Fig. 5.
Effect of CD39, CD73 or A2a adenosine receptor inhibition on the lytic activity of cytotoxic effector cells against OvCA cells. a, b Polyclonal NK cells (pNK) were stained with PKH-26 and co-incubated for 4 h with CFDA-SE labelled OvCa cell lines SK-OV-3 or OAW42 at different effector to target cell (E:T) ratios [45]. Ectonucleotidase activity was either downregulated by transient transfection with siRNA or stable plasmid-based shRNA expression (a) or blocked by addition of ARL67156 or APCP as indicated (both used at 100 μM) (b). SCH58261 (SCH) was used at 100 nM to inhibit ADORA2A. Target cell lysis was determined in modified FATAL assays. Depicted are exemplarily the E:T ratios 10:1 for SK-OV-3 (left) and 5:1 for OAW42 (right). Lower E:T ratios that resulted in reduced target cell lysis showed a similar trend (data not shown). c Peripheral blood mononuclear cells (PBMC) were co-incubated with siRNA- or shRNA-transfected SK-OV-3 or OAW42 cells. On day 10, recall lysis by primed PBMC was evaluated by modified FATAL assays. d PBMC were co-cultured for 10 days with SK-OV-3 OvCA cells in the absence or presence of CD39 and CD73 inhibitors ARL67156 or APCP (used at 100 μM each). ADORA2A signalling was blocked with SCH58261 (100 nM). The lytic activity of the PBMC was then evaluated in a luciferase-based cytotoxicity assay against fresh SK-OV-3 cells that were stably transfected with a firefly luciferase plasmid. As we were unable to obtain suitable luciferase-expressing OAW42 targets, this cell line was not included in the assay. Representative and independent experiments are shown (n = 3)