Extracellular IFNγ signaling sustains chromatin accessibility and ISG expression even after cytokine washout. (A) Schematic of experiments: Human macrophages were stimulated with 100 ng/ml IFNγ for 8 h, washed, and then cultured for an additional 88 h in regular media or media with 1 µM ruxolitinib for an additional 88 h. Cells were collected for ATACseq and RNAseq at the indicated time points. (B) Heatmap of Z-scored reads within ATAC peaks induced by IFNγ (L2FC > 2, FDR < 0.01) after 8 h of stimulation for 4 days after washout when cultured in regular media or media with 1 µM ruxolitinib. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (C) Boxplot of log2CPM of reads within each peak for each cluster in B. (D) Heatmap of Log2 fold change in RNAseq reads of genes induced at least fivefold after 8 h of IFNγ stimulation. Log2 fold changes are shown after washout for cells cultured in regular media and media containing 1 µM ruxolitinib. Genes are clustered by persistent level of expression after washout (CPM after wash as percent of CPM at 8-h simulation). (E) Boxplot showing Log2 fold changes of individual genes by cluster in D. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. (F) Macrophages were stimulated and washed as above in A; after washout, cells were cultured in media alone, media with 1 µM ruxolitinib, or 10 µg/ml anti-IFNγ neutralizing antibody for 88 h. Cells were collected 88 h after washout, and qPCR was performed for IDO1. Boxplots indicate 2ΔΔCt normalized to HPRT. Error bars indicate standard deviation. Statistical tests determined by ordinary one way ANOVA. (G) qPCR for IRF1 as in F. ** P < 0.01; ***P < 0.001; ****P < 0.0001.