扩大目标，脐血细胞毒性T淋巴细胞，巨细胞病毒，EB病毒，腺病毒
1Center for Cell and Gene Therapy,Baylor College of Medicine , 2Pathology and Immunology,Baylor College of Medicine , 3Department of Stem Cell Transplantation and Cellular Therapy,University of Texas M.D. Anderson Cancer Center, 4의학,Baylor College of Medicine , 5Department of Pediatrics,Baylor College of Medicine
Summary
在这里，我们描述的第一个好生产质量管理规范（GMP）标准的方法从脐带血幼稚T细胞的来源主要是病毒特异性细胞毒性T淋巴细胞（CTL）。
Abstract
干细胞移植后的病毒感染是最常见的死亡原因，尤其是在脐带血（CB）移植（CBT），CB不包含相当数量的病毒有经验的T细胞可以保护收件人感染。1 –我们和其他人表明，病毒特异性CTL产生的血清学阳性的捐助者，并注入到收件人的安全和保护。5-8然而，直到最近，病毒特异性T细胞不能产生脐带血，可能是由于病毒特异性记忆T细胞的缺乏。
为了更好地模仿体内吸幼稚T细胞的条件，我们建立了一个方法，该方法使用CB来源的树突状细胞（DC）的腺病毒载体（Ad5f35pp65）含有的免疫CMV抗原病毒pp65转，因此驱动T细胞特异性对巨细胞病毒和腺病毒。在开始，我们利用这些马捕获区议会，以及CB来源的T细胞的细胞因子的存在下，IL-7，IL-12和IL-15。[10]在所述第二刺激我们使用EBV-转化的B细胞，或EBV-LCL，同时表达潜在的和裂解EB病毒抗原。 ad5f35pp65转导的EBV-LCL是用于刺激的T细胞中的IL-15的存在，在第二次刺激。后续的刺激使用Ad5f35pp65转导的EBV-LCL和IL-2。
从50×10 6 CB单核细胞中，我们能够产生向上的150×10 6病毒特异性T细胞，裂解抗原脉冲的目标和释放细胞因子响应于抗原刺激11将这些细胞在GMP兼容的方式制造，使用仅分馏脐带血单元分数的20％，并已被翻译供临床使用。
Protocol
1。单核细胞分离（0天） 插入秒杀到出口的20％部分的脐带血阴鲁尔适配器，连接注射器和清除血液中。解冻血液转移到20毫升温热在50ml离心管中的RPMI。用5毫升的RPMI冲洗脐血袋和转移到相同的离心管中。 离心细胞以400×g进行10分钟。吸出上清液。 在20毫升的温暖的RPMI重悬细胞。到在50ml离心管中的15毫升淋巴制剂层细胞。离心40分钟@ 400×克。 含有单核细胞的收获的接?…
Discussion
目的是在控制病毒感染后，CBT目前的战略是有效的，但他们都与显著的毒性，价格昂贵，并且不授予长期保护，对以后的感染。事实上，一些抗病毒药物的使用可能会限制病毒特异性T细胞，否则将保护的膨胀。14另一种选择是来自供体的病毒特异性T细胞的输注。我们和其他人表明，这种T细胞是安全，有效和符合成本效益的。15日至17日在这里，我们表明，这种方法也可以扩展…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了一个丹·邓肯的合作研究资助（CMB和EJS），美国国家心脏，肺，和血液研究所（US4HL081007），白血病和淋巴瘤协会临床研究学者奖（CMB），美国国家癌症研究所（RO1 CA06150816 EJS）。
Materials
| Name of reagent | Company | Catalogue number |
| RPMI 1640 | Invitrogen | 21870-076 |
| DC media | CellGenix | 20801-0500 |
| EHAA (Click’s Medium) | Irvine Scientific | 9195 |
| Human Serum | Gemini Bio Products | 100-110 |
| Gas Permeable Cultureware18 | Wilson-Wolf | 80040S |
| IL-2 | Chiron (TCH Pharmacy) | |
| IL-12 | NCI/CTEP | |
| IL-15 | CellGenix | 1013-050 |
| IL-7 | R&D | AFL207 |
| IL-1beta | R&D | AFL201 |
| IL-6 | CellGenix | 1004-050 |
| GM-CSF | TCH Pharmacy | |
| IL-4 | R&D | AFL204 |
| TNF-alpha | R&D | AFL210 |
| Ad5f35pp65 | BCM CAGT Vector Production Facility | |
| Plasma transfer set with female luer adapter | Charter Medical | 89-550-66j |
| Lymphoprep | Nycomed | 1114550 |
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Cite This Article
Hanley, P. J., Lam, S., Shpall, E. J., Bollard, C. M. Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus. J. Vis. Exp. (63), e3627, doi:10.3791/3627 (2012).