GrB is endogenously expressed in pDCs and is strongly induced upon IL-21 stimulation. (A) Human pDCs purified from sources as indicated were stained for intracellular GrB expression (black lines) and analyzed by flow cytometry using a GrB-PE–conjugated (thymus and tonsil) or a GrB-APC–conjugated (peripheral blood) antibody. Gray-filled histograms represent appropriate isotype control stainings. (B) GrB RNA levels were measured by qPCR in pDCs after incubation in the presence (black bars) or absence (white bars) of IL-21 for 4 hours and 6 hours as indicated. Expressions of the housekeeping genes (β-actin, GAPDH, and HPRT) were used to control for the amount of RNA used. Values were normalized to cells incubated without IL-21, which was set to 1. (C) Freshly isolated pDCs from tonsil (n = 5) and blood (n = 2) were cultured overnight in the presence or absence of IL-21 in serum-free medium. Culture supernatants were analyzed for the presence of GrB by ELISA. Shown are the mean GrB levels of the pDC donors tested +/− standard deviation. *P < .05. (D,E) Freshly isolated pDCs from 2 tonsil donors (D, upper and lower panels) and from blood (E) were cultured for 1 day (D) or for 2 days (E) in medium (dark gray–shaded histograms) or stimulated (black lines) with either IL-21 alone or with CpG-B (10 μg/mL) or R848 (10 μg/mL) in the presence or absence of IL-21 as indicated. Intracellular GrB expression was analyzed by flow cytometry. Isotype control stainings are shown as light gray–filled histograms. The numbers in E indicate the differences in mean fluorescence intensity (ΔMFI), which were calculated by subtracting the MFI of medium-cultured pDCs from the MFI of stimulated (IL-21, R848, or both) pDCs.