Integrated ATAC‐seq and RNA‐seq analysis identifies key regulatory elements in NK cells activated with feeder cells and IL‐2

Natural killer (NK) cells are in development for allogeneic immunotherapy; however, for such use as off-the-shelf medicines, NK cells need to undergo ex vivo expansion, typically through activation with feeder cells and cytokines, to generate sufficient cells for clinical applications. Upon stimulation with feeder cells in the presence of cytokines, NK cells undergo profound changes in gene expression, altering their metabolic activity, cell cycle progression, and growth behavior, but the precise changes that drive this transformation remain poorly understood. In this study, we identified significant differences in the transcriptome and chromatin accessibility of NK cells 7 days after feeder cell and cytokine activation, with the changes even more pronounced in genome regions closer to enhancers. Several transcription factors, including AP-1, IRF4, STATs, T-bet, Eomes, and bHLHE40, which play key roles in NK cell development and immune response, exhibited differential binding activity between unstimulated and day 7 NK cells. Gene sets composed of target genes downstream of these transcription factors were also enriched at day 7, implying their involvement in NK cell activation. Moreover, we compared potential super-enhancer regions in NK cells before and after activation, combined with the transcriptional activity of nearby genes. We identified stable and transcriptionally active super-enhancers in unstimulated and day 7 NK cells, as well as those that form or disappear after co-culture initiation. The transcriptomic and epigenetic characterization of NK cells presented in this study could facilitate the ex vivo expansion and engineering of functionally superior NK cells.