Murine Melanoma Cell Lines for Immunotherapy

Female C57BL/6 (H-2^b) mice, 6–10 wk old, obtained from Frederick Cancer Research Center (Frederick, MD) and maintained in a barrier facility, were used for all experiments. EL-4 thymoma (H-2^b) and the derived β-galactosidase (β-gal)–transfected clone E22 have been described previously (12 ). B16 (H-2^b), hereafter named B16.WT, is a spontaneous murine melanoma expressing gp100, MART-1, tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 by FACS^® and Western blot analysis (data not shown). B16.B7-1 is a hyperpigmented clone of B16.WT that was stably transfected using a Moloney mouse leukemia virus encoding the gene for B7-1 driven by a LTR promoter. JB/MS is a pigmented, chemically induced melanoma expressing gp100, provided by Dr. Vincent Hearing (National Cancer Institute, NIH, Bethesda, MD). 293K^b and 293K^bD^b are highly transfectable human renal 293 cells stably transfected with K^b and K^b plus D^b, respectively. RMA/S (H-2^b) is a cell line deficient in endogenous peptide loading (13 (link)). EL-4, B16.WT, RMA/S, MCA205, and MC38, a C57BL/6-derived colon carcinoma, were maintained in complete medium (CM; RPMI 1640 with 10% heat-inactivated fetal bovine serum [FBS; Biofluids, Rockville, MD], 0.03% l-glutamine, 100 μg/ml streptomycin, 100 μg/ml penicillin, and 50 μg/ml gentamycin sulfate [NIH Media Center, Bethesda, MD]). B16.B7-1 and E22 were maintained in CM with 400 μg/ml of bioactive G418. 293K^b and 293K^bD^b were maintained in DMEM with 10% heat-inactivated FBS, 0.03% l-glutamine, 100 μg/ml streptomycin, 100 μg/ml penicillin, 50 μg/ml gentamycin sulfate, and 400 μg/ml of bioactive G418.