淋巴细胞共刺激信号和凋亡信号异常与RA免疫效应亢进的研究

目的：探究T淋巴细胞表面多种细胞信号分子所介导的细胞活化或凋亡信号在RA患者免疫功能紊乱中的作用。方法：采用流式细胞术检测RA患者外周血T细胞亚群及其表面共刺激分子cD154（cD40L）、CD30和凋亡受体CD95（Fas）的表达。结果：RA患者外周血T细胞亚群偏移，CD4^＋T细胞增加，CD8^＋T细胞减少；共刺激分子CD154在CD4^＋和CD8^＋T细胞上的表达均上调，但CD30分子的表达均降低，并以CD4^＋T细胞降低更为明显。同时，凋亡受体CD95分子在T细胞亚群上的表达均明显增加。结论：RA患者T淋巴细胞表面多种信号分子表达异常，共同导致了RA患者免疫功能紊乱。     

环孢菌素通过FLIP影响再生障碍性贫血患者CD8+T细胞亚群CD28的表达

目的研究共刺激分子CD28在再生障碍性贫血（AA）患者的不同T细胞亚群中的表达变化，及其与凋亡抑制蛋白FLIP的关系。方法取21例AA患者环孢菌素A（CyA）治疗前后的外周血，应用流式细胞仪检测T细胞CIMCD28、CD8CD28的表达；磁珠分选患者治疗前后外周血的CD8^＋T细胞，RT-PCR检测治疗前后FLIP、Caspase-8表达变化；分选获得的治疗前的CD8^＋ T细胞体外经CyA处理后检测其CD28、FLIP、Caspase-8的表达变化。结果AA患者的CD8^＋ CD28^＋ T细胞的比例较正常人显著升高，治疗有效者该亚群的细胞比例在治疗后趋于正常；患者CD8^＋ T细胞的FLIP表达显著上调，但Caspase-8的表达无明显变化；CyA能下调患者CD8^＋T细胞的FLIP的表达。结论AA患者CD8^＋CD28^- T细胞亚群比例升高与FLIP表达增加有关，CyA能抑制FLIP的表达，降低CD8^＋CD28^-T细胞的比例。     

移植免疫耐受中调节性T细胞的研究进展

调节性T细胞（Tr）在诱导和维持移植免疫耐受过程中的作用越来越受到人们的重视。根据不同的细胞表型和功能已将Tr分为若干亚类，首先是胸腺内T细胞发育过程中自然产生的CD4^＋T细胞亚群，能够构成性表达IL-2受体的仅链（CD25）存在于外周，体内体外均可抑制效应性T细胞的增殖和功能；其次是产生于正常的免疫应答过程中的诱导性Tr亚群，包括Tr1和Th3通过分泌细胞因子如IL-10和TGF—β发挥抑制作用。除此之外，也有报道CD8^＋Tr、双阴性T（DNT）细胞和NKT细胞在移植耐受的不同模型中亦发挥重要作用。因而研究各种Tr亚群的生物学特征、调节机制及其在移植耐受中的作用非常必要。     

T细胞凋亡受体和共刺激分子的表达变化与终末期肾功能衰竭患者细胞免疫功能的关系研究

目的：探究终末期肾功能衰竭患者T细胞亚群的凋亡受体CD95分子与共刺激分子CD28、CDl52（CTLA-4）的表达与细胞免疫功能的关系。方法：采用流式细胞术检测外周血T细胞的凋亡受体CD95（Fas）与共刺激分子CD28／CDl52表达。结果：终末期肾功能衰竭患者CD3^＋T细胞和CD4^＋T细胞比例明显高于健康对照组，CD4／CD8比值增加（P=0．008），CD4^＋T细胞和CD8^＋T细胞上CD95分子表达均上调（P=0．001），以CD8^＋T细胞上CD95分子增加更为明显；CD28和CD152分子在不同T细胞亚群上的表达均上调（P〈0．05），然而，CD4^＋T细胞以CD28分子表达增加为主，而CD8^＋T细胞则CD152分子表达增加为主。结论：终末期肾功能衰竭患者的细胞亚群失衡，共刺激分子CD28和CD152表达异常增加，提示T细胞活化与抑制性调节发生紊乱。T细胞亚群上凋亡受体CD95分子表达增加，以CD8^＋T细胞为主，说明终末期肾功能衰竭者淋巴细胞的减少可能通过两种途径——受体配体途径和负性共刺激分子CD152抑制信号途径，其中以CD8^＋T细胞减少为主，造成患者细胞免疫缺陷。     

SEB诱导的CD4+ T细胞无能、凋亡及MHC-I类分子表达下调

目的：探讨超抗原金黄色葡萄球菌肠毒素（SEB）体外诱导外周T细胞免疫耐受的作用机制。方法：采用SEB体外刺激C57BL/6J（B6）小鼠的脾细胞后，以MTT比色法检测脾细胞的增殖，并用PI染色后以流式细胞术（FCM）分析不同时间段处于S期，G0-G1期的细胞及无能T细胞的凋亡，测定T细胞亚群及MHC-I（H-2K^b)表达的变化。用琼脂糖凝胶电泳，观察不同时间段凋亡T细胞的DNA特征。结果：部分去除CD8^ T细胞后。SEB可刺激B6小鼠脾细胞中CD4^ T细胞大量增殖，在SEB刺激后第3天，CD4^ T细胞中处于S期的比率最大，此后开始下降；而处于G0-G1期的CD4^ T细胞变化则相反，在初次刺激后第3天，增殖的CD4^ T细胞出现无能，FCM检测及用琼脂糖凝胶电泳检查DNAladder证实，在第7天，无能CD4^ T细胞出现凋亡，且凋亡细胞的比率逐渐增多，不因加入抗CD3抗体或CoN A而逆转，在SEB刺激后，CD4^ T细胞表面MHC-I类分子（H-2K^b)的表达，随细胞无能的出现而明显下调。结论：SEB诱导的T细胞免疫耐受，可能与CD4^ T细胞的无能，凋亡及细胞表面分子MHC-I的表达下调有关。     

CD4+ CD25+调节性T细胞AICD机制的研究

目的探讨CD4^＋CD25^＋调节性T细胞活化诱导的细胞死亡（AICD）发生的机制。方法CD4^＋CD25^＋T细胞以磁性细胞分离器（MACS）从BALB／c小鼠或DO11．10小鼠的静息T细胞分离纯化。体外细胞增殖抑制实验证实其免疫调节作用。CD4^＋CD25^＋T细胞的AICD以CD3／CD28单克隆抗体活化或以特异性OVA323-339肽、抗原提呈细胞活化等两种方法获得。CD4^＋CD25^＋T细胞凋亡相关基因的表达通过实时定量PCR检测。流式细胞仪检测细胞的凋亡率。进一步观察FasL中和抗体、TRAIL中和抗体及caspase抑制剂zVAD-fmk对CD4^＋CD25^＋T细胞凋亡的影响。结果MACS成功分离CD4^＋CD25^＋T细胞，纯度可达98％，该细胞可特异性表达Foxp3基因，能明显抑制效应性T细胞的体外增殖。CD3／CD28抗体以及OVA特异性抗原活化8d的CD4^＋CD25^＋调节性T细胞AICD达39％～45％。活化前后的CD4^＋CD25^＋调节性T细胞死亡受体家族表达发生明显变化；FasL、TRAIL中和抗体及zVAD-fmk可明显抑制CD4^＋CD25^＋调节性T细胞的凋亡。结论FasL／Fas及其他凋亡相关分子可能参与了CD4^＋CD25^＋调节性T细胞的凋亡。     

CD4^＋CD25^＋T细胞：一类新被认识的免疫调节细胞

CD4^ CD25^ T细胞是最近才被认识的一类免疫调节细胞，在胸腺产生，主要发挥抑制性免疫调节功能，表达IL-10mRNA，细胞表面表达IL-2受体α链(CD25)，本身不产生IL-2，但在体内、外增殖需要外源性IL-2，在体外为无能细胞(anergy cell)，CD4^ CD25^ T细胞发挥免疫抑制作用是通过细胞-细胞接触依赖方式而非细胞因子依赖方式，CD4^ CD25^ T细胞在调控自身免疫性疾病的发生、炎症反应和T细胞稳态中发挥重要作用。本文拟对CD4^ CD25^ T细胞的生物学特性及其应用研究的最新进展作一综述。     

CD4^＋CD25^＋调节性T细胞和肿瘤免疫

近期研究发现一个有独特免疫调节功能的T细胞亚群：CD4^ CD25^ 调节性T细胞，不仅能抑制自身免疫性疾病发生，还可能参与肿瘤免疫的调节。这群细胞具有免疫无能和免疫抑制特性，通过与细胞直接接触发挥作用，而不依赖于其分泌的细胞因子。肿瘤环境中CD4^ CD25^ 调节性T细胞比例增加，导致肿瘤免疫失调，去除这群细胞可有效诱导肿瘤免疫，为肿瘤治疗提供了一种新的方法。     

人CD4+CD25+调节性T细胞系的建立与功能分析

目的：建立可在体外长期培养的人CD4^ CD25^ 调节性T细胞系并研究其免疫生物学特性。方法：用流式分选的方法从健康人外周血淋巴细胞中得到CD4^ CD25^ T细胞，并对其进行体外长期培养、扩增；淋巴细胞转化实验分析其免疫抑制功能，流式细胞法分析其表型。结果：经对人CD4^ CD25^ T细胞的长期培养扩增，获得具有免疫抑制功能的调节性T细胞系。该T细胞系对经TCR的刺激不敏感，且能抑制同一来源或同种异型CD4^ CD25^ T细胞的活化，大剂量IL－2可以逆转其抑制功能。长期培养的CD4^ CD25^ T细胞，膜表面CD25和CTLA－4分子持续高表达，而CD4^ CD25^ T细胞CD25和CTLA－4分子的表达呈周期性变化。结论：将CD4^ CD25^ T细胞体外扩增培养成系，其功能和表型与同一来源的CD4^ CD25^ T细胞显著不同。     

介导CD4+CD25+调节性T细胞发育与功能的一个关键基因--Foxp3

CD4^＋CD25^＋调节性T细胞是天然产生的调节性T细胞的重要亚群之一，在维持免疫系统稳态中发挥重要的作用。目前有关CD4^＋CD25^＋调节性T细胞分化发育的确切机制还不清楚。最近的研究发现Foxp3基因与CD4^＋CD25^＋调节性T细胞的分化发育和免疫调节功能的关系密切，进而提示Foxp3可能是CD4^＋CD25^＋调节性T细胞的一个特征性标志。     

环孢素A治疗再障和骨髓增生异常综合征T淋巴细胞异常激活的变化

研究再生障碍性贫血(AA)用环孢素A(CsA)治疗前后和骨髓增生异常综合征(MDS)患者外周血CD4+、CD8+T淋巴细胞培养前后早期激活标志CD69的表达及其意义。将外周血在PHA20μg/ml条件下进行全血细胞培养,于0h和4h分别用双色免疫荧光标记流式细胞仪对CD4+、CD8+T淋巴细胞CD69的表达进行分析。发现PHA刺激前初治SAA和MDS-RA+MDS-RAS患者CD4+、CD8+细胞CD69的表达率增高,CAA与RAEB+RAEB-T患者CD8+细胞CD69的表达率增高;PHA刺激后AA与MDS患者CD4+、CD8+细胞表达CD69明显增强,AA患者CD4+细胞CD69的表达率高于CD8+细胞。CsA治疗后SAA患者PHA刺激前CD4+、CD8+细胞CD69的表达率较治疗前明显减低,CAA患者CD8+细胞CD69的表达率较治疗前明显减低。治疗后AA患者PHA刺激后CD4+、CD8+细胞CD69的表达率较治疗前明显减低。CsA治疗有效的AA患者治疗前PHA刺激前后CD4+、CD8+细胞CD69的表达率明显增高,治疗后明显减低。初治AA患者PHA刺激前后CD4+细胞CD69的表达率均明显高于MDS患者。说明T细胞早期活化及其活化潜能增强,以及产生针对自身造血干/祖细胞的细胞毒效应在AA和MDS发病中起重要作用,CsA能抑制AA患者T细胞的早期激活。     

Activation of human intraepithelial lymphocytes reduces CD3 expression

The aim of this study was to examine in detail the low functional capacity of human intraepithelial lymphocytes (IELs) in response to phytohaemagglutinin (PHA) and CD3 ligation. Human IELs were extracted from jejunal mucosa obtained from patients undergoing gastric bypass operations for morbid obesity and compared to peripheral blood (PB) lymphocytes composed predominantly of CD8+ T cells. Calcium influx ([Ca2+]i) was analysed using Fura-2-loaded cells; IL-2 receptor expression was measured by immunofluorescence and flow cytometry; IL-2 binding was determined using radiolabelled IL-2; IL-2 production was quantified by ELISA; and apoptosis was detected with Apo 2.7 staining. Compared to naive PB CD8+ T lymphocytes, calcium influx by IELs was only transient with CD3 ligation and low in amplitude with PHA. IL-2 receptor expression was reduced after CD3 ligation, yet normal in numbers and affinity after PHA stimulation. Both cell types secreted similar amounts of IL-2. CD3 expression on IELs, but not PB CD8+ T cells, declined upon activation, due partly to incomplete reexpression after modulation. Little apoptosis was found. The partial activation of IELs in response to PHA and CD3 ligation, as manifested by diminished [Ca2+]i, resulted in a decline in CD3 expression.     

Kinetics of cytokine gene expression in human CD4+ and CD8+ T-lymphocyte subsets using quantitative real-time PCR

The time kinetics of five cytokines [interleukin-2 (IL-2), IL-5, interferon-gamma (IFN-gamma), granulocyte macrophage-colony stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNF-alpha)] and one cytotoxic effector protein (granzyme B) was analysed by real-time quantitative polymerase chain reaction (PCR) following in vitro stimulation of human CD4 and CD8 T lymphocytes. Two stimuli were used, a mitogen [phytohemagglutinin (PHA)] and a recall antigen [purified protein derivative (PPD)]. The pattern of cytokine mRNA expression was found to be dependent on the T-cell subset and stimulus used. A wide interindividual variability in the cytokine gene expression pattern was demonstrated. Two expression patterns were observed. A bell-shaped expression profile was seen for most cytokines upon PHA activation in both subsets and PPD-activated CD4 T cells, whereas a biphasic/multiphasic expression pattern was noted in CD8 T cells upon PPD stimulation. For most cytokines, the time to induction was within 30 min of activation, and maximum accumulation seemed to be obtained after 4-8 h of activation. A sustained high level could, however, be noticed for up to 24 h. Granzyme B gene expression was also induced within 30 min of activation but showed a continuous gradual increase and late maximal accumulation (48-72 h). The findings of the present study are of importance when designing studies using the cytokine gene expression profile as a marker for antigen-specific T lymphocytes. It might be recommended that cytokine gene expression (IL-2, IL-5 and IFN-gamma) should be measured after 4-8 h of specific activation but also up to 24 h of stimulation is acceptable. Granzyme B should preferentially be measured after 48-72 h of activation.     

慢性乙型肝炎患者外周血CD8^＋ T细胞的KIR3DL1表达的探讨

目的：检测慢性乙型肝炎患者外周血CD8＋T细胞的KIR3DL1表达情况。方法：：采用流式细胞术检测慢性乙型肝炎患者外周血CD8＋T细胞的KIR3DL1分子表达,并与正常对照组比较。结果：慢性乙型肝炎患者外周血CD8＋T细胞的KIR3DL1分子表达明显高于对照组。结论：慢性乙型肝炎患者CD8＋T细胞的KIR3DL1表达显著增加。     

Activation-induced changes in peptides presented by T cells.

We compared the sets of peptides presented via HLA Class I in a CD4 + T cell line (Jurkat) before and after activation with PMA plus PHA.

We found that cell activation resulted in the de n vo presentation of some peptides.

Sequence analysis revealed that one of the newly presented peptides derived from IL-1 receptor antagonist (IL-1ra).

Since IL-1ra was not known to be expressed by lymphocytes, we also investigated ist pattern of expression in lymphocytes of the T lineage.

RT-PCR analysis allowed us to demonstrate that IL-1ra is expressed upon activation in Jurkat cells, in CD4+ lymphocytes from peripheral blood, but not in CD8+ ones, and in thymocytes.

This suggests that activation in CD4+ T cells is followed by the novo presentation of peptides der ved from proteins expressed only upon activation. Interestingly, of the two forms of IL-1ra expre sed in different cell lineages, the intracellular one and the secreted one, only the former is expre sed in activated CD4+ cells.     

Induction of CD95 ligand expression on T lymphocytes and B lymphocytes and its contribution to apoptosis of CD95-up-regulated CD4+ T lymphocytes in macaques by infection with a pathogenic simian/human immunodeficiency virus

Using an established SIV/HIV-C2/1-infected cynomolgus monkey model displaying stable CD4+ T cell depletion, the kinetics of apoptosis and the levels of expression of CD95 membrane-associated CD95L on lymphocytes were investigated to test the involvement of the CD95/CD95L system in CD4+ T lymphocyte loss in vivo. Rapid depletion of CD4+ T cells occurred up to 2 weeks after infection, with chronic CD4+ T lymphopenia thereafter. During the initial CD4+ T cell loss, which was accompanied by viraemia, about 90% of the peripheral CD4+ T cell subset underwent spontaneous apoptotic cell death during 24 h of culture. Increased expression of CD95 was observed on both CD4+ and CD8+ T cell subsets, with CD95 expression on CD8+ cells declining rapidly, but high CD95 expression being maintained on CD4+ cells. Since CD95L was expressed on CD8+ T cells, B cells and to a lesser extent on CD4+ T cells, this suggests that CD95-mediated apoptosis might be controlled in an autocrine/paracrine fashion.     

PHA对人扁桃体T淋巴细胞亚群变化及活化抗原表达的效应

应用FITC和PE双色荧光试剂并经流式细胞计分析了人扁桃腺T淋巴细胞亚群组成,以及用PHA刺激后T淋巴细胞亚群的变化和活化抗原的表达情况。结果表明:1)人扁桃体T淋巴细胞以CD4~+细胞占优势,CD4~+与CD8~+细胞比值约为5.32±0.55;2),经PHA刺激培养72小时,CD4~+CD8~+细胞明显增加;3)经PHA刺激培养后,IL-2受体(CD25)和HLA-DR抗原表达增加。本实验结果为深入研究人扁桃体T淋巴细胞的表型变化提供了客观指标,对进一步探讨其在免疫系统中的作用具有一定的意义。     

Deficiencies in CD4+ and CD8+ T cell subsets in ataxia telangiectasia

Chronic sinopulmonary infections that are associated with immunodeficiency are one of the leading causes of death in the multi-systemic disease ataxia telangiectasia (AT). Immunological investigations of AT patients revealed a broad spectrum of defects in the humoral and the cellular immune system. Based on their important role in host defence the aim of our study was an extensive analysis of cell distribution and function of CD4+ and CD8+ T lymphocytes and NK cells. We found that naive (CD45RA+) CD4+ lymphocytes, as well as CD8+/CD45RA+ lymphocytes, are decreased, whereas NK cells (CD3-/CD16+CD56+) are significantly elevated in AT patients. In our culture system proliferation and cytokine production was normal in purified memory (CD45RO+) lymphocytes after stimulation with phorbol-12,13-dibutyrate (PBu2) and after PHA activation, indicating that differences in proliferation and cytokine production are due solely to reduced numbers of CD45RA+ lymphocytes. However, activation, and especially intracellular interferon production of AT lymphocytes, seem to follow different kinetics compared to controls. In contrast to polyclonal activation, stimulation via the T cell receptor results consistently in a reduced immune response. Taken together, our results suggest that deficiency of immunocompetent cells and an intrinsic immune activation defect are responsible for the immunodeficiency in AT.     

罗格列酮上调成人隐匿性自身免疫糖尿病患者CD4+T细胞Foxp3 mRNA的表达

目的 观察罗格列酮对成人隐匿性自身免疫糖尿病(LADA)患者CD4+调节性T细胞的影响,旨在探讨罗格列酮的免疫调节机制.方法 采用磁珠分离LADA患者CD4+T细胞,1、10和100 μmol/L罗格列酮干预CIM+T细胞.MTT法检测细胞活性,3H-TdR掺人法检测增殖抑制率.流式细胞术检测CD4+CD25+T细胞比值.RT-PCR检测过氧化物酶体增殖激活受体-γ(PPARγ)mRNA、TGF-β1 mRNA表达,实时荧光定量PCR检测Foxp3 mRNA表达.结果 CD4+T细胞表达PPARγmRNA.罗格列酮抑制植物凝集素(PHA)刺激的CD4+T细胞增殖.1μmol/L和10μmol/L组罗格列酮作用的CD4+CD25+T细胞比例无明显变化,100 μmol/L组CD4+CD25+T细胞比例降低.10μmol/L罗格列酮上调CD4+T细胞的Foxp3 mRNA表达,但TGF-β1 mRNA表达无显著变化.小剂量IL-2参与下,1μmol/L罗格列酮(药理浓度范围内)升高CD4+T细胞Foxp3mRNA表达.结论 罗格列酮上调LADA患者CD4+T细胞Foxp3 mRNA表达,改善自身免疫耐受缺陷.     

原发性胆汁性肝硬化患者共刺激分子B7-H4的检测及临床意义

目的 探讨原发性胆汁性肝硬化(primary biliary cirrhosis,PBC)患者共刺激因子B7-1t4的表达及其与疾病发病机制的关系.方法 分别采用荧光实时定量PCR法(real-time PCR)、酶联免疫吸附试验(ELISA)及流式细胞术(FCM)检测65名PBC患者外周血单个核细胞(PBMC)B7-H4mRNA表达水平、血清IL-2水平以及CD4+、CD8+ T细胞亚群和T细胞表面B7-H4表达百分率,同时监测抗线粒体抗体(anti-mitochondrial antibody,AMA)及临床各项生化指标的关系.结果 (1)PBC患者PBMC B7-144 mRNA水平及T细胞表面B7-H4表达百分率显著低于非PBC肝硬化组及健康对照组(P<0.01).(2)活化72 h后各实验组及对照组IL-2含量及CD4+、CD8+、CD4+ CD8+T淋巴细胞表达水平均低于活化前,以非PBC肝硬化组与健康对照组降低显著(P<0.05);PBC组IL-2含量及CD4+、CD4+ CD8+ T淋巴细胞表达水平高于非PBC肝硬化组与健康对照组(P<0.01).(3)AMA-M2阳性患者血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)及γ-谷氨酰转肽酶(GGT)水平升高,其中ALP及GGT升高显著(P<0.05);AMA-M2阳性患者与阴性患者T细胞表面B7-H4表达百分率差异无统计学意义(P>0.05).结论 共刺激因子B7-H4对PBC患者体内T细胞活化增殖及细胞因子分泌的抑制作用减弱,为研究PBC的发生发展和阐明PBC的发病机制提供了试验资料.