Fig 2: Mechanisms of mTORC2 repression of mTORC1 activity in NK cells. a Intracellular flow cytometric analysis depicting the phosphorylation (p-) of Akt at Thr308 (p-AktThr308) in total splenic NK cells (CD3-CD19-NK1.1+). b The expression of p-S6ser235/236 in splenic NK cells from control versus Rictor?/? mice after treatment with DMSO (set as the solvent control) or 2 µM AKTi-1/2 for 6 h ex vivo. c The abundance of CD122 expression on splenic NK cells (CD3-CD19-NK1.1+NKp46+) from control versus Rictor?/? mice, analyzed by flow cytometry. d Statistical quantification of p-STAT5Y694 in splenic NK cells (CD3-CD19?NK1.1+) with or without IL-15 stimulation for 4 h ex vivo. e–g, i Statistical quantification of p-S6ser235/236 (e), perforin and granzyme B (f), frequencies of CD107a+ NK cells (g), SLC7A5 expression (i) in splenic NK cells from control versus Rictor?/? mice after treatment with DMSO or 100 µM STAT5-IN for 6 h ex vivo. h Statistical quantification of SLC7A5 expression on both total NK cells and the indicated subsets. j The expression of p-S6ser235/236 in splenic NK cells from control versus Rictor?/? mice after treatment with DMSO or 25 mM BCH for 6 h ex vivo. The MFI was relative to the littermates of control + DMSO group (b, e, f, i, j). Each dot represents one mouse, and all experiments were replicated 3 (a, b, e–j), 5 (d), 6 (c) times; error bars represent SD; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired two-tailed Student’s t test (a, c, h); one-way ANOVA (b, d–g, i, j)

Fig 3: N-820 enhanced the expression of NK activating receptors and proliferation of exPBNK with enhanced Ki67, p-Stat5, and CD25 levels. The percentages of NK activating and inhibitory receptors on exPBNK surface were monitored by flow cytometry analysis at day 3 (A). The NK proliferation was monitored by CellTiter 96 AQueous One solution cell proliferation assay (Promega) according to the manufacturer’s instruction (B). Intracellular Ki67 (C), Phosphorylated STAT5 (p-STAT5) (D) and phosphorylated Akt (p-AKT) (E) were monitored by flow cytometry analysis at day 3. Ki67 (p<0.01) and p-STAT5 (p<0.02) were significantly increased following N-820 versus rituximab (Ritux)+N-803. Average values are reported as the mean±SEM. Representative histograms illustrate the expression levels of Ki67, p-STAT5, and p-AKT (F). Purified exPBNK cells were co-cultured with Raji, Raji-2R, Raji-4RH cells or without tumor cells in medium with 10 nM N-820, 10 nM N-803, or 10 nM IgG for 3 days. The percentage of CD25 on exPBNK surface was monitored by flow cytometry analysis at day 3. CD25 expression was significantly enhanced on exPBNK cells by N-820 versus rituximab+N-803 (Ritux+N-803) against Raji-2R and Raji-4RH, p=0.0012 and p=0.0016, respectively (G). Average values are reported as the mean ± SEM on the left panel. The representative histograms illustrate the expression levels of CD25 on exPBNK cells that were co-cultured with Raji, Raji-2R, Raji-4RH cells or without tumor cells in medium with 10 nM N-820 or 10 nM IgG for 3 days on the right panel. n=4 (G). *p<0.05, **p<0.01, ***p<0.001, ritux=rituximab.

Fig 4: 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. ß-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)