笔者想感谢安德烈斯·萨拉萨尔（Oncovir公司）聚ICLC的礼物。

1。外周血单个核细胞的分离和冷冻保存<ol><li>无菌操作 - 尖峰之一中的白细胞分离袋的接入端口使用的等离子转移集。用60毫升的注射器，转移到无菌的500毫升瓶装获得了患者的白细胞分离。 </li><li>调整音量的白细胞分离的2倍原体积，使用室温下的RPMI。调匀。 </li><li>轻轻混合的一瓶聚蔗糖 - 帕确PLUS。 12毫升聚蔗糖 - 帕确PLUS添加到无菌的50毫升锥形管。 </li><li>轻轻层30毫升稀释后的白细胞分离产品每个无菌的50毫升锥形管含聚蔗糖。要小心，不要打扰聚蔗糖和细胞悬液之间的接口。 </li><li>重复，直到所有的白细胞分离已分层的步骤1.3和1.4。 </li><li>没有制动器20分钟，在室温下，在1,000×g离心50毫升锥形管。 </li><li>小心地收获阴天从每个管和transfe的外周血单个核细胞层r到新的无菌50ml试管。 </li><li>每个试管中加入RPMI至终体积为50ml。轻轻混匀反转。 </li><li>细胞在500×g离心10分钟，在4℃下用全制动。 </li><li>从每管中取出上清液。从每管中重悬细胞沉淀，加的RPMI至终体积为50ml。轻轻混匀反转。 </li><li>细胞在500×g离心6分钟，在4℃下</li><li>从每管中取出上清液。重悬，汇集成一个50毫升的锥形管细​​胞悬液。 </li><li>将汇集的外周血单个核细胞的体积到50ml带更多的RPMI。轻轻混匀反转。 </li><li>细胞在300×g离心6分钟，在4℃下</li><li>去除上清。在50毫升的RPMI重悬细胞沉淀。轻轻混匀，以保证均匀的细胞悬液。 </li><li>的细胞数进行计数，并确定细胞存活率。计算存活细胞的总数。 </li><li>离心机的细胞在300×g下6分钟，在4℃下准备冻结的媒体。凝固介质由10％人AB血清（谷生物）的二甲基亚砜（DMSO;美天旎生物技术）。 </li><li>去除上清。重悬细胞，使最终浓度为2×10 8个细胞/ ml在寒冷的冷冻介质。 </li><li> 1毫升，1.8毫升离心管中。 </li><li>冻存管转移到的控制率冰柜，开始冻结的运行，程序1。在运行结束时，立即冷冻离心管中转移到汽相中的液氮冷冻库。 </li></ol> 2。 0天:从单核细胞分化为树突状细胞<ol><li>到无菌的50毫升锥形管中加入30毫升的RPMI / 1％自体血浆。 </li><li>解冻等份冷冻外周血单个核细胞，在37℃水浴中轻轻摇动。完全解冻时，小瓶的内容转移到无菌的50毫升锥形管。调匀。 </li><li>离心细胞6分钟在500×g下在室温下。去除上清，重悬细胞沉淀，在小体积的RPMI / 1％自体血浆。添加更多的RPMI / 1％自体血浆，至终体积为50ml。调匀。 </li><li> 6分钟，在500×g下在室温下再次离心细胞。吸出上清液，在50毫升的RPMI / 1％自体血浆和悬浮颗粒。调匀。 </li><li>的细胞数进行计数，并确定细胞存活率。计算存活细胞的总数。 </li><li>板1.40×10 8个细胞在40毫升的RPMI / 1％自体血浆到225厘米2 EasyFlask。重复以上步骤，直到所有的细胞已被镀。 </li><li>平放在其一侧上孵育EasyFlasks，1 - 2小时，在37℃在含5％CO 2的培养箱，使单核细胞附着到该烧瓶中的组织培养。 </li><li>当孵化完成后，取出EasyFlask从孵化器和洗两次，以除去非贴壁细胞USI毫微克30毫升预热的RPMI。 </li><li>第二次洗涤后，加40毫升的RPMI / 1％自体血浆400-1,000 IU / ml的IL-4和100-1,000 IU / ml的GM-CSF的5-8。对于这个协议，我们使用的是400 IU / ml的IL-4和100 IU / ml的GM-CSF。 </li><li>孵育EasyFlasks 2天在37℃，在组织培养孵化含有5％的CO 2。 </li></ol> 3。第2天:饲养的树突状细胞与IL-4和GM-CSF <ol><li>加入4毫升的RPMI / 1％自体血浆与400-1,000 IU / ml的IL-4和100-1,000 IU / ml的GM-CSF的每个EasyFlask。混合在其一侧旋转和摇摆。 </li><li>在组织培养孵化37℃，含5％CO 2孵育EasyFlasks 3天以上（直到第5天）。 </li></ol> 4。第5天:未成熟树突状细胞的收获<ol><li>收获的文化，大力摇动烧瓶和上下吹打悬浮非贴壁和松散贴壁细胞。转移收获细胞新的50毫升锥形管。 </li><li>为6分钟，在室温下，在500×g离心管。取出上清，重悬细胞沉淀在50毫升的RPMI / 1％自体血浆。调匀。 </li><li>的细胞数进行计数，并确定细胞存活率。计算存活细胞的总数。 </li><li>为6分钟，在室温下，在500×g离心管。板1-2×10 6细胞在3毫升的RPMI / 1％自体血浆，每孔用400 IU / ml的IL-4和100 IU / ml的GM-CSF在6孔组织培养板中。 </li><li>孵育板，在37℃组织培养孵化器中含5％CO 2下过夜。 </li></ol> 5。第6天:树突状细胞的成熟和抗原装载<ol><li>加入10微克/毫升KLH 6孔培养板的孔中，以1/3。旋流板混合。 KLH仅添加到孔中的1/3，因为KLH仅用于所述第一D​​C疫苗。随后的DC疫苗CON泰恩肿瘤抗原肽。 </li><li>区议会可以刺激成熟使用不同的刺激。已在临床研究中使用的最常见的成熟刺激了细胞因子（IL-1β，TNF-α和IL-6）和前列腺素E 2（PGE 2）9,10的鸡尾酒。最近，Toll样受体（TLR）激动剂的使用已经获得了普及11,12。在这个协议中，加入2毫克/毫升聚肌胞苷酸聚-L-赖氨酸羧甲基纤维素（聚ICLC）井，拌匀。聚ICLC]是稳定的聚-L-赖氨酸和羧甲基纤维素（由Oncovir，Inc制造）的聚集成电路临床级配方。 </li><li> 100微克/毫升的长肽抗原（NY-ESO-1和/或Melan-A/MART-1）添加到其指定的井，每口井接收只有一个单一的肽，以避免交叉竞争13。 </li><li>孵育板在37℃的组织培养箱含5％CO &lt;子&gt; 2 O / N </li></ol> 6。第7天:树突状细胞的冷冻保存<ol><li>准备DC冷冻解决方案采用自体血浆和10％DMSO。直流凝解，在2000×g下离心20分钟，在4℃下，以除去任何颗粒材料。上清转移到一个新的标记15毫升锥形管。储存于4℃直至使用。 </li><li>当孵育过夜后，收获和池中的所有井到50毫升锥形管含有肽树突。 </li><li> 6分钟，在500×g离心管。取出上清液。重悬细胞沉淀，在5毫升的RPMI / 1％自体血浆。使总体积至50ml用RPMI / 1％自体血浆。调匀。 </li><li>的细胞数进行计数，并确定细胞存活率。计算存活的细胞的总数。 </li><li>在500×g离心管在室温为6分钟。取出上清液，每个颗粒和悬浮在5毫升免缝LE 0.9％氯化钠，USP转移到新的15毫升锥形管。使每个细胞悬液14毫升无菌0.9％氯化钠，USP。盖颠倒混匀。 </li><li>重复上述步骤两次。最后离心后，除去上清液，得到尽可能接近的细胞沉淀的不扰乱颗粒的情况下。 </li><li>在直流冻结解重悬细胞沉淀，在4 - 20×10 6细胞/ ml等分试样1毫升每1.8毫升冻存管形瓶中。 </li><li>使用速率控制冰柜，程序1，冻结DC疫苗的等分试样，并立即转移到液氮冷冻长期储存的汽相。 </li></ol> 7。批签发测试<ol><li> DC疫苗每批必须进行评估，并满足特定的大量释放标准（ 表1），然后再注入患者的研究中，疫苗的制备通常为5〜6周后，它被释放。 </li><li> 可行性:评估使用自动细胞计数的DC疫苗的可行性。可接受的最低的可行性规范是&gt; 70％。 </li><li> 身份:评估成熟的DCS（CD11c +细胞CD14-CD83 +）的身份，通过流式细胞仪。的CD11c +细胞的百分比应为&gt; 50％。 CD11c +细胞和CD14 +细胞的百分比应为&lt;30％的百分比的CD11c +，CD14，和CD83 +细胞&gt; 50％。 </li><li> 无菌试验:测试DC疫苗的存在下厌氧和好氧细菌和真菌直接培养和革兰氏污渍。支原体的存在下，使用直接培养体系和指标细胞株的DNA的荧光染料的染色法的评估。对于这些测试，最小规格集是从所有的培养物中观察到没有增长。 </li><li> 内毒素:评估采用的动力学-QCL动力学的显色LAL检测内毒素含量，而且它必须是欧盟50个/毫升，以满足最低标准。 </li><l&gt; 功能:评估DC功能孵化同种异体T细胞混合淋巴细胞反应。比未受刺激的DC的存在下增殖（ 图3）。 </li></ol>

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作者接受财政支持以下内容:美国国立卫生研究院（AI061684，AI071078，AI044628，K08 AI084578（米勒）），比尔和梅林达·盖茨基金会，多丽丝公爵慈善基金会，癌症研究所，狼疮研究联盟，和翡翠基金会。尼娜BHARDWAJ是一个共同发明人的专利，涉及到操纵免疫力的树突状细胞的制备方法和用途。作者有没有其他任何组织或实体的财务权益或财务冲突稿件除所披露者外讨论的题材或材料相关背景或资金介入。

I期和II期临床试验已经表明，他们的单核细胞来源的DC诱导免疫反应，患者临床成功但已被限制1。这可能是部分原因是由于缺乏共识，如何产生最佳的DC肿瘤免疫治疗应用。虽然有许多方法来产生临床级的DC，这些细胞因子抗原负载的单核细胞，刺激诱导成熟和方法用来区分使用方法不尽相同。的公式产生最佳的DC仍然被定义2。 最近的体外研究表明，成熟的DCs用鸡尾酒的炎性细胞因子10已被用于临床试验的大多数，特别是前列腺素E2（PGE2）的存在，可诱导分化的调节性T细胞和Th2应答17，快递氧酶18，以及缺乏IL-12p70的生产19。这些效果显着破坏疫苗诱发免疫反应的能力，因此支持需要评估，以优化他们的影响在体内成熟的DCS替代方法。 TLR激动剂的使用，特别是Toll样受体3激动剂聚-IC成熟的区议会，区议会可能提高临床疗效， 在体外研究显示，保利IC成熟DC保持稳定的高表达MHCⅡ类分子和CD83共刺激分子CD40，CD80，和CD86 14,15,20。此外，聚集成电路（IC）成熟的DC产生高水平的IL-12的14,16,20，一个重要的细胞因子的产生的抗肿瘤反应21，以及其他促炎细胞因子，如TNF-α，IL-6， IL-1β，IP-10，I型干扰素14。更重要的是，已在小鼠肿瘤模型中显示，与人类乳头状瘤病毒（HPV）抗原的树突和成熟瓦特第i个聚集成电路（IC）催芽细胞毒性T细胞反应能够铲除成立HPV16表达肿瘤22。作为成熟状态的区议会和IL-12的存在似乎与在临床试验中的疗效23,24，使用IC聚到成熟的DC是临床成功达到目标迈出的重要一步。

<table><tbody><tr><td>RPMI-1640 medium with L-glutamine</td><td>BioWhittaker</td><td>12-702F</td><td></td></tr><tr><td>1M HEPES buffered saline</td><td>BioWhittaker</td><td>17-737E</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>BioWhittaker</td><td>17-516F</td><td></td></tr><tr><td>Human albumin, 25% solution USP</td><td>Aventis Behring</td><td></td><td></td></tr><tr><td>Ficoll-Hypaque PREMIUM</td><td>GE Healthcare</td><td>17-5442-03</td><td></td></tr><tr><td>Human AB serum</td><td>Valley Biomedical</td><td>HP1022</td><td></td></tr><tr><td>Sterile saline USP</td><td>Hospira</td><td></td><td></td></tr><tr><td>CryoMACS DMSO </td><td>Miltenyi Biotec</td><td>170-076-303</td><td></td></tr><tr><td>Leukine GM-CSF, 0.5 mg/ml</td><td>Berlex</td><td>A02266</td><td></td></tr><tr><td>MACS GMP IL-4</td><td>Miltenyi Biotec</td><td>170-076-101</td><td></td></tr><tr><td>Hiltonol, Poly-ICLC, 2 mg/ml</td><td>Oncovir</td><td>NA</td><td></td></tr><tr><td>VACMUNE KLH</td><td>Biosyn</td><td></td><td></td></tr><tr><td>225 sq cm EasyFlasks</td><td>Nalgene Nunc</td><td>159934</td><td></td></tr><tr><td>Falcon 6-well tissue culture plates</td><td>Becton Dickinson</td><td>353046</td><td></td></tr><tr><td>1.8 ml CryoTube vials</td><td>Nalgene Nunc</td><td>377267</td><td></td></tr><tr><td>Controlled Rate Freezer</td><td>Thermo</td><td>CryoMed</td><td></td></tr></tbody></table>

preparation of tumor antigen-loaded mature dendritic cells for immunotherapy

虽然临床研究已证实，抗原负载DC疫苗是安全的，有前途的治疗肿瘤1，其临床疗效还有待建立。下面介绍的方法，符合良好的制造工艺（GMP）指南编制，是一种最常见的体外制备方法产生大量临床研究2区议会优化。 我们的方法利用了合成TLR 3激动剂聚肌胞苷酸聚-L-赖氨酸羧甲基纤维素（聚ICLC），以刺激区议会。我们以前的研究聚ICLC是最有力的个人所评估的上调CD83和CD86，诱导白细胞介素12（IL-12），肿瘤坏死因子（TNF），干扰素γ诱导人DCs的成熟刺激蛋白10（IP-10），interleukmin 1（IL-1），I型干扰素（IFN），以及最小的白细胞介素10（IL-10）的生产。 </p&gt; 区议会从冷冻的外周血单核细胞（PBMCs）白细胞分离得到的是有区别的。聚蔗糖梯度离心分离外周血单个核细胞，并冻结在等分。在第1天，外周血单个核细胞解冻并镀上选择坚持到塑料表面的单核细胞，经过1-2小时的潜伏期在37°C在组织培养孵化器的组织培养瓶中。孵育后，细胞被洗掉粘附单核细胞培养5天的白细胞介素4（IL-4）的存在下，和粒细胞巨噬细胞集落刺激因子（GM-CSF），分化为未成熟DCs。在第6天，未成熟DC脉冲与匙孔血蓝蛋白（KLH）的蛋白质，它作为一个控制疫苗质量，并可能提高疫苗的免疫原性。议会刺激成熟，装有肽抗原，孵育过夜。第7天，将细胞洗涤，并在1毫升等分试样中含有冻结4 - 20×10 6个细胞，使用一个受控速率冷冻。大量释放试验区议会的批次进行，必须符合最低规格之前，他们被注入患者。

之间的10 - 20％的起始外周血单个核细胞分化成区议会在培养期结束。成熟的DC的CD11c +，CD14-，CD83 +，CD40 +，CCR7 +（ 图1）。它们表达高水平的MHC I类和II类分子和共刺激分子CD80和CD86。聚ICLC也诱导了较低水平的PDL-1相比，其他TLR激动剂14。此外，这些聚集成电路成熟的区议会分泌大量IL-12（ 图2和图 15,16），诱导增殖的异基因T细胞（ 图3）。 <img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/50085/50085fig1highres.jpg" src="/files/ftp_upload/50085/50085fig1.jpg" /> 图1。完全分化的议会代表型聚IC到期。所有区议会选通正向和侧面散点图。区议会确定了ŞCD11c +细胞和CD14。聚ICLC响应DC成熟（粗线）进行评估的基础上表达CD83，CD40和CCR7比未受刺激的DCs（灰色阴影）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50085/50085fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/50085/50085fig2.jpg" /> 图2。区议会的成熟与保利IC产生高水平的细胞因子。隔夜聚IC的DC成熟后收集上清区议会的分化从3个健康献血者。产生的细胞因子，细胞因子珠阵列（CBA）测定人的炎性细胞因子试剂盒（BD Biosciences公司），它测量IL-12p70的，肿瘤坏死因子，IL-10，IL-6，IL-1β和IL-8。 <img alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/50085/50085fig3highres.jpg" s<html> RC ="/ files/ftp_upload/50085/50085fig3.jpg"/&gt; 图3。区议会成熟聚ICLC诱导同种异体T细胞的增殖。聚ICLC成熟的区议会从健康献血者用二醋酸盐琥珀酰亚胺酯（CFSE）标记的同种异体T细胞孵育1:10。经过6天，流式细胞仪检测细胞增殖（A）门控CD3 + T细胞人口。 X轴显示CFSE稀释，作为测定增殖，在不同的共培养条件:单独的T细胞，未刺激的DC，聚ICLC刺激的DC，植物血球凝集素（PHA）刺激的T细胞。增殖过程中细胞因子的分泌（B）进行评价从在使用BD CBA人力Th1/Th2细胞因子试剂盒II（BD Biosciences公司），测定IFNγ，TNF，IL-10，IL-6，IL-4的培养物的上清液，并的IL-2。只有IFNγ所示其他细胞因子可测量的水平检测中未检测到。/ / www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50085/50085fig3large.jpg"目标="_blank"&gt;点击这里查看大图。 <table border="1"><tbody><tr><td> 测试 </td><td> 方法 </td><td> 标准 </td></tr><tr><td>可行性</td><td>番石榴个人细胞分析ViaCount试剂</td><td> &gt; 70％ </td></tr><tr><td rowspan="3">特性</td><td>流式细胞仪 - ％的CD11c +细胞</td><td> &gt; 50％ </td></tr><tr><td>流式细胞仪 - ％的CD11c + CD14 +细胞</td><td> &lt;30％ </td></tr><tr><td>流式细胞术 - ％的CD11c + CD14-CD83 +细胞</td><td> &gt; 50％ </td></tr><tr><td rowspan="2">不育症</td><td>细菌和真菌培养</td><td>负</td></tr><tr><td>支原体细胞直接培养</td><td>负</td></tr><tr><td>内毒素</td><td>动力学的显色鲎含量</td><td> &lt;欧盟50个/毫升</td></tr><tr><td>功能</td><td>混合淋巴细胞反应</td><td>报告结果</td></tr></tbody></table> 表1中。批签发标准。

Watch this Scientific Journal Video about Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy at JoVE.com

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

用于产生在肿瘤免疫治疗中使用的大量的自体树突状细胞（DC）的最常用的方法进行说明。该方法使用的IL-4和GM-CSF分化的单核细胞的树突状。未成熟DC刺激才成熟，然后装入抗原注射到病人。

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 ...

preparation-tumor-antigen-loaded-mature-dendritic-cells-for

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

18.2K 次观看.  NYU Langone Medical Center. 用于产生在肿瘤免疫治疗中使用的大量的自体树突状细胞（DC）的最常用的方法进行说明。该方法使用的IL-4和GM-CSF分化的单核细胞的树突状。未成熟DC刺激才成熟，然后装入抗原注射到病人。

视频: Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

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

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

小鼠单核细胞衍生树突状细胞的分离及与肿瘤免疫配合物的后体外活化

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

癌症研究

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

文化髓系树突状细胞从骨髓前体

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

生物学

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

代骨髓衍生的双光子成像使用的小鼠树突状细胞

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

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 ...

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

The protocol presents a cancer immunotherapy model using cell-based tumor vaccination with Flt3L-expressing B16-F10 melanoma. This protocol demonstrates the procedures, including preparation of cultured tumor cells, tumor implantation, cell irradiation, measurement of tumor growth, isolation of intratumoral immune cells, and flow cytometry analysis.

使用造血细胞因子接种肿瘤的实验性黑色素瘤免疫治疗模型

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been ...

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

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and 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. ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

一种新的生理免疫原树突状细胞生成方法

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

小鼠致耐受性树突状细胞的发育及功能表征

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

医学

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...