The B16 melanoma tumor line was derived from a malignant melanoma that arose spontaneously in C57BL/6 mice at the Jackson Laboratories (Bar Harbor, Me). This tumor was obtained from the Tumor Depository, Division of Cancer Treatment, National Cancer Institute (Frederick, Md) and is characterized as being nonimmunogenic. The B16 cell lines were free of mycoplasma (tested by ATCC, Rockville, Md) and free of the following viruses and microorganisms: pneumonia virus of mice, Sendai, mouse hepatitis virus, Theiler, minute virus of mice, parvovirus, lymphocytic choriomeningitis, epizootic diarrhea of infant mice, lactate dehydrogenase–elevating virus, reo type 3, polyoma, ectromelia, K virus, mouse adenovirus, mouse cytomegalovirus, Mycoplasma pulmonis, and cilium-associated respiratory bacillus (tested by Microbiological Associates, Rockville). The cells were grown in vitro in Dulbecco Modified Eagle Medium (high glucose) supplemented with 10% fetal calf serum (Hyclone, Logan, Utah), 2mM of glutamine, 100 U/mL of penicillin G sodium, 100 µg/mL of streptomycin sulfate, 0.25 µg/mL of amphotericin B, and 50µM 2-mercaptoethanol in 6% carbon dioxide to 95% air at 37°C. Cells were passaged biweekly by removing the old medium, rinsing the cells with Hank balanced salt solution (HBSS) (calcium- and magnesium-free), and briefly exposing them to 0.05% trypsin in 0.53mM edetic acid solution at 37°C for 2 to 3 minutes. The flask was tapped sharply against a solid surface to dislodge the cells, Dulbecco Modified Eagle Medium was added, and the cell suspension was repeatedly pipetted to produce a single cell suspension.