小剂量氨茶碱对支气管哮喘患者外周血CD4+CD25+调节性T细胞凋亡的影响

目的 观察小剂量氨茶碱对分离培养的健康人和支气管哮喘(简称哮喘)患者外周血CD4+CD25+调节性T细胞(T regulatory cells,Treg)凋亡的影响.方法 经密度梯度离心法、尼龙棉柱法、磁珠分离法分离出健康人和哮喘患者外周血CD4+CD25+Treg,分小剂量氨茶碱(1.13 mg/L)、及空白组培养72 h后,用流式细胞仪检测凋亡率变化.结果 ①健康人外周血CD4+CD25+Treg的纯度为77.4%～92.3%,哮喘患者CD4+CD25+Treg的纯度为75.2%～93.8%.②CD4+CD25+Treg占外周血CD4+T细胞的比例在健康组为4.12%～7.98%,在哮喘组为4.51%～8.68%.两者差异无统计学意义.③小剂量氨茶碱均可以诱导健康组及哮喘组外周血CD4+CD25+Treg凋亡率增加(P<0.05).结论 小剂量氨茶碱(1.13 mg/L)可能通过促进CD4+CD25+Treg凋亡来发挥免疫调节作用.     

结核病患者外周血CD4+CD25+ T淋巴细胞凋亡及FOXp3表达情况观察

目的初步观察分离培养的结核病患者外周血CD4+CD25+ T淋巴细胞凋亡及其表面特征性标志物FOXp3的表达情况。方法采用密度梯度离心法及尼龙棉柱法分离结核病患者外周血T淋巴细胞,磁性细胞分离器(MACS)分离得到CD4+CD25+ T淋巴细胞,体外培养72 h后分别利用电镜及流式细胞仪观察、检测各分组(结核病组、健康对照组)细胞的凋亡率及FOXp3的表达。结果 MACS可成功分离CD4+CD25+ T淋巴细胞,纯度可达81.1%～84.7%。结核病组较健康对照组外周血CD4+CD25+ T淋巴细胞计数增多,凋亡率增加,FOXp3表达增高(P均<0.05)。结论 FOXp3高表达的CD4+CD25+ T淋巴细胞在结核病的免疫调节过程中起着重要作用,并且与CD4+CD25+ T淋巴细胞的凋亡密切相关。     

刚地弓形虫排泄-分泌抗原体外诱导的IFN-γ促CD4~+CD25~+调节性T细胞凋亡作用

目的研究刚地弓形虫(Toxoplasma gondii)RH株和Tg Ctwh3株排泄-分泌抗原(ESA)体外诱导小鼠CD4~+CD25~+调节性T细胞凋亡和IFN-γ分泌方面的差异。方法分别制备刚地弓形虫RH株和Tg Ctwh3株的ESA。将分离的野生型C57BL/6小鼠脾单个核细胞随机分为3组(每孔2×10~6细胞),分别加入RH株ESA、Tg Ctwh3株ESA(均为10μg/ml)和鸡卵清蛋白(OVA)进行诱导刺激,流式细胞术检测各组诱导刺激48 h和72 h的CD4~+CD25~+调节性T细胞的早期凋亡情况,ELISA检测各组诱导刺激72 h细胞培养上清中的γ干扰素(IFN-γ)分泌水平。另外,将分离的野生型C57BL/6小鼠脾单个核细胞随机分为2大组(每孔2×10~6细胞),其中一大组在分别加入两株ESA(10μg/ml)和OVA的同时加入抗IFN-γ中和抗体(10μg/ml)进行诱导刺激72 h,流式细胞术检测各组CD4~+CD25~+调节性T细胞早期凋亡情况。用两虫株ESA(10μg/ml)及OVA分别诱导刺激IFN-γ基因敲除型和野生型C57BL/6小鼠的脾单个核细胞(每孔2×10~6细胞)72 h后,流式细胞术检测各组CD4~+CD25~+调节性T细胞早期凋亡情况。结果制备的刚地弓形虫RH株和Tg Ctwh3株ESA抗原蛋白浓度分别为0.54 mg/ml和2.14 mg/ml。流式细胞术检测结果显示,RH株和Tg Ctwh3株ESA组诱导刺激48 h后,CD4~+CD25~+调节性T细胞的早期凋亡率分别为(12.90±1.26)%和(9.71±1.04)%,均显著高于OVA对照组(4.48±0.48)%(P0.01);诱导刺激72 h后,RH株ESA组诱导CD4~+CD25~+调节性T细胞的凋亡率为(15.21±1.11)%,仍明显高于Tg Ctwh3株ESA组(11.02±0.92)%(P0.05)和OVA对照组(10.10±1.49)%(P0.01)。ELISA检测结果显示,RH株和Tg Ctwh3株ESA组诱导刺激脾单个核细胞72 h的培养上清中分泌的IFN-γ水平分别为(4 764.0±118.7)pg/ml和(3 629.0±33.6)pg/ml(P0.01),均显著高于OVA对照组的(679.4±30.6)pg/ml(P0.01)。流式细胞术检测结果显示,RH株ESA加抗IFN-γ中和抗体组诱导CD4~+CD25~+调节性T细胞的早期凋亡率为(10.44±1.44)%,明显低于未加抗IFN-γ中和抗体组(14.96±0.83)%(P0.05);但Tg Ctwh3株ESA加抗IFN-γ中和抗体与否诱导CD4~+CD25~+调节性T细胞的早期凋亡率变化不大(P0.05)。RH株ESA诱导IFN-γ基因敲除小鼠的CD4~+CD25~+调节性T细胞凋亡率为(10.64±0.55)%,明显低于野生型小鼠的(15.21±1.11)%(P0.01);Tg Ctwh3株ESA诱导两组小鼠的CD4~+CD25~+调节性T细胞凋亡率的差异无统计学意义(P0.05)。结论刚地弓形虫RH株ESA在体外诱导CD4~+CD25~+调节性T细胞凋亡及促IFN-γ分泌强于Tg Ctwh3株ESA。弓形虫ESA诱导机体产生IFN-γ可通过介导调节性T细胞凋亡来促进弓形虫感染早期抗感染免疫力的形成。     

沙利度胺对类风湿关节炎T淋巴细胞的免疫调节作用

目的 探讨沙利度胺(Thd)对类风湿关节炎(RA)患者外周血T淋巴细胞的免疫调节作用.方法 在植物血凝素(PHA)刺激下,RA患者外周血T淋巴细胞与不同浓度的Thd(2.5、25、100、300、500μg/ml)共同培养.用四甲基噻唑蓝(MTT)比色法检测T淋巴细胞的增殖,用流式细胞术检测T淋巴细胞的凋亡和T淋巴细胞CD152、CD28的表达情况,用Real-time聚合酶链反应(PCR)检测T淋巴细胞的自细胞介素(IL)-6、IL-10、肿瘤坏死因子(TNF)-α mRNA表达水平.结果 在体外,与阴性对照组相比,500μg/ml,Thd组显著抑制T淋巴细胞的增殖,促进T淋巴细胞早期凋亡,抑制T淋巴细胞CD3+CD28+表达,促进CD8+CD152+表达,而≤300μg/ml,对T淋巴细胞未见明显影响.与对照组相比,(100～500)μg/ml剂量组可抑制T淋巴细胞IL-6、TNF-α mRNA表达,(2.5～500)μg/ml可促进IL-10 mRNA的表达.结论 Thd可能通过影响RA患者外周血T淋巴细胞增殖、早期凋亡、CD3*CD28+和CD8+CD152+表达,以及IL-6、IL-10、TNF-α mRNA表达,反转RA患者的免疫平衡失控.     

抗CD3单克隆抗体对支气管哮喘患者CD4＋CD25＋T细胞凋亡和自噬的影响

目的探讨抗CD3单克隆抗体对分离培养的支气管哮喘（简称哮喘）患者外周血CD4＋CD25＋T细胞凋亡和自噬及其分泌的代表性因子转化生长因子p（TGF—β）的影响。方法采用密度梯度离心法及尼龙棉柱法分离32例哮喘患者（哮喘组）及30名健康者（对照组）外周血T细胞,磁性细胞分离器分离得到CD4＋CD25＋T细胞,分别利用电镜及流式细胞仪观察、检测抗CD3单克隆抗体干预72h的细胞凋亡率、自噬率。用EI,ISA法检测细胞培养上清液中细胞因子TGF-β的水平。结果抗CD3单克隆抗体干预72h后两组外周血CD4＋CD25＋T细胞凋亡率、自噬率及TGF-β均增加（P值均〈0．01）,但哮喘组凋亡率、自噬率均低于健康对照组（P值均〈0．01）;两组间 TGF-β水平无显著差异（P〉0．01）。哮喘组外周虹CD4＋CD25＋T调节细胞在CD3单克隆抗体的干预下自噬与TGF-β的分泌呈显著负相关（r=-0．38,P〈0．01）。结论抗CD3单克隆抗体可促进CD4＋CD25＋T细胞凋亡和自噬及TGF-β分泌。     

恶性疟原虫抗原对健康人外周血T细胞免疫功能的影响

目的 目的 探讨恶性疟原虫抗原对健康人外周血T淋巴细胞免疫功能的影响。方法 方法 健康成人外周血单个核细胞 （PBMC） 体外分别用恶性疟原虫 （P. f） 抗原 （5 μg/ml） 和正常红细胞 （nRBC） 抗原 （5 μg/ml） 进行刺激, 同时给予IL?2维持细 胞增殖, 另外设立只加IL?2的阴性对照组进行培养。培养12 d时, 刺激组细胞再用对应的抗原刺激20 h, 用流式细胞仪 检测T淋巴细胞亚群分泌IL?4和IFN?γ的情况, 并用羧基荧光素乙酰乙酸琥珀酰亚胺酯 （Carboxyfluorescein diacetate, suc? cinimidyl ester, CFSE） 标记法检测T细胞增殖反应。结果 结果 健康人PBMC经P. f抗原刺激扩增后CD4+ T细胞的增殖指数 （PI） 明显高于nRBC抗原刺激组和阴性对照组 （P均＜0.05）, 但3组γδ T的PI差异无统计学意义 （P＞0.05）。P. f抗原组 分泌IL?4的CD4+ T细胞百分率明显高于nRBC抗原组和阴性对照组 （P均＜0.05）, 但3组分泌IFN?γ的CD4+ T细胞百分 率差异无统计学意义 （P＞0.05）。结论 结论 P. f抗原在体外可刺激健康人外周血CD4+ T细胞增殖活化, 后者通过优先分泌 IL?4而发挥免疫调节作用。     

抗CD137单克隆抗体对哮喘患者外周血CD4^＋CD25^＋T淋巴细胞死亡方式的影响

目的探讨抗CD啪单克隆抗体对哮喘患者外周血CD4^＋CD25^＋T淋巴细胞的影响。方法采用密度梯度离心法及尼龙棉柱法分离16例健康志愿者（对照组）及12例哮喘患者（哮喘组）外周血T淋巴细胞,磁性细胞分离器（MACS）分离得到CD4^＋CD25^＋T淋巴细胞,分别利用电镜及流式细胞仪观察、检测抗CD137单克隆抗体干预72h的细胞自噬率、凋亡率、胀亡率及FOXp3的表达。结果抗CD137单克隆抗体干预后两组外周血CD4^＋CD25^＋T淋巴细胞自噬率及凋亡率均增加,但哮喘组均低于对照组。结论抗CD137单克隆抗体可促进CD4^＋CD25^＋T淋巴细胞凋亡和自噬。     

日本血吸虫虫卵抗原诱导CD4+CD25+T细胞及其细胞因子表达

目的 探讨日本血吸虫虫卵抗原诱导宿主免疫应答下调的机制.方法 6～8周龄雌性队LB/c小鼠分为2组,实验组小鼠口服血吸虫虫卵10 000个以及尾静脉注射可溶性虫卵抗原(SEA)200μg,每周免疫1次,共4次;对照组注射PBS.流式细胞仪检测SEA免疫小鼠CD4+CD25+T细胞数量;与CD4+CD25-T细胞共同培养,检测CD4+CD25+T细胞抑制功能;流式细胞仪检测CD4+CD25+T细胞表达IL-4、IL-10与IFN-γ水平;酶联免疫吸附试验检测静脉血IL-10和转化生长因子-β表达水平.结果 实验组小鼠CD4+CD25+T细胞数量为14.7%,对照组为7.4%;实验组IL-10为29.2 pg/ml,对照组为11.0 pg/ml.与CD4+CD25-T细胞相比,CD4+CD25+T细胞主要合成IL-10及少量IFN-γ.CD4+CD25+T细胞显著抑制CD4+T细胞增殖. 结论 日本血吸虫虫卵抗原可能通过诱导CD4+CD25+T细胞和IL-10下调机体免疫应答.     

特发性血小板减少性紫癜患者CD3^＋、CD4^＋和CD8^＋T淋巴细胞凋亡率的研究

目的:通过检测CD3+、CD4+、CD8+T淋巴细胞的凋亡率,探讨T淋巴细胞凋亡在特发性血小板减少性紫癜(ITP)免疫发病机制中的作用。方法:应用流式细胞仪检测ITP患者外周血CD3+、CD4+、CD8+T淋巴细胞的凋亡率;分离ITP患者和正常人外周血单个核细胞(PBMC),分成A、B 2组,A组加入白介素2(IL-2),B组加入IL-2和地塞米松共培养,分别于24和48 h收获细胞,应用流式细胞仪检测CD3+、CD4+、CD8+T淋巴细胞的凋亡率。结果:①ITP患者组CD3+、CD4+、CD8+T淋巴细胞凋亡率均明显低于正常对照(均P<0.01);②细胞培养24 h时ITP患者组A、B 2组间CD3+、CD4+、CD8+T淋巴细胞凋亡率间均差异无统计学意义(均P>0.05),而48 h时B组CD3+、CD4+、CD8+T淋巴细胞的凋亡率均明显高于A组(P<0.05或0.01);正常对照组24和48 h B组CD3+、CD4+、CD8+T淋巴细胞凋亡率均明显高于A组(均P<0.05);③ITP患者组细胞培养24 h后A、B 2组间CD3+、CD4+、CD8+T淋巴细胞凋亡率的差值均明显低于正常对照组(P<0....     

日本血吸虫虫卵抗原诱导CD4+CD5+T细胞及其细胞因子表达

目的 探讨日本血吸虫虫卵抗原诱导宿主免疫应答下调的机制.方法 6～8周龄雌性队LB/c小鼠分为2组,实验组小鼠口服血吸虫虫卵10 000个以及尾静脉注射可溶性虫卵抗原(SEA)200μg,每周免疫1次,共4次;对照组注射PBS.流式细胞仪检测SEA免疫小鼠CD4+CD25+T细胞数量;与CD4+CD25-T细胞共同培养,检测CD4+CD25+T细胞抑制功能;流式细胞仪检测CD4+CD25+T细胞表达IL-4、IL-10与IFN-γ水平;酶联免疫吸附试验检测静脉血IL-10和转化生长因子-β表达水平.结果 实验组小鼠CD4+CD25+T细胞数量为14.7%,对照组为7.4%;实验组IL-10为29.2 pg/ml,对照组为11.0 pg/ml.与CD4+CD25-T细胞相比,CD4+CD25+T细胞主要合成IL-10及少量IFN-γ.CD4+CD25+T细胞显著抑制CD4+T细胞增殖. 结论 日本血吸虫虫卵抗原可能通过诱导CD4+CD25+T细胞和IL-10下调机体免疫应答.     

Biology of T lymphocytes

T cells constitute one arm of the adaptive immune system. The accumulating information on various aspects of T-cell biology shows the intricacies in the regulation of immune responses. How we translate the cellular and molecular details of this regulation into innovation and development of therapies for disease management remains a fundamental, but exciting, challenge.     

Regulation of human gut B lymphocytes by T lymphocytes

The aims of this study were first, to assess whether or not immunoglobulin secretion from human gut mucosal B lymphocytes can be modified by T lymphocytes, and second whether human gut mucosal T lymphocytes are capable of regulating mucosal B lymphocyte function. T and B lymphocyte enriched cell populations were isolated from gut mucosa and co-cultured in varying proportions. Addition of T lymphocytes to B enriched mucosal cell populations (ratio B:T = 2:1) showed that mucosal B lymphocytes were responsive to T cell 'help'. Addition of more T cells (ratio B:T = 2:10) suppressed immunoglobulin synthesis.     

The activation of T lymphocytes

The events involved in T cell activation are initiated at the cell surface by the interaction of ligands with specific cell surface receptors on the T cell. Central to antigen-induced activation is the CD3/Ti complex, a complex multi-chain receptor responsible for antigen/MHC recognition and signal transduction. Triggering the CD3/Ti complex results in the generation of intracellular second messengers, IP3 and DG, which are derived from PI metabolism. The second messengers lead to increases in [Ca2+]i and activation of pkC, events causally linked to various cellular responses, including the production of IL-2 through as yet poorly defined pathways. Little is known about how other cell surface molecules that may provide an accessory function participate in such events. However, future genetic and biochemical studies are likely to shed light upon the mechanisms of signal transduction by the CD3/Ti complex and accessory molecules and the details of the intracellular events involved in the activation of a host of cellular genes associated with activation.     

Signal transduction in T lymphocytes

T lymphocytes utilize a variety of surface receptors to transmit environmental signals across the plasma membrane and initiate biochemical events leading to responses such as proliferation, anergy, cytokine secretion, and death. The T cell receptor complex, CD28, IL-2 receptor, and CD95 each couple to distinct sets of cytoplasmic signaling events to modulate the biological responses of T cells. Deficiency or defective function of proteins involved in signaling through these receptors are associated with murine and human disease.     

Cytotoxic T lymphocytes and autoimmunity

PURPOSE OF REVIEW: The possibility of the recognition by cytotoxic T lymphocytes of tissue autoantigens has been largely ignored in explaining organ-specific autoimmune diseases. Recent advances in the understanding of human leukocyte antigen class I-binding peptides motifs have led to the detection and the characterization of those autoreactive CD8(+) cytotoxic T lymphocytes involved in organ-specific autoimmune diseases. The purpose of this review is to discuss recent studies that shed light on the implication of cytotoxic T lymphocytes in several autoimmune disorders as well as the mechanisms underlying their stimulation. RECENT FINDINGS: Significant progress has been made in the characterization of autoantigens targeted by cytotoxic T lymphocytes in several class I-restricted autoimmune diseases, including Beh?et's disease and ankylosing spondylitis, and their implication in systemic autoimmune disease such as systemic lupus erythematosus. Moreover, the signals involved in the activation of autoreactive cytotoxic T lymphocytes have been better characterized, particularly the molecular requirements of the antigen presentation at the surface of the dendritic cell system, mainly because of a better understanding of the Toll-like receptor-induced signals or the discovery of a defect in regulatory T cells. SUMMARY: New findings in the pathophysiology of cytotoxic T lymphocytes in autoimmunity and especially a better comprehension of their activation may give a new impetus for the development of targeted immunologic therapies in various autoimmune disorders.     

Resistance of cytotoxic T lymphocytes to lysis by a clone of cytotoxic T lymphocytes.

To investigate how cytotoxic T lymphocytes (CTL) avoid killing themselves when they destroy target cells, we compared 20 different cell lines as target cells, including several CTL cell lines, for their susceptibility to lysis by CTL. Variations in recognition of this diverse set of target cells was circumvented by attaching to all of them a monoclonal antibody to the antigen-specific receptor of a cloned CTL cell line (clone 2C) and using the 2C cell line as the standard aggressor or effector cell. All of the nine tumor cell lines and the four noncytolytic T-helper cell lines tested as targets were highly susceptible to lysis by the aggressor CTL, but seven cytotoxic T-cell lines (six CTL and one T-helper cell line with cytotoxic activity) were largely resistant. These results, and the use of the lectin Con A as an alternative means for triggering CTL activity, point clearly to a level of resistance that could enable CTL to avoid their own destruction when they lyse target cells. The resistance of the cytolytic T cells did not appear to be accompanied by a similar resistance to complement-mediated lysis, indicating that mechanisms of CTL-mediated and complement-mediated lysis are not identical.     

Cord blood T lymphocytes lack constitutive perforin expression in contrast to adult peripheral blood T lymphocytes

Perforin is the cytolytic pore-forming protein, which alone can be responsible for the lethal hit in one of the killing mechanisms used by natural killer (NK) cells or cytotoxic T lymphocytes. In this study, perforin expression was investigated in cord blood (CB) lymphocytes to determine their killing potential in vivo. The majority of CB CD3- NK cells had the protein. Compared with adult perforin-positive NK cells, a significantly lower percentage of cells expressing CD56 and CD57, the related neural cell adhesion molecules, was found (P = .0001). Perforin was also present in a unique immature CB NK-cell subset, characterized by cytoplasmic CD3 antigen (Ag) expression. In CB, very few CD8 perforin-positive T lymphocytes could be detected, but they were in significant numbers in adult peripheral blood (P = .02). A substantial proportion of these cells (70% +/- 23%) lacked the CD28 T-cell coactivation Ag, and they were able to exert NK-like, major histocompatibility complex nonrestricted cytolytic activity. CD4+ and gamma delta-T cells expressing perforin were absent from CB, but low numbers of such cells were detected in adult peripheral blood (P = .0001). Therefore, the spontaneous cytolytic activity of CB lymphocytes appeared to be dependent on well-represented perforin-positive NK cells, which were shown to efficiently lyse NK-sensitive target cells.     

Cytotoxic T lymphocytes against HIV.

HIV-1 infection has clearly been shown to induce a vigorous CTL response in infected people, and this response is present at a time when immune function otherwise is globally impaired. HIV-1-specific CTL are detectable both in peripheral blood and tissues of infected people, and are aimed at multiple viral proteins. The precise epitopes recognized by these CTL are now being defined, and the establishment of CTL clones should facilitate further functional analysis of these cells. However, the central question as to the clinical relevance of HIV-1-specific CTL remains. By analogy with animal model systems of virus infection, it is reasonable to postulate that HIV-1-specific CTL serve a protective role as a host defense. In this regard, in vitro data indicate that HIV-1-specific CTL can suppress viral replication, and longitudinal clinical studies indicate that the vigorous CTL activity seen in the early stages of infection declines with disease progression. Alternatively, the presence of HIV-1-specific CTL in tissues such as the lung and brain have to at least raise the possibility that these cells may be contributing to the pathologic consequences of infection. In addition, the relative protective effects of virus-specific CTL compared to other effector mechanisms such as ADCC and neutralizing antibodies remain to be determined. Nevertheless, recent data in the SIV vaccine model give reason for encouragement that a state of protective immunity can be achieved in AIDS-like illness caused by retroviruses. The search continues presently not only for the parameters which define protective immunity in HIV-1 infection, but also for the ideal HIV-1 immunogens to be used for vaccination of human populations.