树突状细胞扩增抗原特异性CD4+ CD25+调节性T细胞研究进展

CD4+CD25+调节性T细胞(Tr)是同时具有免疫低反应性和免疫抑制性功能两大特征的T细胞.研究证实,CD4+ CD25+ Tr在抑制器官特异性自身免疫性疾病及GVHD是抗原特异性的,因此,应用器官特异性而不是多克隆性的Tr将大大促进以Tr为基础的免疫治疗.而具有调节活性的CD4+ CD25+ Tr仅占人类外周血CIM+ T细胞的1%～2%,因此,研究体外大量扩增的方法 对于以Tr基础的治疗至关重要.研究表明,树突状细胞(DC)作为机体强有力的专职抗原递呈细胞可以扩增具有抗原特异性的CD4+ CD25+ Tr且能增加后者的抑制活性,这为治疗自身免疫性疾病及GVHD提供了新的治疗前景.     

CD4+ CD25+ regulatory T cells in human pregnancy: development of a Treg-MLC-ELISPOT suppression assay and indications of paternal specific Tregs

The current study was aimed at developing a one-way mixed leucocyte culture-enzyme-linked immunospot (MLC-ELISPOT) assay for the study of CD4(+) CD25(+) regulatory T (T(reg)) cells and applying this method in the study of antifetal immune reactions during human pregnancy. Twenty-one pregnant women and the corresponding fathers-to-be, and 10 non-pregnant control women and men, participated in the study. CD4(+) CD25(+) cells were isolated from peripheral blood mononuclear cells (PBMC) by immunomagnetic selection. Maternal/control PBMC were stimulated with paternal or unrelated PBMC in MLC. Secretion of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) from responder cells, with or without the presence of autologous T(reg) cells, was analysed by ELISPOT. PBMC from pregnant women showed increased secretion of IL-4 compared to controls. In pregnant and non-pregnant controls, T(reg) cells suppressed IFN-gamma reactivity against paternal and unrelated alloantigens. Interestingly, T(reg) cells suppressed IL-4 secretion against paternal but not unrelated alloantigens during pregnancy. We have successfully developed a model for studying T(reg) cells in antifetal cytokine reactions during pregnancy. Results indicate that T(reg) cells contribute to strict regulation of both T helper type 1-like and type 2-like antifetal immune reactions. Interestingly, T helper type 2-like cells specific to unrelated alloantigens are able to escape the suppression of T(reg) cells, which would allow for IL-4, alongside CD4(+) CD25(+) T(reg) cells, to control potentially detrimental IFN-gamma reactions during pregnancy.     

Dendritic cells partially abrogate the regulatory activity of CD4+CD25+ T cells present in the human peripheral blood

The factors that influence the functionality of human CD4(+)CD25(+) regulatory T cells are not well understood. We sought to characterize the effects of dendritic cells (DCs) on the in vitro regulatory activity of CD4(+)CD25(+) T cells obtained from peripheral blood of healthy human donors. Flow cytometry showed that a higher proportion of CD4(+)CD25(+(High)) T cells expressed surface glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) and CTL-associated antigen 4 than CD4(+)CD25(-) or CD4(+)CD25(+(Med-low)) T cells. Intracellular Foxp3 was equivalently expressed on CD4(+)CD25(+(All)), CD4(+)CD25(+(High)), CD4(+)CD25(+(Med-low)) and CD4(+)CD25(-) T cell populations, irrespective of GITR and CTL-associated antigen 4 expression. CD4(+)CD25(+) T cells were isolated and then cultured in vitro with CD4(+)CD25(-) responder T cells and stimulated with anti-CD3 antibodies, and immature dendritic cells (iDCs), mature dendritic cells (mDCs), PBMCs or PBMCs plus anti-CD28 antibodies to provide co-stimulation. In addition, secretion of the T(h)1 cytokine IFN-gamma, IL-2 and the immunoregulatory cytokines, IL-10 and transforming growth factor (TGF)-beta, were also assessed in these cultures. We found that iDCs and mDCs were capable of reversing the suppression of proliferation mediated by CD4(+)CD25(+) regulatory T cells. However, the reversal of suppression by DCs was not dependent upon the increase of IFN-gamma and IL-2 production or inhibition of IL-10 and/or TGF-beta production. Therefore, DCs are able to reverse the suppressive effect of regulatory T cells independent of cytokine production. These results suggest for the first time that human DCs possess unique abilities which allow them to influence the functions of regulatory T cells in order to provide fine-tuning in the regulation of T cell responses.     

Depletion of CD4+CD25+CD127lo regulatory T cells does not increase allergen‐driven T cell activation

Background It has been suggested that allergic diseases are caused by defective suppression of allergen‐specific Th2 cells by CD4⁺CD25⁺ regulatory T cells. However, such studies have been hampered by the difficulty in distinguishing regulatory T cells from CD25‐expressing activated T cells. Recently, it was shown that conventional T cells expressed high levels of CD127, whereas regulatory T cells were CD127^lo, allowing discrimination between these distinct T cell subpopulations. Objective The aim of this study was to study whether the putative regulatory subset defined as CD4⁺CD25⁺CD127^lo was involved in grass pollen‐reactive T cell responses. Methods Peripheral blood mononuclear cells (PBMCs) were obtained from allergic donors and non‐atopic controls out of season. Grass pollen‐induced cytokine production and proliferation were compared in cultures of undepleted cells and cells depleted of CD4⁺CD25⁺, CD4⁺CD25⁺CD127^hi or CD4⁺CD25⁺CD127^lo T cells. Results Undepleted cell cultures from allergic patients showed significantly increased proliferation and Th2 cytokine production compared with non‐atopic controls. Depletion of all CD25⁺ T cells did not increase cytokine production or proliferation, and more importantly, no increase in Th2 cytokine production or proliferation was observed in cell cultures depleted of CD4⁺CD25⁺CD127^lo cells (putative regulatory T cells) compared with undepleted PBMCs in both the allergic and the non‐atopic group. Conclusion Our study showed that T cells from grass pollen‐allergic patients and non‐atopic controls responded very differently to grass pollen extract, but this difference could not be explained by differences in regulatory T cell function. Further studies are needed to understand the importance of regulatory T cells in allergy.     

Regulatory T cell phenotype and function 4 years after GAD–alum treatment in children with type 1 diabetes

Glutamic acid decarboxylase (GAD)₆₅ formulated with aluminium hydroxide (GAD‐alum) was effective in preserving insulin secretion in a Phase II clinical trial in children and adolescents with recent‐onset type 1 diabetes. In addition, GAD‐alum treated patients increased CD4⁺CD25^hi forkhead box protein 3⁺ (FoxP3⁺) cell numbers in response to in‐vitro GAD₆₅ stimulation. We have carried out a 4‐year follow‐up study of 59 of the original 70 patients to investigate long‐term effects on the frequency and function of regulatory T cells after GAD‐alum treatment. Peripheral blood mononuclear cells were stimulated in vitro with GAD₆₅ for 7 days and expression of regulatory T cell markers was measured by flow cytometry. Regulatory T cells (CD4⁺CD25^hiCD127^lo) and effector T cells (CD4⁺CD25^–CD127⁺) were further sorted, expanded and used in suppression assays to assess regulatory T cell function after GAD‐alum treatment. GAD‐alum‐treated patients displayed higher frequencies of in‐vitro GAD₆₅‐induced CD4⁺CD25⁺CD127⁺ as well as CD4⁺CD25^hiCD127^lo and CD4⁺FoxP3⁺ cells compared to placebo. Moreover, GAD₆₅ stimulation induced a population of CD4^hi cells consisting mainly of CD25⁺CD127⁺, which was specific of GAD‐alum‐treated patients (16 of 25 versus one of 25 in placebo). Assessment of suppressive function in expanded regulatory T cells revealed no difference between GAD‐alum‐ and placebo‐treated individuals. Regulatory T cell frequency did not correlate with C‐peptide secretion throughout the study. In conclusion, GAD‐alum treatment induced both GAD₆₅‐reactive CD25⁺CD127⁺ and CD25^hiCD127^lo cells, but no difference in regulatory T cell function 4 years after GAD‐alum treatment.     

CD4+CD25+调节性T细胞的发育与胸腺CD4-CD25+细胞关联性的探讨

目的:探讨CD4 CD25 调节性T细胞(CD4 CD25 regulatoryTcells,CD4 CD25 Tr)的发育及其与胸腺CD4-CD25 细胞的关系。方法:以流式细胞术检测小鼠从出生至发育成熟过程中,胸腺、脾脏、淋巴结和外周血中CD4 CD25 Tr比例变化,以及胸腺CD4-CD25 细胞比例变化;通过磁激活细胞分选(MACS)从小鼠淋巴结纯化CD4 CD25 T和CD4 CD25-T细胞,经CFDA-SE标记,以多种刺激形式诱导增殖。结果:小鼠出生1d到10周的发育过程中,胸腺CD4 CD25 Tr比例一直比较恒定,但在脾脏、淋巴结和外周血,随鼠龄增加而不断升高,从1d龄到1周时升高最迅速,其后的升高速度逐渐减慢,10周龄时达平台期。胸腺中CD4-CD25 细胞在出生1d的小鼠比例非常高,1d龄到1周龄期间迅速下降,10周龄时达平台期。ConA不能诱导CD4 CD25 Tr和CD4 CD25-T细胞增殖,但CD4 CD25 Tr出现一过性细胞增大;佛波醇酯加离子霉素能诱导CD4 CD25 Tr和CD4 CD25-T细胞增殖;包被的抗CD3抗体加可溶性抗CD28抗体能刺激CD4 CD25-T细胞增殖,但CD4 CD25 Tr不增殖,加入高浓度IL-2,CD4 CD25-T细胞增殖更强,CD4 CD25 Tr出现增殖。结论:胸腺CD4-CD25 细胞很有可能是CD4 CD25 Tr的前体。     

Cat allergen peptide immunotherapy reduces CD4(+) T cell responses to cat allergen but does not alter suppression by CD4(+) CD25(+) T cells: a double-blind placebo-controlled study

BACKGROUND: We have previously described both modification of allergen immunotherapy using peptide fragments, and reduced regulation of allergen stimulated T cells by CD4(+) CD25(+) T cells from allergic donors when compared with nonallergic controls. It has been suggested that allergen immunotherapy induces regulatory T cell activity: we hypothesized that allergen peptide immunotherapy might increase suppressive activity of CD4(+) CD25(+) T cells. OBJECTIVE: To examine cat allergen-stimulated CD4 T cell responses and their suppression by CD4(+) CD25(+) T cells before and after cat allergen peptide immunotherapy in a double-blind placebo-controlled study. METHODS: Peripheral blood was obtained and stored before and after peptide immunotherapy or placebo treatment. CD4(+) and CD4(+) CD25(+) were then isolated by immunomagnetic beads and cultured with allergen in vitro. RESULTS: Comparing cells from blood taken before with that after peptide immunotherapy there was a significant reduction in both proliferation and IL-13 production by allergen-stimulated CD4+ T cells, whereas no change was seen after placebo. CD4(+) CD25(+) T cells suppressed both proliferation and IL-13 production by CD4(+) CD25(-) T cells before and after therapy but peptide therapy was not associated with any change in suppressive activity of these cells. CONCLUSION: Allergen peptide immunotherapy alters T cell response to allergen through mechanisms other than changes in CD4(+) CD25(+) T cell suppression.     

天然CD4+CD25+Treg细胞的研究进展

天然CD4+ CD25+ Treg细胞在针对自身抗原和外来抗原的免疫应答中起关键控制作用,其缺乏或功能性的缺陷将导致多重病理性的失调.本文就近年在其产生、作用机制以及与免疫耐受的诱导关系等方面的研究进展进行了综述.     

CD4+CD25+调节性T细胞在肿瘤免疫中的作用

CD4+CD25+调节性T细胞(Tr)是体内自然发生的调节性T细胞的重要亚群,具有无反应性和免疫抑制两大特性,主要通过与靶细胞的直接接触而起作用,其在体内不仅参与自身免疫性疾病、移植排斥反应等,还在肿瘤的发生、发展及免疫治疗中发挥重要作用.近几年来,Tr在肿瘤免疫中的作用倍受关注.     

CD4+CD25+调节性T细胞对B细胞免疫应答的抑制作用

CD4 CD25 调节性T细胞(CD4 CD25 Treg细胞)主要来源于胸腺,在体内外抑制CD4 或CD8 T细胞的活化及增殖,是维持自身免疫耐受的重要机制之一。近来研究发现该调节性T细胞除了能够抑制T细胞的免疫应答外,还能够抑制B细胞免疫应答,包括抑制B细胞活化和抗体生成,从而抑制主要由抗体介导的自身免疫性疾病的发生。     

肺炎支原体感染患儿外周血CD4+CD25+调节性T细胞的变化及其临床意义

探讨肺炎支原体(MP)感染患儿外周血CD4+ CD25+调节性T细胞(Treg)频率变化及其意义.选取32例肺炎支原体感染患儿,其中肺炎支原体肺炎18例,肺炎支原体肺炎合并肺外症状14例;26名健康儿童,分离其外周血单个核细胞,以CD4、CD25单克隆抗体标记细胞后,用流式细胞仪(FACS)检测Treg细胞频率.FAC...     

晚期肺癌患者血CD4+CD25+调节T细胞水平的变化

为探讨晚期肺癌患者CD 4CD 25去调节T细胞水平的变化及其与其它T细胞亚群和NK细胞的相互关系,对86例肺癌组患者和64名对照组测定和比较CD 4CD 25调节T细胞、T细胞亚群(CD 3、CD 4、CD 8、CD 8CD 28)和NK细胞的水平,探讨其临床意义.CD 4CD 25细胞和T细胞亚群均用流式细胞仪检测.结果表明,晚期肺癌患者血CD 4CD 25细胞明显高于对照组(18.4%±6.2%vs7.1%±0.4%,P<0.01),CD 4细胞、CD 8CD 28细胞和NK细胞明显低于对照组(32.4%±8.7%vs44.9%±8.4%,P<0.01;7.4%±3.5%vs16.5%±2.7%,P<0.01;10.2%±4.1%vs18.5%±7.2%,P<0.01),CD 8细胞明显高于对照组(36.7%±7.5%vs31.8%±5.1%,P<0.01).CD 4CD 25细胞与CD 8CD 28细胞、NK细胞呈明显负相关.表明CD 4CD 25细胞增多是晚期肺癌患者免疫功能紊乱的一个证据.     

小鼠脾细胞CD4+CD25+调节性T细胞的表型特征

观察CD4+CD25+T和CD4+CD25-T细胞的表型和细胞因子的表达。自小鼠脾脏制备单个细胞悬液,分离CD4+T细胞、CD4+CD25+和CD4+CD25-T细胞,进行细胞表面标记,激活后进行细胞内细胞因子染色,利用流式细胞仪在单个细胞水平上分析细胞表面分子、转录因子和细胞因子表达之间的关系。结果:在CD4+T细胞中,约有7.8%的细胞同时表达CD25分子。与CD4+CD25-T细胞相比,CD4+CD25+T细胞CD44的表达略有增加,CD45RB的表达明显下降,CTLA-4和Foxp3明显增加。以同时表达CTLA-4和Foxp3的细胞为主,其次为单独表达Foxp3的细胞。细胞因子的研究结果表明,与CD4+CD25-T细胞相比,CD4+CD25+T细胞IL-2、IFN-γ明显减少,而只产生IL-10的细胞略有增加。CD4+CD25+调节性T细胞无论在表型、转录因子的表达以及细胞因子表达方面均于非调节性T细胞不同。     

CD4+ CD25+调节性T细胞对树突状细胞的杀伤作用

目的 探讨CD4^＋CD25^＋调节性T细胞是否对树突状细胞发挥免疫调节作用及其可能的机制。方法 用MACS（magnetic cell sorting）从BALB／c小鼠静息T细胞分离纯化CD4^＋CD25^＋T细胞，体外细胞增殖实验观察其对CD4^＋CD25^＋T细胞的免疫抑制作用；GM-CSF／IL-4培养自体小鼠骨髓来源DC，FACS（fluorescence-activated cell sorting）鉴定其表面分子特性；以CD3／CD28单克隆抗体活化CD4^＋CD25^＋调节性T细胞，FACS体外杀伤实验研究其对自体DC的调节作用，并观察穿孔素抑制剂EGTA对上述作用的影响。结果 用MACS法成功分离出CD4^＋CD25^＋T细胞，纯度可达98％，特异性表达而Faxp3基因，能明显抑制CD4^＋CD25^＋T细胞的体外增殖；骨髓来源的DC表达CDllc、MHCⅡ及少量协同刺激分子CD80、CD86；FACS体外杀伤实验证实以CD3／CD28抗体体外活化的CD4^＋CD25^＋调节性T细胞对自体DC有显著杀伤作用（P〈0．05），穿孔素抑制剂EGTA能部分抑制该杀伤效应（P〈0．05）。结论 CD4^＋CD25^＋调节性T细胞可通过杀伤作用对自体DC发挥免疫调节作用，穿孔素／颗粒酶杀伤途径可能参与其中。     

CD4+CD25+调节性T细胞及血小板参数在重症肺炎患者中表达的意义

目的 探讨CD4+ CD25+调节性T细胞及血小板参数在重症肺炎患者中表达的意义.方法 收集2013年5月至2015年10月我院诊断为肺炎的患者,共计80例,进一步分为重症肺炎组28例和肺炎组52例.对照组为同期健康体检者,共50例.对比重症肺炎组、肺炎组和对照组入院时CD4+ CD25+调节性T细胞及血小板参数水平,并且分析重症肺炎组死亡患者与存活患者入院时CD4+ CD25+调节性T细胞及血小板参数水平.结果 ①重症肺炎组、肺炎组和对照组入院时CD4+ CD25+调节性T细胞水平、MPV及PLT结果比较有差异(P＜0.05);重症肺炎组、肺炎组和对照组入院时PDW、PCT结果比较无差异(P＞0.05).②重症肺炎组死亡患者与存活患者入院时CD4+ CD25+调节性T细胞水平、MPV及PLT结果比较有差异(P＜0.05);重症肺炎组死亡患者与存活患者入院时PDW、PCT结果比较无差异(P＞0.05).结论 CD4+ CD25+调节性T细胞、MPV及PLT可作为反应肺炎严重程度的敏感指标,并且可作为评价重症肺炎患者预后的客观指标.     

肺癌患者CD4+ CD25+调节性T细胞的检测及临床意义

目的:检测肺癌患者外周血CD4 CD25 调节性T细胞的分布并探讨相关机制.方法:采用流式细胞仪分析66例肺癌患者外周血CD4 CD25 调节性T细胞占CD4 T淋巴细胞的比例.结果:66例肺癌患者外周血中CD4 CD25 调节性T细胞占CD4 T淋巴细胞的比例为(16.2±2.4)%,与对照组(6.19±1.5)%比较差异有显著性(P＜0.05). 25例鳞癌、29例腺癌、12例小细胞癌患者外周血中CD4 CD25 调节性T细胞比例分别为(18.3±2.9)%、(15.6±1.8)%、(17.3±2.2)%,各组间比较差异无显著性(P＞0.05);均显著高于对照组(6.19±1.5)%,P＜0.05;34例Ⅲ期,14例Ⅳ期肺癌患者外周血中CD4 CD25 调节性T细胞比例为(15.3±2.6)%,(20.4±3.1%),均显著高于18例Ⅱ期患者(9.4±1.3)%,P均＜0.05.结论:肺癌患者外周血中有CD4 CD25 调节性T细胞比例增高,且与分期有关.它可能与肺癌患者免疫功能受损有关,可作为评估肺癌患者预后的一项指标.     

CD4+CD25+调节性T细胞在自身免疫病中作用的研究进展

CD4 CD25 调节性T细胞具有独特的免疫抑制功能,通过细胞接触和细胞因子机制抑制自身反应性T细胞的活化与增殖,在自身免疫病中发挥着重要作用。通过研究CD4 CD25 调节性T细胞在自身免疫病中的作用,将会揭示这类疾病的发病机制,从而为治疗自身免疫病提供一个新途径。     

CD4+ CD25+调节性T细胞在肾脏疾病中的作用

CD4+ CD25+ 调节性T细胞(Tr)是一个具有独立功能的T细胞亚群,是机体维持自身免疫耐受的重要组成部分,在免疫病理、移植物耐受、阻止自身免疫反应和维持机体免疫平衡方面都有一定作用,其T细胞亚群在多种免疫性疾病中发挥重要的调节作用,但国内外有关该群细胞在肾脏疾病中作用的报道较少.     

CD4+CD25+免疫调节性T细胞对CD4+T细胞介导的移植物抗宿主病预防作用的研究

目的研究CD4+CD25+免疫调节性T(Treg)细胞在小鼠骨髓移植后,对移植物抗宿主病的预防作用及其作用机制.方法用C3H(H-2k)小鼠骨髓作为供体,提取C3H(H-2k)小鼠CD4+T及CD4+CD25+T细胞,C3H×B6(H-2k/b)F1小鼠为骨髓移植的受者.在受者接受致死量全身放射后,输注供者去除T细胞的骨髓(ATBM),使其造血功能重建(ATBM组).于不同的实验组给予CD4+(CD4组)T细胞,CD4+CD25+T(CD25组)或二者同时输注(CD4/CD25组).观察各组小鼠移植物抗宿主病(GVHD)的发生率.结果所有10只ATBM组小鼠至骨髓移植后60天仍全部存活,无GVHD发生;所有10只CD4组小鼠在骨髓移植10天内全部死于GVHD(P＜0.01);所有5只CD25组小鼠于骨髓移植后60天仍全部存活,无明显GVHD发生(P＞0.05);同样,所有6只CD4/CD25组小鼠至骨髓移植后仍全部存活,无明显GVHD发生(P＞0.05).结论在同种异基因小鼠的骨髓移植模型中,CD4+CD25+T不诱导GVHD的发生,并有预防CD4+T细胞介导的GVHD发生的作用.