Kurachi Makoto, Kurachi Junko, Chen Zeyu, Johnson John, Khan Omar, Bengsch Bertram, Stelekati Erietta, Attanasio John, McLane Laura M, Tomura Michio, Ueha Satoshi, Wherry E John
Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Institute for Immunology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Nat Protoc. 2017 Sep;12(9):1980-1998. doi: 10.1038/nprot.2017.083. Epub 2017 Aug 31.
Retroviral (RV) expression of genes of interest (GOIs) is an invaluable tool and has formed the foundation of cellular engineering for adoptive cell therapy in cancer and other diseases. However, monitoring of transduced T cells long term (weeks to months) in vivo remains challenging because of the low frequency and often poor durability of transduced T cells over time when transferred without enrichment. Traditional methods often require additional overnight in vitro culture after transduction. Moreover, in vitro-generated effector CD8 T cells enriched by sorting often have reduced viability, making it difficult to monitor the fate of transferred cells in vivo. Here, we describe an optimized mouse CD8 T-cell RV transduction protocol that uses simple and rapid Percoll density centrifugation to enrich RV-susceptible activated CD8 T cells. Percoll density centrifugation is simple, can be done on the day of transduction, requires minimal time, has low reagent costs and improves cell recovery (up to 60%), as well as the frequency of RV-transduced cells (∼sixfold over several weeks in vivo as compared with traditional methods). We have used this protocol to assess the long-term stability of CD8 T cells after RV transduction by comparing the durability of T cells transduced with retroviruses expressing each of six commonly used RV reporter genes. Thus, we provide an optimized enrichment and transduction approach that allows long-term in vivo assessment of RV-transduced T cells. The overall procedure from T-cell isolation to RV transduction takes 2 d, and enrichment of activated T cells can be done in 1 h.
逆转录病毒（RV）介导的目的基因（GOI）表达是一种非常有价值的工具，它构成了癌症及其他疾病过继性细胞治疗中细胞工程的基础。然而，在体内长期（数周乃至数月）监测转导的T细胞仍然具有挑战性，因为在未进行富集转移时，转导的T细胞频率较低且随着时间推移其耐久性往往较差。传统方法通常在转导后需要额外进行过夜体外培养。此外，通过分选富集的体外产生的效应性CD8 T细胞活力常常降低，这使得在体内监测转移细胞的命运变得困难。在此，我们描述了一种优化的小鼠CD8 T细胞RV转导方案，该方案使用简单快速的Percoll密度离心法来富集对RV敏感的活化CD8 T细胞。Percoll密度离心法操作简单，可在转导当天进行，所需时间最少，试剂成本低，能提高细胞回收率（高达60%），以及RV转导细胞的频率（与传统方法相比，在体内数周内提高约六倍）。我们已使用该方案通过比较用表达六种常用RV报告基因的逆转录病毒转导的T细胞的耐久性，来评估RV转导后CD8 T细胞的长期稳定性。因此，我们提供了一种优化的富集和转导方法，可对RV转导的T细胞进行长期体内评估。从T细胞分离到RV转导的整个过程需要2天，活化T细胞的富集可在1小时内完成。
