作者姓名钱程

作者姓名：钱程论文题目：Fas信号和TLR信号促进调节性树突状细胞负向调控 CD4+ T细胞反应及相关机制研究作者简介：钱程，女，1978 年11月出生，2005 年9 月师从于第 二军医大学曹雪涛教授，于2008 年6 月获博士学位。中文摘要作为功能独特的专职性抗原提呈细胞(antigen-presenting cell, APC),树突状 细胞(dendritic cell, DC )是目前发现的唯一能直接活化初始型(naive) T细胞的 APC, 其在诱导 T 细胞免疫应答或免疫耐受的过程中发挥了十分重要的作用。 DC 的功能与其不同的亚群和所处于的功能状态(非成熟、成熟)密切相关。一 般认为，成熟DC激活T细胞以启动免疫应答，而非成熟DC参与免疫耐受的 诱导。近年来研究表明, DC 具有异质性,包含了很多表型各异和功能不同的 细胞亚群，其中，具有负向免疫调控作用的DC亚群的发现及其功能特点与作用 机制的研究是近年来的重要进展之一，此类具有负向免疫调控作用的DC亚群被 成为调节性DC(regulatory DC )。虽然DC对于T细胞功能的研究取得了很大 进展，但到目前为止，对于调节性DC亚群和T细胞相互调控的方式和机制仍不 十分清楚，有待于进一步深入研究。获得性/适应性免疫应答的效应与调控以及免疫耐受的维持多发生于免疫器 官中。学者们在过去的研究中往往关注免疫细胞及其亚群之间的相互作用，而很 少研究免疫器官微环境(包括免疫器官基质细胞和细胞外基质及多种膜分子和可 溶性细胞因子)对免疫细胞的功能以及免疫应答的调控作用。鉴于此，本实验室 开展了脾脏基质外环境对于DC的功能的调控作用以及对于DC调控T细胞反应 的影响，结果发现，以往均被认为终末分化细胞的成熟DC，与脾基质细胞共培 养后能够进一步增殖并分化为一类能够通过分泌NO而抑制T细胞增殖的新型 调节性树突状细胞，我们将之命名为“分化样树突状细胞(differentiated dendritic cell，diffDC)，从而提出了成熟DC并非在终末分化细胞的的观点，其在完成了 抗原递呈任务之后，在次级淋巴器官微环境的作用下，仍能够进一步增殖分化， 发挥免疫负向调控功能。但是，对于调节性DC (diffDC)如何识别危险信号、如 何与T细胞相互精密调控、其发挥负向免疫调控的相关机制是什么？我们并不 十分清楚。考虑到diffDC与已经活化的T细胞相互作用而抑制T细胞增殖，而活化型 T细胞也可能反馈地作用于diffDC，已知活化型T细胞表达一定水平的FasL, 而我们的预实验表明diffDC在胞膜和胞浆内均表达更高水平的Fas，那么，我 们提出的科学问题之一是Fas信号对于diffDC的功能调控如何？ FasL/Fas系统 在活化型T细胞与diffDC的相互调控中起什么作用？如果起作用，其作用机制 是什么？另外一方面，Toll样受体（Toll-like receptors, TLR）是目前发现的极为重 要的一类免疫识别受体，已知DC表达丰富的TLR并通过TLR识别不同的病原 体如LPS、LTA、CpG等病源微生物成分，DC 一旦识别TLR的配体，其功能将 被活化，从而触发一系列免疫应答效应，但是，对于不同DC亚群表达TLR的 种类及其功能意义，研究尚不深入，由此，我们提出的科学问题之二是 diffDC 表达TLR的种类与水平，其对于TLR配体的刺激的反应性和功能变化是什么？ TLR 信号刺激在 diffDC 与 T 细胞的相互调控中起什么作用， 其相关的作用机 制是什么？鉴于目前尚未见Fas信号和TLR信号参与调节性DC的功能调控、特别是 参与调节性DC与T细胞相互调控的报道，我们在本研究中分两部分内容，分 别研究了 Fas信号和TLR信号对调节性DC（diffDC）的调控作用和相关机制， 并在此基础上研究了 Fas信号和TLR信号刺激后的diffDC与T细胞相互调控的 意义。一、Fas信号促进调节性树突状细胞diffDC分泌免疫调控因子及相关机制研究Fas为肿瘤坏死因子受体（TNFR）超家族的成员之一，一般认为Fas与其 配体（FasL）结合后向靶细胞内传递凋亡信号但是，研究表明，并非所有表达 Fas的细胞在Fas信号启动后都发生凋亡，即存在Fas信号的非凋亡效应。我们 以往的实验曾经证明Fas信号不能诱导DC凋亡，反而诱导DC成熟并增强其 抗原提呈功能。在本部分实验中，我们研究了 Fas信号对于调节性DC（diffDC） 功能的调控作用并探讨了其调控作用的相关机制。结果发现，与非成熟DC（immature DC, imDC）和成熟DC （mature DC, maDC） 相比，diffDC能够表达更高水平的Fas，但在脾脏基质细胞ESSC/maDC共培养以 诱导产生diffDC的体系中加入TGF-P中和性抗体，发现能够减弱diffDC上Fas的表 达和ERK的活化。实验表明，diffDC内ERK的活化程度较高，用ERK抑制剂 PD98059抑制diffDC中ERK的活化，发现可以抑制diffDC表达Fas，结果提示，来 源于基质细胞ESSC的TGF-P通过增强diffDC内ERK的活化而促进了 diffDC高表 达Fas。接下去我们想知道Fas信号是否会影响diffDC独特细胞因子谱的表达和对 于CD4 T细胞增殖的抑制作用，结果发现，与imDC相比，Fas活化型抗体Jo2能够 呈剂量依赖和时间依赖性地刺激diffDC分泌更多的IL-10和IP-10，表明Fas信号能 够促进diffDC分泌IL-10和IP-10。Western blot结果显示，与imDC相比，diffDC在 胞浆和核内高表达p-catenin, Jo2刺激可增加diffDC胞浆内和核内卩-catenin水平。 为了进一步确证p-cateni n在Fas信号促进diffDC高分泌IL-10和IP-10中的作用，我 们培养了正常野生型小鼠和p-catenin缺陷小鼠来源的diffDC，结果发现，p-catenin 缺陷diffDC在Jo2刺激后，其分泌IL-10和IP-10的水平显著下降，结果提示Fas信 号上调了diffDC内p-catenin然后由p-catenin促进了其IL-10和IP-10的高分泌。有文 献报道ERK可能做为GSK-3p的骨架蛋白，从而维持p-catenin的稳定。考虑到 diffDC中ERK高度活化，而且在Jo2刺激下diffDC内ERK的磷酸化更加显著，因 此，我们应用ERK抑制剂PD98059预处理diffDC然后再用Jo2刺激diffDC，结果表 明，ERK抑制后能够抑制GSK-3和p-catenin的活化并随之抑制了diffDC分泌IL-10 和IP-10。因此，这些结果表明Fas信号诱导diffDC高分泌IL-10和IP-10是通过活化 了 ERK/GSK-3/p-catenin 信号途径。在免疫反应的动态过程中，DC与周围很多免疫细胞存在相互作用，彼此精 密调节，其中最为重要的便是DC和T细胞的相互作用。目前对两种细胞相互作用 的研究主要集中在DC对T细胞的调控上，而很少关注T细胞（包括活化型T细胞） 反馈性地对DC尤其是调节性DC的调控作用。由此我们想知道是否表达FasL的活 化型CD4 T细胞也能调控diffDC的免疫调节功能。我们检测了在不同diffDC/T细 胞比例的共培养体系的上清中的IL-10和IP-10的分泌，结果发现活化型T细胞在 不同的比例下均能诱导共培养体系分泌更高水平的IL-10和IP-10。胞内染色流式 检测确证IL-10主要来源于diffDC。进一步用transwell实验，发现共培养体系分泌 更高水平的IL-10和IP-10需要diffDC与活化型T细胞之间细胞直接接触。随后我们 使用Fas缺陷小鼠制备diffDC、应用FasL缺陷小鼠制备活化型T细胞，结果发现， 一旦diffDC缺失Fas或活化型T细胞缺失FasL，两者共培养体系中的IL-10和IP-10 的产生显著下降， 表明活化型T细胞通过FasL诱导了高表达Fas的diffDC分泌 IL-10和IP-10。在检测Fas信号对diffDC分泌细胞因子的影响的过程中，我们意 外地发现diffDC高分泌IL-6并且Fas信号刺激能够呈剂量依赖和时间依赖性地促 进diffDC分泌更多的IL-6。Western blot检测和AG490抑制实验发现Jo2刺激促进 diffDC的ERK/STAT3的活化而促进了 IL-6的分泌。同样，活化型T细胞能诱导共 培养体系中的diffDC在较短的时间内快速高分泌IL-6，但此促进效应并不是通 过FasL/Fas信号所介导综上所述，在本部分实验中，我们发现diffDC高表达Fas但抵抗Fas信号诱 导的凋亡。来源于基质细胞ESSC的TGF-p通过活化ERK而促进了diffDC高表达 Fas。Fas信号可促进diffDC高分泌IL-10和IP-10，其机制可能是通过ERK活 化导致GSK-3失活从而上调p-catenin所介导Fas信号也可促进diffDC高分泌IL-6, 其机制可能是通过ERK活化诱导STAT3高度磷酸化有关。Fas/FasL信号参与了 diffDC和活化型T细胞的相互作用后诱导diffDC更高分泌IL-10和IP-10，但是不参 与diffDC更高分泌IL-6的过程。进一步研究表明，Fas信号能够增强diffDC对于抗 原特异性T细胞增殖的负向调节作用。我们的结果提示脾脏内活化的T细胞和 diffDC间的“ cross-talk”可能在免疫应答反应的后期促进diffDC更好地发挥免疫负 向调节作用，从而有利于维持机体免疫功能的稳态。二、TLR信号促进调节性树突状细胞diffDC趋化Th1细胞并抑制其增殖的相关机制研究TLR是一类重要的免疫识别受体，APC通过TLR能够识别病源微生物的特 异性成分，从而启动免疫应答以清除外源异物，因此APC和TLR构成了防御 很多病原体入侵的第一道防线，在固有免疫的活化和适应性免疫启动中起着重要 的作用。TLR对激活固有免疫的重要性已被大家所认同，而TLR对适应性免疫 应答的调控机制也越来越受到人们的关注，那么这群新型调节性DC在各类病原 体的入侵的反应时是如何与免疫效应细胞相互作用，从而发挥其独特的免疫调节 作用呢？ DC和趋化因子都是连接固有免疫和适应性免疫的重要介质，我们对于 炎症情况下 diffDC 的趋化因子的表达知之甚少。通过筛选， 我们发现 diffDC 能够分泌趋化因子IP-10。接下去我们观察了 TLR激动剂对于diffDC分泌IP-10 的影响及其可能的生物学意义。我们发现，与imDC相比，在TLR2配体(LTA)、 TLR3 配体(PolyI:C)、TLR4 配体(LPS)、TLR9 配体(CpG ODN)刺激下，diffDC 可分泌更高水平的IP-10,因为IP-10的分泌与干扰素有关，我们发现TLR激动 剂能够诱导diffDC分泌I型干扰素IFN-a/p，而阻断IFN-a/卩能够抑制TLR激动 剂刺激的diffDC高分泌IP-10，进一步研究表明，IFN-P依赖的IP-10高分泌与 自分泌IFN-a/p促进了 diffDC中IRF-3活性和STAT1的高度活化有关。尤为重 要的是, 趋化和增殖实验提示, TLR 激动剂可以通过诱导高分泌 IP-10 而促进 diffDC有效地趋化Th1而非Th2细胞，并能够显著抑制Th1增殖。综上所述，在本部分实验中，我们发现diffDC在TLR激动剂刺激后可分 泌更高水平的 IP-10， 其机制是由于 TLR 激动剂诱导 diffDC 分泌 I 型干扰素 (IFN-a/p)，然后自分泌I型干扰素促进了 diffDC的IRF-3表达和STAT1磷酸 化，最终导致TLR激动剂促进diffDC高分泌IP-10。进一步研究表明，diffDC 通过高分泌IP-10选择性地趋化Th1细胞并抑制Th1细胞的增殖，从而提出了调 节性DC参与T细胞应答反应的负向调控的新方式新机制。总之，我们的实验结果表明，脾脏基质细胞来源的TGF-P诱导的ERK活化促 进了 diffDC高表达Fas。Fas信号通过ERK所介导的GSK-3的失活而上调了 P-catenin表达，从而诱导diffDC高分泌IL-10和IP-10；活化型T细胞能够通过 FasL/Fas促进diffDC高分泌IL-10和IP-10,有意义的是，Fas能够增强diffDC对于 抗原特异性CD4 T细胞的增殖反应。另外一方面的研究表明，TLR激动剂刺激能 够通过IRF3/IFNP/STAT1通路促进diffDC更高地分泌IP-10,使得diffDC有效地趋 化Thl细胞并显著抑制Thl细胞的增殖。可见，Fas信号和TLR信号能够通过不同 的信号转导通路而促进调节性DC负向调控CD4 +T细胞反应，参与了调节性DC 与T细胞的相互调控作用。本实验结果为今后进一步研究新型调节性DC（diffDC） 在维持机体免疫稳态中的生理作用，以及在病理状态下对免疫反应的负向调控提 供了一定的实验基础，为探索肿瘤、自身耐受和自身免疫性疾病等的发生机制及 免疫治疗的应用提供了新的研究途径。关键词：调节性树突状细胞，T细胞，免疫微环境，Fas, TLR,信 号传导，免疫调节Fas and TLR signaling promotes suppression of CD4 T cellresponse by regulatory dendritic cells and the underlyingmechanismsQian ChengABSTRACTDendritic cells (DCs), the most potent professional antigen-presenting cells (APCs), are critical initiator of the immune response and regulator of immune tolerance because of their unique ability to directly prime nai ve T cells. The functions of DCs depend on the mature state or different subsets. Generally accepted, mature DCs (maDCs) prefer to initiate T cell response, and immature DCs (imDCs) living in the peripheral non-lymphoid organ are involved in the induction of immune tolerance. However, once capture of the invaded microbial antigens, imDCs will become maturation and migrate to the peripheral lymphoid organs where they prime T cell immune response. Recent studies show that DCs are heterogeneous cell population, with many kinds of DC subsets exhibiting different phenotype and functions. Among the DCs subsets, one population of DCs with negative immune regulation, so-called regulatory DCs, attracts much attention, and more evidences show that the regulatory DCs play important roles in the regulation of immune response and also the pathogenesis of immunological disorders. However, the detailed mechanisms by which regulatory DCs control the T cell response, and whats about the reciprocal interaction between regulatory DCs and T cells remain to be fully understood.The control of adaptive immune effector cells and maintenance of immune tolerance are exerted mainly in the lymph organs. For example, DCs uptake antigens from peripheral tissues and then present antigens to T cells and trigger the immune response in the lymphoid organs. In the past, the researchers have already found that DCs present antigens to T cells, meanwhile DCs receive signals from T cells which may lead to apoptosis of DCs to downregulate the immune response. However, the most researchers focused the interaction of DCs and T cells, but had ignored the effect of the lymphoid microenvironment on the interaction of DCs and T cells. There are abundance of cytokines and protein molecules expressed by stromal cells in the microenvironment, and the soluble factors and cell-to-cell contact of stromal microenvironment may play important roles to regulate the function of immune cells. During investigation of the fate of the maDCs after their antigen presentation and T cell activation in the secondary lymphoid organs, we observed the effect of splenic stromal cells on fate and functions of maDCs. Our previous study demonstrate that endothelial-like splenic stromal cells (ESSCs), which mimic the secondary lymph organ microenvironment, can drive maDCs to proliferate and further differentiate into a unique subset of regulatory DCs (we designated this new subset of DCs as diffDCs, the differentiated DCs). diffDCs have been shown to inhibit CD4 T cell proliferation via secretion of NO, exerting negative feedback control of T cell immune response. Although the role of immature and mature DCs in regulating T cell tolerance and T cell activation has been abundantly documented, however, little is known about the mechanisms how regulatory DCs (diffDCs) suppress CD4 T cell proliferation and whats reciprocal regulation of diffDCs and T cells, especially activated CD4 T cells at the late stage of immune response.Considering that the activated T cells may directly interact with regulatory diffDCs and the activated T cells can express high level of FasL, together with our previous study showing Fas ligation may induce DCs maturation but not apoptosis, so, we are wondering whether Fas signal can regulate the function of diffDCs, and whether FasL-expressing T cells can feedback regulate the functions of diffDCs? If so, whats the underlying mechanisms? In other hand, DCs, as professional APCs, can express abundant Toll-like receptors (TLRs) to recognize the pathogenic components. Once triggered by TLR ligands, DCs will be activated to produce large amount cytokines and chemokines, upregulate expression of molecules for antigen presentation and T cell activation. Up to now, the profile of TLR expression by DC subsets and what s the biological significance of the TLR expression for DC subsets need to be fully investigated. Therefore, we are wondering whats the response of regulatory diffDCs to TLR ligands, and whats the effect of TLR ligands on the function of diffDCs to regulate T cell response. Further, whats the underlying mechanisms for the regulation of diffDCs through ligation of TLR ligands?Up to now, there is no report about the functional regulation of diffDCs by Fas and TLR signals. Also, there is no report about the role of Fas and TLR signals in the reciprocal regulation of diffDCs and T cells. Therefore, in this study, we have two parts to investigate the effects of Fas signal or TLR signal on the function of regulatory diffDCs and their reciprocal interaction with the activated T cells.Part I Fas signaling promotes regulatory dendritic cells (diffDCs) to secrete cytokines with immune regulatory functions and the underlying mechanismsFas and FasL are typical members of the TNF receptor and TNF ligand family, respectively. They play a pivotal role in the regulation of apoptotic processes, including activation-induced cell death, T-cell-induced cytotoxicity, immune privilege and escape. Our previous study showed that Fas ligation could trigger DCs to secrete proinflammatory cytokines like IL-lp, resulting in DC maturation instead of DC apoptosis. However, we don t know what s the effect of spleen stromal cells on the Fas expression of regulatory diffDC. And we are wondering whether Fas ligation can affect the unique cytokine secretion and T cell-suppressive capability of diffDCs. On the basis of our preliminary observation that diffDC could spontaneously express significantly higher level of Fas than imDCs and maDCs as demonstrated by screening the membrane molecules expression on regulatory diffDCs by flow cytometry, we stimulated diffDCs with Jo2 (the agonist mAb to Fas) to observe the effect of the Fas signaling on the cytokine expression profiles and function of diffDCs, and then investigated the related mechanisms.Our results demonstrate that the diffDCs could spontaneously express significantly higher level of Fas than imDCs and maDCs. When the neutralizing antibody to TGF-0 was added to the coculture system of endothelial-like splenic stromal cells (ESSCs) and maDCs to prepare diffDCs, we found that blockade of TGF-p could decrease Fas expression and ERK activation in diffDCs. In addition, ERK overactivation was observed in diffDCs at the steady state, so we assayed the levels of Fas by diffDCs cultured in the presence of ERK inhibitor PD98059, and found that Fas expression in diffDCs was decreased. These results demonstrate that increased ERK activation in diffDCs by stroma-derived TGF-p contributes to the high level of Fas expression in diffDCs. Next, we analyzed whether Fas ligation would influence the unique cytokine secretion of diffDCs. We found that Jo2 could stimulate diffDC to produce more IL-10 and IP-10 in the dose- and time-dependent manners, and Fas-ligated diffDC could inhibit antigen-specific CD4+T cell proliferation more significantly. Furthermore, after stimulation with Jo2, higher level of p-catenin could be detected in nuclear and cytoplasmic extracts of diffDCs than imDCs. As expected, diffDCs expressed sharply higher nuclear p-catenin at resting state, and the nuclear translocation of p-catenin was more markedly following Jo2 stimulation as comapred to that in imDCs. Although Jo2 stimulation could induce p-catenin cytoplasmic expression early at 30min in imDCs, the amounts of p-catenin in cytoplasm of diffDCs was enchanced more than that in imDCs. To further confirm the role of p-catenin in the elevated production of IL-10 and IP-10 in diffDCs, we assayed IL-10 and IP-10 production by diffDCs and imDCs prepared from p-catenin deficient mice. We found that p-catenin deficiency impaired the secretion of IL-10 and IP-10 in Fas-ligated diffDCs. Recent evidences show that ERK may serve as a scaffold to hold GSK-3p which affect s the stability of p-catenin. As ERK was more significantly phosphorylated in diffDCs following Jo2 stimulation, to further investigate the relationship between ERK and p-catenin, diffDCs and imDCs were stimulated with Jo2 in the presence of PD98059. PD98059 pretreatment could inhibit the activation of GSK-3p and p-catenin. Together, these results suggest that diffDCs with or without Fas ligation could preferentially secret IL-10 and IP-10 by activating ERK/GSK-3p/p-catenin pathways.During the induction phase and effector phase of immune response, DCs can interact with many kinds of immune cells. Among the cross-talks, the reciprocal regulation between DCs and T cells attract much attention, however, most of studies focused on the effect of DCs on the function of T cells, up to now, few reports were described about the feedback effect of the activated T cells on the function of DCs, especially, there is no report about the regulatory effect of the activated CD4 T cells on the function of regulatory DC. As the activated T cells express FasL and the diffDCs express Fas, next, we wondered whether the activated T cells regulate the function of diffDCs via FasL/Fas system? We examined IL-10 and IP-10 production in supernatants of coculture of diffDCs-T cells at different diffDC/T ratios. IL-10 and IP-10 production was increased by the activated T cells at different ratios of diffDC/T, and the increased IL-10 secretion was mainly from diffDC as demonstrated by intracellular staining. The induction of IL-10 and IP-10 by the activated T cells in diffDCs/T coulture was dramatically higher than that in imDCs/T or maDCs/T coulture. IL-10 and IP-10 production in diffDCs /T coculture could be induced rapidly. These results suggest that the activated T cells could condition diffDCs to enhance the unique function during an immune response. We then investigated whether cell-to-cell direct contact from the activated T cells was required for the increased IL-10 and IP-10 production by diffDCs. By using a transwell system, we found IL-10 and IP-10 production by diffDCs reduced significantly once diffDCs were separated from the activated T cells in the coculture system, however, IL-10 and IP-10 production by diffDCs in Transwell system was still higher than that by diffDCs cultured alone. With the diffDCs prepared from Fas-deficient mice and the activated T cells prepared the FasL-deficient mice, we found IL-10 and IP-10 production in the coculture decreased significantly, thus convincingly conforming the critical role of Fas/FasL in the cytokines production of diffDCs induced by the activated T cells.During our investigation of the influence of Fas ligation on the unique cytokine production of diffDCs, we surprisingly found that diffDCs could secrete significantly high level of IL-6, and Jo2 stimulation could enhance IL-6 production of diffDCs in the dose- and time-dependent manner. Interestingly, diffDCs express sharply higher STAT-3 at resting state, and this molecule is more significantly phosphorylated in diffDCs following Jo2 stimulation as compared to that in imDCs. After diffDCs were stimulated with Jo2 in the presence of STAT3 inhibitor AG490, we found that the production of IL-6 in diffDCs decreased. Meanwhile, the ERK inhibitor PD98059 could also decrease the phosphorylation of STAT3 in diffDCs stimulated with Jo2. The data suggest that Fas ligation-increased IL-6 production by diffDCs is STAT3 and ERK dependent. We then examined IL-6 production in supernatant of diffDC-T cocultures at different diffDC/T ratios and found that IL-6 production by diffDCs was enhanced by the activated T cells, but Fas/FasL is not involved in the increased IL-6 production of diffDCs induced by the activated T cells.In summary of this part of our study, we demonstrate that induction of ERK activation by endothelial-like splenic stromal cell (ESSC)-derived TGF- 0 is responsible for the high Fas expression of diffDCs derived from maDCs cocultured with ESSCs. Fas ligation induces preferential secretion of IL-10 and IP-10 by diffDC though ERK-mediated inactivation of GSK-30 leading to the uprgulation of 0 -catenin. Interestingly, diffDCs secrete high level of IL-6 through STAT3 activation mediated by ERK overactivation. Importantly, FasL-expressing the activated T cells can promote Fas-expressing diffDCs to secret more IL-10 and IP-10 via FasL/Fas interaction. Fas-ligated diffDCs could suppress maDC-initiated antigen-specific CD4 T cell proliferation more significantly. Therefore, Fas signal can promote regulatory DCs to secrete more cytokines for the potent regulation of T cell immune response.Part II TLR agonists promote regulatory dendritic cells (diffDCs) to attract more Th1 cells via IP-10 and subsequently inhibit Th1 proliferationTLR are abundantly expressed on professional antigen-presenting cells such as macrophages and DCs, and serve as an important link between the innate and adaptive immune responses by discriminating among molecular patterns associated with microbial components and subsequently initiate immune response. DCs and chemokines are important mediators linking innate and adaptive immunity upon activation by TLR agonists. Its well accepted that TLR-triggered signaling plays important roles in the innate immunity, the also, the role of TLR ligands in the regulation of adaptive immunity attract much more attention then before now. Up to now, little is know about the expression profile of TLR and chemokines by regulatory DCs, whats the responsiveness of regulatory DCs to the stimulation with TLR ligands, and how about the secretion of chemokines by regulatory DC in response to TLR ligands.By screening the chemokine expression pattern by diffDCs, we found diffDCs preferentially express CXCR3 chemokine IFN- -induced protein-10 (IP-10). In this part of our study, we investigated the mechanisms for the increased IP-10 production by regulatory diffDCs after stimulated with TLR agonists, and what s the biological significance of the increased IP-10 production in the reciprocal regulation between DCs and T cells. We found that stimulation with TLR 2, 4, 3, 9 ligands induced di