这项工作是由局科技与Reasearch核心资金（JEC到）的支持。

这项研究得到批准的机构审查委员会（NUS-IRB 10-250）。 1.分离外周血单个核细胞（PBMC中） <ol><li> 试剂的制备 <ol><li>制备的PBS / EDTA:磷酸盐缓冲盐水溶液（PBS）中，并补充有2mM乙二胺四乙酸（EDTA）。消毒通过0.2微米的过滤该溶液通过过滤。注意存储的PBS / EDTA，在4℃，使用前温热至室温。 </li><li>制备染色缓冲液:磷酸盐缓冲盐水溶液（PBS）中，用2％胎牛血清（FBS）补充，10毫4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）和2mM乙二胺四乙酸（EDTA）。消毒通过0.2微米的过滤该溶液通过过滤。 </li></ol></li><li> 从血液锥采集血液 注意:血液锥体包含板后收集白色血细胞成分letpheresis出院。如果血液在肝素或EDTA试管收集，在1稀释血液用PBS:1的比例，并进行到步骤1.3;如果接收到白膜层，稀释用PBS捉鬼外套在1:2的比例并继续执行步骤1.3。 <ol><li>切割锥体的两端，以允许血液流出到50毫升管中。注意，锥形，通常含有10毫升血液。 </li><li>使用含有30毫升的PBS / EDTA至洗锥体，并收集在50ml管中的钝端的注射器。 </li><li>用PBS / EDTA进一步稀释血液至80ml的终体积。 </li></ol></li><li> 通过密度离心 16 外周血单个核细胞的分离 <ol><li>分装15毫升菲各4新鲜的50毫升管。 </li><li>用25毫升血清吸管给在菲层加20毫升稀释血液。请留意以45°角持有50毫升管，并小心不打扰相间。 </li><li>离心在805 xg离心管而不制动器30分钟，20℃。 </li><li>去除等离子层，并收集外周血单个核细胞躺在只是一个巴斯德吸管等离子层以下的环。外周血单个核细胞结合四管成两个50毫升管。注意避免收集的PBMC下方的透明层。 </li><li>添加的PBS / EDTA，以每PBMC中的管和离心机为50ml终体积在548 xg离心与制动器10分钟，20℃。 </li><li>吸上清，重悬沉淀在每个管用25ml染色缓冲液中，合并成一个50ml管中。 </li><li>离心367 XG带刹车5分钟，4℃。 </li><li>吸去上清液，并用10毫升染色缓冲液重悬沉淀。 </li></ol></li></ol> 2.单核细胞富集磁选17 <ol><li> 确定细胞数 <ol><li>采取20微升的PBMC细胞悬浮液，并用20微升台盼蓝的混合计数用流式细胞仪的活细胞的数目。 </li><li>在367 XG带刹车离心细胞悬液为10分钟，4℃。 </li><li>吸去上清液完全重悬细胞沉淀到每80微升染色缓冲液10 7个细胞的浓度。 </li></ol></li><li> 磁标记 <ol><li>加入20μlCD14微珠每10 7个细胞，拌匀孵育在2 15分钟- 8°C。 </li><li>通过加入1ml每10 7个细胞染色缓冲液并离心以367 xg离心与制动器10分钟，4℃洗涤细胞。 </li><li>在每50μl染色缓冲液10 7个细胞的浓度吸上清完全重悬细胞沉淀。 </li></ol></li><li> 磁选 <ol><li>最多2×10 8个细胞的使用中等大小列。 注意:选择一个适当的列和分离器根据总细胞数和在数据表中，建议CD14 +细胞的数目。 </li><li>将列在磁场Ø发适当隔板和通过用500μl染色缓冲液漂洗制备柱。 </li><li>吸管细胞悬液到柱上，收集穿过用15毫升管列未标记细胞。 </li><li>柱下更换新的15ml试管并用500μl染色缓冲液洗柱3次。确保列水库增加洗涤之间新的染色缓冲区之前空。 </li><li>从分离器中取出柱，并将其放置在一个新的15ml试管。 </li><li>吸管1毫升染色缓冲液上柱。立即用力推动柱塞（在试剂盒中提供）到柱冲洗掉磁标记的细胞。 </li><li>使用一个新的列中的洗脱级分，以增加CD14 +细胞的纯度，重复步骤2.3.2至2.3.6。需要注意的是珠将被从细胞中自动培养步骤3.2中释放出来。 </li></ol></li></ol>树突状细胞分化为不同的激活小号tates <ol><li> 试剂的制备（内毒素水平必须在所有试剂小于0.1 EU / ml）的 <ol><li>制备细胞培养基:RPMI 1640补充有10％胎牛血清（FBS），1％非必需氨基酸（NEAA）和0.05毫摩尔2-巯基乙醇（2ME）。消毒通过0.2微米的过滤该溶液通过过滤。 </li><li>制备细胞因子:重组的IL-4和GM-CSF中的细胞培养级水中的0.25毫克/ mL的浓度分别在无菌条件下。分装成细胞因子200μl的离心管中，并储存于-80℃。 </li><li>准备维生素D3的股票:在重组细胞培养级水维生素D3，以在无菌条件下100毫米的浓度。分装维生素D3成的1.5 ml离心管中并储存在-20°C。 </li><li>准备地塞米松的股票:在重组细胞培养级水地塞米松在无菌条件下10毫米的浓度。分装dexamethasone成的1.5 ml离心管中并储存在-20°C。 </li><li>制备脂多糖:重组在细胞培养级水脂多糖（LPS），以在无菌条件下的1毫克/毫升的浓度。分装成LPS的1.5 ml离心管中并储存在-20°C。 </li></ol></li><li> 产生不同的moDCs <ol><li>种子4套CD14 +单核细胞的在0.3浓度- 0.5×10 6 / ml的补充有200纳克/毫升GM-CSF的和在6孔板200纳克/毫升的IL-4的细胞培养基。注意，这是第0天和介质在每个6孔体积为2毫升</li><li>孵育在组织培养箱细胞在37℃，5％的CO 2。 </li><li>从培养在第4天，离心，在300 xg离心除去850微升培养基的5分钟，4℃。吸去上清液，并在1ml含有（200 ng / ml的GM-CSF的200纳克/毫升的IL-4）2×细胞培养基重悬沉淀。收藏此细胞混合物回向培养。注意添加了对GM-CSF和IL-4的2倍浓度时，1ml培养基中加入回文化将成为1倍。 </li><li>加入1微升维生素D3的股票和每毫升中的地塞米松股票1μl的两集的第5天，以产生耐受性moDCs。注意，维生素D3的最终浓度为100nM的地塞米松为10nm;另外的GM-CSF和IL-4的不需要在第5天。 </li><li> GM-CSF的200纳克/毫升和200纳克/毫升的IL-4添加到所有集合上6天。 </li><li> 1微克/毫升的LPS添加到具有唯一的GM-CSF和IL-4在第6天处理以产生成熟moDCs的集之一。 </li><li> 1微克/毫升的LPS添加到一组致耐受性moDCs中的第6天以产生脂多糖致耐受性moDCs。 </li><li>用PBS，EDTA冲洗培养皿流式细胞仪或其他研究收获了不同类型的7天moDCs的。请注意，只有非粘附细胞收获。注意到因CD14 + monoc这一比例产量对于未成熟moDCs，成熟moDCs，致耐受性moDCs和LPS-致耐受性树突ytes分别约90％，50％，60％和60％和献血者和FBS很多之间变化。 </li></ol></li></ol> 4.流式细胞仪<ol><li> 在moDCs细胞表面标志特征 <ol><li>吹打分离从培养皿中的细胞。洗细胞一次，用PBS / EDTA和悬浮细胞在每50μl染色缓冲液5×10 5个细胞的浓度在1.5 ml离心管。 </li><li>孵育0.5×10 6个细胞的等分试样用PerCP缀合HLADR（1:100），PE缀合的CD80（1:50），PE缀合的CD83（1点25分），PE缀合的CD86（1:50），APC-共轭的CD11c（1:50），PE缀合的CD14（1:50）和PE缀合的BDCA3（1:50）和APC缀合的ILT3（1点25分）在黑暗中在4℃下30分钟。同种型匹配PerCP缀合的单克隆抗体（1:20），PE缀合的单克隆抗体（1点11），APC标记的单克隆抗体（1点11）将作为阴性对照。 </li><li>检测用流式细胞仪18的流动表面标志物的表达水平。 </li></ol></li><li> moDCs的细胞线粒体膜电位分析 <ol><li>由每冻干红CMXRos 50微克加入94.1微升二甲基亚砜（DMSO）的制备1mM的红色氯-X-rosamine（CMXRos）的储备溶液。 </li><li>在15毫升试管孵育2×10 5 moDCs用100nM红色CMXRos在1ml Hank氏平衡盐溶液（HBSS）中30分钟的37℃。 </li><li> 2毫升的PBS / EDTA添加到细胞和离心机在300×g离心5分钟，室温。重复2次。吸去上清液，并在300μl的PBS中，EDTA 2％FCS的流式细胞仪分析悬浮细胞沉淀。请留意用PE通道分析红CMXRos信号。 </li></ol></li><li> T-细胞的标志物表征 <ol><li>孵育50微升的1.2×10 6 CFSE标记的异基因CD4 + T细胞（从生成步骤5.6）与PerCP缀合的CD3（1:200），PE / Cy7的共轭的CD4（1:400）和APC / Cy7的缀合的CD25（1:在黑暗中在4℃，30分钟100）。使用在黑暗中与Foxp3的-的Alexa Fluor 647（1:50）商业试剂盒在室温下1小时染色T细胞。 </li></ol></li></ol> 5.同种异体反应研究<ol><li>通过加入18微升的DMSO至冻干的CFSE制备羧基琥珀酰亚胺酯（CFSE）的5mM储备溶液，按照制造商的协议。 </li><li> 净化从PBMC中的CD4 + T-细胞 <ol><li>通过采取20微升PBMC细胞悬浮液的测定细胞数，并用20微升台盼蓝的混合计数使用血细胞计数活细胞的数量。 </li><li>在367 XG与制动器10分钟，4℃离心细胞悬浮液。 </li><li>在5×10 7个细胞每1ml染色缓冲液的浓度吸上清完全重悬细胞沉淀在5ml的POLYSTY刘若英管。 </li><li>补充人体CD4 + T细胞富集的鸡尾酒在50微升/毫升细胞。拌匀并在室温下孵育（15 - 25℃）10分钟。 </li><li>涡旋磁性颗粒30秒，并在100微升/毫升的细胞添加的磁性粒子。拌匀并在室温下孵育5分钟。 </li><li>染色缓冲液至细胞悬浮液添加到2.5毫升总体积。 3次 - 轻轻吹打上下2混合在管内的细胞。放置管进入磁体和孵育5分钟。 </li><li>倒置磁铁用管，倒出悬浮液（含有T细胞）到一个新的5毫升聚苯乙烯管中。 </li></ol></li><li>确定细胞数，孵育1.2×10 6个 CD4 + T细胞用5μM的CFSE在1ml PBS中20分钟，在37℃，避光。 </li><li>添加五倍的细胞培养基（根据步骤3.1.1的方法制备）与细胞的原始染色体积并孵育5分钟。 </li><li> CentrifUGE在300×g离心5分钟，4℃，重悬沉淀在细胞培养基中以2×10 5个每75微升的浓度。 </li><li>添加75微升2×10 5个 CFSE标记的CD4 + T细胞对含75微升细胞培养基与0每MODC培养，2.5×10 3，5×10 3，10×10 3，20×10 3和40×10 3 moDCs在96孔U型底平板。培养在组织培养培养箱6天在37℃，5％的CO 2。 </li><li>吹打CD4 + T细胞分化成15ml试管并离心，在300×g离心5分钟，4℃的收获。收集细胞因子分析，重悬细胞沉淀上清在2ml的PBS / EDTA和离心机在300×g离心在4℃下5分钟。 </li></ol> 6.细胞因子分析19 <ol><li>如步骤5.5中所述收集来自同种异体反应研究上清液。 </li><li> 试剂的制备人细胞因子/趋化因子Ë磁板分析<ol><li>为了制备混合的对珠粒单独小瓶抗体固定化珠瓶（包括在试剂盒），1分钟涡流。从各抗体珠子小瓶到混合瓶（包括在kit中）添加60微升并提请3毫升与珠稀释液的最终体积（提供）。 </li><li>对于制剂的质量控制，重建质量控制1和质量控制2（包括在kit中）与250微升去离子水。 </li><li>为制备洗涤缓冲液中，取30 ml高10倍的洗涤缓冲液（含在试剂盒中）具有270毫升去离子水。 </li><li>用于制备人细胞因子标准的，重构用250微升去离子水，得到10,000微克/毫升浓度的人细胞因子标准（包括在kit中）。 <ol><li>在1.5ml微量离心管中加入50微升10,000微克/毫升的人细胞因子标准200微升测定缓冲液（在试剂盒中提供的），以使2000微克/毫升的工作标准。转移50微升在1.5ml微量离心管2000微克/毫升的人细胞因子标准200微升测定缓冲液（在试剂盒中提供的），以使400微克/毫升的工作标准。 </li><li>转印在1.5ml微量离心管中的400微克/毫升的人细胞因子标准200微升测定缓冲液（在试剂盒中提供）的50微升，使80微克/毫升的工作标准。转印在1.5ml微量离心管中的80微克/毫升的人细胞因子标准200微升测定缓冲液（在试剂盒中提供）的50微升，使16微克/毫升的工作标准。 </li><li>转印在1.5ml微量离心管中，以使3.2微克/毫升的工作标准的16微克/毫升的人细胞因子标准200微升测定缓冲液（在试剂盒中提供）的50微升。 0微克/毫升的工作标准仅具有200在1.5ml微量离心管微升测定缓冲液中。 </li></ol></li></ol></li><li>预湿过滤板（包括在kit中）吹打200微升测定缓冲液（提供）到过滤器的各孔盘子。密封，并放置在过滤板在板振荡器上在室温下10分钟。 </li><li>吸分析缓冲液，加入25微升每个标准或质量控制到合适的井。 </li><li>添加25微升的细胞培养基的背景，标准和对照孔（根据步骤3.1.1制备）。 </li><li>添加25微升测定缓冲液中的样品孔中，并添加25微升样品放入合适的样品孔中。 </li><li>涡旋含有抗体固定化珠的混合瓶，并添加25微升的混合珠每个孔中。密封板并在4℃下在板振荡器上搅拌孵育过夜。 </li><li>吸液和洗涤板2次，加入200μl/孔洗涤缓冲液和抽吸流体。 </li><li>加入25微升检测抗体（提供）到每口井。密封并在板振荡器上搅拌孵育在室温下1小时。 </li><li>加入链霉藻红蛋白的25微升（省被提供的），以每孔含有25μl的抗体的检测。密封并在板振荡器上搅拌孵育在室温下30分钟。 </li><li>如步骤6.8中所述吸液和洗涤板。 </li><li>在板振荡器上添加150微升/孔护套的流体到所有孔中，重悬珠子5分钟。 </li><li>检测的使用3D系统20珠的荧光强度。使用五参数日志逻辑曲线拟合方法用于样品21计算细胞因子/趋化因子的浓度分析的中位荧光强度数据。 </li></ol> 7.实时耗氧率（OCR）和细胞外酸化速率（ECAR）的测量<ol><li> 试剂的制备/材料 <ol><li>制备的OCR培养基:补充有25毫米的葡萄糖和1mM丙酮酸钠（pH为7.35）测定培养基。消毒通过0.2微米的过滤该溶液通过过滤。注意到，这个媒体是公关epared没有FBS因为FBS成分非常复杂，从一个很多很多花药变化。 </li><li>制备ECAR介质:基础培养基补充了2mM L-谷氨酰胺（pH值7.35）。消毒通过0.2微米的过滤该溶液通过过滤。需注意，该介质被无碳酸氢盐，葡萄糖，丙酮酸和FBS制备。注意，FBS具有缓冲能力，并且将与ECAR读干扰。 </li><li>准备为化合物OCR测量:重悬寡630微升OCR中，使100微米的原液，用OCR中稀释至16微米的工作浓度。悬浮羰基氰化物4-（三氟甲氧基）苯腙（FCCP）与720微升OCR中，使100微米的原液，用OCR中稀释至4.5微米的工作浓度。鱼藤酮悬浮/ 540微升OCR介质的抗霉素A，以50微米的原液，用OCR中稀释至10μm工作浓度。 </li><li>准备对于ECAR测量化合物:悬浮葡萄糖用3ml ECAR介质，使100毫米原液稀释至80毫米的工作浓度。重悬寡用720微升ECAR介质，使100微米的原液稀释至18毫米的工作浓度。悬浮2-脱氧-D-葡萄糖用1.5ml ECAR介质，以使1000毫米原液。 </li><li>吸管200微升校准的到盒并储存在37℃的各孔用测量之前没有CO 2的一天。 </li></ol></li><li> 实时测量OCR <ol><li>收获moDCs和在60×10 3每150微升的OCR介质的浓度重悬每种类型中的OCR介质moDCs的。 </li><li>种子60×10 3个细胞/孔在聚D-赖氨酸包被的96孔平底板中，在37℃下在非CO 2培养箱1小时孵育。需注意使用50微升的50ng / ml的聚-D-赖氨酸涂覆的板1小时并用洗涤无菌水。保持板干燥，在室温下在使用前2小时。 </li><li>吸取25微升OCR介质进入所有井注入口;包含所有井注入B口寡16μm的25微升OCR介质;含注入端口C（FCCP）为4.5μm的所有水井和25个含10毫米鱼藤酮/在所有孔注射D口抗霉素A微升OCR媒体25微升OCR网上平台。请注意，寡最后阱浓度为2微米，FCCP为0.5μm和鱼藤酮/抗霉素A为1微米。 </li><li>将板进入细胞外流量分析仪和连续四个阶段同时运行所有moDCs一个完整的OCR研究:基础呼吸，线粒体复合物V抑制（从B口药物注射后），最大呼吸感应（从A口剂注射后）（后从C口药物注射）和电子传递链的抑制（从D口药）22后。注意到，有3Çycles在注射和测量之间6分钟间隔之间。 </li></ol></li><li> 实时测量ECAR <ol><li>收获moDCs和在60×10 3每150微升ECAR介质的浓度重悬每种类型ECAR介质moDCs的。 </li><li>种子6×10 4个细胞/孔在聚D-赖氨酸包被的96孔平底板中，在37℃下在非CO 2培养箱1小时孵育。 </li><li>吸取25微升ECAR介质进入所有井注入口;含有葡萄糖在所有孔中喷射口B的10mM的25微升ECAR介质;包含所有孔18微米的注射端口C的寡和25含有1000毫在注射端口D的2-脱氧-D-葡萄糖对所有孔微升的OCR介质25微升ECAR介质。请注意，对于葡萄糖的最后阱浓度为10μM，寡为2μm和2-脱氧-D-葡萄糖为100mM。 </li><li>将板进入细胞外流量分析仪和运行完整ECAR研究，在连续的四个阶段，同时所有moDCs:基础呼吸（从A口剂注射后），糖酵解感应（从B口药物注射后），最大糖酵解感应和糖酵解抑制（从C口药物注射后）（后从药D口）22。 </li><li>分析使用单向ANOVA与杜克多重比较测试后的统计差异。 </li></ol></li></ol>

Open access fees for this article were provided by, Agilent Technologies.

本文介绍了单核细胞不成熟moDCs，耐受性moDCs和成熟moDCs产生的方法。在这个协议中的重要步骤中详细在下面的段落中讨论。要注意的是人外周血用作这个协议和用于处理人血应实行普遍预防起始材料是重要的。虽然在技术上是可行的推导在人中24从骨髓的DC，从外周血中发现的细胞， 在体外 DC分化优选由于相比骨髓外周血的可用性。间在外周血中发现的细胞，造血CD34 +干细胞和单核细胞通常用于在体外生成DC的。造血CD34 +干细胞是与GM-CSF和TNF-α培养，以得到其然后进一步分化成朗格汉斯的CD1a +和CD14 +亚群样细胞和树突状细胞。相反地​​，单核细胞是在GM-CSF和IL-4中培养，以产生未成熟moDCs。几个协议用于从外周血单核细胞的富集;例如，通过加入塑料盘中，淘析和分离试剂盒25,26的粘附协议的优点是对细胞损害最小和相对成本效益但细胞纯度可能受到影响。和一个额外的步骤是必需的，以分离细胞用于进一步的实验。淘析是分隔根据它们的大小和密度的细胞的技术。淘洗的优点是细胞活力和单核细胞可容易地用于进一步的实验;然而这种技术是通过淘析器的可用性和无法分离具有相似沉淀参数细胞不同群体（T细胞和单核细胞）的限制。市售提取试剂盒采用磁珠来无论是正面还是选择单核细胞人口负选择。一些协议使用的是负向选择单核细胞隔离偏置为孤立的单核细胞保持"不变"（不标记或微珠的约束）。在这个协议中，CD14珠用于从PBMC中阳性选择人单核细胞。 CD14缺乏胞质域和CD14抗体的结合不会触发的信号转导。此外，该微珠将从培养后的单核细胞中分离，因此不会妨碍分化过程。此外，CD14的强烈大部分的单核细胞和弱对中性粒细胞和某些骨髓树突状细胞，因此这在更高的细胞纯度比其他方法17隔离结果的方法来表示。 血单核细胞可以分化成树突状或巨​​噬细胞和单核细胞的命运在很大程度上取决于细胞因子环境。在本文中，通过加入粒细胞巨噬细胞集落刺激因子（GM生成moDCs-CSF）和白细胞介素-4（IL-4）的人外周血单核细胞。 GM-CSF是必需的单核细胞的生存和IL-4上施加巨噬细胞分化的抑制活性;和组合加成的GM-SCSF和IL-4的单核细胞产生相比，个别的细胞因子27未成熟moDCs的比例更高。有迹象表明，通过添加肿瘤坏死因子α（TNF-α），干扰素α（IFN-α）和白细胞介素13（IL-13）到外周血单核细胞28，29，30生成moDCs其他协议。 GM-CSF和IL-4的组合在20世纪90年代进行了优化，现在公认的协议产生分化成免疫原性或耐受性moDCs和极化为Th1，Th2细胞或Th17细胞促进moDCs塑料未成熟DC。 未成熟moDCs通过加入的维生素D3和地塞米松分化为致耐受性moDCs。有几个协议产生耐受性例如树突状，通过核因子-κB（NF-KB）抑制，β连环蛋白活化，维生素D3，地塞米松和雷帕霉素31，32，33，34，35，9，36，37。虽然两者单独维生素D3和地塞米松已经报道以诱导对DC，维生素D3和地塞米松结果在alloproliferation更大抑制当使用各药物比组合的耐受性的效果。因此，用于产生致耐受性DC的现有协议被修改，以维生素D3和地塞米松的组合。这种方法目前正在接受为有治疗效用的人树突状耐受性的典范。同样重要的是要注意，重构维生素D3和地塞米松具有短的保质期。 在这个协议中，脂多糖（LPS）的溶液中加入作为DC成熟诱导剂。未成熟moDCs也可以使用诱导促炎症鸡尾酒成熟:（TNF-α），白细胞介素1β（IL-1β），白细胞介素-6（IL-6）和前列腺素E2）或促炎细胞因子（TNF-α和干扰素γ（IFN-Γ））。促炎性鸡尾酒产生具有高共刺激和迁移功能成熟moDCs但它们产生IL-12的38的相对较低的水平。 TNF-α或IFN-Γ单独是不能诱导稳定树突表型39。 LPS刺激Toll样受体4（TLR4），介导的NF-κB和丝裂原活化蛋白激酶（的MAPKs）的活化来诱导DC成熟。 LPS诱导的DC成熟示出的DC成熟标志物（CD83，CD86，HLA-DR）的上调和还导致生产的IL-12p70的。此外，该步骤可以被进一步修饰以配对TLR3激动剂LPS以产生用于临床癌症疫苗的成熟DCs。在本文中，耐受性的DC被证明是对LPS处理成熟性。这些半成熟树突状都没有免疫原性和不releaSE促炎细胞因子40。 此协议的局限性在于分化过程。这个过程需要从第0天8天的时间这对被改编成高通量分析困难7天。在协议的修改需要缩短分化过程然而，在不同的状态产生高数可行的DC。其次，通过增加细胞因子在这个协议产生的DC和这些细胞因子不支持DC人口的时间长。此外，细胞因子的浓度使用比体内要高得多，并可能会导致不属于生理相同体内的DC通路偏置发展。例如在直流前体的体外培养物已被证明对GM-CSF的，这是不用于体内 41正常DC分化的重要的细胞因子反应。然而，细胞因子刺激可以是基因的有效方法在体外进行实验率高的数字直流。以经受从这个协议来的其他分析，如免疫荧光染色产生的这些细胞，流式细胞仪的能力，异体反应的研究和代谢研究增加了这种方法的有效性。 这些体外的DC作为一个很好的模式，以提高直流发育，成熟和抗原呈递的知识，这是以前难以在体内 DC的罕见数字做。 DC的调节免疫的免疫耐受性与能力，使得他们在对抗癌症和自身免疫性疾病的42，43，44，45疗法有吸引力的候选人。在这个协议中产生免疫原性的DCs可以用来改进对感染性疾病和肿瘤疫苗功效;同时致耐受性树突可用于控制不需要的T细胞应答和防止移植后排斥反应。错综复杂免疫力和耐受性之间的平衡非常依赖于DC的分化状态。 DC分化是受多种信号途径和代谢命运管辖协调蜂窝方案。 DC的不同分化状态的生物能量和生物合成需求不同;例如，相比于在静止状态的DC活化DC需要更有活力的代谢适应生存和迁移是非常重要。要注意的是维生素D3，地塞米松和雷帕霉素为它们诱导耐受性树突能力已知它是重要的，已经描述了以影响直流代谢。在本文中，从不同的分化状态moDCs的能量代谢使用外流量分析仪进行了表征和耐受性moDCs表现出最高的代谢可塑性和LPS诱导的成熟降低这种可塑性。同化代谢支持DCs成熟，而分解代谢的影响耐受性DC功能46。区议会从这个协议产生可用于评估是否改变DC的代谢状态按住键修改在治疗免疫力和宽容。总之，我们提出了一个协议，为研究DC的免疫功能至关重要的代不成熟，耐受性和成熟moDCs的。

DC最初是由保罗·朗格汉斯（朗格汉斯细胞）在十九世纪后期描述的Jolles 1作为引用，并在1973年谁承认他们是专职抗原提呈细胞2特点是拉尔夫斯坦曼和Zanvil科恩。的DC在外周血和在体内，在暴露于外部环境的组织尤其丰富的大多数组织如皮肤（本如朗格汉斯细胞）和在鼻，肺，胃的衬里和肠这使发现他们遇到的外来抗原。未成熟DC具有内吞能力，但相对较低的能力，以刺激T细胞3。未成熟DC表达各种模式识别受体（的PRRs），该捕获病原体相关分子模式（PAMP），或损害相关分子模式（D-AMPS）4。激活危险信号驱动向成熟树突状免疫原性，而自身分子导致T细胞unresponsiveneSS和凋亡5。免疫原性的DC是由MHC分子和共刺激表面分子和他们的总理幼稚T细胞的能力6,7 ​​的上调为特征。 未成熟DC也可以朝向Treg细胞诱导或致耐受性状态中响应于维生素D3代谢物1α，25（O）2 D 3和某些免疫抑制剂象白细胞介素-10（IL-10），地塞米松和雷帕霉素8-9成熟。致耐受性树突由它们含有表面受体和配体免疫受体基于酪氨酸的抑制基序（ITIMs）的表达为特征。含ILT家族成员，ILT3和ILT4的耐受性树突状ITIMs的信号转导抑制alloproliferation和驱动器的Foxp3 + Treg细胞膨胀10,11。耐受性DC的这些独特的性质导致它们在体内的深刻效力，即诱导持久的耐受移植异体移植物和suppr的能力ESS自身免疫疾病的发展。致耐受性树突可以因此被看作其中在免疫激活的抑制功能成熟偏振光的DC的子类型。 目前，有树突状细胞在人外周血的两种通用子集:浆树突和骨髓DC 12。循环DC是罕见构成对人体血液中的白细胞的小于2％，这造成了困难的DC适当数量的分离研究其免疫调节功能。为了克服这个问题，单核细胞分化的DC用作树突细胞功能的研究的体外模型。相比在体内的DC 这些体外 DC具有相似的受体和功能。 体内的DC和体外产生的单核细胞衍生的DC（moDCs）详细的比较是由其他实验室13，14，15的影响。它也有报道说moDCs和CD1C +的DC分别相当于在抗原提呈和诱导T细胞的功能15。 在本文中，我们描述了产生自外周血单核细胞的未成熟moDCs，然后将它们分化成免疫原性和耐受性DC的方法。这些单核细胞来源的树突细胞（moDCs）由它们的表面标记，细胞因子分布，免疫调节功能和代谢状态，其特征。免疫原性和耐受性的树突细胞产生其导致要么同种异体T细胞或调节性T细胞的膨胀不同的细胞因子。在本文中，细胞因子分析与采用多重技术系统进行。细胞的生长培养基中培养用抗体固定化颜色编码珠和在一个紧凑的分析器读取。 DC的代谢状态使用测量氧气消耗速率，细胞呼吸的一个指标，和细胞外酸化率反映糖酵解外通量分析仪分析通量树突状细胞。这些生物能学率的测量提供跟踪细胞代谢这是在树突状细胞的发育和功能的重要变化的手段。

<table>
	<tbody>
		<tr>
			<td>Ficoll</td>
			<td>GE Healthcare</td>
			<td>17-1440-03</td>
			<td>PBMC isolation</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Becton, Dickinson</td>
			<td>302832</td>
			<td>PBMC isolation</td>
		</tr>
		<tr>
			<td>1.5 mL centrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td>PBMC isolation</td>
		</tr>
		<tr>
			<td>15 mL falcon tube</td>
			<td>Falcon</td>
			<td>352096</td>
			<td>PBMC isolation</td>
		</tr>
		<tr>
			<td>50 mL falcon tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>PBMC isolation</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>PBMC isolation</td>
		</tr>
		<tr>
			<td>0.2 &#181;m filter</td>
			<td>Sartorius stedim biotech</td>
			<td>17597</td>
			<td>PBMC isolation</td>
		</tr>
		<tr>
			<td>MACs kit</td>
			<td>Miltenyi biotec</td>
			<td>130-042-201</td>
			<td>Monocyte enrichment</td>
		</tr>
		<tr>
			<td>MiniMACS Separator</td>
			<td>Miltenyi biotec</td>
			<td>130-042-102</td>
			<td>Monocyte enrichment</td>
		</tr>
		<tr>
			<td>Cell culture grade water</td>
			<td>Invitrogen, Life Technologies</td>
			<td></td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco, Life Technologies</td>
			<td>11875-093</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>SH30070103</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco, Life Technologies</td>
			<td>15140</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Phosphate 
			Buffered Saline</td>
			<td>Gibco, Life Technologies</td>
			<td>10010-031</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Gibco, Life Technologies</td>
			<td>11140-040</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Gibco, Life Technologies</td>
			<td>15575</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco, Life Technologies</td>
			<td>15630-080</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Gibco, Life Technologies</td>
			<td>11360-070</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>GM-CSF</td>
			<td>Miltenyi biotec</td>
			<td>130-093-868</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>IL-4</td>
			<td>Miltenyi biotec</td>
			<td>130-093-924</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Vitamin D3</td>
			<td>Sigma</td>
			<td>D1530</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D2915</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>LPS</td>
			<td>Sigma</td>
			<td>l2755</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>trypan blue</td>
			<td>Gibco, Life Technologies</td>
			<td>15250-061</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>PerCP-conjugated HLADR&#160;</td>
			<td>BioLegend</td>
			<td>307628</td>
			<td>Cytometry</td>
		</tr>
		<tr>
			<td>PE-conjugated CD80</td>
			<td>BD Biosciences</td>
			<td>557227</td>
			<td>Cytometry</td>
		</tr>
		<tr>
			<td>PE-conjugated CD83&#160;</td>
			<td>BD Biosciences</td>
			<td>556855</td>
			<td>Cytometry</td>
		</tr>
		<tr>
			<td>PE-conjugated CD86&#160;</td>
			<td>BD Biosciences</td>
			<td>555665</td>
			<td>Cytometry</td>
		</tr>
		<tr>
			<td>APC-conjugated CD11c</td>
			<td>BD Biosciences</td>
			<td>340544</td>
			<td>Cytometry</td>
		</tr>
		<tr>
			<td>PE-conjugated CD14&#160;</td>
			<td>Miltenyi biotec</td>
			<td>130-091-242</td>
			<td>Cytometry</td>
		</tr>
		<tr>
			<td>PE-conjugated BDCA3&#160;</td>
			<td>Miltenyi biotec</td>
			<td>130-090-514</td>
			<td>Cytometry</td>
		</tr>
		<tr>
			<td>APC-conjugated ILT3&#160;</td>
			<td>eBioscience</td>
			<td>12-5139-73</td>
			<td>Cytometry</td>
		</tr>
		<tr>
			<td>Isotype matched 
			PerCP- conjugated Mab</td>
			<td>BioLegend</td>
			<td>400250</td>
			<td>Cytometry</td>
		</tr>
		<tr>
			<td>Isotype matched 
			PE- conjugated Mab</td>
			<td>Miltenyi biotec</td>
			<td>130-091-835</td>
			<td>Cytometry</td>
		</tr>
		<tr>
			<td>Isotype matched 
			APC- conjugated Mab</td>
			<td>Miltenyi biotec</td>
			<td>130-091-836</td>
			<td>Cytometry</td>
		</tr>
		<tr>
			<td>BD LSR II Flow Cytometer</td>
			<td>BD Pharmingen</td>
			<td>BD LSR II&#160;</td>
			<td>Cytometry</td>
		</tr>
		<tr>
			<td>cytofix/cytoperm</td>
			<td>BD Biosciences</td>
			<td>554714</td>
			<td>Cytometry</td>
		</tr>
		<tr>
			<td>APC/CY7-conjugated CD25</td>
			<td>BD pharmingen</td>
			<td>557753</td>
			<td>Cytometry</td>
		</tr>
		<tr>
			<td>PE/CY7-conjugated CD4</td>
			<td>Biolegend</td>
			<td>300512</td>
			<td>Cytometry</td>
		</tr>
		<tr>
			<td>PerCP-conjugated CD3</td>
			<td>Biolegend</td>
			<td>300428</td>
			<td>Cytometry</td>
		</tr>
		<tr>
			<td>EasySep Human CD4+ T cell enrichment kit</td>
			<td>STEMCELL Technologies</td>
			<td>19052</td>
			<td>Alloreaction study</td>
		</tr>
		<tr>
			<td>EasySep magnet&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>18000</td>
			<td>Alloreaction study</td>
		</tr>
		<tr>
			<td>Cell Trace CFSE cell proliferation kit</td>
			<td>Molecular probes</td>
			<td>C34554</td>
			<td>Alloreaction study</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco, Life Technologies</td>
			<td>14025092</td>
			<td>Alloreaction study</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647-conjugated FoxP3</td>
			<td>BD Biosciences</td>
			<td>560889</td>
			<td></td>
		</tr>
		<tr>
			<td>Milliplex MAP Human 
			Cytokine/Chemokine 
			magnetic bead panel</td>
			<td>Millipore</td>
			<td>HCYTOMAG-60K</td>
			<td>Cytokine analysis</td>
		</tr>
		<tr>
			<td>5 mL Polystyrene tube</td>
			<td>Falcon</td>
			<td>352058</td>
			<td>Cytokine analysis</td>
		</tr>
		<tr>
			<td>Luminex Sheath Fluid&#160;</td>
			<td>Millipore</td>
			<td>SHEATHFLUID</td>
			<td>Cytokine analysis</td>
		</tr>
		<tr>
			<td>FLEXMAP 3D system with xPONENT software</td>
			<td>Luminex Corporation</td>
			<td>FLEXMAP 3D</td>
			<td>Cytokine analysis</td>
		</tr>
		<tr>
			<td>MitoTracker Red CMXRos</td>
			<td>Cell Signalling</td>
			<td>9082</td>
			<td>Mitochondrial activity</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D2650</td>
			<td>Mitochondrial activity</td>
		</tr>
		<tr>
			<td>XF Assay Medium (OCR)</td>
			<td>Seahorse Bioscience</td>
			<td>102352-000</td>
			<td>Metabolic adaptation</td>
		</tr>
		<tr>
			<td>Glucose&#160;</td>
			<td>Sigma Aldrich</td>
			<td>G8769</td>
			<td>Metabolic adaptation</td>
		</tr>
		<tr>
			<td>XF Base Medium (ECAR)</td>
			<td>Seahorse Bioscience</td>
			<td>102353-100</td>
			<td>Metabolic adaptation</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco, Life Technologies</td>
			<td>25030-081</td>
			<td>Metabolic adaptation</td>
		</tr>
		<tr>
			<td>Calibrant</td>
			<td>Seahorse Bioscience</td>
			<td>100840-000&#160;</td>
			<td>Metabolic adaptation</td>
		</tr>
		<tr>
			<td>XF Cell Mito Stress kit</td>
			<td>Seahorse Bioscience</td>
			<td>103015-100</td>
			<td>Metabolic adaptation</td>
		</tr>
		<tr>
			<td>XF Glycolysis Stress kit</td>
			<td>Seahorse Bioscience</td>
			<td>103020-100</td>
			<td>Metabolic adaptation</td>
		</tr>
		<tr>
			<td>Seahorse&#160;</td>
			<td>Seahorse Bioscience</td>
			<td>XFe96</td>
			<td>Metabolic adaptation</td>
		</tr>
	</tbody>
</table>

generation of immature, mature and tolerogenic dendritic cells with differing metabolic phenotypes

免疫反应的结果，从所述抗原的非特异性先天免疫系统和抗原特异性适应性免疫系统之间的复杂的相互作用。免疫系统是在维持耐受自身分子和迅速反应的病原体的恒定的平衡。树突细胞（DC）是先天免疫系统连结至适应性免疫系统和平衡自我和非自我之间的适应性反应有力专业的抗原呈递细胞。根据不同的成熟信号，未成熟树突状细胞可被选择性地刺激分化成免疫原性或耐受性的DC。免疫原性树突状细胞的抗原特异性T细胞克隆扩增提供扩散信号;同时致耐受性树突细胞调节由抗原特异性T细胞缺失或调节性T细胞的克隆扩增公差。由于这种独特的属性，树突细胞是高度为癌症和自身免疫DISE作为治疗剂追捧ASES。树突细胞可装载在体外特异性抗原和注入人体装入一个特定的免疫应答二者的免疫原性和耐受性。这项工作提出从在表面标志物表达，功能和代谢表型差异的单核细胞，单核细胞成熟树突状细胞（moDCs），耐受性和成熟moDCs 体外 ，产生一种手段。

单核细胞纯化和树突状细胞分化 单核细胞由外周血（ 图1A）密度离心从外周血纯化，随后CD14 +在GM-CSF和IL-4的存在下，得到未成熟树突状细胞的阳性选择磁分离（ 图1B）和培养在完全培养基（ 图2A）。维生素D3和地塞米松后GM-CSF和IL-4的添加导致未成熟moDCs成致耐受性moDCs（ 图2B）的分化。加入脂多糖诱导未成熟moDCs成熟成熟moDCs（ 图2C）和致耐受性moDCs用LPS刺激来验证到成熟（ 图2D）的阻力。在这项工作中使用的血液样品从与先前的健康供体知情得到同意。 MODC表征 表面标志特征流式细胞仪 的直流表面标记分析表明，成熟moDCs表达相比LPS处理耐受性moDCs，致耐受性moDCs和未成熟moDCs（ 图3）成熟标记的HLA-DR，CD83和CD86的水平最高。这些结果表明，相比于以下LPS刺激未成熟moDCs致耐受性moDCs分别到成熟抗性。此外，LPS治疗耐受性moDCs和耐受性moDCs显示相比，未成熟和成熟moDCs CD14，BDCA3（CD141）和免疫球蛋白样成绩单（ILT）3的表达增加。我们在这里产生的耐受性moDCs与以前的报告相一致23。 moDCs的功能特性 诱导成熟moDCs成为免疫原性，并释放促进CD4 + T细胞的增殖的细胞因子。我们通过测定共培养的T细胞的增殖评估了不同的MoDC亚型的免疫原性。成熟moDCs相比致耐受性moDCs是弱免疫原性，如由CD4 + T细胞的低alloproliferation（ 图4A）。致耐受性moDCs由其低的IFN-Γ，低IL-12p40的，和高的IL-10细胞因子的产生在同种异体反应共培养的CD4 + T细胞（ 图4B），其特征。此外，成熟moDCs的增加致耐受性moDCs数量共培养诱导allospecific CD4 + T细胞增加的CD25 高 Foxp3 +调节性T细胞（ 图4C）的频率。 <p类="jove_content"FO:保together.within页="1"&gt; 线粒体活性分析 红色CMXRos用于反映线粒体活性来分析在moDCs线粒体膜电位水平。观察致耐受性moDCs与其他的MoDC分化亚型（ 图5A）相比，具有更高的线粒体活性。接着，线粒体氧消耗（OCR）的速率被评估为使用生物分析仪的不同的MoDC亚型。 OCR测量允许高分辨率见解代谢分布，提供信息，包括但不限于基础呼吸，备用呼吸容量，质子泄漏和非线粒体呼吸。 OCR的测量提供，以评估细胞对应激反应的能力的装置。将细胞通过加入相继三个不同的化合物代谢扰动。在第一次注射是01igomycin（ATP耦合器），其抑制电子传递链（ETC）的络合物V时，抑制ATP的合成。此步骤区分消耗ATP合成的氧的百分比和氧的消耗跨线粒体膜克服质子泄漏的百分比。在第二次注射是FCCP（ETC加速器），通过在线粒体膜，而不是通过复杂的五线粒体膜电位的崩溃导致能量和氧的快速消耗的质子通道输送氢离子破坏ATP合成，无新一代的ATP。 FCCP治疗可用于计算细胞的备用呼吸容量。胁迫条件下的备用产能的呼吸是维持细胞生存的关键。这种能力是由几个因素，包括基片的可用性和功能的能力涉及的等酶的测定在第三次注射是鱼藤酮，一个器Comp1的组合恩I抑制剂，和抗霉素A，一个复合物III抑制剂。这种组合关闭线粒体呼吸和OCR被观察到降低线粒体功能受损（ 图5C）的结果。致耐受性moDCs显示较高的基础的OCR水平比成熟moDCs（ 图5D）。此外，致耐受性，脂多糖致耐受性，和未成熟moDCs表现出与成熟moDCs（ 图5E）相比增加备用呼吸的能力。 moDCs代谢表征 因为乳酸和质 ​​子从细胞糖酵解过程中释放，我们通过进行细胞外酸化（ECAR）（ 图6B）的速率的实时分析来分析moDCs的糖酵解活性。在葡萄糖的存在下，所有moDCs的糖酵解速率增加与基础阶段相比，具有成熟moDCs呈现更高的糖酵解速度比未成熟moDCs（ 图6D）。用LPS处理的moDCs（ 图6B）相比，耐受性和未成熟moDCs表现出更高的最大糖酵解（通过在葡萄糖存在下寡诱导）。耐受性和不成熟的moDCs的糖酵解能力比成熟moDCs（ 图6E）高。相反，他们的高糖酵解率，糖酵解储备是成熟moDCs（ 图 6F）的最低值。 <img alt="图1" src="/files/ftp_upload/54128/54128fig1.jpg" /> 图1:从外周血单核细胞纯化 （A）将25ml血液小心离心前铺在15毫升每50毫升管菲的。的PBMC都集中在密度离心后下等离子体的层。（B）中的PBMC一起培养与那些连词微珠<html> ugated单克隆人CD14抗体（同型:小鼠IgG2a），再装上这是摆在一个分离器的磁场分离CD14 +单核细胞一列<a href="https://www.jove.com/files/ftp_upload/54128/54128fig1large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54128/54128fig2.jpg" /> 图2:moDCs形态表征。 （A）的 200 ng / ml的GM-CSF和IL-4加入到在第0天，第4和第6纯化的CD14 +单核细胞，以产生未成熟moDCs;和（B）刺激的用100nM维生素D3和第5天为10nM地塞米松的额外步骤产生致耐受性moDCs。未成熟moDCs和耐受性moDCs与6日1微克/毫升LPS刺激生成（C）成熟moDCs和（D）LPS-耐受性moDCs。TTPS://www.jove.com/files/ftp_upload/54128/54128fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54128/54128fig3.jpg" /> 图3:通过流式细胞术表面标记的HLA-DR，CD80，CD83，CD86，CD11，CD14，BDCA3和LT3在致 ​​耐受性的表达水平（绿），脂多糖致耐受性（黑色），未成熟的（红色） 表面标记表征成熟（蓝色）moDCs。同种型对照以灰色显示。对于每种细胞类型个人直方图绘制与抗荧光团的强度和重叠的X轴日志Y轴细胞计数。所有直方图代表四个独立的实验。 10.4049 / jimmunol.1303316（2015年6月1日）:该图已经从免疫学杂志 194（11），5174-5186，DOI修改。转载和版权许可转载版权所有2015年的美国协会免疫学家，公司<a href="https://www.jove.com/files/ftp_upload/54128/54128fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54128/54128fig4.jpg" /> 图4:moDCs的功能特性。 （A）和致耐受性的越来越多的诱导共培养的CD4 + T-细胞的alloproliferation频率的定量（TOL;绿色），脂多糖致耐受性（L-TOL;黑色），未成熟的（IMM;红色）和成熟（MAT ;蓝色）moDCs。该数据是从四个独立实验汇集;意味着所有moDCs与成熟moDCs通过双向ANOVA进行分析以及Dunnett多重比较测试后在+ SEM的统计学差异。（B）中的IFN-Γ（左面板），IL-12p40的（中图）和IL-的细胞因子分析10（右窗格升）在从CD4 + T细胞用任一致耐受性（绿色），未成熟的（红色）或成熟的（蓝色）moDCs的越来越多的共培养之间alloreactions上清。数据来自六个独立实验汇集;平均值±SEM（C）的 CD4 + T细胞alloproliferation以及提高致耐受性moDCs的号码的存在诱导共培养成熟moDCs调节性T细胞的扩增。左侧Y轴，CD4 + T细胞增殖的频率。右Y轴，CD25 高 Foxp3的+栅上增殖的CD4 + T细胞的细胞频率。该数据来自三次独立实验汇集;平均值±SEM存在与不存在耐受性moDCs通过单向ANOVA进行分析以及Dunnett多重比较测试后的之间的统计学差异（D）的CD4 + T细胞alloproliferation（左）和CD25highFoxP3 +细胞的频率（右）诱导通过共文化与耐受性moDCs在增加成熟moDCs数量的存在。该数据是从两到三个独立实验汇集;平均值±SEM的存在之间的统计学差异与缺乏成熟moDCs的通过双向ANOVA以及Dunnett多重比较测试后进行分析。 10.4049 / jimmunol.1303316（2015年6月1日）:该图已经从免疫学杂志 194（11），5174-5186，DOI修改。转载和版权许可。 版权所有2015年免疫学家协会再版，公司 <a href="https://www.jove.com/files/ftp_upload/54128/54128fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54128/54128fig5.jpg" /> 图5:moDCs的线粒体活性分析。 （A）是用f得到moDCs线粒体膜电位的水平（红色CMXRos）低流式细胞分析。的四个独立实验的数据合并。平均值±SEM（B）的一个实时线粒体呼吸的示意图。 OCR分析，从基础呼吸和增加寡（复合物V抑制），FCCP（最大呼吸感应），鱼藤酮和/抗霉素A混合物（电子传递链[ETC]抑制）后启动。线粒体SRC（从最大呼吸减去最大基底）被从OCR曲线的。（C）的线粒体的OCR（皮摩尔/分钟）在致耐受性（TOL，绿色）的代表性动力学研究，脂多糖致耐受性（L-TOL，黑色）未成熟（IMM，红色）和成熟（MAT，蓝）通过使用顺序加成寡霉素（OLIG），FCCP，和鱼藤酮/抗霉素A（腐-AA）（D）的OCR moDCs和的基础呼吸的量化moDCs（ E）备用moDCs的呼吸能力。这些数据是从五个独立的实验汇总。平均值±SE10.4049 / jimmunol.1303316（2015年6月1日）:M。该图是从免疫学杂志 194（11），5174-5186，DOI修改。转载和版权许可。 版权所有2015年免疫学家协会再版，公司 <a href="https://www.jove.com/files/ftp_upload/54128/54128fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54128/54128fig6.jpg" /> 图6:moDCs代谢表征。 （一）实时糖酵解的示意图。 ECAR分析从基底ECAR，其中，所述细胞在无葡萄糖的培养基，然后加入葡萄糖（糖酵解诱导）的培养开始时，寡霉素（其诱导最大细胞糖酵解和复合物V抑制），并最后2-脱氧-D-葡萄糖（糖酵解抑制）。糖酵解率（糖酵解感应减去基础ECAR），糖酵解能力（减去基础ECAR最大糖酵解）和糖酵解储备（最大糖酵解减去糖酵解感应）从ECAR曲线的。（B）代表动力学研究糖酵解依赖在致耐受性ECAR（MPH /分钟）（TOL，绿色），脂多糖致耐受性（L-TOL，黑色），未成熟的（IMM，红），和成熟的（MAT，蓝）通过使用顺序添加葡萄糖（GLUC）的moDCs，寡霉素（OLIG），和2-DG。（℃）酒吧显示基底ECAR水平（D）的糖酵解速率（E）糖酵解容量，和（F）moDCs的糖酵解储备。该数据来自三次独立实验汇集;平均6 SEM。统计差异通过单向ANOVA与杜克多重比较测试后进行分析。 10.4049 / jimmunol:这一数字已经从免疫学杂志 194（11），5174-5186，DOI修改0.1303316（2015年6月1日）。转载和版权许可。 版权所有2015年免疫学家协会再版，公司 <a href="https://www.jove.com/files/ftp_upload/54128/54128fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes at JoVE.com

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

不成熟，成熟和耐受性树突状细胞的生成与代谢不同表型

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

generation-immature-mature-tolerogenic-dendritic-cells-with-differing

Research

JoVE Journal

Immunology and Infection

22.9K 次观看.  Agency for Science, Technology and Research. Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

视频: Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

高效转染树突状细胞的无细胞的成熟优化的协议

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

科研

免疫与感染

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

小鼠肺树突状细胞的分离

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies 2.
Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production.
DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 &deg;C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine 3. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4 - 20 x 106 cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.

The most commonly used method for generating large numbers of autologous dendritic cells (DCs) for use in tumor immunotherapy is described. The method uses IL-4 and GM-CSF to differentiate DCs from monocytes. The immature DCs are stimulated to mature and then loaded with antigens before they are injected back into the patient.

制备肿瘤抗原负载成熟树突状细胞免疫治疗

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

使用黑色素瘤和鳞状癌干细胞生成新型支气干细胞疫苗

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

性T细胞Transinfection从树突状细胞捕捉细菌

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

在体外产生的常见淋巴祖使用AC-6馈线系统的小鼠浆细胞样树突细胞

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

代和GM-CSF的鉴定源性肺泡状巨噬细胞和树突状细胞从小鼠骨髓

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...