Tumor-derived sGITRL down-regulates NK cell cytotoxicity. Cytotoxicity of NK cells was evaluated by chromium release assays with the indicated GITRL-positive or -negative tumor cells. (A) Cytotoxicity in the absence (diamonds) or presence of 10 ng/mL sGITRL derived from C1R-GITRL supernatants (filled squares) with an equal volume of C1R-neo supernatant as control (open squares). (B) Cytotoxicity in the absence or presence of the indicated concentrations of sGITRL derived from C1R-GITRL supernatants. As control, C1R-neo supernatant corresponding in volume to the highest concentration of C1R-GITRL supernatant was included. (C) Cytotoxicity assays in control medium (diamonds) or with 10 ng/mL sGITRL derived from C1R-GITRL supernatants in the absence (filled squares) or presence of 10 ng/mL human IgG1 (circles) or GITR-Ig fusion protein (triangles). (D) HCT116 and NCCIT tumor cells that express GITRL were cultured for 24 hours alone (diamonds) or on immobilized GITR-Ig (triangles) with human IgG1 (squares) as control to induce GITRL signaling. Subsequently cytotoxicity of NK cells was determined. Means of triplicates with SD of one representative experiment each of a total of at least 3 experiments with similar results are shown.