FIGURE 4 from Impact of Surgery-Induced Myeloid-derived Suppressor Cells and the NOX2/ROS Axis on Postoperative Survival in Human Pancreatic Cancer

M-MDSC suppress NK cells by generating ROS and show enhanced NK cell suppression after surgery. A–C, NK cells were co-cultured with or without PANC-1 cells and freshly isolated healthy donor (HD) monocytes in the presence or absence of 100 µmol/L HDC, 200 U/mL catalase (Cat), 10 µmol/L GSK2795039 (GSK) or 1% DMSO (control). NK cell activation and degranulation were measured by flow cytometry and are shown by frequencies of NK cells expressing CD69⁺CD16⁺ (A) or CD107a⁺CD16⁺ (B), respectively. C, NK cell cytotoxicity against PANC-1 cells was determined by frequency of LIVE/DEAD stained PANC-1 cells. NK cell cytotoxicity against PANC-1 cells was measured in four independent experiments, that each included two to three combinations of NK cells and monocyte from different donors. D, ROS formation was measured by CL from CD33⁺ cells isolated 3–5 days after surgery in the absence of stimulation (PDAC Ctrl, orange), in the presence of rituximab (PDAC RTX, red), in the presence of rituximab and HDC (PDAC RTX + HDC, green) and from 2 HDs stimulated with rituximab (mean ROS formation shown, HD RTX, black). NK cells were cocultured with K562 cells and CD33⁺ cells isolated from HDs or patients before surgery (pre-op) or 3–5 days after surgery (days 3–5) in the presence (HDC) or absence of HDC. NK cell activation and degranulation were measured by flow cytometry as frequencies of CD16⁺ NK cells expressing CD69 (E) or CD107a (F) in NK:K562:Myeloid cell cocultures. NK cell cytotoxicity was measured in 10 independent experiments that were normalized by setting the mean NK cell cytotoxicity against K562 in the absence of myeloid cells to 1 in each experiment. Statistics by mixed-effects analysis followed by Šídák multiple comparison test (P-values indicated by *). HD and cancer samples were compared using Kruskal–Wallis test followed by Dunn multiple comparison test (P-values indicated by #).*, P < 0.05; **/##, P < 0.01; ***, P < 0.001; ****, P < 0.0001.