Functional characterization of the effects of sGITRL on NK cell reactivity. (A) NK cells were cultured for 24 hours in the absence or presence of 10 ng/mL IL-15 and afterward incubated for an additional 24 hours or 48 hours with 10 ng/mL sGITRL derived from C1R-GITRL supernatants with an equal volume of C1R-neo supernatant as control. Subsequently, determination of apoptosis was performed by FACS using propidium iodide (PI) and annexin V–fluorescein isothiocyanate. The percentage of dead cells is indicated. One representative experiment each of a total of 3 experiments with similar results is shown. (B) NK cells were cultured for 24 hours with or without of 10 ng/mL IL-15 in the presence or absence of control supernatant or 10 ng/mL sGITRL. Where indicated, 10 ng/mL GITR-Ig or IgG1 had been added to sGITRL-containing supernatant. Subsequently, nuclear extracts were prepared and RelB protein was analyzed by Western blot. (C) Intensity of bands was densitometrically determined using ImageJ software, and results obtained with IL-15–stimulated NK cells were defined as 100%. Columns represent means with SD of 4 independent experiments.