CTLA-4基因多态性与Graves病

细胞毒性T淋巴细胞相关抗原4（cytotoxie Tlymphocyte associated antigen4，CTLA-4）是激活的T细胞表达的一种膜蛋白，属免疫球蛋白超家族成员，对T细胞增生起负性调节作用。与抗原呈递细胞（APC）表面的B7分子结合，作为协同刺激信号抑制T细胞增生、活化，诱导T细胞耐受。CTLA-4功能和（或）表达缺陷参与了T细胞介导的自身免疫性疾病的发生发展。Graves病（GD）是一种由于抑制性T淋巴细胞（TS）功能缺陷所导致的器官特异性自身免疫病。近年来研究结果认为，CTLA4基因的多态性与GD有关，CTLA4基因作为GD的易感候选基因已成为研究的热点。     

转染人CTLA-4细胞的对树突状细胞B7分子表达的影响

从PHA活化的人外周血T细胞中抽提总RNA,经RT-PCR扩增出CTLA-4的全长基因,构建逆转录病毒载体pEGZ-Term/CTLA-4,经PCR及电泳鉴定后感染包装细胞293T。收集培养上清感染L929细胞。用Zeocin选择培养。经免疫荧光及流式细胞术进行筛选,获取稳定表达CTLA-4分子的细胞株CTLA-4/L929。将CTLA-4/L929经丝裂霉素处理后与体外细胞因子诱导的树突状细胞共同孵育。分别于24、48及72h,用直接免疫荧光法和流式细胞术分析树突状细胞B7-1和B7-2分子的表达。研究结果显示,构建的重组逆转录病毒载体pEGZ-Term/CTLA-4的PCR产物长约700 bp,与人CTLA-4基因的长度一致。经Zeocin选择及多次亚克隆化培养,CTLA-4基因转染细胞CTLA-4/L929稳定表达膜型CTLA-4分子。以其为刺激细胞与树突状细胞共同培养后,可明显下调树突状细胞B7-1的表达,但对B7-2的表达没有影响。本研究获得的CTLA-4基因转染细胞株,为进一步探讨CTLA-4及其配基分子在免疫应答中的作用与机制提供了手段。     

CTLA-4基因启动子区单核苷酸多态性与特发性扩张型心肌病的关联研究

目的探讨特发性扩张型心肌病（idiopathic dilated cardiomyopathy，IDC）患者细胞毒性T淋巴细胞相关抗原-4（cytotoxic T lymphocyte assoccated antigen 4，CTLA-4）表达状况及由CTLA-4基因启动子区单核苷酸多态性（single nucleotide polymorphism，SNP）导致的不同遗传易感性机制。方法采用限制性片段长度多态性分析151例IDC患者，120名正常健康人CTLA-4基因启动区-1772、-1661及-318位点SNP；免疫酶联吸附测定法检测血清sCTLA-4、干扰素-7及白介素-4水平；综合分析CTLA-4启动区基因型、等位基因频率及与sCTLA-4、干扰素-γ／白介素一4的相关性。结果IDC患者sCTLA-4水平与CTLA一4基因启动区SNP相关，携带-1772T／C变异者sCTLA-4表达增高。-1772TC基因型频率在IDC组尤其低射血分数亚组显著高于对照组，IDC组-1661G和-1661GG频率显著降低，具有-1772TC-1661AA及-1772TC-1661AG单倍型IDC患者sCTLA-4显著升高。结论IDC患者CTLA-4表达异常，CTLA-4基因启动区-1772C／T和-1661A／GSNP与IDC遗传易感性相关。-1772T／C变异可能影响CTLA-4基因剪接，干扰蛋白表达和功能，阻止负性调节信号传递而导致对IDC的易感。     

特发性扩张型心肌病CTLA-4基因3''非翻译区(AT)n微卫星多态性研究

目的 特发性扩张型心肌病（idiopathic dilated cardiomyopathy，IDC）的发病机制与T细胞免疫应答密切相关。细胞毒性T淋巴细胞相关抗原-4（cytotoxic T lymphocyte antigen-4，CTLA-4）主要在已激活的T细胞上表达，通过与CIY28竞争结合B7，抑制T细胞过度激活，维持免疫系统内环境稳定。CTLA-4基因3’非翻译区（AT）n微卫星多态性影响CTLA-4功能。本研究旨在探讨外周血单个核细胞（PBMC）CTLA-表达状况及3’非翻译区（AT）n微卫星基因多态性与IDC的相关性。方法分别用原位杂交、免疫组化、序列特异性引物PCR等方法检测38例无血缘关系的北方汉族IDC患者以及50例正常对照者的CTLA-4mRNA、蛋白质表达、CTLA-4基因外显子3’末端非翻译区（AT）n重复序列多态性，并对PCR扩增产物进行序列分析。结果IDC组与对照组相比，PBMC经金黄色葡萄球菌肠毒素B刺激后CTLA-4mRNA、蛋白质表达强度显著减弱，且无一定规律；3’末端非翻译区共发现18种CTLA-4等位基因，与对照组比较，106bp等位基因频率在IDC患者中显著增高[22．22％ vs 1％，P=0．0002．OR=23．56，95％可信区间（CI）：9．65～83．74]。结论IDC患者CTLA-4基因转录和表达缺陷，该缺陷与CTLA-4基因3’末端非翻译区（AT）n重复序列多态性存在关联。     

一对重要的免疫调节分子:CTLA-4及其抗体

杀伤性T细胞淋巴细胞相关抗原4(CTLA-4)分子是淋巴细胞表面的一种与免疫信号传递有关的配体蛋白分子，又称CD152，它属于免疫球蛋白超家族成员，为Ⅰ型膜蛋白分子。CTLA-4和CD28是同源性蛋白，它们在结构上相似，且都可以特异性结合B7-1／B7—2分子，但CD28与B7的结合，正调节T细胞的活化，而CTLA-4与B7结合后，负调节T细胞的活化，可减缓细胞周期的进程，降低IL-2的产生，使T细胞活化增殖受到抑制，从而使免疫达到平衡状态，保持外周的免疫平衡。而CTLA-4分子作为一种免疫活化的制动剂，表现为较少的量即可发挥强有力的免疫抑制功能。近几年来的研究结果使人们不断认识到此分子及其抗体在免疫调节中的重要作用。     

CTLA-4基因多态性调节溃疡性结肠炎患者CTLA-4 mRNA稳定性和蛋白水平

目的 研究细胞毒T淋巴细胞相关抗原4(cytotoxic T-lymphocyte associated antigen 4,CTLA-4)基因3'非转录区(AT)n重复序列多态性对溃疡性结肠炎(ulcerative colitis,UC)患者CTLA-4mRNA稳定性和基因表达的影响.方法 采用实时定量PCR方法检测膜型CTLA-4(full length CTLA-4,flCTLA-4)和可溶性CTLA-4(soluble CTLA-4,sCTLA-4)mRNA表达,半衰期法分析其mRNA稳定性.免疫组化检测flCTLA-4蛋白表达.酶联免疫吸附实验测定sCTLA-4蛋白水平.荧光PCR-毛细管电泳技术检测300例UC患者和700名健康对照者CTLA-4基因(AT)n重复序列多态性.结果 活动期UC患者肠黏膜sCTLA-4 mRNA的表达显著低于缓解期UC患者(P=0.004).在UC患者中,(AT)n重复序列长等位基因携带者表达低水平的flCTLA-4和sCTLA-4 mRNA以及sCTLA-4蛋白(均P＜0.01).携带长等位基因的UC患者CTLA-4 mRNA的稳定性明显降低.UC患者CTLA-4基因(AT)n重复序列长等位基因携带者(≥116 bp)频率显著高于正常对照组(P＜0.01),且与广泛型结肠炎相关(P=0.008).结论 UC患者CTLA-4基因(AT)n重复序列多态性与CTLA-4基因表达水平相关,携带(AT)n重复序列长等位基因的UC患者CTLA-4 mRNA及蛋白的表达降低,提示CTLA-4基因在UC遗传免疫发病机制中起重要作用.     

中国人群CTLA-4基因多态性与系统性红斑狼疮及类风湿性关节炎的相关性研究

系统性红斑狼疮(SLE)与类风湿性关节炎(RA)是常见的自身免疫性疾病,病因未明.研究发现,除了HLA基因的相关性以外,还可能存在其它重要的敏感基因[1].近年来发现CTLA-4基因在自身免疫性疾病发病中的作用,本文检测SLE及RA病人CTLA-4基因多态性,进一步探讨SLE及RA的发病机制.     

儿童Graves病与CTLA-4基因多态性的初步研究

Graves’病（简称GD）是一种常见的器官特异性自身免疫性甲状腺疾病,其确切病因不明,但已肯定GD是一种多基因遗传的自身免疫性疾病。近年来儿童GD与成人GD一样有发病率逐渐增加的趋势,已成为一种危害儿童健康的常见病。最新研究认为候选基因中HLA和CTLA-4两个基因位点占GD遗传易感性的50％,而CTLA-4基因作为T细胞活化的一种重要的负性调节因子,已成为导致自身免疫性内分泌疾病的一个非常重要的易感位点,也成为国内外学者研究的热点,儿童起病的GD与成人起病的GD在发病机制上截然不同,本研究对于CTLA-4基因第二外显子的多态性进行初步研究。     

特发性扩张型心肌病CTLA-4基因3′非翻译区(AT)n微卫星多态性研究

目的特发性扩张型心肌病(idiopathic dilated cardiomyopathy,IDC)的发病机制与T细胞免疫应答密切相关。细胞毒性T淋巴细胞相关抗原24(cytotoxic Tlymphocyte antigen-4,CTLA-4)主要在已激活的T细胞上表达,通过与CD28竞争结合B7,抑制T细胞过度激活,维持免疫系统内环境稳定。CTLA-4基因3′非翻译区(AT)n微卫星多态性影响CTLA-4功能。本研究旨在探讨外周血单个核细胞(PBMC)CTLA-4表达状况及3′非翻译区(AT)n微卫星基因多态性与IDC的相关性。方法分别用原位杂交、免疫组化、序列特异性引物PCR等方法检测38例无血缘关系的北方汉族IDC患者以及50例正常对照者的CTLA-4 mRNA、蛋白质表达、CTLA24基因外显子3′末端非翻译区(AT)n重复序列多态性,并对PCR扩增产物进行序列分析。结果IDC组与对照组相比,PBMC经金黄色葡萄球菌肠毒素B刺激后CTLA-4 mRNA、蛋白质表达强度显著减弱,且无一定规律;3′末端非翻译区共发现18种CTLA24等位基因,与对照组比较,106bp等位基因频率在IDC患者中显著增高[22.22%vs1%,P=0.0002,OR=23.56,95%可信区间(CI):9.65～83.74]。结论IDC患者CTLA-4基因转录和表达缺陷,该缺陷与CTLA-4基因3′末端非翻译区(AT)n重复序列多态性存在关联。     

细胞间黏附分子-1基因多态性与疾病易感性

细胞间黏附分子-1(ICAM-1)属于免疫球蛋白超家族成员之一，是一种细胞表面单链糖蛋白，它表达于多种细胞表面，通过识别其受体LFA-1、MAC-1、P150、P95介导细胞-细胞间的黏附，参与多种炎症反应及免疫过程。不同种族和地区研究证实ICAM-1基因多态性与人类多种疾病相关。     

共刺激分子CTLA-4的研究进展及在风湿病治疗中的应用

细胞毒T淋巴细胞抗原-4(CTLA-4)是免疫应答反应起始阶段T细胞活化的重要共同活化分子,它与T细胞表面的配体结合后可以抑制T细胞功能,使T细胞不能被激活,从而抑制免疫反应的发生。在多种风湿病中都发现有CTLA-4等位基因异常和蛋白表达异常。CTLA-4免疫球蛋白(CTLA-4 Ig)是针对CTLA-4的融合蛋白,能选择性地抑制免疫反应,改善患者的临床症状,达到控制疾病进展的目的。但目前的临床研究尚仅限于类风湿关节炎。临床研究的初步结果表明CTLA-4 Ig治疗类风湿关节炎是有效而安全的,但由于其临床应用时间短,在风湿病治疗中的作用还有待进一步的长期临床观察来证实。     

CTLA-4: a key regulatory point in the control of autoimmune disease

Summary: Chronic autoimmune disease in humans is the result of a failure to control autoreactive immune cells in the periphery. This control is largely achieved by inhibition of newly activated and memory cells. A number of negative immune regulatory pathways have been characterized. The cell surface coreceptor cytotoxic T-lymphocyte antigen-4 (CTLA-4) has emerged as a critical attenuator of T-cell activation and an essential component of the regulatory systems that serve to maintain peripheral tolerance. CTLA-4 expression is induced on the surface of T cells after they have received a costimulatory signal from antigen-presenting cells (APCs) via engagement of CD28 on the T-cell surface. CTLA-4 attenuates this costimulation by competing for CD28 ligands and through direct effects on APCs via the same ligands utilized by CD28. A large number of genetic association studies suggest that the CTLA-4 gene is a locus of susceptibility to autoimmune disease. However, specific functional defects in the CTLA-4 gene in patients have not been identified to date. Elucidating the role of CTLA-4 in immune tolerance has also led to a number of therapeutic applications, particularly in the treatment of malignancy and autoimmune disease.     

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.     

CTLA-4 regulates cell cycle progression during a primary immune response.

Engagement of CTLA-4 is critical for inhibiting T cell immune responses. Recent studies have shown that CTLA-4 plays a key role in regulating peripheral T cell tolerance. It has been suggested that one mechanism by which CTLA-4 performs this function is by regulating cell cycle progression. Here, we investigate in depth the role of CTLA-4 in regulating cell cycle progression in naive T cells by comparing the immune responses in the absence or presence of CTLA-4. In the absence of CLTA-4, T cells exhibit marked increases in T cell proliferation, IL-2 mRNA and protein secretion, and cells cycling in the S and G2-M phase. Analyses of cyclins, cyclin-dependent kinases, and cell cycle inhibitors involved in the transition from the G1 to S phase reveal that cell cycle progression is prolonged in the absence of CTLA-4. This is due to the early exit from the G1 phase, entry into the S phase, and prolonged S phase period. Re-expression of the cell cycle inhibitor p27(kip1) is delayed in the absence of CTLA-4. These studies demonstrate that the B7 : CTLA-4 pathway exerts its major effects on T cell immune responses via regulation of the cell cycle.     

CTLA-4 gene polymorphism and its association with Graves' disease in the Lebanese population.

Graves' disease is an organ-specific autoimmune disease that has a female predominance. It is probably the result of a complex interaction of genetic and environmental factors. This disease is characterized by immune system activation, evidenced by elevated serum thyroid-specific autoantibodies and lymphocytic infiltration of the target organ (the thyroid gland), associated with raised levels of circulating activated T lymphocytes. Several reports have demonstrated genetic linkage and association between the genetic markers of the CTLA-4 gene on chromosome 2q33 and Graves' disease. In order to confirm this association in the Lebanese population, a bi-allelic A/G polymorphism at position 49 of CTLA-4 exon 1 was studied in 34 patients with Graves' disease, and in 38 healthy individuals, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The results showed a significant increase in allele and genotype frequencies in patients with Graves' disease compared to controls. This suggests that the CTLA-4 gene might play a role in the development of Graves' disease in the Lebanese population.     

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.     

Identification of CTLA-4 isoforms produced by alternative splicing and their association with myasthenia gravis

Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness induced by autoantibodies against the acetylcholine receptor. CTLA-4 (CD152) plays an inhibitory role in the immune response and has been suggested to be involved in the pathophysiology of MG. In this study, we focused on alternative CTLA-4 mRNA expression in PBMCs from MG patients. We defined two new isoforms of CTLA-4 mRNA that arise due to alternative splicing. By semi-quantitative RT-PCR analysis, we observed a lower expression of sCTLA-4 mRNA relative to the membrane form in MG patients. In addition, the MG patients had lower levels of sCTLA-4 mRNA in PBMCs compared to healthy controls, as assessed by real-time PCR. One of the spliced isoforms (LCTLA-4) was more prevalent in MG patients compared to healthy controls. The alternative splicing was not associated with sex, thymectomy, serum levels of anti-AChR, immunosuppressive treatment or the four CTLA-4 gene polymorphisms analyzed. This study reveals an abnormal spectrum of mRNA expression of CTLA-4 in MG patients, which marks the importance of studying gene expression of alternative splicing.     

Expression and functional significance of CTLA-4, a negative regulator of T cell activation

The generation of an effective immune response involves antigen-specific T cell expansion and differentiation of effector function. T cell activation requires at least two distinct signals, including signaling via the antigen-specific T cell receptor (TCR) and a costimulatory pathway. Antigen stimulation of T cells can lead either to a productive immune response, characterized by proliferation, differentiation, clonal expansion and effector function, or, in the absence of an appropriate costimulation, to a state of long-lasting unresponsiveness, termed anergy. Anergic T cells fail to proliferate and secrete cytokines in response to secondary stimulation. The interaction between the costimulatory molecule CD28 on T cells and members of the B7 family on antigen-presenting cell results in upregulation of T cell proliferation and cytokine production and induces the expression of the anti-apoptotic protein Bcl-xl. Based of these findings, the two-signal requirement model for T cell activation is generally accepted today. The negative regulatory mechanisms during T cell activation are not well understood, but they are crucial for the maintainance of lymphocyte homeostasis. For several years the functional role of the enigmatic CD28 homologue cytotoxic T lymphocyte antigen-4 (CTLA-4) in T cell activation has been both obscure and controversial. CTLA-4 was initially supposed to provide a costimulatory signal in conjunction with TCR/CD3 signaling. Today we know that CD28 and CTLA-4 molecules may have diametrically opposed functions: signaling via CD28, in conjunctive with TCR, is required for T cell activation, while signaling via CTLA-4 is a negative signal that inhibits T cell proliferation. How the T cell integrates signals through the TCR/CD3 complex, CD28 and CTLA-4 to initiate, maintain and terminate antigen-specific immune response is in fact not fully clarified. In this review, we will focus on the emerging role of CTLA-4 as a negative regulator of T lymphocyte activation and its role in the dynamic interplay of activatory and inhibitory signals.     

Control of peripheral T-cell tolerance and autoimmunity via the CTLA-4 and PD-1 pathways

Summary: Classically, the CD28/cytotoxic T-lymphocyte antigen-4 (CTLA-4) and B7 families of cell surface molecules regulate complex signaling pathways that profoundly affect T-cell responses. The recent identification and characterization of additional CD28 and B7 family members including programmed death-1 (PD-1), programmed death ligand-1 (PD-L1) (B7-H1), and PD-L2 (B7-DC) has added to the complexity and greater appreciation of how surface molecules control T-cell activation and peripheral tolerance. CD28/B7 interactions mediate co-stimulation and significantly enhance peripheral T-cell responses. CTLA-4, in contrast, interacting with the same B7 molecules, results in decreased T-lymphocyte activity and regulates the immune response. Similarly, PD-1 interactions with PD-L1 and PD-L2 downmodulate T-cell immune responses. Despite these similarities, the regulatory roles of the CTLA-4 and PD-1 pathways are distinct. This may be due, at least in part, to the differential expression patterns of the CTLA-4 and PD-1 ligands both temporally and spatially. This article examines the role of CTLA-4 and PD-1 in limiting autoreactivity and establishing peripheral self-tolerance with the hypothesis that CTLA-4 signals are required early in the lymph node during initiation of an immune response and PD-1 pathways act late at the tissue sites to limit T-cell activity.     

早孕期外周血及蜕膜淋巴细胞CD28/CTLA-4的表达

目的：探讨人早孕期外周血及蜕膜淋巴细胞协同刺激分子表达水平在母．胎免疫调节中的作用。方法：以15例正常育龄妇女黄体期外周血为对照，应用流式细胞仪分析15例正常人早孕期外周血及蜕膜淋巴细胞CD28和CTLA-4的表达水平。结果：各组淋巴细胞几乎不表达表面CTLA-4分子；而表达细胞内CTLA-4分子（CTLA-4i）。蜕膜组织淋巴细胞CTLA-4i^＋／CD28^＋的比例显著高于早孕期及黄体期外周血。早孕期与黄体期外周CTLA-4i^＋／CD28^＋的百分率比较无显著性差异。结论：母胎界面局部高表达细胞内CTLA-4在母-胎免疫耐受中起重要作用。