Granulysin activates human Mo-iDCs. Mo-iDCs were cultured at 1 × 10⁶ cells/mL in the presence or absence of 10 μg/mL 15-kDa GNLY and 1 μg/mL ultrapure LPS for 48 hours. Cells were then analyzed for (A) expression of costimulatory molecules on Mo-DC by fluorescence-activated cell sorting (FACS) and (B) production of cytokines by Mo-DC. To further confirm whether DC activation was specifically due to GNLY, Hut-78 (WT) or Hut 78-520 (transfected with 15 kDa GNLY cDNA) cells were cultured at 2 × 10⁶/mL in complete RPMI 1640 for 48 hours. Cells were centrifuged, and supernatants collected. Mo-iDCs were treated with 5% of supernatants in the presence or absence of anti-GNLY (C). Cells were then analyzed for expression of costimulatory molecules on Mo-DC by FACS. Gray lines represent isotype control. Data are shown as geometric mean fluorescence of intensity. One representative experiment of 4 is shown. To assess functional activation of DCs by 15-kDa GNLY, proliferation of allogeneic, human peripheral blood T lymphocytes by GNLY-treated Mo-DCs were compared with that of untreated DCs (D). T cells (10⁵/well) were cultured in triplicate in the absence or presence of DCs at the concentrations specified in 96-well plates for 6 days with the addition of ³H-TdR (0.5 μCi/well) in the last 18 hours of incubation. The cells were harvested and measured for the incorporation of ³H-TdR (mean ± SD). DCs were treated with or without (untreated) 10 μg/mL 15-kDa GNLY for 48 hours at 37°C in humidified air with 5% CO₂ before use in the allogeneic MLR experiments. **P < .001. One representative experiment of 3 is shown.