Figure 3. IL-2 target gene expression and STAT5 activation are reduced in Mst1–Mst2-deficient Treg cells.

(A) Number of differentially expressed genes (> 0.5 log₂ difference) from the transcriptome analysis of WT and Mst1–Mst2-deficient Treg cells isolated from mixed BM chimeras (Foxp3^Cre, n = 5; Foxp3^CreStk4^f/fStk3^f/f, n = 4). (B) Functional enrichment of downregulated genes in Mst1–Mst2-deficient Treg cells using the Hallmarks database (MSigDB). BH_FDR, Benjamini-Hochberg false positive rate. (C) Empirical cumulative distribution function for the change in expression (log₂ values) of all genes (black line) expressed in WT Treg cells (change relative to that in Mst1–Mst2-deficient Treg cells) and for subsets of genes upregulated (red line) or downregulated (blue line) by IL-2Rβ deficiency in Treg cells (Yu et al., 2009). (D) Flow cytometry analysis and statistics of p-STAT5⁺ cell frequency in WT and Mst1–Mst2-deficient YFP⁺ Treg cells from mixed BM chimeras (n = 4). Numbers in gates indicated percentage of cells. (E) Statistics of p-STAT5⁺ cell frequency in WT or Mst1-deficient Treg cells (n = 3) stimulated with IL-2 for 30 min. (F) Immunoblot analysis of p-STAT5, STAT5, p-S6, p-ERK and p-AKT in WT and Mst1-deficient Treg cells upon 1 U/ml IL-2 stimulation. (G) WT or Mst1–Mst2-deficient naïve CD4+ T cells (from Cd4^Cre or Cd4^CreStk4^f/fStk3^f/f mice) were stimulated by plate-bound anti-CD3-CD28 for 3 days and rested for another 3 days in IL-2 to generate T cell blasts. T cell blasts were rested without IL-2 for 8 h at 37°C followed by 1 U/ml IL-2 stimulation, and analyzed by immunoblot. (H) Activated CD4⁺ T cell blasts were generated as in (G). Mst1 protein and p-Mob1 were examined in activated T cell blasts and freshly isolated Treg cells from WT mice by immunoblot (n = 4). (I) Representative images of p-STAT5 nuclear translocation in WT and Mst1–Mst2-deficient Treg cells from mixed BM chimeras in response to IL-2 stimulation for 0 and 30 min (left). Right, quantification of p-STAT5 nuclear immunofluorescence signal in WT and Mst1–Mst2-deficient Treg cells with IL-2 stimulation for 0 and 30 min (n > 30). (J) Flow cytometry analysis and statistics of CD25 expression on WT and Mst1-deficient Treg cells (n = 4). Numbers in graph indicate MFI. The relative CD25 MFI was calculated by normalizing the value in WT Treg cells to 1. (K) Statistical analysis of p-STAT5 vs. CD25 expression in WT and Mst1-deficient Treg cells upon 1 U/ml IL-2 stimulation for 30 min (n = 3). Data in plots indicate means ± s.e.m. NS, not significant; *P < 0.05, **P < 0.01, ****P < 0.0001; two-tailed unpaired Student’s t test (D, E, H, J, K), one-way ANOVA (I). Data are from one experiment (A–C), representative of at least 2 independent experiments (D, F–J) or pooled from 3 experiments (E, K). See also Figure S3.