风湿病患者肺功能降低与BTLA阳性细胞及调节性T细胞的数量减少有关

目的探讨B、T淋巴细胞衰减因子(BTLA)、调节性T细胞(Treg)与风湿病患者肺功能降低的关系。方法选取482例风湿病患者,包括类风湿性关节炎(RA)198例,强直性脊柱炎(AS)114例、干燥综合征(SS)102例、骨关节炎(OA)68例,应用肺功能仪检测患者肺功能水平,采用流式细胞术检测患者外周血B、T淋巴细胞衰减因子(BTLA)和调节性T细胞(Treg)表达。结果与正常组比较,风湿病患者肺功能降低,外周血BTLA、Treg表达降低;与风湿病BTLA、Treg正常组比较,BTLA、Treg异常组肺功能参数第1秒用力呼气容积(FEV1)、最大呼气流量(PEF)、50%肺活量位的最大呼气流量(FEF50)、FEF75降低(P0.05或P0.01);相关分析显示,肺功能参数用力肺活量(FVC)、最大通气量(MVV)、FEV1、FEF25、FEF50分别与BTLA、Treg呈正相关,FVC、FEV1、FEF50、FEF75分别与IgA、IgM呈负相关(P0.05或P0.01)。结论风湿病患者肺功能降低可能与BTLA、Treg表达降低,促使T细胞、B细胞过度异常活化有关。     

Abp1在调节性T细胞的发育及功能中的作用

目的 探讨肌动蛋白连接蛋白1(actin-binding protein 1,Abp1)对调节性T细胞(Treg)发育及功能的影响.方法 通过流式细胞术检测野生型小鼠(WT组)和Abp1基因敲除小鼠(KO组)的胸腺、脾脏、浅表淋巴结及肠系膜淋巴结中T淋巴细胞及Treg细胞的比例、数量以及相关功能性分子的表达,并利用体外...     

调节性DC与调节性T细胞的相互作用

近年来研究发现调节性树突状细胞(Dendritic cells,DC)能够下调免疫应答和介导外周免疫耐受,调节性DC诱导的免疫耐受与其未成熟或半成熟状态密切相关。大量研究表明调节性DC和调节性T细胞(Regulatory T cells,Treg)之间存在着复杂的双向调控:调节性DC可扩增和诱导产生Treg,还可影响Treg向局部组织和外周淋巴器官归巢;Treg则妨碍DC与非调节性T细胞的结合并抑制DC的活化、成熟和刺激T细胞增殖的能力。总之,调节性DC与Treg相互协同以精细调控机体的免疫应答。     

地塞米松体外短期处理影响Ⅰ型调节性T 细胞与自然调节性T 细胞平衡

目的:本研究拟通过地塞米松体外短期处理外周血淋巴细胞探讨地塞米松对调节性T 细胞包括自然调节性T 细胞(Natural regulatory T cell,Treg)和Ⅰ型调节性T 细胞(Type I regulatory T cell,Tr1) 的影响。方法:取健康人外周血淋巴细胞,分为地塞米松处理组和阴性对照组,处理3 d 后按多色分析法进行染色,用流式细胞仪检测并分析CD25、CD127、淋巴细胞活化因子3(Lymphocyte-activation 3,LAG-3)和叉头样转录因子3(Forkhead box P3,Foxp3)的表达及Treg 和Tr1 的频率变化。结果:相对于对照组,地塞米松处理3 d 后CD4+ T 细胞频率增加且呈剂量依赖性;CD4+ T 细胞上CD25 和Foxp3 的表达显著降低(P =0.006,P<0.000 1),而CD127 和LAG-3 表达显著升高(P<0.000 1、P =0.011);Treg 频率显著降低(P<0.001),而Tr1频率显著升高(P =0.051),且增加Tr1/ Treg 细胞比率(P =0.044)。结论:地塞米松体外短期处理上调Tr1 比率,下调Treg 比率,改变Tr1 与Treg 细胞平衡。     

调节性T细胞的研究进展

调节性T细胞对于维持机体免疫自稳、防止自身免疫病具有极其重要的作用,也涉及抗感染免疫、肿瘤免疫、移植免疫、变态反应等许多病理性免疫过程。CD4+CD25+调节性T细胞是调节性T细胞中最为重要的一群,此外,Tr1,Th3等细胞也归属于调节性T细胞。调节性T细胞是外周免疫耐受的新的重要机制,它可通过细胞接触或者抑制性细胞因子作用于抗原呈递细胞或效应性T细胞,从而发挥调节作用。     

小鼠BTLA慢病毒表达载体的构建及鉴定

目的 构建小鼠BTLA慢病毒表达载体,并对表达产物进行鉴定.方法 以pET-28a-mBTLA质粒为模板,通过PCR技术构建pMD18-T-mBTLA质粒,将小鼠BTLA基因全长编码序列克隆到慢病毒转移载体,通过脂质体转染293T细胞包装成小鼠BTLA慢病毒颗粒.PCR技术和基因测序对重组质粒进行鉴定.荧光显微镜观察重组慢病毒感染293T细胞形态学变化.RT-PCR和Western blot法鉴定BTLA在重组慢病毒感染293T细胞中的表达.50％组织培养感染剂量法(TCID5o法)检测重组慢病毒滴度.结果 成功构建的pMD18-T-mBTLA质粒和pSL6-mBTLA质粒,经双酶切电泳后均出现大小约为1 kb的条带与mBTLA编码序列的大小相符.基因测序并比对分析进一步证实mBTLA编码序列成功整合到质粒载体.病毒上清PCR扩增和293T细胞形态学观察证实Lenti-mBTLA慢病毒包装成功.TCID50法测定Lenti-mBTLA慢病毒滴度为1.3×108 pfu/mL.RT-PCR和Western blot法证实Lenti-mBTLA慢病毒能有效表达mBTLA mRNA和蛋白质.结论 成功构建了表达小鼠BTLA的慢病毒.     

调节性T细胞的研究进展

调节性T细胞对于维持机体免疫自稳、防止自身免疫病具有极其重要的作用，也涉及抗感染免疫、肿瘤免疫、移植免疫、变态反应等许多病理性免疫过程。CD4^ CD25^ 调节性T细胞是调节性T细胞中最为重要的一群，此外，Tr1，Th3等细胞也归属于调节性T细胞。调节性T细胞是外周免疫耐受的新的重要机制，它可通过细胞接触或者抑制性细胞因子作用于抗原呈递细胞或效应性T细胞，从而发挥调节作用。     

调节性T细胞

调节性T细胞具有免疫低反应性和免疫调节功能两大特征。它们在自身免疫性疾病的治疗、肿瘤的治疗以及移植耐受的诱导等方面具有潜在的应用价值。本对近年来献报道的调节性T细胞进行了总结，并就其诱导、分化、膜表面分子标志、功能以及可能的作用机制进行了综述。     

调节性T细胞研究进展

调节性T细胞是不同于Th1和Th2的具有调节功能的T细胞群体 ,因其具有免疫抑制作用 ,近来受到人们的广泛关注 ,本文综述调节性T细胞近来的研究进展     

抗原特异性调节性T细胞的诱导及其对动脉粥样硬化的影响

目的探讨热休克蛋白（HSP）60抗原特异性CD4^＋CD25^＋T细胞的体外诱导及其对动脉粥样硬化斑块形成的影响。方法分离apoE^-/-小鼠骨髓单个核细胞，经阿司匹林处理培养出未成熟树突状细胞；体外诱导HSP60特异性调节性T细胞分化，检测其百分比和分泌功能；通过混合淋巴细胞反应研究CD4^＋CD25^＋T细胞的特异性抑制效应；过继转移CD4^＋CD25^＋T细胞后，观察其对小鼠动脉粥样斑块形成的影响。结果阿司匹林处理的树突状细胞共刺激分子CD80和CD86表达减少，形态学表现为未成熟树突状细胞；未成熟树突状细胞比成熟树突状细胞能诱导更多的特异性CD4^＋CD25^＋T细胞，培养体系中IL-10和TGF-β1水平明显升高。CD4^＋CD25^＋T细胞体外明显抑制效应性T细胞的增殖以及IFN-γ的分泌。体内，过继转移HSP60特异性CD4^＋CD25^＋T细胞组小鼠动脉粥样斑块面积显著小于对照组。结论未成熟树突状细胞可诱导出HSP60抗原特异性CD4^＋CD25^＋T细胞，后者在体内能明显抑制动脉粥样斑块的形成。     

Distinct expression and inhibitory function of B and T lymphocyte attenuator on human T cells

B and T lymphocyte attenuator (BTLA) has been recently identified as a new inhibitory receptor of the CD28 superfamily, with similarities to cytotoxic T lymphocyte activation antigen (CTLA)-4 and programmed death (PD)-1. Engagement of BTLA on T lymphocytes can profoundly reduce the T cell receptor (TCR)-mediated activation. In this study, we generated four monoclonal antibodies (mAbs) against human BTLA. Using the produced mAb 8H9, the BTLA molecule was found to distinctly express on many subgroups of immunocytes and show a regulatory expression, which was in accordance with its unique ligand herpes virus entry mediator (HVEM) in the process of T cell activation. In addition, the expression of BTLA was increased in the CD4(+) and CD8(+) T cells of pleural fluid in lung cancer patients. Furthermore, we showed that the BTLA-induced negative signals could be triggered by mAb 7D7. Cross-linking of BTLA with mAb 7D7 suppressed T lymphocyte proliferation, downregulated the expression of T cell activation marker CD25, and inhibited the production of interferon (IFN)-gamma, interleukin (IL)-2, IL-4, and IL-10.     

可溶性小鼠BTLA对树突状细胞表面B7-1和B7-2表达的影响

目的研究可溶性小鼠B/T淋巴细胞弱化因子（murine B and T lymphocyte attenuator ，mBTLA）-人IgG1 Fc融合蛋白（mBTLA-hIg）对树突状细胞表面共刺激分子表达的影响。方法克隆mBT／A基因，构建mBTLA胞外功能区和人IgG1Fc融合基因的真核表达载体（pmBTLA-hIg），并采用脂质体转染法，将pmBTLA-hIg质粒转染小鼠树突状细胞系（DC2．4）。采用RT-PCR、ELISA和Westem blot分别检测pmBTLA-hIg质粒转染DC中mBTLA基因mBNA的表达及细胞培养上清中mBTLA-hIg融合蛋白的表达；采用流式细胞术检测pmBTLA-hIg质粒转染对DC2．4表面共刺激分子B7-1（CD80）和B7-2（CD86）表达的影响。结果成功克隆了小鼠BT／A基因，并构建了真核表达载体；mBTLA-hIg融合基因转染的DC高表达mBTLA-hIg融合蛋白，并可结合到DC表面。表达可溶性mBTLA-hIg融合蛋白的DC2．4表面B7．1的表达上调，B7-2的表达下调，而且这种改变可被兔抗GST-mBTLA血清阻断。结论可溶性mBTLA-hIg融合蛋白对DC细胞表面研分子的表达具有调节作用，可能是BTLA反向信号作用于DC的结果。     

基于B、T淋巴细胞衰减因子探讨膝骨关节炎患者肺功能变化的机制及相关性分析

目的 通过观察膝骨关节炎(knee osteoarthritis,KOA)患者肺功能参数、B、T淋巴细胞衰减因子(B and T lymphocyte attenuator,BTLA)及血清细胞因子的变化情况,探讨KOA患者肺功能变化的可能分子机制.方法 选取2011年10月-2012年7月安徽中医学院第一附属医院风湿病科收治的住院、门诊患者47例.应用肺功能仪检测患者肺功能指标;流式细胞术检测BTLA;ELISA法检测白介素(interleukin,IL)-1β、IL-10、基质金属蛋白酶9(matrix metalloproteinase-9,MMP-9)、基质金属蛋白酶组织抑制物-1(tissue inhibitor of matrix metalloprotease-1,TIMP-1);魏氏法测定血沉(erythrocyte sedimentation rate,ESR);日立7060型全自动生化分析仪进行测定超敏C反应蛋白(high-sensitivityC-reactive protein,hs-CRP).结果 (1)与正常组比较,KOA患者用力肺活量(forced vital capacity,FVC)、一秒用力呼气容积(forced expiratory volume in 1 second,FEV1)、FEV1/FVC、用力最大呼气流量(peak expiratory flow,PEF)、最大呼气中段流量(maximal mid-expiratory flow,MEF25-75)、用力呼气50％流量(Maximal mid-expiratory flow velocity in pulmonary vital capacity 50％,MEF50)、用力呼气25％流量(maximal mid-expiratory flow velocity in pulmonary vital capacity 25％,MEF25)、CD3+ BTLA+T细胞、CD4+BTLA+T细胞、IL-10、TIMP1明显降低,IL-1β、MMP9明显升高.(2)相关性分析显示,FVC与Lequesne MG、症状分级量化总分、病程、MMP9呈负相关,与CD3+ BTLA+T细胞、IL-10、TIMP1呈正相关;FEV1与CD3+ BTLA+T细胞、CD4+BTLA+T细胞、TIMP1呈正相关,与病程呈负相关;MEF50与CD3+ BTLA+T细胞、CD4+ BTLA+T细胞呈正相关.结论 KOA患者在发生关节软骨病变的同时,伴有肺功能的下降.其机制可能与BTLA表达水平下降,IL-1β、MMP9明显升高,IL-10、TIMP1明显降低,诱发异常免疫应答,从而介导气道损伤,导致KOA患者肺功能下降有关.     

Mechanisms regulating the development and function of natural regulatory T cells

The key of the immune system is to protect the host from foreign threat posed by pathogens and from the internal threat posed by self-attacking lymphocytes. The ability to discriminate self versus non-self ensures that only “non-self” pathogens, but not the self antigens, are attacked. Such tolerance to “self” arises from the central tolerance mechanisms that include the deletion of thymocytes with high reactivity to self antigens and also the induction of unresponsiveness of autoreactive T cells in the periphery. Natural regulatory T cells (nTregs) directly inhibit effector T cells, and keep their proliferation in control. Apart from preventing autoimmune reactions, Tregs also contribute to peripheral immune homeostasis as evidenced by the excessive lymphocyte accumulation in peripheral lymphoid organs and intestinal inflammation in the absence of nTregs. Here we discuss the molecular aspects of the development and suppressive function of naturally occurring Tregs. Accumulating evidence shows the importance of these Tregs in autoimmunity, tumor immunity, organ transplantation, allergy, and microbial immunity.     

B/T淋巴细胞间抑制信号研究进展

B7-CD28家族共刺激信号分子在T淋巴细胞激活、抑制机体耐受及产生理想的T细胞免疫应答等方面起着关键作用。B7-CD28家族共信号分子已经被集中的研究,并且引起免疫调控领域充分的重视。CD80/CD86/CD28/CTLA-4经典途径的新功能的发现以及这一家族包括B7-H1/B7-DC/PD-1,B7-H2/ICOS,B7-H4和BTLA的新途径的确定,都拓宽了我们对T细胞介导的免疫应答和免疫耐受调控的理解。本文就CTLA-4、PD-1和BTLA对T细胞应答的负性调节以及在维持外周耐受中起作用的研究进展作一综述。     

BTLA associates with increased Foxp3 expression in CD4+ T cells in dextran sulfate sodium-induced colitis

Ulcerative colitis (UC) is an inflammatory bowel disease, and its pathogenesis involves a variety of genetic, environmental, and immunological factors such as T helper cells and their secreted cytokines. B and T lymphocyte attenuator (BTLA) is an immunoregulatory receptor that has a strong suppressive effect on T-cell function. However the role of BTLA in UC remains poorly understood. Here we demonstrated that the frequency of BTLA-expressing CD3⁺ T cells, especially CD4⁺ T cells, increased in blood and mucosa in mice with DSS-induced colitis. The frequency of Foxp3-expressing cells in BTLA+ CD4⁺ T cell from lamina propria mononuclear cells (LPMCs) was much higher in DSS-treated mice than that in controls. Similarly, the proportion of IL-17+ cells in BTLA+ CD4⁺ T cells from LPMCs in DSS-treated mice is much higher than that in controls, while no perceptible difference for the proportion of IFN-γ+ cells in BTLA+ CD4⁺ T cells was noted between DSS-treated mice and controls. Treatment of mesalazine, an anti-ulcerative colitis drug, down-regulated Foxp3 and IL-17 expression in BTLA positive T cells along with attenuated severity for colitis. Our findings indicate that BTLA may be involved in the control of inflammatory responses through increasing Foxp3 expression, rather than attenuating IL-17 production, in DSS-induced colitis.     

转染人BTLA基因细胞株的建立及其生物学功能的初步研究

目的:构建B、T淋巴细胞衰减子(BTLA)基因转染的细胞作为免疫原,并探讨该基因转染细胞在体外的生物学功能。方法:通过RT-PCR法从经PHA活化的人外周血T细胞中克隆出人BTLA编码区全长基因,经EcoR I和BamHI双酶切后插入逆转录病毒载体pEGZ-Term中构建成重组载体pEGZ-Term/BTLA。用脂质体法以重组逆转录病毒载体转染293T细胞,并用Zeocin抗生素进行长期筛选;流式细胞术分析BTLA在基因转染细胞膜上的表达;通过MTT法和流式细胞术探讨基因转染细胞在体外对T淋巴细胞增殖与活化的影响。结果:流式细胞术检测表明BTLA基因转染的293T细胞膜上能稳定地高表达人BTLA蛋白。BTLA基因转染的293T细胞和T细胞体外共培养显示,与未转染的293T细胞相比,该基因转染的细胞能部分地抑制抗人CD3单克隆抗体(mAb)刺激的T细胞增殖;流式细胞术和ELISA法分析揭示,该基因转染的细胞能够下调T细胞表面活化标志CD25的表达并降低IFN-γ和IL-10的分泌。结论:获得稳定高表达人BTLA基因转染的细胞株。该细胞株在体外对抗人CD3 mAb刺激的T细胞的增殖与活化具有部分地抑制作用。     

Emerging possibilities in the development and function of regulatory T cells

CD25+CD4+ Regulatory T cells (Treg) represent a unique population of lymphocytes capable of powerfully suppressing immune responses. A large body of experimental data have now confirmed the essential role played by these cells in a host of clinically relevant areas such as self-tolerance, transplantation, allergy and tumor/microbial immunity. Despite this mass of knowledge, significant gaps in our understanding of fundamental Treg biology remain, particularly regarding their development and mechanisms of suppression. In this review we attempt to highlight the current controversies and directions in which this exciting field is moving.     

慢性乙型肝炎患者外周血CD4~+CD25~+Treg与CD4~+和CD8~+ T淋巴细胞亚群的相关研究

目的:探讨慢性乙型肝炎患者外周血中CD4+CD25+调节性T细胞的含量和CD4+CD8+T淋巴细胞亚群分布,两者之间相关性及与HBV的相关性。方法:采用流式细胞术检测50例慢性乙型肝炎患者和20例健康对照者外周血中CD4+CD25high、CD4+CD25+Foxp3+Treg细胞表达及CD3/CD4/CD8 T淋巴细胞亚群,荧光定量PCR法检测HBV DNA含量。结果:慢性乙型肝炎患者外周血中CD4+CD25highTreg明显高于健康对照组(P0.01),且随HBV DNA载量增加,患者外周血中CD4+CD25highTreg细胞的水平逐渐升高。慢性乙型肝炎患者外周血中CD4+CD25+Foxp3+Treg细胞也相应增高,且与CD4+CD25highTreg细胞的变化成正相关(r=0.890,P0.001)。与健康对照组比较,患者组CD4+T细胞百分率及CD4+/CD8+比值均降低,而CD3+T细胞和CD8+T细胞百分率差异无显著性(P0.05)。CD4+CD25highTreg细胞与HBV DNA取对数后成正相关(r=0.782,P0.001),与谷丙转氨酶(ALT)成正相关(r=0.432,P0.005);与CD3+、CD4+、CD8+T细胞水平及CD4+/CD8+比值均无相关性(P0.05)。CD3+、CD4+、CD8+T淋巴细胞及CD4+/CD8+比值与HBV DNA载量之间亦无相关性(P0.05)。结论:慢性乙型肝炎患者外周血中CD4+CD25+Treg细胞增高,且与HBV的复制水平及ALT增高具有一致性,而T细胞亚群是否可作为监测CHB患者免疫状态的指标需进一步探讨。     

CD4 regulatory T cells

There is now compelling evidence that CD4(+) T cells that specialize in the suppression of immune responses play a key role in the control of immune pathology. Recently, there have been a number of reports that have provided information on the generation of CD4(+) regulatory T cells in the thymus and in the periphery. These cells have also been identified in humans, paving the way for analysis of the function of CD4(+) regulatory T cells in immune-mediated disease.