Efficient Simultaneous Presentation of NY-ESO-1/LAGE-1 Primary and Nonprimary Open Reading Frame-Derived CTL Epitopes in Melanoma

Abstract

Recent studies have shown that CTL epitopes derived from tumor-associated Ags can be encoded by both primary and nonprimary open reading frames (ORF). In this study we have analyzed the HLA-A2-restricted CD8+ T cell response to a recently identified CTL epitope derived from an alternative ORF product of gene LAGE-1 (named CAMEL), and the highly homologous gene NY-ESO-1 in melanoma patients. Using MHC/peptide tetramers we detected CAMEL1–11-specific CD8+ T cells in peptide-stimulated PBMC as well as among tumor-infiltrated lymph node cells from several patients. Sorting and expansion of tetramer+ CD8+ T cells allowed the isolation of tetramerbright and tetramerdull populations that specifically recognized the peptide Ag with high and low avidity, respectively. Remarkably, only high avidity CAMEL-specific CTL were able to recognize Ag-expressing tumor cells. A large series of HLA-A2-positive melanoma cell lines was characterized for the expression of 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-1157–165) encoded by the primary ORF products of the LAGE-1 and NY-ESO-1 genes. This analysis revealed that tumor-associated CD8+ T cell epitopes are simultaneously and efficiently generated from both primary and nonprimary ORF products of LAGE-1 and NY-ESO-1 genes and, importantly, that this occurs in the majority of melanoma tumors. These findings underscore the in vivo immunological relevance of CTL epitopes derived from nonprimary ORF products and support their use as candidate vaccines for inducing tumor specific cell-mediated immunity against cancer.

Proteasome-Assisted Identification of a SSX-2-Derived Epitope Recognized by Tumor-Reactive CTL Infiltrating Metastatic Melanoma

“…4A). As previously reported (10,25,26), CTL clones specific for several tumor Ags (NY-ESO-1 157-165 , Camel [1][2][3][4][5][6][7][8][9][10][11] , and MAGE-A10 254 -262 ) have been derived from this TILN. On the basis of these data, we performed an ELISPOT to assess the recognition of the above predicted SSX-2-derived peptides (Table I) by the short term-cultured TILN population obtained from LAU 50 in 1995.…”

NY-ESO-1 Expression and Immunogenicity in Malignant and Benign Breast Tumors

“…Fifty g lysates of cells or tissues prepared with HEPES buffer (HEPES 50 mM, NaCl 150 mM, EGTA 2.5 mM, EDTA 1.0 mM, Tween 20 0.1%, Glycerol 10%, DTT 1 mM, leupeptin 10 g/ml, aprotinin 10 g/ml, and phenylmethylsulfonyl fluoride 1.0 mM) were subjected to 12.5% polyacrylamide gel (Readygels J; Bio-Rad Laboratories, Hercules, CA) under reducing conditions. After electrophoresis for 35 min, Western blot was performed as described previously (18). Briefly, after transfer for 90 min, the transferred membrane was blocked by 5% skim milk/Tween 20-Tris-buffered saline.…”

Immunohistochemical and Molecular Analysis of Human Melanomas for Expression of the Human Cancer-Testis Antigens NY-ESO-1 and LAGE-1

“…In addition, HLA-A2-restricted antigenic peptides have been identified from LAGE-1 and alternative putative proteins. CTLs, specific for these epitopes, have been found (8,9).…”

“…Their biological function is unknown. There are two known splice variants of the LAGE-1 antigen, "LAGE long" and "LAGE short" (6), and both the NY-ESO-1 and LAGE-1 genes are able to use an alternative open reading frame (8,9). Both humoral and cellular responses to NY-ESO-1 have been described in patients with NY-ESO-1-positive tumors (10,11), and a number of HLA class I-restricted epitopes have been identified (11,12).…”