Summary
本文介绍了一个简单的方法诱导人外周血单个核细胞同种异体抗原特异性无能。该技术可应用于临床，产生非同种异体的供体细胞。这些细胞输注可以提高免疫功能重建和减少异基因造血干细胞移植后的毒性。
Abstract
异基因造血干细胞移植（AHSCT）提供许多先天和后天的血液病病人治愈的最佳机会。不幸的是，承认并损害健康的病人组织的同种供体T细胞移植可以导致移植物抗宿主病（GVHD）1 。成功AHSCT的挑战之一是预防GVHD没有相关的捐助者的T细胞，尤其是免疫重建和预防复发的有利影响的减值。 GVHD是可以预防的供者T细胞从干细胞移植或由行政药理免疫抑制非特异性枯竭。不幸的是，这些方法增加感染和疾病的复发2-4。另一种策略是有选择性地消耗同种异体捐助者的T细胞后，由收件人抗原提呈细胞（APC）的移植前allostimulation。这些allodepletion战略的早期临床试验改进没有多余的GVHD 5，6 HLA不相合造血干细胞移植后免疫重建。然而，一些allodepletion技术要求专门收件人APC生产6，7，脱靶效应，包括捐助病原体特异性T细胞和CD4枯竭调节性T细胞的一些方法可能有一个替代的方法是同种异体捐助者的T细胞的失活通过诱导对同种异体抗原特异性低反应。这是通过与受援国APC的刺激体细胞，同时提供CD28介导的共刺激信号 10的封锁。这种“alloanergization”的做法降低了1-2日志alloreactivity，同时保留在体外培养11的病原体和肿瘤相关抗原的T细胞反应。该战略已成功地采用了2完成和正在进行的临床试验alloanergized捐助者的T细胞被注入导致快速的免疫重建，感染少，比历史控制收件人的不太严重的急性和慢性GVHD的HLA不相合造血干细胞移植期间或之后的研究unmanipulated HLA不相合移植12。在这里，我们描述当前的一代已alloanergized HLA不相合的无关刺激外周血单核细胞的外周血单个核细胞（PBMC）的协议。 Alloanergization是实现配体单克隆抗体的存在allostimulation B7.1和B7.1阻断CD28介导的共刺激。这种技术并不需要专门刺激APC的生产，是简单的执行，要求只有一个相对短暂的体外孵育阶段。因此，该方法可以很容易地供临床使用，捐助者的T细胞生成减少alloreactivity但保留病原体特异性免疫过继转移AHSCT改善没有过多的移植物抗宿主病的免疫重建的设置标准。
Protocol
Discussion
Disclosures
没有利益冲突的声明。
Acknowledgments
Materials
| Name | Company | Catalog Number | Comments |
| Ficoll-Hypaque PLUS | GE Healthcare | 17-1440-02 | |
| RPMI 1640 | Invitrogen | 11875-093 | |
| Hepes 1M | Invitrogen | 15630-080 | |
| L-Glutamine 200mM | Invitrogen | 25030-081 | |
| Gentamicin | Invitrogen | 15750-060 | |
| Human AB serum (heat inactivated) | Gemini Bio Products | 100-512 | Use validated batches |
| Complete Culture Media (500ml) | 440ml RPMI 1640, 50ml AB serum, 5ml Hepes, 5 ml Glutamine, 167uL Gentamicin | ||
| Irradiator | GammaCell 1000 | ||
| T-25 Cell Culture Flasks with gas permeable caps | Corning | 3056 | |
| Humanized Monoclonal anti-B7.1 and anti B7.2 antibodies | See protocol text | ||
| U bottomed 96 well culture plates | BD Biosciences | 353077 | |
| Monoclonal CD3 antibody | Beckman Coulter Inc. | IM0178 | Reconstitute with 200ml distilled water |
| Monoclonal CD28 antibody | Beckman Coulter Inc. | IM1376 | Reconstitute with 200ml distilled water |
| CMV grade 2 antigen | Microbix | EL-01-02 | |
| Tritiated Thymidine | NEN | NET027 | |
| Scintillation Fluid | PerkinElmer, Inc. | 1205-440 | |
| Harvester | Tomtec | ||
| Printed Filtermat A | PerkinElmer, Inc. | 1450-421 | |
| Filter Bag | PerkinElmer, Inc. | 1450-432 | |
| 1450 MicroBeta TriLux Scintillation Counter | Wallac | ||
| null | |||
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