Tumor cells release a soluble form of GITRL. (A) Sandwich ELISA. Serial dilutions of rGITRL were analyzed using a polyclonal and a monoclonal anti-GITRL antibody followed by anti–mouse IgG1-horseradish peroxidase. X indicates 100 ng/mL MHC class I chain-related protein A as negative control. The means of 4 replicates are shown. (B) Cell surface expression of GITRL on the indicated tumor cell lines and transfectants was determined by FACS using anti-GITRL mAb (shaded peaks) with mouse IgG1 (open peaks) as isotype control followed by anti–mouse PE. (C) C1R-GITRL and C1R-neo transfectants and the indicated GITRL-positive or -negative tumor cell lines were cultured for 48 hours; then the supernatants were analyzed for the presence of sGITRL by ELISA. (D) In addition, supernatants from monocytes, macrophages, DCs, B cells, and T cells cultured in the absence or presence of LPS, CpG oligodeoxynucleotide, and PHA, with C1R transfectant supernatants as control were investigated. The data are means of triplicates with SD. One representative experiment each from a total of at least 3 with similar results is shown.