制备肿瘤抗原负载成熟树突状细胞免疫治疗
Summary
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。
Abstract
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>
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。
Protocol
1。外周血单个核细胞的分离和冷冻保存无菌操作 – 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。 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。 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。 轻轻层30毫升稀释后的白细胞分离…
Representative Results
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）…
Discussion
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，?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
笔者想感谢安德烈斯·萨拉萨尔（Oncovir公司）聚ICLC的礼物。
Materials
| Reagent/Supplies/Equipment | Manufacturer | Catalog No. | |
| RPMI-1640 medium with L-glutamine | BioWhittaker | 12-702F | |
| 1M HEPES buffered saline | BioWhittaker | 17-737E | |
| Phosphate buffered saline (PBS) | BioWhittaker | 17-516F | |
| Human albumin, 25% solution USP | Aventis Behring | ||
| Ficoll-Hypaque PREMIUM | GE Healthcare | 17-5442-03 | |
| Human AB serum | Valley Biomedical | HP1022 | |
| Sterile saline USP | Hospira | ||
| CryoMACS DMSO | Miltenyi Biotec | 170-076-303 | |
| Leukine GM-CSF, 0.5 mg/ml | Berlex | A02266 | |
| MACS GMP IL-4 | Miltenyi Biotec | 170-076-101 | |
| Hiltonol, Poly-ICLC, 2 mg/ml | Oncovir | NA | |
| VACMUNE KLH | Biosyn | ||
| 225 sq cm EasyFlasks | Nalgene Nunc | 159934 | |
| Falcon 6-well tissue culture plates | Becton Dickinson | 353046 | |
| 1.8 ml CryoTube vials | Nalgene Nunc | 377267 | |
| Controlled Rate Freezer | Thermo | CryoMed |
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Diesen Artikel zitieren
Sabado, R. L., Miller, E., Spadaccia, M., Vengco, I., Hasan, F., Bhardwaj, N. Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy. J. Vis. Exp. (78), e50085, doi:10.3791/50085 (2013).