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Blood, Vol. 94 No. 4 (August 15), 1999: pp. 1372-1381

Induction of Epstein-Barr Virus-Specific Cytotoxic T-Lymphocyte Responses Using Dendritic Cells Pulsed With EBNA-3A Peptides or UV-Inactivated, Recombinant EBNA-3A Vaccinia Virus

Marion Subklewe, Ann Chahroudi, Alan Schmaljohn, Michael G. Kurilla, Nina Bhardwaj, and Ralph M. Steinman

From the Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, NY; the Virology Division, USAMRIID, Fort Detrick, Frederick, MD; and the Department of Pathology and Microbiology, University of Virginia, Charlottesville, VA.

Cell-mediated immunity, especially the cytotoxic T lymphocyte (CTL), provides resistance to Epstein-Barr virus (EBV), as is demonstrated by the occurrence of posttransplant lymphoproliferative disease in immunosuppressed patients. We set out to use dendritic cells (DCs) to elicit anti-EBV-specific CTLs in culture. In unselected, HLA-B8+ donors, monocyte-derived mature DCs were pulsed with the HLA-B8-restricted EBNA-3A peptide, FLRGRAYGL, and added to autologous T cells for 7 days at a DC:T ratio of 1:5 to 1:60. The cultured cells specifically lysed EBNA-3A peptide-pulsed, HLA-B8+, B-lymphoblastoid cell lines in a 5-hour 51Cr-release assay. The generation of CTLs did not require the addition of interleukin-2. In comparison, monocytes were weak antigen-presenting cells. DCs were then infected with recombinant vaccinia-EBNA-3A. Vaccinia infection significantly decreased the viability of immature DCs after 3 days of culture (to 25% to 45%) but had a smaller effect on mature DC recovery (40% to 70%). To decrease these cytopathic effects and to expand the potential use of vaccinia vectors for DC therapy in immunocompromised patients, we successfully used psoralen and UV-inactivated virus. Mature DCs pulsed with either live or inactivated vaccinia EBNA-3A virus could elicit strong EBNA-3A-specific CTLs. Therefore, mature DCs are powerful stimulators of EBV-specific CTLs and their major histocompatibility complex class I products can even be charged with UV-inactivated recombinant vaccinia.


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