Efficient simultaneous presentation of NY-ESO-1/LAGE-1 primary and nonprimary open reading frame-derived CTL epitopes in melanoma

Recent studies have shown that CTL epitopes derived from tumor-associated A gs can be encoded by both primary and nonprimary open reading frames (ORF), In this study we have analyzed the HLA-AZ-restricted CD8(+) T cell respons e to a recently identified CTL epitope derived from an alternative ORF prod uct of gene LACE-1 (named CAMEL), and the highly homologous gene NY-ESO-I i n melanoma patients. Using MHC/peptide tetramers we detected CAMEL(1-11)-sp ecific CD8(+) T cells in peptide-stimulated PBMC as well as among tumor-inf iltrated lymph node cells from several patients, Sorting and expansion of t etramer(+) CD8(+) T cells allowed the Isolation of tetramer(bright) and tet ramer(dull) populations that specifically recognized the peptide Ag with hi gh and low avidity, respectively, Remarkably, only high avidity CAMEL-speci fic CTL were able to recognize Ag-expressing tumor cells. A large series of HLA-A2-positive melanoma cell lines was characterized for the expression o f LAGE-1 and NY-ESO-1 mRNA and protein and tested for recognition by CAMEL- specific CTL as well as CTL that recognize a peptide (NY-ESO-1(127-165)) en coded by the primary ORF products of the LAGE-1 and NY-ESO-1 genes, This an alysis revealed that tumor-associated CD8(+) T cell epitopes are simultaneo usly and efficiently generated from both primary and nonprimary ORF product s of LAGE-1 and NY-ESO-1 genes and, importantly, that this occurs in the ma jority of melanoma tumors. These findings underscore the in vivo immunologi cal relevance of CTL epitopes derived from nonprimary ORF products and supp ort their use as candidate vaccines for inducing tumor specific cell-mediat ed immunity against cancer.