Fig. 3. Stimulation with IL-15 plus IL-21 enhances cytotoxic activity of NK-EVs.

A CFSE-labeled K562 cells (2 × 10⁴) were cultured with PBS or 3 μg of NK-EVs from cells cultured with no cytokine, IL-15, IL-21, or IL-15 + IL-21 for 12 h. The graph represents the means + SEM of percentages of CFSE⁺/PI⁺ K562 cells. Data are shown as means + SEM of three samples. B CFSE-labeled K562 cells (5 × 10³) were co-cultured with NK-92 cells (5 × 10⁴) cultured with no cytokine, IL-15, IL-21, or IL-15 + IL-21 for 4 h. The graph represents the means + SEM of percentages of CFSE⁺/PI⁺ K562 cells. Data are shown as means + SEM of three samples. C qRT-PCR was performed with RNA from NK-92 cells cultured with no cytokine, IL-15, IL-21, or IL-15 + IL-21 for 5 days. Data are shown as means + SEM of three samples. D Lysates of NK-EVs and NK-92 cells cultured with no cytokine, IL-15, IL-21, or IL-15 + IL-21 for 5 days were immunoblotted with anti-granzyme B and CD81 (loading control) Ab. M, protein marker. All data shown are representative of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.