Cytotoxic activity of TWEAK against tumor cell lines. (A) Cytotoxic activity of CD8–TWEAK. Target tumor cells (5 × 10³) were cultured with the indicated concentrations of CD8–TWEAK in the presence (•) or absence (○) of IFN-γ (20 ng/ml). After 36-h culture, viability was estimated by the WST assay. Data represent mean ± SD of triplicate samples. Similar results were obtained in three independent experiments. (B) Cytotoxic activity of hTWEAK, hTRAIL, and hFasL transfectants against HSC3 cells. Cytotoxic activity of the indicated transfectants was tested against HSC3 cells in 8- (white bars) or 18-h (gray bars) ⁵¹Cr-release assay at an E/T ratio of 10. Data represent mean ± SD of triplicate samples. Similar results were obtained in three independent experiments. (C) Cytotoxic activity of hTWEAK or hTRAIL transfectants against KATO-III and HSC3 cells. Cytotoxic activity of hTWEAK/2PK-3 (•), hTRAIL/2PK-3 (○), or 2PK-3 (▵) was tested against KATO-III cells in the presence of IFN-γ (20 ng/ml) or HSC3 cells in 18-h ⁵¹Cr-release assay at the indicated E/T ratios. Data represent mean ± SD of triplicate samples. Similar results were obtained in three independent experiments.

Expression of TWEAK on human PB T cells and monocytes. (A) Expression of TWEAK on human PB T cells. PBMCs were cultured for 24 h on plates precoated with anti-CD3 mAb (10 μg/ml) or in the presence of PHA (20 μg/ml), IFN-α (200 U/ml), or IFN-γ (100 ng/ml). The cells were then stained with biotinylated CARL-1, followed by PE-labeled avidin and FITC-labeled anti-CD3 mAb. White histograms indicate the staining with CARL-1 on electronically gated CD3⁺ T cells. Dark histograms indicate the background staining with biotinylated control mAb and PE-labeled avidin. Similar results were obtained with PBMCs from three individuals. (B) Expression of TWEAK and TRAIL on human PB monocytes. Purified monocytes were stimulated with IFN-α (200 U/ml), IFN-γ (100 ng/ml), LPS (20 μg/ml), or GM-CSF (100 ng/ml) for 12 h and then stained with biotinylated CARL-1 and PE–avidin (white histograms). Dark histograms indicate the background staining with biotinylated control mAb and PE–avidin. Similar results were obtained with monocytes from three individuals. (C) Time course of TWEAK expression on monocytes after stimulation with IFN-γ (100 ng/ml). After the indicated time periods, the cells were stained with biotinylated CARL-1 and PE–avidin (white histograms). Dark histograms indicate the background staining with biotinylated control mAb and PE–avidin. Similar results were obtained with monocytes from three individuals.

Characterization of anti-hTWEAK mAbs. (A) Inhibition of TWEAK cytotoxicity. Cytotoxic activity of CD8–TWEAK (100 ng/ml) was tested against HSC3 cells in the presence of serially diluted anti-hTWEAK mAbs, CARL-1 (•) or CARL-2 (○), by the WST assay. Data represent mean ± SD of triplicate samples. Similar results were obtained in three independent experiments. (B) Cell surface staining of hTWEAK transfectant. 2PK-3 and hTWEAK/2PK-3 cells were stained with biotinylated CARL-1 or CARL-2, followed by PE-labeled avidin (white histograms). Dark histograms indicate background staining with biotinylated control mAb plus PE-labeled avidin.

Contribution of TWEAK, TRAIL, and TNF-α to IFN-γ–stimulated monocyte cytotoxicity. Purified monocytes were stimulated with IFN-γ (100 ng/ml) for 12 h. Cytotoxic activity was then tested against HSC3 and IFN-γ–treated KATO-III cells by 18-h ⁵¹Cr-release assay at an E/T ratio of 50. Data represent mean ± SD of triplicate samples. Similar results were obtained in three independent experiments.