儿童癫痫患者T淋巴细胞活化并产生促炎因子

目的检测儿童癫痫患者外周T淋巴细胞活化状态以及细胞因子水平变化。方法选取20名儿童癫痫患者和19名健康对照儿童,采集外周血,分离外周血单个核细胞(PBMC),用细胞膜表面标记抗体流式细胞术检测T淋巴细胞表面共刺激分子CD69、CD25和细胞毒性T淋巴细胞相关蛋白4(CTLA4)的表达,用细胞内因子染色结合流式细胞术检测T细胞细胞因子γ干扰素(IFN-γ)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)和IL-17A的表达,利用细胞内因子染色结合流式细胞术检测调节性T淋巴细胞(Treg)表达IL-10的情况。结果与对照组相比,儿童癫痫患者外周血中T淋巴细胞表面高表达共刺激分子CD69、CD25和CTLA4,活化的CD4+T细胞高表达IFN-γ、TNF-α、IL-6和IL-17 A。在儿童癫痫患者高表达IL-10的Treg数目增加。结论儿童癫痫患者外周T淋巴细胞活化并产生细胞因子。     

CTLA4-Ig在树突状细胞中的表达及效应研究

目的 获得CTLA4-Ig转基因修饰的DC细胞,分析表达产物CTLA4-Ig对T细胞活化功能的影响。方法 采用脂质体转染方法将CTLA4-Ig表达载体转入DC细胞,通过ELISA法鉴定转染DC细胞48和72h培养上清液及稳定表达的培养上清液,分离Balb/c小鼠和C57BL/10J小鼠淋巴细胞为反应细胞和刺激细胞,观察CTLA4-Ig对同种细胞混合培养反应的影响。结果 将鼠CTLA4-Ig表达载体转入DC细胞,获得瞬时表达及稳定表达,CTLA4-Ig可抑制单向混合淋巴细胞反应,且效应亦呈剂量依赖性。结论 鼠CTLA4-Ig表达产物对同种细胞刺激的增殖反应有抑制作用,其抑制作用随表达载体转染细胞培养上清液的增加而增强。同时获得了稳定表达CTLA4-Ig融合蛋白的CTLA4-Ig转基因修饰的DC细胞,为以后的工作奠定了基础。     

人CD4+CD25+调节性T细胞系的建立与功能分析

目的：建立可在体外长期培养的人CD4^ CD25^ 调节性T细胞系并研究其免疫生物学特性。方法：用流式分选的方法从健康人外周血淋巴细胞中得到CD4^ CD25^ T细胞，并对其进行体外长期培养、扩增；淋巴细胞转化实验分析其免疫抑制功能，流式细胞法分析其表型。结果：经对人CD4^ CD25^ T细胞的长期培养扩增，获得具有免疫抑制功能的调节性T细胞系。该T细胞系对经TCR的刺激不敏感，且能抑制同一来源或同种异型CD4^ CD25^ T细胞的活化，大剂量IL－2可以逆转其抑制功能。长期培养的CD4^ CD25^ T细胞，膜表面CD25和CTLA－4分子持续高表达，而CD4^ CD25^ T细胞CD25和CTLA－4分子的表达呈周期性变化。结论：将CD4^ CD25^ T细胞体外扩增培养成系，其功能和表型与同一来源的CD4^ CD25^ T细胞显著不同。     

CTLA4-Ig基因修饰的树突状细胞体外对Th1/Th2平衡的影响

目的:探讨细胞毒性T淋巴细胞相关抗原4免疫球蛋白(CTLA4 Ig)基因修饰的树突状细胞(CTLA4 Ig-DCs)体外对Th1/Th2平衡的影响。方法:通过腺病毒载体将目的基因(CTLA4 Ig)转染至小鼠骨髓来源的树突状细胞(DC)。采用流式细胞术(FCM)检测DC表面分子和胞内CTLA4 Ig的表达;采用混合淋巴细胞反应检测DC刺激同种异体T细胞的能力,ELISA法检测DC抗原提呈反应中Th1和Th2类细胞因子IFN-γ和IL-4分泌水平。结果:CTLA4 Ig基因成功转染至DC,转染率约为80%,制备的CTLA4 Ig-DCs稳定表达CTLA4Ig,表面分子CD86呈现低表达;CTLA4 Ig-DCs可有效抑制T细胞增殖,降低抗原提呈反应上清中IFN-γ和IL-4的分泌,并增加IFN-γ/IL-4比值。结论:通过腺病毒将CTLA4 Ig转染DC并且高效表达,可有效降低DC表面CD86分子,抑制同种异体T细胞反应,并能影响体外Th1/Th2水平。     

霉酚酸及其衍生物对人T淋巴细胞及混合淋巴细胞免疫应答的影响

目的探讨霉酚酸及霉酚酸衍生物对人T淋巴细胞和混合淋巴细胞免疫应答的影响。方法从正常人外周血中分离单个核淋巴细胞(PBMC),在抗CD3刺激下和混合淋巴细胞反应体系中,加入或不加霉酚酸及其衍生物共培养,应用ELISA和流式细胞术检测CD4+和CD8+T细胞细胞因子的产生、细胞活化和增殖情况。结果经抗CD3刺激后,PBMC产生的细胞因子明显增加,加入霉酚酸及其衍生物后,IFN-γ及TNF-α明显降低,IL-2产生略有增加。在混和淋巴细胞反应体系中,3种细胞因子产生皆被抑制。抗CD3刺激和混和淋巴细胞反应后,T细胞表面活化分子CD25及CD69表达增加,加入霉酚酸及其衍生物共培养24、48 h后,T细胞CD25及CD69的表达并未被抑制。霉酚酸及其衍生物可抑制抗CD3刺激下和混和淋巴细胞反应后T细胞的增殖。结论霉酚酸及其衍生物对T淋巴细胞和混合淋巴细胞体系中因子的产生和细胞增殖都有抑制作用,为其在临床自身免疫疾病及移植排斥中的广泛应用提供了理论依据。     

IL-2调节人外周血T淋巴细胞CTLA-4表达

目的 探讨人外周血T淋巴细胞CTLA-4的表达情况及IL-2对其表达的调节作用。方法 采用流式细胞仪定量测定人外周血T淋巴细胞内及细胞膜上CTLA-4的水平，半定量RT-PCR检测T淋巴细胞内CTLA-4mRNA的水平，并在体外用IL-2刺激T淋巴细胞后观察CTLA-4及CTLA-4mRNA水平的变化。结果 人外周血T淋巴细胞膜表面几乎不表达CTLA-4，7．6％-18．0％的T淋巴细胞有胞内表达，CD4^ T淋巴细胞表达CTLA-4的阳性比例略高于CD8^ T淋巴细胞；人T淋巴细胞可溶性形式的CTLA-4mRNA半衰期短于全长CTLA-4mRNA；IL-2可以通过诱导人T淋巴细胞CTLA-4mRNA的转录上调CTLA-4的表达，IL-2诱导的细胞多为CD25^ T淋巴细胞。结论 CTLA4多存在于人外周血T淋巴细胞内，参与T淋巴细胞活化过程的调节。IL-2的免疫抑制作用可能与其诱导T淋巴细胞内CTLA-4mRNA转录，从而上调CTLA-4的表达有关。     

B7H1-Ig诱导Tr1细胞向CD4~+CD25~+Foxp3~+Treg细胞转化的研究

为探讨Ⅰ型调节性T细胞(Tr1)与CD4+CD25+Foxp3+Treg之间的转化和相互关系,以预包被而固相化的B7H1-Ig融合蛋白加抗CD3单抗刺激初始CD4+CD62L+T细胞,分析细胞因子及Foxp3表达水平的变化,检测细胞功能;在B7H1-Ig开始刺激时或诱导细胞分化结束后加入重组人TGF-β,观察其对细胞分化的影响。结果显示,B7H1-Ig激活的CD4+T细胞产生高水平IL-10、IFN-γ和IL-5,极低水平的IL-2和IL-4,不表达Foxp3,通过分泌抑制性细胞因子IL-10发挥免疫抑制功能,证实B7H1-Ig可诱导Tr1细胞的产生。同时发现TGF-β不影响B7H1-Ig刺激的初始CD4+T的分化,却可促进B7H1-Ig诱导的已分化Tr1细胞向CD4+CD25+Foxp3+Treg转化,提示在特定条件下,Tr1细胞可转化的CD4+CD25+Foxp3+Treg。研究结果为将来临床应用CD4+Treg治疗免疫失调性疾病奠定了基础。     

白藜芦醇对活化小鼠T淋巴细胞IL-2和CD25表达的影响

目的：研究白藜芦醇(RSV)对活化的小鼠T淋巴细胞分泌IL-2和CD25的影响，并探讨其免疫抑制机制。方法：无菌分离小鼠淋巴结细胞，与不同浓度的白藜芦醇共同孵育1小时后，用多克隆刺激剂佛波醇酯和离子霉素刺激T细胞活化，继续培养6小时后收获细胞，进行胞内细胞因子染色，流式细胞仪检测IL-2的分泌情况，RT-PCR检测IL-2 mRNA的表达：24小时后检测CD25表达。结果：RSV对IL-2的分泌具有抑制作用，并呈剂量依赖性，同时RSV也能抑制T细胞表面活化分子CD25。结论：RSV对活化T细胞分泌的细胞因子IL-2及IL-2α链CD25的表达可能是RSV对T细胞具有免疫抑制作用的机制之一，并可能与T细胞活化通路的PKC-NF-κB信号传导途径相关。     

CTLA4Ig对丝裂原诱导的淋巴细胞反应的影响

本研究证明CTLA4Ig 与B7 分子结合后, 可以阻断B7 与CD28 的结合。CTLA4Ig 可以抑制丝裂原所触发淋巴细胞的增殖反应, 同时抑制细胞因子IL 2 ,IL 4 的产生以及IL 2R 的表达。表明CTLA4Ig 可以通过不同的途径影响T淋巴细胞的活化     

雪胆素乙对小鼠淋巴细胞体外活化及增殖的影响

目的分析雪胆素乙(Cucurbitacin IIb,CuIIb)对丝裂原刺激的小鼠淋巴细胞体外活化、增殖及促炎因子表达的影响,探讨其潜在的抗炎效应及作用机制。方法以WST法检测CuIIb对刀豆蛋白A(Con A)刺激的淋巴细胞增殖的作用,利用流式细胞术分析小鼠淋巴细胞活化抗原CD69和CD25以及促炎因子肿瘤坏死因子(TNF-α)和白细胞介素6(IL-6)表达的影响。结果 CuIIb能明显抑制Con A刺激引起的淋巴细胞的增殖作用,并呈现剂量依赖性;经CuIIb处理后,T细胞早期活化标志分子CD69和中期活化分子CD25的表达受到明显抑制;同时,CuIIb以剂量依赖方式抑制Con A诱导的促炎因子TNF-α和IL-6在CD3+T细胞中的表达。结论 CuIIb对小鼠淋巴细胞的活化、增殖均具有明显抑制作用,同时减少炎症相关细胞因子的表达,提示其通过对适应性免疫的调控发挥抗炎效应。     

Differential inhibition of autoreactive memory- and alloreactive naive T cell responses by soluble cytotoxic T lymphocyte antigen 4 (sCTLA4), CTLA4Ig and LEA29Y

Cytotoxic T lymphocyte antigen 4 (CTLA4) is a potent inhibitory co-stimulatory molecule believed to be involved in type 1 diabetes and other autoimmune diseases. An association has been reported of both mRNA expression and serum levels of the soluble splice variant of CTLA4 (sCTLA4) with type 1 diabetes. Furthermore, recombinant fusion proteins CTLA4Ig and LEA29Y have been proposed as therapies for type 1 diabetes. We studied the role of (s)CTLA4 in islet autoimmunity. Binding capacity of the proteins to antigen-presenting cells was determined by flow cytometry in competition and binding assays. Functionality of sCTLA4 as well as the therapeutic inhibitory fusion proteins CTLA4Ig and LEA29Y was measured in a dose-response lymphocyte stimulation test, using a panel of diabetes-associated T cell clones reactive to islet autoantigens. As controls, mixed lymphocyte reactions (MLR) were performed to assess functionality of these proteins in a primary alloreactive setting. All three CTLA4 molecules were able to bind to antigen-presenting cells and inhibit the expression of CD80/CD86. sCTLA4 was able to suppress proliferation of different committed autoreactive T cell clones in a dose-dependent manner, whereas CTLA4Ig and LEA29Y were not. Conversely, CTLA4Ig and LEA29Y, rather than sCTLA4, were able to suppress naive alloreactive proliferation in a MLR. Our results indicate a differential role for sCTLA4, CTLA4Ig and LEA29Y proteins in memory versus primary immune responses with implications for efficacy in intervention therapy.     

腺病毒介导CTLA4Ig基因修饰骨髓间质干细胞体外抑制免疫应答的研究

目的:通过腺病毒载体介导CTLA4Ig在大鼠骨髓间质干细胞(MSC)中的表达,探讨MSC作为基因转移靶细胞的可行性和转染效率,研究其在体外对免疫应答的抑制作用。方法:构建含有CTLA4Ig基因的重组腺病毒载体pAd-CTLA4Ig,按照不同的感染倍率(MOI)转染大鼠MSC,用荧光显微镜和流式细胞仪分析转染效率,流式细胞术、Westernblotting等方法检测目的蛋白CTLA4Ig在MSC中的表达。将转基因MSC加入混合淋巴细胞反应(MLR)体系,观察其抑制淋巴细胞增殖的生物学效应。结果:重组腺病毒载体pAd-CTLA4Ig对MSC的最大转染率为80.7%±4.7%,转基因MSC可检测到目的蛋白的表达。基因修饰的MSC能有效抑制MLR体系中淋巴细胞的增殖,4d达到最大抑制效率51.46％;再次MLR证实这种抑制作用是供者特异性的。结论:MSC是一种较理想的基因转移靶细胞,其表达的转基因产物CTLA4Ig可在体外特异性地抑制免疫应答。     

Upregulation of CD4+CD25+ T lymphocyte by adenovirus-mediated gene transfer of CTLA4Ig fusion protein in experimental autoimmune myocarditis

OBJECTIVE: To explore the effects of adenovirus vector-mediated gene transfer of CTLA4Ig fusion protein on CD4+CD25+ T cells in experimental autoimmune myocarditis (EAM). METHODS: EAM was induced by porcine cardiac myosin as previously described. Adenovirus vector-mediated CTLA4Ig gene was administrated intravenously in EAM rats on days 1, 4 and 7, with EGFP as control. On day 21, myocardium histopathology was examined and CD4+CD25+ T cells were isolated. Proliferation and suppression assays were used to evaluate the suppressive capacity of CD4+CD25+ T cells in vitro. Relative mRNA level of Foxp3 and TGF-beta was determined by quantitative real-time RT-PCR; expression of CTLA-4, B7-1 and B7-2 protein was compared with Western blot in CD4+CD25+ Tregs. RESULTS: Severe inflammatory lesions were observed in the hearts of EGFP-treated EAM rats and the untreated ones, while Ad-CMV-CTLA4Ig alleviated the myocarditis histologically. Adenovirus vector-mediated CTLA4Ig gene transfer up-regulated the proportion of CD4+CD25+ Tregs significantly. T cell proliferation was greatly inhibited in the CTLA4Ig group compared with the untreated and EGFP-treated groups in vitro. CTLA-4 and B7-2 proteins were down-regulated in the CTLA4Ig group, Foxp3 and TGF-beta mRNA was up-regulated significantly by CTLA4Ig treatment. CONCLUSIONS: Adenovirus vector-mediated CTLA4Ig gene transfer alleviated inflammation in EAM, one of the potential mechanisms is up-regulation of CD4+CD25+ Tregs.     

Negative feedback of T cell activation through inhibitory adapters and costimulatory receptors

Summary: Antigen recognition by the T cell receptor (TCR) complex induces the formation of a TCR signalosome by recruiting various signaling molecules, generating the recognition signals for T cell activation. The activation status and functional outcome are positively and negatively regulated by dynamic organization of the signalosome and by costimulation signals. We have studied the negative regulation of T cell activation, particularly through inhibitory adapters and costimulation receptors that are little expressed in resting cells but are induced upon T cell activation. We described Grb‐associated binder 2 (Gab2) and cytotoxic T lymphocyte antigen‐4 (CTLA‐4) as a representative inhibitory adapter and a negative costimulation receptor, respectively, both of which exhibit negative feedback. Gab2 functions as a signal branch for activation vs. inhibition, as phosphorylation of either Src homology 2 (SH2) domain‐containing leukocyte phosphoprotein of 76 kDa (SLP‐76) or Gab2 by zeta‐associated protein of 70 kDa (ZAP‐70) determines the fate of the response. As a professional inhibitory receptor, CTLA‐4 inhibits T cell response by competition of ligand binding with positive costimulator receptor CD28, and also induces inhibitory signaling. The trafficking and the cell surface expression of CTLA‐4 are dynamically regulated and induced. CTLA‐4 is accumulated in lysosomes and secreted to the T cell–APC contact site upon TCR stimulation. As T cell activation proceeds, these inhibitory adapters and costimulation receptors are induced and suppress/regulate the responses as negative feedback.     

Immunological potential of cytotoxic T lymphocyte antigen 4 immunoglobulin in murine autoimmune cholangitis

Cytotoxic T lymphocyte antigen 4 (CTLA‐4) immunoglobulin (Ig) is an important regulator of T cell activation and a fusion protein directed at CD80 and CD86; it blocks co‐stimulatory signalling and T cell activation. We have taken advantage of a murine model of human primary biliary cirrhosis (PBC), mice expressing a transforming growth factor (TGF)‐β receptor II dominant negative (dnTGF‐βRII) transgene to address the potential therapeutic efficacy of CTLA‐4 Ig. To mimic patients with PBC at different stages or duration of disease, we treated mice with either CTLA‐4 Ig or control IgG three times weekly from 3 to 12 or 24 weeks of age, or from 12 to 24 weeks of age. CTLA‐4 Ig treatment from 3 weeks of age significantly reduced liver inflammation to 12 weeks of age. Treatment initiated at 12 weeks of age also ameliorated the autoimmune cholangitis at 24 weeks of age. However, in mice treated at 3 weeks of age, suppression of liver inflammation was not sustained and colitis was aggravated when treatment was extended to 24 weeks of age. Our data indicate that, in dnTGF‐βRII mice, CTLA‐4 Ig treatment has short‐term beneficial effects on autoimmune cholangitis, but the effect varies according to duration of treatment and the time in which therapy was initiated. Further dissection of the events that lead to the reduction in therapeutic effectiveness of CTLA‐4 Ig will be critical to determining whether such efforts can be applied to human PBC.     

CTLA4Ig Primed Donor Lymphocyte Infusion: A Novel Approach to Immunotherapy after Haploidentical Transplantation for Advanced Leukemia

CTLA4Ig attenuates T cell activation by co-stimulation blockade, but natural killer (NK) cells are not only resistant to CTLA4Ig, they also may demonstrate better antileukemia effect in the presence of CTLA4Ig. To explore this phenomenon we used sequential CTLA4Ig primed donor lymphocyte infusion (DLI) after post-transplant cyclophosphamide–based haploidentical transplantation. Thirty patients (CTLA4Ig-DLI group) with advanced leukemia received CTLA4Ig on day –1 and subsequently on days +7, +21, and +35, followed 12hours later by DLI of 1 to 10?×?10⁶ CD3⁺ T cells/kg containing .1 to 3.27?×?10⁶/kg CD56⁺ NK cells, with low dose cyclosporine for 60days. The incidences of acute graft-versus-host disease (GVHD), chronic GVHD and nonrelapse mortality (NRM) were 6.7%, 21%, and 4.5 %, respectively, with disease progression of 23.3% and overall survival of 79% at 18 months. Patients without disease progression had a significant early surge in CD56^dimCD16⁺NK cells with lower NKG2A expression. CTLA4Ig primed DLI was associated with an upregulation of CD86 in mature NK cells that was not witnessed with CTLA4Ig administration alone. Thus, CTLA4Ig primed DLI resulted in early proliferation of mature NK cells with cytotoxic potential enabling early institution of adoptive immunotherapy to mitigate the risk of relapse in advanced leukemia with reduced GVHD and NRM.     

CTLA-4Ig抑制嗜酸性粒细胞的抗原呈递过程

探讨嗜酸性粒细胞在体外培养条件下抗将原呈递给致敏Ｔ淋巴细胞的机制。方法应用流式细胞仪检测了小鼠ＥＯＳ于组粒细胞－巨噬细胞集落刺激因子刺激前后表达作为协同刺激因子主要成员的ＣＤ８０和ＣＤ８６分子的水平;并观察应用细胞毒性Ｔ淋细胞相关蛋白４融合蛋白（ＣＴＬＡ－４Ｉｇ）封闭ＣＤ８０和ＣＤ８６分子以阻断协同刺激通路对ＥＯＳ呈递抗原的影响。结果小鼠ＥＯＳ即使新鲜分离也可达表达ＣＤ８０和ＣＤ８６,经粒细胞－巨     

CTLA4Ig融合蛋白体外免疫抑制作用机制探讨

本文在建立EAEWistar大鼠模型的基础上 ,借助MBP特异反应的T细胞 ,体外探讨CTLA4Ig免疫抑制作用。实验结果发现 ,CTLA4Ig (10 μg/ml)可完全抑制MBP反应的大鼠T细胞增殖 ,而外源性IL 2的同时加入可恢复T细胞针对MBP的增殖反应。FACS检测经CTLA4Ig处理的T细胞表面仍有少量IL 2R的表达 ,且CTLA4Ig能诱导T细胞凋亡。RT PCR结果显示CTLA4Ig能明显抑制IL 2mRNA的表达 ,轻度抑制IFN γmRNA和TNF αmRNA的表达 ,而对IL 10mRNA的表达无明显影响。本研究旨在为CTLA4Ig进一步实验室及临床应用提供依据