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INTEGRATED ATAC-SEQ AND RNA-SEQ ANALYSIS IDENTIFIES KEY REGULATORY ELEMENTS IN FEEDER CELL-ACTIVATED NK CELLS [RNA-seq]

ABSTRACT: NK cells can potentially be used in 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, to generate enough cells for clinical applications. Upon stimulation with feeder cells, NK cells undergo profound changes in gene expression, altering their metabolic activity, cell cycle progression, and growth behavior. After examining the transcriptome and chromatin accessibility of NK cells before and 7 days after feeder cell activation, significant alterations were seen. These changes are 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 coculture, 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 coculture 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE268341 | GEO | 2025/05/22

REPOSITORIES: GEO

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Project description:Ex vivo activation and expansion of natural killer (NK) cells is a strategy to produce sufficient numbers of these effector cells for adoptive immunotherapy. Therefore we aimed at the development of an automated, clinical scale NK cell expansion process and compared NK cells after manual and automated expansion.We found only a small subset of 124 genes that significantly varied between both sample groups. These genes were associated with movement of leukocytes were identified including a group of genes for NK cell movement (CMKLR1, CX3CR1, S1PR5, GNLY and CXCR1) which were slightly higher expressed after automated compared to manual expansion. Nevertheless, the expansion profiles of NK cells after automated or manual expansion were rather similar in comparison to the remarkable difference between primary and expanded NK cells in general represented by the vast majority of regulated genes between the analyzed sample groups. Pathway analysis revealed that most of regulated genes upon expansion were associated with cell cycle regulation, DNA replication, DNA recombination and DNA repair, regulation of apoptosis and cell survival as well as cytokine signaling. Furthermore, the expression of many NK cell relevant markers was changed upon expansion with the strongest effects on up regulation of TRAIL and FASL, inhibitory TIGIT and chemokine receptors CCR2, CCR5 and CXCR6. In addition, granzyme M was down regulated but other important effector molecules like TNFa, perforin and granzymes A, B and K were up regulated. In summary we could show that manual and automatically expanded NK cells show a similar expansion profile while the differ significantly from primary NK cells. Up regulation of many NK cell relevant markers indicate for an enhanced NK cell functionaility after ex vivo activation and expansion. 24 samples were analyzed in total. 6 biological replicates represented by cells from different donors. 4 different samples per donor: freshly isolated NK cells (d0) and NK cells expanded for 14 days under 3 different conditions: in co-culture with EBV-LCL feeder cells and 500 U/mL IL2 either cultivated manually in T75 flasks (T) or automatically using the CliniMACS Prodigy system (P); NK cells cultured with 500 U/ml IL2 without feeder cells (I)

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INTEGRATED ATAC-SEQ AND RNA-SEQ ANALYSIS IDENTIFIES KEY REGULATORY ELEMENTS IN FEEDER CELL-ACTIVATED NK CELLS [RNA-seq]

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