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.     

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.     

CTLA-4 x Ig converts naive CD4+CD25- T cells into CD4+CD25+ regulatory T cells

CTLA-4 x Ig was originally designed as an immunosuppressive agent capable of interfering with the co-stimulation of T cells. In the present study, we demonstrate that CTLA-4 x Ig, in combination with TCR ligation, has the additional capacity to convert naive CD4+CD25- T cells into Foxp3+ regulatory T (T(reg)) cells, as well as to expand their numbers. The CD4+CD25+Foxp3+ T(reg) generated by CTLA-4 x Ig treatment in vitro potently suppress effector T cells. Extending this in vivo, we show that systemic administration of CTLA-4 x Ig increases the percentage of CD4+CD25(hi)Foxp3+ cells within mixed lymphocyte reaction-induced murine lymph nodes. Significantly, the in vitro conversion of naive CD4+CD25- T cells into T(reg) cells is antigen-presenting cell (APC) dependent. This finding, together with the further observation that this conversion can also be driven in vitro by an antibody that engages B7-2 ligand, suggests that CTLA-4 x Ig-driven T(reg) induction may be predicated upon active CTLA-4 x Ig to B7-2 signaling within APC, which elicits from them T(reg)-inducing potential. These findings extend CTLA-4 x Ig's functional repertoire, and at the same time, reinforce the concept that T cell anergy and active suppression are not entirely distinct processes and may be linked by some common molecular triggers.     

Increased cytotoxicity of CD4+ invariant NKT cells against CD4+CD25hiCD127lo/- regulatory T cells in allergic asthma

CD4+CD25(hi)CD127(lo/-) regulatory T cells (Treg) have been implicated in the resolution of asthma-associated inflammation while the opposite role of CD4+ invariant NKT (iNKT) cells has been the subject of recent investigations. Studies here focused on mechanisms of interaction between CD4+ iNKT cells and Treg to further explore their roles in allergic asthma (AA). Flow cytometry analysis revealed a significant increase in the expression of the natural cytotoxicity receptors NKp30 and NKp46 by CD4+ iNKT cells in AA subjects compared to healthy controls (HC) and non-allergic asthmatics (NA). Subsequent intracellular staining showed that CD4+ iNKT cells also expressed higher levels of granzyme B and perforin in AA than HC. In in vitro killing assays, AA CD4+ iNKT cells selectively killed autologous Treg, but not CD4+CD25- T cells, more potently than HC and NA counterparts. This increased cytotoxicity positively correlated with asthma severity and granzyme B/perforin expression of CD4+ iNKT cells. Furthermore, it could be abrogated by either inhibition of the granzyme B-/perforin-dependent cell death pathway or oral corticosteroid administration. Altogether, these findings suggest that increased cytotoxicity of CD4+ iNKT cells against Treg might contribute to dysfunctional cellular interactions in AA.     

CD4+ CD25+ [corrected] regulatory T cells render naive CD4+ CD25- T cells anergic and suppressive

CD4(+) CD25(+) Foxp3(+) naturally occurring regulatory T cells (nTreg) are potent inhibitors of almost all immune responses. However, it is unclear how this minor population of cells is capable of exerting its powerful suppressor effects. To determine whether nTreg mediate part of their suppressor function by rendering naive T cells anergic or by converting them to the suppressor phenotype, we cocultured mouse nTreg with naive CD4(+) CD25(-) T cells from T-cell receptor (TCR) transgenic mice on a RAG deficient (RAG(-/-)) background in the presence of anti-CD3 and interleukin-4 (IL-4) to promote cell viability. Two distinct responder cell populations could be recovered from the cocultures. One population remained undivided in the coculture and was non-responsive to restimulation with anti-CD3 or exogenous IL-2, and could not up-regulate IL-2 mRNA or CD25 expression upon TCR restimulation. Those responder cells that had divided in the coculture were anergic to restimulation with anti-CD3 but responded to restimulation with IL-2. The undivided population was capable of suppressing the response of fresh CD4(+) CD25(-) T cells and CD8(+) T cells, while the divided population was only marginally suppressive. Although cell contact between the induced regulatory T cell (iTreg) and the responders was required for suppression to be observed, anti-transforming growth factor-beta partially abrogated their suppressive function. The iTreg did not express Foxp3. Therefore nTreg are not only able to suppress immune responses by inhibiting cytokine production by CD4(+) CD25(-) responder cells, but also appear to modulate the responder cells to render them both anergic and suppressive.     

CD4+ CD25+调节性T细胞AICD机制的研究

目的探讨CD4^＋CD25^＋调节性T细胞活化诱导的细胞死亡（AICD）发生的机制。方法CD4^＋CD25^＋T细胞以磁性细胞分离器（MACS）从BALB／c小鼠或DO11．10小鼠的静息T细胞分离纯化。体外细胞增殖抑制实验证实其免疫调节作用。CD4^＋CD25^＋T细胞的AICD以CD3／CD28单克隆抗体活化或以特异性OVA323-339肽、抗原提呈细胞活化等两种方法获得。CD4^＋CD25^＋T细胞凋亡相关基因的表达通过实时定量PCR检测。流式细胞仪检测细胞的凋亡率。进一步观察FasL中和抗体、TRAIL中和抗体及caspase抑制剂zVAD-fmk对CD4^＋CD25^＋T细胞凋亡的影响。结果MACS成功分离CD4^＋CD25^＋T细胞，纯度可达98％，该细胞可特异性表达Foxp3基因，能明显抑制效应性T细胞的体外增殖。CD3／CD28抗体以及OVA特异性抗原活化8d的CD4^＋CD25^＋调节性T细胞AICD达39％～45％。活化前后的CD4^＋CD25^＋调节性T细胞死亡受体家族表达发生明显变化；FasL、TRAIL中和抗体及zVAD-fmk可明显抑制CD4^＋CD25^＋调节性T细胞的凋亡。结论FasL／Fas及其他凋亡相关分子可能参与了CD4^＋CD25^＋调节性T细胞的凋亡。     

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。     

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.     

Targeting cytokines secreted by CD4+CD25highCD127low regulatory T cells inhibits ovarian cancer progression

Epithelial ovarian cancer (EOC) is one of the major malignant cancers with high rates of early metastasis in which regulatory T cells (Tregs) play an important role. Tregs suppress immune responses and promote the development of tumours in patients with EOC. However, the underlying mechanisms remain unclear. In this study, we found higher levels of CD4⁺CD25^highCD127^low Tregs in patients with EOC than in patients with benign ovarian tumours and healthy donors. The immune inhibitory effect of Tregs functions by maintaining high levels of immunosuppressive cytokines in EOC. The high levels of Tregs and related cytokines (TGF‐β1 or IL‐10) were associated with lymphatic metastasis and FIGO stages of patients with EOC. Expression of matrix metalloproteinase (MMP)‐2 and tissue inhibitors of metalloproteinase (TIMP)‐2 in EOC cell lines were significantly regulated in the coculture experiment with CD4⁺CD25^highCD127^low Tregs sorted from EOC patients. Levels of MMP‐2 and TIMP‐2 conversely changed after blocking IL‐10R and TGF‐β1R in EOC cells. The invasion ability of EOC cells was also significantly downregulated in this process. The metastasis of EOC cells was correlated with the levels of TGF‐β1 or IL‐10. These findings suggested that immunosuppressive cytokines secreted by CD4⁺ Tregs could be a novel target for inhibiting EOC progression.     

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.     

替比夫定对慢性乙型肝炎患者外周血CD4+CD25+CD127lowT和CD8+CD25+T细胞频率的影响及临床意义

目的探讨核苷类药物替比夫定对慢性乙肝患者(CHB)外周血中CD4+CD25+CD127lowT细胞和CD8+CD25+T细胞比例的影响,并结合临床指标分析其临床意义。方法替比夫定抗病毒治疗22例CHB患者,在治疗前及治疗后3,6个月时,分别以流式细胞仪检测外周血中CD4+CD25+CD127lowT细胞和CD8+CD25+T细胞比例,实时荧光定量RT-PCR检测Foxp3 mRNA的表达水平,荧光定量PCR检测血清HBV DNA水平,酶联免疫吸附法检测HBV标志物,全自动生化分析仪检测ALT水平。结果 CHB患者外周血中CD4+CD25+CD127lowT细胞和CD8+CD25+T细胞比例显著高于对照组。替比夫定治疗3个月时,这两群细胞比例显著下降,Foxp3 mRNA的表达也显著下降;HBV DNA水平降至检测水平以下的CHB患者,其CD4+CD25+CD127lowT细胞也降至正常水平。治疗3、6个月时,HBeAg阴转率分别为9.1%和18.2%,发生HBeAg血清学转换者的CD4+CD25+CD127lowT细胞和CD8+CD25+T细胞比例均降至正常水平。结论替比夫定能快速有效抑制CHB患者的病毒复制...     

Chemokine responsiveness of CD4+ CD25+ regulatory and CD4+ CD25− T cells from atopic and nonatopic donors

Background:  Allergic inflammation is associated with Th2-type T cells, which can be suppressed by CD4+ CD25+ regulatory T cells (Tregs). Both express chemokine receptors (CCR) 4 and CCR8, but the dynamics of expression and effect of atopic status are unknown.
Objective:  To examine the expression of chemokine receptors by CD4+ CD25+ and CD4+ CD25− T cells from atopic and nonatopic donors, and document response to allergen stimulation in vitro .
Methods:  Chemokine receptor expression was examined by flow cytometry and quantitative PCR of CD4+ CD25hi and CD4+ CD25− T cells from atopics and nonatopics. Responsiveness to chemokines was by actin polymerization. Dynamics of chemokine receptor expression in 6-day allergen-stimulated cultures was analysed by carboxyfluoroscein succinimidyl ester labelling.
Results:  CD4+ CD25hi Tregs preferentially expressed CCR3, CCR4, CCR5, CCR6 and CCR8. CD4+ CD25hi Tregs responded to the chemokine ligands for CCR4, CCR6 and CCR8 (CCL17, 22, 20 and 1 respectively), with no differences between atopic and nonatopic donors. Over 6-day allergen stimulation, CD4+ CD25+ T-cells downregulated CCR4 and upregulated CCR7, in contrast to CD4+ CD25− effector cells, which downregulated CCR7 and upregulated CCR4.
Conclusions:  CCR4, CCR6 and CCR8 have potential roles in localization of both CD4+ CD25+ regulatory and CD4+ CD25− effector T cells to sites of allergic inflammation. Upregulation of CCR7 and downregulation of CCR4 upon allergen stimulation of Tregs may allow their recirculation from sites of inflammation, in contrast to retention of effector T cells.     

Expansion of regulatory CD8+ CD25+ T cells after neonatal alloimmunization

Transplantation tolerance induced by neonatal injection of semi-allogeneic spleen cells is associated with a pathological syndrome caused by T helper type 2 (Th2) differentiation of donor-specific CD4(+) T lymphocytes. We have shown previously that this Th2-biased response is inhibited by host CD8(+) T cells. Herein, we demonstrate that upon neonatal immunization with (A/J × BALB/c)F(1) spleen cells, BALB/c mice expand a population of CD8(+) T cells expressing both CD25 and forkhead box P3 (FoxP3) markers. In this setting, CD8(+) CD25(+) T cells predominantly produce interferon (IFN)-γ and interleukin (IL)-10 and are efficient in controlling IL-4, IL-5 and IL-13 production by donor-specific CD4(+) T cells in vitro. CD8(+) FoxP3(-) T cells are single producers of IFN-γ or IL-10, whereas CD8(+) FoxP3(+) T cells are double producers of IFN-γ and IL-10. We further demonstrate that IFN-γ and IL-10 are two major cytokines produced by CD8(+) T cells involved in the in vivo regulation of Th2-type pathology. In this setting, we conclude that neonatal alloimmunization induces the expansion of several regulatory CD8(+) T cells which may control Th2 activities via IFN-γ and IL-10.     

CD4+CD25+ regulatory T cells in the small intestinal lamina propria show an effector/memory phenotype

CD4⁺CD25⁺ regulatory T cells (Tregs) have been implicated inthe suppression of pathogenic responses to both self- and non-self-antigensin the intestine. However, their precise properties and functionsin the gut, as well as the molecular basis of their recruitmentto the gut, are poorly understood. Here, we found that mostof the CD4⁺CD25⁺ T cells in the small intestinal lamina propria(LP) express Foxp3 and exhibit an ‘effector/memory’phenotype, CD44^hiCD45RB^loCD62L^–, whereas only a minorityof the Foxp3⁺CD4⁺CD25⁺ T cells in the spleen and mesentericlymph nodes showed this phenotype. The Tregs in the small intestinalLP (LP-Tregs) expressed higher levels of CCR4 and CCR9 and asubstantially lower level of CCR7 than the Tregs in the spleen.In vitro, the LP-Tregs showed chemotaxis to CCL25/thymus-expressedchemokine. In addition, they showed efficient chemotaxis tothe CCR4 ligands, CCL17/thymus and activation-regulated chemokineand CCL22/macrophage-derived chemokine, which are abundantlyexpressed by dendritic cells (DCs) in the small intestinal LP.In vivo, 50% of the LP-Tregs were closely associated or in directcontact with LP-DCs. These findings demonstrate that LP-Tregsare phenotypically and functionally unique and raise the possibilitythat they are retained in the small intestinal LP through theaction of CCL17 and CCL22, which are locally produced by LP-DCs.     

Daily subcutaneous injections of peptide induce CD4+ CD25+ T regulatory cells

Peptide immunotherapy is being explored to modulate varied disease states; however, the mechanism of action remains poorly understood. In this study, we investigated the ability of a subcutaneous peptide immunization schedule to induce of CD4(+) CD25(+) T regulatory cells. DO11.10 T cell receptor (TCR) transgenic mice on a Rag 2(-/-) background were injected subcutaneously with varied doses of purified ovalbumin (OVA(323-339)) peptide daily for 16 days. While these mice have no CD4(+) CD25(+) T regulatory cells, following this injection schedule up to 30% of the CD4(+) cells were found to express CD25. Real-time quantitative polymerase chain reaction (QPCR) analysis of the induced CD4(+) CD25(+) T cells revealed increased expression of forkhead box P3 (FoxP3), suggesting that these cells may have a regulatory function. Proliferation and suppression assays in vitro utilizing the induced CD4(+) CD25(+) T cells revealed a profound anergic phenotype in addition to potent suppressive capability. Importantly, co-injection of the induced CD4(+) CD25(+) T cells with 5,6-carboxy-succinimidyl-fluorescence-ester (CFSE)-labelled naive CD4(+) T cells (responder cells) into BALB/c recipient mice reduced proliferation and differentiation of the responder cells in response to challenge with OVA(323-339) peptide plus adjuvant. We conclude that repeated subcutaneous exposure to low-dose peptide leads to de novo induction of CD4(+) CD25(+) FoxP3(+) T regulatory cells with potent in vitro and in vivo suppressive capability, thereby suggesting that one mechanism of peptide immunotherapy appears to be induction of CD4(+) CD25(+) Foxp3(+) T regulatory cells.     

抗CD25单克隆抗体对CD4+CD25high调节性T细胞的影响

目的 评价抗CD25单克隆抗体对活体肾移植受者外周血CD4 CD25high调节性T细胞(Treg)的影响.方法 观察14例活体肾移植受者外周血CD4 CD25high T细胞百分比和FoxP3mRNA表达水平的变化,应用流式细胞仪FACS Calibur和CellQuest软件行双色流式细胞计数和结果分析,半定量逆转录-聚合酶链反应(RT-PCR)对肾移植受者外周血单个核细胞(MNC)的FoxP3基因水平进行检测.结果 在移植术后3d、13d,CD4 CD25high T细胞占CD4 比例显著升高;在移植后1个月内,CD4 CD25high T细胞比例升高约至原来的3倍.移植术后13d、17d、27d时外周血FoxP3 mRNA表达明显高于移植术后3d.在肾移植术后的不同时间抗CD25单克隆抗体使用组受者的CD25 T细胞百分比明显低于对照组(未使用抗CD25单克隆抗体).抗CD25单克隆抗体使用组与对照组两组间比较,术后3d、13d,抗CD25单克隆抗体组CD4 CD25high T细胞占CD4 的比例明显降低,术后3d对照组、抗CD25单克隆抗体使用组分别为1.24%±0.15%、2.25%±0.24%,P=0.00;术后13d为3.75%±0.38%、7.28%±0.51%,P=0.00.然而,术后17d、27d后,抗CD25单克隆抗体使用组的CD4 CD25high T恢复到对照组水平,两组间CD4 CD25high T细胞占CD4 的比例无统计学差异,即第17d为3.72%、0.26%、4.43%、0.44%,P=0.17,27d为9.00%±0.30%、8.31%±0.33%,P=0.16.在肾移植术后不同时间,受者外周血FoxP3mRNA表达水平在两组之间相似,无统计学差异,说明FoxP3mRNA表达水平并没有受到体内注射抗CD25单克隆抗体影响.结论 抗CD25单克隆抗体的使用对活体肾移植受者外周血CD4 CD25high调节性T细胞在一定时间具有暂时性的影响,而FoxP3 mRNA表达水平没有受到影响.     

诱导CD4+CD25+自然调节性T细胞发育的免疫识别

CD4+ CD25+ 自然调节性T细胞(nTreg)对维持自身免疫耐受极为重要,了解其发育途径具有重大理论和实用价值.最近一些证据表明在胸腺内nTreg的发育可能由不同于常规CD4+ T细胞发育的免疫受体刺激类型诱导,存在特殊的细胞内信号转导途径.这提示CD4+T细胞的不同免疫识别类型或机制在T细胞分化或免疫功能形成中有重要作用,并为深入理解复杂的免疫现象提供了一种新观念.