Fig 2: mTORC2 and mTORC1 promote NK cell maturation by controlling the expression of Tbx21 and Eomes in a cooperative and nonredundant manner. a Intracellular flow cytometric analysis and cumulative results for the phosphorylation (p-) of S6 at ser235/236 (p-S6ser235/236) (top) and Akt at Ser473 (p-AktSer473) (bottom) in splenic NK cells (CD3−CD19−NK1.1+) from mice of the indicated genotype. b Cumulative ratio and enumeration of NK cells (CD3−CD19−NK1.1+) in the BM, spleen, and peripheral lymph nodes (pLNs) from mice of the indicated genotype. c Flow cytometric analysis and cumulative frequencies of subpopulations of NK cells (CD3−CD19−NK1.1+NKp46+) in the BM, spleen, and pLNs (left) and the calculated ratio of CD27− versus CD27+ cells among CD11b+ NK cells (right). d The cumulative frequencies depicting the CD43+KLRG1+ subset of NK cells (CD3−CD19−NK1.1+NKp46+) in the BM, spleen, and pLNs from mice of the indicated genotype. e, f Tbx21 and Eomes mRNA and protein expression in control versus Rictor∆/∆ mice (e) and control versus Raptor∆NK mice (f), as assessed by quantitative RT-PCR and flow cytometry, respectively. Purified splenic NK cells were used for quantitative RT-PCR (left). Cumulative data for Tbx21 and Eomes protein expression in total splenic NK cells (CD3−CD19−NK1.1+NKp46+) and the indicated subpopulations thereof were analyzed by flow cytometry (right). g Cumulative data for Tbx21 and Eomes protein expression in total splenic NK cells (CD3−CD19−NK1.1+NKp46+) and the indicated subpopulations thereof from control, Rictor∆NK and Rictor∆NKRaptorfl/+ mice. The control represents Rictorfl/+/Ncr1-Cre+ mice; Rictor∆NK represents Rictorfl/fl/Ncr1-Cre+ mice; and Rictor∆NKRaptorfl/+ represents Rictorfl/fl/Rptorfl/+/Ncr1-Cre+ mice. The dashed line indicates the control group, and the solid line indicates the gene knockout group. The MFI was calculated relative to total NK cells or to the CD27SP subset of NK cells from control littermates. The subpopulations of NK cells are distinguished by surface expression of CD27 and CD11b, and CD27SP, DP, and CD11bSP represent the CD27+CD11b−, CD27+CD11b+, and CD27−CD11b+ NK cell subsets, respectively. Each dot represents one mouse, and all experiments were replicated 3 (a, e left, f, g), 4 (e right), or 5 (b–d) times; error bars represent SD; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; one-way ANOVA (a–d, g), unpaired two-tailed Student’s t test (e, f)

Fig 3: Crosstalk between mTORC1 and mTORC2 during NK cell activation. a Immunoblotting to detect the phosphorylation (p-) of S6 at ser235/236 (p-S6ser235/236) and Akt at Ser473 (p-Aktser473) in NKL cells after stimulation with IL-15 (10 ng/ml) (left) or IL-15 (100 ng/ml) (right) for the indicated time periods. β-Actin was used as an internal control. The data shown are representative of three independent experiments. Uncropped gels are shown in Supplementary Fig. 8. b Intracellular flow cytometric analysis of p-S6ser235/236 and p-Aktser473 in murine splenic NK cells (CD3−CD19−NK1.1+) after stimulation with IL-15 (100 or 10 ng/ml) for the indicated time periods. The MFI of p-S6 ser235/236 or p-Aktser473 at each time point was normalized to the baseline reading at the 0-h time point for each individual mouse. Four independent individual mice were used. c–e Flow cytometric analysis and cumulative results for p-AktSer473 (c), CD122 (d), and p-STAT5Y694 (e) in splenic NK cells (CD3−CD19−NK1.1+) from control versus Raptor∆NK mice. f Intracellular flow cytometric analysis and cumulative results for p-S6 ser235/236 in total splenic NK cells (CD3−CD19−NK1.1+) and the indicated subpopulations thereof from control versus Rictor∆/∆ (top) or control versus Rictor∆NK (bottom) mice. g Statistical quantification of p-S6ser235/236 (MFI relative to the untreated control littermates) in splenic NK cells (CD3−CD19−NK1.1+) with or without IL-15 stimulation for 4 h ex vivo. The dashed line indicates the control group, and the solid line indicates the gene knockout group. The MFI was calculated relative to total NK cells or to the CD27SP subset of NK cells from control littermates. CD27SP, DP, and CD11bSP represent CD27+CD11b−, CD27+CD11b+, and CD27−CD11b+ NK cell subsets, respectively. Each dot represents one mouse, and all the experiments were replicated 3 (c–e, f bottom, g), or 7 (f top) times; error bars represent SD; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; one-way ANOVA (b, g); unpaired two-tailed Student’s t test with Welsh’s correction (c–f)