Interactions Between TLR7 and TLR9 Agonists and Receptors Regulate Innate Immune Responses by Astrocytes and Microglia

Toll样受体7(TLR7)和Toll样受体9(TLR9)是固有免疫反应中的重要介质。它们均定位于内涵体间隔,识别核酸和经过髓样分化因子88(MyD88)传导来的信号。本文分析TLR7和TLR9导致的在中枢神经系统固有免疫反应中起作用的星形胶质细胞和小胶质细胞的活化。TLR7和TLR9激动剂引起星形胶质细胞和小胶质细胞相似的细胞因子反应,但二者仍有区别,小胶质细胞产生抗炎因子IL-10、抗凋亡因子G-CSF及IL-9,但星形胶质细胞不产生。对2种细胞的共刺激研究均显示,TLR7激动剂——咪喹莫特,能浓度依赖性地抑制TLR9激动剂引起的固有免疫反应。令人惊讶的是,这种抑制并非由TLR7介导,因为在TLR7的缺乏的情况下,TLR9激动剂导致的固有免疫反应仍然被抑制。固有免疫反应的抑制也并非由于抑制TLR9激动剂摄取所致。这提示咪喹莫特的抑制效应可能是直接作用,也许是通过阻滞CpG-ODN结合和/或TLR9介导的信号转导所致,从而限制细胞活化。这种拮抗关系也存在于小胶质细胞中的这2类受体之间,TLR7的缺乏导致CpG-ODN刺激的细胞因子反应增强。因此,在胶质细胞中,TLR7及其激动剂对TLR9介导的细胞因子反应有抑制作用。     

Toll样受体4在免疫相关细胞中的作用

Toll样受体(TLRs)是参与固有免疫的重要蛋白分子,也是连接固有免疫和适应性免疫的桥梁。TLR4是TLRs家族的重要成员之一。TLR4通过调控中性粒细胞和单核巨噬细胞的活化,可促进细胞因子的释放,启动固有免疫应答。同时TLR4能促进树突状细胞的成熟,激活初始T细胞,启动适应性免疫应答。在T细胞和B细胞的适应性免疫应答中,TLR4可促进细胞分泌细胞因子,介导全身炎症反应,并调控记忆性细胞的生成。本文就TLR4在中性粒细胞、树突状细胞、单核巨噬细胞、T淋巴细胞和B淋巴细胞中的作用进行综述。     

miR-363-3p促进人浆细胞样树突细胞干扰素α分泌的机制研究

目的 :观察miR-363-3p对人浆细胞样树突细胞(plasmacytoid dendritic cells,pDCs)分泌干扰素α(interferon,IFN-α)的影响,探讨其在Ⅰ型IFN通路中的调控作用及其可能的作用机制。方法:通过磁珠分选获得高纯度的人外周血pDCs,予TLR7刺激剂活化。采用体外脂质体转染使pDCs高表达miR-363-3p,检测其活化后分泌的IFN-α。采用生物信息学预测miR-363-3p可能作用的靶蛋白,并用双荧光报告基因系统证实其与靶蛋白间的相互作用。结果:磁珠分选获得的pDCs纯度大于95%,其活化后分泌高浓度的IFN-α。体外过表达miR-363-3p可促进pDCs分泌IFN-α。生物信息学预测miR-363-3p作用的靶蛋白可能为信号调节蛋白α(signal-regulatory protein alpha,SIRPα);双荧光报告实验结果提示,miR-363-3p过表达能显著抑制重组SIRPα3′UTR荧光素酶表达载体的荧光素酶活性。结论:miR-363-3p可能通过作用于其靶基因SIRPα,进而促进pDCs分泌IFN-α。本研究进一步阐明了Ⅰ型IFN通路的调控机制,为Ⅰ型IFN相关自身免疫病的诊断、治疗提供了新的生物学靶点。     

α-干扰素诱导外周血单核细胞向树突状细胞分化

背景：研究表明，树突状细胞接受刺激的性质和微环境及其起源是决定免疫反应中初始CD4^＋T细胞发生Th1或Th2应答的关键因素，树突状细胞是否成熟对其功能的发挥影响很大。 目的：观察广泛应用的细胞因子α-干扰素对人外周血单核细胞向树突状细胞分化成熟的影响。 设计、时间及地点：细胞水平的观察对照实验，于2007-05／12在深圳市人民医院临床医学研究中心实验室完成。 材料：取10名健康成人志愿者的外周血。 方法：采集外周血10mL，肝素抗凝，常规淋巴细胞分离液分离单核细胞，然后将单核细胞与粒-巨噬细胞集落刺激因子、白细胞介素4体外培养7d。分3组进行树突状细胞培养，对照组不加α-干扰素；100U／L α-干扰素组于培养第5天加入100U／L的α-干扰素；300U／Lα-干扰素组于培养第5天加入300U／L的α-干扰素。主要观察指标：培养第7天用流式细胞术检测树突状细胞膜表面CD83和MHC—DR表达水平；应用四氮唑盐法测定树突状细胞刺激同种异体T细胞增殖的能力：采用酶联免疫吸附法检测树突状细胞培养上清中白细胞介素12p40＋p70含量。 结果：100，300U／Lα-干扰素组中CD83^＋和MHC—DR^＋树突状细胞的数量、刺激同种异体T细胞增殖的能力及培养上清液中白细胞介素12p40＋p70水平均显著高于对照组（P〈0．01），并以300U／Lα-干扰素组作用更强。 结论：α-干扰素可以有效促进单核细胞源树突状细胞的功能成熟，并呈剂量依赖效应。     

急性乙型肝炎患者pDCs上CD86表达变化及意义

目的 探讨外周血浆细胞样树突状细胞(pDCs)在急性乙型肝炎(AHB）患者临床转归中的变化及作用。方法 纳入2015年6月至2017年5月收治的急性乙型肝炎(AHB组)患者40例， 给予退黄、降酶、保肝等药物，并加用恩替卡韦0．5 mg空腹口服，1次/d。另纳入健康体检者26例为健康对照组（ HC组）。HC组于体检当天、AHB组于治疗前及治疗后6周采集外周静脉全血，采用ELISA法检测2组血浆HBV DNA、HBsAg、HbeAg及肝功能指标水平，并应用流式细胞仪检测pDCs的频数及其功能分子CD86的表达水平。结果 治疗6周后，AHB组临床症状显著缓解，血清学指标和乙型肝炎标志物水平显著下降（P<0.01）。治疗6周后，AHB组HBV DNA转阴率为82.5%，HBsAg清除率为72.5%，HBeAg血清学转换率为75.0%，与治疗前比较差异均有统计学意义（P<0.01）, HC组pDCs频数显著高于AHB组治疗前。AHB组治疗后CD86+pDCs显著高于治疗前。HC组CD86ABC显著低于AHB组治疗前。结论 在急性乙型肝炎患者外周血pDCs的数量降低。急性乙型肝炎患者在治疗后CD86+pDCs增多。急性乙型肝炎患者pDCs上CD86表达上调。     

皮肤的免疫功能

皮肤和皮肤免疫细胞为机体遭受的损伤和感染提供免疫保护。角质形成细胞、皮肤树突状细胞和T细胞组成皮肤重要的免疫细胞。角质形成细胞通过Toll样受体和炎症复合体等预警系统感知危险因素侵扰,角质形成细胞活化后能释放细胞因子、趋化因子和抗菌肽,帮助启动皮肤免疫应答,同时招募其它固有免疫细胞参与早期固有免疫反应。皮肤树突状细胞,如表皮内分布的朗格汉斯细胞和真皮树突状细胞,能捕获摄入抗原并游走至皮肤引流区淋巴结将抗原递呈给幼稚T细胞从而启动皮肤特异性免疫应答。近年来的研究改变了人们对朗格汉斯细胞作为经典的树突状细胞在皮肤免疫反应作用的认识。越来越多的证据表明皮肤常驻T细胞比外周血中循环的T细胞在维持皮肤免疫稳态和皮肤病理状态下发挥更重要的作用。本文将着重介绍皮肤免疫细胞研究的最新进展以及皮肤免疫细胞在皮肤稳态和疾病中的作用。     

韦格纳肉芽肿患者外周血免疫细胞TLR_4的表达

目的检测韦格纳肉芽肿(WG)患者外周血免疫细胞特别是树突状细胞(DC)上Toll样受体4(TLR4)的表达有无异常。方法流式细胞仪检测正常人及WG患者外周血免疫细胞表面TLR4蛋白的表达。结果 TLR4在WG患者外周血中性粒细胞(PMN)、单核细胞、BDCA3+DC、CD11c+DC和DC样单核细胞-CD14+CD16+单核细胞上的表达与正常人无显著差异,但在淋巴细胞上的表达显著高于正常人;活动期与非活动期患者、环磷酰胺和甲氨蝶呤治疗组患者TLR4的表达无显著差异。结论 TLR4在WG患者外周血DC、DC样单核细胞及免疫细胞的表达无明显异常。     

小鼠骨髓来源树突状细胞吲哚胺2,3-过氧化酶表达对T细胞增殖的影响

目的:诱导树突状细胞产生吲哚胺2,3-过氧化酶,从而调节T细胞反应,可能是诱导器官移植免疫耐受的理想途径.观察小鼠骨髓来源的树突状细胞本身及其在多种因素刺激活化状态下吲哚胺2,3-过氧化酶的表达变化,分析其对T细胞增殖的影响.方法:实验于2005-01/08在解放军第二军医大学免疫研究所完成,动物实验方法符合动物伦理学要求.①培养C57BL/6小鼠骨髓来源的树突状细胞,并分别利用脂多糖80 μg/L、CD40L500 μg/L、γ-干扰素50 μg/L及三者联合作用于培养7 d的上述细胞,利用反转录聚合酶链反应检测各组树突状细胞中吲哚胺2,3-过氧化酶mRNA的表达.②培养BALB/C小鼠脾脏来源的T淋巴细胞做为静息T淋巴细胞,并采用抗CD3单抗及抗CD28单抗各1 mg/L刺激T淋巴细胞作为活化T淋巴细胞.将树突状细胞及γ–干扰素作用下的树突状细胞与静息T淋巴细胞及活化T淋巴细胞共同培养,利用混合淋巴细胞反应法测定其刺激T细胞增殖的能力.结果:①培养7 d的树突状细胞及在脂多糖、CD40L刺激下的树突状细胞均未见吲哚胺2,3-过氧化酶mRNA的表达,而γ–干扰素及γ–干扰素 脂多糖 CD40L三者联合刺激下的树突状细胞可检测到吲哚胺2,3-过氧化酶 mRNA的表达.②与树突状细胞相比,γ–干扰素活化的树突状细胞可明显抑制T细胞增殖(P < 0.01),加入色氨酸可使对T细胞增殖的抑制作用有所恢复,同树突状细胞比较差异亦具有显著性意义(P < 0.01).结论:活化的树突状细胞可以产生吲哚胺2,3-过氧化酶,通过降解色氨酸发挥抑制T细胞增殖的作用,可能在移植排斥反应中发挥效应.     

韦格纳肉芽肿患者外周血免疫细胞TLR4的表达

目的 检测韦格纳肉芽肿（WG）患者外周血免疫细胞特别是树突状细胞（DC）上Toll样受体4（TLR4）的表达有无异常.方法 流式细胞仪检测正常人及WG患者外周血免疫细胞表面TLR4蛋白的表达.结果 TLR4在WG患者外周血中性粒细胞（PMN）、单核细胞、BDCA3＋DC、CD11c＋DC和DC样单核细胞-CD14＋CD16＋单核细胞上的表达与正常人无显著差异,但在淋巴细胞上的表达显著高于正常人;活动期与非活动期患者、环磷酰胺和甲氨蝶呤治疗组患者TLR4的表达无显著差异.结论 TLR4在WG患者外周血DC、DC样单核细胞及免疫细胞的表达无明显异常.     

由外周血单个核细胞衍生的树突状细胞抗癌作用研究

在身体内复杂的免疫细胞网络系统中,树突状细胞是最新近才被认识的。淋巴系造血细胞已被全面了解,具有向T淋巴细胞递呈抗原能力的树突状细胞被称作专职抗原递呈细胞,并在启动免疫反应时起关键作用。已公认存在骨髓树突状细胞(DCI)和淋巴系树突状细胞(DC2),其可在异类抗原出现时诱导机体产生免疫和免疫耐受,因为这些潜在的免疫调节功能,树突状细胞已被用于免疫治疗,尤其用于肿瘤免疫接种方面。抱着完全应用于癌症治疗的目的,作者实验室完成了关于人类树突状细胞的实验研究。简介如下:     

Imiquimod clears tumors in mice independent of adaptive immunity by converting pDCs into tumor-killing effector cells

Imiquimod is a synthetic compound with antitumor properties; a 5% cream formulation is successfully used to treat skin tumors. The antitumor effect of imiquimod is multifactorial, although its ability to modulate immune responses by triggering TLR7/8 is thought to be key. Among the immune cells suggested to be involved are plasmacytoid DCs (pDCs). However, a direct contribution of pDCs to tumor killing in vivo and the mechanism of their recruitment to imiquimod-treated sites have never been demonstrated. Using a mouse model of melanoma, we have now demonstrated that pDCs can directly clear tumors without the need for the adaptive immune system. Topical imiquimod treatment led to TLR7-dependent and IFN-α/β receptor 1-dependent (IFNAR1-dependent) upregulation of expression of the chemokine CCL2 in mast cells. This was essential to induce skin inflammation and for the recruitment of pDCs to the skin. The recruited pDCs were CD8α+ and induced tumor regression in a TLR7/MyD88- and IFNAR1-dependent manner. Lack of TLR7 and IFNAR1 or depletion of pDCs or CD8α+ cells from tumor-bearing mice completely abolished the effect of imiquimod. TLR7 was essential for imiquimod-stimulated pDCs to produce IFN-α/β, which led to TRAIL and granzyme B secretion by pDCs via IFNAR1 signaling. Blocking these cytolytic molecules impaired pDC-mediated tumor killing. Our results demonstrate that imiquimod treatment leads to CCL2-dependent recruitment of pDCs and their transformation into a subset of killer DCs able to directly eliminate tumor cells.     

Flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing T helper type 1 and 2 cell development: dependency on antigen dose and differential toll-like receptor ligation

Distinct dendritic cell (DC) subsets have been suggested to be preprogrammed to direct either T helper cell (Th) type 1 or Th2 development, although more recently different pathogen products or stimuli have been shown to render these DCs more flexible. It is still unclear how distinct mouse DC subsets cultured from bone marrow precursors, blood, or their lymphoid tissue counterparts direct Th differentiation. We show that mouse myeloid and plasmacytoid precursor DCs (pDCs) cultured from bone marrow precursors and ex vivo splenic DC subsets can induce the development of both Th1 and Th2 effector cells depending on the dose of antigen. In general, high antigen doses induced Th1 cell development whereas low antigen doses induced Th2 cell development. Both cultured and ex vivo splenic plasmacytoid-derived DCs enhanced CD4(+) T cell proliferation and induced strong Th1 cell development when activated with the Toll-like receptor (TLR)9 ligand CpG, and not with the TLR4 ligand lipopolysaccharide (LPS). The responsiveness of plasmacytoid pDCs to CpG correlated with high TLR9 expression similarly to human plasmacytoid pDCs. Conversely, myeloid DCs generated with granulocyte/macrophage colony-stimulating factor enhanced Th1 cell development when stimulated with LPS as a result of their high level of TLR4 expression. Polarized Th1 responses resulting from high antigen dose were not additionally enhanced by stimulation of DCs by TLR ligands. Thus, the net effect of antigen dose, the state of maturation of the DCs together with the stimulation of DCs by pathogen-derived products, will determine whether a Th1 or Th2 response develops.     

Plasmacytoid dendritic cells induce NK cell-dependent, tumor antigen-specific T cell cross-priming and tumor regression in mice

A prerequisite for strong adaptive antiviral immunity is the robust initial activation of the innate immune system, which is frequently mediated by TLR-activated plasmacytoid DCs (pDCs). Natural antitumor immunity is often comparatively weak, potentially due to the lack of TLR-mediated activation signals within the tumor microenvironment. To assess whether pDCs are capable of directly facilitating effective antitumor immune responses, mice bearing established subcutaneous B16 melanoma tumors were administered TLR9-activated pDCs directly into the tumor. We found that TLR9-activated pDCs induced robust, spontaneous CTL cross-priming against multiple B16 tumor antigens, leading to the regression of both treated tumors and untreated tumors at distant contralateral sites. This T cell cross-priming was mediated by conventional DCs (cDCs) and was completely dependent upon the early recruitment and activation of NK cells at the tumor site. NK cell recruitment was mediated by CCR5 via chemokines secreted by pDCs, and optimal IFN-gamma production by NK cells was mediated by OX40L expressed by pDCs. Our data thus demonstrated that activated pDCs are capable of initiating effective and systemic antitumor immunity through the orchestration of an immune cascade involving the sequential activation of NK cells, cDCs, and CD8(+) T cells.     

A crucial role for plasmacytoid dendritic cells in antiviral protection by CpG ODN-based vaginal microbicide

Topical microbicides represent a promising new approach to preventing HIV and other sexually transmitted infections. TLR agonists are ideal candidates for microbicides, as they trigger a multitude of antiviral genes effective against a broad range of viruses. Although vaginal application of CpG oligodeoxynucleotides (ODNs) and poly I:C has been shown to protect mice from genital herpes infection, the mechanism by which these agents provide protection remains unclear. Here, we show that plasmacytoid DCs (pDCs) are required for CpG ODN-mediated protection against lethal vaginal challenge with herpes simplex virus type 2 (HSV-2). Moreover, we demonstrate that cells of both the hematopoietic and stromal compartments must respond to CpG ODN via TLR9 and to type I IFNs through IFN-alphabeta receptor (IFN-alphabetaR) for protection. Thus, crosstalk between pDCs and vaginal stromal cells provides for optimal microbicide efficacy. Our results imply that temporally and spatially controlled targeting of CpG ODN to pDCs and epithelial cells can potentially maximize their effectiveness as microbicides while minimizing the associated inflammatory responses.     

HIV-activated human plasmacytoid DCs induce Tregs through an indoleamine 2,3-dioxygenase-dependent mechanism

Plasmacytoid DCs (pDCs) have been implicated as crucial cells in antiviral immune responses. On recognizing HIV, they become activated, secreting large amounts of IFN-α and inflammatory cytokines, thereby potentiating innate and adaptive antiviral immune responses. Here, we have shown that HIV-stimulated human pDCs can also induce the differentiation of naive CD4⁺ T cells into Tregs with suppressive function. This differentiation was independent of pDC production of IFN-α and primarily dependent on pDC expression of indoleamine 2,3-dioxygenase, which was induced through the TLR/MyD88 pathway, following binding of HIV to CD4 and triggering of TLR7 by HIV genomic RNA. Functionally, the Tregs induced by pDCs were shown to inhibit the maturation of bystander conventional DCs. This study therefore reveals what we believe to be a novel mechanism by which pDC may regulate and potentially limit anti-HIV immune responses.     

Tumoricidal activity of TLR7/8-activated inflammatory dendritic cells

Imiquimod (IMQ), a synthetic agonist to Toll-like receptor (TLR) 7, is being successfully used for the treatment of certain skin neoplasms, but the exact mechanisms by which this compound induces tumor regression are not yet understood. While treating basal cell carcinoma (BCC) patients with topical IMQ, we detected, by immunohistochemistry, sizable numbers of both myeloid dendritic cells (mDCs) and plasmacytoid DCs (pDCs) within the inflammatory infiltrate. Surprisingly, peritumoral mDCs stained positive for perforin and granzyme B, whereas infiltrating pDCs expressed tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The biological relevance of this observation can be deduced from our further findings that peripheral blood-derived CD11c(+) mDCs acquired antiperforin and anti-granzyme B reactivity upon TLR7/8 stimulation and could use these molecules to effectively lyse major histocompatibility complex (MHC) class I(lo) cancer cell lines. The same activation protocol led pDCs to kill MHC class I-bearing Jurkat cells in a TRAIL-dependent fashion. While suggesting that mDCs and pDCs are directly involved in the IMQ-induced destruction of BCC lesions, our data also add a new facet to the functional spectrum of DCs, ascribing to them a major role not only in the initiation but also in the effector phase of the immune response.     

Spatiotemporal trafficking of HIV in human plasmacytoid dendritic cells defines a persistently IFN-α-producing and partially matured phenotype

Plasmacytoid DCs (pDCs) are innate immune cells that are specialized to produce IFN-α and to activate adaptive immune responses. Although IFN-α inhibits HIV-1 replication in vitro, the production of IFN-α by HIV-activated pDCs in vivo may contribute more to HIV pathogenesis than to protection. We have now shown that HIV-stimulated human pDCs allow for persistent IFN-α production upon repeated stimulation, express low levels of maturation molecules, and stimulate weak T cell responses. Persistent IFN-α production by HIV-stimulated pDCs correlated with increased levels of IRF7 and was dependent upon the autocrine IFN-α/β receptor feedback loop. Because it has been shown that early endosomal trafficking of TLR9 agonists causes strong activation of the IFN-α pathway but weak activation of the NF-κB pathway, we sought to investigate whether early endosomal trafficking of HIV, a TLR7 agonist, leads to the IFN-α-producing phenotype we observed. We demonstrated that HIV preferentially traffics to the early endosome in human pDCs and therefore skews pDCs toward a partially matured, persistently IFN-α-secreting phenotype.     

Plasmacytoid dendritic cells sense skin injury and promote wound healing through type I interferons

Plasmacytoid dendritic cells (pDCs) are specialized type I interferon (IFN-α/β)-producing cells that express intracellular toll-like receptor (TLR) 7 and TLR9 and recognize viral nucleic acids in the context of infections. We show that pDCs also have the ability to sense host-derived nucleic acids released in common skin wounds. pDCs were found to rapidly infiltrate both murine and human skin wounds and to transiently produce type I IFNs via TLR7- and TLR9-dependent recognition of nucleic acids. This process was critical for the induction of early inflammatory responses and reepithelization of injured skin. Cathelicidin peptides, which facilitate immune recognition of released nucleic acids by promoting their access to intracellular TLR compartments, were rapidly induced in skin wounds and were sufficient but not necessary to stimulate pDC activation and type I IFN production. These data uncover a new role of pDCs in sensing tissue damage and promoting wound repair at skin surfaces.     

Type I interferon dependence of plasmacytoid dendritic cell activation and migration

Differential expression of Toll-like receptor (TLR) by conventional dendritic cells (cDCs) and plasmacytoid DC (pDCs) has been suggested to influence the type of immune response induced by microbial pathogens. In this study we show that, in vivo, cDCs and pDCs are equally activated by TLR4, -7, and -9 ligands. Type I interferon (IFN) was important for pDC activation in vivo in response to all three TLR ligands, whereas cDCs required type I IFN signaling only for TLR9- and partially for TLR7-mediated activation. Although TLR ligands induced in situ migration of spleen cDC into the T cell area, spleen pDCs formed clusters in the marginal zone and in the outer T cell area 6 h after injection of TLR9 and TLR7 ligands, respectively. In vivo treatment with TLR9 ligands decreased pDC ability to migrate ex vivo in response to IFN-induced CXCR3 ligands and increased their response to CCR7 ligands. Unlike cDCs, the migration pattern of pDCs required type I IFN for induction of CXCR3 ligands and responsiveness to CCR7 ligands. These data demonstrate that mouse pDCs differ from cDCs in the in vivo response to TLR ligands, in terms of pattern and type I IFN requirement for activation and migration.     

NK cell activation in visceral leishmaniasis requires TLR9, myeloid DCs, and IL-12, but is independent of plasmacytoid DCs

Natural killer (NK) cells are sentinel components of the innate response to pathogens, but the cell types, pathogen recognition receptors, and cytokines required for their activation in vivo are poorly defined. Here, we investigated the role of plasmacytoid dendritic cells (pDCs), myeloid DCs (mDCs), Toll-like receptors (TLRs), and of NK cell stimulatory cytokines for the induction of an NK cell response to the protozoan parasite Leishmania infantum. In vitro, pDCs did not endocytose Leishmania promastigotes but nevertheless released interferon (IFN)-alpha/beta and interleukin (IL)-12 in a TLR9-dependent manner. mDCs rapidly internalized Leishmania and, in the presence of TLR9, produced IL-12, but not IFN-alpha/beta. Depletion of pDCs did not impair the activation of NK cells in L. infantum-infected mice. In contrast, L. infantum-induced NK cell cytotoxicity and IFN-gamma production were abolished in mDC-depleted mice. The same phenotype was observed in TLR9(-/-) mice, which lacked IL-12 expression by mDCs, and in IL-12(-/-) mice, whereas IFN-alpha/beta receptor(-/-) mice showed only a minor reduction of NK cell IFN-gamma expression. This study provides the first direct evidence that mDCs are essential for eliciting NK cell cytotoxicity and IFN-gamma release in vivo and demonstrates that TLR9, mDCs, and IL-12 are functionally linked to the activation of NK cells in visceral leishmaniasis.