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Class I HLA Folding and Antigen Presentation in β₂-Microglobulin-Defective Daudi Cells¹

Aline Martayan^*, Leonardo Sibilio^*, Elisa Tremante^*, Elisa Lo Monaco^*, Arend Mulder, Doriana Fruci, Agata Cova, Licia Rivoltini and Patrizio Giacomini^2,*

^* Laboratory of Immunology, Regina Elena Cancer Institute Centro della Ricerca Sperimentale, Rome, Italy; Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands; Research Center "Ospedale Bambino Gesù," Rome, Italy; and Unit of Immunotherapy of Human Tumors, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico, Istituto Nazionale Tumori, Milan, Italy

To present virus and tumor Ags, HLA class I molecules undergo a complex multistep assembly involving discrete but transient folding intermediates. The most extensive folding abnormalities occur in cells lacking the class I L chain subunit, called β₂-microglobulin (β₂m). Herein, this issue was investigated taking advantage of eight conformational murine mAbs (including the prototypic W6/32 mAb) to mapped H chain epitopes of class I molecules, four human mAbs to class I alloantigens, as well as radioimmunoprecipitation, in vitro assembly, pulse-chase, flow cytometry, and peptide-pulse/ELISPOT experiments. We show that endogenous (HLA-A1, -A66, and -B58) as well as transfected (HLA-A2) heavy chains in β₂m-defective Burkitt lymphoma Daudi cells are capable of being expressed on the cell surface, although at low levels, and exclusively as immature glycoforms. In addition, HLA-A2 is: 1) partially folded at crucial interfaces with β₂m, peptide Ag, and CD8; 2) receptive to exogenous peptide; and 3) capable of presenting exogenous peptide epitopes (from virus and tumor Ags) to cytotoxic T lymphocytes (bulk populations as well as clones) educated in a β₂m-positive environment. These experiments demonstrate a precursor-product relationship between novel HLA class I folding intermediates, and define a stepwise mechanism whereby distinct interfaces of the class I H chain undergo successive, ligand-induced folding adjustments in vitro as well as in vivo. Due to this unprecedented class I plasticity, Daudi is the first human cell line in which folding and function of class I HLA molecules are observed in the absence of β₂m. These findings bear potential implications for tumor immunotherapy.

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 the Italian Ministry of Health agreements 2007 (to L.R. and P.G.), the European Community Contract N. 518234 and Associazione Italiana Ricerca sul Cancro funds (to L.R.), and the Macropa Foundation (to A.M.).

² Address correspondence and reprint requests to Dr. Patrizio Giacomini, Immunology Laboratory, Regina Elena Institute for Cancer Research, Via delle Messi D’Oro, 156, Rome, Italy. E-mail address: giacomini{at}ifo.it

³ Abbreviations used in this paper: β₂m, β₂-microglobulin; CTL, cytotoxic T lymphocyte; IEF, isoelectrofocusing.