Tumor- and cytokine-primed human natural killer cells exhibit distinct phenotypic and transcriptional signatures [RNA-seq]

An emerging cellular immunotherapy for cancer is based on the cytolytic activity of natural killer (NK) cells against a wide range of tumors. Although in vitro activation or 'priming' of NK cells by exposure to pro-inflammatory cytokines such as interleukin (IL)-2 has been extensively studied, the biological consequences of NK cell activation in response to target cell interactions are less characterized. Herein, we investigate the consequences of co-incubation with K562, CTV-1, Daudi and RPMI-8226 tumor cell lines on the phenotype, cytokine expression profile and transcriptome of human NK cells. We observe the downregulation of several activation receptors including CD16, CD62L, C-X-C chemokine receptor (CXCR)-4, natural killer group 2 member D (NKG2D), DNAX accessory molecule (DNAM)-1 and NKp46 following tumor-priming. Although this NK cell phenotype is typically associated with NK cell dysfunction in cancer, we show upregulation of NK cell activation markers such as CD69 and CD25, secretion of pro-inflammatory cytokines including macrophage inflammatory proteins (MIP-1) a /ß and IL-1ß/6/8 and overexpression of numerous genes associated with enhanced NK cell cytotoxicity and immunomodulatory functions such as FAS, TNFSF10, MAPK11, TNF and IFNG. Key signaling molecules exclusively affected by tumor-priming include MAP2K3, MARCKSL1, STAT5A, and TNFAIP3, which are specifically associated with NK cell cytotoxicity against tumor targets. Collectively, these findings help define the phenotypic and transcriptional signature of NK cells following their encounter with tumor cells, independent of cytokine stimulation, and provide insight into tumor-specific NK cell responses to inform the transition towards harnessing the therapeutic potential of NK cells in cancer. Overall design: The mRNA profiles of freshly isolated healthy donor-derived human NK cells after stimulation with tumor cells (K562 or CTV-1 also referred to as INB16) or cytokine (IL-2) were analyzed through RNA-sequencing and compared to unstimulated controls (NK alone)