S. Alijagic et al., DENDRITIC CELLS GENERATED FROM PERIPHERAL-BLOOD TRANSFECTED WITH HUMAN TYROSINASE INDUCE SPECIFIC T-CELL ACTIVATION, European Journal of Immunology, 25(11), 1995, pp. 3100-3107
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.