Selective histocompatibility leukocyte antigen (HLA)-A2 loss caused by aberrant pre-mRNA splicing in 624MEL28 melanoma cells

Histocompatibility leukocyte antigen (HLA)-A2 is used as a restricting elem ent to present several melanoma-associated antigen (MAA)-derived peptides t o cytotoxic T lymphocytes (CTLs). HLA-A2 antigen is selectively lost in pri mary melanoma lesions and more frequently in metastases. Only scanty inform ation is available about the molecular mechanisms underlying this abnormali ty, in spite of its potentially negative impact on the clinical course of t he disease and on the outcome of T cell-based immunotherapy. Therefore, in this study we have shown that the selective HLA-A2 antigen loss in melanoma cells 624MEL28 is caused by a splicing defect of HLA-A2 pre-mRNA because o f a base substitution at the 5' splice donor site of intron 2 of the HLA-A2 gene. As a result, HLA-A2 transcripts are spliced to two aberrant forms, o ne with exon 2 skipping and the other with intron 2 retention. The latter i s not translated because of an early premature stop codon in the retained i ntron. In contrast, the transcript with exon 2 skipping is translated to a truncated HLA-A2 heavy chain without the cr, domain. Such a polypeptide is synthesized in vitro but is not detectable in cells, probably because of th e low steady state level of the corresponding mRNA and the low translation efficiency. These results indicate that a single mutational event in an HLA class I gene is sufficient for loss of the corresponding allele. This may account, at least in part, for the high frequency of selective HLA class I allele loss in melanoma cells. Our conclusion emphasizes the need to implem ent active specific immunotherapy with a combination of peptides presented by various HLA class I alleles. This strategy may counteract the ability of melanoma cells with selective HLA class I allele loss to escape from immun e recognition.