LFA-1/ICAM-1单抗对ConA诱导脾淋巴细胞增殖及分泌细胞因子的影响

通过研究ＬＦＡ－１／ＩＣＡＭ－１单抗对ＣｏｎＡ诱导的脾淋巴细胞活化增殖及其分泌细胞因子的影响　，探　讨了ＬＦＡ－１／ＩＣＡＭ－１分子在Ｔ细胞活化过程中所起的共刺激作用。结果表明，单独应用ＬＦＡ－１α链单抗（Ｍ１７／４．４．１１．９）或ＩＣＡＭ－１单抗（ＹＮ１／１．７．４）均不能引起脾淋巴细胞的增殖，但在加入ＣｏｎＡ诱导脾淋巴细胞增殖反应的最初８小时内加入ＬＦＡ－１单抗可以剂量依赖性地抑制ＣｏｎＡ诱导的脾淋巴细胞增殖反应及脾淋巴细胞分泌ＩＬ－２、ＩＰＮ－γ，而加入ＩＣＡＭ－１单抗却无此效应。说明ＬＰＡ－１在ＣｏｎＡ诱导的Ｔ细胞活化过程中起着重要的共刺激作用，ＬＰＡ－１通过与除ＩＣＡＭ－１以外的其它配体分子（如ＩＣＡＭ－３）相互识别，提供Ｔ细胞活化所必需的共刺激信号。     

仙茅多糖对小鼠免疫功能调节作用实验研究

仙茅多糖体外单独能刺激小鼠脾淋巴细胞增殖，而单独对小鼠胸腺细胞无作用，但在ＣｏｎＡ存在条件下对胸腺细胞增殖有协同作用，体外对尼龙毛柱分离小鼠脾Ｔ细胞富含部分有明显刺激增殖作用：体外能对抗由氢化可的松（ＨＣ）抑制ＣｏｎＡ诱导脾Ｔ细胞增殖，体内对ＨＣ诱导免疫受抑小鼠胸腺及脾脏重量降低，胸腺细胞及脾Ｔ、Ｂ细胞增殖降低有明显对抗作用。     

小鼠胸腺基质细胞单抗对胸腺细胞及脾细胞增殖的抑…

试验两种大鼠抗小鼠胸腺基质细胞ＭｃＡｂｓ：Ｐｆ１８－３、Ｒｓ２１－Ｃ６的生物作用。Ｐｆ１８－３ＭｃＡｂ识别分子表达于胸腺细胞、脾脏Ｔ及Ｂ细胞；Ｒｓ２１－Ｃ６只表达于ＭＴＳＣ。加Ｐｆ１８－３或Ｒｓ２１－Ｃ６于ＭＴＥＣ１与胸腺细胞或脾细胞的体外培养中，在ＣｏｎＡ存在及不存在下，均可抑制ＭＴＥＣ１诱导胸腺细胞及脾细胞的增殖作用。ＣｏｎＡ活化下，其对胸腺细胞增殖的抑制率Ｐｆ１８－３为９６％；Ｒｓ２１－Ｃ６     

神经酰胺对结核杆菌低分子多肽抗原诱导的γδT细胞活化及凋亡作用

目的：研究神经酰胺对结核杆菌低分子多肽抗原（Ｍｔｂ）诱导的γδＴ细胞活化及凋亡作用。方法：用结核杆菌低分子多肽抗原刺激正常人ＰＢＭＣ，并用磁珠阳性分选法获得高纯度的γδＴ细胞。通过ＭＴＴ试验观察神经酰胺、鞘磷脂酰以及神经酰胺合成酶抑制剂ＦｕｍｏｎｉｓｉｎＢ１对γδＴ细胞的活化作用；ＦＡＣＳ检测其对γδＴ细胞的亡作用。结果：Ｍｔｂ刺激获得的γδＴ细胞可达７３％。磁珠分选后可高达９８％；高浓度神经酰胺及鞘磷脂酶能抑制γδＴ细胞的增殖。而出现凋亡，ＦｕｍｏｎｉｓｉｎＢ１则对γδＴ细胞的增殖及凋亡无明显影响。结论：鞘磷脂水解产生的社经酰胺对γδＴ细胞的有致凋亡作用。     

ConA激活的小鼠T细胞CD69表达动力学的体内外研究

目的 为明确ＣＤ６９体内外表达动力学并进一步探讨其作用。方法 ＣｏｎＡ体内外刺激小鼠Ｔ细胞后,选取不同时间点,流式细胞仪观察Ｔ细胞ＣＥ６９表达率。结果ＣｏｎＡ刺激后２ｈＣＤ６９即有明显表达,６～８ｈ达到峰值,ＣＤ８＾＝与ＣＤ８＾＋Ｔ细胞相比无明显差异。体外条件对ＣＤ６９的表达有显著的影响。结论 结果提示ＣＤ８＾－和ＣＤ８＾＋Ｔ细胞的活化均需要ＣＤ６９的参与,Ｔ细胞可能在活化早期一过性高表达ＣＤ６９     

小鼠胸腺基质细胞单抗对胸腺细胞及脾细胞增殖的抑制作用

试验两种大鼠抗小鼠胸腺基质细胞（ＭＴＳＣ）ＭｃＡｂｓＰｆ１８－３、Ｒｓ２１－Ｃ６的生物作用。Ｐｆ１８－３ＭｃＡｂ识别分子表达于胸腺细胞、脾脏Ｔ及Ｂ细胞；Ｒｓ２１－Ｃ６只表达于ＭＴＳＣ。加Ｐｆ１８－３或Ｒｓ２１－Ｃ６于ＭＴＥＣＩ与胸腺细胞或脾细胞的体外培养中，在ＣｏｎＡ存在及不存在下，均可抑制ＭＴＥＣ１诱导胸腺细胞及脾细胞的增殖作用。ＣｏｎＡ活化下，其对胸腺细胞增殖的抑制率Ｐｆ１８－３为９６％；Ｒｓ２１－Ｃ６为９１％。对脾细胞的抑制率Ｐｆ１８－３为７５％；Ｒｓ２１－Ｃ６为５１％。Ｐｆ１８－３对单纯ＣｏｎＡ活化的胸腺细胞增殖也有显著的抑制作用，而Ｒｓ２１－Ｃ６无此作用。包被于平皿的固相Ｐｆ１８－３ＭｃＡｂ，对胸腺细胞、脾细胞均有轻度但显著的促增殖作用。故Ｐｆ１８－３ＭｃＡｂ识别的抗原分子（及其配基）参与Ｔ细胞的活化过程。鉴于Ｐｆ１８－３＋细胞的分布有其特异性，分析此抗原分子的功能有重要的意义。     

小鼠胸腺树突状细胞系MTSC4诱导早期T细胞株C320表达IL－2R

通过流式细胞仪（ＦＡＣＳ）分析了我室自建的小鼠胸腺树突状细胞系ＭＴＳＣ４与小鼠肿瘤性早期Ｔ细胞株Ｃ３２０细胞共育后，Ｃ３２０细胞表达ＩＬ－２Ｒ的状态，分析了ＣｏｎＡ和ＰＨＡ对Ｃ３２０及与ＭＴＳＣ４共育后的Ｃ３２０表达ＩＬ－２Ｒ的诱导促进作用以及ＩＬ－２Ｒ＋Ｃ３２０细胞在脱离外源性刺激后其比例的变化。ＭＴＳＣ４及ＣｏｎＡ和ＰＨＡ在不同程度上抑制Ｃ３２０细胞的无限制增殖，ＣｏｎＡ和ＰＨＡ对与ＭＴＳＣ４共育后的Ｃ３２０细胞的抑制增殖作用更强。     

小鼠胸腺树突状细胞系MTSC4诱导早期T细胞株C320…

通过流式细胞仪分析了我室自建的小鼠胸腺树突状细胞系ＭＴＳＣ４与小鼠肿瘤性早期Ｔ细胞株Ｃ３２０细胞共育后，Ｃ３２０细胞表达ＩＬ－２Ｒ的状态，分析了ＣｏｎＡ和ＰＨＡ对Ｃ３２０及与ＭＴＳＣ４共育后的Ｃ３２０表达ＩＬ－２Ｒ的诱导促进作用以及ＩＬ－２Ｒ＾＋Ｃ３２０细胞在脱离外源性刺激后其比例的变化，ＭＴＳＣ４及ＣｏｎＡ和ＰＨＡ在不同程度上抑制Ｃ３２０细胞的无限制增殖，ＣｏｎＡ和ＰＨＡ对与ＭＴＳＣ４共育后的Ｃ     

T细胞疫苗抗同种移植排斥反应的研究

应用Ｔ细胞疫苗于同种心脏移植的研究，用Ｃｏｎ Ａ和ＣＤ３单抗诱导受同种特异性抗原（Ｃ５７ＢＬ／６（Ｈ－２＾ｂ）小鼠脾细胞）免疫的ＢＡＬＢ／Ｃ（Ｈ－２＾ｄ）小鼠，取后者脾细胞所制备的Ｔ细胞疫苗具有延长同种心脏移植物的存活期。受Ｔ细胞疫苗接种的ＢＡＬＢ／Ｃ（Ｈ－２＾ｄ）鼠脾细胞对经Ｃｏｎ Ａ激活或未激活的Ｃ５７ＢＬ／６小鼠脾细胞皆表现出特异的强烈的增殖反应，而对同系脾细胞呈低的反应。Ｔ细胞疫苗表型分析     

LFA—1／ICAM—1单抗对ConA诱导脾淋巴细胞增殖及分…

通过研究ＬＦＡ－１／ＩＣＡＭ－１单抗对Ｃｏｎ Ａ诱导的脾淋巴细胞活化增殖及其分泌细胞因子的影响，探讨了ＬＦＡ－１／ＩＣＡＭ－１分子在Ｔ细胞活化过程中所起的共刺激作用。结果表明，单独应用ＬＦＡ－１α链单抗（Ｍ１７／４．４．１１．９）或ＩＣＡＭ－１单抗（ＹＮ１／１．７．４）均不能引起脾淋巴细胞的增殖，但在加入ＣｏｎＡ诱导脾淋巴细胞增殖反应的最初８小时内加入ＬＦＡ－１单抗可以剂量依赖性地抑制ＣｏｎＡ诱导     

Apoptosis of T Cells in the First Trimester Human Decidua

PROBLEM: Apoptosis has been accepted as a mechanism for maintaining tolerance in the immune system. The induction of apoptotic cell death can also be a possible outcome of the lymphocyte activation. Expression of Fas ligand (FasL) by the human trophoblast has been proposed as a mechanism providing protection against the lytic action of decidual immune cells. The aim of this study was to determine whether decidual T cells undergo apoptosis during abortion. METHOD OF STUDY: We studied apoptosis of T cells isolated from the first-trimester decidua in 12 women after spontaneous or elective abortion. We used gel electrophoresis to detect DNA fragmentation. Cells undergoing DNA fragmentation also were identified by DNA analysis using flow cytometry. This method was based on the accumulation of ethanol-fixed apoptotic cells in the sub-GO/G1 peak of the DNA content as a result of the loss of DNA fragments from the cells and because of a reduced DNA ability to be stained by propidium iodide. In addition, the expression of Fas antigen on the surface of decidual T cells (CD3⁺) also was determined. RESULTS: We did not detect apoptosis by the “ladder” technique. However, the apoptotic index (the percentage of positive cells per total number of cells) ranged from 2% to 24% using flow cytometry. CONCLUSIONS: Trophoblast cells usually fail to stimulate alloantigen-specific T cells, but they may express nonclassical major histocompatibility complex alloantigens to which mothers can produce immunoglobulin G alloantibody, which requires T helper cell activation. The apoptosis of T cells in the human decidua, probably through Fas-FasL signaling, may be a defense mechanism against rejection of the fetal allograft by the maternal immune system.     

Involvement of tumour necrosis factor-alpha-related apoptosis-inducing ligand in enhanced cytotoxicity of lipopolysaccharide-stimulated dendritic cells to activated T cells

Dendritic cells (DC) are potent antigen-presenting cells (APC) specialized in T-cell mediated immune responses, and also play critical roles in the homeostasis of T cells for controlling immune responses. In the present study, we demonstrated that during mouse bone-marrow-derived DC activation of ovalbumin (OVA)-specific Ia-kb-restricted T hybridoma cells, MF2.2D9 and OVA257-264-specific H-2kb-restricted RF33.70 T cells, respectively, both hybridomas undergo cell death, partially mediated via apoptotic ligand-tumour necrosis factor-alpha (TNF-alpha)-related apoptosis-inducing ligand (TRAIL). Lipopolysaccharide enhanced the cytotoxic effect on the two activated T hybridoma cells, which was correlated with up-regulation of TRAIL-expression on DC to some extent. The activation of caspase-3 in activated T hybridoma cells cocultured with DC contributed to the programmed cell death pathway T cells underwent. Therefore, our results show that activation-induced cell death of T hybridoma cells can be influenced by DC, suggesting that DC may be involved in elimination of activated T cells at the end of primary immune responses.     

大剂量γ线照射对小鼠免疫功能近期、远期的影响

目的 :观察大剂量γ线照射对小鼠免疫功能近期、远期的影响。方法 :将 2 2 5只清洁级C5 7小鼠 ,随机分为 0、6、9、12、15和 2 0Gy 6个剂量组 ,经γ线全身 1次照射后 ,于照后 1～2 8d和 3～ 12月活杀取材 ,用原位末端标记 (TUNEL)和May Grunwald Giemsa(MGG)染色 ,检测细胞凋亡并观察其与照射剂量的关系。用流式细胞仪检测外周血T细胞亚群的变化。结果 :(1)照后早期 (1～ 14 )d外周血淋巴细胞的凋亡率即出现明显升高 ,随着照射剂量的增加凋亡率的升高更为显著 ;而T细胞亚群经不同剂量照射后持续下降 ,剂量为 2 0Gy时降至最低 ,其中以CD8 T细胞对辐射的敏感性最高 ,因而推测早期的严重损伤是急性辐射免疫损伤的重要特点之一。 (2 )照射后 1月淋巴细胞的凋亡率降低 ,T细胞及其亚群也逐渐恢复。然而直至照后 6～ 12月 ,无论是淋巴细胞的凋亡率还是CD3 T细胞及CD8 T细胞亚群 ,均未恢复到对照的水平 ,提示大剂量辐射对机体免疫系统的损伤呈现较重的远期效应。 (3)本实验还发现 ,12≤Gy照射后 ,外周血淋巴细胞的凋亡率与照射剂量呈明显的剂量效应关系 ,15～ 2 0Gy照射后未观察到明显的量效关系。结论 :照射后早期外周血淋巴细胞的大量凋亡 ,可能是导致T细胞亚群的百分率急剧下降和后期免疫功能受损的重要原     

Immune deficiency following thermal trauma is associated with apoptotic cell death

Thermal injury-associated specific immune deficiency occurs despite indicators of systemic activation of the lymphoid compartment. We investigated the possibility that postburn immune failure and T cell activation are causally related through activation-induced (apoptotic) cell death. The relationship between the cellular immune response and cell mortality was examined in cultures of peripheral blood mononuclear cells (PBMC) from 14 immunosuppressed patients with extensive burns (35–90% total body surface area). Impaired cellular immunity coincided with significantly reduced cell viability as ascertained by propidium iodide staining and dye reduction assays. Following stimulation with the mitogenic lectin, phytohemagglutinin (PHA), the majority of DNA in patient cultures was fragmented, suggesting the occurrence of apoptotic cell death. Even without stimulation a portion of patient cells was apoptotic as indicated by oligonucleosomal bands on agarose gel electrophoresis. Exogenous interleukin-2 or phorbol ester markedly reduced constitutive as well as PHAinduced DNA fragmentation.In situ demonstration of DNA strand breaks in freshly isolated patient PBMC, by a TdT-based labeling technique, confirmed that a larger fraction (up to 60%) of circulating lymphocytes was undergoing apoptosis on the periphery. These novel observations suggest that apoptosis may play a major role in thermal injury-related cellular immunodeficiency.     

Elevation of Immune Activation in Kenyan Women is Associated with Alterations in Immune Function: Implications for Vaccine Development

The infectious burden leading to immune activation can vary between different populations and lead to various immune dysfunctions. We compared the effect of immune activation on apoptosis and T cell function in HIV uninfected individuals from Nairobi, Kenya (n=34), and Winnipeg, Canada (n=10). Women from Nairobi had a significantly greater number of CD8+ T cells expressing the activation markers CD38 and HLA DR. Kenyan women also had significantly higher levels of CTLA-4+ CD4 and CD8+ T cells, and reduced levels of CD28+ CD8+ cells. Levels of CD95+ CD4+ T cells were higher in Kenyan women and, correspondingly, showed higher levels of spontaneous apoptosis. Kenyan women also demonstrated hyper-responsiveness to T cell activation as assessed by interferon gamma production. This study demonstrates that in a population of Kenyan women with high levels of T cell activation, there were also elevated levels of T cell apoptotic death and hyper-responsiveness. These differences may influence the efficacy of immune responses to pathogens and must be considered when testing candidate vaccines.     

Superantigen-induced T cell death by apoptosis: analysis on a single cell level and effect of IFN-gamma and IL-4 treatment

BACKGROUND: A role of bacterial superantigens in several chronic inflammatory diseases has previously been proposed. Many of these diseases are associated with an imbalance of the T helper cell subsets and their cytokine production. METHODS: Peripheral blood mononuclear cells from healthy donors were incubated with various concentrations of staphylococcal enterotoxin B (SEB) with or without IL-4 or IFN-gamma. After different time points cell activation, proliferation, Fas expression, cytokine release and cell death via apoptosis were detected. RESULTS: SEB treatment resulted in sequential T cell activation, proliferation, Fas expression, cytokine release, subsequently followed by cell death via apoptosis in a dose- and time-dependent manner. This biphasic effect occurred preferentially in SEB-responsive cells represented by the expression of Vbeta3 and Vbeta12 on T cells. A strong relationship between T cell activation and apoptosis was observed. The amplitude between these events increased with the dose of SEB. The highest rate of apoptotic T cells was observed at a dose of 1,000 ng/ml SEB. Addition of IFN-gamma to SEB-treated cells significantly reduced the rate of apoptotic cells, whereas IL-4 prevented apoptosis only in SEB-untreated cells. CONCLUSION: These results support the concept that the dose of superantigen exposure determines the rate of T cell proliferation and subsequent cell death. This T cell immune response is modulated by the presence and the type of cytokines.     

高三尖杉酯碱通过激活Caspase-3诱导T-淋巴细胞白血病细胞Molt-3凋亡

目的：研究高三尖杉酯碱（ＨＨＴ）诱导Ｔ－淋巴细胞白血病细胞凋亡的作用机制。方法：采用流式细胞仪等技术对ＨＨＴ诱导Ｔ－淋巴细胞白血病细胞（Ｍｏｌｔ－３）的凋亡作用进行探讨。结果：ＨＨＴ诱导Ｍｏｌｔ－３凋亡,凋亡率与作用浓度和时间成正比;细胞被阻滞在Ｇ１期。ＨＨＴ作用２４ｈ后细胞内Ｃａｓｐａｓｅ－３活性增高,Ｃａｓｐａｓｅ－３抑制剂Ｚ－ＤＥＶＤ－ＦＭＫ能特异地阻断ＨＨＴ对Ｍｏｌｔ－３凋亡的诱导;Ｆａｓ     

Apoptotic cells actively inhibit the expression of CD69 on Con A activated T lymphocytes

Although apoptosis is commonly viewed as a silent cell death without damage to adjacent tissues, the effect of apoptosis on immunity has been unclear. We have investigated the influence of apoptotic cells on T-cell activation. The K562 or HL-60 human leukemia cell lines that had been induced apoptosis by FTY720 or cycloheximide (CHX) were added into the culture of mouse spleen cells stimulated with Con A. Six to 20 h later, the expression of CD69, an early T-cell activation antigen, was detected using flowcytometry. Living cells and necrotic cells served as control groups. Apoptotic K562 or HL-60 cells induced by either FTY720 or CHX unanimously inhibited CD69 expression on the CD3+ mouse T cells while living and necrotic cells did not. The inhibition was proportional to the number of apoptotic cells and was different in the T-cell subsets, showing a rapid and transient inhibition on the CD3+CD8+ T-cell activation but with a slow and continuous inhibition on CD3+CD8- T-cell activation. In conclusion, the apoptotic cells actively inhibit a T-cell activation that is independent of the cell lines or the apoptotic inducers, indicating that the apoptotic cells dominantly regulate T-cell immunity.     

P-glycoprotein decreases with T cell maturation but is not responsible for resistance to CD95-induced apoptosis

P-glycoprotein (Pgp) is a membrane transporter responsible for resistance to chemotherapy in cancer cells. Its presence in T cells is very well documented, but its function in the immune system is still poorly understood. Recent findings suggest that Pgp may be involved in regulating programmed cell death by inhibiting caspase 8 and caspase 3. Utilising antigenically-activated T cells and the physiologically relevant apoptotic ligand, membrane CD95-L, we have previously reported that while T cells are generally resistant to CD95-induced death at early stages of activation, their susceptibility to apoptosis increases with successive activation and clonal expansion. In this study we investigated whether changes in apoptotic susceptibility were related to T cell Pgp function. Results showed that Pgp expression and function in T cells decreases with maturation, with CD8 cells having the highest Pgp function. However, although Pgp function inversely correlated with caspase 3 activity, no difference was observed between apoptotic susceptible CD25- cells and resistant CD25+ cells. In addition sorting of cells with high and low Pgp function showed no correlation with apoptotic capability. Therefore, whilst Pgp modulates caspase activity, it is not responsible for resistance to apoptosis of early activated T cells nor the increased susceptibility observed at the later stages of maturation in antigenically activated cells.     

Survivin expression correlates with apoptosis resistance after lymphocyte activation and is found preferentially in memory T cells

The prevention of apoptosis may be critical for immunological function. Survivin is a recently cloned member of the inhibitor of apoptosis protein family. We analyzed survivin expression before and after lymphocyte activation in isolated cell populations. Prior to activation, survivin was undetectable. After activation with IL-2 and OKT-3, CD3(+) cells expressed survivin. Next, we correlated survivin expression with Fas, FasL and the amount of apoptosis over time in culture. After activation, survivin was readily detected by day 2 and decreased thereafter. Prior to activation (day 0), Fas was present on 60% of the cells and on 100% by days 2-6. Peak FasL mRNA expression was at day 2. During peak survivin expression (days 2-4) the apoptotic fraction was low, but when survivin expression decreased the apoptotic fraction increased rapidly. Finally, we found that CD45RO(+) memory T cells showed a higher expression of survivin than did CD45RA(+) naive T cells after activation. These results suggest that survivin may contribute to T-cell survival early in T-cell responses as well as in memory immune responses.