Overexpression of miR-17∼92 leads to compromised polyfunctionality of antigen-specific CD8 T cells. Congenically mismatched antigen-specific CD8^WT (Thy1.1⁺) and CD8^CKO (Thy1.2⁺) or CD8^CTg (Thy1.2⁺) cells were cotransferred in equal numbers (10³) into congenically mismatched naïve B6 mice (Ly5.1⁺), which were infected with LCMV. Polyfunctionality was assessed by (A) measuring the frequencies of donor cells coproducing IFN-γ, TNF-α, and IL-2 and by (B) determining the extent of degranulation, as marked by cell surface expression of CD107a/b by intracellular cytokine staining (ICCS) after 5 hours of in vitro stimulation of splenocytes with GP33-41 peptide in the presence of brefeldin A and monensin and CD107a/b antibodies. CD8^WT and CD8^CKO or CD8^CTg cells were gated using CD8, Thy1.1, Thy1.2, and Ly5.1 markers. (A) Representative histogram plots (gated on IFN-γ⁺ donors) are shown for day 8 (peak) and day >30 (memory) after infection. Open histograms represent unstimulated controls, and filled histograms represent GP33-41–stimulated samples. Bold numbers in histograms indicate % IFN-γ⁺ donors that also produce TNF-α or IL-2. Italicized numbers indicate MFI of IFN-γ and TNF-α expression. Pie charts show mean ± SEM of %donor cells producing IFN-γ only (single producers), IFN- γ and TNF-α (double producers), or IFN- γ, TNF-α, and IL-2 (triple producers) at peak (day 8) and memory (>day 30). Results are representative of 4 independent experiments, with n ≥ 3 in each group for peak time point and 5 independent experiments, with n ≥ 3 in each group for memory time point. (B) Representative histogram plots and bar graphs of mean ± SEM of CD107a/b MFI on degranulating CD8 T cells are shown. Open histograms represent unstimulated controls, and filled histograms represent GP33-41–stimulated samples. Numbers in histograms indicate MFI of CD107a/b. Plots are representative of 2 independent experiments, with n ≥ 3 in each group for peak and memory. Unpaired Student t test was used for statistical significance.