BAG6 is released from CLL cells and cell lines as a soluble factor and inhibits NK cell cytotoxicity. (A) BAG6 was quantified in plasma (pl), soluble (so), and exosomal (exo) fraction of healthy donors and CLL patients using ELISA. Mean and standard error of the mean (SEM) from patients (n = 6) or healthy donors (n = 3) are depicted. (B) BAG6 ELISA of the soluble (so) and exosomal (exo) fraction of supernatant derived from cultured primary CLL or healthy B cells. Mean and SEM of 4 experiments are indicated. The amount of soluble BAG6 is significantly higher in CLL-derived supernatant in comparison with healthy B cell supernatant (P = .0001). The differences among the exosomal fractions are not significant (P = .0967); unpaired Student t test. (C) BAG6 concentration in fractionated supernatants of the CLL cell lines JVM-3, MEC-1, the human embryonic kidney cell line 293T, and the breast cancer cell line MCF7 was determined with ELISA. The differences of BAG6 distribution in the soluble vs the exosomal fraction are significant (mean and SEM, n = 4) between JVM/MEC in comparison with 293T/MCF7; unpaired Student t test. (D) NK cells were incubated with healthy donor plasma with or without soluble BAG6 protein (10 ng/mL) and their cytotoxicity was assessed in an NK cell cytotoxicity assay using the target cell line Raji. Normalized lysis is shown (mean and SEM of 4 independent experiments). (E) NK cells were incubated with control (gray bars) or CLL patient plasma (25% overnight, black bars) and the cytotoxicity against Raji cells (effector:target ratio, 10:1) was measured in the presence of isotype antibody (-), NKG2D blocking antibody (clone 1D11), NKp30 blocking antibody (clone P30-15), Fc control protein (-), NKG2D-Fc, or NKp30-Fc. NK cells were incubated with blocking constructs (10 μg/mL). Depletion of BAG6 from CLL patient plasma was performed using the monoclonal 3E4 antibody. Mean and SEM of 3 independent experiments are shown; significant differences are indicated; unpaired Student t test.