Name: preparation of tumor antigen-loaded mature dendritic cells for immunotherapy
Uploaded: 2013-08-01T12:00:00
Description: Watch this Scientific Journal Video about Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy at JoVE.com

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...

<ol>
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B., Bhardwaj, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+agonists:+are+they+good+adjuvants?.">Toll-like receptor agonists: are they good adjuvants?.</a> Cancer J. 16, 382-391 (2010).</li><li>Kedl, R. M., Kappler, J. W., Marrack, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitope+dominance,+competition+and+T+cell+affinity+maturation.">Epitope dominance, competition and T cell affinity maturation.</a> Curr. Opin. Immunol. 15, 120-127 (2003).</li><li>Bogunovic, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR4+engagement+during+TLR3-induced+proinflammatory+signaling+in+dendritic+cells+promotes+IL-10-mediated+suppression+of+antitumor+immunity.">TLR4 engagement during TLR3-induced proinflammatory signaling in dendritic cells promotes IL-10-mediated suppression of antitumor immunity.</a> Cancer Research. 71, 5467-5476 (2011).</li><li>Verdijk, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyriboinosinic+polyribocytidylic+acid+(poly(I:C))+induces+stable+maturation+of+functionally+active+human+dendritic+cells.">Polyriboinosinic polyribocytidylic acid (poly(I:C)) induces stable maturation of functionally active human dendritic cells.</a> J. Immunol. 163, 57-61 (1999).</li><li>Rouas, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(I:C)+used+for+human+dendritic+cell+maturation+preserves+their+ability+to+secondarily+secrete+bioactive+IL-12.">Poly(I:C) used for human dendritic cell maturation preserves their ability to secondarily secrete bioactive IL-12.</a> Int. Immunol. 16, 767-773 (2004).</li><li>Jongmans, W., Tiemessen, D. M., van Vlodrop, I. J., Mulders, P. F., Oosterwijk, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th1-polarizing+capacity+of+clinical-grade+dendritic+cells+is+triggered+by+Ribomunyl+but+is+compromised+by+PGE2:+the+importance+of+maturation+cocktails.">Th1-polarizing capacity of clinical-grade dendritic cells is triggered by Ribomunyl but is compromised by PGE2: the importance of maturation cocktails.</a> J. Immunother. 28, 480-487 (2005).</li><li>Krause, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandin+E2+is+a+key+factor+for+monocyte-derived+dendritic+cell+maturation:+enhanced+T+cell+stimulatory+capacity+despite+IDO.">Prostaglandin E2 is a key factor for monocyte-derived dendritic cell maturation: enhanced T cell stimulatory capacity despite IDO.</a> J. Leukoc. Biol. 82, 1106-1114 (2007).</li><li>Morelli, A. E., Thomson, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+under+the+spell+of+prostaglandins.">Dendritic cells under the spell of prostaglandins.</a> Trends Immunol. 24, 108-111 (2003).</li><li>Adams, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+(DC)+based+therapy+for+cervical+cancer:+use+of+DC+pulsed+with+tumour+lysate+and+matured+with+a+novel+synthetic+clinically+non-toxic+double+stranded+RNA+analogue+poly+[I]:poly+[C(12)U]+(Ampligen+R).">Dendritic cell (DC) based therapy for cervical cancer: use of DC pulsed with tumour lysate and matured with a novel synthetic clinically non-toxic double stranded RNA analogue poly [I]:poly [C(12)U] (Ampligen R).</a> Vaccine. 21 (12), 787-790 (2003).</li><li>Colombo, M. P., Trinchieri, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-12+in+anti-tumor+immunity+and+immunotherapy.">Interleukin-12 in anti-tumor immunity and immunotherapy.</a> Cytokine Growth Factor Rev. 13, 155-168 (2002).</li><li>Mayordomo, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow-derived+dendritic+cells+pulsed+with+synthetic+tumour+peptides+elicit+protective+and+therapeutic+antitumour+immunity.">Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity.</a> Nat Med. 1, 1297-1302 (1995).</li><li>Dhodapkar, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+generation+of+broad+T-cell+immunity+in+humans+after+a+single+injection+of+mature+dendritic+cells.">Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells.</a> J. Clin. Invest. 104, 173-180 (1999).</li><li>Schuler-Thurner, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+induction+of+tumor-specific+type+1+T+helper+cells+in+metastatic+melanoma+patients+by+vaccination+with+mature,+cryopreserved,+peptide-loaded+monocyte-derived+dendritic+cells.">Rapid induction of tumor-specific type 1 T helper cells in metastatic melanoma patients by vaccination with mature, cryopreserved, peptide-loaded monocyte-derived dendritic cells.</a> J. Exp. Med. 195, 1279-1288 (2002).</li></ol>

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，?...

<table border="1">
<tbody>
 <tr>
 <td fo:width="2in">Reagent/Supplies/Equipment</td>
 <td>Manufacturer</td>
 <td>Catalog No.</td>
 <td></td>
 </tr>
 <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）...

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

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高效转染树突状细胞的无细胞的成熟优化的协议

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