BAG6-positive exosomes trigger NK cell cytotoxicity. (A) BAG6 was detected on exosomes released by 293T cells either constitutively or in response to cellular stress, including treatment with heat shock (HS, 30 minutes, 42°C and 2 hours recovery), doxorubicine (50 nM), TNF-α (50 ng/mL), and valproate (100 μg/mL). Mean and standard error of the mean (SEM) are indicated (n = 3), the enhanced BAG6 release was significant compared with untreated cells (HS: P = .0158; doxo: P = .0142, TNF-α: 0.0037, valproate: P = .001, unpaired Student t test). (B) Flow cytometry demonstrated binding of a BAG6-specific antibody (3E4) and a CD9-specific antibody to exosomes from wt cells (dotted line, endosomes) and exosomes from BAG6-transfected cells (+BAG6, black line). Gray-filled histogram: isotype control. FACS, fluorescence-activated cell sorter. (C) Western blot (WB) analysis to detect BAG6 and exosome marker (HSP70, CD9, CD81, CD63) in exosomes lysates of wt and BAG6-transfected cells (+BAG6). (D) BAG6-expressing exosomes enhanced the NK cell killing activity in cytotoxicity assays against the NK cell-resistant target cell lines 697 and NALM6 because untreated NK cells were less cytolytic than NK cells treated with exosomes purified from wt 293T cells (+exo). NK cells treated with exosomes from BAG6-transfected 293T cells (+BAG6-exo) demonstrated the highest killing activity. One representative of 3 experiments is shown. Blocking of NKp30 (clone P30-15 and IgG1-isotype control from BioLegend) reduced the target cell killing significantly (mean and SEM [n = 3]); 697: P = .0200 and Nalm6: P = .0123, unpaired Student t test; effector:target ratio, 10:1).