Tregs were isolated from the lungs of unchallenged Foxp3^EGFP mice or from the lungs and spleens from Foxp3^EGFP mice 7 days after LPS (3 mg/kg i.t.) challenge. (A) Body weight relative to baseline measured daily before and after injury (n = 19 females; 39 males; 58 total). Data are expressed as the mean ± SEM. (B) Principal component analysis (PCA) of mRNA expression demonstrating the contribution of the top 3 principal components to the variance in the 3 Treg sets. Each symbol represents 1 sort of pooled Tregs from mice (15–27 mice/experiment); 3 arrays/set. (C) Venn diagram showing the overlap of significantly differentially expressed transcripts between sets (adjusted P < 0.05, fold change > 2). Area of the circles reflects the relative number of differentially expressed transcripts for each comparison. Numbers in parentheses indicate the total from the respective pairwise comparison. These transcripts are listed in Supplemental Tables 1–3. (D) Unsupervised hierarchical clustering with Euclidean distance metric of 1,599 differentially expressed transcripts identified in all 3 comparisons. The dendrogram identifies clustering across transcripts (top) or across experimental samples (right). The heatmap depicts the log₂ normalized intensity, and each row represents Treg RNA from a pooled set of mice. As described in the Methods, transcripts between sorted Treg sets were identified by filtering at adjusted P < 0.05 and fold change > 2 (bright blue for lower expression levels and bright red for high expression levels). (E) Differentially expressed genes (DEGs) between resolving lung Tregs and unchallenged lung Tregs. Unsupervised hierarchical clustering of the unique 78 DEGs between lung Treg samples. The heatmap depicts the log₂ normalized intensity across samples. Each column represents Treg RNA from pooled Tregs, and each row represents a DEG. The dendrogram identifies clustering across experimental samples (top) or DEGs (right). (F) Select list of transcripts with a >2-fold change between resolving lung Tregs and unchallenged lung Treg comparisons. (G) Select list of transcripts with a >2-fold change between resolving lung Tregs and resolving splenic Treg comparisons.