YTHDF2 expression in NK cells, generation of Ythdf2^fl/fl mice, Ythdf2 deletion efficiency in Ythdf2 ^ΔNK mice, and assessment of T cell responses in Ythdf2^ΔNK mice implanted with B16F10 tumor cells. (A) Representative immunoblot of YTHDF2 in immune cell subsets of CD4⁺ T cells (CD3⁺CD4⁺), CD8⁺ T cells (CD3⁺CD8⁺), NK cells (CD3⁻NK1.1⁺), B cells (CD3⁻NK1.1⁻CD19⁺), macrophages (CD3⁻NK1.1⁻CD19⁻CD11b⁺F4/80⁻), and DCs (CD3⁻NK1.1⁻CD19⁻CD11b⁺F4/80⁻CD11c⁺) isolated from the spleen of Ythdf2^WT mice. (B) Representative immunoblot of m⁶A writers, readers, and erasers in NK cells isolated from the spleen of Ythdf2^WT mice stimulated with or without IL-15 (50 ng/ml) overnight. (C) Representative immunoblot (left) and quantified protein levels of YTHDF2 normalized to actin (right) in splenic NK cells from WT and IL-15Tg mice. (D) RNA-seq analysis of the database GSE25672 showing the expression of m⁶A enzymes and readers in splenic NK cells during the MCMV infection. (E) Representative immunoblot (left) and quantified protein levels (right) of YTHDF2 in NK cells isolated from the spleens of mice infected with MCMV at the indicated time. (F) Representative plots showing the NK cells (CD3⁻NK1.1⁺) in the lung tissues from B16F10 tumor-bearing mice on days 0, 12, and 24 after tumor injection. (G) Representative immunoblot (left) and quantified protein levels (right) of YTHDF2 in NK cells isolated from the lung of mice injected i.v. with B16F10 at the indicated time. (H) Schematic diagram showing the targeting strategy for generating Ythdf2^fl/fl mice. The mouse Ythdf2 gene consists of five exons (blue rectangles). The En2SA-IRES-LacZ-pA-hBactP-Neo-pA cassette, which is flanked by two Flp-recognition target (FRT) sites, is inserted into intron 3 by homologous recombination. The fourth exon of Ythdf2 is flanked by two LoxP sites (red triangles). Mice with the floxed allele were generated by crossing the F1 offspring mice with ROSA26-FlpE mice. Subsequently, Ythdf2^fl/flmice were bred with mice expressing Ncr1-iCre to produce NK cell–specific Ythdf2 knockout mice. (I) PCR genotyping demonstrated germline transmission of the Ythdf2 allele. (J and K) The deletion of Ythdf2 in sorted NK cells (CD3⁻NKp46⁺) from Ythdf2^ΔNK mice versus Ythdf2^WT mice was verified by qPCR (J) and immunoblotting (K). (L–O) The percentages of infiltrating CD4⁺ T cells (L) and CD8⁺ T cells (M) as well as expression levels of IFN-γ (N and O) in the lung-infiltrated NK cells from Ythdf2^WT and Ythdf2^ΔNK mice were analyzed on day 14 after B16F10 injection (n = 5 mice per group). Each symbol represents an individual mouse. Data are shown as mean ± SD and were analyzed by unpaired two-tailed t test (C, J, and L–O) or one-way ANOVA (E and G). Data are representative of at least two independent experiments. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Het, heterozygous; Ho, homozygous; Unstim, unstimulated.