Identification of new NY‐ESO‐1 epitopes recognized by CD4+ T cells and presented by HLA‐DQ B1 03011

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‐γ and TNF‐α 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. © 2005 Wiley‐Liss, Inc.