1. ¹Institute of Molecular Immunology, GSF National Research Center for Environment and Health, 81377 Munich, Germany
2. ²Clinical Cooperation Group "Immune Monitoring," GSF National Research Center for Environment and Health, 81377 Munich, Germany
3. ³GSF Clinical Cooperation Group "Urological Tumors," Laboratory for Tumor Immunology, Department of Urology, Ludwig-Maximilians-University, Munich, Germany
4. ⁴Third Department of Medicine, Hematology, and Oncology, Johannes Gutenberg-University of Mainz, Mainz, Germany

Correspondence: Miran Javorovic, Institute of Molecular Immunology, GSF National Research Center for Environment and Health, Marchioninistrasse 25, 81377 Munich, Germany. Fax: +49 89 7099300. E-mail: miran.javorovic@gsf.de

Received 14 September 2004; Accepted 21 March 2005.

Abstract

Previous studies have analyzed transfer of RNA-encoded tumor-associated antigens (TAAs) into immature dendritic cells (DCs) because of their exceptional ability to internalize antigens. Concerns have been raised regarding the use of immature DCs in clinical studies because of their capacity to tolerize T cells. Therefore, we focused on optimizing RNA transfer into mature DCs using the method of electroporation and obtained high protein expression in 90% of mature DCs. Particular emphasis was placed on quantifying RNA transfer. Reconstitution of peptide–MHC (pMHC) ligands on RNA-pulsed DCs was measured with the help of effector-memory cytotoxic T lymphocytes (CTLs) specific for the melanoma-associated antigens tyrosinase and mutated cyclin-dependent kinase 4. In contrast to single-species RNA, transfer of native tumor-derived RNA or amplified tumor RNA into DCs resulted in very low or no capacity to reactivate these CTLs. A correlation was found between TAA message levels in tumor-derived RNA and pMHC ligand reconstitution on DCs. These results demonstrate that even TAAs with highly immunogenic mutated epitopes are not necessarily transferred when tumor-derived RNA is transfected into the DCs, thereby resulting in a lack of DC capacity to reactivate some preexisting effector-memory CTLs.