title: Dendritic cells generated from peripheral blood transfected with human tyrosinase induce specific T cell activation.
abstract: Peptides of melanosomal proteins have recently been shown to be recognized in an HLA-restricted mode by specific cytolytic T lymphocytes in melanoma patients. Dendritic antigen-presenting cells (DC) are considered 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, generated DC from peripheral blood of normal donors in the presence of granulocyte/macrophage colony-stimulating factor and interleukin-4. Flow cytometric analysis of the cells during a 2-week culture revealed a loss of CD14 and CD34 expression, a concomittent increase of CD1a, CD11a,b and c, CD44, CD45, CD54, HLA-class I and II, and intermediate levels of CD26, CD80 and CD86. Cultured DC stimulated proliferation of allogeneic T cells and induced a marked, up to 20-fold, stimulation of T cell proliferation after pulsing with tetanus toxoid. To achieve independence of already-identified antigenic peptides presented in HLA class I-restricted fashion, which limits the general applicability of such peptides for vaccination of melanoma patients, we tested whether DC are transfectable with eukaryotic expression plasmids. DC transfected with two reporter genes (CAT, beta-galactosidase) using a liposome-based transfection technique, exhibited only low levels of enzymatically active 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 reverse-transcriptase-polymerase chain reaction, and enzyme activities became measurable. Furthermore, DC transfected with the tyrosinase gene were able to induce specific T cell activation in vitro, indicating appropriate peptide processing and presentation in DC after transfection. These data suggest new approaches to future tumor vaccination strategies.
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002450:Cell Communication / Q000276:immunology; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003713:Dendritic Cells / Q000201:enzymology / Q000378:metabolism*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008213:Lymphocyte Activation; D007959:Lymphocyte Culture Test, Mixed; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000235:genetics / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology*; D013745:Tetanus Toxoid / Q000494:pharmacology; D014162:Transfection / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: D013745:Tetanus Toxoid; D014409:Tumor Necrosis Factor-alpha; D014442:Monophenol Monooxygenase; D015500:Chloramphenicol O-Acetyltransferase