Gene expression profiles of primary human NK cells before and after expansion on CSTX002 feeder cells, with and without IL-21 stimulation

NK cell development, maturation, and activation by cytokines is driven by alterations in gene expression mediated by activation and repression or transcriptional programs. In particular, we have extensively studied the role of STAT3 in human NK cells. This was based in part on a method we developed for in vitro expansion of large numbers of highly active NK cells using a genetically-modified feeder cell expressing 4-1BBL and membrane-bound IL-21. To dissect the various gene expression profiles induced by IL-21 from the various other signals received from the feeder cell, we purified peripheral NK cells from 4 healthy subjects (naïve, N), expanded NK cells for 14 days using CSTX002 feeder cells (expanded, E), and extracted RNA from the cells without (Neg) or after (Pos) the cells were activated with IL-21. We then performed RNA sequencing on each sample. Overall design: NK cells were purified from buffy coats obtained from 4 normal healthy blood-bank donors using RosetteSep NK for negative depletion of other cell subsets. NK cells were expanded by weekly stimulation with irradiated CSTX002 feeder cells. Naïve or expanded NK cells were stimulated for 30 minutes with 20 ng/ml recombinant human IL-21. Total RNA was prepared using the Total RNA Purification Plus Kit (Norgen Biotek, Ontario, ON, Canada). Libraries were prepared using the TruSeq RNA Sample Preparation Kit (Illumina Inc., San Diego, CA). 60–80 million paired-end 150 bp sequence reads per library were generated using the Illumina HiSeq4000 platform.

NK细胞（natural killer cell）的发育、成熟以及细胞因子介导的活化过程，均由激活、抑制或转录程序所调控的基因表达改变所驱动。其中，我们已对STAT3（signal transducer and activator of transcription 3）在人NK细胞中的作用展开了深入研究，该研究的部分依据来自我们开发的一种体外扩增大量高活性NK细胞的方法：使用表达4-1BBL与膜结合型IL-21的基因修饰饲养层细胞进行扩增。为剖析IL-21诱导的基因表达谱与饲养层细胞传递的其他信号之间的差异，我们从4名健康受试者体内纯化外周血NK细胞（未处理组，N），并利用CSTX002饲养层细胞将NK细胞扩增14天（扩增组，E）；随后分别从未经IL-21激活的细胞（阴性对照组，Neg）与经IL-21激活后的细胞（阳性组，Pos）中提取RNA，并对所有样本开展RNA测序。
整体实验设计如下：采用RosetteSep NK细胞分选试剂盒对4名健康献血者的血沉棕黄层（buffy coat）中的NK细胞进行纯化，通过阴性耗竭去除其他细胞亚群。通过每周使用辐照后的CSTX002饲养层细胞进行刺激，实现NK细胞的扩增。将未处理或扩增后的NK细胞以20 ng/ml的重组人IL-21刺激30分钟。总RNA采用Total RNA Purification Plus试剂盒（Norgen Biotek公司，加拿大安大略省）进行提取。文库构建采用TruSeq RNA样本制备试剂盒（Illumina公司，美国加利福尼亚州圣地亚哥）。每个文库通过Illumina HiSeq4000平台生成6000万至8000万条150 bp双端测序读段。