单抗可以有效地减少移植肾急性排斥反应的发生率,作用途径可能与其增加外周血中Foxp3mRNA、CD4+CD25+调节性T细胞和白细胞介素2表达,减少血浆可溶性白细胞介素2受体水平有关.     

Tyrosine phosphorylation of CD6 by stimulation of CD3: augmentation by the CD4 and CD2 coreceptors

When T cells are activated via the T cell receptor (TCR) complex a number of cellular substrates, including some cell surface proteins, become phosphorylated on tyrosine (Tyr) residues. Phosphorylation of cytoplasmic Tyr renders these cell surface receptors competent to interact with proteins that link cell surface receptors to protein in the intracellular signaling pathways. Here we show that Tyr residues in the cytoplasmic domain of CD6 become phosphorylated upon T cell activation via the TCR complex. Tyr phosphorylation was observed when the T cells were activated by crosslinking CD3 or by cocrosslinking CD3 with CD2 or CD4, but not when the cells were stimulated by crosslinking CD2, CD4, or CD28 alone. Unlike other Tyr kinase substrates, such as the phospholipase C gamma 1-associated pp35/36 protein, whose level of Tyr phosphorylation is highest when T cells are activated by cocrosslinking CD3 with CD2, the levels of CD6 Tyr phosphorylation are highest when T cells were activated by cocrosslinking CD3 with CD4.     

CD137 enhances cytotoxicity of CD3CD56 cells and their capacities to induce CD4 Th1 responses

CD137 (4-1BB) is a TNFR superfamily member that mediates the costimulatory signal resulting in T cells and NK cells proliferation and cytokines production, but the effects of CD137 signaling on CD3⁺CD56⁺ cell subpopulation have not been well-documented. The aim of this study was to investigate the effects of CD137 signaling on regulation of CD3⁺CD56⁺ cell function. Anti-CD137 mAb or mouse IgG1 isotype control was added to CIK cell culture to determine the effects of proliferation and anti-tumor effects on CD3⁺CD56⁺ cells. We observed that anti-CD137 mAb could dramatically promote proliferation of CIK cells. And CD137–CIK cells and CD3⁺CD56⁺ cell subpopulation within them possessed higher ability to kill tumor cell line A549. The SCID mice engrafted with A549 cells and treated with CD137–CIK cells have prolonged survival. Further studies revealed that the percentages of CD3⁺CD56⁺ cells were elevated significantly in CD137–CIK cells. The expression of NKG2D was up-regulated on CD3⁺CD56⁺ cells from CD137–CIK cells. The expression of IFN-γ, IL-2 and TNF-α increased significantly whereas the production of TGF-β₁, IL-4 and IL-10 decreased in CD3⁺CD56⁺ cells from CD137–CIK cells. In addition, anti-CD137 mAb can elevate the capacity of CD3⁺CD56⁺ cells to induce CD4⁺ Th1 responses. We further showed that the anti-CD137 mAb also had the same effects on CD3⁺CD56⁺ cells expanded from the PBMCs of patients with NSCLC. We concluded that CD137 signaling could enhance the abilities of CIK cells to kill tumor cells in vitro and in vivo via increasing the proportion of CD3⁺CD56⁺ cells and their cytotoxicity. Furthermore, CD137 signaling can elevate the capacity of CD3⁺CD56⁺ cells to induce CD4⁺ Th1 responses which may enhance their anti-tumor activity indirectly. Taken together, our studies could be considered as valuable in CIK cells-based cancer immunotherapy.     

CD4+CD25+和CD8+调节性T细胞的作用机制

调节性T细胞（Treg）主要在机体免疫系统中发挥负向调节作用,既能抑制不恰当的免疫反应,又能限定免疫应答的范围、程度及作用时间,对CD4^＋和CD8^＋效应性T淋巴细胞的增殖起抑制作用,因此在移植物抗宿主病、自身免疫病、过敏性疾病等的发病机制和临床治疗中有潜在的应用价值.本文重点介绍CD4^＋CD25^＋Treg和CD8^＋Treg的作用机制,并简述调节性T细胞研究面临的挑战与展望.     

Differential increase of CD34, KDR/CD34, CD133/CD34 and CD117/CD34 positive cells in peripheral blood of patients with acute myocardial infarction

Objective Circulating progenitor cells (CPC) may contribute to cardiac regeneration and neovascularization after acute myocardial infarction (AMI). For potential therapeutic use, understanding the endogenous mechanisms after ischemia is inevitable. We investigated the absolute number, but also the subset composition of CD34+ CPC after AMI. Methods CD34+, KDR+/ CD34+, CD133+/CD34+ and CD117+/CD34+ CPC were analyzed by FACS in peripheral blood of 10 patients with acute MI (59±5 yrs, m/f=8/2) at day of AMI (day 0) and days 1–5. For comparison patients with stable coronary artery disease (CAD, n=12, 66±2 yrs, m/f=10/2) and young healthy volunteers (n=7, 26±2 yrs, m/f=3/4) were studied. Results CD34 and KDR/CD34, CD133/CD34, CD117/CD34 were increased day 3 and 4 after AMI. KDR+ fraction within CD34+ population remained unchanged (58.3±7.8% vs 55.3±10.6%), whereas CD133+ (64.9±3.1% vs 43.5±5.9%, P=0.006) and CD117+ fractions (71.7±5.6% vs 50.1±5.5%, P=0.02) were elevated. In CAD, all CPC and fractions were similar as AMI day 0. Healthy volunteers had more CD34+ than CAD and AMI day 0. Double positive CPC were also higher, but fractions were unchanged vs CAD with more KDR/CD34 in trend (72.8±10.6% vs 50.5±5.6%, P=0.058). After AMI both absolute numbers of CD34+ and their subset composition change, suggesting selective mobilization of CPC. Increased CPC after AMI never reach numbers of young healthy volunteers.     

CD28／CD152：CD80／CD86分子在强直性脊柱炎患者外周血淋巴细胞上的表达

目的探讨共刺激分子CD28／CD152：CD80／CD86在强直性脊柱炎（AS）患者外周血淋巴细胞上的表达及与免疫功能的关系。方法流式细胞仪检测AS患者及健康体检者T细胞表面标志CD3、CD4、CD8、CD19的表达及CD28／CD152和CD80／CD86在外周血T、B淋巴细胞上的表达水平；采用速率散射比浊法、酶联免疫吸附试验（ELISA）测定血清中免疫球蛋白IgG、IgA、IgM及C-反应蛋白（CRP）和白细胞介素-6（IL-6）水平。结果CD3^＋CD4^＋T细胞及CD4^＋／CD8^＋比值明显高于健康对照组（P〈0．05），而CD3^＋CD8^＋T细胞则明显低于健康对照组（P〈0．05）；CD4^＋和CD8^_T细胞上CD28和CD19^＋B细胞上CD80、CD86的表达均较健康对照组增高（P〈0．05），而CD152在CD4^＋T淋巴细胞上的表达也比健康对照组显著增加，但在CD8%＋T淋巴细胞上的表达却显著降低（P〈0.05）；血清IgG、IgA及CRP、IL-6水平均较健康对照组显著增高（P〈0．05）。结论AS患者外周血淋巴细胞CD28／CD152：CD80／CD86分子的异常表达与其免疫功能紊乱有密切关系。     

哮喘患儿CD8+CD28+、CD8+CD28-T淋巴细胞检测及其临床意义

目的通过对哮喘患儿CD4、CD8和CD28的联合检测,探讨哮喘患儿淋巴细胞免疫功能状态及其临床意义.方法采用流式细胞术检测哮喘患儿外周血的总T细胞(CD3+)、辅助/诱导T淋巴细胞(CD4+)、抑制/细胞毒T淋巴细胞(CD8+)、细胞毒T细胞(CD8+CD28+)、抑制T细胞(CD8+CD28-).结果哮喘患儿组与对照组比较;CD3+、CD4+、CD8+CD28-细胞均低于对照组(P＜0.01,P＜0.05),CD8+CD28+细胞增高(P＜0.05),CD4+/CD8+比值、CD8+与对照组比较均无显著性差异(P＞0.05).结论哮喘患儿存在T淋巴细胞亚群免疫功能紊乱,而CD8+CD28+、CD8+CD28-T细胞失衡可能是导致机体免疫功能紊乱的主要因素.     

Contribution of CD4+, CD8+CD28+, and CD8+CD28- T cells to CD3+ lymphocyte homeostasis during the natural course of HIV-1 infection.

The relationship between the number of circulating CD4+ T cells and the presence of particular CD8+ T cell subsets was analyzed by flow cytometry on PBL from asymptomatic HIV-1-infected patients whose specimens were collected every 2 mo for a total period of 32 mo. Only slight variations were detected in the absolute number of lymphocytes and percentage of CD3+ lymphocytes, whereas both CD4+ and CD8+ T cell subsets showed wide intrapatient variation. Variations in the number of CD8+CD28+ cells paralleled those of the CD4+ T cell subset in each patient tested, while the presence of CD8+CD28- T cells correlated inversely with CD4+ and CD8+CD28+ T cells. These data show that changes in the number of circulating CD4+-and CD8+CD28+ T cells are strongly related to the presence of CD8+CD28- T cells in these patients. Insight into the significance of CD8+CD28- T cell expansion will allow us to understand the mechanisms and significance of the HIV-1- driven change in CD4+CD8+ T cell homeostasis and the basic immunopathology of HIV disease.