CD137 impairs antitumor reactivity of human NK cells. (A) CD137L surface expression on HEK cells transfected with human CD137L (HEK-CD137L) and the respective mock controls (HEK-mock) was determined by FACS using CD137L mAb (shaded peaks) and isotype control (open peaks). (B) Cytotoxicity of polyclonal NK cells in cultures with the HEK transfectants was evaluated by 4-hour chromium release assays. (C) IFN-γ levels in cultures of polyclonal NK cells and HEK transfectants were determined after 24 hours by ELISA. (D) Granule mobilization of polyclonal NK cells after 3-hour cultures with HEK transfectants in the presence or absence of 10 μg/mL mouse IgG1 or blocking CD137 mAb was analyzed by FACS. Numbers in top right quadrants indicate the percentage of CD107a⁺ NK cells. (E) Freshly isolated NK cells were cultured for 24 hours with or without 10 ng/mL IL-15 followed by incubation for additional 24 hours with or without IL-15 in the presence or absence of immobilized agonistic (4B4-1) and blocking (BBK-2) CD137 mAb or isotype control. Subsequently, IFN-γ levels in culture supernatants were determined by ELISA. (F) Polyclonal NK cells were incubated for 24 hours in medium alone or on immobilized agonistic CD137 mAb or isotype control. The respective culture supernatants (untreated, IgG1, and αCD137, respectively) were then harvested, and fresh polyclonal NK cells of the same donor were incubated with HEK-mock cells using the different supernatants as culture medium. Cytotoxicity was analyzed after 4 hours by chromium release assays (left), and IFN-γ production was determined after 24 hours by ELISA (right). (G) Apoptosis of polyclonal NK cells was determined after culture with HEK transfectants for 48 hours by FACS for Annexin V/PI. Numbers in dot plots indicate the percentage of Annexin V/PI⁺ CD56⁺CD3⁻ cells. One representative experiment each of a total of at least 3 with similar results is shown. Results shown in panels B, C, E, and F represent means of triplicates with SDs. *Statistically significant differences.