Fig. 1: Characterization of Nr2f6-deficient NK cells via RNA-Seq analysis.

A Volcano plot of differentially expressed genes (DEGs) between wild-type (Nr2f6^+/+) or Nr2f6-deficient (Nr2f6 ^−/−) splenic NK cells. Genes were considered DEG if the adjusted p value (padj) after DESeq2 normalization was <0.05. Downregulated genes are depicted in blue, and upregulated genes in red, NK cell-relevant genes are labeled (n = 3 per genotype). B Heatmap of the selected cluster of NK cell-defining genes in mice, as defined by the group of Vivier [34] in wild-type (Nr2f6^+/+) or Nr2f6-deficient (Nr2f6^−/−) splenic NK cells. All genes were z-score normalized, and DEGs were defined by DESeq2 (adjusted p value (padj) < 0.05) (n = 3 per genotype). C Quantification of total splenocytes in wild-type (Nr2f6^+/+) or Nr2f6-deficient (Nr2f6^−/−) mice. D Representative dot plots and quantification of splenic NK cell frequencies and total cell numbers (CD3^-CD19^-NK1.1⁺NKp46⁺) from wild-type (Nr2f6^+/+) or Nr2f6-deficient (Nr2f6^−/−) mice. E Representative histogram of NKp46 expression and quantification of the MFI of NKp46 in wild-type (Nr2f6^+/+) or Nr2f6-deficient (Nr2f6^−/−) splenic NK cells (CD3^-CD19^-NK1.1⁺NKp46⁺). F Representative histogram of CCR5 expression and quantification of the MFI of CCR5 in wild-type (Nr2f6^+/+) or Nr2f6-deficient (Nr2f6^−/−) splenic NK cells (CD3^-CD19^-NK1.1⁺NKp46⁺). G Quantification of the frequencies of CCR5⁺ splenic NK cells (CD3^-CD19^-NK1.1⁺NKp46⁺) in wild-type (Nr2f6^+/+) or Nr2f6-deficient (Nr2f6^−/−) mice. H Quantification of the frequencies of DNAM-1⁺ splenic NK cells (CD3^-CD19^-NK1.1⁺NKp46⁺) in wild-type (Nr2f6^+/+) or Nr2f6-deficient (Nr2f6^−/−) mice. I Quantification of the frequencies of NKG2D⁺ splenic NK cells (CD3^-CD19^-NK1.1⁺NKp46⁺) in wild-type (Nr2f6^+/+) or Nr2f6-deficient (Nr2f6^−/−) mice. J Quantification of the MFI of NK1.1, DNAM-1 and NKG2D in wild-type (Nr2f6^+/+) or Nr2f6-deficient (Nr2f6^−/−) splenic NK cells (CD3^-CD19^-NK1.1⁺NKp46⁺). A, B RNA sequencing and all downstream analyses were performed on splenic NK cells from n = 3 per genotype. C, D, H–J Representative data is shown as pooled experiments of at least three independent experiments n = 10. E Representative data is shown as one of three independent experiments with n = 5 per genotype and experiment. F Representative data is shown as one of three independent experiments with n = 5 wild-type (Nr2f6^+/+) and n = 4 Nr2f6-deficient (Nr2f6^−/−) mice. Each dot represents the data of an individual mouse. Results are shown as mean ± SD. The normality of data was evaluated by the Shapiro–Wilk test. An asterisk indicates statistically significant differences between genotypes calculated using Student’s t-test. A p value < 0.05 was considered statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001 ****p < 0.0001.