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Expression Hierarchy of T Cell Epitopes from Melanoma Differentiation Antigens: Unexpected High Level Presentation of Tyrosinase-HLA-A2 Complexes Revealed by Peptide-Specific, MHC-Restricted, TCR-Like Antibodies¹

Yael Michaeli^*, Galit Denkberg, Keren Sinik^*, Liz Lantzy, Chiang Chih-Sheng, Celine Beauverd, Tamar Ziv^*, Pedro Romero and Yoram Reiter^2,*

^* Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel; Applied Immune Technologies Ltd, Haifa, Israel; MannKind Corporation, Valencia, CA 91534; and Division of Clinical Onco-Immunology, University Hospital (CHUV), Ludwig Institute for Cancer Research, Lausanne Branch, Lausanne, Switzerland

Peptide Ags presented by class I MHC molecules on human melanomas and that are recognized by CD8⁺ T cells are the subjects of many studies of antitumor immunity and represent attractive candidates for therapeutic approaches. However, no direct quantitative measurements exist to reveal their expression hierarchy on the cell surface. Using novel recombinant Abs which bind these Ags with a peptide-specific, MHC-restricted manner, we demonstrate a defined pattern of expression hierarchy of peptide-HLA-A2 complexes derived from three major differentiation Ags: gp100, Melan-A/Mart-1, and tyrosinase. Studying melanoma cell lines derived from multiple patients, we reveal a surprisingly high level of presentation of tyrosinase-derived complexes and moderate to very low expression of complexes derived from other Ags. No correlation between Ag presentation and mRNA expression was found; however, protein stability may play a major role. These results provide new insights into the characteristics of Ag presentation and are particularly important when such targets are being considered for immunotherapy. These results may shed new light on relationships between Ag presentation and immune response to cancer Ags.

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 study was supported by National Institutes of Health-National Cancer Institute R01 Grant 5R01CA115550 administered (to Y.R.). P.R. was funded in part by a grant from the European Union 6th FP Cancer Immunotherapy.

² Address correspondence and reprint requests to Dr. Yoram Reiter, Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel. E-mail address: reiter{at}tx.technion.ac.il

³ Abbreviations used in this paper: scMHC, single-chain MHC; IPTG, isopropyl β-D-thiogalactoside; ER, endoplasmic reticulum; MS, mass spectrometry; L-DOPA, 3,4-dihydroxy-L-phenylalanine; MFI, mean fluorescence intensity.

⁴ The online version of this article contains supplemental material.