Figure 3. Altered gene expression in DKI T cells.

(A) Preactivated splenic T cells were rested overnight and re-stimulated with IL-2 for 1 day prior to determining CD25 expression in CD4⁺ (top row) and CD8⁺ (bottom row) T cells. IL-2-induced CD25 expression levels are indicated as mean fluorescence (MFI). Isotype control (IgG), IL-2, and untreated samples are shown in gray, red, and blue, respectively. This experiment was performed three times. (B) On the left is a hierarchical clustering analysis of gene expression profiles regulated by IL-2 in WT T cells. Also shown is a pie graph of the number of mRNAs not regulated (red) or regulated (blue) by IL-2. For IL-2-regulated mRNAs, the linked bar graph indicates the number of mRNAs whose expression was induced (open bars) or repressed (solid bars) at each time point. (C) Shown on the left is the hierarchical clustering analysis of IL-2-regulated mRNAs in DKI as compared with WT T cells. Also shown is a pie graph of the number of IL-2-regulated mRNAs whose expression was not affected (orange) or affected (blue) in DKI T cells. For the 506 mRNAs whose expression was affected, the linked bar graph indicates the number of genes whose expression was higher (open bars) or lower (solid bars) in the DKI T cells. For the experiments in panels B and C, 5 replicates of WT and DKI splenic T cells were analyzed for each treatment. (D and E) Validation of (D) STAT5 tetramer-dependent IL-2-induced gene expression and (E) STAT5 tetramer-independent IL-4-induced gene expression. The experiments described in panel D were performed 4 times and in panel E were performed twice. The cDNA inputs were normalized after RT-PCR based on the cycle number for Rpl7 primers, as Rpl7 expression was constant under our experimental conditions. Shown are the relative expression of triplicate samples of a representative experiment.