Tumor-derived sGITRL diminishes IFN-γ production of NK cells. NK cells were incubated for 24 hours with or without the indicated GITRL-positive or -negative tumor cell lines; afterward, supernatants were harvested and analyzed for IFN-γ by ELISA. (A) Cultures in the absence or presence of 10 ng/mL sGITRL derived from C1R-GITRL supernatants with an equal volume of C1R-neo supernatant as control. (B) Cultures in the absence or presence of the indicated concentrations of sGITRL with C1R-neo supernatant corresponding in volume to the highest concentration of C1R-GITRL supernatant as control. (C) Cultures with or without 10 ng/mL sGITRL derived from C1R-GITRL supernatants in the absence or presence of 10 ng/mL human IgG1 or GITR-Ig fusion protein. (D) GITRL-positive HCT116 and NCCIT tumor cells were cultured for 24 hours alone or on immobilized GITR-Ig with human IgG1 as control to induce GITRL signaling before addition of NK cells. Means of triplicates and SD of one representative experiment each of a total of 4 experiments with similar results are shown.