NY-ESO-1 is one of the most immunogenic cancer antigens eliciting strong humoral and cellular immune responses in patients with NY-ESO-1-expressing malignancies. Since CD4+ T cells play a critical role in generating and maintaining antigen-specific cellular and humoral immune responses, we searched for new NY-ESO-1 epitopes presented by MHC class II molecules. CD4+ T cells of patients with NY-ESO-1-expressing cancer were presensitized with 18-mer overlapping synthetic peptides spanning the entire sequence of NY-ESO-1. Two partly overlapping NY-ESO-1 epitopes p49-66 and p55-72 were identified as targets for NY-ESO-1-specific CD4+ T cells. Peptide-specific CD4+ T-cell clones were generated by repetitive stimulation with NY-ESO-1 p49-66 and p55-72. Further experiments confirmed distinct specificities for the CD4+ T-cell clones indicating that at least 2 different CD4+ T-cell epitopes are located in the region p49-72 of the NY-ESO-1 sequence. Using a set of partially histocompatible EBV-B cell lines and MHC class II-specific antibodies, we found that both CD4+ T-cell epitopes were presented in the context of HLA-DQ B1 03011(DQ7). Natural processing and presentation of these epitopes was demonstrated by recognition of an HLA-DQ B1 03011- and NY-ESO-1-expressing lymphoma cell line and by recognition of dendritic cells (DC) exogenously loaded with NY-ESO-1 protein or infected with recombinant NY-ESO-1 adenoviral constructs. The specific production of IFN-gamma and TNF-alpha suggests that the NY-ESO-1-specific CD4+ T-cell clones belong to the Th1 subtype. The characterization of the new HLA-DQ B1 03011-restricted NY-ESO-1 peptides broadens the repertoire of epitopes that can be used to monitor NY-ESO-1-specific spontaneous and vaccine-induced T-cell responses in cancer patients.
Copyright 2005 Wiley-Liss, Inc.