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。     

共刺激信号分子表达异常与自身免疫性疾病的关系

目的 分析类风湿性关节炎（RA）和系统性红斑狼疮（SLE）细胞免疫异常中共刺激活化信号途径的异同点,探讨细胞活化的共刺激信号途径对于RA和SLE发病的意义。方法 采用流式细胞术分析SLE和RA患者T细胞表面共刺激分子CD28、CD152／CTLA-4、CD154／CD40L和ICOS表达情况。结果 ①RA患者CD4^＋T细胞比例明显增加,SLE患者以CD8^＋T细胞增加显著。②SLE患者CD4^＋和CD8^＋T细胞上CD28和CD152的表达增加;RA患者T细胞亚群上CD28分子的表达减少,CD152分子的表达增加。③RA患者CD4^＋T细胞上CD154分子表达增加,CD8^＋T细胞上CD154分子表达减少,T细胞亚群上可诱导共刺激分子（ICOS）的表达无明显变化;SLE患者CD4^＋和CD8^＋T细胞上CD1254或ICOS分子的表达均减少。结论 RA和SLE患者机体的免疫异常中存在着明显的分子信号途径差异。     

CD28协同刺激分子对T细胞受体介导的胸腺细胞亚群凋亡的影响

目的：分析anti-TCRαβmAb anti-CD28mAb诱导小鼠胸腺淋巴细胞不同亚群的凋亡及凋亡程度，分析CD28协同刺激分子对TCR受体介导的胸腺细胞亚群凋亡的影响。方法：新鲜分离胸腺细胞，加入anti-TCRαβmAb-anti-TCRαβmAb anti-CD28mAb等培养20h，进行多重染色，流式细胞仪分析。结果：与胸腺细胞自发凋亡的结果相比较；（1）双信号刺激可明显增加胸腺细胞凋亡的数目，尤其是CD4^ CD8^ 胸腺细胞的凋亡数目。（2）凋亡的CD4^ CD8^ 亚群，CD4^ CD8^-亚群细胞表面CD28的表达均增多。结论：CD28共刺激分子对TCR受体介导的胸腺细胞亚群凋亡的影响与细胞的成熟程度有关，CD28共刺激分子能明显增强不成熟皮质胸腺细胞的凋亡。     

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。     

他克莫司与环孢菌素A对肝移植受者CD4/CD8 T淋巴细胞亚群及共刺激分子的调节作用

目的:探讨他克莫司(Tacrolimus, FK506)与环孢菌素A(CsA)对肝移植受者T淋巴细胞亚群共刺激分子的调节作用.方法:采用荧光标记单克隆抗体(mAb)结合流式细胞技术, 测定移植术后使用FK506或CsA治疗2月末的肝移植受者外周血T细胞亚群及其表面共刺激分子CD28、CD152 和ICOS的表达情况.以健康志愿者(健康对照组)和患终末期肝脏疾病拟进行肝移植者(疾病对照组)为对照.结果:疾病对照组T细胞亚群平衡紊乱、共刺激分子表达异常(P＜0.05).治疗组肝移植受者T淋巴细胞亚群表达恢复至健康对照水平, T细胞表面CD28和ICOS分子表达显著降低(P＜0.05)而CD152分子表达明显升高(P＜0.05).比较不同药物治疗组:CsA治疗组CD4 T细胞表达和CD8 T细胞表面CD28、CD152分子表达均明显高于FK506治疗组(P＜0.05);其他指标无统计学意义(P＞0.05).结论:在常规血药浓度条件下FK506和CsA的对CD4/CD8T细胞亚群及共刺激分子的免疫调节作用存在差异.FK506对T细胞亚群的调节作用强于CsA.FK506可同时抑制正性共刺激分子CD28和ICOS表达并促进负性共刺激分子CD152表达, 而CsA对T细胞免疫抑制作用主要是通过促进CD152分子的高表达介导.     

神经营养素受体在人外周血T细胞中表达的研究

目的 研究人外周血CD4^ /CD8^ T细胞4种神经营养素受体基因的转录。方法 应用尼龙手法分离出T细胞，磁式细胞分离法（MACS）分离CD4^ /CD8^ T细胞亚群，再以RT－PCR法研究4种神经营养素受体在两种T细胞亚群上的表达。结果 未经刺激的CD4^ /CD8^ T细胞亚群不表达任何神经营养素受体。经PHA或PPD刺激后，CD4^ /CD8^ T细胞亚群表达trkA,CD8^ T细胞亚群表达trkC；而在各种状态下的T细胞上均未见表达trkB及p75^NGFR。结论神经营养素受体在两种T细胞亚群中有不同的表达格局，提示不同T细胞亚群受神经营养素调节的模式可能各不相同。     

重症肌无力患者外周血B7：CD28／CTLA4共刺激分子表达的动态变化

目的：研究重症肌无力（MG）患者B7：CD28／CTLA4共刺激通路相关分子表达动态变化及其与MG发病的关系。方法：用流式细胞仪检测18例MG患者和16例健康对照者外周血单个核细胞（PBMC）在经PMA（佛波酯）＋ionomycin(钙离子导入剂）刺激的0h,6h,24h,48h时B7－1、B7－2、CD28、CTLA4分子在CD4^ T细胞和CD8^ T细胞表面的表达。结果：（1）0h,MG患者B7－1、B7－2分子总体表达增加，CD4^ 、CD8^ T细胞表面B7－1、B7－2的表达未见明显增加，PMA＋ionomycin刺激后B7－1、B7－2在CD4^ 、CD8^ T细胞表面也无增加；（2）0h,CD28、CTLA4的表达增强，其中CD28^ 细胞的增多主要表现在CD4^ T细胞亚群，CTLA4的表达增强主要在CD8^ T细胞，在PMA＋ionomycin激活后，CD28表达明显增强，持续到48h都处于较高水平（与对照组相比P＜0．01），CTLA4表达出现短暂增强，6h即达高峰，以后下降，与对照组相比无显著增强（P＞0．05）。结论：MG患者外周血中B7：CD28／CTLA4通路共刺激相关分子表达增高，持续时间延长，检测共刺激分子的表达可反映机体的免疫激活状态，B7：CD28／CTLA4通路在MG发病中可能起重要作用。     

共刺激分子与类风湿性关节炎患者T细胞的平衡失控

共刺激分子和凋亡受体CD95(Fas)分子的异常表达与T细胞的异常活化及其亚群平衡偏移密切相关，本研究拟通过对类风湿性关节炎(RA)患者T细胞亚群上表面CD30和CD95分子表达的检测，探讨细胞信号分子在参与RA免疫紊乱中的作用。     

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。     

SLE患者外周血淋巴细胞凋亡异常及血清中IL-10的影响

目的：研究SLE患者外周血单个核细胞(PBMCs)中淋巴细胞亚群的凋亡特点。方法：采用三色荧光流式细胞术检测CD3、CD4、CD8、CD19细胞亚群的早期凋亡；ELISA法检测PBMCs培养上清中的sFas、sFasL；实时荧光半定量RT-PCR方法检测细胞内FasmRNA的表达水平。结果：SLE患者PBMCs中CD3^ 细胞凋亡明显增多；CD4^ 和CD8^ 细胞亚群凋亡均有明显增加，但以CD4^ 细胞凋亡增多更为明显；相应地，其PBMCs中CD4^ 、CD8^ 细胞百分率下降，CD4／CD8比值降低。SLE患者血清可诱导CD3^ 、CD4^ 、CD8^ T细胞亚群凋亡增多，sFas、sFasL水平增高及Fas mRNA表达增加；抗-IL-10抗体则可中和SLE血清的上述作用。结论：SLE患者体内T细胞凋亡增多，以CD4^ T细胞凋亡增加更为显著，导致CD4／CD8比值下降；SLE血清中高水平的IL-10可能通过诱导Fas、FasL表达增高而促进T细胞凋亡的发生。     

Enhanced expression of CTLA-4 (CD152) on CD4+ T cells in HIV infection

CTLA-4 (CD152) is a surface molecule of activated T cells with sequence homology to CD28. Both molecules bind to the same ligands, B7.1 (CD80) and B7.2 (CD86) but have antagonistic functions. While CD28 is an important costimulator, CTLA-4 has an essential inhibitory function in maintaining the homeostasis of the immune system. Down-regulation of CD28 predominantly on CD8+ T cells has been described in HIV infection, but analysis of CTLA-4 is complicated by its low expression levels. Here we have used potent signal enhancement to study CTLA-4 on peripheral blood mononuclear cells (PBMC) during HIV infection. CTLA-4 was expressed only on T cells. Expression levels were significantly increased selectively on CD4+ T cells during all stages of HIV infection, while CTLA-4 expression on CD8+ T cells was always low. In contrast, after stimulation with the mitogen phytohaemagglutinin (PHA), CTLA-4 levels were strongly increased on T cells from controls but in T cells from HIV patients this response was severely impaired. Our data suggest that in HIV infection CD4+ and CD8+ T cells may be less responsive to B7 costimuli due to two different mechanisms: increase in CTLA-4 expression by CD4+ cells and down-regulation of CD28 by CD8+ cells.     

Expression of CTLA-4 (CD152) in peripheral blood T cells of children with influenza virus infection including encephalopathy in comparison with respiratory syncytial virus infection

Influenza virus and respiratory syncytial virus (RSV) are the most common causes of acute severe respiratory infection in children during the winter. There have been few reports about peripheral blood T cell activation in vivo in influenza virus infection and conflicting results concerning peripheral blood T cells activation in RSV infection. Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4, CD152) is a receptor present on T cells that plays a critical role in the down-regulation of antigen-activated immune responses. To clarify the status of peripheral blood T cells, we investigated intracellular CTLA-4 expression in T cells in patients with influenza virus and RSV infection. We collected blood samples from 15 patients with influenza virus infection, including three with complications of influenza virus-associated encephalopathy and 18 patients with RSV infection, as well as 44 healthy children. We determined the intracellular expression of CTLA-4 in CD4+ and CD8+ T cells by flow cytometry. There were no significant differences in the percentages of intracellular CTLA-4-positive CD4+ T cells and CD8+ T cells by age. The percentages of intracellular CTLA-4-positive CD4+ T cells in the patients with influenza virus infection were significantly higher than those in healthy children (P < 0.01). In particular, the patients with influenza virus-associated encephalopathy had sevenfold higher percentages of CTLA-4-positive CD4+ T cells than influenza patients without encephalopathy (P < 0.05). The patients with influenza virus-associated encephalopathy had increased percentages of CTLA-4-positive CD8+ cells at the acute stage in comparison with the convalescent stage and in control subjects (P < 0.01, respectively). RSV patients showed no increase in CTLA-4-positive CD4+ T cells or CD8+ T cells. The immunological status of peripheral T cell activation is substantially different in influenza virus infection and RSV infection. The patients with RSV infection did not show any increase in CTLA-4-positive peripheral blood T cells. There was a remarkable increase in intracellular CTLA-4 in CD4+ and CD8+ T cells in influenza virus-associated encephalopathy. Down-regulation of antigen-activated peripheral blood T cell activation might play an important role in the pathogenesis of influenza virus-associated encephalopathy and host defence against influenza virus infection.     

Increased expression of CTLA-4 (CD152) by T and B lymphocytes in Wegener's granulomatosis.

CTLA-4 (CD152) is a surface molecule of activated T cells with sequence homology to CD28. Both molecules bind to the same ligands, B7.1 (CD80) and B7.2 (CD86) but have antagonistic functions. While CD28 is an important costimulator, CTLA-4 has an essential inhibitory function in maintaining the homeostasis of the immune system. Furthermore, CTLA-4 has a role in inducing a Th1 response and suppressing Th2 cytokines, an effect which is antagonized by CD28. Many autoimmune diseases are characterized by an overwhelming production of Th1 cytokines. Recently, the predominance of the Th1 cytokine pattern has been directly observed in the granulomatous inflammation of patients with Wegener's granulomatosis. The balance between CD28 and CTLA-4 expression by T lymphocytes could be a factor in the pathogenesis of autoimmune diseases. Down regulation of CD28 predominantly on CD8+ T cells has been described in Wegner's granulomatosis; however, analysis of CTLA-4 is complicated by its low expression levels. Here we have used potent signal enhancement to study CTLA-4 on PBMC in patients with Wegener's granulomatosis (n = 25) in comparison with healthy controls (n = 19). Expression levels of CTLA-4 were significantly increased selectively on CD4+ and possibly also on CD4-/CD8- T cells in Wegener's granulomatosis. High CTLA-4 expression by T lymphocytes was associated with more severe disease. In contrast, after stimulation with the mitogen PHA, CTLA-4 levels were strongly increased on T cells from controls but in T cells from Wegener's granulomatosis patients this response was severely impaired. Interestingly, while CTLA-4 was seen exclusively on T cells in control individuals, about half of the Wegener's patients showed CTLA-4 expression by a fraction of peripheral B lymphocytes. CTLA-4 positive B cells in the periphery were associated with less acute disease.     

重症肌无力患者外周血CD28~-T细胞亚群变化及临床意义

检测重症肌无力(MG)患者外周血CD28-T细胞亚群的变化,并探讨其临床意义。收集46例MG患者和35例健康对照(HC)外周血标本,采用免疫荧光染色和流式细胞术检测外周血CD4+CD28-和CD8+CD28-T细胞亚群。结果显示,MG患者和HC比较,前者CD28表达下调,CD4、CD8表达无差异;MG患者CD4+CD28-T细胞亚群(13.53%±6.31%)较HC(9.24%±4.62%)异常升高(P=0.001),而CD8+CD28-T细胞亚群(39.22%±11.91%)较HC(48.41%±13.63%)显著下降(P=0.002);全身型MG(GMG)和并发胸腺异常的MG患者中,CD4+CD28-T细胞亚群比例分别较眼肌型MG(OMG)和正常胸腺MG患者升高,而CD8+CD28-T细胞亚群百分比明显下降;QMGS评分与CD4+CD28-T细胞亚群呈正相关(r=0.4113,P=0.0045),而与CD8+CD28-T细胞亚群呈负相关(r=-0.3989,P=0.0060);激素治疗后伴随CD4+CD28-T细胞比例下降(P=0.018)和CD8+CD28-T细胞比例升高(P=0.018)。本研究发现,MG患者外周血CD4+CD28-T细胞亚群比例升高和CD8+CD28-T细胞亚群比例下降与疾病严重程度、治疗反应密切相关,且在不同临床分型的MG患者中存在差异性变化。上述提示CD28-T细胞亚群的异常变化可能参与了MG的免疫病理进程。     

Accumulation of CTLA-4 expressing T lymphocytes in the germinal centres of human lymphoid tissues.

CTLA-4, a coreceptor with sequence homology to CD28 is expressed on T cells after activation. Mice deficient for CTLA-4 die young from massive infiltration of many organs by activated T cells, which highlights the essential inhibitory role the coreceptor plays in the regulation of the immune response. To study the prevalence and distribution of CTLA-4 in situ immunohistological analyses were carried out on human tonsils and lymph nodes. Expression of CTLA-4 was restricted to alpha beta T cells, and CTLA-4+ B cells were not observed. In T-cell areas, 2-10% of T cells were positive for CTLA-4 with similar percentages in the CD4+ and CD8+ subpopulations. In the germinal centres (GC) the fraction of CTLA-4+ T cells was much higher (70-90%). This was due to frequent expression of CTLA-4 on the CD4+ helper subpopulation. GC CD8+ T cells were rare and mostly did not express the coreceptor. The CTLA-4+ T-cell fraction was also over-represented among intraepithelial tonsillar T cells. Cycling (Ki-67+) and apoptotic (TUNEL+) T cells were never positive for CTLA-4, while a subset of CD25+ cells did express the coreceptor. Since CTLA-4 is essential for the physiological limitation of the immune response, GC T cells, which are mostly CTLA-4 positive, might be important in this process.     

The CD28/HLA-DR expressions on CD4+T but not CD8+T cells are significant predictors for progression to AIDS

To investigate the changes of CD28 and HLA-DR molecules on CD4+ and CD8+ T cells during HIV infection, we classified 130 HIV-infected Koreans into four groups by the CD4 level as follows: group I (> or = 500 cells/mm3), group II (201-499 cells/mm3), group III (51-200 cells/mm3), and group IV (< or = 50 cells/mm3). In CD4+ T cells, the proportion of CD28 expression decreased significantly with the CD4 level while the proportion of HLA-DR expression increased gradually. In particular, the changes of HLA-DR expressions on CD4+ T cells were parallel to the loss of CD28 molecules from stage III to IV. However, the CD28 expression on CD8+ T cells decreased dramatically in the early stage of HIV infection, and the sum and pattern of CD28 and HLA-DR expressions on CD8+ T cells was stable after the first stage. Even though CD28 down-regulation on CD8+ T cells was very severe from the early stage of HIV infection, it might not influence the survival time of HIV-infected Koreans. The sum of the CD28+ subsets and HLA-DR subsets in each T cell was stable in all stages of disease progression. The sums of the CD28+ subsets and HLA-DR+ subsets in CD4+ T and CD8+ T cells were constant as approximately 100% and 55-60% of each T cell. These results suggested that the changes of CD28/HLA-DR expressions on CD4+ T cells were more predictable than those on CD8+ T cells in the evaluation of the disease progression during HIV-infected periods. However, we need further studies to understand why the sum of two molecules in each T cell are constant.     

中国HIV/AIDS患者T细胞及调节性T细胞CTLA-4表达与疾病进展关系研究

目的 对中国HIV感染者T细胞及凋节性T细胞CTLA-4表达与HIV疾病进展的相关性进行研究,探讨CTLA-4在HIV感染中的作用.方法 选取58名HIV/AIDS患者(长期不进展组、无症状HIV组、AIDS组),应用流式细胞仪胞内染色技术检测T细胞及CD4+CD25+Foxp3+调节性T细胞内CTLA-4表达水平,分析其与CD4+T细胞、病毒载量、淋巴细胞活化凋亡水平的相关性.结果 长期不进展组、无症状HIV组、AIDS组CD4+T细胞内CTLA-4表达水平依次增岛(P<0.05);与CD+T细胞显著负相关(P<0.01),与CD8+T细胞活化(CD38表达)、凋亡水平(CD95表达)及CD4+T细胞凋亡水平显著相关(P<0.05),与病毒载量无显著相关性.长期不进展组、无症状HIV组、AIDS组CD8+T细胞内CTLA-4表达水平差异无统计学意义;与CD4+T细胞、病毒载量、CD4,'+>、CD8+T细胞活化及凋亡水平均无显著相关性.CD4+CD25+Foxp3+T细胞内CTLA-4表达水平长期不进展组明显低于无症状HIV组及AIDS纽(P<0.05);与CD4+T细胞显著负相关(P<0.05);与CD4+、CD8+T淋巴细胞活化(HLA-DR表达)显著相关(P<0.01).结论 中国HIV感染者CD4+T细胞及CD4+CD25+Foxp3+调节性T细胞内CTLA-4表达水平与疾病进展及免疫活化状态显著相关,参与HIV感染免疫平衡的调节.