Fig. 4

Alteration in effector function and receptor expression of human primary NK cells by the ADSC secretome. Human primary NK cells isolated from PBMCs of a healthy donor were cultured for 48 h with 25 (blue) or 150 μg/ml ADSC secretome (red), or without (gray) in the presence of 10 ng/ml of rhIL-2. Unstimulated NK cells were also cultured for negative control (white). A The production of effector cytokines (IFN-γ and GM-CSF) and cytolytic granules (granzyme B and perforin) in the supernatant was measured by ELISA. B Cytotoxicity of primary NK cells against K562 target cells at the indicated NK:K562 (E:T, effector:target) ratios of 1:1, or 2:1 (upper). Stimulated primary NK cells were treated with 150 μg/ml ADSC secretome for 48 h, followed by co-culture with K562 target cells at the indicated ratios for 4 h (bottom). C Degranulation marker from activated primary NK cells was assessed using flow cytometry (CD3⁻ CD56⁺ CD107a⁺) as shown dot blot (at a 1:1 ratio, upper), frequency of CD107a⁺ NK cells (at the indicated ratios, bottom), and the expression level of CD107a on primary NK cells (at the indicated ratios, right side). D to E Flow cytometry analysis of activating receptors, such as NKG2D, NKp30, and NKp46 (D; histogram, MFI, and frequencies), inhibitory receptor CD96 (E; histogram, MFI, and frequencies). Error bars represent mean ± SEM of five to seven healthy donors. Statistical tests were determined by Student’s paired two-tailed t-test; not significant (ns), *P < 0.05, **P < 0.01, ***P < 0.001