NY-ESO-1, a member of the cancer-testis family of antigens, is expressed in a subset of a broad range of different human tumor types. Patients with advanced NY-ESO-1-expressing tumors frequently develop humoral immunity to NY-ESO-1, and three HLA A2-restricted peptides were defined previously as targets for cytotoxic CD8(+) T cells in a melanoma patient with NY-ESO-1 antibody. The objectives of the present study were (i) to develop enzyme-linked immunospot (ELISPOT) and tetramer assays to measure CD8(+) T cell responses to NY-ESO-1, (ii) to determine the frequency of CD8(+) T cell responses to NY-ESO-1 in a series of HLA-A2 patients with NY-ESO-1 expressing tumors, (iii) to determine the relation between CD8(+) T cell and humoral immune responses to NY-ESO-1, and (iv) to compare results of NY-ESO-1 ELISPOT assays performed independently in two laboratories with T cells from the same patients. NY-ESO-1 ELISPOT and tetramer assays with excellent sensitivity, specificity, and reproducibility have been developed and found to correlate with cytotoxicity assays. CD8(+) T cell responses to HLA-A2-restricted NY-ESO-1 peptides were detected in 10 of 11 patients with NY-ESO1 antibody, but not in patients lacking antibody or in patients with NY-ESO-1-negative tumors. The results of ELISPOT assays were concordant in the two laboratories, providing the basis for standardized monitoring of T cell responses in patients receiving NY-ESO-1 vaccines. DOI: https://doi.org/10.1073/pnas.97.9.4760 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-113858 Originally published at: Jäger, E; Nagata, Y; Gnjatic, S; Wada, H; Stockert, E; Karbach, J; Dunbar, P R; Lee, S Y; Jungbluth, A; Jäger, D; Arand, M; Ritter, G; Cerundolo, V; Dupont, B; Chen, Y T; Old, L J; Knuth, A (2000). Monitoring CD8 T cell responses to NY-ESO-1: correlation of humoral and cellular immune responses. Proceedings of the National Academy of Sciences of the United States of America, 97(9):4760-4765. DOI: https://doi.org/10.1073/pnas.97.9.4760 Monitoring CD8 T cell responses to NY-ESO-1: Correlation of humoral and cellular immune responses Elke Jäger*†‡, Yasuhiro Nagata†‡§, Sacha Gnjatic§, Hisashi Wada§, Elisabeth Stockert§, Julia Karbach*, P. Rod Dunbar¶, Sang Yull Leei, Achim Jungbluth§, Dirk Jäger**, Michael Arand††, Gerd Ritter§, Vincenzo Cerundolo¶, Bo Duponti, Yao-Tseng Chen**, Lloyd J. Old§, and Alexander Knuth* *II. Medizinische Klinik, Hämatologie-Onkologie, Krankenhaus Nordwest, 60488 Frankfurt, Germany; §Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan–Kettering Cancer Center, New York, NY 10021; ¶Institute of Molecular Medicine, John Radcliffe Hospital, OX3 7TN, Oxford, United Kingdom; iHuman Immunogenetics, Memorial Sloan–Kettering Cancer Center, New York, NY 10021; ††Institut für Toxikologie, Johannes Gutenberg Universität Mainz, D-55131 Mainz, Germany; and **Cornell University Medical College, New York, NY 10021 Contributed by Lloyd J. Old, February 22, 2000 NY-ESO-1, a member of the cancer–testis family of antigens, is expressed in a subset of a broad range of different human tumor types. Patients with advanced NY-ESO-1-expressing tumors frequently develop humoral immunity to NY-ESO-1, and three HLA A2-restricted peptides were defined previously as targets for cytotoxic CD81 T cells in a melanoma patient with NY-ESO-1 antibody. The objectives of the present study were (i) to develop enzyme-linked immunospot (ELISPOT) and tetramer assays to measure CD81 T cell responses to NY-ESO-1, (ii) to determine the frequency of CD81 T cell responses to NY-ESO-1 in a series of HLA-A2 patients with NY-ESO-1 expressing tumors, (iii) to determine the relation between CD81 T cell and humoral immune responses to NY-ESO-1, and (iv) to compare results of NY-ESO-1 ELISPOT assays performed independently in two laboratories with T cells from the same patients. NY-ESO-1 ELISPOT and tetramer assays with excellent sensitivity, specificity, and reproducibility have been developed and found to correlate with cytotoxicity assays. CD81 T cell responses to HLA-A2-restricted NY-ESO-1 peptides were detected in 10 of 11 patients with NY-ESO-1 antibody, but not in patients lacking antibody or in patients with NY-ESO1-negative tumors. The results of ELISPOT assays were concordant in the two laboratories, providing the basis for standardized monitoring of T cell responses in patients receiving NY-ESO-1 vaccines. M progress in the identification and characterization of human tumor antigens has occurred over the past decade (1). The development of approaches to analyzing humoral (2) and cellular (3) immune reactivity to cancer in the context of the autologous host has led to the molecular characterization of tumor antigens recognized by CD81 T cells (4) and antibody (5). As a consequence of these advances, it is now possible to classify human tumor antigens, and the majority of antigens defined to date fall into one or more of the following categories: (i) differentiation antigens, e.g., tyrosinase (6), Melan-AyMART-1 (7, 8), and gp100 (9); (ii) mutational antigens, e.g., CDK4 (10), b-catenin (11), caspase-8 (12), and p53 (13); (iii) amplification antigens, e.g., HER2yneu (14) and p53 (15); (iv) splice variant antigens (16); (v) viral antigens, e.g., HPV (17) and EBV (18); and (vi) cancer–testis (CT) antigens, e.g., MAGE (19) and NY-ESO-1 (20). The defining characteristics of CT antigens are high levels of expression in male germ cells but generally not in other normal tissues and aberrant expression in a variable proportion of a wide range of different cancer types. CT antigens first were recognized as targets for autologous cytotoxic T lymphocytes in a melanoma patient with an unusual clinical course (21). Analysis of humoral immune reactivity to human cancer by SEREX (serological screening of cDNA expression libraries) (5) also has uncovered a number of human tumor antigens with characteristics of CT antigens (22). To date, 10 genes or gene families encoding CT antigens have been recognized: MAGE (19), BAGE (23), GAGE (24), SSX (5), NYESO-1yLAGE-1 (20, 25), SCP-1 (26), CT7yMAGE-C1 (27, 28), CT8 (U. Sahin, personal communication), CT9 (29), and CT10y MAGE-C2 (30). Six of these CT systems are known to be coded for by genes on the X chromosome. The only CT antigen with a known function is SCP-1, a synaptonemal complex protein involved in chromosome reduction in meiosis (26). NY-ESO-1, the focus of the present study, was identified during a SEREX analysis of an esophageal cancer (20). The gene for NY-ESO-1 maps to Xq28 (31) and codes for an 18-kDa protein (20). NY-ESO-1 mRNA expression is found in 20–30% of melanomas, lung, breast, ovarian, and bladder cancers, and other tumor types but, like other CT antigens, rarely in colon cancer or renal cancer (20). In a survey of sera from normal individuals and cancer patients, antibodies to NY-ESO-1 were found in 40–50% of patients with advanced NY-ESO-1expressing tumors (32). One patient with high-titered NYESO-1 antibodies was found to have HLA-A2-restricted cytotoxic T lymphocytes against autologous NY-ESO-1-expressing melanoma cells, and three HLA-A2-restricted NY-ESO-1 peptides were identified as the target epitopes recognized by these cytotoxic T lymphocytes (33). In the present study, we have analyzed CD81 T cell responses to NY-ESO-1 by enzyme-linked immunospot (ELISPOT), cytotoxicity, and tetramer assays and the humoral immune responses by ELISA and Western blots. Our findings show that NY-ESO-1 elicits a strong, integrated humoral and cellular immune response in a high proportion of patients with NYESO-1-expressing tumors. Materials and Methods Tumor Typing for NY-ESO-1 mRNA. Expression of NY-ESO-1 mRNA in tumor specimens was assessed by reverse transcription–PCR, using previously described primers (20). Assays for NY-ESO-1 Antibody. NY-ESO-1 serum antibodies were assayed by ELISA and Western blots, using NY-ESO-1 recombinant protein purified from Escherichia coli (32). Abbreviations: ELISPOT, enzyme-linked immunospot; PBL, peripheral blood lymphocyte; SEREX, serological screening of cDNA expression libraries; CT, cancer–testis. †E.J. and Y.N. contributed equally to this work. ‡To whom reprint requests should be addressed at: E.J., II. Medizinische Klinik, Hämatologie–Onkologie, Krankenhaus Nordwest, 60488 Frankfurt, Germany; or Y.N., Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan–Kettering Cancer Center, 1275 York Avenue, Box 32, New York, NY 10021. E-mail: 100333.1434@compuserve.com or