oIL-2 stimulation in oIL-2Rβ Tregs reveals upregulation of transcripts involved in activation and IL-2/STAT5 signaling during GVHD suppression. (A) Schematic representation of the workflow for the RNA-seq (created with BioRender.com). (B) PCA of transcriptome based on the top DEGs across oIL-2Rβ Tregs treated with oIL-2 (green) or PBS (blue) and tEGFR Tregs treated with oIL-2 (red). (C) Heatmap and hierarchical clustering of the differential expression from all the samples. Genes related with activation and Treg suppression are highlighted. Expression for each gene is scaled across single rows. (D) Volcano plots reveal significant and log2 fold change of transcripts from oIL-2Rβ Tregs treated with oIL-2 compared with PBS (left) or oIL-2 stimulation (right). Vertical dashed lines on volcano plots indicate a fold change of ±1.5; horizontal dashed line indicates an adjusted P = .05. (E) Gene set enrichment analysis of significant upregulated pathways (hallmark) shown in purple of oIL-2Rβ Tregs stimulated with oIL-2 compared with those stimulated with PBS. (F) The expression levels of different activator markers (CD62L, PD1, KLRG1, CD103, ICOS, and Lag3) on the FoxP3⁺ CD4⁺ GFP⁺ population were analyzed 7 days after allo-HSCT on cells in the spleen and mLNs. Pooled data from 3 independent experiments including 4 to 5 mice per group per each experiment. ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001 between indicated groups. Student t test with Bonferroni correction for multiple comparisons was used for statistical analysis. FC, fold change; padj, adjusted P value.