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note
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5 cells were analyzed by EPICS-ELITE (Coulter Electronics, Hialeah, FL) with a logarithmic amplifier as described (5).
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48
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note
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6 B16 cells for subcutaneous tumor growth on day O. Recombinant murine IL-12 (2400 U/mouse) or phosphate-buffered saline (PBS) was injected on days 3, 5, 7, and 9. On day 14, the mice were killed and either metastatic nodules counted or GM3 melanoma antigens measured by radioimmunoassay in the liver or lung as described (22). For subcutaneous tumor growth, injection of IL-12 or PBS was initiated on day 5, and the mice were treated five times per week. The diameters of tumors were measured every day with calipers. The sizes of tumors were expressed as the products of the longest diameter times the shortest diameter (in square millimeters).
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49
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note
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51Cr-release was calculated by the following formula: percent of specific lysis = (sample cpm -spontaneous cpm) × 100/(maximum cpm - spontaneous cpm). Spontaneous cpm was calculated from the supernatant of the target cells alone, and the maximum release was obtained by adding 1N HCl to target cells.
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50
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note
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β8 (MR5-2), NK1.1 (PK136), Ly49C (5E6), IFN-γ (R4-6A2), Fas (Jo2), and Fas ligand (K10) (Pharmingen) were used for blocking experiments.
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note
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We thank M. Kobayashi and S. F. Wolf, Genetic Institute Incorporated, for recombinant murine IL-12 and H. Tanabe for secretarial assistance. Supported in part by the Grant-in-Aid for Scientific Research on Priority Areas (08282103) from the Ministry of Education, Culture, and Science, Japan and the Taisho Pharmaceutical Company.
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