Tumor-Specific CTL Kill Murine Renal Cancer Cells Using Both Perforin and Fas Ligand-Mediated Lysis In Vitro, But Cause Tumor Regression In Vivo in the Absence of Perforin

Renca was cultured overnight in the presence or absence of IFN-γ (500 U/ml), TNF-α (400 U/ml), and the combination of these two cytokines. These cells were then stained with PE-labeled anti-Fas, or FITC-labeled anti-H-2K^d or ICAM-1 mAb (solid lines). Dotted lines show background staining with isotype-matched control mAbs. Solid lines show staining with test Abs.

Inhibition of CTL activities to Renca by blocking of LFA-1/ICAM-1 interactions. A, Renca pretreated for 18 h with IFN-γ (500 U/ml) and TNF-α (400 U/ml) were used as targets in a 6 h [¹¹¹In]Ox release assay (E:T = 20:1) in the presence or absence of anti-LFA-1, anti-ICAM-1, and anti-FasL mAbs (10 μg/ml), or the combination of anti-LFA-1 and anti-FasL mAbs. B, Release of BLT esterase by CTL. CTL (1 × 10⁵) were incubated with Renca or IFN-γ/TNF-α-treated Renca (1 × 10⁴) for 4 h in 96-well tissue culture plates with or without anti-LFA-1 mAb (10 μg/ml). Next, the BLT activity in the supernatants was measured. Data represent mean ± SD of triplicate samples. Similar results were obtained in three independent experiments.

Regulation of FasL and TRAIL expression on CTL by various stimuli. CTL were cocultured with Renca, Harvey BALB fibroblasts, or the combination of PMA (20 ng/ml) and ionomycin (1 μg/ml) for 6 h. These CTL were stained with biotinylated anti-FasL or anti-TRAIL mAb and PE-avidin (solid lines). Dotted lines show background staining with biotinylated isotype control and PE-avidin; solid lines show staining with test Abs.

A, Adoptive transfer of CTL in 3-day experimental metastasis model. Renca cells (1.5 × 10⁵) were injected i.v. to BALB/c mice on day 0. CTL or Con A blasts (10⁷ cells/day) with IL-2 (10,000 U/day) or IL-2 alone were administered on days 3, 4, and 5. On day 17, half of the mice were sacrificed and lung metastasis was quantified. •, A single mouse. A horizontal bar shows the average number of metastases in each group. A vs B, p < 0.01; A vs C, p < 0.01; B vs C, p < 0.05. B, Survival of the remainder of the mice was monitored. The data shown are representative of two independent experiments.

Activity of CTL generated from pfp^−/− mouse. A, Cytotoxic activities of CTL generated from WT mouse and pfp^−/− mouse against Renca were tested in an 18 h [¹¹¹In]Ox release assay (E:T = 20 or 4:1). B, Specific IFN-γ production. CTL generated from WT or pfp^−/− mice were incubated with Renca, A20, or medium alone for 18 h at an E:T of 4:1 in the presence or absence of anti-CD4 or anti-CD8 mAb (10 μg/ML). The IFN-γ in the supernatant was measured by ELISA. C, Cytotoxic activities of pfp^−/− CTL were tested in an 18 h [¹¹¹In]Ox release assay in the presence or absence of anti-FasL mAb (10 μg/ml). D, Cytotoxic activities of pfp^−/− CTL against Renca pretreated with IFN-γ (500 U/ml) and TNF-α (400 U/ml) were tested in an 8 h [¹¹¹In]Ox release assay (E:T = 10 or 2:1) in the presence or absence of anti-LFA-1, anti-ICAM-1 mAbs (10 μg/ml), or the combination of anti-LFA-1 and anti-FasL mAbs. Data represent mean ± SD of triplicate samples. Similar results were obtained in three independent experiments.

Adoptive transfer of pfp^−/− CTL in 3-day experimental metastasis model. The mice were injected i.v. with Renca cells (1.5 × 10⁵) on day 0. Three days later, CTL or vehicle were transferred to these tumor-bearing mice. WT CTL or pfp^−/− CTL (6 × 10⁶ or 2 × 10⁶) were administered to WT BALB/c recipients. A or B vs E, p < 0.01; C or D vs E, p < 0.05.