[CANCER RESEARCH 62, 213–218, January 1, 2002] NY-ESO-1 119 –143 Is a Promiscuous Major Histocompatibility Complex Class II T-Helper Epitope Recognized by Th1- and Th2-Type Tumor-reactive CD4 T Cells 1 Hassane M. Zarour, 2 Bernard Maillere, Vladimir Brusic, Kara Coval, Eileen Williams, Sandra Pouvelle-Moratille, Florence Castelli, Stephanie Land, Jaafar Bennouna, Theodore Logan, and John M. Kirkwood Department of Medicine and Melanoma Center, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213 [H. M. Z., K. C., E. W., J. M. K.]; Protein Engineering and Research Department, CEA-Saclay, 91191 Gif sur Yvette, France [B. M., S. P-M., F. C.]; Kent Ridge Digital Labs, Singapore 119613 [V. B.]; Biostatistics, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213 [S. L.]; Immunologic Monitoring and Cellular Products Laboratory, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213 [J. B.]; and Indianapolis Cancer Research Institute, Indianapolis, Indiana 46202 [T. L.] ABSTRACT The NY-ESO-1 gene product is expressed by a range of human tumors and is recognized by antibodies from sera of cancer patients with NY- ESO-1-expressing tumors. The NY-ESO-1 gene also encodes several MHC class I- and MHC class II-restricted tumor epitopes recognized by T lymphocytes. In particular, we previously reported that the NY-ESO-1 119 –143 peptide contains at least two HLA-DRB1*0401-presented epitopes that are recognized by melanoma-reactive CD4 T cells. Here we report that the NY-ESO-1 119 –143 peptide can be presented in the context of multiple HLA-DR alleles to stimulate tumor-reactive CD4 T cells. The NY-ESO-1 119 –143 peptide is able to bind to several DR molecules. The NY-ESO-1 119 –143 peptide is also capable of inducing specific CD4 T cells in vitro from peripheral blood lymphocytes of normal donors and patients with melanoma who express these HLA-DR alleles. These CD4 T cells recognize NY-ESO-1  , HLA-matched or autologous melanoma cell lines, as well as autologous antigen-presenting cells fed with the NY-ESO-1 protein. We also demonstrate that the NY-ESO-1 119 –143 peptide stimulates in vitro both Th1-type and Th2- type CD4 T-cell responses from peripheral blood lymphocytes of normal donors and melanoma patients. Taken together, these data suggest a key role of the NY-ESO-1 119 –143 peptide sequence in the induction of cellular and humoral responses against NY-ESO-1-expressing tumors. They support the relevance of cancer vaccine trials with the NY-ESO-1 119 –143 peptide in the large number of cancer patients with NY-ESO-1- expressing tumors. INTRODUCTION The NY-ESO-1 antigen is expressed by many tumors of different histological types, including breast, prostate, lung, and melanoma, and by male germ-line cells, but it is silent in normal tissues. Because of this expression pattern, NY-ESO-1 is classified in the group of TAAs 3 alternatively designated cancer-testis antigens (1), cancer-germ line antigens (2), or tumor-specific shared antigens (3). The NY-ESO-1 antigen was initially identified by serological analysis of a recombi- nant cDNA library from a human esophageal cancer (4). NY-ESO-1 has subsequently been shown to encode class I-restricted peptides expressed by a diverse range of cancers (1, 5). More recently, multiple NY-ESO-1-derived epitopes presented by HLA-DRB1*0401, HLA- DRB4*0101, and HLA-DRB1*0401 and capable of stimulating CD4+ T cells, have been reported (6 – 8). NY-ESO-1 appears to be very immunogenic, inducing both spontaneous cellular and humoral responses in 50% of patients with NY-ESO-1 + tumors (1, 9). The induction of primary NY-ESO-1-specific CD8+ T-cell responses has been reported after intradermal peptide vaccination in patients with NY-ESO-1 + tumors (10). We have previously reported a peptide-based strategy to identify novel DR4-restricted tumor-derived peptides recognized by CD4+ T cells (11). In doing so, we have already identified several DR4- restricted peptide sequences, including peptide NY-ESO-1 119 –143 (7). Here we develop a strategy to identify those sequences that can be broadly presented by multiple DR alleles among peptide sequences initially identified as DR4-restricted. In particular, we demonstrate that peptide NY-ESO-1 119 –143 not only binds to multiple DR alleles, but also stimulates CD4+ T-cell responses when presented at the surface of these molecules. We also observed that peptide NY- ESO-1 119 –143, which stimulated in vitro strong Th1-type T-cell responses, could also induce Th2-type CD4+ T-cell responses from PBLs of normal donors and melanoma patients. These findings support the use of the NY-ESO-1 119 –143 peptide as a cancer vaccine for a large number of patients with NY-ESO-1- expressing tumors. MATERIALS AND METHODS Cell Lines, Media, and Antibodies. Tissues and blood samples used for all studies reported here were obtained under UPCI Institutional Review Board-approved protocol 96-99. Patients UPCI-MEL 527, UPCI-MEL 285, and UPCI-MEL 598 are long-lived patients who have remained disease free several years after successful therapy for distant NY-ESO-1-expressing metastatic melanoma. The UPCI-MEL 527.1, UPCI-MEL 285.1, and UPCI-MEL 591.8 cell lines were derived from metastatic lesions of these patients. Patients UPCI-MEL 527, UPCI-MEL 285, and UPCI-MEL 598 have been genotyped as HLA-DRB1*0401, HLA-DRB1*0101/ DRB1*0401, and HLA-DRB1*0701/DRB1*1101, respectively. HLA-DR genotyping was performed with a commercial DR typing panel of PCR primers, according to the manufacturer’s instructions (Dynal, Oslo, Norway). High-titer anti-NY-ESO-1 antibodies have been consistently observed in the serum of patient UPCI-MEL 527. The T2.DR4 cell line was generated through transfection of HLA- DRB1*0401 cDNA into T2 cells (18). The T2.DR4 cell line is HLA-DM deficient, making its cell surface DRB1*0401 complexes receptive to loading by exogenous peptides. The homozygous B-EBV-transformed cell lines MGAR and POER were provided by Dr. Penelope Morel (UPCI, Pittsburgh, PA). DR-transfected mouse cells, i.e., L.DR1, L.DR3, L.DR7, and L.DR53 were a gift of Dr. Robert Karr (Searle, Inc., St. Louis, MO). All cell lines were cultured in RPMI 1640 (Life Technologies) supplemented with 10% FCS, L-arginine (116 mg/liter), L-asparagine (36 mg/liter), and L-glutamine (216 mg/liter). The HB55 and HB95 hybridomas, which secrete the L243 anti- HLA-DR (class II) mAb and the W6/32 anti-HLA-A, B, C (class I) mAb, Received 7/30/01; accepted 11/1/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by NIH Grant CA 56774 (to J. M. K.), a Clinical Trial Grant/Melanoma Initiative from the Cancer Research Institute (to J. M. K.), the Compet- itive Medical Research Fund of the University of Pittsburgh Medical Center, and a Cancer Research Institute/Elaine R. Shepard Memorial Fellowship (to H. M. Z.). 2 To whom requests for reprints should be addressed, at the University of Pittsburgh Cancer Institute, Biomedical Science Tower, E 1051, 211 Lothrop Street, Pittsburgh, PA 15213-2582. Phone: (412) 648-9119; Fax: (412) 624-7794; E-mail: zarourhm@msx. upmc.edu. 3 The abbreviations used are: TAA, tumor-associated antigen; Th, T-helper; PBL, peripheral blood lymphocyte; UPCI, University of Pittsburgh Cancer Institute; EBV; Epstein Barr virus; mAb, monoclonal antibody; HPLC, high-performance liquid chroma- tography; ELISPOT, enzyme-linked immunospot; IL, interleukin; DC, dendritic cell; APC, antigen-presenting cell. 213 Research. on February 4, 2016. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from