Expression of human GITRL on pDCs and GITR on NK cells. (A,D) Quantification of GITRL (A) and GITR (D) mRNA in human primary cells was performed by human Affymatrix microarray gene expression analyses. Expression of GITRL or GITR in each population was analyzed at the 20-hour time point. (B) Isolated pDCs were stimulated with each stimuli for 20 hours. Following stimulation, total mRNA was prepared for GITRL mRNA evaluation by RT-PCR. (C) Purified pDCs were stimulated with each stimuli for 18 and 36 hours. Filled histograms represent isotype control; open histograms, staining of GITRL on pDCs. (E) Freshly isolated resting NK cells or NK cells stimulated with IL-2, IL-12, IL-15, IFN-α, or TNF-α for the indicated times were stained with anti-GITR mAb. Open histograms represent isotype control staining; filled histograms, GITR expression on NK cells. Numbers on histograms indicate the mean fluorescence intensity (MFI) of GITR expression. All data shown are representative of 4 experiments.