口服卡介菌诱导免疫耐受的CD4^＋CD25^＋调节性T细胞机制研究

目的：研究口服卡介菌诱导免疫耐受对CD4^＋CD25^＋调节性T细胞的影响。方法：采用口服MPB制备EAE大鼠模型,随机分为BCG组（0.5mg/kg）和EAE模型组（PBS）,每组各15只,连续经口灌服给药14d,同时选取15只健康大鼠作为对照组。分别于免疫后15d、27d流式细胞术检测外周血、胸腺及脾脏中CD4^＋CD25^＋T淋巴细胞百分率,ELISA检测血清IL-6、TGF-β、IgE、IgG含量。结果：与EAE模型组相比,免疫后BCG组大鼠外周血、胸腺及脾脏中CD4^＋CD25^＋T淋巴细胞百分率增加,血清IL-6、TGF-β含量上升,血清IgE、IgG抗体水平下降。结论：口服BCG通过上调淋巴器官中CD4^＋CD25^＋T淋巴细胞比例,抑制效应性T细胞活性,发挥免疫耐受作用。     

口服卡介菌治疗实验性自身免疫性脑脊髓炎的研究

目的:观察口服卡介菌对实验性变态反应性脑脊髓炎(EAE)的治疗效果。方法:制备EAE大鼠模型,随机分为BCG高、中、低剂量组和PBS对照组,每组各15只,对大鼠治疗后的临床症状及病理组织学进行评估,提取脾脏淋巴细胞,流式细胞术检测T淋巴细胞亚群,3H-TdR掺入法检测淋巴细胞增殖能力。结果:BCG组EAE大鼠与对照组相比,临床症状减轻,发病时间延迟,炎性细胞浸润数减少;急性期,口服BCG各组CD4+、CD8+T细胞的数量随剂量增加而增加,缓解期CD4+、CD8+T细胞数量减少;口服BCG可促进EAE大鼠T淋巴细胞增殖能力;高、中剂量组上述变化均较其它分组明显。结论:口服BCG可很好的诱导免疫耐受,延迟EAE发病,减轻炎症反应,改善临床症状。     

幽门螺杆菌感染者CD4~+ CD25~+调节性T细胞的检测及其相关性分析

目的检测幽门螺杆菌(Helicobacter pylori,H.pylori)感染阳性的胃部疾病患者外周血中CD4+CD25+调节性T细胞(Treg细胞)的百分含量及转化生长因子-β1(transforming growth factor-β1,TGF-β1)的水平,探讨CD4+CD25+调节性T细胞在H.pylori感染中的免疫调节作用及意义。方法采用流式细胞术检测H.pylori感染的慢性浅表性胃炎、胃癌前病变和胃癌患者外周血中CD4+CD25+调节性T细胞的含量、CD4+CD25+T细胞中表达FOXP3的细胞比例;并采用ELISA方法检测H.pylori感染者血清中TGF-β1的含量,无H.pylori感染的患者作为阴性对照。结果 H.pylori感染的患者外周血中CD4+CD25+调节性T细胞的百分含量及TGF-β1的水平较不伴有H.pylori感染的患者显著升高(P<0.05);H.pylori感染的浅表性胃炎、胃癌前病变及胃癌患者外周血中CD4+CD25+T淋巴细胞的百分含量及CD4+CD25+T细胞中表达FOXP3的细胞比例随病变严重程度的进展逐渐升高,差异有统计学意义(P<0.05);H.pylori感染的患者血清中TGF-β1水平也随病变严重程度的进展逐渐升高,差异有统计学意义(P<0.05)。结论 H.pylori感染可增加CD4+CD25+调节性T细胞的含量和TGF-β1的水平;随着病变严重程度的进展,CD4+CD25+调节性T细胞的含量和TGF-β1的水平逐渐升高,CD4+CD25+调节性T细胞百分含量和TGF-β1水平可作为临床判断病情进展的指标。     

口服卡介菌治疗实验性自身免疫性脑脊髓炎的研究

目的：观察口服卡介菌对实验性变态反应性脑脊髓炎（EAE）的治疗效果。方法：制备EAE大鼠模型,随机分为BCG高、中、低剂量组和PBS对照组,每组各15只,对大鼠治疗后的临床症状及病理组织学进行评估,提取脾脏淋巴细胞,流式细胞术检测T淋巴细胞亚群,3H—TdR掺入法检测淋巴细胞增殖能力。结果：BCG组EAE大鼠与对照组相比,临床症状减轻,发病时间延迟,炎性细胞浸润数减少;急性期,口服BCG各组CD4^＋、CD8^＋T细胞的数量随剂量增加而增加,缓解期CD4^＋、CD8^＋T细胞数量减少;口服BCG可促进EAE大鼠T淋巴细胞增殖能力;高、中剂量组上述变化均较其它分组明显。结论：口服BCG可很好的诱导免疫耐受,延迟EAE发病,减轻炎症反应,改善临床症状。     

CD4~+CD25~+ Treg细胞与移植免疫耐受

诱导器官移植受者对供者抗原的免疫耐受是防治同种异型移植排斥反应的最理想途径。目前认为,免疫耐受形成的主要机制包括:胸腺及骨髓阴性选择引起的克隆清除(Clonal deletion)、组织特异性自身抗原低表达引起的克隆忽视(Clonal ignorance)、阻断T细胞共刺激信号引起的克隆无能(Clonal anergy)、嵌合体(Chimerism)的形成、调节性T细胞(Regulatory Tcell,Treg)介导的克隆抑制(Clonal suppression)等。近年来CD4+CD25+ Treg细胞的研究已成为免疫学界的热门课题之一。已知CD4+CD25+ Treg细胞存在于小鼠、大鼠和人体中,是机体自然存在的具有主动调节活性的T细胞,对维持自我耐受和控制自身免疫病发挥着重要作用。本文着重就CD4+CD25+ Treg细胞的免疫调节机制及其在诱导移植免疫耐受方面的研究进展做一综述。     

CD4+CD25+调节性T细胞在自身免疫性肝炎发病中的作用

目的:比较自身免疫性肝炎(autoimmune hepatitis,AIH)患者与健康对照者(healthy controls,HCs)外周血CD4+CD25+调节性T细胞(CD4+CD25+Tregs)数量、免疫抑制功能的变化,探讨CD4+CD25+Tregs参与AIH发病的可能机制.方法:采用流式细胞仪检测8例AIH患者及15例健康对照组的外周血CD4+CD25+Tregs数量的百分比及绝时数量;采用共同培养方法检测AIH患者外周血CD4+CD25+Tregs的免疫抑制功能的变化;实时荧光定量聚合酶链反应(RT-FQ-PCR)检删AIH患者外周血CD4+CD25+Tregs中FoxP3mRNA的表达.结果:AIH患者外周血CD4+CD25+Tregs数量明显低于HCs(p<0.01);混合淋巴细胞共同培养结果显示,AIH患者外周血CD4+CD25+Tregs抑制功能明显低于HCs组(p<0.01);AIH患者外周血CD4+CD25+Tregs的FoxP3 mRNA相对表达量显著降低,与HCs组比较有显著性差异(p<0.01).结论:CD4+CD25+Tregs细胞的数量的减少和Foxp3表达的降低所造成的CD4+CD25+Tregs细胞免疫抑制功能受损可能是AIH发病的一个因素.     

CD4+CD25+FoxP3+ 调节性T 细胞与黑龙江省HIV/AIDS 患者病情 相关性的研究

目的:比较黑龙江省HIV/AIDS患者与健康对照者(healthy controls,HCs)外周血CD4+CD25+FoxP3+调节性T细胞数量、免疫抑制功能的变化,探讨CD4+CD25+FoxP3+调节性T细胞在HIV/AIDS感染过程中的作用。方法:采用流式细胞仪检测21例HIV/AIDS患者及20例健康对照组的外周血CD4+CD25+FoxP3+调节性T细胞数量的百分比及绝对数量;采用共同培养方法检测HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞免疫抑制功能的变化;实时荧光定量聚合酶链反应(RT-FQ-PCR)检测HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞中FoxP3mRNA的表达。结果:黑龙江省HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞比率明显高于HCs(P<0.01),而CD4+CD25+FoxP3+调节性T细胞的绝对计数显著下降,且与CD4+T细胞绝对计数成反比;混合淋巴细胞共同培养结果显示,HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞的抑制功能无明显变化;HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞的FoxP3 mRNA相对表达量无显著变化。结论:黑龙江省HIV/AIDS患者CD4+CD25+FoxP3+调节性T细胞的数量变化与病情相关。     

非霍奇金淋巴瘤患者外周血中CD4+CD25+ 调节性T 细胞 亚群变化初探

目的:检测非霍奇金淋巴瘤(non-Hodgkin’s lymphoma,NHL)患者外周血中CD4+CD25+调节性T细胞(CD4+CD25+regulatoryT cell,Treg)的改变,探讨Treg与NHL的相关性。方法:病例组(n=60)为本院收治的初诊NHL患者,对照组(n=60)为本院健康体检者,用流式细胞技术联合标记CD4、CD25检测对照组及病例组化疗前、化疗后的外周血中CD4+CD25+调节性T细胞的分布特点。结果:(1)病例组化疗前外周血中CD4+细胞比例显著低于对照组(P<0.05),CD4+CD25+调节性T细胞比例显著高于对照组(P<0.05);(2)病例组化疗后,CD4+细胞比例明显高于化疗前(P<0.05),CD4+CD25+调节性T细胞比例明显低于化疗前(P<0.05);(3)病例组化疗后CD4+细胞比例与对照组无显著差异(P>0.05),而CD4+CD25+调节性T细胞比例显著高于对照组(P<0.05)。结论:非霍奇金淋巴瘤患者外周血中CD4+CD25+调节性T细胞比例升高,存在机体免疫抑制,化疗可降低CD4+CD25+调节性T细胞比例。     

CD8~+CD122~+T细胞在脑缺血过程中作用的研究

目的:探讨CD8+CD122+T细胞在脑缺血过程中的作用及其机制。方法:线栓法建立小鼠大脑中动脉栓塞模型(MCAO);激光共聚焦显微镜检测小鼠脑缺血组织中CD8+CD122+T细胞浸润情况;流式细胞仪检测脑缺血组织中CD8+CD122+T细胞/CD3+T细胞的比例及脾和胸腺中CD8+CD122+T细胞数量变化;RT-PCR方法检测CD8+CD122+T细胞对氧糖剥夺(Oxygen-glucose deprivation,OGD)条件下星形胶质细胞表达TNF-α,IL-1β,IFN-γ的影响。结果:各时间点脑缺血组织中均有CD8+CD122+T细胞浸润,且随脑缺血时间延长,缺血侧脑组织中CD8+CD122+T细胞/CD3+T细胞比例逐渐增加,5 d和7 d组差异显著,与非缺血侧相比,P5d0.05,P7d0.05;MCAO小鼠脾及胸腺中CD8+CD122+T细胞呈现先增高后降低的趋势。星形胶质细胞经OGD处理后,与对照组相比,IFN-γ、TNF-α、IL-1β表达显著增高,PIFN-γ0.01、PTNF-α0.001、PIL-1β0.01;CD122-blocked组与CD8+组相比,IFN-γ、TNF-α、IL-1β表达明显增高,PIFN-γ0.05、PTNF-α0.05、PIL-1β0.01;CD8+组与HBSS组相比,IFN-γ表达降低,P0.05;IL-1β表达有降低的趋势。结论:CD8+CD122+T细胞在脑缺血过程中发挥保护性作用,其保护作用通过CD122抑制星形胶质细胞TNF-α,IL-1β,IFN-γ炎症因子表达实现的。     

黄芪多糖对免疫抑制模型小鼠Treg细胞及Th17细胞亚群的影响

本研究旨在探讨黄芪多糖对免疫抑制模型小鼠CD4+CD25+调节性T细胞(Treg细胞)和CD4+Th17细胞亚群功能的影响。通过小鼠腹腔注射环磷酰胺建立免疫抑制动物模型,应用黄芪多糖对免疫抑制小鼠进行治疗,采用流式细胞仪分析脾脏中CD4+T细胞、CD4+CD25+Treg细胞的比例,ELISA检测血清IL-17的水平。结果表明:与模型对照组比较,黄芪多糖高、中剂量明显降低小鼠脾脏CD4+T细胞数的比例(P0.05),同时黄芪多糖上调小鼠脾脏CD4+CD25+Treg细胞数的比例(P0.05),降低免疫抑制小鼠血清IL-17的水平,尤其1.0 g/(kg·d)剂量组的IL-17水平下降较为明显(P0.01)。提示黄芪多糖具有提高免疫抑制小鼠CD4+CD25+Treg细胞数的比例、降低CD4+T细胞的数量及抑制IL-17分泌活性的作用。     

Role for thymic and splenic regulatory CD4+ T cells induced by donor dendritic cells in allograft tolerance by LF15-0195 treatment

A 20-day treatment with LF15-0195, a deoxyspergualine analogue, induced allograft tolerance in a fully MHC-mismatched heart allograft model in the rat. Long-term allografts displayed minimal cell infiltration with no signs of chronic rejection. CD4+ spleen T cells from tolerant LF15-0195-treated recipients were able to suppress in vitro proliferation of allogeneic CD4+ T cells and to transfer tolerance to second syngeneic recipients, demonstrating dominant suppression by regulatory cells. A significant increase in the percentage of CD4+CD25+ T cells was observed in the thymus and spleen from tolerant LF15-0195-treated recipient. In vitro direct stimulation with donor APCs demonstrated that CD4+ regulatory T cells proliferated weakly and expressed low levels of IFN-gamma, IL-10, and IL-2. CD4+CD25+ cell depletion increased IL-2 production by CD4+CD25- thymic cells, but not splenic cells. Moreover, tolerance was transferable with splenic and thymic CD4+CD25+ cells, but also in 50% of cases with splenic CD4+CD25- cells, demonstrating that CD25 can be a marker for regulatory cells in the thymus, but not in the periphery. In addition, we presented evidences that donor APCs were required to induce tolerance and to expand regulatory CD4+ T cells. This study demonstrates that LF15-0195 treatment induces donor APCs to expand powerful regulatory CD4+CD25+/- T cells present in both the central and peripheral compartments.     

CD4+CD25+ 调节性T 细胞与冠状动脉粥样硬化病变的研究进展

CD4+CD25+调节性T细胞是一个具有独特免疫调节功能的T细胞亚群,人体主要通过CD4+CD25+调节性T细胞以免疫负向调节的方式来抑制自身反应性T细胞的作用,减少免疫性疾病的发生,从而维持机体内环境的稳定,维持免疫耐受。CD4+CD25+Treg已被证实其与肿瘤、感染、自身免疫病、移植免疫等多种疾病的发生、发展及转归均相关。随着社会的进步和人民生活水平的提高冠状动脉粥样硬化性病变作为一种慢性病变,其发病率越来越高,已经成为严重危害人类健康的常见病,近年来越来越多的证据表明炎症及免疫反应机制在冠状动脉粥样硬化性心脏病的发生、发展及预后过程中具有重要的作用。而CD4+CD25+调节性T细胞在冠状动脉粥样硬化性病变中所起的作用也受到越来越多的关注。本文就CD4+CD25+调节性T细胞与冠状动脉粥样硬化病变之间的关联做一综述。     

Regulatory T cell properties of chicken CD4+CD25+ cells

Chicken CD4(+)CD25(+) cells were characterized for mammalian regulatory T cells' suppressive and cytokine production properties. Anti-chicken CD25 mAb was produced in mice and conjugated with a fluorescent tag. The specificity of the Ab against chicken CD25 was confirmed by evaluating Con A-induced CD25 upregulation in thymocytes and by quantifying the CD25 mRNA content of positive and negative cells identified by anti-chicken CD25 Ab. The percentage of CD4(+)CD25(+) cells, expressed as a percentage of CD4(+) cells, in thymus and blood was ～3-7%, in spleen was 10%, and in cecal tonsil, lung, and bone marrow was ～15%. Bursa had no detectable CD4(+)CD25(+) cells. CD25(+) cells were mostly CD4(+) in the thymus, whereas in every other organ studied, CD25(+) cells were distributed between CD4(+) and CD4(-) cells. Chicken thymic CD4(+)CD25(+) cells did not proliferate in vitro in the absence of recombinant chicken IL-2 (rCIL-2). In the presence of rCIL-2, PMA plus ionomycin or Con A stimulated CD4(+)CD25(+) cell proliferation, whereas anti-CD3 plus CD28 did not stimulate CD4(+)CD25(+) cell proliferation. Naive CD4(+)CD25(+) cells had 29-fold more IL-10 mRNA and 15-fold more TGF-β mRNA than the naive CD4(+)CD25(-) cells. Naive CD4(+)CD25(+) had no detectable IL-2 mRNA. Both naive and PMA plus ionomycin-stimulated thymic CD4(+)CD25(+) cells suppressed naive T cell proliferation. The suppressive properties were partially contact dependent. Supplementing CD4(+)CD25(+) cell coculture with rCIL-2 reversed the suppressive properties of CD4(+)CD25(+) cells. Chicken CD4(+)CD25(+) cells have suppressive properties similar to that of mammalian regulatory T cells.     

CD7 and CD28 are required for murine CD4+CD25+ regulatory T cell homeostasis and prevention of thyroiditis

CD7 and CD28 are T cell Ig superfamily molecules that share common signaling mechanisms. To determine roles CD7 and CD28 might play in peripheral lymphocyte development and function, we have generated CD7/CD28-double-deficient mice. CD7- and CD28-single-deficient and CD7/CD28-double-deficient mice had normal levels of CD4 and CD8-single-positive T cells in thymus and spleen. However, CD28-deficient mice had decreased CD4+CD25+ T cells in spleen compared with wild-type mice, and CD7/CD28-double-deficient mice had decreased numbers of CD4+CD25+ T cells in both thymus and spleen compared with both wild-type and CD28-deficient mice. Functional studies demonstrated that CD4+CD25+ T cells from CD28-deficient and CD7/CD28-double-deficient mice could mediate suppression of CD3 mAb activation of CD4+CD25- wild-type T cells, but were less potent than wild-type CD4+CD25+ T regulatory cells. Thyroiditis developed in aged CD7/CD28-double-deficient mice (>1 year) that was not seen in age-matched control mice or single CD7- or CD28-deficient mice, thus suggesting in vivo loss of T regulatory cells allowed for the development of spontaneous thyroiditis. Taken together, these data demonstrated collaborative roles for both CD7 and CD28 in determination of number and function of CD4+CD25+ T regulatory cells in the thymus and peripheral immune sites and in the development of spontaneous thyroiditis.     

双歧杆菌完整肽聚糖对食物过敏小鼠调节性T细胞影响的研究

目的以食物过敏小鼠为动物模型,通过灌喂双歧杆菌完整肽聚糖(Whole Peptidoglycan,WPG),观察其对调节性T细胞的作用。方法 4～6周龄SPF级无鸡蛋喂养BALB/c雌鼠随机分为3组,每组8只。取其中2组建立食物过敏模型,分别为OVA致敏阳性对照组、OVA激发后灌喂双歧杆菌WPG治疗组和生理盐水对照组。采用ELISA法检测各组血清中OVA特异性IgE、IL-10和TGF-β1水平;流式细胞术分析脾脏单个核细胞悬液中CD4+CD25+调节性T细胞的数量变化。结果 OVA组IL-10、TGF-β1水平显著高于对照组(P分别0.05和0.01);WPG组血清IL-10和TGF-β1水平显著低于OVA组(P0.05)。OVA组脾CD4+CD25+T淋巴细胞的百分比低于对照组(P0.05),WPG组与OVA组相比有升高趋势,差异有统计学意义(P0.05);OVA组脾Foxp3+CD4+CD25+调节性T细胞的百分比显著低于对照组(P0.01),WPG组与OVA组相比有升高趋势,差异有统计学意义(P0.01)。结论双歧杆菌WPG可以增加食物过敏小鼠CD4+CD25+调节性T细胞数量,刺激细胞因子IL-10和TGF-β分泌。     

Orally-Induced Intestinal CD4+ CD25+ FoxP3+ Treg Controlled Undesired Responses towards Oral Antigens and Effectively Dampened Food Allergic Reactions

The induction of peripheral tolerance may constitute a disease-modifying treatment for allergic patients. We studied how oral immunotherapy (OIT) with milk proteins controlled allergy in sensitized mice (cholera toxin plus milk proteins) upon exposure to the allergen. Symptoms were alleviated, skin test was negativized, serum specific IgE and IgG1 were abrogated, a substantial reduction in the secretion of IL-5 and IL-13 by antigen-stimulated spleen cells was observed, while IL-13 gene expression in jejunum was down-regulated, and IL-10 and TGF-β were increased. In addition, we observed an induction of CD4⁺CD25⁺FoxP3⁺ cells and IL-10- and TGF-β-producing regulatory T cells in the lamina propria. Finally, transfer experiments confirmed the central role of these cells in tolerance induction. We demonstrated that the oral administration of milk proteins pre- or post-sensitization controlled the Th2-immune response through the elicitation of mucosal IL-10- and TGF-β-producing Tregs that inhibited hypersensitivity symptoms and the allergic response.     

Cutting edge: the phosphoinositide 3-kinase p110 delta is critical for the function of CD4+CD25+Foxp3+ regulatory T cells

CD4+CD25+Foxp3+ regulatory T cells (Tregs) contribute to the maintenance of peripheral tolerance by inhibiting the expansion and function of conventional T cells. Treg development and homeostasis are regulated by the Ag receptor, costimulatory receptors such as CD28 and CTLA-4, and cytokines such as IL-2, IL-10, and TGF-beta. Here we show that the proportions of Tregs in the spleen and lymph nodes of mice with inactive p110delta PI3K (p110deltaD910A/D910A) are reduced despite enhanced Treg selection in the thymus. p110deltaD910A/D910A CD4+CD25+Foxp3+ Tregs showed attenuated suppressor function in vitro and failed to secrete IL-10. In adoptive transfer experiments, p110deltaD910A/D910A T cells failed to protect against experimental colitis. The identification of p110delta as an intracellular signaling protein that regulates the activity of CD4+CD25+Foxp3+ Tregs may facilitate the further elucidation of the molecular mechanisms responsible for Treg-mediated suppression.     

Natural recovery and protection from autoimmune encephalomyelitis: contribution of CD4+CD25+ regulatory cells within the central nervous system

Immune regulation of autoimmune disease can function at two sites: at the secondary lymphoid organs or in the target organ itself. In this study, we investigated the natural resolution of autoimmune pathology within the CNS using murine experimental autoimmune encephalomyelitis (EAE). Recovery correlates with the accumulation of IL-10-producing CD4+CD25+ T cells within the CNS. These CD4+CD25+ cells represent as many as one in three of CD4+ cells in the CNS during recovery, they are FoxP3+ and express other markers associated with regulatory cells (CTLA-4, GITR, and alpha(E)beta7), and they have regulatory function ex vivo. Depletion of CD25+ cells inhibits the natural recovery from EAE. Also, depletion of CD25+ cells after recovery removes the resistance to reinduction of EAE observed in this model. Furthermore, passive transfer of CNS-derived CD4+CD25+ cells in low numbers provides protection from EAE in recipient mice. These are the first data demonstrating the direct involvement of CD4+CD25+ regulatory T cells in the natural resolution of autoimmune disease within the target organ.