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Raffaella Lupetti^*, Patrizia Pisarra^*, Alessandro Verrecchia, Cinthia Farina^*, Gabriella Nicolini^*, Andrea Anichini^*, Claudio Bordignon, Marialuisa Sensi^*, Giorgio Parmiani^*, and Catia Traversari

From the ^* Division of Experimental Oncology D, Istituto Nazionale Tumori, 20133 Milan, Italy; and the Gene Therapy Program, Istituto Scientifico San Raffaele Hospital, 20132 Milan, Italy

We report here the identification of a new shared human melanoma antigen recognized by a human leukocyte antigen (HLA)-A*68011–restricted cytotoxic T lymphocyte clone (CTL 128). The cDNA encoding this antigen is composed of a partially spliced form of the melanocyte differentiation antigen tyrosinase-related protein (TRP)-2, containing exons 1–4 with retention of intron 2 and part of intron 4 (TRP-2–INT2). The sequence coding for the antigenic epitope is located at the 5' end of intron 2 and is available for translation in the same open reading frame of the fully spliced TRP-2 mRNA. This peptide is also recognized by CTL 128 when presented by the HLA-A*3301, a member of the HLA-A3–like supertype that includes the HLA-A*68011. Quantitative reverse transcription PCR analysis carried out on total and/or cytoplasmic mRNA demonstrated that, in contrast to the fully spliced TRP-2 mRNA expressed in melanomas, normal skin melanocytes, and retina, the TRP-2–INT2 mRNA could be detected at significant levels in melanomas but not in normal cells of the melanocytic lineage. Instead, in these normal samples, both the spliced and the unspliced transcript of gp100 were expressed at high levels. Absence of endogenous TRP-2–INT2 expression in melanocytes was also confirmed by lack of recognition of HLA-A*68011–transduced, TRP-2⁺ melanocyte lines by CTL 128. These results indicate that a partially spliced form of a differentiation antigen mRNA, present in the cytoplasmic compartment of neoplastic but not normal cells of the melanocytic lineage, can be the source of a melanoma-restricted T cell epitope.

Key Words: melanoma • antigen • T cell • specificity • intron-encoded epitope

R. Lupetti is the recipient of a fellowship from the Italian Association for Cancer Research (AIRC), Milan. This work was supported in part by grants from the AIRC and from the European Community Biomed2 Program (BMH4-CT95-1627).

Abbreviations used: LCL, lymphoblastoid cell line; LNGFR, low-affinity nerve growth factor receptor; nt, nucleotide(s); ORF, open reading frame; RT, reverse transcription; TRP, tyrosinase-related protein; UTR, untranslated region.

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