Intracellular CD70 reverse signaling pathway. (A-C) FACS-sorted NK cells were stimulated for 4 hours with 0.5 μg/mL α-Ly49D or control rat IgG (rIgG, unstimulated) in the presence of Armenian Hamster IgG (IgG; 5 μg/mL) or activating anti-CD70 (6D8; 5 μg/mL). NK cells were stained intracellularly for IFN-γ and CD107α. (A) Representative FACS plots showing frequencies of NK cell subsets in anti-Ly49D–activated NK cells. (B) Frequency of IFN-γ⁺ CD107α⁺ NK cells. (C) Δ MFI [MFI-stimulated – MFI-unstimulated] of IFN-γ staining in NK cells. (D) FACS-sorted NK cells (10⁵) were cocultured with irradiated 4 × 10⁴ MC57 or MC57-CD27-trunc cells (35 Gy), in the presence of 40 U/mL interleukin-2. IFN-γ concentration in supernatants after 2 days is shown. NK cells alone supplemented with 50 ng/mL interleukin-18 were used as positive controls. (E-F) NK cells were stained with Annexin-V (AnxV) and viability dye (VD) after 16 hours of activation with anti-Ly49D and anti-CD70 or isotype control and analyzed by FACS. (E) Frequency of AnxV⁻VD⁻ viable cells. (F) Representative FACS plot showing AnxV and VD staining out of 3 is shown. Data are shown as mean ± SEM of 3 wells and represent 3 independent experiments. (G-I) FACS-sorted NK cells were stimulated with 5 μg/mL anti-Ly49D antibody in the presence of 10 μg/mL anti-CD70 antibody (6D8) or control IgG for 0, 5, 15, 30, and 45 minutes. Phosphorylation of AKT and ERK was assessed by FACS. (G-H) MFI of p-AKT⁺ and p-ERK⁺ NK cells. (I) Representative FACS plots of p-AKT and p-ERK expression in NK cells 15 minutes after stimulation. Data are shown as mean ± SEM of triplicates and are representative of 3 different experiments. Statistics: Student t test (B-E), 2-way ANOVA (G-H). stim, stimulation.