Tregs from lung and spleen single cell suspensions from male and female Foxp3^YFP-Cre and Kdm6b^ΔTreg mice, 8–12 weeks of age, were sorted at either steady state or after being challenged with intratracheal PR8 influenza as previously described (38) at day 15 after PR8 influenza infection and used for either H3K27me3 CUT&RUN-Seq or RNA-Seq. (A) Frequency of H3K27me3^hi Tregs from the spleen or lung of Foxp3^YFP-Cre and Kdm6b^ΔTreg mice (n = 6–11 mice per strain; a combination of 2 separate experiments). (B) Log₂ FPKM of H3K27me3 signal across the entire genome of lung Tregs from naive and influenza virus–infected Foxp3^YFP-Cre and Kdm6b^ΔTreg mice. Triplicates per condition. (C and D) H3K27me3 modification tracks in lung Tregs from naive and influenza virus–infected Foxp3^YFP-Cre and Kdm6b^ΔTreg mice. Data from triplicates per condition were combined for analysis and presentation. (E and F) Volcano plots showing differential gene expression between pairwise conditions of sorted lung Tregs from either Foxp3^YFP-Cre or Foxp3^ΔKdmb6 at either (E) steady state (naive) or (F) day 15 after influenza (Flu) are shown. DEGs significantly (P < 0.01) upregulated (red) or downregulated (blue) are shown. Data from triplicates per condition were combined for analysis. (G) Gene-set enrichment analysis identified several Gene Ontology biological pathways in sorted Foxp3^YFP-Cre or Foxp3^ΔKdmb6 lung Tregs at day 15 after influenza infection. reg, regulation; proc, process. (H) Cross-comparison of RNA-Seq and H3K27me3 CUT&RUN results from lung Tregs. Red dots highlight differentially expressed transcripts showing increased expression and decreased H3K27me3 marks in Tregs from Foxp3^YFP-Cre mice compared with Foxp3^ΔKdmb6 mice.