超抗原SEA对外周血T细胞的活化作用

目的 研究超抗原SEA对外周血T淋巴细胞的活化作用。方法 用SEA刺激人外周血T淋巴细胞,观察细胞的DNA合成、形态、IL-2的产生、细胞表型以及凋亡情况。结果 SEA对T淋巴细胞的促增殖作用的浓度为100mg/mL最强,在第2、3d达高峰;首次或多次刺激不改变T淋巴细胞的CD4^ CD8^ 的表型;首次刺激24h内未见明显凋亡;但再次刺激24h内即有明显凋亡;加入rhIL-22d凋亡现象消失。结论 超抗原SEA对T淋巴细胞的促增殖作用与其浓度、作用时间有关,并且SEA首次或多次刺激对CD4^ T淋巴细胞和CD8^ T淋巴细胞起同等的活化作用。     

Fas／FasL在超抗原SEA诱导T细胞凋亡中的作用

目的超抗原能诱导 T细胞凋亡 ,但其作用机制尚未阐明 ,Fas系统与 Ca2 在其中的作用尚待进一步研究。方法超抗原金黄色葡萄球菌肠毒素 A(SEA)诱导人外周血建立的短期 SEA反应 T细胞系凋亡 ,1.8%琼脂糖凝胶 DNA电泳于不同时间观察凋亡的特征条带 ;FCM检测 Fas、Fas L 的表达量变化 ,Fura- 2 / AM荧光指示剂检测胞浆游离 [Ca2 ]i的变化。结果使用 FCM特异性检测凋亡亚 G0 / G1峰大小及 1.8%琼脂糖凝胶电泳检测凋亡 DNA梯状图谱证明 ,SEA能诱导短期 SEA反应 T细胞凋亡 ,其凋亡的时间与程度与 SEA的剂量有关。SEA1μg/ ml建立的短期 SEA反应 T细胞对 SEA1μg/ml的再次刺激 ,凋亡量随作用时间的延长而增多 ,在作用的第 16 h最高 ,达 5 8%。 FCM间接荧光染色法检测发现 ,Fas及Fas L 亦随 SEA作用时间的延长而表达增多 ,16 h时表达最多 ,分别为 92 %和 6 0 %。用 Fura- 2 / AM荧光指示剂检测 ,此时细胞胞浆 [Ca2 ]i已从刺激前的 (2 90± 33) nm ol/ L 上升到 (6 80± 16 ) nmol/ L。结论 Fas系统与 SEA诱导的 T细胞凋亡密切相关。换言之 ,Fas系统在超抗原诱导 T细胞凋亡中起作用 ,而胞浆 [Ca2 ]i升高与 SEA诱导的凋亡 T细胞 DNA断裂及其它凋亡形态变化有关。     

CTLA-4在超抗原诱导T细胞无能中的作用研究

目的 探讨CTLA—4对T细胞无能的诱导作用。方法 通过使用超抗原SEA作为一种无能诱导剂，建立体外无能模型，检测了无能T细胞在受到SEA的再次刺激时其膜分子CD28和CTLA—4的表达。结果 与活化T细胞相比，无能T细胞在免疫应答的后期阶段表面表达高水平的CTLA—4分子，而CD28的表达则只较活化组T细胞稍高一些。结论 CTLA—4表面表达水平的升高很可能与T细胞无能状态的诱导有关。     

超抗原SEA诱导T细胞失能体外实验模型的建立

目的 建立超抗原诱导T细胞失能的体外实验模型。方法 SEA刺激人外周血淋巴细胞,再加入外源性的rIL-2维持,建立短期SEA反应性T细胞系;进行Ficoll-Hypaque密度梯度离心获取经SEA多次刺激过SEA反应性T细胞;加入处理过的PBMC作APCs,建立超抗原SEA诱导T细胞的化组和失能组。结果 SEA和rIL-2共同作用可得到静息SEA反应性T细胞系;SEA反复刺激使T细胞处于一低反应状态,与活化组相比,其DNA合成能力明显下降,IL－2分泌显著减少,静息一段时间后,其增殖能力慢慢恢复。结论 超抗原SEA多次刺激诱导SEA反应性T细胞处于失能状态。     

超抗原诱导T细胞克隆缺失的机制

超抗原诱导Ｔ细胞克隆缺失受到环境中多方面因素的影响。超抗原诱导Ｔ细胞Ｆａｓ系统（Ｆａｓ／ＦａｓＬ）表达升高，可导致Ｔ细胞经过Ｆａｓ／ＦａｓＬ途径而诱导凋亡，这是超抗原诱导Ｔ细胞克隆缺失的重要原因。细胞因子、抗原递呈细胞、年龄等均与超抗原诱导的Ｔ细胞克隆缺失有关，其中ＩＬ－２对Ｔ细胞克隆缺失起保护作用     

CTLA-4与超抗原对SEA诱导T细胞无能的关联研究

在建立超抗原SEA诱导T细胞无能的体外模型基础上 ,观察了无能T细胞受SEA刺激时共刺激分子CD2 8和CTLA 4的表达。结果发现 ,与活化组相比 ,在SEA加入后的不同时相点无能T细胞上CD2 8的表达都是正常的 ,而CTLA 4分子在SEA加入的第 60小时细胞表面有高水平的表达。这些结果表明 ,超抗原SEA诱导的这种无能状态与CTLA 4所介导的抑制作用增强有关     

APC辅助超抗原活化T细胞的机制

超抗原是ＭＨＣ非限制性及ＴＣＲＶβ特异性方式激活Ｔ细胞的一种新的蛋白质抗原分子。现已发现它所激活的Ｔ细胞可引发人体的多种急、慢性疾病。ＡＰＣ以与辅助普通抗原不同的方式辅助超抗原是超抗原活化Ｔ细胞的关键，了解ＡＰＣ辅助超抗原活化Ｔ细胞的机制有助于理解超抗原激活Ｔ细胞的机理及预防超抗原所引发的疾病。     

超抗原诱导T细胞凋亡或增殖的体外研究

研究抗原递呈细胞在超抗原诱导人外周血特异性Ｔ细胞反应中的作用。方法金黄色葡萄球菌Ａ肠毒素刺激短期人外周血ＳＥＡ反应Ｔ细胞系的作用中,通过去除或加入Ａｐｃ观察细胞形态,ＦＣＭ检测细胞亚ＧＯ／Ｇ１峰大小及１．８％ＤＮＡ琼脂糖凝胶电泳观察凋亡特征条带。     

APC辅助超抗原活化T细胞的机制

超抗原是ＭＨＣ非限制性及ＴＣＲＶＯ特异性方式激活Ｔ细胞的一种新的蛋白质抗原分子。现已发现它所激活的Ｔ细胞可引发人体的多种急、慢性疾病。ＡＰＣ以与辅助普通抗原不同的方式辅助超抗原是超抗原化Ｔ细胞的关键，了解ＡＰＣ辅助超抗原活化Ｔ细胞的机制有助于理解超抗原激活Ｔ细胞的机理及预防超抗原所引发的疾病。     

超抗原SEA联合PML-RARа对外周血T细胞TCR Vβ亚家族基因表达的影响

目的 探讨超抗原SEA联合PML-RARa多肽对外周血T细胞TCR Vβ亚家族基因表达的影响.方法 分别将SEA、PML-RARa多肽以及SEA联合PML-RARa多肽与正常人外周血单个核细胞共同培养,20 d后收集增殖细胞,利用RT-PCR及基因扫描技术分析诱导后增殖T细胞TCR Vβ亚家族的利用和克隆性增殖的特点.结果 单纯SEA诱导后,T细胞表达了11个TCR Vβ亚家族,且仍为多克隆增殖.单纯PML-RARa多肽诱导后,T细胞限制性表达8个Vβ亚家族,其中、Vβ13、Vβ14表现出寡克隆或寡克隆趋势.PML-RARa多肽联合SEA共同诱导,其T细胞表达TCR Vβ亚家族依然呈明显的限制性,且Vβ13、Vβ14亚家族呈寡克隆、双克隆及寡克隆趋势.结论 SEA能协同PML-RARa多肽诱导的T细胞克隆性活化与增殖.     

Clonal analysis of differential lymphokine production in peptide and superantigen induced T cell anergy

A failure of T lymphocytes to produce interleukin 2 (IL-2) on restimulation may, in part, account for the specific unresponsiveness that accompanies incomplete activation. The evidence to support this has been derived predominantly from the investigation of the molecular basis of anergy in murine type 1 T cells. In this study, the effects of different tolerogenic signals delivered by specific peptide or Staphylococcus aureus enterotoxin on the ability of antigen-specific human T cells to produce lymphokines, both in the induction phase and in established antigen-specific non-responsiveness, have been examined. Although T cell proliferation was decreased by supraoptimal concentrations of specific peptide in the presence or absence of antigen presenting cells, IL-2, IL-4, and interferon gamma (IFN-gamma) synthesis were comparable to that of activated T cells. The different tolerogenic signals, all capable of inhibiting phase of unresponsiveness. Restimulation of anergic T cells with an antigenic challenge failed to induce lymphokine production, with the exception of allergen-reactive T cells that secreted IFN-gamma. This latter observation is relevant to the desensitization of specific responsiveness in allergic disease.     

T细胞耐受的诱导及其机理研究

目的　阐明抗原特异性 Ｔ 细胞无能的诱导条件、无能细胞的特性及其耐受的机理。方法　抗 Ｂ７１ 单抗与 Ｃｓ Ａ 联用诱导抗原特异性 Ｔ 细胞无能, 通过３ Ｈ Ｔｄ Ｒ 掺入法测定 Ｔ 细胞增殖和 Ｍ Ｌ Ｒ, 利用 Ｒ Ｔ Ｐ Ｃ Ｒ 检测细胞因子基因表达。结果　耐受 Ｔ 细胞与异体淋巴细胞比例为０ ．０１∶１时, 可显著抑制 Ｍ Ｌ Ｒ, 转染 Ｂ７１ 分子的 Ｍ Ｄ Ａ４５３ 和３ Ａ Ｏ 能协同刺激 Ｃ Ｄ３ 诱导的 Ｔ 细胞增殖,不表达 Ｂ７ 分子的 Ｍ Ｄ Ａ４５３ 和３ Ａ Ｏ 无此作用。 Ｐ Ｈ Ａ、 Ｃ Ｄ３ 单抗、 Ｐ Ｍ Ａ＋ Ａ２３１８７ 可以逆转本试验所用诱导方法所致的 Ｔ 细胞的耐受状态。无能 Ｔ 细胞 Ｉ Ｌ２ 和 Ｉ Ｆ Ｎγｍ Ｒ Ｎ Ａ 不表达, 而 Ｉ Ｌ４ 和 Ｉ Ｌ１０ ｍ Ｒ Ｎ Ａ可表达。无能 Ｔ 细胞活化后, Ｉ Ｌ２ 和 Ｉ Ｆ Ｎγｍ Ｒ Ｎ Ａ 能够表达。结论　抗原特异性 Ｔ 细胞耐受是可以人为诱导的, 无能 Ｔ 细胞细胞因子基因格局向 Ｔ Ｈ２ 细胞偏离。     

T cell anergy is programmed early after exposure to bacterial superantigen in vivo

We have investigated the effects of the protein synthesis inhibitor,cycloheximide (CHX), on the induction of post-thymic T celltolerance in mice primed with the bacterial superantigen, Staphylococcusaureus enterotoxin B (SEB). A single injection of 1 mg CHX preventedprotein synthesis in splenic cells for <6 h in vivo. Theconcomitant administration of SEB and CHX prevented inductionof SEB-specific anergy, but did not interfere with the deletionof SEB-speclfic V_ß8⁺ T cells by activation-induced,programmed cell death. When CHX was given 24 h after SEB administrationthe expression of anergy was not affected. These findings suggestthat anergy and deletion represent independent processes. Furthermore,these observations, together with the fact that SEB retainsthe potential to induce anergy in specific T cells 8 h afterpriming in vivo, imply that the determination of alternate fates(anergy or death) occurs at early time points after SEB injection     

Peripheral tolerance in T cell receptor-transgenic mice: Evidence for T cell anergy

T cell tolerance can be induced in adult mice by injection of soluble antigenic peptide. The underlying mechanism has been difficult to establish in normal mice due to the low precursor frequency of T cells specific for any given antigen. Therefore, we examined peripheral tolerance in mice transgenic for a T cell receptor specific for a cytochrome c peptide bound to I-E^k. Antigen-specific hyporesponsiveness could be induced in the transgenic mice. We followed the transgene-bearing T cells with a clonotypic monoclonal antibody and found similar numbers of clonotypic T cells in tolerized and control mice. To prevent de novo differentiation of T cells we analyzed thymectomized mice in which antigen-specific hyporesponsiveness was induced. Our analysis of thymectomized transgenic mice showed that antigen-specific T cell hyporesponsiveness following injection of peptide intravenously is not caused by gross elimination of T cells. These data provide evidence for the role of anergy in peripheral tolerance.     

Increased c-Fos/activator protein-1 confers resistance against anergy induction on antigen-specific T cell

We have studied the contribution of c-Fos/activator protein-1 (AP-1) to antigen-specific T cell response with reference to T cell anergy by increasing c-Fos/AP-1 in vivo and in vitro. First, after injection of a high dose of staphylococcus enterotoxin B (SEB), clonal deletion of SEB-reactive V(beta)8(+) CD4 T cells occurred both in control B6 and H2-c-fos transgenic (fos) mice, whereas proliferation of T cells against SEB was profoundly depressed in B6 but not in fos mice. Second, the keyhole limpet hemocyanin-specific CD4 T(h)1 cell clone produced decreasing amounts of IL-2 in response to increasing amounts of concanavalin A (Con A) in vitro, whereas the decrease was less significant in the T(h)1 clones stably transfected with c-fos gene. Electrophoretic mobility shift assay with nuclear protein from the transformants showed that overexpression of the c-fos gene compensated the amounts of AP-1 in the nuclei of Con A-treated T(h)1 clones. Thus, increased c-Fos/AP-1 confers resistance against anergy induction on antigen-specific T cells.     

T细胞无能与凋亡关系初探

目的：探讨无能Ｔ细胞与凋亡的关系。方法：利用Ｂ７－１单抗和环孢素Ａ（ＣｓＡ）联用处理ＡＰＣ即Ｔ细胞系统,在体外诱致抗原特异性Ｔ细胞无能,用ＰＩ染色法经ＦＡＣＳ分析了无能Ｔ细胞的凋亡,用ＲＴ－ＰＣＲ法检测了凋亡细胞Ｆａｓ ｍＲＮＡ的表达水平。结果：无能Ｔ细胞在１、３、５、１０ｄ细胞凋亡的百分率依次为２．１６％１１．２８％、１９．２７％、４１．２２％。当加入１００Ｕ／ｍｌ ＩＬ－２维培养,无能Ｔ细胞为     

Molecular underpinning of B-cell anergy

Summary: A byproduct of the largely stochastic generation of a diverse B-cell specificity repertoire is production of cells that recognize autoantigens. Indeed, recent studies indicate that more than half of the primary repertoire consists of autoreactive B cells that must be silenced to prevent autoimmunity. While this silencing can occur by multiple mechanisms, it appears that most autoreactive B cells are silenced by anergy, wherein they populate peripheral lymphoid organs and continue to express unoccupied antigen receptors yet are unresponsive to antigen stimulation. Here we review molecular mechanisms that appear operative in maintaining the antigen unresponsiveness of anergic B cells. In addition, we present new data indicating that the failure of anergic B cells to mobilize calcium in response to antigen stimulation is not mediated by inactivation of stromal interacting molecule 1, a critical intermediary in intracellular store depletion-induced calcium influx.