Abstract
Recently, major advances have been made in the identification of antigens from human melanoma which are recognized by T cells. In spite of this, little is known about the optimal ways to use these antigens to treat patients with cancer. Progress in this area is likely to require accurate preclinical animal models, but the availability of such models has lagged behind developments in human tumor immunology. Whereas many of the identified human melanoma antigens are normal tissue differentiation proteins, analogous murine tumor antigens have not yet been identified. In this paper we identify a normal tissue differentiation antigen, tyrosinase-related protein 2 (TRP-2), expressed by the murine B16 melanoma which was found by screening a cDNA library from B16 with tumor-reactive cytotoxic T lymphocytes (CTL). A peptide conforming to the predicted MHC class I H2-Kb binding motif, TRP-2181-188, was identified as the major reactive epitope within TRP-2 recognized by these anti-B16 CTLs. By site-directed mutagenesis, it was shown that alteration of this epitope eliminated recognition of TRP-2. It was further demonstrated that a CTL line raised from splenocytes by repeated stimulation in vitro with this peptide could recognize B16 tumor and was therapeutic against 3-d-old established pulmonary metastases. The use of TRP-2 in a preclinical model of tumor immunotherapy may be helpful in suggesting optimal vaccination strategies for cancer therapy in patients.
Highlights
We report that T cells reactive with the B16 murine melanoma recognize a nonmutated product of the tyrosinaserelated protein 2 (TRP-2) gene
When adoptively transferred into mice bearing 3-d-old pulmonary metastases, line A significantly reduced the number of metastases compared to mice given IL-2 alone (P Ͻ0.001) or mice administered both IL-2 and cytotoxic T lymphocytes (CTL) that recognized the -gal protein (P Ͻ0.01) (Table 1)
To identify the antigens of B16 which were being recognized by line A, a cDNA library from B16 tumor was screened by transiently transfecting pools of ف25 cDNA clones into 293KbDb cells and coincubating these cells with line A CTLs
Summary
(Biofluids, Inc., Rockville, MD) supplemented with the following: 10% heat-inactivated fetal calf serum (Biofluids, Inc.), 0.1 mM nonessential amino acids and 1 mM sodium pyruvate (Biofluids, Inc.), 5 ϫ 10Ϫ5 M 2-mercaptoethanol (GIBCO BRL, Gaithersburg, MD), 0.3% glutamine (National Institutes of Health Media Unit, Bethesda, MD), 100 U/ml penicillin, 100 g/ml streptomycin, 50 g/ml gentamicin, and 0.5 g/ml fungizone. The transformed human embryonic kidney line 293 was purchased from the American Type Culture Collection (Rockville, MD), and grown in DMEM medium containing 5% heat-inactivated fetal calf serum, 0.3% glutamine, 0.01 M Hepes (National Institutes of Health Media Unit), 100 U/ml penicillin, and 100 g/ml streptomycin. Hearing (National Cancer Institute, Bethesda, MD), and was grown in Bennett’s medium, pH 6.9, which consisted of MEM (GIBCO BRL), 5% heat-inactivated fetal calf serum, 1 mM sodium pyruvate, 25 mM sodium bicarbonate, 100 M 2-mercaptoethanol, 200 nM PMA (Sigma Chemical Co., St. Louis, MO), 50 U/ml penicillin, and 50 g/ml streptomycin
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