TGF-β诱导的CD4~+CD25~+Foxp3~+调节性T细胞对胶原诱导性关节炎的抑制作用及机制研究
|
摘要
类风湿性关节炎(Rheumatoid Arthritis, RA)是以慢性、进展性和侵蚀性关节炎为特点的一类自身免疫性疾病,最终表现为骨质的破坏、关节的畸形,致残率极高。目前RA的治疗以免疫抑制剂为主,但治疗的副作用较大。
已知CD4+CD25+Foxp3+的天然调节性T细胞(Natural Ocurring Regulatory T Cells, nTregs)数量的减少和/或功能的降低与RA的发病有关。有学者发现,在RA小鼠模型输注nTregs可以预防关节炎的发生和减轻关节炎的严重程度。但是,在已发病的RA经典小鼠模型——胶原诱导性关节炎(collagen-induced arthritis, CIA)中,即使额外输注nTregs,对关节炎也没有起到改善作用。近来,本研究小组发现,TGF-β诱导的调节性T细胞(Induced Regulatory T cells, iTregs)不仅具有与nTregs相同的表型和抑制关节炎发病的功能,而且在体外促炎症因子白介素-6 (Interleukin 6, IL-6)的作用下能表现出更强的稳定性和对效应性T淋巴细胞的抑制力。因此,本论文第一部分的研究旨在比较nTregs和iTregs对CIA预防与治疗作用的差异。
关节破坏是RA患者最常见的症状和转归,破骨细胞在RA患者的骨质重建中发挥重要作用；破骨细胞是一种多核巨细胞,主要功能是骨质吸收,生理条件下与成骨细胞一起维持骨代谢的平衡：而破骨细胞的过度激活和功能异常被认为是炎性关节病人骨质侵蚀的重要诱因：已有报道在RA的CIA小鼠模型中观察到破骨细胞前体细胞(Osteoclast precursors, OCPs)的数量增多。破骨细胞起源于骨髓,其前体细胞在转录因子-KB受体激活因子配体(Receptor Activator of Nuclear Factor-κB, RANKL)和巨噬细胞集落刺激因子(Macrophage Colony-stimulating Factor, M-CSF)的共同作用下,进一步分化为成熟的破骨细胞；RANKL和M-CSF是破骨细胞生成过程所必须的两个细胞因子,任何一个缺失或信号途径的阻断都会影响破骨细胞的分化。因此,破骨细胞的生成已成为关节炎关节破坏及治疗的重要靶点。已有报道证实,激活的nTregs可以抑制破骨细胞的生成,而iTregs具有与nTregs相似的表型和抑制功能。因此,本论文第二部分研究是在第一部分研究的基础上,探讨iTregs是否可以通过抑制破骨细胞的生成而发挥其对CIA模型动物的骨质侵蚀及破坏的抑制能力。
实验结果：
第一部分：TGF-p诱导的CD4CD25+Foxp3+调节性T细胞制备、鉴定及对胶原诱导性关节炎的抑制、治疗作用
1. TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞的制备、鉴定
自DBA1/J小鼠的脾脏分离出幼稚的CD4+细胞。以牛CⅡ型胶原(bovine collagenⅡ, CⅡ)多肽、白介素-2(Interleukin-2,IL-2)和TGF-β诱导Tregs (iTregs),单用IL-2培养的细胞为效应性T淋巴细胞,作为Tregs的对照(Tcon)。nTregs分离自同种小鼠的胸腺,在培养液中加入IL-2进行体外扩增,流式细胞仪、Western Blotting和PCR法分别检测Foxp3的表达；同时nTregs和iTregs分别与效应性T淋巴细胞共培养(绿色荧光蛋白CFSE标记法和增生实验),比较两者对效应性T淋巴细胞增生的抑制能力,实验设立基线值(baseline)、nTregs组、Tcon组和iTregs组。
1.1 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞Foxp3的表达
流式细胞仪的结果显示,经过扩增的nTregs与诱导的iTregs, Foxp3的表达相近,多在60%以上,而Tcon Foxp3的表达仅为10%左右。计数培养孔内的CD25+Foxp3+细胞数,Tcon组的细胞数显著低于iTregs组。Western Blotting检测CD4+ CD25-细胞、nTregs、Tcon和iTregs Foxp3蛋白的表达水平,nTregs与iTregs Foxp3蛋白的表达相近并均显著高于CD4＋CD25－细胞和Tcon。PCR法检测Foxp3 mRNA的水平,nTregs与iTregs Foxp3 mRNA的水平相近并均显著高于CD4+ CD25-细胞和Tcon。
1.2 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞对效应性T淋巴细胞的
抑制能力
体外CFSE标记法与增生实验法均证实,与基线值、Tcon组相比,iTregs拥有与nTregs相似的对效应性T淋巴细胞增生的抑制功能,而Tcon则与基线值相近,无法抑制T淋巴细胞的增生。
2 TGF-β诱导的CD4CD25+Foxp3+调节性T细胞对CIA的抑制和治疗作
用采用牛Ⅱ型胶原(4mg/mL)和完全弗式佐剂(体积比1：1)乳化液50ul于小鼠尾根部皮下注射诱导关节炎小鼠模型,在免疫后的第0天、第14天和第28天分别自小鼠尾静脉输注3×106 nTregs或iTregs,小鼠随机分为4组：CIA模型组、nTregs注射组、Tcon组和iTregs组,发病后隔天观察小鼠关节炎的发病率及临床评分。检测血清抗CⅡ特异性抗体的水平以及关节组织学染色,进一步评估疾病的严重程度。
2.1 TGF-P诱导的CD4+CD25+Foxp3+调节性T细胞对CIA的预防作用
在DBAl/J小鼠免疫后的第0天分别注射nTregs和iTregs,两者都可显著抑制关节炎的发生和发展；在免疫后的第14天分别注射nTregs和iTregs, iTregs仍可降低关节炎的发生,两者均可显著降低关节炎的临床评分；并且检测血清抗CII特异性抗体水平发现,两种Tregs均可显著抑制抗CII IgG2b的水平。
2.2 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞对CIA的治疗作用
在DBAl/J小鼠免疫后的第28天(即关节炎发病后)分别注射nTregs和iTregs, iTregs而非nTregs可以显著抑制关节炎的发生和严重程度,以及血清抗CII IgG2a和IgG2b的水平；病变关节组织学染色显示,iTregs可以显著抑制关节炎性细胞的浸润及骨质破坏的发生；分离自关节炎小鼠体内的淋巴细胞体外增生实验显示,iTregs尚可抑制淋巴细胞的增生程度和促炎症因子的水平；而nTregs则完全丧失了对关节炎的保护作用。
小结：
以上实验结果表明,iTregs拥有与nTregs相似的表型和对T淋巴细胞增生的抑制能力；同时,对CIA具有与nTregs相似的预防作用；但在已发病的关节炎小鼠,iTregs而非nTregs具有明确的治疗作用且抑制了关节破坏的发生,nTregs则完全丧失了对关节炎的保护作用。
第二部分：TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞预防和治疗CIA的机制研究
基于第一部分的研究结果,iTregs较nTregs在已发病的关节炎小鼠具有稳定的治疗作用,并可抑制骨质破坏的发生,我们在本部分将进一步探究其作用机制。
3 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞在体内、外的稳定性
按照1中的描述分别制备nTregs与iTregs,进行如下实验：
3.1 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞在体外的稳定性
采用与1.2相同的CFSE标记法与增生实验鉴定两者对效应性T淋巴细胞的抑制作用,同时设立IL-6添加组(IL-6+)与非添加组(IL-6-),检测在促炎症因子IL-6存在的情况下,两种Tregs抑制力的变化。建立Th17细胞培养体系即IL-6+TGF-P诱导幼稚的CD4+细胞向Th17细胞转化,在培养液中同样分别加入nTregs、iTregs (Tregs:幼稚CD4+细胞=1：4),比较两组IL-17A生成的水平。
结果显示,在IL-6作用下,nTregs而非iTregs显著地丧失了其对效应性T淋巴细胞的抑制作用,并且无法抑制Th17细胞的分化及IL-17A的生成；而iTregs则始终保持着良好的抑制能力。
3.2 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞在体内的稳定性
来自GFP-Foxp3转基因小鼠的nTregs和iTregs分别注射入免疫缺陷(CD3-/-)的小鼠体内,分别在注射后4周和8周观察Foxp3水平的变化；绿色荧光蛋白CFSE标记来自正常DBA1/J小鼠的两种Tregs并注射到同种小鼠体内,分别在注射后的1周和3周,检测两种Tregs Foxp3水平的变化；另将标记了CFSE的两种Tregs分别注射入已发病的关节炎小鼠(炎性小鼠)体内,观察两种Tregs在炎性环境下的稳定性和转归。
3.2.1 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞在免疫缺陷和正常小鼠体内环境下的稳定性
在注射后4周和8周,nTregs和iTregs同样可以维持Foxp3的表达,Foxp3表达占CD3+细胞的百分比为50%左右。在正常体内环境下,nTregs和iTregs同样可以维持Foxp3的表达在50%左右(Foxp3占CFSE+细胞的百分比)。
3.2.2 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞在关节炎小鼠炎性环境下的稳定性
实验结果显示,相对于iTregs, nTregs在细胞注射后1周的关节炎小鼠的引流淋巴结内,更易于凋亡、Foxp3的表达丢失并向Th2和Th17细胞转化；相反,iTregs在相似的炎性环境下能够维持Foxp3的表达且不会向辅助性T淋巴细胞转化。在nTregs注射组的关节炎小鼠淋巴结细胞中,Th17细胞的数量是iTregs组的10倍,而CFSE+Foxp3+细胞的数量为iTregs组的八分之一,提示了iTregs能够在炎性环境下诱导出更多的Foxp3+的调节性T细胞的产生。
4 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞通过抑制破骨细胞的生成发挥对CIA的保护作用
nTregs可以抑制破骨细胞的生成,而破骨细胞的生成过多是造成关节炎骨质破坏的主要因素；由此,我们将探究iTregs是否也通过抑制破骨细胞的生成而发挥对关节炎的保护作用。
分离自正常DBA1/J小鼠骨髓的CDllc+细胞即破骨细胞前前体细胞(Osteoclast Precursors, OCPs),以RANKL和M-CSF共同诱导破骨细胞的生成；同时在培养液中分别加入CD4+CD25-细胞、nTregs, Tcon和iTregs,抗酒石酸磷酸酶(Tartrate-risistant acid phosphatase,TRAP)染色,观察各种细胞对破骨细胞生成的影响。
nTregs、iTregs分别在免疫后14天注射入关节炎小鼠体内,观察期(56天)后,病变关节行CT扫描,观察关节骨质侵蚀、破坏的发生与不同。
4.1 TGF-p诱导的CD4+CD25+Foxp3+调节性T细胞在体外对破骨细胞生成的抑制作用实验结果显示,与基线值(baseline)、CD4+CD25-细胞组和Tcon组相比,nTregs和iTregs均可显著抑制破骨细胞的生成；减少加入的iTregs比例,破骨细胞的生成有所增加,但与Tcon相比,破骨细胞的生成仍受到显著抑制。提示iTregs对破骨细胞生成的抑制为剂量相关性的。
4.2 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞在体内对骨质侵蚀和破坏的抑制作用
CT扫描结果显示,CIA模型组小鼠的关节呈慢性多关节炎的表现,关节表面骨质侵蚀明显,关节腔融合,关节强直,骨量减少；而iTregs仅表现为一侧关节的轻度关节腔狭窄,无骨质侵蚀的发生,关节腔间隙和骨量与正常的小鼠关节无异。
5 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞抑制破骨细胞生成的机制研究
实验设立OCPs阳性对照组,和iTregs组,在iTregs与OCPs共培养组中分别加入同型对照(cIgG)组、抗TGF-β单抗组和抗IL-10R抗体组,观察iTregs是否通过TGF-β和IL-10信号途径发挥对破骨细胞生成的抑制作用；同时设立双层细胞培养板实验,观察iTregs对破骨细胞的抑制是否为细胞接触所依赖的；另外设立OCPs组、Tcon组和iTregs组,收集细胞,提取总蛋白,Western Blotting检测各组NF-κB亚单位P65和P50的表达水平,明确iTregs是否通过NF-κB途径发挥其抑制作用。
5.1 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞对破骨细胞生成的抑制为细胞接触而非细胞因子接触所依赖的
实验结果显示,在加入了抗TGF-β和IL-10R中和抗体后,并未影响iTregs对破骨细胞生成的影响；但双层细胞板实验显示,iTregs一旦与OCPs分离培养,即无法抑制其分化成破骨细胞,提示,iTregs的抑制作用为细胞接触所依赖的。
5.2 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞通过NF-κB信号途径发挥对破骨细胞生成的抑制
实验结果显示,iTregs与OCPs共培养组,NF-κB亚单位P65和P50的表达水平显著低于Tcon组及OCPs阳性对照组。
小结
结合上述实验结果,iTregs在免疫缺陷和正常小鼠体内环境下,都具有很好的稳定性,与nTregs相似；但在已发病的关节炎小鼠的炎性体内环境下,nTregs易于凋亡并向Th17细胞转化,同时还丧失了其对T淋巴细胞的抑制力；与之相反的是,iTregs能稳定存在,并维持了其对T淋巴细胞的抑制功能。此外,iTregs对CIA小鼠关节的保护作用,一定程度上得益于其对破骨细胞生成的抑制作用,从而减少了破骨细胞引起的骨质侵蚀和破坏；iTregs对破骨细胞的抑制为细胞接触和NF-κB信号途径所依赖的,并且表现为iTregs剂量相关性的。
综合以上研究,所得结论如下：
1.在体外,iTregs具有与nTregs相似的Foxp3的表达和对效应性T淋巴细胞的抑制能力。
2.对CIA小鼠,iTregs具有与nTregs (?)目似的预防发病和减轻关节炎严重程度的作用。
3. iTregs而非nTregs能保持对已发病关节炎小鼠的保护作用。
4. iTregs对已发病的关节炎小鼠的治疗作用的可能机制如下：
1).在体外促炎症因子IL-6存在的情况下,iTregs仍可保持其抑制力,而nTregs则丧失了大部分的抑制力。
2).在体外,iTregs而非nTregs可以抑制Th17细胞的分化。
3).在免疫缺陷和正常体内环境下,nTregs和iTregs具有相似的稳定性。
4).在炎性环境下,iTregs而非nTregs稳定存在,且能显著降低促炎症因子IL-17A的水平。
5).iTregs通过抑制破骨细胞的生成实现对关节炎小鼠骨质破坏的抑制作用,该作用为细胞接触和NF-kB信号途径所依赖的,并且表现为iTregs剂量相关性。
5.本研究为将iTregs用于类风湿性关节炎的细胞治疗提供了理论依据。
Rheumatoid Arthritis (RA) is one of autoimmune diseases with chronic, progressive and aggressive arthritis, which end is bone destruction, joint disability and high deformity. Its therapy is mainly focus on immune suppressive agents that have severe side effects.
It is reported that diminished frequency and/or dysfunction of CD4+CD25+Foxp3+ regulatory T cells (natural regulatory T cells, nTregs) may contribute to the pathogenesis of rheumatoid arthritis (RA). However, adaptive transfer of nTregs to the mouse model of RA, collagen induced arthritis (CIA) failed to protect established arthritis. Recently, our group found that induced CD4+CD25+Foxp3+regulatory T cells (iTregs) have the same phenotypes and suppressive function as nTregs, but show more stability and maintain suppressive function on T effector cells in the presence of IL-6 (Interleukin-6) in vitro. In the first part of our research, we transfer both Tregs to arthritic mice and investigate their preventive and therapeutic effects on the development of disease.
Joint damage is the commonest consequence of RA patients. It's well-known that osteoclasts play a crucial role in bone remodeling in RA. Osteoclasts are multinucleated giant cells and responsible for the bone resorption. The balance between osteoclasts and osteoblasts maintains bone metabolism homeostasis. Osteoclast activation and function are thought to be the key causer of bone erosion in inflammatory arthritic patients. And the elevated number of osteoclasts precursors (OCPs) can also be found in RA's murine model CIA. Osteoclasts come from bone marrows and OCPs are able to convert into mature osteoclasts governed by the differentiation factor receptor activator of NF-kB ligand (RANKL) with the assistance of macrophage colony stimulating factor (M-CSF) that supports cell survival during differentiation.These two cytokines are crucially necessary for osteoclastogenesis. Lack of any of them or blockage of these signal pathways can impede the differentiation of osteoclasts. Therefore, osteoclasts formation has been considered as a crucial therapeutic target for RA.
Previous study has demonstrated that activated nTregs can inhibit osteoclastogenesis. Since we and other groups have identified that iTregs have the similar phenotypic and functional characteristics as nTregs, here we try to learn if iTregs also have the suppressive capacity on osteoclastogenesis and CIA based on the results of the first part.
Results
Part I. The preparation and identification of TGF-βinduced CD4+CD25+Foxp3+ regulatory T cells and their effects on CIA
1. The preparation and identification of TGF-βinduced CD4+CD25+Foxp3+ regulatory T cells
Naive CD4+cells isolated from spleens of normal DBA1/J mice were stimulated with collagen II (CII) peptide, IL-2 and TGF-βfor four days. Tcon (IL-2 stimulated only), which means effector T lymphocytes, are as control of iTregs. nTregs were isolated from thymus of the same mice and expanded with IL-2 for five days. Detect the expression of Foxp3 with Flow Cytometry, Western Blotting and PCR after culture. Meanwhile, co-culture nTregs and iTregs with T effector cells respectively and identify the suppressive function of both Tregs on the proliferation of T effector cells (CFSE labeled method or proliferation assay). The experiment is set up four groups: baseline, nTregs group, Tcon group and iTregs group.
1.1 The expression of Foxp3 in TGF-βinduced CD4+CD25+Foxp3+regulatory T cells and nTregs after induction
The expression of Foxp3 among cultured cells in nTregs and iTregs is almost the same. The percentage is always above sixty percent while that of Tcon is only ten percent. The total number of CD25+Foxp3+ in Tcon wells is obviously lower than in iTregs wells. Then we detected the expression of Foxp3 protein in CD4+CD25- cells, nTregs, Tcon and iTregs group with western blotting. The expression of Foxp3 protein in both nTregs and iTregs is much higher than CD4+CD25- and Tcon group. The similar result was found in level of Foxp3 mRNA.
1.2 The suppressive function of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells on the proliferation of T effector cells
Both Tregs had the similar suppressive function on the proliferation of effector T lymphocytes compared by baseline and Tcon group.
2. The effects of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells on CIA
CIA was induced by subcutaneous injection of 50μl of emulsion containing bovine collagen II (4mg/mL) and complete Freund's adjuvant (1:1 ratio).3×106 of nTregs and iTregs were adaptively transferred to DBA1/J mice on d0, d14 or d28 after immunization. Mice were divided into four groups including CIA model, nTregs group, Tcon group and iTregs group at random. Investigate the prevalence and clinic scores every other day after onset of arthritis. Detect the level of anti-CII specific antibodies and do HE staining of involved joints to evaluate the severity of arthritis.
2.1 The preventive effects of TGF-P induced CD4+CD25+Foxp3+regulatory T cells on CIA
Both Tregs can suppress the onset and development of CIA when adaptively transferred to mice on day0. When transferred on d14, only iTregs can decrease the prevalence of arthritis, but both can decrease the clinic scores and anti-CII specific IgG2b significantly.
2.2 The therapeutic effects of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells on CIA
When adaptively transferred to DBAl/J mice on d28 after immunization, only iTregs but not nTregs can significantly suppress the prevalence, severity of arthritis and the level of anti-CII specific IgG2a and IgG2b. HE staining of involved joints showed that iTregs can dramatically suppress inflammatory cells infiltration and bone destruction. The proliferation assay of lymphocytes from each group showed that iTregs could suppress the proliferation of lymphocytes and also the production of pro-inflammatory factors. However, nTregs lost their suppressive function.
Summary
All the results verified that iTregs owned the same phenotypes and suppressive function as nTregs. Meanwhile, iTregs have the similar preventive effects on CIA, but only iTregs maintain their therapeutic function on established arthritis while nTregs lost their function completely.
Part II. The mechanism of TGF-βinduced CD4+CD25+Foxp3+ regulatory T cells therapeutic effects on CIA
Based on the results mentioned above, iTregs are much stable and functional than nTregs in established arthritic mice and can suppress the development of bone destruction. We will study further to clarify its mechanism.
3. The stability of TGF-P induced CD4+CD25+Foxp3+regulatory T cells in vitro and in vivo
To expand nTregs and induce iTregs as mentioned in part I.
3.1 Investigate the stability of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells in vitro
Detect both Tregs's suppressive functions on effector T lymphocytes with or without IL-6 (10ng/mL). Naive CD4+cells were induced to Th17 cells with IL-6 and TGF-P, and nTregs or iTregs were added to some wells (Tregs:naive CD4+cells=1:4) to compare the production of IL-17A.
Results showed that nTregs dramatically lost their suppressive function on the proliferation of effector T lymphocytes, the differentiation of Th17 cells and the generation of IL-17A. But iTregs maintained ail these functions.
3.2 Investigate the stability of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells in vivo
Both Tregs from GFP-Foxp3 transgenic mice were adaptively transferred to immune deficient(CD3-/-)mice and detect the changes of Foxp3 expression after four and eight weeks transfer. CFSE labeled Tregs were transferred to normal DBA1/J mice and the expression of Foxp3 in both Tregs was detected after one or three weeks transfer. These CFSE labeled Tregs were also injected into established arthritic mice to investigate their fate in inflammatory milieu.
3.2.1 The stability of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells in immune deficient and normal mice
iTregs maintained the same level of Foxp3 expression as nTregs in immune deficient mice even after eight weeks transfer. The percentage is about fifty percent (GFP+cells among CD3+cells). The same results can be found in normal mice.
3.2.2 The stability of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells in established arthritic mice (inflammatory milieu)
Compared to iTregs, nTregs were prone to apoptosis, lost Foxp3 expression and convert to Th2 and Th17 cells in the draining lymph nodes in established arthritic mice. Conversely, iTregs can maintain Foxp3 expression and resistant to the conversion to other helper T lymphocytes. At the same time, we found that injection of iTregs but not nTregs altered the balance of Foxp3+/Th17 cells in arthritis mice. The number of Th17 cells in lymph nodes in CIA mice received nTregs were 10-fold greater than those seen in mice received iTregs. However, the number of CD4+Foxp3+ cells in LNs 8-fold less in mice received nTregs than in mice received iTregs. This phenomenon revealed that iTregs can induce more Foxp3+regulatory T cells in inflammatory milieu.
4. TGF-βinduced CD4+CD25+Foxp3+regulatory T cells protect CIA from bone destruction through osteoclastogenesis suppression
nTregs can suppress osteoclastogenesis and over generated osteoclasts are the key factors for bone destruction in inflammatory arthritis. Here, we will investigate if iTregs can protect arthritis through osteoclastogenesis suppression.
CD11C+ cells as Osteoclast Precursors (OCPs) were isolated from bone marrows of normal DBA1/J mice and stimulated with Receptor Activator of NF-κB Ligand and Mcrophage Colony-stimulating Factor to induce osteoclasts. CD4+CD25-cells. nTregs, Tcon and iTregs were added to some wells respectively and did Tartrate-risistant acid phosphatase (TRAP) staining to investigate their effects on osteoclasts generation.
nTregs and iTregs were adaptively transferred to CIA mice on d14 after immunization. The involved joints from each group were accepted CT scanning after observation to evaluate bone erosion and destruction.
4.1 The suppressive effects of TGF-P induced CD4+CD25+Foxp3+ regulatory T cells on osteoclastogenesis in vitro
The results showed that both nTregs and iTregs can significantly suppress osteoclasts generation compared to baseline, CD4+ CD25-cells and Tcon. The generation of osteoclasts elevated with decrease of the number of iTregs added to OCPs, but osteoclastogenesis was also suppressed compared with Tcon. This phenomenon revealed that the suppressive function of iTregs was dose dependent.
4.1 The suppressive effects of TGF-0 induced CD4+CD25+Foxp3+regulatory T cells on bone erosion and destruction in vivo
We can find from the results of CT scanning that The CIA mice showed the chronic destructive phase of polyarthritis in the feet compared to normal mice. The joint surface showed clear bone erosion, joint integration, ankylosis and loss of bone. However, joints from iTregs group only showed milder joint space narrow without bone erosion compared to normal ones with normal joint space and bone volume.
5. The mechanism of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells suppression on osteoclastogenesis
We have five groups as follows, OCPs group as positive control, iTregs+OCPs group, iTregs+ OCPs+ isotype control cIgG group, iTregs+ OCPs+ anti-TGF-P antibody group, iTregs+ OCPs+ anti-IL-10R antibody group. This experiment was set up to detect the role of TGF-βand IL-10 in iTregs suppressive function. Then transwell experiments were done to detect that if iTregs function on osteoclastogenesis is cell-contact dependent. After that, collecting cells from OCPs, Tcon+OCPs and iTregs+OCPs to extract total proteins and doing western blotting to evaluate the level of NF-kB subunits P65/P50.
5.1 TGF-βinduced CD4+CD25+Foxp3+regulatory T cells suppressed osteoclastogenesis in a cell-contact but not cytokine dependent way.
We observed that addition of anti-TGF-βand anti-IL-10 receptor did not alter the suppressive effects of iTregs on osteoclast formation compared to addition of similar istotypes of control IgG. Conversely, transwell experiments that saperate iTregs from OCPs completely abolished the suppression of CD4+ iTregs against osteoclast formation.
5.2 TGF-βinduced CD4+CD25+Foxp3+regulatory T cells blocked osteoclastogenesis via a NF-kB-P65/P50 pathway.
The express of NF-kB/P65 and NF-kB/P50 was significantly decreased in non-T cells that had been treated with iTregs compared with those cells treated with Tcon cells or no CD4+ T cells (OCPs).
Summary
Based on the results in Part II, iTregs maintain good suppressive functions as nTregs in immune deficient and normal mice. Conversely, nTregs are prone to apoptosis, convert to Th17 cells and lose their functions on T effector cells proliferation while iTregs are stable and sustain their suppressive functions in inflammatory milieu in established arthritic mice.The suppressive functions of iTregs on CIA mice are partially through their work on osteoclasts formation. Therefore, iTregs can protect CIA mice from bone erosion and destruction. iTregs suppress osteoclastogenesis via a cell-contact way but not cytokine dependent way, a NF-κB-P65/P50 signal pathway and also a dose-dependent way.
In brief, we got conclusions as follows,
1. iTregs have the same Foxp3 expression and suppressive function on the proliferation of T lymphocytes as nTregs in vitro.
2. iTregs have the same preventive effects on CIA mice as nTregs in vivo.
3. iTregs but not nTregs can maintain the therapeutic effects on established arthritic mice.
4. The possible mechanism of iTregs suppressive effects can be concluded as
follows,
4.1 iTregs can maintain suppressive function in the presence of pro-inflammatory factor IL-6 in vitro, but nTregs lost their function.
4.2 iTregs but not nTregs can suppress Th17 cells differentiation in vitro.
4.3 iTregs are as stable as nTregs in immune deficient and normal mice.
4.4 iTregs are stable and can decrease the production of IL-17A in inflammatory milieu in vivo but nTregs cannot.
4.5 iTregs protect arthritic mice from bone destruction by their suppression on osteoclasts formation. This effect is via a cell-contact and NF-kB-P65/P50 signal pathway and also a dose-dependent way. 5. This research offered theoretical supports for iTregs cellular treatment of
rheumatoid arthritis.
已知CD4+CD25+Foxp3+的天然调节性T细胞(Natural Ocurring Regulatory T Cells, nTregs)数量的减少和/或功能的降低与RA的发病有关。有学者发现,在RA小鼠模型输注nTregs可以预防关节炎的发生和减轻关节炎的严重程度。但是,在已发病的RA经典小鼠模型——胶原诱导性关节炎(collagen-induced arthritis, CIA)中,即使额外输注nTregs,对关节炎也没有起到改善作用。近来,本研究小组发现,TGF-β诱导的调节性T细胞(Induced Regulatory T cells, iTregs)不仅具有与nTregs相同的表型和抑制关节炎发病的功能,而且在体外促炎症因子白介素-6 (Interleukin 6, IL-6)的作用下能表现出更强的稳定性和对效应性T淋巴细胞的抑制力。因此,本论文第一部分的研究旨在比较nTregs和iTregs对CIA预防与治疗作用的差异。
关节破坏是RA患者最常见的症状和转归,破骨细胞在RA患者的骨质重建中发挥重要作用；破骨细胞是一种多核巨细胞,主要功能是骨质吸收,生理条件下与成骨细胞一起维持骨代谢的平衡：而破骨细胞的过度激活和功能异常被认为是炎性关节病人骨质侵蚀的重要诱因：已有报道在RA的CIA小鼠模型中观察到破骨细胞前体细胞(Osteoclast precursors, OCPs)的数量增多。破骨细胞起源于骨髓,其前体细胞在转录因子-KB受体激活因子配体(Receptor Activator of Nuclear Factor-κB, RANKL)和巨噬细胞集落刺激因子(Macrophage Colony-stimulating Factor, M-CSF)的共同作用下,进一步分化为成熟的破骨细胞；RANKL和M-CSF是破骨细胞生成过程所必须的两个细胞因子,任何一个缺失或信号途径的阻断都会影响破骨细胞的分化。因此,破骨细胞的生成已成为关节炎关节破坏及治疗的重要靶点。已有报道证实,激活的nTregs可以抑制破骨细胞的生成,而iTregs具有与nTregs相似的表型和抑制功能。因此,本论文第二部分研究是在第一部分研究的基础上,探讨iTregs是否可以通过抑制破骨细胞的生成而发挥其对CIA模型动物的骨质侵蚀及破坏的抑制能力。
实验结果：
第一部分：TGF-p诱导的CD4CD25+Foxp3+调节性T细胞制备、鉴定及对胶原诱导性关节炎的抑制、治疗作用
1. TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞的制备、鉴定
自DBA1/J小鼠的脾脏分离出幼稚的CD4+细胞。以牛CⅡ型胶原(bovine collagenⅡ, CⅡ)多肽、白介素-2(Interleukin-2,IL-2)和TGF-β诱导Tregs (iTregs),单用IL-2培养的细胞为效应性T淋巴细胞,作为Tregs的对照(Tcon)。nTregs分离自同种小鼠的胸腺,在培养液中加入IL-2进行体外扩增,流式细胞仪、Western Blotting和PCR法分别检测Foxp3的表达；同时nTregs和iTregs分别与效应性T淋巴细胞共培养(绿色荧光蛋白CFSE标记法和增生实验),比较两者对效应性T淋巴细胞增生的抑制能力,实验设立基线值(baseline)、nTregs组、Tcon组和iTregs组。
1.1 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞Foxp3的表达
流式细胞仪的结果显示,经过扩增的nTregs与诱导的iTregs, Foxp3的表达相近,多在60%以上,而Tcon Foxp3的表达仅为10%左右。计数培养孔内的CD25+Foxp3+细胞数,Tcon组的细胞数显著低于iTregs组。Western Blotting检测CD4+ CD25-细胞、nTregs、Tcon和iTregs Foxp3蛋白的表达水平,nTregs与iTregs Foxp3蛋白的表达相近并均显著高于CD4＋CD25－细胞和Tcon。PCR法检测Foxp3 mRNA的水平,nTregs与iTregs Foxp3 mRNA的水平相近并均显著高于CD4+ CD25-细胞和Tcon。
1.2 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞对效应性T淋巴细胞的
抑制能力
体外CFSE标记法与增生实验法均证实,与基线值、Tcon组相比,iTregs拥有与nTregs相似的对效应性T淋巴细胞增生的抑制功能,而Tcon则与基线值相近,无法抑制T淋巴细胞的增生。
2 TGF-β诱导的CD4CD25+Foxp3+调节性T细胞对CIA的抑制和治疗作
用采用牛Ⅱ型胶原(4mg/mL)和完全弗式佐剂(体积比1：1)乳化液50ul于小鼠尾根部皮下注射诱导关节炎小鼠模型,在免疫后的第0天、第14天和第28天分别自小鼠尾静脉输注3×106 nTregs或iTregs,小鼠随机分为4组：CIA模型组、nTregs注射组、Tcon组和iTregs组,发病后隔天观察小鼠关节炎的发病率及临床评分。检测血清抗CⅡ特异性抗体的水平以及关节组织学染色,进一步评估疾病的严重程度。
2.1 TGF-P诱导的CD4+CD25+Foxp3+调节性T细胞对CIA的预防作用
在DBAl/J小鼠免疫后的第0天分别注射nTregs和iTregs,两者都可显著抑制关节炎的发生和发展；在免疫后的第14天分别注射nTregs和iTregs, iTregs仍可降低关节炎的发生,两者均可显著降低关节炎的临床评分；并且检测血清抗CII特异性抗体水平发现,两种Tregs均可显著抑制抗CII IgG2b的水平。
2.2 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞对CIA的治疗作用
在DBAl/J小鼠免疫后的第28天(即关节炎发病后)分别注射nTregs和iTregs, iTregs而非nTregs可以显著抑制关节炎的发生和严重程度,以及血清抗CII IgG2a和IgG2b的水平；病变关节组织学染色显示,iTregs可以显著抑制关节炎性细胞的浸润及骨质破坏的发生；分离自关节炎小鼠体内的淋巴细胞体外增生实验显示,iTregs尚可抑制淋巴细胞的增生程度和促炎症因子的水平；而nTregs则完全丧失了对关节炎的保护作用。
小结：
以上实验结果表明,iTregs拥有与nTregs相似的表型和对T淋巴细胞增生的抑制能力；同时,对CIA具有与nTregs相似的预防作用；但在已发病的关节炎小鼠,iTregs而非nTregs具有明确的治疗作用且抑制了关节破坏的发生,nTregs则完全丧失了对关节炎的保护作用。
第二部分：TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞预防和治疗CIA的机制研究
基于第一部分的研究结果,iTregs较nTregs在已发病的关节炎小鼠具有稳定的治疗作用,并可抑制骨质破坏的发生,我们在本部分将进一步探究其作用机制。
3 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞在体内、外的稳定性
按照1中的描述分别制备nTregs与iTregs,进行如下实验：
3.1 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞在体外的稳定性
采用与1.2相同的CFSE标记法与增生实验鉴定两者对效应性T淋巴细胞的抑制作用,同时设立IL-6添加组(IL-6+)与非添加组(IL-6-),检测在促炎症因子IL-6存在的情况下,两种Tregs抑制力的变化。建立Th17细胞培养体系即IL-6+TGF-P诱导幼稚的CD4+细胞向Th17细胞转化,在培养液中同样分别加入nTregs、iTregs (Tregs:幼稚CD4+细胞=1：4),比较两组IL-17A生成的水平。
结果显示,在IL-6作用下,nTregs而非iTregs显著地丧失了其对效应性T淋巴细胞的抑制作用,并且无法抑制Th17细胞的分化及IL-17A的生成；而iTregs则始终保持着良好的抑制能力。
3.2 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞在体内的稳定性
来自GFP-Foxp3转基因小鼠的nTregs和iTregs分别注射入免疫缺陷(CD3-/-)的小鼠体内,分别在注射后4周和8周观察Foxp3水平的变化；绿色荧光蛋白CFSE标记来自正常DBA1/J小鼠的两种Tregs并注射到同种小鼠体内,分别在注射后的1周和3周,检测两种Tregs Foxp3水平的变化；另将标记了CFSE的两种Tregs分别注射入已发病的关节炎小鼠(炎性小鼠)体内,观察两种Tregs在炎性环境下的稳定性和转归。
3.2.1 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞在免疫缺陷和正常小鼠体内环境下的稳定性
在注射后4周和8周,nTregs和iTregs同样可以维持Foxp3的表达,Foxp3表达占CD3+细胞的百分比为50%左右。在正常体内环境下,nTregs和iTregs同样可以维持Foxp3的表达在50%左右(Foxp3占CFSE+细胞的百分比)。
3.2.2 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞在关节炎小鼠炎性环境下的稳定性
实验结果显示,相对于iTregs, nTregs在细胞注射后1周的关节炎小鼠的引流淋巴结内,更易于凋亡、Foxp3的表达丢失并向Th2和Th17细胞转化；相反,iTregs在相似的炎性环境下能够维持Foxp3的表达且不会向辅助性T淋巴细胞转化。在nTregs注射组的关节炎小鼠淋巴结细胞中,Th17细胞的数量是iTregs组的10倍,而CFSE+Foxp3+细胞的数量为iTregs组的八分之一,提示了iTregs能够在炎性环境下诱导出更多的Foxp3+的调节性T细胞的产生。
4 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞通过抑制破骨细胞的生成发挥对CIA的保护作用
nTregs可以抑制破骨细胞的生成,而破骨细胞的生成过多是造成关节炎骨质破坏的主要因素；由此,我们将探究iTregs是否也通过抑制破骨细胞的生成而发挥对关节炎的保护作用。
分离自正常DBA1/J小鼠骨髓的CDllc+细胞即破骨细胞前前体细胞(Osteoclast Precursors, OCPs),以RANKL和M-CSF共同诱导破骨细胞的生成；同时在培养液中分别加入CD4+CD25-细胞、nTregs, Tcon和iTregs,抗酒石酸磷酸酶(Tartrate-risistant acid phosphatase,TRAP)染色,观察各种细胞对破骨细胞生成的影响。
nTregs、iTregs分别在免疫后14天注射入关节炎小鼠体内,观察期(56天)后,病变关节行CT扫描,观察关节骨质侵蚀、破坏的发生与不同。
4.1 TGF-p诱导的CD4+CD25+Foxp3+调节性T细胞在体外对破骨细胞生成的抑制作用实验结果显示,与基线值(baseline)、CD4+CD25-细胞组和Tcon组相比,nTregs和iTregs均可显著抑制破骨细胞的生成；减少加入的iTregs比例,破骨细胞的生成有所增加,但与Tcon相比,破骨细胞的生成仍受到显著抑制。提示iTregs对破骨细胞生成的抑制为剂量相关性的。
4.2 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞在体内对骨质侵蚀和破坏的抑制作用
CT扫描结果显示,CIA模型组小鼠的关节呈慢性多关节炎的表现,关节表面骨质侵蚀明显,关节腔融合,关节强直,骨量减少；而iTregs仅表现为一侧关节的轻度关节腔狭窄,无骨质侵蚀的发生,关节腔间隙和骨量与正常的小鼠关节无异。
5 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞抑制破骨细胞生成的机制研究
实验设立OCPs阳性对照组,和iTregs组,在iTregs与OCPs共培养组中分别加入同型对照(cIgG)组、抗TGF-β单抗组和抗IL-10R抗体组,观察iTregs是否通过TGF-β和IL-10信号途径发挥对破骨细胞生成的抑制作用；同时设立双层细胞培养板实验,观察iTregs对破骨细胞的抑制是否为细胞接触所依赖的；另外设立OCPs组、Tcon组和iTregs组,收集细胞,提取总蛋白,Western Blotting检测各组NF-κB亚单位P65和P50的表达水平,明确iTregs是否通过NF-κB途径发挥其抑制作用。
5.1 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞对破骨细胞生成的抑制为细胞接触而非细胞因子接触所依赖的
实验结果显示,在加入了抗TGF-β和IL-10R中和抗体后,并未影响iTregs对破骨细胞生成的影响；但双层细胞板实验显示,iTregs一旦与OCPs分离培养,即无法抑制其分化成破骨细胞,提示,iTregs的抑制作用为细胞接触所依赖的。
5.2 TGF-β诱导的CD4+CD25+Foxp3+调节性T细胞通过NF-κB信号途径发挥对破骨细胞生成的抑制
实验结果显示,iTregs与OCPs共培养组,NF-κB亚单位P65和P50的表达水平显著低于Tcon组及OCPs阳性对照组。
小结
结合上述实验结果,iTregs在免疫缺陷和正常小鼠体内环境下,都具有很好的稳定性,与nTregs相似；但在已发病的关节炎小鼠的炎性体内环境下,nTregs易于凋亡并向Th17细胞转化,同时还丧失了其对T淋巴细胞的抑制力；与之相反的是,iTregs能稳定存在,并维持了其对T淋巴细胞的抑制功能。此外,iTregs对CIA小鼠关节的保护作用,一定程度上得益于其对破骨细胞生成的抑制作用,从而减少了破骨细胞引起的骨质侵蚀和破坏；iTregs对破骨细胞的抑制为细胞接触和NF-κB信号途径所依赖的,并且表现为iTregs剂量相关性的。
综合以上研究,所得结论如下：
1.在体外,iTregs具有与nTregs相似的Foxp3的表达和对效应性T淋巴细胞的抑制能力。
2.对CIA小鼠,iTregs具有与nTregs (?)目似的预防发病和减轻关节炎严重程度的作用。
3. iTregs而非nTregs能保持对已发病关节炎小鼠的保护作用。
4. iTregs对已发病的关节炎小鼠的治疗作用的可能机制如下：
1).在体外促炎症因子IL-6存在的情况下,iTregs仍可保持其抑制力,而nTregs则丧失了大部分的抑制力。
2).在体外,iTregs而非nTregs可以抑制Th17细胞的分化。
3).在免疫缺陷和正常体内环境下,nTregs和iTregs具有相似的稳定性。
4).在炎性环境下,iTregs而非nTregs稳定存在,且能显著降低促炎症因子IL-17A的水平。
5).iTregs通过抑制破骨细胞的生成实现对关节炎小鼠骨质破坏的抑制作用,该作用为细胞接触和NF-kB信号途径所依赖的,并且表现为iTregs剂量相关性。
5.本研究为将iTregs用于类风湿性关节炎的细胞治疗提供了理论依据。
Rheumatoid Arthritis (RA) is one of autoimmune diseases with chronic, progressive and aggressive arthritis, which end is bone destruction, joint disability and high deformity. Its therapy is mainly focus on immune suppressive agents that have severe side effects.
It is reported that diminished frequency and/or dysfunction of CD4+CD25+Foxp3+ regulatory T cells (natural regulatory T cells, nTregs) may contribute to the pathogenesis of rheumatoid arthritis (RA). However, adaptive transfer of nTregs to the mouse model of RA, collagen induced arthritis (CIA) failed to protect established arthritis. Recently, our group found that induced CD4+CD25+Foxp3+regulatory T cells (iTregs) have the same phenotypes and suppressive function as nTregs, but show more stability and maintain suppressive function on T effector cells in the presence of IL-6 (Interleukin-6) in vitro. In the first part of our research, we transfer both Tregs to arthritic mice and investigate their preventive and therapeutic effects on the development of disease.
Joint damage is the commonest consequence of RA patients. It's well-known that osteoclasts play a crucial role in bone remodeling in RA. Osteoclasts are multinucleated giant cells and responsible for the bone resorption. The balance between osteoclasts and osteoblasts maintains bone metabolism homeostasis. Osteoclast activation and function are thought to be the key causer of bone erosion in inflammatory arthritic patients. And the elevated number of osteoclasts precursors (OCPs) can also be found in RA's murine model CIA. Osteoclasts come from bone marrows and OCPs are able to convert into mature osteoclasts governed by the differentiation factor receptor activator of NF-kB ligand (RANKL) with the assistance of macrophage colony stimulating factor (M-CSF) that supports cell survival during differentiation.These two cytokines are crucially necessary for osteoclastogenesis. Lack of any of them or blockage of these signal pathways can impede the differentiation of osteoclasts. Therefore, osteoclasts formation has been considered as a crucial therapeutic target for RA.
Previous study has demonstrated that activated nTregs can inhibit osteoclastogenesis. Since we and other groups have identified that iTregs have the similar phenotypic and functional characteristics as nTregs, here we try to learn if iTregs also have the suppressive capacity on osteoclastogenesis and CIA based on the results of the first part.
Results
Part I. The preparation and identification of TGF-βinduced CD4+CD25+Foxp3+ regulatory T cells and their effects on CIA
1. The preparation and identification of TGF-βinduced CD4+CD25+Foxp3+ regulatory T cells
Naive CD4+cells isolated from spleens of normal DBA1/J mice were stimulated with collagen II (CII) peptide, IL-2 and TGF-βfor four days. Tcon (IL-2 stimulated only), which means effector T lymphocytes, are as control of iTregs. nTregs were isolated from thymus of the same mice and expanded with IL-2 for five days. Detect the expression of Foxp3 with Flow Cytometry, Western Blotting and PCR after culture. Meanwhile, co-culture nTregs and iTregs with T effector cells respectively and identify the suppressive function of both Tregs on the proliferation of T effector cells (CFSE labeled method or proliferation assay). The experiment is set up four groups: baseline, nTregs group, Tcon group and iTregs group.
1.1 The expression of Foxp3 in TGF-βinduced CD4+CD25+Foxp3+regulatory T cells and nTregs after induction
The expression of Foxp3 among cultured cells in nTregs and iTregs is almost the same. The percentage is always above sixty percent while that of Tcon is only ten percent. The total number of CD25+Foxp3+ in Tcon wells is obviously lower than in iTregs wells. Then we detected the expression of Foxp3 protein in CD4+CD25- cells, nTregs, Tcon and iTregs group with western blotting. The expression of Foxp3 protein in both nTregs and iTregs is much higher than CD4+CD25- and Tcon group. The similar result was found in level of Foxp3 mRNA.
1.2 The suppressive function of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells on the proliferation of T effector cells
Both Tregs had the similar suppressive function on the proliferation of effector T lymphocytes compared by baseline and Tcon group.
2. The effects of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells on CIA
CIA was induced by subcutaneous injection of 50μl of emulsion containing bovine collagen II (4mg/mL) and complete Freund's adjuvant (1:1 ratio).3×106 of nTregs and iTregs were adaptively transferred to DBA1/J mice on d0, d14 or d28 after immunization. Mice were divided into four groups including CIA model, nTregs group, Tcon group and iTregs group at random. Investigate the prevalence and clinic scores every other day after onset of arthritis. Detect the level of anti-CII specific antibodies and do HE staining of involved joints to evaluate the severity of arthritis.
2.1 The preventive effects of TGF-P induced CD4+CD25+Foxp3+regulatory T cells on CIA
Both Tregs can suppress the onset and development of CIA when adaptively transferred to mice on day0. When transferred on d14, only iTregs can decrease the prevalence of arthritis, but both can decrease the clinic scores and anti-CII specific IgG2b significantly.
2.2 The therapeutic effects of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells on CIA
When adaptively transferred to DBAl/J mice on d28 after immunization, only iTregs but not nTregs can significantly suppress the prevalence, severity of arthritis and the level of anti-CII specific IgG2a and IgG2b. HE staining of involved joints showed that iTregs can dramatically suppress inflammatory cells infiltration and bone destruction. The proliferation assay of lymphocytes from each group showed that iTregs could suppress the proliferation of lymphocytes and also the production of pro-inflammatory factors. However, nTregs lost their suppressive function.
Summary
All the results verified that iTregs owned the same phenotypes and suppressive function as nTregs. Meanwhile, iTregs have the similar preventive effects on CIA, but only iTregs maintain their therapeutic function on established arthritis while nTregs lost their function completely.
Part II. The mechanism of TGF-βinduced CD4+CD25+Foxp3+ regulatory T cells therapeutic effects on CIA
Based on the results mentioned above, iTregs are much stable and functional than nTregs in established arthritic mice and can suppress the development of bone destruction. We will study further to clarify its mechanism.
3. The stability of TGF-P induced CD4+CD25+Foxp3+regulatory T cells in vitro and in vivo
To expand nTregs and induce iTregs as mentioned in part I.
3.1 Investigate the stability of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells in vitro
Detect both Tregs's suppressive functions on effector T lymphocytes with or without IL-6 (10ng/mL). Naive CD4+cells were induced to Th17 cells with IL-6 and TGF-P, and nTregs or iTregs were added to some wells (Tregs:naive CD4+cells=1:4) to compare the production of IL-17A.
Results showed that nTregs dramatically lost their suppressive function on the proliferation of effector T lymphocytes, the differentiation of Th17 cells and the generation of IL-17A. But iTregs maintained ail these functions.
3.2 Investigate the stability of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells in vivo
Both Tregs from GFP-Foxp3 transgenic mice were adaptively transferred to immune deficient(CD3-/-)mice and detect the changes of Foxp3 expression after four and eight weeks transfer. CFSE labeled Tregs were transferred to normal DBA1/J mice and the expression of Foxp3 in both Tregs was detected after one or three weeks transfer. These CFSE labeled Tregs were also injected into established arthritic mice to investigate their fate in inflammatory milieu.
3.2.1 The stability of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells in immune deficient and normal mice
iTregs maintained the same level of Foxp3 expression as nTregs in immune deficient mice even after eight weeks transfer. The percentage is about fifty percent (GFP+cells among CD3+cells). The same results can be found in normal mice.
3.2.2 The stability of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells in established arthritic mice (inflammatory milieu)
Compared to iTregs, nTregs were prone to apoptosis, lost Foxp3 expression and convert to Th2 and Th17 cells in the draining lymph nodes in established arthritic mice. Conversely, iTregs can maintain Foxp3 expression and resistant to the conversion to other helper T lymphocytes. At the same time, we found that injection of iTregs but not nTregs altered the balance of Foxp3+/Th17 cells in arthritis mice. The number of Th17 cells in lymph nodes in CIA mice received nTregs were 10-fold greater than those seen in mice received iTregs. However, the number of CD4+Foxp3+ cells in LNs 8-fold less in mice received nTregs than in mice received iTregs. This phenomenon revealed that iTregs can induce more Foxp3+regulatory T cells in inflammatory milieu.
4. TGF-βinduced CD4+CD25+Foxp3+regulatory T cells protect CIA from bone destruction through osteoclastogenesis suppression
nTregs can suppress osteoclastogenesis and over generated osteoclasts are the key factors for bone destruction in inflammatory arthritis. Here, we will investigate if iTregs can protect arthritis through osteoclastogenesis suppression.
CD11C+ cells as Osteoclast Precursors (OCPs) were isolated from bone marrows of normal DBA1/J mice and stimulated with Receptor Activator of NF-κB Ligand and Mcrophage Colony-stimulating Factor to induce osteoclasts. CD4+CD25-cells. nTregs, Tcon and iTregs were added to some wells respectively and did Tartrate-risistant acid phosphatase (TRAP) staining to investigate their effects on osteoclasts generation.
nTregs and iTregs were adaptively transferred to CIA mice on d14 after immunization. The involved joints from each group were accepted CT scanning after observation to evaluate bone erosion and destruction.
4.1 The suppressive effects of TGF-P induced CD4+CD25+Foxp3+ regulatory T cells on osteoclastogenesis in vitro
The results showed that both nTregs and iTregs can significantly suppress osteoclasts generation compared to baseline, CD4+ CD25-cells and Tcon. The generation of osteoclasts elevated with decrease of the number of iTregs added to OCPs, but osteoclastogenesis was also suppressed compared with Tcon. This phenomenon revealed that the suppressive function of iTregs was dose dependent.
4.1 The suppressive effects of TGF-0 induced CD4+CD25+Foxp3+regulatory T cells on bone erosion and destruction in vivo
We can find from the results of CT scanning that The CIA mice showed the chronic destructive phase of polyarthritis in the feet compared to normal mice. The joint surface showed clear bone erosion, joint integration, ankylosis and loss of bone. However, joints from iTregs group only showed milder joint space narrow without bone erosion compared to normal ones with normal joint space and bone volume.
5. The mechanism of TGF-βinduced CD4+CD25+Foxp3+regulatory T cells suppression on osteoclastogenesis
We have five groups as follows, OCPs group as positive control, iTregs+OCPs group, iTregs+ OCPs+ isotype control cIgG group, iTregs+ OCPs+ anti-TGF-P antibody group, iTregs+ OCPs+ anti-IL-10R antibody group. This experiment was set up to detect the role of TGF-βand IL-10 in iTregs suppressive function. Then transwell experiments were done to detect that if iTregs function on osteoclastogenesis is cell-contact dependent. After that, collecting cells from OCPs, Tcon+OCPs and iTregs+OCPs to extract total proteins and doing western blotting to evaluate the level of NF-kB subunits P65/P50.
5.1 TGF-βinduced CD4+CD25+Foxp3+regulatory T cells suppressed osteoclastogenesis in a cell-contact but not cytokine dependent way.
We observed that addition of anti-TGF-βand anti-IL-10 receptor did not alter the suppressive effects of iTregs on osteoclast formation compared to addition of similar istotypes of control IgG. Conversely, transwell experiments that saperate iTregs from OCPs completely abolished the suppression of CD4+ iTregs against osteoclast formation.
5.2 TGF-βinduced CD4+CD25+Foxp3+regulatory T cells blocked osteoclastogenesis via a NF-kB-P65/P50 pathway.
The express of NF-kB/P65 and NF-kB/P50 was significantly decreased in non-T cells that had been treated with iTregs compared with those cells treated with Tcon cells or no CD4+ T cells (OCPs).
Summary
Based on the results in Part II, iTregs maintain good suppressive functions as nTregs in immune deficient and normal mice. Conversely, nTregs are prone to apoptosis, convert to Th17 cells and lose their functions on T effector cells proliferation while iTregs are stable and sustain their suppressive functions in inflammatory milieu in established arthritic mice.The suppressive functions of iTregs on CIA mice are partially through their work on osteoclasts formation. Therefore, iTregs can protect CIA mice from bone erosion and destruction. iTregs suppress osteoclastogenesis via a cell-contact way but not cytokine dependent way, a NF-κB-P65/P50 signal pathway and also a dose-dependent way.
In brief, we got conclusions as follows,
1. iTregs have the same Foxp3 expression and suppressive function on the proliferation of T lymphocytes as nTregs in vitro.
2. iTregs have the same preventive effects on CIA mice as nTregs in vivo.
3. iTregs but not nTregs can maintain the therapeutic effects on established arthritic mice.
4. The possible mechanism of iTregs suppressive effects can be concluded as
follows,
4.1 iTregs can maintain suppressive function in the presence of pro-inflammatory factor IL-6 in vitro, but nTregs lost their function.
4.2 iTregs but not nTregs can suppress Th17 cells differentiation in vitro.
4.3 iTregs are as stable as nTregs in immune deficient and normal mice.
4.4 iTregs are stable and can decrease the production of IL-17A in inflammatory milieu in vivo but nTregs cannot.
4.5 iTregs protect arthritic mice from bone destruction by their suppression on osteoclasts formation. This effect is via a cell-contact and NF-kB-P65/P50 signal pathway and also a dose-dependent way. 5. This research offered theoretical supports for iTregs cellular treatment of
rheumatoid arthritis.
引文
1. Van Amelsfort, J. M., Jacobs, K. M., Bijlsma, J. W., Lafeber, F. P.& Taams, L. S. CD4+CD25+ regulatory T cells in rheumatoid arthritis:differences in the presence, phenotype, and function between peripheral blood and synovial fluid. Arthritis Rheum.2004.50,2775-2785.
2. Ehrenstein, M. R. et al. Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNF-α therapy. J. Exp. Med.2004.200, 277-285.
3. Mottonen, M. et al. CD4+CD25+ T cells with the phenotypic and functional characteristics of regulatory T cells are enriched in the synovial fluid of patients with rheumatoid arthritis. Clin. Exp. Immunol.2005.140,360-367.
4. Flores-Borja, F., Jury, E. C., Mauri, C.& Ehrenstein, M. R. Defects in CTLA-4 are associated with abnormal regulatory T cell function in rheumatoid arthritis. Proc. Natl Acad. Sci.2008.105,19396-19401.
5. Cao, D. et al. Isolation and functional haracterization of regulatory CD25bright CD4+ cells from the target organ of patients with rhumatoid arthritis. Eur. J. Immunol.2003.33,5-223.
6. Lawson, C. A. et al. Early rheumatoid arthritis is ssociated with a deficit in the CD4+CD25high regulatory T cell population in peripheral blood. Rheumatology (Oxford).2006.45,1210-1217.
7. Valencia, X. et al. TNF down modulates the function of human CD4+CD25hi T-regulatory cells. Blood.2006.108,253-261.
8. Mary E. Morgan, Roger P. M. Sutmuller, Hendrik J. Witteveen, Leonie M. van Duivenvoorde,Eric Zanelli, Cornelis J. M. Melief, et al. CD25+ Cell Depletion Hastens the Onset of Severe Disease in Collagen-Induced Arthritis. Arthritis Rheum.2003.48,1452-1460.
9. H Kelchtermans,L Geboes,T Mitera,D Huskens,G Leclercq,P Matthys. Activated CD4+CD25+ regulatory T cells inhibit osteoclastogenesis and collagen-induced arthritis. Ann Rheum Dis.2009.68;744-750
10. Morgan ME, Flierman R, van Duivenvoorde LM, Witteveen HJ, van Ewijk W, van Laar JM, et al. Effective treatment of collagen-induced arthritis by adoptive transfer of CD25+ regulatory T cells. Arthritis Rheum.2005.52:2212-2.
11. Miyara, M. et al. Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FOXP3 transcription factor. Immunity.2009.30, 899-911.
12. Michel, L. et al. Patients with relapsing-remitting multiple sclerosis have normal TREG function when cells expressing IL-7 receptor a-chain are excluded from the analysis. J. Clin. Invest.2008.118,3411-3419.
13. Liu, W. et al. CD127 expression inversely correlates with FOXP3 and suppressive function of human CD4+ TREG cells. J. Exp. Med.2006.203, 1701-1711.
14. Jonathan H. Esensten, David Wofsy and Jeffrey A. Bluestone.Regulatory T cells as therapeutic targets in rheumatoid arthritis. Nat.Rev. Rheumatol.2009.5,560-565.183.
15. Alwi Shatry, Jackeline Chirinos, Michael A. Gorin, Monica Jones, Robert B. Levy. Targeting Treg Cells In Situ:Emerging Expansion Strategies for (CD4+CD25+) Regulatory T Cells. Biol Blood Marrow Transplant 2009.15:1239-1243.
16. Song Guo Zheng, Juhua Wang, David A. Horwitz. Cutting Edge:Foxp3+CD4+ CD25+Regulatory T Cells Induced by IL-2 and TGF-β Are Resistant to Th17 Conversion by IL-6. The Journal of Immunology.2008.180:7112-7116.
17. Xu L, Kitani A, Fuss I, Strober W. Cutting edge:regulatory T cells induce CD4+ CD25-Foxp3- T cells or are self-induced to become Th17 cells in the absence of exogenous TGF-beta. J Immunol.2007.178:6725-6729.
18. dhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B.TGF-beta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity.2006.24(2):179-89.
19. gan PR, Harrington LE, O'Quinn DB, Helms WS, Bullard DC, Elson CO, Hatton RD, Wahl SM, Schoeb TR, Weaver CT. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature.2006.441(7090):231-4.
20. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature.2006.441(7090):235-8.
21. Michel ML, Keller AC, Paget C et al. Identification of an IL-17-producing NK1.1neg iNKT cell population involved in airway neutrophilia. J Exp Med. 2007.204:995.
22. Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B.TGF-beta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity.2006. Feb;24(2):179-89
23. Mangan PR, Harrington LE, O'Quinn DB, Helms WS, Bullard DC, Elson CO, Hatton RD, Wahl SM, Schoeb TR, Weaver CT. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature.2006.441(7090):231-4.
24. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature.2006.441(7090):235-8.
25. Heremans H, Dillen C, Groenen M, Martens E, Billiau A. Chronic relapsing experimental autoimmune encephalomyelitis (CREAE) in mice:enhancement by monoclonal antibodies against interferon-gamma. Eur J Immunol.1996.26(10): 2393-8.
26. Ferber IA, Brocke S, Taylor-Edwards C, Ridgway W, Dinisco C, Steinman L, Dalton D, Fathman CG. Mice with a disrupted IFN-gamma gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J Immunol.1996.156(1):5-7.
27. Lubberts E, Koenders MI, Oppers-Walgreen B, van den Bersselaar L, Coenen-de Roo CJ, Joosten LA, van den Berg WB. Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of collagen-induced arthritis reduces joint inflammation, cartilage destruction, and bone erosion. Arthritis Rheum.2004.50(2):650-9.
28. Zhang Z, Zheng M, Bindas J, Schwarzenberger P, Kolls JK. Critical role of IL-17 receptor signaling in acute TNBS-induced colitis. Inflamm Bowel Dis. 2006.12(5):382-8.
29. Koenders MI, Kolls JK, Oppers-Walgreen B, van den Bersselaar L, Joosten LA,Schurr JR, Schwarzenberger P, van den Berg WB, Lubberts E. Interleukin-17 receptor deficiency results in impaired synovial expression of interleukin-1 and matrix metalloproteinases 3,9, and 13 and prevents cartilage destruction during chronic reactivated streptococcal cell wall-induced arthritis. Arthritis Rheum. 2005.52:3239-3247.
30. Lubberts E, Koenders MI, Oppers-Walgreen B, van den Bersselaar L, Coenen-de Roo CJ, Joosten LA, van den Berg WB. Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of collagen-induced arthritis reduces joint inflammation, cartilage destruction, and bone erosion. Arthritis Rheum.2004.50(2):650-9.
31. Zheng SG, Wang JH, Gray JD, Soucier H, Horwitz DA. Natural and induced CD4+CD25+ cells educate CD4+CD25- cells to develop suppressive activity:the role of IL-2, TGF-beta, and IL-10.J Immunol 2004.172:5213-21.
32. Fantini MC, Becker C, Monteleone G, Pallone F, Galle PR, Neurath MF. Cutting edge:TGFbeta induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and downregulation of Smad7. J Immunol 2004.172:5149-53.
33. Horwitz DA, Zheng SG, Gray JD. Natural and TGF-binduced Foxp3+CD4+ CD25+regulatory T cells are not mirror images of each other. Trends Immunol 2008.29:429-435.
34. Zheng SG, Wang J, Wang P, Gray JD, Horwitz DA. IL-2 is essential for TGF-(3 to convert naive CD4+CD25= cells to CD25+Foxp3+ regulatory T cells and for expansion of these cells. J Immunol 2007.178:2018-2027.
35. Read S, Malmstro"m V, Powrie F. Cytotoxic T lymphocyteassociated antigen 4 plays an essential role in the function of CD25+CD4+regulatory cells that control intestinal inflammation. J Exp Med 2000.192:295-302
36. Kohm AP, Carpentier PA, Anger HA, Miller SD. Cutting edge:CD4+CD25+ regulatory T cells suppress antigenspecific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis. J Immunol 2002.169:4712-4716.
37. Salomon B, Lenschow DJ, Rhee L, Ashourian N, Singh B, Sharpe A, Bluestone JA. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 2000.12: 431-440.
38. You S, Leforban B, Garcia C, Bach JF, Bluestone JA, Chatenoud L. Adaptive TGF-β-dependent regulatory T cells control autoimmune diabetes and are a privileged target of anti-CD3 antibody treatment. Proc Natl Acad Sci USA 2007. 104:6335-6340.
39. Zheng SG, Gray JD, Ohtsuka K, Yamagiwa S, Horwitz DA. Generation ex vivo of TGF-beta-producing regulatory T cells from CD4+CD25- precursors. J Immunol 2002.15;169(8):4183-9.
40. Thornton AM, Shevach EM. CD4+CD25+immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J Exp Med 1998.188:287-296.
41. Nakamura K, Kitani A, Strober W. Cell contact-dependent immunosuppression by CD4+CD25+regulatory T cells is mediated by cell surface-bound transforming growth factor β.J Exp Med 2001.194:629-644.
42. Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+regulatory T cells by TGF-β induction of transcription factor Foxp3. J Exp Med 2003.198: 1875-1886.
43. Piccirillo CA, Letterio JJ, Thornton AM, McHugh RS, Mamura M, Mizuhara H, Shevach EM. CD4+CD25+regulatory T cells can mediate suppressor function in the absence of transforming growth factor b1 production and responsiveness. J Exp Med 2002.196:237-246.
44. Fontenot JD, Rasmussen JP, Gavin MA, Rudensky AY. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat Immunol 2005.6:1142-1151.
45. Floess S, Freyer J, Siewert C, Baron U, Olek S, Polansky J, Schlawe K, Chang HD, Bopp T, Schmitt E, Klein-Hessling S, Serfling E, Hamann A, Huehn J. Epigenetic control of the Foxp3 locus in regulatory T cells. PLoS Biol 2007.5: e38.
46. Lai G, Zhang N, van der Touw W, Ding Y, Ju W, Bottinger EP, Reid SP, Levy DE, Bromberg JS. Epigenetic regulation of Foxp3 expression in regulatory T cells by DNA methylation. J Immunol 2009.182:259-273.
47. Zheng SG, Wang JH, Koss MN, Quismorio F Jr, Gray JD, Horwitz DA. CD4+ and CD8+ regulatory T cells generated ex vivo with IL-2 and TGF-beta suppress a stimulatory graft versus-host disease with a lupus-like syndrome. J Immunol. 2004.172:1531-9.
48. Weber SE, Harbertson J, Godebu E, Mros GA, Padrick RC, Carson BD, Ziegler SF, Bradley LM.Adaptive islet-specific regulatory CD4 T cells control autoimmune diabetes and mediate the disappearance of pathogenic Thl cells in vivo. J Immunol 2006.176:4730-9.
49. Selvaraj RK, Geiger TL. Mitigation of experimental allergic encephalomyelitis by TGF-beta induced Foxp3+ regulatory T lymphocytes through the induction of anergy and infectious tolerance. J Immunol.2008.180:2830-8.
50. Wan YY, Flavell RA. Regulatory T-cell functions are subverted and converted owing to attenuated Foxp3 expression. Nature 2007.445:766-770.
51. Lu L, Wang J, Zhang F, Chai Y, Brad DD, Wang XH, Horwitz DA, Shi W, Zheng SG. Role of Smad and non-Smad signals in the development of Th 17 and regulatory T cells. J Immunol 2010.184(8):4295-4306.
52. Tsuji M, Komatsu N, Kawamoto S, Suzuki K, Kanagawa O, Honjo T, Hori S, Fagarasan S. Preferential generation of follicular B helper T cells from Foxp3+T cells in gut Peyer's patches. Science 2009.323:1488-1492.
53. Zhou X, Kong N, Wang J, Fan H, Zou H, Horwitz D, Brand D, Liu Z, Zheng SG. Cutting edge:all-trans retinoic acid sustains the stability and function of natural regulatory T cells in an inflammatory milieu.J Immunol.2010.185(5):2675-9.
54. Fossiez F, Djossou O, Chomarat P, Flores-Romo L, Ait-Yahia S, Maat C, et al. T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines. J Exp Med.1996.183(6):2593-603.
55. Chen W, Jin W, Lei K-j, Hardegen, N, Li L, Marinos N et al. Conversion of peripheral CD4+CD25- regulatory T cells by TGF-Pinduction of transcription factor Foxp3. J Exp Med 2003.198:1875-1886.
56. Fantini MC,Becker C, Monteleone G, Pallone F, Galle PR, Neurath MF. Cutting edge:TGF-beta induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and down-regulation of Smad7. J Immunol 2004.172:5149-5153.
57. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T,et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998.93:165-76.
58. Redlich K, Hayer S, Ricci R, David JP, Tohidast-Akrad M, Kollias G, et al. Osteoclasts are essential for TNF-aamediated joint destruction. J Clin Invest 2002.110:1419-27.
59. S Pettit AR, Ji H, von Stechow D, Muller R, Goldring SR, Choi Y, et al. TRANCE/RANKL knockout mice are protected from bone erosion in a serum transfer model of arthritis. Am J Pathol 2001.159:1689-99.
60. Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 1999.20:345-357.
61. Yoshida H, Hayashi S, Kunisada T, Ogawa M, Nishikawa S,Okamura H, et al. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 1990.345:442-4.
62. Wiktor-Jedrzejczak W, Bartocci A, Ferrante AW Jr, et al. Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse. Proc Natl Acad Sci U S A 1990.87:4828-4832.
63. Bim Nicolas Houde, Estelle Chamoux, Martine Bisson, and Sophie Roux. Transforming growth factor-beta1 (TGF-beta1) induces human osteoclast apoptosis by up-regulating Bim. J Biol Chem 2009.284:23397-23404.
64. Manolagas SC, Jilka RL. Mechanisms of disease:bone marrow, cytokines, and bone remodeling-emerging insights into the pathophysiology of osteoporosis. N Engl J Med 1995.332:305-311.
65. Nishimoto N, Kishimoto T. Interleukin 6:from bench to bedside. Nat Clin Pract Rheumatol 2006.2:619-26.
66. McInnes I, Schett G. Cytokines in the pathogenesis of rheumatoid arthritis. Nat Rev Immunol 2007.7:429-42.
67. Sugita T, Furukawa O, Ucno M, Murakami T, Takata I, Tosa T. Enhanced expression of interleukin 6 in rat and murine arthritis models. Tnt J Tmmunopharmecol 1993.15:469-76.
68. Lee SK, Lorenzo J.Cytokines regulating osteoclast formation and function. Curr Opin Rheumatol 2006.18:411-418.
69. Strand V, Kavanaugh AF. The IL-1 family and inflammatory diseases. Clin Exp Rheumatol 2004.20:S1-S13.
70. Strand V, Kavanaugh AF. The role of interleukin-1 in bone resorptionin rheumatoid arthritis. Rheumatology (Oxford) 2004.43 Suppl 3:ⅲ10-ⅲ16.
71. Trebec-Reynolds DP, Voronov I, Heersche JN, Manolson MF. IL-1 alpha and IL-1beta Have Different Effects on Formation and Activity of Large Osteoclasts. J Cell Biochem 2010.109(5):975-82.
72. Sato K, Suematsu A, Okamoto K, Yamaguchi A, Morishita Y, Kadono Y,et al. Th17 functions as an osteoclastogenic helper T cell subset that links T cell activation and bone destruction. J Exp Med 2006.203:2673-2682.
73. Adamopoulos IE, Chao CC, Geissler R, Laface D, Blumenschein W, Iwakura Y, et al. Interleukin-17A upregulates receptor activator of NF-kappaB on osteoclast precursors. Arthritis Res Ther 2010.12(1):R29.
74. Geboes L, De Klerck B, Van Balen M, Kelchtermans H, Mitera T, Boon L, et al. Freund's complete adjuvant induces arthritis in mice lacking a functional interferon-creceptor by triggering tumor necrosis factor alpha-driven osteoclastogenesis. Arthritis Rheum 2007.56:2595-607.
75. Li P, Schwarz EM, O'Keefe RJ, Ma L, Looney RJ, Ritchlin CT, et al. Systemic tumor necrosis factor alpha mediates an increase in peripheral CD1lbhigh osteoclast precursors in tumor necrosis factor alpha-transgenic mice. Arthritis Rheum 2004.50:265-76.
76. De Klerck B, Carpentier I, Lories RJ, Habraken Y, Piette J, Carmeliet G, et al. Enhanced osteoclast development in collagen-induced arthritis in interferon-γ receptor knock-out mice as related to increased splenic CD11b+ myelopoiesis. Arthritis Res Ther 2004.6:221-31.
77. Brand DD, Latham KA, Rosloniec EF. Collagen-induced arthritis. Nat Protoc 2007.2 (5):1269-75.
78. Kelchtermans H, Struyf S, De Klerck B, Mitera T, Alen M, Geboes L, et al. Protective role of IFN-c in collagen-induced arthritis conferred by inhibition of mycobacteriainduced granulocyte chemotactic protein-2 (GCP-2) production. J Leukoc Biol 2007.81:1044-53.
79. Pasare C, Medzhitov R. Toll pathway-dependent blockade of CD4+CD25+T cell-mediated suppression by dendritic cells. Science.299:1033-1036,2003
80. Valencia X, Stephens G, Goldbach-Mansky R, Wilson M, Shevach EM, Lipsky PE. TNF downmodulates the function of human CD4+CD25hi T-regulatory cells. Blood.2006.108:253-261.
81. S. Kotake, IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis, J. Clin. Invest.1999.103:1345-1352.
82. S.Y. Hwang, J.P. Kim, M.K. Park, Y. Moon, W.U. Kim and H.Y. Kim, IL-17 induce production of IL-6 and IL-18 in rheumatoid arthritis synovial fibroblasts via NF-Kappa Beta and PI-3Kinase/AK 1-dependent pathways, Arthritis Res. Ther.2004.6:120-128.
83. Zaiss MM, Axmann R, Zwerina J, Polzer K, Guckel E, Skapenko A, et al. Treg cells suppress osteoclast formation:a new link between the immune system and bone. Arthritis Rheum 2007.56(12):4104-12.
84. Kong YY, Feige U, Sarosi I, Bolon B, Tafuri A, Morony S, et al. Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 1999.402(6759):304-9.
85. Takayanagi H, Ogasawara K, Hida S, Chiba T, Murata S, Sato K, et al. T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-gamma. Nature 2000.408(6812):600-5.
86. Gao Y, Grassi F, Ryan MR, Terauchi M, Page K, Yang X, et al. IFN-y stimulates osteoclast formation and bone loss in vivo via antigen-driven T cell activation. J Clin Invest 2007.117(1):122-132.
87. Koenders MI, Joosten LA, van den Berg WB. Potential new targets in arthritis therapy:interleukin (IL)-17 and its relation to tumour necrosis factor and IL-1 in experimental arthritis. Ann Rheum Dis 2006.65 Suppl 3:iii29-33.
88. Zaiss MM, Frey B, Hess A, Zwerina J, Luther J, Nimmerjahn F, et al. Regulatory T cells protect from local and systemic bone destruction in arthritis. J Immunol 2010.184(12):7238-46.
89. Zaiss MM, Sarter K, Hess A, Engelke K, Bohm C, Nimmerjahn F, et al. Increased bone density and resistance to ovariectomy-induced bone loss in FoxP3-transgenic mice based on impaired osteoclast differentiation. Arthritis Rheum 2010.62(8):2328-38.
90. Kim YG, Lee CK, Nah SS, Mun SH, Yoo B, Moon HB. Human CD4+CD25+ regulatory T cells inhibit the differentiation of osteoclasts from peripheral blood mononuclear cells. Biochem Biophys Res Commun 2007.357(4):1046-52.
91. Shevach EM, Davidson TS, Huter EN, Dipaolo RA, Andersson J. Role of TGF-Beta in the induction of Foxp3 expression and T regulatory cell function. J Clin Immunol 2008.28(6):640-6.
92. Powrie F, Carlino J, Leach MW, Mauze S, Coffman RL. A critical role for transforming growth factor-beta but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RB(low) CD4+T cells. J Exp Med 1996. 183(6):2669-74.
93. Soysa NS, Alles N. NF-kappaB functions in osteoclasts. Biochem Biophys Res Commun 2009.378(1):1-5.
94. Weitzmann MN, Pacifici R. The role of T lymphocytes in bone metabolism. Immunol Rev 2005.208:154-68.
1. Sakaguchi S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 2004.22:531-562.
2. Shevach EM. CD4+CD25+ suppressor T cells:More questions than answers. Nat Rev Immunol 2002.2:389-400.
3. Gershon RK, Kondo K. Cell interactions in the induction of tolerance:The role of thymic lymphocytes. Immunology 1970.18:723-737.
4. Kojima A, Prehn RT. Genetic susceptibility to postthymectomy autoimmune diseases in mice. Immunogenetics 1981.14:15-27.
5. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor a-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995.155:1151-1164.
6. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003.299:1057-1061.
7. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 2003.4:330-336.
8. Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD4+ CD25+T regulatory cells. Nat Immunol 2003.4:337-342.
9. Fontenot JD, Rasmussen JP, Williams LM, Dooley JL, Farr AG, Rudensky AY. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 200.22:329-341.
10. Baecher-Allan C, Brown JA, Freeman GJ, Hafler DA. CD4+CD25high regulatory cells in human peripheral blood. J Immunol 2001.167:1245-1253.
11. Morgan ME, van Bilsen JH, Bakker AM, Heemskerk B,Schilham MW, Hartgers FC, Elferink BG, van der Zanden L,de Vries RR, Huizinga TW, Ottenhoff TH, Toes RE.Expression of FOXP3 mRNA is not confined to CD4+CD25+T regulatory cells in humans. Hum Immunol 2005.66:13-20.
12. Allan SE, Crome SQ, Crellin NK, Passerini L, Steiner TS, Bacchetta R, Roncarolo MG, Levings MK. Activation induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production. Int Immunol 2007. 19:345-354.
13. Yu JN, Ma JL, Wang JH, Tao XJ, Li XP, Zou HJ, Zheng SG. FOXP3, is still a specific marker for human regulatory T cells? Chin J Allergy Clin Immunol 2008. 2:5-10.
14. Chatila TA, Blaeser F, Ho N, Lederman HM, Voulgar opoulos C, Helms C, Bowcock AM. JM2, encoding a forkhead-related protein, is mutated in X-linked autoimmunity allergic disregulation syndrome. J Clin Invest 2000.106:R75-R81.
15. Wildin RS, Ramsdell F, Peake J, Faravelli F, Casanova JL,Buist N, Levy-Lahad E, Mazzella M, Goulet O, Perroni L,Bricarelli FD, Byrne G, McEuen M, Proll S, Appleby M,Brunkow ME. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet 200.27:18
16. Bennett CL, Christie J, Ramsdell F, Brunkow ME, Ferguson PJ, Whitesell L, Kelly TE, Saulsbury FT, Chance PF, Ochs HD. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet 2001.27:20-21.
17. Tang Q, Bluestone JA. The Foxp3+ regulatory T cell:A jack of all trades, master of regulation. Nat Immunol 2008.9:239-244.
18. Horwitz DA, Zheng SG, Gray JD, Wang JH, Ohtsuka K, Yamagiwa S. Regulatory T cells generated ex vivo as an approach for the therapy of autoimmune disease. Semin Immunol 2004.16:135-143.
19. Hahn BH, Singh RP, La Cava A, Ebling FM. Tolerogenic treatment of lupus mice with consensus peptide induces Foxp3-expressing, apoptosis-resistant, TGF-β-secreting CD8+ T cell suppressors. J Immunol 2005.175:7728-7737.
20. Ochi H, Abraham M, Ishikawa H, Frenkel D, Yang K, Basso AS, Wu H, Chen ML, Gandhi R, Miller A, Maron R, Weiner HL. Oral CD3-specific antibody suppresses autoimmune encephalomyelitis by inducing CD4+CD25- LAP+T cells. Nat Med 2006.12:627-635.
21. Kang HK, Michaels MA, Berner BR, Datta SK. Very low-dose tolerance with nucleosomal peptides controls lupus and induces potent regulatory T cell subsets. J Immunol 2005.174:3247-3255.
22. Brand DD, Myers LK, Whittington KB, Latham KA, Stuart JM, Kang AH, Rosloniec EF. Detection of early changes in autoimmune T cell phenotype and function following intravenous administration of type II collagen in a TCR-transgenic model. J Immunol 2002.168:490-498.
23. Vieira PL, Christensen JR, Minaee S, O'Neill EJ, Barrat FJ, Boonstra A, Barthlott T, Stockinger B, Wraith DC, O'Garra A. IL-10-secreting regulatory T cells do not express Foxp3 but have comparable regulatory function to naturally occurring CD4+CD25+regulatory T cells. J Immunol 2004.172:5986-5993.
24. Zheng SG, Gray JD, Ohtsuka K, Yamagiwa S, Horwitz DA. Generation ex vivo of TGF-β producing regulatory T cells from CD4(?) CD252 precursors. J Immunol 2002.169:4183-4189.
25. Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-β induction of transcription factor Foxp3. J Exp Med 2003. 198:1875-1886.
26. Zheng SG, Wang JH, Gray JD, Soucier H, Horwitz DA. Natural and induced CD4+CD25+ cells educate CD4+CD25- cells to develop suppressive activity:The role of IL-2, TGF-β, and IL-10. J Immunol 2004.172:5213-5221.
27. Fantini MC, Becker C, Monteleone G, Pallone F, Galle PR, Neurath MF. Cutting edge:TGF-β induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and downregulation of Smad7. J Immunol 2004.172: 5149-5153.
28. Chatenoud L, Bluestone JA. CD3-specific antibodies:A portal to the treatment of autoimmunity. Nat Rev Immunol 2007.7:622-632.
29. Battaglia M, Gregori S, Bacchetta R, Roncarolo MG. Trl cells:From discovery to their clinical application. Semin Immunol 2006.18:120-127.
30. Horwitz DA, Zheng SG, Gray JD. Natural and TGF-binduced Foxp3+ CD4+ CD25+ regulatory T cells are not mirror images of each other. Trends Immunol 2008.29:429-435.
31. Thornton AM, Shevach EM. CD4+ CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J Exp Med 1998.188:287-296.
32. Nakamura K, Kitani A, Strober W. Cell contact-dependent immunosuppression by CD4+CD25+regulatory T cells is mediated by cell surface-bound transforming growth factor β. J Exp Med 2001.194:629-644.
33. Zheng SG, Wang J, Horwitz DA. Cutting edge:Foxp3+CD4+CD25+regulatory T cells induced by IL-2 and TGF-β are resistant to Th17 conversion by IL-6. J Immunol 2008.180:7112-7116.
34. DiPaolo RJ, Glass DD, Bijwaard KE, Shevach EM. CD4+ CD25+ T cells prevent the development of organ-specific autoimmune disease by inhibiting the differentiation of autoreactive effector T cells. J Immunol 2005.175:7135-7142.
35. Read S, Malmstro"m V, Powrie F. Cytotoxic T lymphocyteassociated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intestinal inflammation. J Exp Med 2000.192:295-302
36. Kohm AP, Carpentier PA, Anger HA, Miller SD. Cutting edge:CD4+ CD25+ regulatory T cells suppress antigenspecific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis. J Immunol 2002,169:4712-4716.
37. Scalapino KJ, Tang Q, Bluestone JA, Bonyhadi ML, Daikh DI. Suppression of disease in New Zealand Black/New Zealand White lupus-prone mice by adoptive transfer of ex vivo expanded regulatory T cells. J Immunol 2006.177:1451-1459.
38. Zheng SG, Wang JH, Koss MN, Quismorio F, Jr, Gray JD, Horwitz DA. CD4+ and CD8+ regulatory T cells generated ex vivo with IL-2 and TGF-β suppress a stimulatory graft versus-host disease with a lupus-like syndrome. J Immunol 2004. 172:1531-1539.
39. Weber SE, Harbertson J, Godebu E, Mros GA, Padrick RC, Carson BD, Ziegler SF, Bradley LM. Adaptive islet-specific regulatory CD4 T cells control autoimmune diabetes and mediate the disappearance of pathogenic Thl cells in vivo. J Immunol 2006.176:4730-4739.
40. Selvaraj RK, Geiger TL. Mitigation of experimental allergic encephalomyelitis by TGF-β-induced Foxp3+ regulatory T lymphocytes through the induction of anergy and infectious tolerance. J Immunol 2008.180:2830-2838.
41. Fahle'n L, Read S, Gorelik L, Hurst SD, Coffman RL, Flavell RA, Powrie F. T cells that cannot respond to TGF-β escape control by CD4+ CD25+ regulatory T cells. J Exp Med 2005.201:737-746.
42. Hara M, Kingsley CI, Niimi M, Read S, Turvey SE, Bushell AR, Morris PJ, Powrie F, Wood KJ. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. J Immunol 2001.166:3789-3796.
43. Zheng SG, Meng L, Wang JH, Watanabe M, Barr ML, Cramer DV, Gray JD, Horwitz DA. Transfer of regulatory T cells generated ex vivo modifies graft rejection through induction of tolerogenic CD4+ CD25+ cells in the recipient. Int Immunol 2006.18:279-289.
44. Jonuleit H, Schmitt E, Kakirman H, Stassen M, Knop J, Enk AH. Infectious tolerance:Human CD25+ regulatory T cells convey suppressor activity to conventional CD4p T helper cells. J Exp Med 2002.196:255-260.
45. Qiao M, Thornton AM, Shevach EM. CD4+ CD25+ regulatory T cells render na(?)ve CD4+ CD25- T cells anergic and suppressive. Immunology 2007.120(4): 447-455.
46. La Cava A. Tregs are regulated by cytokines:Implications for autoimmunity. Autoimmun Rev 2008.8:83-87.
47. Zheng SG, Wang J, Wang P, Gray JD, Horwitz DA. IL-2 is essential for TGF-p to convert naive CD4+ CD25- cells to CD25+ Foxp3+ regulatory T cells and for expansion of these cells. J Immunol 2007.178:2018-2027.
48. Davidson TS, DiPaolo RJ, Andersson J, Shevach EM. Cutting Edge:IL-2 is essential for TGFb-mediated induction of Foxp3+ T regulatory cells. J Immunol 2007.178:4022-4026.
49. Huber S, Stahl FR, Schrader J, Lu"th S, Presser K, Carambia A, Flavell RA, Werner S, Blessing M, Herkel J, Schramm C. Activin a promotes the TGF-β-induced conversion of CD4+CD25- T cells into Foxp3+ induced regulatory T cells. J Immunol 2009.182:4633-4640.
50. Lu L, Ma J, Wang X, Wang J, Zhang F, Yu J, He G, Xu B, Brand DD, Horwitz DA, Shi W, Zheng SG. Synergistic effect of TGF-β superfamily members on the induction of Foxp3+ Treg. Eur J Immunol 2010.40(1):142-152.
51. Piccirillo CA, Letterio JJ, Thornton AM, McHugh RS, Mamura M, Mizuhara H, Shevach EM. CD4+ CD25+ regulatory T cells can mediate suppressor function in the absence of transforming growth factor b1 production and responsiveness. J Exp Med 2002.196:237-246.
52. Fontenot JD, Rasmussen JP, Gavin MA, Rudensky AY. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat Immunol 2005.6:1142-1151.
53. Salomon B, Lenschow DJ, Rhee L, Ashourian N, Singh B, Sharpe A, Bluestone JA. B7/CD28 costimulation is essential for the homeostasis of the CD4+ CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 2000.12: 431-440.
54. You S, Leforban B, Garcia C, Bach JF, Bluestone JA, Chatenoud L. Adaptive TGF-β-dependent regulatory T cells control autoimmune diabetes and are a privileged target of anti-CD3 antibody treatment. Proc Natl Acad Sci USA 2007. 104:6335-6340.
55. Zheng SG. Wang JH, Stohl W, Kim KS, Gray JD, Horwitz DA. TGF-β requires CTLA-4 early after T cell activation to induce FoxP3 and generate adaptive CD4+ CD25+ regulatory cells. J Immunol 2006.176:3321-3329.
56. Herman AE, Freeman GJ, Mathis D, Benoist C. CD4+CD25+ T regulatory cells dependent on ICOS promote regulation of effector cells in the prediabetic lesion. J Exp Med 2004.199:1479-1489.
57. So T, Croft M. Cutting edge:OX40 inhibits TGF-β-and antigen-driven conversion of naive CD4 T cells into CD25+Foxp3+ T cells. J Immunol 2007. 179:1427-1430.
58. Vu MD, Xiao X, Gao W, Degauque N, Chen M, Kroemer A, Killeen N, Ishii N, Chang Li X. OX40 costimulation turns off Foxp3+ Tregs. Blood 2007. 110:2501-2510.
59. Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B. TGF-β in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17 producing T cells. Immunity 2006.24:179-189.
60. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006.441:235-238.
61. Annunziato F, Cosmi L, Liotta F, Maggi E, Romagnani S. Type 17 T helper cells-origins, features and possible roles in rheumatic disease. Nat Rev Rheumatol 2009.5:325-331.
62. Lu L, Wang J, Zhang F, Chai Y, Brad DD, Wang XH, Horwitz DA, Shi W, Zheng SG. Role of Smad and non-Smad signals in the development of Th17 and regulatory T cells. J Immunol 2010.184(8):4295-4306.
63. Manel N, Unutmaz D, Littman DR. The differentiation of human T(H)-17 cells requires transforming growth factor-P and induction of the nuclear receptor RORgt. Nat Immunol 2008.9:641649.
64. Xu L, Kitani A, Fuss I, Strober W. Cutting edge:Regulatory T cells induce CD4+ CD25-Foxp3- T cells or are self-induced to become Th17 cells in the absence of exogenous TGF-β. J Immunol 2007.178:6725-6729.
65. Wan YY, Flavell RA. Regulatory T-cell functions are subverted and converted owing to attenuated Foxp3 expression. Nature 2007.445:766-770.
66. Tsuji M, Komatsu N, Kawamoto S, Suzuki K, Kanagawa O, Honjo T, Hori S, Fagarasan S. Preferential generation of follicular B helper T cells from Foxp3+T cells in gut Peyer's patches. Science 2009.323:1488-1492.
67. Kong N, Zhou XH, Fan HM, Liu ZM, Brand DD, Zou HJ, Zheng SG. TGF-β-induced CD4+Foxp3+ Cells but not natural CD4+ CD25+ cells suppress established collagen induced arthritis. Arthritis Rheum 2009.60:S4.
68. Floess S, Freyer J, Siewert C, Baron U, Olek S, Polansky J, Schlawe K, Chang HD, Bopp T, Schmitt E, Klein-Hessling S, Serfling E, Hamann A, Huehn J. Epigenetic control of the Foxp3 locus in regulatory T cells. PLoS Biol 2007.5: e38.
69. Lal G, Zhang N, van der Touw W, Ding Y, Ju W, Bottinger EP, Reid SP, Levy DE, Bromberg JS. Epigenetic regulation of Foxp3 expression in regulatory T cells by DNA methylation. J Immunol 2009.182:259-273.
70. Zhou XH, Kong N, Wang J, Zou HJ, Fan HM, Brand DD, Liu ZM, Zheng SG. All-trans retinoid acid restores the stability and functionality of nTregs in the Inflammatory milieu. Arthritis Rheum 2009.60:S736.
71. Polansky JK, Kretschmer K, Freyer J, Floess S, Garbe A, Baron U, Olek S, Hamann A, von Boehmer H, Huehn J. DNA methylation controls Foxp3 gene expression. Eur J Immunol 2008.38:1654-1663.
72. Mottet C, Uhlig HH, Powrie F. Cutting edge:Cure of colitis by CD4+CD25+ regulatory T cells. J Immunol 2003.170:3939-3943.
73. Morgan ME, Flierman R, van Duivenvoorde LM, Witteveen HJ, van Ewijk W, van Laar JM, de Vries RR, Toes RE. Effective treatment of collagen-induced arthritis by adoptive transfer of CD25+ regulatory T cells. Arthritis Rheum 2005. 52:2212-2221.
74. Bardos T, Czipri M, Vermes C, Finnegan A, Mikecz K, Zhang J. CD4+ CD25+ immunoregulatory T cells may not be involved in controlling autoimmune arthritis. Arthritis Res Ther 2003.5:R106-113.
75. Huter EN, Stummvoll GH, DiPaolo RJ, Glass DD, Shevach EM. Cutting edge: Antigen-specific TGF-β-induced regulatory T cells suppress Th17-mediated autoimmune disease. J Immunol 2008.181:8209-8213.
76. Chai JG, Tsang JY, Lechler R, Simpson E, Dyson J, Scott D. CD4+CD25+T cells as immunoregulatory T cells in vitro. Eur J Immunol 2002.32:2365-2375.
77. Monk CR, Spachidou M, Rovis F, Leung E, Botto M, Lechler RI, Garden OA. MRL/Mp CD4+CD25- T cells show reduced sensitivity to suppression by CD4+ CD25+regulatory T cells in vitro:A novel defect of T cell regulation in systemic lupus erythematosus. Arthritis Rheum 2005.52:1180-1184.
78. Pasare C, Medzhitov R. Toll pathway-dependent blockade of CD4+CD25+T cell-mediated suppression by dendritic cells. Science 2003.299:1033-1036.
79. Valencia X, Stephens G, Goldbach-Mansky R, Wilson M, Shevach EM, Lipsky PE. TNF downmodulates the function of human CD4+CD25hi T-regulatory cells. Blood 2006.108:253-261.
80. Linker-Israeli M, Deans RJ, Wallace DJ, Prehn J, Ozeri-Chen T, Klinenberg JR. Elevated levels of endogenous IL-6 in systemic lupus erythematosus. A putative role in pathogenesis. J Immunol 1991.147:117-123.
81. Aricha R, Feferman T, Fuchs S, Souroujon MC. Ex vivo generated regulatory T cells modulate experimental autoimmune myasthenia gravis. J Immunol 2008.180: 2132-2139.
82. Su H, Ye DQ, Wang BL, Fang XH, Chen J, Wang Q, Li WX, Zhang N. Transforming growth factor-(31-induced CD4+CD25+regulatory T cells in vitro reverse and prevent a murine lupus-like syndrome of chronic graft-versus-host disease. Br J Dermatol 2008.158:1197-1209.
83. Tran DQ, Ramsey H, Shevach EM. Induction of FOXP3 expression in naive human CD4+FOXP3 T cells by T-cell receptor stimulation is transforming growth factor-β dependent but does not confer a regulatory phenotype. Blood 2007.110: 2983-2990.
84. Yamagiwa S, Gray JD, Hashimoto S, Horwitz DA. A role for TGF-β in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood. J Immunol 2001.166:7282-7289.
85. Riley JL, June CH, Blazar BR. Human T regulatory cell therapy:Take a billion or so and call me in the morning. Immunity 2009.30:656-665
2. Ehrenstein, M. R. et al. Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNF-α therapy. J. Exp. Med.2004.200, 277-285.
3. Mottonen, M. et al. CD4+CD25+ T cells with the phenotypic and functional characteristics of regulatory T cells are enriched in the synovial fluid of patients with rheumatoid arthritis. Clin. Exp. Immunol.2005.140,360-367.
4. Flores-Borja, F., Jury, E. C., Mauri, C.& Ehrenstein, M. R. Defects in CTLA-4 are associated with abnormal regulatory T cell function in rheumatoid arthritis. Proc. Natl Acad. Sci.2008.105,19396-19401.
5. Cao, D. et al. Isolation and functional haracterization of regulatory CD25bright CD4+ cells from the target organ of patients with rhumatoid arthritis. Eur. J. Immunol.2003.33,5-223.
6. Lawson, C. A. et al. Early rheumatoid arthritis is ssociated with a deficit in the CD4+CD25high regulatory T cell population in peripheral blood. Rheumatology (Oxford).2006.45,1210-1217.
7. Valencia, X. et al. TNF down modulates the function of human CD4+CD25hi T-regulatory cells. Blood.2006.108,253-261.
8. Mary E. Morgan, Roger P. M. Sutmuller, Hendrik J. Witteveen, Leonie M. van Duivenvoorde,Eric Zanelli, Cornelis J. M. Melief, et al. CD25+ Cell Depletion Hastens the Onset of Severe Disease in Collagen-Induced Arthritis. Arthritis Rheum.2003.48,1452-1460.
9. H Kelchtermans,L Geboes,T Mitera,D Huskens,G Leclercq,P Matthys. Activated CD4+CD25+ regulatory T cells inhibit osteoclastogenesis and collagen-induced arthritis. Ann Rheum Dis.2009.68;744-750
10. Morgan ME, Flierman R, van Duivenvoorde LM, Witteveen HJ, van Ewijk W, van Laar JM, et al. Effective treatment of collagen-induced arthritis by adoptive transfer of CD25+ regulatory T cells. Arthritis Rheum.2005.52:2212-2.
11. Miyara, M. et al. Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FOXP3 transcription factor. Immunity.2009.30, 899-911.
12. Michel, L. et al. Patients with relapsing-remitting multiple sclerosis have normal TREG function when cells expressing IL-7 receptor a-chain are excluded from the analysis. J. Clin. Invest.2008.118,3411-3419.
13. Liu, W. et al. CD127 expression inversely correlates with FOXP3 and suppressive function of human CD4+ TREG cells. J. Exp. Med.2006.203, 1701-1711.
14. Jonathan H. Esensten, David Wofsy and Jeffrey A. Bluestone.Regulatory T cells as therapeutic targets in rheumatoid arthritis. Nat.Rev. Rheumatol.2009.5,560-565.183.
15. Alwi Shatry, Jackeline Chirinos, Michael A. Gorin, Monica Jones, Robert B. Levy. Targeting Treg Cells In Situ:Emerging Expansion Strategies for (CD4+CD25+) Regulatory T Cells. Biol Blood Marrow Transplant 2009.15:1239-1243.
16. Song Guo Zheng, Juhua Wang, David A. Horwitz. Cutting Edge:Foxp3+CD4+ CD25+Regulatory T Cells Induced by IL-2 and TGF-β Are Resistant to Th17 Conversion by IL-6. The Journal of Immunology.2008.180:7112-7116.
17. Xu L, Kitani A, Fuss I, Strober W. Cutting edge:regulatory T cells induce CD4+ CD25-Foxp3- T cells or are self-induced to become Th17 cells in the absence of exogenous TGF-beta. J Immunol.2007.178:6725-6729.
18. dhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B.TGF-beta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity.2006.24(2):179-89.
19. gan PR, Harrington LE, O'Quinn DB, Helms WS, Bullard DC, Elson CO, Hatton RD, Wahl SM, Schoeb TR, Weaver CT. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature.2006.441(7090):231-4.
20. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature.2006.441(7090):235-8.
21. Michel ML, Keller AC, Paget C et al. Identification of an IL-17-producing NK1.1neg iNKT cell population involved in airway neutrophilia. J Exp Med. 2007.204:995.
22. Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B.TGF-beta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity.2006. Feb;24(2):179-89
23. Mangan PR, Harrington LE, O'Quinn DB, Helms WS, Bullard DC, Elson CO, Hatton RD, Wahl SM, Schoeb TR, Weaver CT. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature.2006.441(7090):231-4.
24. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature.2006.441(7090):235-8.
25. Heremans H, Dillen C, Groenen M, Martens E, Billiau A. Chronic relapsing experimental autoimmune encephalomyelitis (CREAE) in mice:enhancement by monoclonal antibodies against interferon-gamma. Eur J Immunol.1996.26(10): 2393-8.
26. Ferber IA, Brocke S, Taylor-Edwards C, Ridgway W, Dinisco C, Steinman L, Dalton D, Fathman CG. Mice with a disrupted IFN-gamma gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J Immunol.1996.156(1):5-7.
27. Lubberts E, Koenders MI, Oppers-Walgreen B, van den Bersselaar L, Coenen-de Roo CJ, Joosten LA, van den Berg WB. Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of collagen-induced arthritis reduces joint inflammation, cartilage destruction, and bone erosion. Arthritis Rheum.2004.50(2):650-9.
28. Zhang Z, Zheng M, Bindas J, Schwarzenberger P, Kolls JK. Critical role of IL-17 receptor signaling in acute TNBS-induced colitis. Inflamm Bowel Dis. 2006.12(5):382-8.
29. Koenders MI, Kolls JK, Oppers-Walgreen B, van den Bersselaar L, Joosten LA,Schurr JR, Schwarzenberger P, van den Berg WB, Lubberts E. Interleukin-17 receptor deficiency results in impaired synovial expression of interleukin-1 and matrix metalloproteinases 3,9, and 13 and prevents cartilage destruction during chronic reactivated streptococcal cell wall-induced arthritis. Arthritis Rheum. 2005.52:3239-3247.
30. Lubberts E, Koenders MI, Oppers-Walgreen B, van den Bersselaar L, Coenen-de Roo CJ, Joosten LA, van den Berg WB. Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of collagen-induced arthritis reduces joint inflammation, cartilage destruction, and bone erosion. Arthritis Rheum.2004.50(2):650-9.
31. Zheng SG, Wang JH, Gray JD, Soucier H, Horwitz DA. Natural and induced CD4+CD25+ cells educate CD4+CD25- cells to develop suppressive activity:the role of IL-2, TGF-beta, and IL-10.J Immunol 2004.172:5213-21.
32. Fantini MC, Becker C, Monteleone G, Pallone F, Galle PR, Neurath MF. Cutting edge:TGFbeta induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and downregulation of Smad7. J Immunol 2004.172:5149-53.
33. Horwitz DA, Zheng SG, Gray JD. Natural and TGF-binduced Foxp3+CD4+ CD25+regulatory T cells are not mirror images of each other. Trends Immunol 2008.29:429-435.
34. Zheng SG, Wang J, Wang P, Gray JD, Horwitz DA. IL-2 is essential for TGF-(3 to convert naive CD4+CD25= cells to CD25+Foxp3+ regulatory T cells and for expansion of these cells. J Immunol 2007.178:2018-2027.
35. Read S, Malmstro"m V, Powrie F. Cytotoxic T lymphocyteassociated antigen 4 plays an essential role in the function of CD25+CD4+regulatory cells that control intestinal inflammation. J Exp Med 2000.192:295-302
36. Kohm AP, Carpentier PA, Anger HA, Miller SD. Cutting edge:CD4+CD25+ regulatory T cells suppress antigenspecific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis. J Immunol 2002.169:4712-4716.
37. Salomon B, Lenschow DJ, Rhee L, Ashourian N, Singh B, Sharpe A, Bluestone JA. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 2000.12: 431-440.
38. You S, Leforban B, Garcia C, Bach JF, Bluestone JA, Chatenoud L. Adaptive TGF-β-dependent regulatory T cells control autoimmune diabetes and are a privileged target of anti-CD3 antibody treatment. Proc Natl Acad Sci USA 2007. 104:6335-6340.
39. Zheng SG, Gray JD, Ohtsuka K, Yamagiwa S, Horwitz DA. Generation ex vivo of TGF-beta-producing regulatory T cells from CD4+CD25- precursors. J Immunol 2002.15;169(8):4183-9.
40. Thornton AM, Shevach EM. CD4+CD25+immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J Exp Med 1998.188:287-296.
41. Nakamura K, Kitani A, Strober W. Cell contact-dependent immunosuppression by CD4+CD25+regulatory T cells is mediated by cell surface-bound transforming growth factor β.J Exp Med 2001.194:629-644.
42. Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+regulatory T cells by TGF-β induction of transcription factor Foxp3. J Exp Med 2003.198: 1875-1886.
43. Piccirillo CA, Letterio JJ, Thornton AM, McHugh RS, Mamura M, Mizuhara H, Shevach EM. CD4+CD25+regulatory T cells can mediate suppressor function in the absence of transforming growth factor b1 production and responsiveness. J Exp Med 2002.196:237-246.
44. Fontenot JD, Rasmussen JP, Gavin MA, Rudensky AY. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat Immunol 2005.6:1142-1151.
45. Floess S, Freyer J, Siewert C, Baron U, Olek S, Polansky J, Schlawe K, Chang HD, Bopp T, Schmitt E, Klein-Hessling S, Serfling E, Hamann A, Huehn J. Epigenetic control of the Foxp3 locus in regulatory T cells. PLoS Biol 2007.5: e38.
46. Lai G, Zhang N, van der Touw W, Ding Y, Ju W, Bottinger EP, Reid SP, Levy DE, Bromberg JS. Epigenetic regulation of Foxp3 expression in regulatory T cells by DNA methylation. J Immunol 2009.182:259-273.
47. Zheng SG, Wang JH, Koss MN, Quismorio F Jr, Gray JD, Horwitz DA. CD4+ and CD8+ regulatory T cells generated ex vivo with IL-2 and TGF-beta suppress a stimulatory graft versus-host disease with a lupus-like syndrome. J Immunol. 2004.172:1531-9.
48. Weber SE, Harbertson J, Godebu E, Mros GA, Padrick RC, Carson BD, Ziegler SF, Bradley LM.Adaptive islet-specific regulatory CD4 T cells control autoimmune diabetes and mediate the disappearance of pathogenic Thl cells in vivo. J Immunol 2006.176:4730-9.
49. Selvaraj RK, Geiger TL. Mitigation of experimental allergic encephalomyelitis by TGF-beta induced Foxp3+ regulatory T lymphocytes through the induction of anergy and infectious tolerance. J Immunol.2008.180:2830-8.
50. Wan YY, Flavell RA. Regulatory T-cell functions are subverted and converted owing to attenuated Foxp3 expression. Nature 2007.445:766-770.
51. Lu L, Wang J, Zhang F, Chai Y, Brad DD, Wang XH, Horwitz DA, Shi W, Zheng SG. Role of Smad and non-Smad signals in the development of Th 17 and regulatory T cells. J Immunol 2010.184(8):4295-4306.
52. Tsuji M, Komatsu N, Kawamoto S, Suzuki K, Kanagawa O, Honjo T, Hori S, Fagarasan S. Preferential generation of follicular B helper T cells from Foxp3+T cells in gut Peyer's patches. Science 2009.323:1488-1492.
53. Zhou X, Kong N, Wang J, Fan H, Zou H, Horwitz D, Brand D, Liu Z, Zheng SG. Cutting edge:all-trans retinoic acid sustains the stability and function of natural regulatory T cells in an inflammatory milieu.J Immunol.2010.185(5):2675-9.
54. Fossiez F, Djossou O, Chomarat P, Flores-Romo L, Ait-Yahia S, Maat C, et al. T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines. J Exp Med.1996.183(6):2593-603.
55. Chen W, Jin W, Lei K-j, Hardegen, N, Li L, Marinos N et al. Conversion of peripheral CD4+CD25- regulatory T cells by TGF-Pinduction of transcription factor Foxp3. J Exp Med 2003.198:1875-1886.
56. Fantini MC,Becker C, Monteleone G, Pallone F, Galle PR, Neurath MF. Cutting edge:TGF-beta induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and down-regulation of Smad7. J Immunol 2004.172:5149-5153.
57. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T,et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998.93:165-76.
58. Redlich K, Hayer S, Ricci R, David JP, Tohidast-Akrad M, Kollias G, et al. Osteoclasts are essential for TNF-aamediated joint destruction. J Clin Invest 2002.110:1419-27.
59. S Pettit AR, Ji H, von Stechow D, Muller R, Goldring SR, Choi Y, et al. TRANCE/RANKL knockout mice are protected from bone erosion in a serum transfer model of arthritis. Am J Pathol 2001.159:1689-99.
60. Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 1999.20:345-357.
61. Yoshida H, Hayashi S, Kunisada T, Ogawa M, Nishikawa S,Okamura H, et al. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 1990.345:442-4.
62. Wiktor-Jedrzejczak W, Bartocci A, Ferrante AW Jr, et al. Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse. Proc Natl Acad Sci U S A 1990.87:4828-4832.
63. Bim Nicolas Houde, Estelle Chamoux, Martine Bisson, and Sophie Roux. Transforming growth factor-beta1 (TGF-beta1) induces human osteoclast apoptosis by up-regulating Bim. J Biol Chem 2009.284:23397-23404.
64. Manolagas SC, Jilka RL. Mechanisms of disease:bone marrow, cytokines, and bone remodeling-emerging insights into the pathophysiology of osteoporosis. N Engl J Med 1995.332:305-311.
65. Nishimoto N, Kishimoto T. Interleukin 6:from bench to bedside. Nat Clin Pract Rheumatol 2006.2:619-26.
66. McInnes I, Schett G. Cytokines in the pathogenesis of rheumatoid arthritis. Nat Rev Immunol 2007.7:429-42.
67. Sugita T, Furukawa O, Ucno M, Murakami T, Takata I, Tosa T. Enhanced expression of interleukin 6 in rat and murine arthritis models. Tnt J Tmmunopharmecol 1993.15:469-76.
68. Lee SK, Lorenzo J.Cytokines regulating osteoclast formation and function. Curr Opin Rheumatol 2006.18:411-418.
69. Strand V, Kavanaugh AF. The IL-1 family and inflammatory diseases. Clin Exp Rheumatol 2004.20:S1-S13.
70. Strand V, Kavanaugh AF. The role of interleukin-1 in bone resorptionin rheumatoid arthritis. Rheumatology (Oxford) 2004.43 Suppl 3:ⅲ10-ⅲ16.
71. Trebec-Reynolds DP, Voronov I, Heersche JN, Manolson MF. IL-1 alpha and IL-1beta Have Different Effects on Formation and Activity of Large Osteoclasts. J Cell Biochem 2010.109(5):975-82.
72. Sato K, Suematsu A, Okamoto K, Yamaguchi A, Morishita Y, Kadono Y,et al. Th17 functions as an osteoclastogenic helper T cell subset that links T cell activation and bone destruction. J Exp Med 2006.203:2673-2682.
73. Adamopoulos IE, Chao CC, Geissler R, Laface D, Blumenschein W, Iwakura Y, et al. Interleukin-17A upregulates receptor activator of NF-kappaB on osteoclast precursors. Arthritis Res Ther 2010.12(1):R29.
74. Geboes L, De Klerck B, Van Balen M, Kelchtermans H, Mitera T, Boon L, et al. Freund's complete adjuvant induces arthritis in mice lacking a functional interferon-creceptor by triggering tumor necrosis factor alpha-driven osteoclastogenesis. Arthritis Rheum 2007.56:2595-607.
75. Li P, Schwarz EM, O'Keefe RJ, Ma L, Looney RJ, Ritchlin CT, et al. Systemic tumor necrosis factor alpha mediates an increase in peripheral CD1lbhigh osteoclast precursors in tumor necrosis factor alpha-transgenic mice. Arthritis Rheum 2004.50:265-76.
76. De Klerck B, Carpentier I, Lories RJ, Habraken Y, Piette J, Carmeliet G, et al. Enhanced osteoclast development in collagen-induced arthritis in interferon-γ receptor knock-out mice as related to increased splenic CD11b+ myelopoiesis. Arthritis Res Ther 2004.6:221-31.
77. Brand DD, Latham KA, Rosloniec EF. Collagen-induced arthritis. Nat Protoc 2007.2 (5):1269-75.
78. Kelchtermans H, Struyf S, De Klerck B, Mitera T, Alen M, Geboes L, et al. Protective role of IFN-c in collagen-induced arthritis conferred by inhibition of mycobacteriainduced granulocyte chemotactic protein-2 (GCP-2) production. J Leukoc Biol 2007.81:1044-53.
79. Pasare C, Medzhitov R. Toll pathway-dependent blockade of CD4+CD25+T cell-mediated suppression by dendritic cells. Science.299:1033-1036,2003
80. Valencia X, Stephens G, Goldbach-Mansky R, Wilson M, Shevach EM, Lipsky PE. TNF downmodulates the function of human CD4+CD25hi T-regulatory cells. Blood.2006.108:253-261.
81. S. Kotake, IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis, J. Clin. Invest.1999.103:1345-1352.
82. S.Y. Hwang, J.P. Kim, M.K. Park, Y. Moon, W.U. Kim and H.Y. Kim, IL-17 induce production of IL-6 and IL-18 in rheumatoid arthritis synovial fibroblasts via NF-Kappa Beta and PI-3Kinase/AK 1-dependent pathways, Arthritis Res. Ther.2004.6:120-128.
83. Zaiss MM, Axmann R, Zwerina J, Polzer K, Guckel E, Skapenko A, et al. Treg cells suppress osteoclast formation:a new link between the immune system and bone. Arthritis Rheum 2007.56(12):4104-12.
84. Kong YY, Feige U, Sarosi I, Bolon B, Tafuri A, Morony S, et al. Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 1999.402(6759):304-9.
85. Takayanagi H, Ogasawara K, Hida S, Chiba T, Murata S, Sato K, et al. T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-gamma. Nature 2000.408(6812):600-5.
86. Gao Y, Grassi F, Ryan MR, Terauchi M, Page K, Yang X, et al. IFN-y stimulates osteoclast formation and bone loss in vivo via antigen-driven T cell activation. J Clin Invest 2007.117(1):122-132.
87. Koenders MI, Joosten LA, van den Berg WB. Potential new targets in arthritis therapy:interleukin (IL)-17 and its relation to tumour necrosis factor and IL-1 in experimental arthritis. Ann Rheum Dis 2006.65 Suppl 3:iii29-33.
88. Zaiss MM, Frey B, Hess A, Zwerina J, Luther J, Nimmerjahn F, et al. Regulatory T cells protect from local and systemic bone destruction in arthritis. J Immunol 2010.184(12):7238-46.
89. Zaiss MM, Sarter K, Hess A, Engelke K, Bohm C, Nimmerjahn F, et al. Increased bone density and resistance to ovariectomy-induced bone loss in FoxP3-transgenic mice based on impaired osteoclast differentiation. Arthritis Rheum 2010.62(8):2328-38.
90. Kim YG, Lee CK, Nah SS, Mun SH, Yoo B, Moon HB. Human CD4+CD25+ regulatory T cells inhibit the differentiation of osteoclasts from peripheral blood mononuclear cells. Biochem Biophys Res Commun 2007.357(4):1046-52.
91. Shevach EM, Davidson TS, Huter EN, Dipaolo RA, Andersson J. Role of TGF-Beta in the induction of Foxp3 expression and T regulatory cell function. J Clin Immunol 2008.28(6):640-6.
92. Powrie F, Carlino J, Leach MW, Mauze S, Coffman RL. A critical role for transforming growth factor-beta but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RB(low) CD4+T cells. J Exp Med 1996. 183(6):2669-74.
93. Soysa NS, Alles N. NF-kappaB functions in osteoclasts. Biochem Biophys Res Commun 2009.378(1):1-5.
94. Weitzmann MN, Pacifici R. The role of T lymphocytes in bone metabolism. Immunol Rev 2005.208:154-68.
1. Sakaguchi S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 2004.22:531-562.
2. Shevach EM. CD4+CD25+ suppressor T cells:More questions than answers. Nat Rev Immunol 2002.2:389-400.
3. Gershon RK, Kondo K. Cell interactions in the induction of tolerance:The role of thymic lymphocytes. Immunology 1970.18:723-737.
4. Kojima A, Prehn RT. Genetic susceptibility to postthymectomy autoimmune diseases in mice. Immunogenetics 1981.14:15-27.
5. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor a-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995.155:1151-1164.
6. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003.299:1057-1061.
7. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 2003.4:330-336.
8. Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD4+ CD25+T regulatory cells. Nat Immunol 2003.4:337-342.
9. Fontenot JD, Rasmussen JP, Williams LM, Dooley JL, Farr AG, Rudensky AY. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 200.22:329-341.
10. Baecher-Allan C, Brown JA, Freeman GJ, Hafler DA. CD4+CD25high regulatory cells in human peripheral blood. J Immunol 2001.167:1245-1253.
11. Morgan ME, van Bilsen JH, Bakker AM, Heemskerk B,Schilham MW, Hartgers FC, Elferink BG, van der Zanden L,de Vries RR, Huizinga TW, Ottenhoff TH, Toes RE.Expression of FOXP3 mRNA is not confined to CD4+CD25+T regulatory cells in humans. Hum Immunol 2005.66:13-20.
12. Allan SE, Crome SQ, Crellin NK, Passerini L, Steiner TS, Bacchetta R, Roncarolo MG, Levings MK. Activation induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production. Int Immunol 2007. 19:345-354.
13. Yu JN, Ma JL, Wang JH, Tao XJ, Li XP, Zou HJ, Zheng SG. FOXP3, is still a specific marker for human regulatory T cells? Chin J Allergy Clin Immunol 2008. 2:5-10.
14. Chatila TA, Blaeser F, Ho N, Lederman HM, Voulgar opoulos C, Helms C, Bowcock AM. JM2, encoding a forkhead-related protein, is mutated in X-linked autoimmunity allergic disregulation syndrome. J Clin Invest 2000.106:R75-R81.
15. Wildin RS, Ramsdell F, Peake J, Faravelli F, Casanova JL,Buist N, Levy-Lahad E, Mazzella M, Goulet O, Perroni L,Bricarelli FD, Byrne G, McEuen M, Proll S, Appleby M,Brunkow ME. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet 200.27:18
16. Bennett CL, Christie J, Ramsdell F, Brunkow ME, Ferguson PJ, Whitesell L, Kelly TE, Saulsbury FT, Chance PF, Ochs HD. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet 2001.27:20-21.
17. Tang Q, Bluestone JA. The Foxp3+ regulatory T cell:A jack of all trades, master of regulation. Nat Immunol 2008.9:239-244.
18. Horwitz DA, Zheng SG, Gray JD, Wang JH, Ohtsuka K, Yamagiwa S. Regulatory T cells generated ex vivo as an approach for the therapy of autoimmune disease. Semin Immunol 2004.16:135-143.
19. Hahn BH, Singh RP, La Cava A, Ebling FM. Tolerogenic treatment of lupus mice with consensus peptide induces Foxp3-expressing, apoptosis-resistant, TGF-β-secreting CD8+ T cell suppressors. J Immunol 2005.175:7728-7737.
20. Ochi H, Abraham M, Ishikawa H, Frenkel D, Yang K, Basso AS, Wu H, Chen ML, Gandhi R, Miller A, Maron R, Weiner HL. Oral CD3-specific antibody suppresses autoimmune encephalomyelitis by inducing CD4+CD25- LAP+T cells. Nat Med 2006.12:627-635.
21. Kang HK, Michaels MA, Berner BR, Datta SK. Very low-dose tolerance with nucleosomal peptides controls lupus and induces potent regulatory T cell subsets. J Immunol 2005.174:3247-3255.
22. Brand DD, Myers LK, Whittington KB, Latham KA, Stuart JM, Kang AH, Rosloniec EF. Detection of early changes in autoimmune T cell phenotype and function following intravenous administration of type II collagen in a TCR-transgenic model. J Immunol 2002.168:490-498.
23. Vieira PL, Christensen JR, Minaee S, O'Neill EJ, Barrat FJ, Boonstra A, Barthlott T, Stockinger B, Wraith DC, O'Garra A. IL-10-secreting regulatory T cells do not express Foxp3 but have comparable regulatory function to naturally occurring CD4+CD25+regulatory T cells. J Immunol 2004.172:5986-5993.
24. Zheng SG, Gray JD, Ohtsuka K, Yamagiwa S, Horwitz DA. Generation ex vivo of TGF-β producing regulatory T cells from CD4(?) CD252 precursors. J Immunol 2002.169:4183-4189.
25. Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-β induction of transcription factor Foxp3. J Exp Med 2003. 198:1875-1886.
26. Zheng SG, Wang JH, Gray JD, Soucier H, Horwitz DA. Natural and induced CD4+CD25+ cells educate CD4+CD25- cells to develop suppressive activity:The role of IL-2, TGF-β, and IL-10. J Immunol 2004.172:5213-5221.
27. Fantini MC, Becker C, Monteleone G, Pallone F, Galle PR, Neurath MF. Cutting edge:TGF-β induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and downregulation of Smad7. J Immunol 2004.172: 5149-5153.
28. Chatenoud L, Bluestone JA. CD3-specific antibodies:A portal to the treatment of autoimmunity. Nat Rev Immunol 2007.7:622-632.
29. Battaglia M, Gregori S, Bacchetta R, Roncarolo MG. Trl cells:From discovery to their clinical application. Semin Immunol 2006.18:120-127.
30. Horwitz DA, Zheng SG, Gray JD. Natural and TGF-binduced Foxp3+ CD4+ CD25+ regulatory T cells are not mirror images of each other. Trends Immunol 2008.29:429-435.
31. Thornton AM, Shevach EM. CD4+ CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J Exp Med 1998.188:287-296.
32. Nakamura K, Kitani A, Strober W. Cell contact-dependent immunosuppression by CD4+CD25+regulatory T cells is mediated by cell surface-bound transforming growth factor β. J Exp Med 2001.194:629-644.
33. Zheng SG, Wang J, Horwitz DA. Cutting edge:Foxp3+CD4+CD25+regulatory T cells induced by IL-2 and TGF-β are resistant to Th17 conversion by IL-6. J Immunol 2008.180:7112-7116.
34. DiPaolo RJ, Glass DD, Bijwaard KE, Shevach EM. CD4+ CD25+ T cells prevent the development of organ-specific autoimmune disease by inhibiting the differentiation of autoreactive effector T cells. J Immunol 2005.175:7135-7142.
35. Read S, Malmstro"m V, Powrie F. Cytotoxic T lymphocyteassociated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intestinal inflammation. J Exp Med 2000.192:295-302
36. Kohm AP, Carpentier PA, Anger HA, Miller SD. Cutting edge:CD4+ CD25+ regulatory T cells suppress antigenspecific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis. J Immunol 2002,169:4712-4716.
37. Scalapino KJ, Tang Q, Bluestone JA, Bonyhadi ML, Daikh DI. Suppression of disease in New Zealand Black/New Zealand White lupus-prone mice by adoptive transfer of ex vivo expanded regulatory T cells. J Immunol 2006.177:1451-1459.
38. Zheng SG, Wang JH, Koss MN, Quismorio F, Jr, Gray JD, Horwitz DA. CD4+ and CD8+ regulatory T cells generated ex vivo with IL-2 and TGF-β suppress a stimulatory graft versus-host disease with a lupus-like syndrome. J Immunol 2004. 172:1531-1539.
39. Weber SE, Harbertson J, Godebu E, Mros GA, Padrick RC, Carson BD, Ziegler SF, Bradley LM. Adaptive islet-specific regulatory CD4 T cells control autoimmune diabetes and mediate the disappearance of pathogenic Thl cells in vivo. J Immunol 2006.176:4730-4739.
40. Selvaraj RK, Geiger TL. Mitigation of experimental allergic encephalomyelitis by TGF-β-induced Foxp3+ regulatory T lymphocytes through the induction of anergy and infectious tolerance. J Immunol 2008.180:2830-2838.
41. Fahle'n L, Read S, Gorelik L, Hurst SD, Coffman RL, Flavell RA, Powrie F. T cells that cannot respond to TGF-β escape control by CD4+ CD25+ regulatory T cells. J Exp Med 2005.201:737-746.
42. Hara M, Kingsley CI, Niimi M, Read S, Turvey SE, Bushell AR, Morris PJ, Powrie F, Wood KJ. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. J Immunol 2001.166:3789-3796.
43. Zheng SG, Meng L, Wang JH, Watanabe M, Barr ML, Cramer DV, Gray JD, Horwitz DA. Transfer of regulatory T cells generated ex vivo modifies graft rejection through induction of tolerogenic CD4+ CD25+ cells in the recipient. Int Immunol 2006.18:279-289.
44. Jonuleit H, Schmitt E, Kakirman H, Stassen M, Knop J, Enk AH. Infectious tolerance:Human CD25+ regulatory T cells convey suppressor activity to conventional CD4p T helper cells. J Exp Med 2002.196:255-260.
45. Qiao M, Thornton AM, Shevach EM. CD4+ CD25+ regulatory T cells render na(?)ve CD4+ CD25- T cells anergic and suppressive. Immunology 2007.120(4): 447-455.
46. La Cava A. Tregs are regulated by cytokines:Implications for autoimmunity. Autoimmun Rev 2008.8:83-87.
47. Zheng SG, Wang J, Wang P, Gray JD, Horwitz DA. IL-2 is essential for TGF-p to convert naive CD4+ CD25- cells to CD25+ Foxp3+ regulatory T cells and for expansion of these cells. J Immunol 2007.178:2018-2027.
48. Davidson TS, DiPaolo RJ, Andersson J, Shevach EM. Cutting Edge:IL-2 is essential for TGFb-mediated induction of Foxp3+ T regulatory cells. J Immunol 2007.178:4022-4026.
49. Huber S, Stahl FR, Schrader J, Lu"th S, Presser K, Carambia A, Flavell RA, Werner S, Blessing M, Herkel J, Schramm C. Activin a promotes the TGF-β-induced conversion of CD4+CD25- T cells into Foxp3+ induced regulatory T cells. J Immunol 2009.182:4633-4640.
50. Lu L, Ma J, Wang X, Wang J, Zhang F, Yu J, He G, Xu B, Brand DD, Horwitz DA, Shi W, Zheng SG. Synergistic effect of TGF-β superfamily members on the induction of Foxp3+ Treg. Eur J Immunol 2010.40(1):142-152.
51. Piccirillo CA, Letterio JJ, Thornton AM, McHugh RS, Mamura M, Mizuhara H, Shevach EM. CD4+ CD25+ regulatory T cells can mediate suppressor function in the absence of transforming growth factor b1 production and responsiveness. J Exp Med 2002.196:237-246.
52. Fontenot JD, Rasmussen JP, Gavin MA, Rudensky AY. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat Immunol 2005.6:1142-1151.
53. Salomon B, Lenschow DJ, Rhee L, Ashourian N, Singh B, Sharpe A, Bluestone JA. B7/CD28 costimulation is essential for the homeostasis of the CD4+ CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 2000.12: 431-440.
54. You S, Leforban B, Garcia C, Bach JF, Bluestone JA, Chatenoud L. Adaptive TGF-β-dependent regulatory T cells control autoimmune diabetes and are a privileged target of anti-CD3 antibody treatment. Proc Natl Acad Sci USA 2007. 104:6335-6340.
55. Zheng SG. Wang JH, Stohl W, Kim KS, Gray JD, Horwitz DA. TGF-β requires CTLA-4 early after T cell activation to induce FoxP3 and generate adaptive CD4+ CD25+ regulatory cells. J Immunol 2006.176:3321-3329.
56. Herman AE, Freeman GJ, Mathis D, Benoist C. CD4+CD25+ T regulatory cells dependent on ICOS promote regulation of effector cells in the prediabetic lesion. J Exp Med 2004.199:1479-1489.
57. So T, Croft M. Cutting edge:OX40 inhibits TGF-β-and antigen-driven conversion of naive CD4 T cells into CD25+Foxp3+ T cells. J Immunol 2007. 179:1427-1430.
58. Vu MD, Xiao X, Gao W, Degauque N, Chen M, Kroemer A, Killeen N, Ishii N, Chang Li X. OX40 costimulation turns off Foxp3+ Tregs. Blood 2007. 110:2501-2510.
59. Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B. TGF-β in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17 producing T cells. Immunity 2006.24:179-189.
60. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006.441:235-238.
61. Annunziato F, Cosmi L, Liotta F, Maggi E, Romagnani S. Type 17 T helper cells-origins, features and possible roles in rheumatic disease. Nat Rev Rheumatol 2009.5:325-331.
62. Lu L, Wang J, Zhang F, Chai Y, Brad DD, Wang XH, Horwitz DA, Shi W, Zheng SG. Role of Smad and non-Smad signals in the development of Th17 and regulatory T cells. J Immunol 2010.184(8):4295-4306.
63. Manel N, Unutmaz D, Littman DR. The differentiation of human T(H)-17 cells requires transforming growth factor-P and induction of the nuclear receptor RORgt. Nat Immunol 2008.9:641649.
64. Xu L, Kitani A, Fuss I, Strober W. Cutting edge:Regulatory T cells induce CD4+ CD25-Foxp3- T cells or are self-induced to become Th17 cells in the absence of exogenous TGF-β. J Immunol 2007.178:6725-6729.
65. Wan YY, Flavell RA. Regulatory T-cell functions are subverted and converted owing to attenuated Foxp3 expression. Nature 2007.445:766-770.
66. Tsuji M, Komatsu N, Kawamoto S, Suzuki K, Kanagawa O, Honjo T, Hori S, Fagarasan S. Preferential generation of follicular B helper T cells from Foxp3+T cells in gut Peyer's patches. Science 2009.323:1488-1492.
67. Kong N, Zhou XH, Fan HM, Liu ZM, Brand DD, Zou HJ, Zheng SG. TGF-β-induced CD4+Foxp3+ Cells but not natural CD4+ CD25+ cells suppress established collagen induced arthritis. Arthritis Rheum 2009.60:S4.
68. Floess S, Freyer J, Siewert C, Baron U, Olek S, Polansky J, Schlawe K, Chang HD, Bopp T, Schmitt E, Klein-Hessling S, Serfling E, Hamann A, Huehn J. Epigenetic control of the Foxp3 locus in regulatory T cells. PLoS Biol 2007.5: e38.
69. Lal G, Zhang N, van der Touw W, Ding Y, Ju W, Bottinger EP, Reid SP, Levy DE, Bromberg JS. Epigenetic regulation of Foxp3 expression in regulatory T cells by DNA methylation. J Immunol 2009.182:259-273.
70. Zhou XH, Kong N, Wang J, Zou HJ, Fan HM, Brand DD, Liu ZM, Zheng SG. All-trans retinoid acid restores the stability and functionality of nTregs in the Inflammatory milieu. Arthritis Rheum 2009.60:S736.
71. Polansky JK, Kretschmer K, Freyer J, Floess S, Garbe A, Baron U, Olek S, Hamann A, von Boehmer H, Huehn J. DNA methylation controls Foxp3 gene expression. Eur J Immunol 2008.38:1654-1663.
72. Mottet C, Uhlig HH, Powrie F. Cutting edge:Cure of colitis by CD4+CD25+ regulatory T cells. J Immunol 2003.170:3939-3943.
73. Morgan ME, Flierman R, van Duivenvoorde LM, Witteveen HJ, van Ewijk W, van Laar JM, de Vries RR, Toes RE. Effective treatment of collagen-induced arthritis by adoptive transfer of CD25+ regulatory T cells. Arthritis Rheum 2005. 52:2212-2221.
74. Bardos T, Czipri M, Vermes C, Finnegan A, Mikecz K, Zhang J. CD4+ CD25+ immunoregulatory T cells may not be involved in controlling autoimmune arthritis. Arthritis Res Ther 2003.5:R106-113.
75. Huter EN, Stummvoll GH, DiPaolo RJ, Glass DD, Shevach EM. Cutting edge: Antigen-specific TGF-β-induced regulatory T cells suppress Th17-mediated autoimmune disease. J Immunol 2008.181:8209-8213.
76. Chai JG, Tsang JY, Lechler R, Simpson E, Dyson J, Scott D. CD4+CD25+T cells as immunoregulatory T cells in vitro. Eur J Immunol 2002.32:2365-2375.
77. Monk CR, Spachidou M, Rovis F, Leung E, Botto M, Lechler RI, Garden OA. MRL/Mp CD4+CD25- T cells show reduced sensitivity to suppression by CD4+ CD25+regulatory T cells in vitro:A novel defect of T cell regulation in systemic lupus erythematosus. Arthritis Rheum 2005.52:1180-1184.
78. Pasare C, Medzhitov R. Toll pathway-dependent blockade of CD4+CD25+T cell-mediated suppression by dendritic cells. Science 2003.299:1033-1036.
79. Valencia X, Stephens G, Goldbach-Mansky R, Wilson M, Shevach EM, Lipsky PE. TNF downmodulates the function of human CD4+CD25hi T-regulatory cells. Blood 2006.108:253-261.
80. Linker-Israeli M, Deans RJ, Wallace DJ, Prehn J, Ozeri-Chen T, Klinenberg JR. Elevated levels of endogenous IL-6 in systemic lupus erythematosus. A putative role in pathogenesis. J Immunol 1991.147:117-123.
81. Aricha R, Feferman T, Fuchs S, Souroujon MC. Ex vivo generated regulatory T cells modulate experimental autoimmune myasthenia gravis. J Immunol 2008.180: 2132-2139.
82. Su H, Ye DQ, Wang BL, Fang XH, Chen J, Wang Q, Li WX, Zhang N. Transforming growth factor-(31-induced CD4+CD25+regulatory T cells in vitro reverse and prevent a murine lupus-like syndrome of chronic graft-versus-host disease. Br J Dermatol 2008.158:1197-1209.
83. Tran DQ, Ramsey H, Shevach EM. Induction of FOXP3 expression in naive human CD4+FOXP3 T cells by T-cell receptor stimulation is transforming growth factor-β dependent but does not confer a regulatory phenotype. Blood 2007.110: 2983-2990.
84. Yamagiwa S, Gray JD, Hashimoto S, Horwitz DA. A role for TGF-β in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood. J Immunol 2001.166:7282-7289.
85. Riley JL, June CH, Blazar BR. Human T regulatory cell therapy:Take a billion or so and call me in the morning. Immunity 2009.30:656-665