Functional analyses of T cells in WT, KI, and KO mice. (A) Relative percentage of B8R_20–27/H-2K^b tetramer⁺ CD8⁺ T cells in the spleen. Cells were isolated 1 month after MVA immunization and prepared for flow cytometry. Data represent the relative percentage of B8R_20–27/H-2K^b tetramer⁺ CD8⁺ T cells out of total CD8⁺ T cells. Data are representative of 2 independent experiments. (B) Functional activity of IL-15Rα^ext/IL-2Rα^int KI versus WT mice by ELISPOT for IFN-γ production (see “Methods”). Means plus or minus SEM are shown (n = 5/group). Data are representative of 2 independent experiments. (C) Proliferation of CD8⁺ T cells. Splenic CD8⁺ T cells were purified from WT, KI, and KO mice and cultured in medium alone, 1 μg/mL of plate-bound anti-CD3ϵ or 1 μg/mL of anti-CD3ϵ plus anti-CD28 supplemented with IL-15 (5 ng/mL or 50 ng/mL) or IL-2 (100 U/mL). Proliferation was assessed by measuring [³H]-thymidine uptake as described in “Methods.” Means plus or minus SEM are shown (n = 3/group). Data are representative of 4 separate experiments. (D) Proliferation of memory and naive CD8⁺ T cells. Splenic effector/memory (CD44^high) and naive (CD44^low) CD8⁺ T cells from WT and KI mice were labeled with CFSE and then cultured for 4 days in medium alone, 100 ng/mL IL-21 alone, or IL-21 plus IL-15 (5 ng/mL or 50 ng/mL) before flow cytometric analysis. Percentage of CFSE dilution is indicated. The experiment was performed twice with similar cooperative effects of IL-15 and IL-21 on CFSE dilution. (E) Intracellular staining of Stat5 phosphorylation. T cells isolated from WT, KI, and KO mice spleen were preactivated in anti-CD3ϵ plus anti-CD28 for 3 days, washed, and rested for 2 days. IL-15 (5 ng/mL or 50 ng/mL) or IL-2 (100 U/mL) was added to the medium. After 15 minutes of stimulation, cells were harvested and stained for CD4⁺/CD8⁺ subsets and intracellular phosphorylated Stat5. The Stat5 phosphorylation level was analyzed from CD8⁺ T cells. Means plus or minus SEM are shown (n = 6/group).