YTHDF2 contributes to IL-15–mediated NK cell survival, proliferation, and effector functions. (A) qPCR showing the expression of Ythdf2 in NK cells at indicated time points following stimulation of IL-15. (B) qPCR showing the expression of Ythdf2 in NK cells from Stat5^WT and Stat5^ΔNK mice under the stimulation of IL-15. (C) Immunoblot showing the YTHDF2 protein levels in splenic NK cells from Stat5^WT and Stat5^ΔNK mice under the stimulation of IL-15. (D and E) Luciferase reporter assay showing that both STAT5a (D) and STAT5b (E) activate Ythdf2 gene transcription. (F) Scheme denoting putative STAT5-binding sites in the Ythdf2 promoter (top). Binding of STAT5 to the Ythdf2 promoter in NK cells as determined by ChIP–qPCR (bottom). (G) Splenic NK cells isolated from Ythdf2^WT and Ythdf2^ΔNK mice were cultured in vitro in the presence of IL-15 (50 ng/ml) followed by enumeration by a trypan blue exclusion assay. (H) Representative histograms (left) and quantification (right) of CTV dilution of NK cells from Ythdf2^WT and Ythdf2^ΔNK mice 5 d after in vitro culture with IL-15 (50 ng/ml). (I) Representative plots (left) and quantification (right) of cell-cycle distribution of NK cells from Ythdf2^WT and Ythdf2^ΔNK mice 5 d after in vitro culture with IL-15 (50 ng/ml). (J) Representative plots (left) and quantification (right) of apoptotic (annexin V⁺Sytox-Blue^+/−) NK cells from Ythdf2^WT and Ythdf2^ΔNK mice 5 d after in vitro culture with IL-15 (50 ng/ml). (K) Mice were treated with IL-15 (2 µg/d) for 5 d (n = 3 per group). On day 4, mice were injected with BrdU (5 mg i.p.) overnight. Splenic NK cells were then isolated followed by Ki67 cell proliferation and annexin V apoptosis assessment, measured by flow cytometry. (L) qPCR showing the expression of Ifng, Gzmb, and Prf1 in NK cells from Ythdf2^WT and Ythdf2^ΔNK mice following stimulation of IL-15 (50 ng/ml) overnight. (M and N) Representative plots and histograms of IFN-γ (M), granzyme B (N), and perforin (N) levels in NK cells from Ythdf2^WT and Ythdf2^ΔNK mice following stimulation with IL-15 (50 ng/ml) overnight. (O) Representative histograms (left) and quantification (right) of phospho-STAT5 in NK cells from Ythdf2^WT and Ythdf2^ΔNK mice after in vitro stimulation of IL-15 (50 ng/ml) for 1 h. Data are shown as mean ± SD and were analyzed by unpaired two-tailed t test (H, J, L, and O), one-way ANOVA (A, B, D, E, and K), or two-way ANOVA with Šídák post-test (F, G, and I). Data are representative of at least two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. 7-AAD, 7-aminoactinomycin D; MFI, median fluorescence intensity; NC, negative control.