Journal: bioRxiv

Article Title: Prolonged TGF-β locks NK cells in a dysfunctional state through persistent epigenetic remodeling of IRF, T-bet and EOMES binding sites

doi: 10.64898/2026.02.11.705354

Figure Lengend Snippet: (A) Experimental design. Enriched NK cells were cultured for 7 days in IL-15 (50 ng/mL) with or without TGF-β (10 ng/mL). On day 7, media were exchanged for all conditions, and cells were maintained in IL-15 (control) or IL-15 + TGF-β. For the withdrawal condition, cells were washed twice on day 7 and cultured in IL-15 for an additional 10 days. Analyses were performed at days 0, 7, and 17. (B) Baseline frequencies of CD103⁺, CD9⁺, and CD49a⁺ NK cells at day 17, shown as bar plots (n = 7; three independent experiments). A representative donor is shown as a dot plot. (C) Baseline frequencies of granzyme A-positive (GZMA⁺), granzyme B-positive (GZMB⁺), and IFN-γ⁺ NK cells at day 17, assessed by flow cytometry and shown as bar plots (n = 7; three independent experiments). A representative donor is shown as a histogram. (D) RNA-seq analysis at the indicated time points. Heatmap shows z-score of regularized counts of selected TR-associated and effector genes across individual donors (n = 5). (E) NK cells were restimulated at day 17 as indicated and analyzed by flow cytometry. Bar plots show mean fluorescence intensity (MFI) of GZMA and IFN-γ (n = 5; three independent experiments). A representative donor is shown as a histogram. Statistical significance was calculated only between control and withdrawal conditions and within the withdrawal group; only significant comparisons are shown. (F) Venn diagram showing overlap of downregulated gene sets (Hallmark database; NES < 0, padj < 0.05) across the indicated conditions (n = 5). Vertical bar graphs depict normalized enrichment scores (NES) for selected effector-associated gene sets. (G) Network representation of connectivity between selected pathways (cnetplot; clusterProfiler), with genes shown as nodes colored by log2(FC) expression values. Across the figure, statistical significance was determined using a Wilcoxon signed-rank test for panels B and C and a paired Student’s t test for panel E (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Article Snippet: Cells were cultured in complete IMDM GlutaMAX (Gibco) medium supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL) (Thermo Fisher), 1× non-essential amino acids (Thermo Fisher), and 10% FCS (Thermo Fisher), and further supplemented with recombinant human IL-15 (50 ng/mL; Miltenyi).

Techniques: Cell Culture, Control, Flow Cytometry, RNA Sequencing, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Prolonged TGF-β locks NK cells in a dysfunctional state through persistent epigenetic remodeling of IRF, T-bet and EOMES binding sites

doi: 10.64898/2026.02.11.705354

Figure Lengend Snippet: NK cells were cultured for 7 days in IL-15 with or without TGF-β. On day 7, media were exchanged for all conditions, and cells were maintained in fresh IL-15 (control) or IL-15 + TGF-β. For the withdrawal condition, cells were washed twice on day 7 and cultured in IL-15 for an additional 10 days. RNA-seq and ATAC-seq were performed at day 0 (d0), day 7 (d7), and day 17 (d17). (A) Euler diagram showing the overlap of differentially expressed genes (DEGs; |log2(FC)| > 0.58, padj < 0.05) between d17 TGF-β–treated NK cells and cells from which TGF-β was withdrawn at d7. Upregulated and downregulated genes are displayed separately and color-coded. (B) Heatmap showing z-score of regularized counts of TGF-β–regulated genes (defined in Fig. S4A; |log2(FC)| > 0.58, padj < 0.05) across individual donors and conditions, with k-means clustering (k = 5). Violin plots depict the same data with donors pooled by condition (n = 5). (C) Euler diagram showing the overlap of differentially accessible regions (DARs; |log2(FC)| > 0.58, padj < 0.05) between d17 TGF-β–treated NK cells and cells from which TGF-β was withdrawn at d7. Regions with increased or decreased accessibility are shown separately and color-coded. (D) Heatmap showing z-score-normalized chromatin accessibility of TGF-β–regulated DARs (defined in Fig. S4A; |log2(FC)| > 0.58, padj < 0.05) across individual donors and conditions, with k-means clustering (k = 5). Violin plots show the same data with donors pooled by condition (n = 5).

Article Snippet: Cells were cultured in complete IMDM GlutaMAX (Gibco) medium supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL) (Thermo Fisher), 1× non-essential amino acids (Thermo Fisher), and 10% FCS (Thermo Fisher), and further supplemented with recombinant human IL-15 (50 ng/mL; Miltenyi).

Techniques: Cell Culture, Control, RNA Sequencing

Journal: bioRxiv

Article Title: Prolonged TGF-β locks NK cells in a dysfunctional state through persistent epigenetic remodeling of IRF, T-bet and EOMES binding sites

doi: 10.64898/2026.02.11.705354

Figure Lengend Snippet: ATAC-seq was performed on NK cells at day 17 following stimulation with IL-15, TGF-β, or TGF-β for 7 days after a 10-day culture in IL-15 (TGF-β withdrawal). (A) TF motif enrichment analysis of ATAC-seq regions showing persistent or transient changes in chromatin accessibility. The Venn diagram depicts the overlap of the top 50 enriched TF motifs (ranked by p value) across the indicated region categories; color intensity reflects the number of TFs assigned to each region. HOMER analysis was performed on pre-filtered regions (persistent down/up; transient down/up). For TFs present in more than one region, the most significant p value is shown. (B) Genome browser tracks of ATAC-seq signal at the IFNG and GZMA loci from one representative donor, shown as normalized read coverage. Predicted TF binding sites within promoter and putative enhancer regions are indicated. Boxes highlight regions of interest. (C) Inferred TF activity based on univariate linear modeling (ULM) using the DoRothEA regulon framework. Heatmap shows weighted TF activity scores derived from target gene expression changes; color intensity reflects activity level. Asterisks indicate statistically significant differences. (D) RNA-seq analysis of NK cells following the indicated stimulation conditions and time points (n = 5). Normalized expression levels of selected transcription factors are shown.

Article Snippet: Cells were cultured in complete IMDM GlutaMAX (Gibco) medium supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL) (Thermo Fisher), 1× non-essential amino acids (Thermo Fisher), and 10% FCS (Thermo Fisher), and further supplemented with recombinant human IL-15 (50 ng/mL; Miltenyi).

Techniques: Binding Assay, Activity Assay, Derivative Assay, Targeted Gene Expression, RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: Prolonged TGF-β locks NK cells in a dysfunctional state through persistent epigenetic remodeling of IRF, T-bet and EOMES binding sites

doi: 10.64898/2026.02.11.705354

Figure Lengend Snippet: (A) Experimental setup. NK cells from human HCC tumors or liver metastases of other cancer entities were analyzed by flow cytometry (n = 10). In parallel, peripheral blood NK cells from HCC patients were enriched, cultured overnight with IL-15 (10 ng/mL) and IL-2 (25 U/mL), restimulated on day 1 with IL-12/IL-18 (50 ng/mL) for 4 h, and analyzed by flow cytometry. (B) Frequency of CD103⁺ NK cells in the indicated tissue compartments shown as bar plots; a representative donor is shown as a dot plot. (C) Frequency of LAMP1⁺ and IFN-γ⁺ NK cells following 3 h IL-12/IL-18 stimulation (n = 12). Statistical significance was calculated only between stimulated healthy controls and patient samples. (D) ATAC-seq analysis of sorted peripheral NK cells from healthy donors and HCC patients with reduced NK-cell functionality (n = 3). Principal component analysis (PCA) shows variance along PC1 and PC2 based on the top 80,000 most variable features. (E) Genome browser tracks of selected loci showing ATAC-seq signal as RPGC-normalized read coverage plotted by genomic position. Dashed boxes indicate regions of interest in which NK cells from HCC patients show chromatin accessibility patterns similar to the day 17 TGF-β withdrawal condition (n = 3). Across the figure, statistical significance was assessed using an Wilcoxon signed-rank test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Article Snippet: Cells were cultured in complete IMDM GlutaMAX (Gibco) medium supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL) (Thermo Fisher), 1× non-essential amino acids (Thermo Fisher), and 10% FCS (Thermo Fisher), and further supplemented with recombinant human IL-15 (50 ng/mL; Miltenyi).

Techniques: Flow Cytometry, Cell Culture