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* Department of Internal Medicine III and
Department of Immunology, Johannes-Gutenberg University, Mainz, Germany
Genetic modification of vaccines by linking the Ag to lysosomal or endosomal targeting signals has been used to route Ags into MHC class II processing compartments for improvement of CD4+ T cell responses. We report in this study that combining an N-terminal leader peptide with an MHC class I trafficking signal (MITD) attached to the C terminus of the Ag strongly improves the presentation of MHC class I and class II epitopes in human and murine dendritic cells (DCs). Such chimeric fusion proteins display a maturation state-dependent subcellular distribution pattern in immature and mature DCs, mimicking the dynamic trafficking properties of MHC molecules. T cell response analysis in vitro and in mice immunized with DCs transfected with Ag-encoding RNA showed that MITD fusion proteins have a profoundly higher stimulatory capacity than wild-type controls. This results in efficient expansion of Ag-specific CD8+ and CD4+ T cells and improved effector functions. We used CMVpp65 and NY-ESO-1 Ags to study preformed immune responses in CMV-seropositive individuals and cancer patients. We show that linking these Ags to the MITD trafficking signal allows simultaneous, polyepitopic expansion of CD8+ and CD4+ T cells, resulting in distinct CD8+ T cell specificities and a surprisingly broad and variable Ag-specific CD4+ repertoire in different individuals.
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 Combined Project Grant SFB432 and Heisenberg Scholarship TU 115/2-1 of the Deutsche Forschungsgemeinschaft, as well as the Immunology Cluster of Excellence at the University Mainz.
2 Ö.T. and U.S. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Ugur Sahin, Division of Experimental and Translational Oncology, Department of Internal Medicine III, Johannes Gutenberg University, Obere Zahlbacherstr. 63, 55131 Mainz, Germany. E-mail address: Sahin{at}mail.uni-mainz.de
4 Abbreviations used in this paper: LAMP, lysosome-associated membrane protein; BMDC, bone marrow-derived dendritic cell; DC, dendritic cell; eGFP, enhanced GFP; ER, endoplasmic reticulum; HA, influenza hemagglutinin; HCMV, human CMV; IVT, in vitro transription; MITD, MHC class I trafficking signal; sec, secretion signal; TM, transmembrane domain; WT, wild type.
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