负载滋养层细胞抗原对小鼠树突状细胞表型及功能的影响

目的 研究负载滋养层细胞抗原对小鼠髓源性树突状细胞(DC)分化成熟过程的影响,获得致耐受性DC.方法 体外使用粒细胞巨细胞集落刺激因子(GM-CSF)诱导小鼠骨髓细胞定向分化、经LPS刺激获得成熟DC;通过外胎盘锥组织块培养法获得滋养层细胞,制备可溶性抗原,加入DC培养体系.流式细胞术检测DC表面共刺激分子及MHC-Ⅱ的表达,ELISA法检测DC分泌IL-10和IL-12的浓度,混合淋巴细胞培养评估 DC刺激同种T细胞增殖、活化的功能.结果 成熟DC表型为CD40high CD80highCD86highMHC-Ⅱhigh,分泌大量的IL-12和极少量的IL-10 ,体外能有效刺激T细胞的增殖;负载滋养层细胞抗原的DC表型为CD40midCD80lo wCD86lowMHC-Ⅱlow,在分泌大量IL-12的同时IL-10也明显升高,不能有效刺激T细胞增殖,并使T细胞分泌细胞因子呈现明显Th2偏倚.结论 负载滋养层细胞抗原后的DC表面共刺激分子及MHC-Ⅱ表达降低,刺激T细胞增殖能力下降;其自分泌和促使T细胞旁分泌的细胞因子呈现Th2偏倚,是一种耐受性DC.     

天花粉蛋白联合CD40信号激发的人MoDC介导Th2细胞分化的研究

探讨天花粉蛋白(Tk)联合CD40信号诱导人单核细胞来源DC(MoDC)的成熟活化及其介导Th2细胞分化的作用和机制。采用GM-CSF和IL-4联合方案体外诱导人MoDC,并利用鼠抗人CD40激发型单抗(5C11)刺激负载了Tk的DC制备成熟DC。采用免疫荧光标记和流式细胞术分析DC表型(CD80、CD83、CD86、CD14、PDL1)及其摄取FITC-Dextran的能力;ELISA法测定DC上清中IL-12p70的含量;胞内染色和流式细胞术检测经成熟DC活化的T细胞中CD4~+IFN-γ~+T和CD4~+IL-4~+T的比例。实验结果显示单独Tk不能刺激DC完全成熟,用Tk负载DC后必须联合外源性成熟刺激信号(如5C11),才能有效促进DC成熟,表现在CD80、CD83、CD86的上调表达,CD14下调表达,DC摄取FITC-Dextran的能力降低。与CD40信号单独诱导组相比,Tk联合CD40信号诱导的成熟DC表面PDL1分子呈下调表达,分泌IL-12的能力显著降低,明显提高T细胞中CD4~+IL-4~+T的比例。由此表明负载了Tk的成熟MoDC体外能有效介导Th2细胞分化。     

Gp130分子的活化在树突状细胞分化和成熟中的意义

目的 :研究激发型抗gp130分子单抗B S12对树突状细胞 (DC)分化成熟和功能的影响。方法 :人外周血单核细胞在GM CSF和IL 4作用下分化为未成熟DC后 ,加入B S12单抗 ,观察它对DC表型、摄取抗原的能力以及DC分泌IL 12、进行混合淋巴细胞反应及趋化T细胞能力的影响 ;同时比较B S12和激发型抗CD4 0单抗 5C11对DC的作用。结果 :激发型抗gp130分子单抗B S12作用于未成熟DC ,可以上调DC上CD1a和共刺激分子CD80、CD86以及成熟DC的标志CD83分子的表达 ,下调CD14的表达 ,降低DC摄取抗原的能力 ,增强DC分泌IL 12、进行混合淋巴细胞反应及趋化T细胞的能力 ,但这些作用都稍弱于 5C11对DC的作用。结论 :未成熟DC上gp130分子的直接活化可以诱导DC的分化和功能成熟。     

不成熟CD8α+DC经同种白血病抗原冲击后移植物抗白血病效应的体外研究

目的: 观察不成熟CD8α⁺树突状细胞(DC)体外经同种异基因白血病细胞全抗原冲击后的移植物抗白血病(GVL)效应。方法: C57BL/6(H-2^b)小鼠的骨髓细胞以GM-CSF +IL-4 +SCF +Flt3L体外诱导不成熟CD8α⁺DC,第3 d加入0 mg/L、2.5 mg/L、5 mg/L、10 mg/L和20 mg/LBALB/c(H-2^d)小鼠来源的同种异基因EL9611红白血病细胞抗原冲击,冲击后DC与同系(H-2^b)T细胞按DC/T 1∶1、2∶1、4∶1比例共培养,MTT法观察T细胞增殖情况,ELISA法检测上清中IFN-γ和IL-10的含量,LDH释放法检测T细胞对EL9611细胞的杀伤活性。以成熟DC为对照,观察同种异基因不成熟CD8α⁺DC体外对EL9611白血病细胞的GVL效应。结果: 同种红白血病全抗原冲击后不成熟CD8α⁺DC可有效刺激同系T细胞增殖,作用随DC/T比例增加而增加,增殖效应在 小剂量抗原(≤5 mg/L)冲击时更明显(P<0.05);Ag冲击后不成熟CD8α⁺DC刺激T细胞分泌IFN-γ和IL-10明显增高(P<0.05),IL-10的分泌与不成熟CD8α⁺DC发挥GVL效应存在一定的负相关关系,Ag冲击浓度较低而IL-10分泌少时,T细胞增殖活性较强。杀伤实验结果显示,白血病特异性T细胞对EL9611靶细胞的杀伤活性随抗原冲击浓度的增加而升高,浓度为2.5 mg/L时,杀伤率即可达90%,非特异性杀伤实验显示白血病特异性T细胞对同种异基因正常脾细胞的杀伤活性低于对照组(P<0.05),表明杀伤作用为抗原特异性杀伤,对正常细胞无明显杀伤。结论: 经同种异基因白血病抗原冲击后不成熟CD8α⁺DC体外能刺激同系T细胞产生一定的GVL效应。     

MBL对树突状细胞体外分化成熟的影响

目的: 探讨甘露聚糖结合凝集素 (MBL)对人外周血单核细胞来源的树突状细胞 (MoDC)分化成熟的影响。方法: 以天然人MBL刺激MoDC, 在倒置显微镜下观察DC的形态; 用FACS分析DC的表型; 用 3H- TdR掺入法测定DC刺激同种异体T细胞增殖的能力; 以酵母多糖颗粒吞噬试验评估DC的抗原摄取能力; 用ELISA检测DC培养上清中IL- 12和TNF- α的含量。结果: MBL刺激的DC表面分子CD1a、CD83、CD40、CD80、CD86和MHC DR的表达均上调,摄取酵母多糖颗粒的能力降低, 激发初始T细胞增殖的能力加强, 分泌的IL- 12增多但几乎不分泌TNF- α。结论: MBL能诱导DC分化成熟, 提示其可能通过调节DC的功能而参与获得性免疫应答。     

IL-15对异基因抗原刺激下人CD8+T 细胞增殖分化影响的初步研究

目的：探讨IL-15对异基因抗原刺激下人CD8+T细胞增殖分化的影响。方法：在IL-15存在的条件下,将从健康志愿者外周血中新鲜分离的CD8+T细胞与HLA-A,-B,-DR完全错配的异体抗原提呈细胞（APCs）进行混合淋巴细胞培养（MLRs）。经9 d培养后,对诱导后产生的CD28-CD8+和CD28+CD8+T细胞亚群进行细胞毒功能测定和表型检测,并对CD8+T细胞增殖分化过程中CD28分子的动态变化规律进行监测。结果：在IL-15诱导及异基因抗原刺激下,CD8+T细胞增殖分化后产生的CD28+CD8+T细胞具有细胞毒作用,与外周血新鲜分离的类似细胞相比,其高表达了FasL、颗粒霉素-B和穿孔素;而CD28-CD8+抑制性T细胞不具有细胞毒作用,与外周血新鲜分离的类似细胞相比,低表达了FasL、颗粒霉素-B和穿孔素。在异基因抗原提呈细胞刺激下,IL-15可诱导CD8+T细胞增殖分化过程中CD28-/CD28+细胞数比例升高（从0.24到1.01）,并可诱导CD28+T细胞上的CD28分子丢失。结论：IL-15通过调节CD28分子的表达影响异基因抗原对CD8+T细胞的增殖分化。     

Sel1L 对小鼠骨髓源树突状细胞的影响

目的:研究Sel1L(Suppressor/ enhancer of Lin-12-like)基因对小鼠骨髓源树突状细胞(BMDCs)分化及其功能的影响。方法:利用Cre-Loxp 重组系统构建Sel1L 基因敲除小鼠模型,用ELISA 和实时荧光定量法检测BMDCs 分泌细胞因子IL-6、IL-12 的表达;用免疫印迹法(Western bolt)检测BMDCs 细胞中Sel1L蛋白的表达;用流式细胞术检测BMDCs 细胞CFSE、细胞表面分子CD80、CD86、MHC-Ⅰ、MHC-Ⅱ及其对特异性CD4⁺ T 细胞抗原提呈能力的影响。结果:Sel1L的缺失抑制BMDC诱导分化过程中的增殖效率,上调DCs 分泌因子的能力和MHC-Ⅰ的表达,减少MHC-Ⅱ的表达,并抑制BMDCs 细胞对OVA323-339 抗原特异性T 细胞的增殖。结论:Sel1L 缺失可以抑制小鼠骨髓源树突状细胞的分化,下调DC 对OVA 特异性的CD4⁺ T细胞的抗原提呈功能。     

凋亡肿瘤细胞负载的树突状细胞对抗原特异性CTL的激活

为探讨凋亡Daudi细胞负载的树突状细胞 (DC )激发诱导肿瘤抗原特异性CTL及其生物学特性 ,采用常规方法从人外周血单个核细胞 (PBMC )诱导DC ,激发型CD40mAb联合 50Gyγ射线辐照诱导Daudi凋亡后负载DC ,然后与自体T细胞共育 ,并联合IL 2激发诱导肿瘤特异性CTL ;采用免疫荧光标记和流式细胞仪测定分析膜分子的表达 ;ELISA检测细胞因子的产生 ;Dextran FITC内吞实验分析DC的抗原摄取能力 ;3H掺入试验和51 Cr释放试验分别测定CTL的增殖和细胞毒效应。结果 :(1 )细胞因子序贯体外诱导 7d的DC ,对Dextran吞噬能力最强 ;(2 )CD40mAb诱导联合γ射线辐照 ,能有效介导Daudi细胞的凋亡 ;(3 )抗原负载联合CD40mAb激发可使DC上调表达CD1a、CD80、CD86和HLA DR ,并能促进IL 1 2的分泌 ;(4)凋亡Daudi负载后的DC在激发型CD40mAb作用下 ,能激发和扩增对Daudi细胞具有高效和特异杀伤作用的细胞毒性T细胞。因而认为凋亡肿瘤细胞负载联合CD40mAb刺激的DC可有效激活和扩增肿瘤特异性CTL。     

人膜型LIGHT分子对Mo-DC成熟和T细胞激活的调节作用

利用我们建立的表达人膜型LIGHT分子的基因转染细胞(L929/LIGHT)探讨LIGHT/HVEM信号体外对Mo-DC诱导分化的影响,并进一步研究其对T细胞活化和抗凋亡的共刺激作用。从健康人外周血中分离的单核细胞经GM-CSF联合IL-4诱导形成Mo-DC,流式细胞术分析Mo-DC诱导过程中HVEM和LIGHT的表达;基因转染细胞L929/mock、L929/LIGHT、L929/CD40L或L929/LIGHT联合L929/CD40L,分别经丝裂霉素处理后,与GM-CSF联合IL-4诱导的Mo-DC共育,流式细胞术检测Mo-DC细胞表面成熟标志CD83和CD86的表达,利用FITC-Dextran分析Mo-DC对抗原的摄取能力;L929/LIGHT或L929/mock经丝裂霉素处理后,与抗人CD3单抗激发的T细胞共育,流式细胞术分析CD4+和CD8+T细胞表面活化标志CD25的表达,Annexin V和PI双标记分析T细胞的凋亡率。结果表明,高表达HVEM的单核细胞在诱导形成成熟Mo-D(iDC)的过程中下调了HVEM的表达,成熟Mo-DC(mDC)又上调表达HVEM,而LIGHT在Mo-DC分化过程中呈短暂的诱导性表达;基因转染细胞L929/LIGHT及其联合L929/CD40L能上调Mo-DC表面共刺激分子CD83和CD86的表达,并下调Mo-DC对FITC-Dextran的摄取能力;L929/LIGHT细胞能上调CD4+、CD8+T细胞CD25的表达,并增强T细胞抗凋亡能力。因此,基因转染细胞L929/LIGHT表面表达的人膜型LIGHT分子介导的LIGHT/HVEM共刺激信号对Mo-DC的诱导成熟和T细胞活化及抗凋亡能力具有促进作用。     

IL-37通过作用树突状细胞调节CD8~+T细胞功能

将小鼠骨髓细胞诱导分化为DC,用IL-37处理后,流式细胞术检测细胞表面共刺激分子(CD86、CD80),RT-PCR法检测TNF-α、IFN-γ、IL-6、IL-12和TGF-βmRNA的表达,多重液相蛋白定量CBA试剂盒及ELISA试剂盒检测细胞上清中TNF-α、IL-6、IL-12、IFN-γ、MCP-1和TGF-β蛋白的表达。Western blotting方法检测DC中磷酸化蛋白的表达。将DC与T细胞共培养,流式细胞术检测CD8+T细胞增殖及活化程度(CFSE、CD69)。结果显示,IL-37能够降低表达CD80+/CD86+的DC数量。促炎因子TNF-α、IL-12和IL-6的表达被明显抑制,而T淋巴细胞抑制因子TGF-β明显增高。IL-37预处理的DC明显降低T淋巴细胞的增殖和活化能力。IL-37能够降低DC中磷酸化ERK和NF-κB的表达。IL-37处理的DC对CD8+T细胞产生明显抑制作用。结果表明,IL-37能够通过影响ERK和NF-κB依赖的信号通路抑制DC的成熟和免疫反应,从而抑制CD8+T细胞的活化及增殖。     

IL-4对DC产生IL-12影响的研究

为了研究白细胞介素 4 (IL 4 )对树突状细胞 (dendriticcell,DC )产生白细胞介素 1 2 (IL 1 2 )p70的调节作用及其机制 ,将小鼠骨髓细胞在含GM CSF和IL 4的培养液中培养 6d ,加入成熟诱导剂脂多糖 (LPS )或TNF α、CpG ODN继续培养 2d以获得成熟DC。流式细胞仪检测DC表面CD80和MHCII类分子 ,混合淋巴细胞反应检测DC促进同种异体T细胞增殖的能力 ,ELISA法检测不同诱导剂诱导DC产生IL 1 2p70的能力 ,RT PCR和荧光定量PCR检测IL 1 2p35和p4 0mRNA的表达及其变化。结果显示小鼠骨髓细胞在含GM CSF、IL 4和成熟诱导剂的培养液中培养后可以获得成熟的DC ,成熟DC高表达CD80和MHCII类分子 ,具有较强的刺激同种异体T细胞增殖的能力 ,LPS、CpG ODN、TNF α、PolyI:C在促进DC成熟的同时能诱导DC产生有活性的IL 1 2 ,IL 4对IL 1 2的产生具有明显的促进作用 ,RT PCR和荧光定量PCR结果显示LPS诱导IL 1 2产生以及IL 4对其的促进作用与IL 1 2p35基因的转录水平增高有关     

Human dendritic cells activate T lymphocytes via a CD40: CD40 ligand-dependent pathway

The CD40: CD40 ligand (CD40L) interaction provides T lymphocyte-mediated help for B lymphocyte and monocyte function but has also been shown to serve as a co-stimulus for T lymphocyte activation. In this report, we studied the regulation of CD40 expression and its functional relevance for the human dendritic cell (DC) stimulation of T lymphocytes. Only a small subpopulation of directly isolated blood DC expressed CD40. However, CD40 was rapidly up-regulated by culture, and its expression was further enhanced by interleukin (IL)-1α, IL-1β, IL-3, tumor necrosis factor-α and granulocyte/macrophage-colony-stimulating factor. Expression of CD40L on DC was not detected. The proliferation of T lymphocytes in an allogeneic mixed leukocyte reaction, stimulated by blood DC or epidermal Langerhans cells, was significantly reduced in the presence of the CD40 immunoglobulin (CD40Ig) fusion protein or CD40L monoclonal antibodies. Cross-linking of CD40 on directly isolated DC with mouse CD40L trimer (mCD40LT) markedly augmented CD80 and CD86 up-regulation. Nevertheless, the same cross-linking mCD40LT inhibited DC stimulated T lymphocyte proliferation. When CD40Ig was added simultaneously with CTLA-4Ig, only minimal and variable additional inhibition of DC-stimulated allogeneic T lymphocyte proliferation and IL-2 secretion was observed, compared to each fusion protein alone. These results suggest that both CD80/CD86-dependent and -independent components of DC-T lymphocyte CD40: CD40L co-stimulation exist and further emphasize that the majority of blood DC have to differentiate or be activated to express co-stimulatory molecules.     

重组人可溶性CD40配体对树突状细胞体外分化、成熟及功能的影响

用Ficoll密度离心及贴壁法获得外周血单个核细胞 (PBMC ) ,PBMC经细胞因子组合诱导分化成树突状细胞 (DC ) :GM CSF (10 0ng/ml)与IL 4 (5 0ng/ml)诱导 5d后 ,分别加入TNF α (10ng/ml)或rhsCD4 0L (2 μg/ml)继续培养 4d ;倒置显微镜下观察DC形态 ,免疫荧光标记和流式细胞术分析DC表型 (CD1a、CD80、CD83、HLA DR、CD14、CD16、CD19)及摄取FITC Dextran抗原的能力 ;3 H TdR掺入法检测DC刺激自体混合淋巴细胞体外增殖反应 (MLR )能力 ;ELISA法分析DC培养上清中IL 12的水平 ;Trans well细胞趋化实验检测DC对自体外周T淋巴细胞的趋化能力。发现经rhsCD4 0L刺激的DC表面分子 (CD1a、CD80、CD83、HLA DR )的表达水平高于经典的细胞因子组合组 (GM CSF +IL 4 +TNF α ) ,同时rhsCD4 0L刺激后的DC摄取FITC Dextran的能力下降而刺激自体MLR和分泌IL 12的能力明显提高 ;而且rhsCD4 0L诱导的DC表面趋化因子受体CXCR4的表达水平及对自体外周T淋巴细胞的趋化能力均强于TNF α或FL激发的DC。rhsCD4 0L在体外不仅具有显著的诱导DC分化 ,促进DC成熟的功能 ,而且经rhsCD4 0L作用的DC能更有效地激发T淋巴细胞     

CpG ODN促进树突状细胞成熟的实验研究

目的：研究CpG ODN对小鼠骨髓来源的树突状细胞(bone marrow—derived dendritic cells,BMDC)分化成熟的影响。方法：以含非甲基化CpG基序的寡核苷酸(unmethylated CpG motif containing oligonucleotides,CpG ODN)刺激BMDC，流式细胞术检测DC膜表面CD80表达，ELISA检测DC分泌IL-l2水平，MTT法测定CpG ODN活化的DC刺激T细胞培殖能力。结果：CpG ODN可显著刺激DC表达CD80，分泌IL-l2，增强DC刺激T细胞增殖的能力。结论：CpG ODN可以有效促进DC的功能成熟。     

腺病毒载体介导EB病毒潜伏期膜蛋白2A基因转染树突状细胞对其功能的影响

目的 探讨腺病毒载体介导EB病毒（EBV）潜伏期膜蛋白2A（LMP2A）基因转染树突状细胞（DC）对其功能的影响。方法 通过同源重组法构建带有EBV－LMP2A基因的重组腺病毒Ad5-LMP2A，用不同感染滴度（MOI）的Ad5-LMP2A转染成熟的DC，用流式细胞术（FACS）检测DC的LPM 2A蛋白表达细胞百分率及用台盼蓝染色计数DC死亡百分率，选择最佳MOI；用最佳MOI的Ad5-LMP2A转染成熟的DC，FACS检测转染前后DC表面分子CD1a、CD83、CD40、CD80及HLA－DR的变化；并用^3H-TdR掺入法检测转染前后DC刺激同种淋巴细胞增殖能力及荧光定量PCR检测表达IL－12 P40 mRNA等功能的改变。结果 MOI 200为Ad5-LMP2A转染DC的最佳滴度，此时约80％的DC表达LMP2S蛋白及92％以上细胞为活细胞。Ad5-LMP2A转染成熟DC前后对细胞表面共刺激分子及特征性表面标志无影响；转染后的DC仍具有较强的刺激同种异体淋巴细胞增殖能力和表达IL－12 P40 mRNA的功能。结论 腺病毒载体能高效介导EBV LMP2A基因在DC中表达，Ad5-LMP2A转染成熟DC对其功能无明显影响，便于进一步用于EBV相关肿瘤的免疫治疗。     

Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL-12

Human plasmacytoid dendritic cells (DC) (PDC, CD123+) and myeloid DC (MDC, CD11c+) may be able to discriminate between distinct classes of microbial molecules based on a different pattern of Toll-like receptor (TLR) expression. TLR1-TLR9 were examined in purified PDC and MDC. TLR9, which is critically involved in the recognition of CpG motifs in mice, was present in PDC but not in MDC. TLR4, which is required for the response to LPS, was selectively expressed on MDC. Consistent with TLR expression, PDC were susceptible to stimulation by CpG oligodeoxynucleotide (ODN) but not by LPS, while MDC responded to LPS but not to CpG ODN. In PDC, CpG ODN supported survival, activation (CD80, CD86, CD40, MHC class II), chemokine production (IL-8, IP-10) and maturation (CD83). CD40 ligand (CD40L) and CpG ODN synergized to activate PDC and to stimulate the production of IFN-alpha and IL-12 including bioactive IL-12 p70. Previous incubation of PDC with IL-3 decreased the amount of CpG-induced IFN-alpha and shifted the cytokine response in favor of IL-12. CpG ODN-activated PDC showed an increased ability to stimulate proliferation of naive allogeneic CD4 T cells, butTh1 polarization of developing T cells required simultaneous activation of PDC by CD40 ligation and CpG ODN. CpG ODN-stimulated PDC expressed CCR7, which mediates homing to lymph nodes. In conclusion, our studies reveal that IL-12 p70 production by PDC is under strict control of two signals, an adequate exogenous microbial stimulus such as CpG ODN, and CD40L provided endogenously by activated T cells. Thus, CpG ODN acts as an enhancer of T cell help, while T cell-controlled restriction to foreign antigens is maintained.     

Th1 cytokines are more effective than Th2 cytokines at licensing anti‐tumour functions in CD40‐activated human macrophages in vitro

CD40 agonists are showing activity in early clinical trials in patients with advanced cancer. In animal models, CD40 agonists synergise with T‐cell‐activating therapies to inhibit tumour growth by driving tumour macrophage repolarisation from an immunosuppressive to a Th1 immunostimulatory, tumouricidal phenotype. We therefore tested the hypothesis that T‐cell‐derived cytokines license anti‐tumour functions in CD40‐activated human macrophages. CD40 ligand (CD40L) alone activated macrophages to produce immunosuppressive IL‐10, in a similar fashion to bacterial LPS, but failed to promote anti‐tumour functions. The Th1 cytokine IFN‐γ optimally licensed CD40L‐induced macrophage anti‐tumour functions, inducing a switch from IL‐10 to IL‐12p70 production, promoting macrophage‐mediated Th1 T‐cell skewing and enhancing tumouricidal activity. We found that even the Th2 cytokines IL‐4 and IL‐13 promoted IL‐12p70 production (albeit without inhibiting IL‐10 production) and enhanced Th1 T‐cell skewing by CD40L‐activated macrophages. However, IL‐4 and IL‐13 did not enhance tumouricidal activity in CD40L‐activated macrophages. Thus, while both Th1 and Th2 cytokines biased macrophages to a Th1 immunostimulatory phenotype, only Th1 cytokines promoted tumouricidal activity in CD40L‐activated macrophages. The presence of tumour‐infiltrating Th1 or Th2 cells might therefore be predictive for patient response to CD40 agonism.     

Switching from a restricted to an effective CD4 T cell response by activating CD8+ murine dendritic cells with a Toll-like receptor 9 ligand

Freshly isolated quiescent splenic dendritic cell (DC) subtypes differ in their capacity to activate naive CD4 T cells in culture. The CD8+ DC showed a reduced capacity to stimulate T cell proliferation compared to either of the CD8- DC subsets, regardless of antigen and DC dose. In contrast to CD8- DC, the quiescent CD8+ DC did not induce IFN-gamma production from CD4 T cells. The difference between the DC subtypes appeared to be at the level of initial surface molecule interactions, but could not be attributed to differences in expression of MHC class II or B7 family molecules, or to the expression of Fas ligand on DC. However, when activated by inclusion of the Toll-like receptor 9 ligand CpG in culture, CD8+ DC became potent stimulators of both CD4 T cell proliferation and IFN-gamma production. In contrast, similar activation of CD8- DC produced a more modest increase in capacity to stimulate CD4 T cell proliferation and no increase in capacity to stimulate IFN-gamma production. The difference between a quiescent and an activated state is therefore more extreme for CD8+ than for CD8- DC. The especially tight regulation of the activity of CD8+ DC may be essential for the maintenance of self tolerance.