日本血吸虫热休克蛋白60 kDa的免疫原性及保护性研究

目的研究日本血吸虫热休克蛋白60 kDa(SjHSP60)免疫原性,评价其免疫保护性及可能机制。方法通过在线生物信息学软件预测SjHSP60的B细胞表位,随后用SjHSP60免疫BALB/c小鼠后检测特异性抗体水平和淋巴细胞增殖以评估其免疫原性,进一步通过攻击感染实验观察其免疫保护效果;最后分选免疫小鼠的CD4~+CD25~+调节性T细胞(Treg)与CD4~+CD25~-T细胞共培养,通过细胞增殖实验检测CD4~+CD25~+Treg细胞的抑制功能。结果生物信息学预测结果显示,SjHSP60中存在多个B细胞表位的优势区段。SjHSP60可在小鼠体内诱导特异性抗体(P0.01),也可诱导小鼠脾细胞发生增殖(P0.01)和分泌IFN-γ(P0.01);但攻击感染实验显示,SjHSP60免疫小鼠体内虫荷(P0.05)、卵荷(P0.05)均未显著减少。体外共培养实验表明,SjHSP60可显著增强小鼠CD4~+CD25~+Treg细胞的免疫抑制功能(P0.01)。结论SjHSP60对宿主免疫系统可发挥"双刃剑"作用,即在诱导特异性抗感染免疫应答的同时又可增强免疫抑制功能,从而削弱了其免疫保护作用。     

慢性丙型肝炎患者CD4+CD25+调节性T细胞表达增加

目的:探讨CD4+CD25+调节性T(Treg)细胞在慢性丙型肝炎患者免疫下调中的意义.方法:流式细胞仪检测慢性丙型肝炎患者外周血中CD4+CD25+Treg细胞的数量;与CD4+CD25-T细胞共同培养,检测其抑制功能;流式细胞仪检测其对CD4+CD25-T细胞合成IFN-γ和IL-4的影响;RT-PCR检测CD4+CD25+Treg细胞中Foxp3的mRNA表达.结果:CD4+CD25+Treg细胞约占慢性丙型肝炎患者外周血中CD4+T细胞的14.1±1.6%,显著高于正常对照5.3±0.8%(P＜0.01),显著抑制CD4+T细胞的增殖(P=0.002),以及合成IFN-γ.CD4+CD25+Treg 细胞高表达Foxp3.结论:持续性HCV感染患者CD4+CD25+Treg细胞表达增加,特异性抑制Th1细胞反应.     

CD+4 CD+25调节性T细胞抑制持续性丙型肝炎病毒感染患者CD+4T细胞反应

目的探讨CD+4 CD+25调节性T细胞(CD+4 CD+25Treg细胞)在持续性HCV感染患者CD+4 T细胞下调中的意义.方法流式细胞术检测慢性丙型肝炎患者外周血中CD+4 CD+25Treg细胞的数量以及细胞内因子的合成;与正常人或患者CD+4 CD-25 T细胞共同培养,检测其抑制功能;RT-PCR检测Foxp3的mRNA表达.结果 CD+4 CD+25Treg细胞约占慢性丙型肝炎患者外周血中CD+4 T细胞的(13.5±1.8)%,高于正常对照(5.3±0.8)% (P=0.004);主要合成IL-10,高表达Foxp3;CD+4 CD+25Treg细胞显著抑制CD+4 T细胞的增殖,以及合成IFNγ,并且抑制活性较正常人增高(P=0.034),这种作用不依赖IL-10和转化生长因子β.结论持续性HCV感染患者CD+4 CD+25Treg细胞表达增加,抑制活性增强,特异性抑制Th1反应.     

CD4+CD25+调节性T细胞与克罗恩病发病关系

目的研究克罗恩病（CD）患者外周血白细胞介素（IL）-6水平与外周CD4＋CD25^＋调节性T细胞（Treg细胞）频率及体外抑制功能的变化在CD发病中的作用。方法应用流式细胞术检测CD患者与正常对照者外周CD4^＋CD25^＋Treg细胞的频率及表型以及特征性标志叉状头／翅膀状螺旋转录因子（Foxp3）的表达，同时通过MACS缓冲液分选出外周血CD4^＋CD25^＋T细胞和CD4＋CD25^-T细胞，应用[^3H]-胸腺嘧啶渗入法研究CD4^＋CD25^＋T细胞对自体CD4^＋CD25^-效应T细胞增殖的抑制能力。酶联免疫吸附法（ELISA）检测外周血IL-6水平。结果活动性CD患者外周血IL-6水平显著高于非活动性患者及正常对照者。活动性CD患者外周CD4^＋T细胞中CD4^＋CD25^＋Foxp3^＋T细胞的频率显著低于非活动性CD患者，差异有统计学意义。体外抑制功能试验同样提示活动性CD患者的CD4^＋CD25^＋Treg细胞抑制功能减弱。结论CD4^＋CD25^＋Treg细胞抑制功能减弱与CD发病可能有关，可初步解释CD患者出现的免疫耐受缺失现象。     

抗病毒治疗对慢性丙型肝炎CD4^＋CD25^＋调节性T细胞频率和功能的影响

目的研究抗病毒治疗前后慢性丙型肝炎患者CD4^＋CD25^＋调节性T细胞（Treg）频率和功能的变化。方法筛选HLA—A2阳性慢性丙型肝炎患者31例，给予聚乙二醇化干扰素α-2a（相对分子质量为40000）180μg每周1次皮下注射，联合口服利巴韦林。分别在治疗前和治疗结束随访24周时应用流式细胞仪检测患者CD4^＋CD25^＋ Treg细胞占外周血CD4^＋T细胞的频率，应用液闪计数仪检测其对HCV特异性CD8^＋T细胞增殖的抑制作用，ELISA法检测细胞培养上清γ干扰素（IFN-γ）水平的变化情况。结果治疗结束随访24周，患者外周血CD4^＋CD25^＋ Treg细胞频率为（9．6±3．0）％，明显低于治疗前的（11．0±2．3）％（t=2．028，P〈0．05）；持续病毒学应答（SVR）组CD4^＋CD25^＋ Treg细胞频率为（8．9±2．7）％，明显低于未获得SVR患者组的（10．4±2．3）％（t=3．324，P〈0．01）。抗病毒治疗后CD4^＋CD25^＋ Treg细胞抑制HCV特异性CD8^＋T细胞增殖的作用减弱。治疗后患者IFN-γ水平为（3959±577）pg／ml，明显高于治疗前的（1965±326）pg／ml（t=16．1，P〈0．01）；获得SVR患者组IFN-γ（6824±568）pg／ml，明显高于未获得SVR患者组的（2219±286）pg／ml（t=29．853，P〈0．001）。结论慢性丙型肝炎患者随着HCV RNA水平的下降，CD4^＋CD25^＋Treg细胞频率降低，抑制HCV特异性CD8^＋T细胞增殖的作用减弱。     

SLE患者外周血CD4+ CD25+调节性T细胞及相关基因Foxp3的变化

目的研究系统性红斑狼疮（SLE）合并狼疮性肾炎患者经肾上腺糖皮质激素（简称激素）冲击治疗后，外周血CD4^＋CD25^＋调节性T细胞（regulatoryTcell，Treg）及相关基因Foxp3表达的变化，从而探讨CD4^＋CD25^＋Treg和Foxp3与SLE发病的相关性。方法采用流式细胞术检测SLE患者外周血单个核细胞（PBMC）中CD4^＋CD25^＋T、CD4^＋CD25highT细胞数量的变化，RTPCR检测PBMC中CD4^＋CD25^＋Treg功能相关基因Foxp3mRNA的表达。结果激素冲击治疗后的SLE患者CD4^＋CD25^＋T／CD4^＋T及CD4^＋CD25highT／CD4^＋T比值高于正常对照组；Foxp3mRNA水平表达与对照组差异不显著。结论CD4^＋CD25^＋Treg数量和功能的变化可能参与SLE的发病，激素可能通过提升CD4^＋CD25^＋Treg治疗SLE。     

CD4+CD25+Treg细胞与支气管哮喘

CD4+ CD25+ Treg细胞的主要作用表现为免疫无能性和免疫抑制性,是外周免疫耐受形成机制的主要组成部分.其主要作用机制为分泌抑制性细胞因子(IL-10和TGF-β)、表达细胞表面分子(CTLA-4、GITR等)及Foxp3等.支气管哮喘患者外周血CD4+ CD25+ Treg功能及数量存在异常,这可能是支气管哮...     

不同免疫抑制诱导治疗对肝移植受者CD4+CD25+high细胞的影响

目的CD4^＋CD25^＋Treg细胞介导的免疫调节对诱导和维持移植物免疫耐受有非常重要的作用，本研究探讨抗胸腺淋巴细胞球蛋白（ATG）和噻呢哌这两种不同作用途径的免疫抑制诱导药物对CD4^＋CD25^＋high／CD4^＋T细胞及其表面标志性因子CTLA-4、Foxp3的影响，为临床合理选择应用免疫诱导药物提供依据。方法选择15例在我院行同种异体肝移植患者，随机分为ATG组（8例）和噻呢哌组（7例），两组除免疫抑制诱导药物不同外，均接受相同治疗。分别于术前、术后3周、6周、8周取血，应用流式细胞仪检测CD4^＋CD25^＋high/CD4^＋T细胞的数量及其表面因子CTLA-4和Foxp3表达状况，比较不同诱导药物对其影响。结果这两种免疫抑制诱导药物对CD4^＋CD25^＋Treg细胞短期内有一定影响，但可逐渐恢复，两者比较，使用ATG后各指标恢复较噻呢哌快，尤其是Foxp3更加明显。结论作为诱导治疗药物，ATG更有利于CD4^＋CD25^＋Treg细胞功能恢复，从而有助于更好形成移植免疫耐受。     

CD4+CD25+调节性T细胞对日本血吸虫病疫苗保护性效果的影响及机制研究

目的 目的 探讨CD4+ CD25+ 调节性T细胞 （Tregs） 对日本血吸虫病疫苗保护性效果的影响及其机制。 方法 方法 雌性 BALB/c小鼠随机分成5组, 即正常对照组、 感染对照组、 抗CD25单克隆抗体 （anti?CD25 mAb） 组、 谷胱甘肽?S?转移酶 （Gluthatione?S?transferase,GST） 免疫组和GST/anti?CD25 mAb联合组。分别在感染后2、 3、 4、 5周剖杀小鼠, 收集脾细胞 及培养上清, 采用流式细胞术检测脾细胞中CD4+ CD25+ Tregs比例, 双抗夹心ELISA法测定脾细胞培养上清中的IFN?γ、 IL?2、 IL?4、 IL?5和TGF?β水平。感染后5周杀鼠, 门静脉冲虫, 统计每只小鼠虫荷及每克肝脏虫卵数; 肝组织石蜡切片HE 染色观察虫卵肉芽肿病理变化。 结果 结果 感染后5周, GST免疫组小鼠减虫率为24.98%, 而GST/anti?CD25 mAb联合组减 虫率达43.13%; GST免疫组小鼠脾细胞中CD4+ CD25+ Foxp3+ 比例显著高于感染对照组 （P < 0.05）, 而anti?CD25 mAb组小 鼠脾细胞中CD4+ CD25+ Foxp3+ 比例显著低于感染对照组 （P < 0.01）。使用anti?CD25 mAb后2周, GST/anti?CD25 mAb联合 组小鼠脾细胞培养上清中IL?4、 IL?5、 IFN?γ和IL?2含量均较其他组高; 各组小鼠肝脏病理变化和脾细胞培养上清中TGF? β水平间差异均无统计学意义 （P 均 > 0.05）。结论 结论 GST疫苗可引起日本血吸虫感染宿主CD4+ CD25+ Tregs 明显上升, 从 而导致其保护性效果欠佳; anti?CD25 mAb部分封闭CD4+ CD25+ Tregs后有利于增强日本血吸虫病疫苗的免疫保护性效 果, 其机制可能与Th1、 Th2型免疫反应增强有关。     

胰岛素抵抗的慢性丙型肝炎患者外周血CD4+CD25+调节性T细胞的数量及其功能

目的 了解胰岛素抵抗的慢性丙型肝炎患者外周血CD4 +CD25+调节性T细胞(Treg)数量和功能的变化.方法 筛选40例HLA-A2+慢性丙型肝炎患者(其中20例合并胰岛素抵抗),流式细胞仪检测患者CD4+CD25+Treg细胞占外周血中CD4+T细胞的频率,液闪计数仪检测对HCV特异性CD8+T细胞增殖的抑制作用,ELISA法检测IFN-y水平.统计学处理采用t检验.结果 胰岛素抵抗的慢性丙型肝炎患者外周血CD4 +CD25+ Treg细胞占CD4+T细胞的(9.5±1.9)％,明显低于慢性丙型肝炎患者的(11.2±2.2)％(t=2.615,P＜0.05).胰岛素抵抗指数(HOMA-IR)≥4患者的CD4+CD25+ Treg细胞比例为(9.0±1.8)％,明显低于HOMA-IR＜4患者的(10.8±2.3)％(t=2.413,P＜0.05).胰岛素抵抗的慢性丙型肝炎患者CD4+CD25+ Treg细胞和去除Treg的外周血单个核细胞(PBMC)共培养上清液中IFN-y为(4 050±580) pg/mL,明显高于慢性丙型肝炎患者的(2 005±330)pg/mL(t=13.705,P＜0.01).HOMA-IR≥4患者IFN-y为(5 682±986)pg/mL,明显高于HOMA-IR＜4患者的(2 819±660) pg/mL(t=7.630,P＜0.01).结论 随着胰岛素抵抗程度加重,慢性丙型肝炎患者外周血CD4+ CD25+ Treg细胞频率减低,对HCV特异性CD8+T细胞增殖的抑制作用减弱.     

Suppressive activity and altered conventional phenotype markers/mediators of regulatory T cells in patients with self‐limiting hepatitis E

Hepatitis E virus (HEV) is a major cause of self‐limiting acute viral hepatitis in several developing countries. Elevated levels of peripheral CD4⁺CD25⁺Foxp3⁺, CD4⁺CD25⁻Foxp3⁺ and rise in IL‐10 in hepatitis E have been associated with the involvement of regulatory T cells (Treg). The functional role of the same is yet elusive. In the current study, we have assessed (i) Foxp3 expression by real‐time PCR and by flow cytometry, (ii) the levels of antigen‐specific IL‐10 and TGF‐β by ELISA, (iii) functional analysis of Treg cells and (iv) expression of Treg‐associated conventional phenotypes by flow cytometry in 54 acute patients, 44 recovered individuals from hepatitis E and in 33 healthy controls. Foxp3 mRNA elevation in the acute compared with recovered group and elevation in Foxp3⁺ cells in both patient groups were significantly elevated. The levels of IL‐10 and TGF‐β in the acute patients and TGF‐β in the recovered individuals were elevated. Significantly higher expression of CTLA‐4, PD1, GITR, CD95, CD103 and CD73 on Treg and T effector (Teff) cells was detected in the patient groups. Treg cells of acute patients and recovered individuals exhibited suppressive activity indicating that the Treg cells of hepatitis E patients are functional. The suppressive capacity of Treg cells in acute hepatitis E patients was significantly higher compared with the recovered individuals. Based on our findings, the suppressive functionality of these key markers associated with hepatitis E Treg function need further exploration to get a better understanding of the mechanisms of Treg‐mediated suppression.     

Elevated frequency and function of regulatory T cells in patients with active chronic hepatitis C

Background

Regulatory T cells (Tregs) play a pivotal role in the persistence of hepatitis C virus infection. The aim of this study was to evaluate the frequency and function of Tregs in patients with chronic hepatitis C (CHC).

Methods

We enrolled 44 CHC patients with elevated alanine aminotransferase (ALT) levels (CH group), 13 CHC patients with persistent normal ALT levels (PNALT group), and 14 age-matched healthy subjects (HS group; controls). Tregs were identified as CD4+, CD25+, and forkhead box P3 (Foxp3)+ T lymphocytes, using three-color fluorescence-activated cell sorting (FACS). The frequency of Tregs was determined by calculating the percentage of CD4+CD25^high T cells among CD4 T cells. CD127 and CD45RA were also analyzed for subsets of Tregs. The levels of serum transforming growth factor (TGF)-β and interleukin (IL)-10 in immunosuppressive assays were detected by enzyme-linked immunosorbent assay (ELISA). The immunosuppressive abilities of Tregs were evaluated by measuring their ability to inhibit the proliferation of effector cells.

Results

Higher proportions of Tregs were found in the CH and PNALT groups compared with the HS group. The populations of CD127 low/negative and CD45RA negative cells were higher in the CH group than in the PNALT group. The expressions of IL-10 and TGF-β in the CH and PNALT groups were significantly higher than those in the HS group. In addition, the immunosuppressive ability of Tregs from the CH group was increased relative to that in the PNALT and the HS group.

Conclusions

CHC patients, irrespective of liver function, had higher frequencies of Tregs than healthy subjects; however, only CHC patients with inflammation showed enhanced immunosuppressive function of Tregs.     

Increased expression of regulatory T cells and down-regulatory molecules in lepromatous leprosy

T regulatory cells (Tregs) play an important role in the mechanism of host's failure to control pathogen dissemination in severe forms of different chronic granulomatous diseases, but their role in leprosy has not yet been elucidated; 28 newly diagnosed patients (16 patients with lepromatous leprosy and 12 patients with tuberculoid leprosy) and 6 healthy Mycobacterium leprae-exposed individuals (contacts) were studied. Tregs were quantified by flow cytometry (CD4+ CD25+ Foxp3+) in peripheral blood mononuclear cells stimulated in vitro with a M. leprae antigenic preparation and phytohemagglutinin as well as in skin lesions by immunohistochemistry. The lymphoproliferative (LPR), interleukin-10 (IL-10), and interferon-γ (IFN-γ) responses of the in vitro-stimulated peripheral blood mononuclear cells and the in situ expression of IL-10, transforming growth factor-β (TGF-β), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) were also determined. We show that M. leprae antigens induced significantly lower LPR but significantly higher Treg numbers in lepromatous than tuberculoid patients and contacts. Mitogen-induced LPR and Treg frequencies were not significantly different among the three groups. Tregs were also more frequent in situ in lepromatous patients, and this finding was paralleled by increased expression of the antiinflammatory molecules IL-10 and CTLA-4 but not TGF-β. In lepromatous patients, Tregs were intermingled with vacuolized hystiocyte infiltrates all over the lesion, whereas in tuberculoid patients, Tregs were rare. Our results suggest that Tregs are present in increased numbers, and they may have a pathogenic role in leprosy patients harboring uncontrolled bacillary multiplication but not in those individuals capable of limiting M. leprae growth.     

The immunosuppressive effects of CD4+CD25+ regulatory T cells on dendritic cells in patients with chronic hepatitis B

The characteristics and functions of CD4⁺CD25⁺ regulatory T cells (Tregs) have been well defined in murine and human systems. However, the interaction or crosstalk between CD4⁺CD25⁺ Tregs and dendritic cells (DCs) remains controversial. In this study, the effects of chronic hepatitis B (CHB) CD4⁺CD25⁺ Tregs on the maturation and function of monocyte‐derived DCs were examined. The results showed that CD4⁺CD25⁺ render the DCs inefficient as antigen‐presenting cells (APCs) despite prestimulation with CD40 ligand. This effect was marginally reverted by applying neutralizing antibodies (Abs) to IL‐10 and TGF‐β. There were an increased IL‐10 and TGF‐β secretion and reduced expression of costimulatory molecules in DC. Thus, in addition to a direct suppressor effect on CD4⁺T cells, CD4⁺CD25⁺ may modulate the immune response through DCs in CHB patients.     

日本血吸虫可溶性成虫抗原和虫卵抗原诱导调节性T细胞能力差异的比较

目的 目的 观察并比较日本血吸虫可溶性成虫抗原 （SWA） 与可溶性虫卵抗原 （SEA） 诱导CD4+ CD25+ Foxp3+ 调节性T （Treg） 细胞水平及其抑制能力的差异。方法 方法 分别以PBS、 SWA和SEA免疫小鼠, 取小鼠脾淋巴细胞, 采用流式细胞术检测各组Treg细胞占CD4+ T细胞的比例, 以及Treg细胞中产IL?10、 TGF?β的细胞比例。采用磁珠分选出各组小鼠的Treg细胞后, 以3 H?TdR掺入法检测其抑制靶细胞增殖的能力。结果 结果 与SWA相比, SEA可诱导更多的Treg细胞 （P < 0.05）, 刺激 Treg细胞免疫抑制功能的能力也更强 （P < 0.05）; SWA、 SEA免疫小鼠后, SEA刺激Treg细胞产生IL?10、 TGF?β的能力更强（P < 0.01）。结论 结论 SEA较SWA能更好地活化诱导Treg细胞发挥免疫抑制作用。     

Characteristics of regulatory T‐cell function in patients with chronic hepatitis B and C coinfection

Regulatory T cells (Tregs) affect the pathogenesis and disease progression of chronic viral hepatitis. This study evaluated the frequency and function of Tregs in patients with chronic HBV/HCV coinfection. Seventy‐four untreated HBV/HCV co‐infected patients were enrolled in this study. These subjects were divided into four subgroups: HBV‐active/HCV‐active (BACA), HBV‐inactive/HCV‐active (BICA), HBV‐active/HCV‐inactive (BACI) and HBV‐inactive/HCV‐inactive (BICI). Treg frequency was calculated as the fraction of CD4⁺Foxp3⁺T cells among CD4⁺T cells. Treg‐mediated inhibition was measured as percent of inhibition of T‐cell proliferation. The expression of interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α and interleukin (IL)‐10 with/without Treg inhibition was also studied. Among the patients, there were 8 cases of BACA (10.8%), 38 of BICA (51.4%), 14 of BACI (18.9%) and 14 of BICI (18.9%). The frequency of CD4⁺Foxp3⁺T cells was comparable between the four groups. The inhibitory function of Tregs among the patients in the BACA and BICA was higher than that in the BICI (BACA vs BICI, P = .0210; BICA vs BICI, P = .0301). Patients in the BACA and BICA had higher fibrosis‐4 (FIB‐4) scores and serum ALT levels and lower serum albumin levels than those of the other groups. ALT abnormality was significantly and independently associated with a higher Treg immunosuppressive ability. The IFN‐γ expression of the effector T cells in the BACA was higher than that of the other groups. In conclusion, the inhibitory function of Tregs is higher among the HBV/HCV co‐infected patients with active HCV infection. ALT abnormality plays a dominant role in Treg function.     

Induction of regulatory T cells by Opisthorchis viverrini

Opisthorchis viverrini causes public health problems in South‐East Asia. Recently, TGF‐β and IL‐10 have been reported to increase in O. viverrini‐infected hamsters but the sources of these cytokines are still unknown. In this study, the CD4⁺ T cells in infected hamsters were investigated. It was demonstrated that IL‐4⁺CD4⁺ T cells were significantly increased in hamster spleens and mesenteric lymph nodes (MLNs) during chronic infection. Interestingly, IL‐10⁺CD4⁺ T cells were also discovered at a significant level while Treg (T regulatory)‐like TGF‐ β⁺CD4⁺ T cells were in MLNs of infected hamsters. Moreover, the CD4⁺CD25⁺Foxp3⁺ Treg cell response was significantly found both in spleens and MLNs in infected hamsters. The findings were then confirmed by development of T‐cell clones against crude somatic antigens (CSAg) in immunized BALB/c mice. Five clones named TCC21, TCC23, TCC35, TCC41 and TCC108 were established. The TCC21 was found to be the TGF‐β⁺CD4⁺ while TCC35, TCC41 and TCC108 were IL‐4⁺CD4⁺ and TCC23 was IFN‐γ⁺CD4⁺. This TGF‐β⁺CD4⁺ T clone showed an inhibitory function in vitro in mononuclear cell proliferation via TGF‐β‐mediated mechanisms. This study indicated that O. viverrini‐infected hamsters could induce TGF‐β⁺ CD4⁺ Treg‐like cells. The CSAg‐specific Tregs secreted high TGF‐β, and limited immune cell proliferation.