DENDRITIC CELLS GENERATED FROM PERIPHERAL-BLOOD TRANSFECTED WITH HUMAN TYROSINASE INDUCE SPECIFIC T-CELL ACTIVATION

Peptides of melanosomal proteins have recently been shown to be recogn ized in an HLA-restricted mode by specific cytolytic T lymphocytes in melanoma patients. Dendritic antigen-presenting cells (DC) are conside red to be the most effective stimulators of T cell responses, and the use of these cells has therefore been proposed to generate therapeutic responses to tumor antigens in cancer patients. We, therefore, genera ted DC from peripheral blood of normal donors in the presence of granu locyte/macrophage colony-stimulating factor and interleukin-4. Flow cy tometric analysis of the cells during a 2-week culture revealed a loss of CD 14 and CD34 expression, a concomittent increase of CD1a, CDlla, b and c, CD44, CD45, CD54, HLA-class I and II, and intermediate levels of CD26, CD80 and CD86. Cultured DC stimulated proliferation of allog eneic T cells and induced a marked, up to 20-fold, stimulation of T ce ll proliferation after pulsing with tetanus toroid. To achieve indepen dence of already-identified antigenic peptides presented in HLA class I-restricted fashion, which limits the general applicability of such p eptides for vaccination of melanoma patients, we tested whether DC are transfectable with eukaryotic expression plasmids. DC transfected wit h two reporter genes (CAT, beta-galactosidase) using a liposome-based transfection technique, exhibited only low levels of enzymatically act ive proteins, but were able to degrade rapidly intracellular proteins and to process peptides efficiently. Chloramphenicol acetyltransferase as well as tyrosinase mRNA were detectable after transfection by reve rse-transcriptase-polymerase chain reaction, and enzyme activities bec ame measurable. Furthermore, DC transfected with the tyrosinase gene w ere able to induce specific T cell activation in vitro, indicating app ropriate peptide processing and presentation in DC after transfection. These data suggest new approaches to future tumor vaccination strateg ies.