Reciprocal effects of gain or loss of expression of miR-17∼92 on antigen-specific CD8 T-cell proliferation and signaling. (A) CD8^WT and CD8^CKO or CD8^CTg P14 cells were labeled with CFSE and stimulated in vitro with GP33 and CD28 for 60 hours. Activation and proliferation of gated donor populations are presented as dot plots of CD44 vs CFSE. Mean ± SEM of antigen-specific CD8 T-cell numbers are plotted as bar graphs. Fold increase relative to unstimulated controls is listed above the respective bars. (B) 10⁶ congenically mismatched CFSE-labeled CD8^WT and CD8^CKO or CD8^CTg P14 cells were cotransferred into naïve mice, followed by LCMV infection. Cell proliferation was assessed by analyzing CFSE dilution at indicated days after infection. The frequencies of CD8 T cells in divisions 0 to 7 are summarized in bar graphs. Data are representative of 2 independent experiments, with n = 2 to 3 mice per experiment. (C) B6 mice containing 10⁴ wild-type and CKO or wild-type and CTg P14 cells were administered 1 mg BrDU intraperitoneally at days 4.75 or 6.75 after infection. The frequency of BrDU incorporation for each population in spleen (SPL) and blood (PBL) was assessed in CD8⁺Thy1.1⁺ wild-type and CD8⁺Thy1.2⁺ CKO or CTg cells at days 5 and 7 after infection. Representative FACS plots of BrdU incorporation are presented, with mean ± SEM data plotted as bar graphs. Results are representative of 4 independent experiments, with n ≥ 3 in each group. Paired Student t test was used in each mouse to assess statistical significance. (D) 10⁵ naive CKO (Thy1.2⁺) or CTg (Thy1.2⁺) P14 cells were adoptively transferred along with equal numbers of wild-type P14 (Thy1.1⁺) cells into naïve C57Bl/6 mice (Ly5.1⁺) that were subsequently infected with LCMV. Representative histogram plots with median fluorescence intensity of PTEN expression at day 7 after infection are presented. Average median fluorescence intensity of PTEN from 2 independent experiments, with n ≥ 3 in each group, is summarized in bar graph. Error bars represent SEM. Relative expression of PTEN mRNA in purified wild-type, CKO, and CTg CD8 T cells at day 7 post infection is also shown. Wild-type data were normalized using GAPDH control. Unpaired Student t test was used for statistical significance. (E) Histogram plots of pAKT and phosphoS6 expression in wild-type, CKO, and CTg P14 cells stimulated in vitro with GP33 and CD28 for 48 hours are presented as filled histograms. Shaded histograms represent expression of pAKT and phosphoS6 in unstimulated cells. Numbers indicate median fluorescence intensity of expression and are representative of 2 independent experiments. Probability binning algorithm in FlowJo was used to determine the T(x) metric for estimating the significance of difference between wild-type and CKO or wild-type and CTg populations (*P < .01).