B7/CD28和ICAM-1/LFA-1共刺激信号对T及B细胞功能影响的研究

目的　研究共刺激途径Ｂ７／ＣＤ２８ 和ＩＣＡＭ１／ＬＦＡ１ 对Ｔ 细胞活化以及Ｂ 细胞效应的作用。方法　在体外建立ＡＰＣＴＢ 细胞反应系统, 用Ｂ７１ 单抗和ＩＣＡＭ１ 单抗分别阻断Ｂ７／ＣＤ２８ 和ＩＣＡＭ１／ＬＦＡ１ 共刺激途径, 利用３ ＨＴｄＲ 法检测Ｔ 细胞增殖,ＥＬＩＳＡ 法测定Ｂ 细胞分泌的抗体, 用ＲＴＰＣＲ 法检测细胞因子基因的表达。结果　Ｂ７１ 单抗和ＩＣＡＭ１ 单抗均可抑制Ｔ 细胞增殖及ＩＬ２ 的产生。Ｂ７１ 单抗可下调Ｂ 细胞抗体的产生（ Ｐ＜ ０ ．０５） , 而ＩＣＡＭ１ 单抗未见明显的抑制（ Ｐ＞ ０ ．０５） 。Ｂ７１ 单抗和ＣｓＡ 联用能阻断Ｔ 细胞增殖活性及Ｂ 细胞的效应, 而ＩＣＡＭ１ 单抗和ＣｓＡ联用则无此作用。Ｂ７１ 单抗能下调ＩＬ２ 和ＩＦＮγｍ ＲＮＡ 表达,Ｂ７１ 单抗和ＣｓＡ 联用则阻断ＩＬ２ 和ＩＦＮγｍ ＲＮＡ 表达,ＩＬ４ 和ＩＬ１０ ｍ ＲＮＡ 仍可表达。结论　Ｂ７／ＣＤ２８ 和ＩＣＡＭ１／ＬＦＡ１ 共刺激途径在Ｔ 细胞活化中具有不同的作用,Ｂ７１ 单抗和ＣｓＡ 联用可导致Ｔ 细胞功能失活即无能。     

Apo—1／Fas受体及其配体在诱导淋巴细胞凋亡中的作用

本文介绍了Ａｐｏ－１／Ｆａｓ受体及其配体的结构及其分布，阐述了Ａｐｏ－１／Ｆａｓ受体及其配体在免疫生物学方面的效应：诱导ＴＣＲ＾ｉｍ／Ａｐｏ－１＾ｈｉ胸腺细胞的阴性选择；清除外周自身反应Ｔ细胞克隆；诱导某些活化Ｔ，Ｂ细胞凋亡；介导某些白血病细胞凋亡，并阐述了Ａｐｏ－１／Ｆａｓ受体及其配体在诱导细胞凋亡中的作用机理。     

Apo－1／Fas受体及其配体在诱导淋巴细胞凋亡中的作用

本文介绍了Ａｐｏ－１／Ｆａｓ受体及其配体的结构及其分布，阐述了Ａｐｏ－１／Ｆａｓ受体及其配体在免疫生物学方面的效应：诱导ＴＣＲ￣（ｉｍ）／Ａｐｏ－１￣（ｈｉ）胸腺细胞的阴性选择；清除外周自身反应Ｔ细胞克隆；诱导某些活化Ｔ，Ｂ细胞凋亡；介导某些白血病细胞凋亡，并阐述了Ａｐｏ－１／Ｆａｓ受体及其配体在诱导细胞凋亡中的作用机理。     

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。     

共刺激对T细胞功能和细胞因子格局的作用

利用Ｂ７－１单抗和环孢素Ａ处理ＡＰＣ：Ｔ细胞反应系统,探讨了Ｂ７／ＣＤ２８芡刺激对Ｔ细胞增殖及细胞因子产生的影响,并用ＲＴ－ＰＣＲ方法分析了细胞因子基因格局的改变。结果表明Ｂ７－１单抗可抑制特异性抗原诱导的Ｔ细胞增殖和ＩＬ－２的产生,Ｂ７－１单抗瑟ＣｓＡ联用可阻断Ｔ细胞增殖和ＩＬ－２产生。Ｂ７－１单抗绎ＩＬ－４的产生未显示明显的影响,而Ｂ７－１单抗与ＣｓＡ联用ＩＬ－４仍可检测到。     

环孢素A体外诱导HL—60细胞凋亡的研究

目的：观察环孢素Ａ是否有诱导白血病细胞凋亡的作用。方法：应用细胞形态学检查、二苯胺法ＤＮＡ片段化定量，ＤＮＡ凝胶电泳及流式细胞术等方法观察细胞凋亡。结果：ＣｓＡ５０ｍｇ／Ｌ作用ＨＬ－６０细胞４ｈ，ＤＮＡ片段率显著增高，达（２８．２±５．８）％，而对照组仅为（１２．５±１．７）％（Ｐ＜０．０１，ｎ＝１０）。光镜及电镜检查见细胞核固缩，碎裂，有凋亡小体形成。ＤＮＡ电泳显示典型的ＤＮＡｌａｄｄｅｒ。流式细胞术检测发现ＣｓＡ５０ｍｇ／Ｌ作用ＨＬ－６０细胞６ｈ凋亡细胞率为４９．７％，而空白组仅为９．１％。ＣｓＡ诱导ＨＬ－６０细胞ＤＮＡ片段化呈剂量和时间依赖性。结论：ＣｓＡ在体外能诱导ＨＬ－６０细胞凋亡。     

AT1R基因3'-非翻译区A1166→C变异和CA重复序列多态性与藏族EH的关联分析

目的：研究血管紧张素Ⅱ的Ⅰ型（ＡＴ１Ｒ）基因３’－端ＣＡ重复序列多态性和Ａ１１６６→Ｃ突变双等位樗是否与藏族原发性高血压（ｅｓｓｅｎｔｉａｌ ｈｙｐｅｒｔｅｎｓｉｏｎ,ＥＨ）的遗传易感性关联。方法：以荧光标记ｄＣＴＰ为底物,应用ＰＣＲ扩增和ＡＢＩｐｒｉｓｍ３７７半自动测序及ＰＣＲ／ＲＦＬＰ技术,通过病例－对照研究、受累同胞对家系连锁分析,鉴定ＡＴ１Ｒ基因３’－端ＣＡ重复序列多态性和Ａ１１６６→Ｃ点突变与葳族ＥＨ的关联。结果：病例－对照研究证明,ＡＴ１Ｒ基因３’－端ＣＡ重复序列多态性与ＥＨ相关联,χ＾２＝２６．４４,Ｐ＜０．００１,该位点杂合度为０．７３,多态信息量为０．７１。ＡＴ１Ｒ基因３’－端ＣＡ重复序列存在１１种等位基因,Ａ７（１３８ｂｐ）为最常见等位基因,Ａ８等位基因与ＥＨ正相关,ＥＨ组和对照组中Ａ８等位     

B7／CD28和ICAM—1／LFA—1共刺激信号对T及B细胞功？…

目的 研究共刺激途径Ｂ７／ＣＤ２８和ＩＣＡＭ－１／ＬＦＡ－１对Ｔ细胞活化以及Ｂ细胞效应的作用。方法 在体外建立ＡＰＣ：Ｔ：Ｂ细胞反应系统,用Ｂ７－１单抗和ＩＣＡＭ－１单抗分别阻断Ｂ７／ＣＤ２８和ＩＣＡＭ－１／ＬＦＡ－１共刺激途径,利用＾３Ｈ－ＴｄＲ法检测Ｔ细胞增殖,ＥＬＩＳＡ法测定Ｂ细胞分泌的杭体,用ＲＴ－ＰＣＲ法检测细胞因子基因的表达。结果 Ｂ７－１单抗和ＩＣＡＭ－１单坑均可抑制Ｔ细胞增殖及ＩＬ     

广州地区汉族人群细胞色素P450 1A1和细胞色素P450 2E1基因多态性

目的 探讨广州地区汉族人群细胞色素Ｐ４５０ １Ａ１（ＣＹＰ１Ａ１）和细胞色素Ｐ４５０ ２Ｅ１（ＣＹＰ２Ｅ１）基因的多态性分布规律。方法 用ＰＣＲ－ＲＦＬＰ和等位基因特异性扩增技术,对１５０名广州地区汉族正常人的ＣＹＰ１Ａ１和ＣＹＰ２Ｅ１基因多态性进行了检测,并与其他人群进行了比较。结果 ＣＹＰ１Ａ１基因３’端非翻译区的Ｍｓｐ１多态位点ｍ１（ＭＳＰⅠ－）、ｍ２（ＭｓｐⅠ＋）等位基因频率分别为６２．３     

Fas/Apo-1(CD95)系统与细胞凋亡研究新进展

Ｆａｓ／Ａｐｏ－１（ＣＤ９５）系统与机体免疫功能和细胞凋亡的关系十分密切,近几年来一直是免疫学家和临床医师关注的热点。本文综述了Ｆａｓ／Ａｐｏ－１ 在Ｉｐｒ突变小鼠和在胸腺Ｔ 细胞发育中研究的新进展,重点阐述了Ｆａｓ／Ａｐｏ－１ 系统诱导凋亡的分子机理。     

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。     

A role for antigen-presenting cells and bacterial superantigens in reversal of human T lymphocyte anergy

The induction of anergy in T lymphocytes generates T cells incapable of proliferation in response to a conventional antigenic stimulus. To investigate the induction and maintenance of anergy in human T cells, we used T cell-T cell presentation of myelin basic protein (MBP) or MBP synthetic peptides to induce anergy in vitro. Although anergic T cells responded normally to interleukin-2 (IL-2), these T cells did not produce IL-2 or IL-4 when peripheral blood mononuclear cells presented MBP or MBP peptides. Proliferation of anergic T cells was reduced by greater than 95% compared to nonanergic, control T cells. However, when autologous B cell lines were used to present MBP, anergy was partially reversed with a proliferation response about 50% of nonanergic levels. Bacterial superantigens also partially restored proliferation in anergic T cells following presentation by either B cell lines or macrophage isolated from peripheral blood mononuclear cells. Anergic, MBP-reactive T cells fully retained antigen-specific cytolytic activity against both B cell and T cell targets presenting MBP. These results suggest that T cell proliferative anergy may be reversible with both the type of antigen-presenting cell and superantigens potentially contributing to the initiation or maintenance of an autoimmune response.     

Regeneration and tolerance factor is expressed during T-lymphocyte activation and plays a role in apoptosis

Regeneration and tolerance factor (RTF) is a protein cloned from the thymus and expressed on B lymphocytes in normal pregnancy, B lymphocytic leukemia lines, and T and B lymphocytes in individuals with HIV infection. Findings, using the Jurkat T-cell model, revealed that RTF is upregulated after activation and anti-RTF antibody-induced apoptosis. In this article anti-RTF antibody-induced apoptosis of both unstimulated and activated T lymphocytes. RTF expression was examined in human PBMC or purified T lymphocytes after their in vitro activation. Kinetic studies indicated maximal RTF cell surface expression on activated T lymphocytes occurred between expression of the early activation antigen CD69 and the IL-2alpha receptor (CD25) by multiparameter flow cytometry. RTF receptor expression correlated with Fas (CD95) and CD25 receptor expression (r2 = 0.6 and 0.5, respectively). RTF surface expression was dependent on the stimuli used to activate T lymphocytes. T lymphocytes obtained maximal RTF expression when activated through the TCR signal complex using anti-CD3epsilon antibody alone when compared with T lymphocytes activated with costimulation provided by anti-CD28 antibody alone or with anti-CD28 and anti-CD3epsilon antibody. RTF is expressed under conditions of both activation and anergy. The RTFs increased concentration on the surface of anergic T cells may protect these cells from apoptosis because increased RTF concentrations inhibited anti-RTF induced apoptosis. These data further characterize the expression of RTF on activated T lymphocytes and the role of anti-RTF antibody in T-lymphocyte apoptosis.     

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。     

The cyclin-dependent kinase inhibitors p27Kip1 and p21Cip1 are not essential in T cell anergy

Recent evidence suggests that the cyclin-dependent kinase (Cdk) inhibitors p27Kip1 and p21Cip1 are important factors in T cell anergy, but it has remained unclear whether anergy can be induced in their absence. We therefore induced anergy by stimulation of purified T cells from wild-type, p21Cip1-/-, and p27Kip1-/- mice with anti-CD3 antibodies. Anergic wild-type T cells were arrested in the G1 phase of the cell cycle with a high p27Kip1 protein level and low Cdk2 activity. In p27-/- and p21-/- T cells, the pattern of protein expression was preserved, but Cdk2 activity was increased. To confirm the in vivo relevance of these data, anergy was induced by repeated injection of mice with staphylococcal enterotoxin B (SEB), which leads to partial deletion of the responsive Vbeta8+ T cell population and anergy in the remaining T cells. p21-/- mice and wild-type mice reacted similarly to this treatment. p27-/- mice showed reduced deletion of SEB-responsive T cells, but persisting T cells were anergic. These data indicate that other cell cycle regulators contribute to the cell cycle arrest of anergic T cells, as neither Cdk inhibitor is required for the induction of anergy.     

P59(fyn) is upregulated in anergic CD8+ T cells

The ultimate goal in clinical transplantation is achievement of graft tolerance. Despite long-term immunosuppression, alloantigens on transplants elicit alloresponses that can initiate organ rejection. Acute rejection is mediated by CD8(+) cytotoxic T cells, whereas chronic rejection is a result of many factors including non-immunological events. The aim of this study was to examine the molecular requirements of T cell anergy, a cellular state that is an integral component of tolerance in vivo. In vitro, the tolerant state is usually best represented by T cell anergy, which is defined by loss of the ability of T cells to produce and secrete interleukin-2 upon restimulation. In the literature, molecular changes in anergic CD4(+) T cells have been studied in great detail, but only little is known about functional and biochemical characteristics of anergic CD8(+) T lymphocytes. In this study, we demonstrate, that CD8(+) T cells are rendered anergic by TCR stimulation without costimulation. They exhibit impaired interleukin-2 production and tyrosine-phosphorylation, but markedly upregulated p59(fyn) expression, which could be shown to be an early event during anergization. Anergic CD8(+) T lymphocytes show elevated surface expression of early activation markers as well as costimulatory molecules, especially that of CTLA4. These results, are an important component for the discovery of potential molecular targets, which contribute to the development and maintenance of tolerance.     

阻断共刺激通路诱导产生的无能细胞的抗原特异性免疫调节作用

目的：了解联合阻断B7-CD28和CD40-CD154通路诱导产生的小鼠免疫无能细胞的免疫调节特性和表型，以及是否具有抗原特异性。方法：Anti-CD154和anti-CD80单克隆抗体加入BALB／C(夏应细胞)和C3H(刺激细胞)的混合淋巴细胞培养(MLR)体系中诱导产生无能细胞。将无能细胞或对照细胞分别加入新的BALB／C和C3H的MLR体系中观察抑制效果。在反应细胞和刺激细胞／第三方刺激细胞(C57BL/6J)之间的MLR体系中加入无能细胞观察抗原特异性。无能细胞中加入刺激细胞或rmIL-2,观察无能细胞的逆转情况。通过流式细胞仪检测无能细胞的表型。结果：无能细胞对反应细胞和刺激细胞之间的MLR具有抑制作用，且呈剂量依赖关系。无能细胞对于反应细胞和第三方刺激细胞之间的MLR无抑制作用。C3H刺激细胞和rmIL-2共同刺激才能逆转无能细胞的无能状态。无能细胞中CD25^ CD4^ T细胞含量上升，而CD45RB^kwCD4^ 和CD28^-CD8^ T细胞含量无改变。结论：联合阻断B7-CD28和CD40-CD154通路诱导产生的小鼠免疫无能细胞具有抗原特异性的免疫调节功能，可以通过感染性耐受的方式抑制淋巴细胞的增殖。     

Control of CD4 T cell fate by antigen re-stimulation with or without CTLA-4 engagement 24 h after priming

After two consecutive inoculations with Staphylococcus enterotoxin B (SEB) at 24 h intervals in vivo, CD4 T cells became anergic to the antigen challenge in vitro. Administration of anti-CTLA-4 mAb in conjunction with the second SEB inoculation 24 h after antigen priming interfered with anergy and CD4 T cells became T(h)2 cells. However, the anergy induction was not ablated when SEB and anti-CTLA-4 mAb were administered 48 or 72 h after antigen priming. Moreover, anti-CTLA-4 mAb without SEB did not interfere with anergy nor promoted the T(h)2 differentiation. T-antigen-presenting cell (APC) interaction in vitro in the presence of high doses of antigen and anti-CTLA-4 mAb induced a T(h)2-polarizing cytokine IL-6 and IL-10. IL-10 then down-modulated a T(h)1-polarizing cytokine IL-12. The results demonstrate that 24 h after the initial antigen stimulation, CD4 T cells enter the critical activation phase where antigen re-stimulation with or without CTLA-4 engagement alters the fate of the cell, anergy or differentiation respectively. Once anergy is interfered with, T(h)2-polarizing cytokines produced upon prolonged T-APC interaction favor the T(h)2 differentiation.     

Fas/FasL mediated apoptosis of thyrocytes in Graves' disease.

We examined in the present study the possible involvement of Fas and its ligand (FasL) in the process of Graves' disease. Immunohistochemical analysis showed that few normal thyrocytes expressed Fas but many thyrocytes in Graves' disease expressed this molecule. The percentage of FasL-positive thyrocytes in Graves' thyroids was, however, less than in normal thyroids. Several apoptotic thyrocytes and infiltrating mononuclear cells (MNCs) were detected scattered throughout Graves' thyroid tissues and abundant proliferating cell nuclear antigen (PCNA)-positive thyrocytes were present. Apoptotic cells, as well as PCNA-positive cells, were scarcely detectable in normal thyroid glands, however. In vitro treatment of thyrocytes by IL-1beta a cytokine found to be expressed in Graves' thyroid glands, increased Fas but reduced FasL expression. IL-1beta-stimulated thyrocytes became sensitive to apoptosis by anti-Fas IgM monoclonal antibody (mAb). Activated T cells, which strongly expressed FasL, showed cytotoxic activity toward IL-1beta-stimulated thyrocytes but not toward unstimulated thyrocytes. This cytotoxic activity involved the Fas/FasL pathway. Importantly, unstimulated thyrocytes could kill activated, but not resting, T cells. IL-1beta-stimulated thyrocytes, with down-regulated FasL expression, could not efficiently kill activated T cells. The cytotoxic activity of unstimulated thyrocytes toward activated T cells was inhibited by anti-FasL mAb. Interestingly, unstimulated thyrocytes induced apoptosis in IL-1beta-stimulated thyrocytes but not in unstimulated thyrocytes. These interactions were also blocked by anti-FasL mAb. Our results suggest that the apoptotic cell death of both thyrocytes and infiltrating MNCs found in Graves' thyroid glands is regulated by IL-1beta through Fas/FasL interactions.