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. 2021 Sep 24;12:732511. doi: 10.3389/fimmu.2021.732511

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Copyright © 2021 Kiekens, Van Loocke, Taveirne, Wahlen, Persyn, Van Ammel, De Vos, Matthys, Van Nieuwerburgh, Taghon, Van Vlierberghe, Vandekerckhove and Leclercq

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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NK cells developing from T-BET- or EOMES-overexpressing HPC undergo terminal NK cell maturation. eGFP⁺CD45⁺CD56⁺CD94⁺ stage 4 (CD16^-) and stage 5 (CD16⁺) NK cells were sorted from day 21 cultures and RNA-seq (n=4) and Fast-ATAC-seq (n=2) was performed. (A) Fold change plots illustrating differential mRNA expression of T-BET or EOMES versus control stage 4 NK cells. Blue and red dots indicate T-BET- and EOMES-specific differential genes, respectively. Orange dots indicate genes differentially regulated by both overexpression constructs. Selected differential genes are highlighted. (B) Charts show the number of differential events of RNA- and ATAC-seq for T-BET or EOMES stage 4 and stage 5 NK cells compared to control NK cells (left panel) and for stage 4 compared to stage 5 NK cells (right panel). (C) Overlap analysis of differential ATAC sites of T-BET vs. control stage 4 NK cells and of EOMES-overexpressing vs. control HPC. Charts demonstrate the absolute number of ATAC sites. (D) Overlap analysis of up- or downregulated mRNA and the indicated subgroups of ATAC sites for T-BET or EOMES versus control stage 4 NK cells. Relative numbers of ATAC sites are shown in the bar charts. (C, D) Up = upregulated sites; down = downregulated sites; both = up- and downregulated sites; no diff = detectable sites without differential expression; none = non-detectable sites. (E, F) Motif enrichment analysis of up- or downregulated, or non-differential ATAC sites of T-BET or EOMES compared to control stage 4 NK cells. (E) The top 5 best matched motifs are indicated. (F) The relative presence of the indicated motif families is shown. (G) Representative Genome Browser views of selected gene loci, showing tracks of ATAC-seq for the indicated stage 4 and stage 5 NK cells, and of H3K27ac and H3K4me3 ChIP-seq of mature NK cells. Differentially regulated ATAC regions are indicated in grey or orange, whereby the orange colored regions contain a T-BOX motif. See also Supplementary Figures 3 and 4 .