Transcriptional targeting of dendritic cells for gene therapy using the promoter of the cytoskeletal protein fascin.

Strong cell-type-specific promoters are basic tools in gene therapy allowing for novel applications and focused strategies by transcriptionally targeting gene expression to selected cells. In immunotherapy, dendritic cells (DC) are of central importance, since they represent the principal inducers of immune responses. Here we describe isolation and use of the promoter of the murine actin-bundling protein fascin to target transcriptionally gene expression to cutaneous DC. Using the reporter gene enhanced green fluorescent protein (EGFP), we demonstrate that the fascin promoter mediates a strong antigen expression that is restricted to mature DC. DNA vaccination with antigen-encoding expression vectors under control of the fascin promoter using a gene gun resulted, consistently, in limited antigen expression by few directly transfected DC. Nevertheless, nearly as many antigen-specific CD8+ T cells directed against the encoded antigens EGFP and beta-galactosidase, respectively, were induced as with expression constructs under control of the ubiquitously expressed CMV promoter. This result impressively underlines the pivotal role of directly transfected DC in DNA vaccination. Immunization using the fascin promoter induced markedly lower levels of antigen-specific antibodies following single or repeated immunization. Thus, our DC-targeted DNA vaccination approach induces qualitatively distinct, predominantly cellular immune responses and provides new opportunities for immunotherapy.

Expression of the actin-bundling protein fascin in cultured human dendritic cells correlates with dendritic morphology and cell differentiation.

Dendritic cells are key players of the immune system as they efficiently induce primary immune responses by activating naive T cells. We generated human dendritic cells from CD14+ blood precursors and investigated expression of the actin-bundling protein fascin during maturation by western blotting, immunofluorescence, and cytofluorometry. Cells obtained by culture of CD14+ blood precursors in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4, which were only weakly positive for the maturation marker CD83, expressed low amounts of fascin. Addition of a cytokine cocktail including tumor necrosis factor alpha, interleukin-1beta, interleukin-6, and prostaglandin E2 induced maturation of the cells and enhanced fascin expression in parallel with CD83 expression. Isolated mature CD83+ cells displayed especially high fascin levels on western blots, as did gated CD83+ dendritic cells in cytofluorometry. Dendritic cells generated from CD34+ blood precursors expressed high levels of fascin as well. Confocal microscopy revealed that location of fascin within the cell was restricted to the area of the submembranous actin cytoskeleton and to the dendritic processes. Suppression experiments using antisense constructs of fascin hint at a retarded morphologic maturation of dendritic cells, supporting the view that fascin expression is pivotal for dendrite formation. Our data suggest that fascin could serve as a marker molecule to monitor the maturation state of in vitro generated dendritic cells for use in clinical trials.

Maturation of epidermal Langerhans cells: increased expression of beta- and gamma-actin isoforms as a basis of specialized cell functions.

Epidermal Langerhans cells (LC) represent immature dendritic cells. During in vitro culture in the presence of keratinocytes they mature into potent immunostimulatory cells for naive T cells. This process is thought to simulate in vivo maturation of LC following activation by antigen contact. Maturation of LC is accompanied by morphological alterations. Applying a differential screening procedure we isolated differentially expressed cDNAs involved in the maturation events including cDNAs of the cytoskeletal actin isoforms beta- and gamma-actin. Stronger signals with hybridization probes derived from cultured LC compared with probes derived from freshly isolated LC indicate upregulation of actin expression. Upregulated expression of actin was confirmed by RT-PCR, Western blot and immunofluorescence analysis. Staining with fluorescence-labelled phalloidin that selectively binds to polymerized F-actin, indicates an increase in F-actin levels in cultured LC. Thus our data show that maturation of LC, which involves formation of dendritic structures and movement of formerly immobile cells, is accompanied by augmented expression of actin and formation of additional actin filaments. Furthermore, actin mRNA, often used as reference to assess mRNA amounts for Northern blotting or competitive RT-PCR because of its high and ubiquitous expression, is an inappropriate standard for the analysis of LC and DC.

Mouse langerhans cells differentially express an activated T cell-attracting CC chemokine.

Epidermal Langerhans cells represent an immature population of dendritic cells, not yet able to prime naïve T cells. Following in vitro culture Langerhans cells mature into potent immunostimulatory cells. We constructed a representative cDNA library of in vitro matured murine Langerhans cells. Applying a differential screening procedure 112 differentially expressed cDNA clones were isolated. Thirty-six clones represented cDNA fragments of the same gene, identifying it to be the most actively expressed gene induced in maturing Langerhans cells. A full-length cDNA was sequenced completely. The open reading frame codes for a protein of 92 amino acids containing a leader peptide of 24 amino acids, yielding a mature protein of 7.8 kDa molecular weight. Database searches revealed 99.4% sequence identity on the nucleotide level to the recently described mouse CC chemokine ABCD-1, as well as 74% sequence identity to the human CC chemokine, the macrophage-derived chemokine/stimulated T cell chemotactic protein. Expression was analyzed by reverse transcriptase-polymerase chain reaction on a large panel of cell types. Unlike the macrophage-derived chemokine, expression was not detected in macrophages stimulated by various cytokines. Expression is restricted to cultured Langerhans cells, in vitro cultured dendritic cells, and lipopolysaccharide-activated B cells. Recombinant protein was expressed in the yeast Pichia pastoris and purified to homogeneity. Whereas no chemotactic activity was observed in chemotaxis assays for naïve T cells, B cells, cultured dendritic cells, and Langerhans cells, a strong chemoattractant activity was exerted on activated T cells. Thus, production of this chemokine by dendritic cells may be essential for the establishment and amplification of T cell responses.

Nonradioactive detection of differentially expressed genes using complex RNA or DNA hybridization probes.

The analysis of differential gene expression has become increasingly important in recent years. Typically, differentially expressed genes are identified in a primary screening procedure, yielding candidate genes whose differential expression has to be verified. We provide a highly sensitive, efficient and nonradioactive differential screening procedure to analyze numerous candidate genes in a single step. This comprises labeling of poly(A)+ RNA of the cell types analyzed with DIG Chem-Link and differential hybridization to the candidate genes fixed on dot blots. DIG Chem-Link allows, to our knowledge, for the first time efficient and direct nonradioactive labeling of RNA in vitro. Advantages of this method include extremely short exposure times and the feasibility to re-use the probes after prolonged storage. Using this procedure, we isolated several genes that are differentially expressed in maturing Langerhans cells.

The actin-bundling protein fascin is involved in the formation of dendritic processes in maturing epidermal Langerhans cells.

Dendritic cells (DC) are characterized by their unique potential to prime naive T cells. Epidermal Langerhans cells (LC), the DC resident in the epidermis, gain this immunostimulatory capacity following Ag contact in vivo or during in vitro culture of epidermal cell suspensions. To analyze differential gene expression in maturing LC, we constructed a highly representative cDNA library of cultivated LC (cLC) in lambda ZAP II containing 18 x 10(6) independent clones. This library was screened with freshly isolated Langerhans cell (fLC)- and cLC-derived probes for cLC-specific cDNAs. The cDNAs identified were sequenced and analyzed by database searches. Two cDNA fragments were identified as fragments of fascin, indicating that fascin is differentially expressed in LC. By competitive RT-PCR, we confirmed that fascin is highly expressed in cLC cultivated for 1, 2, and 3 days, while no signals were obtained with fLC. Western blot and immunofluorescence analysis revealed cLC-specific expression of fascin on the protein level as well. Fascin is known to be involved in the organization of the actin cytoskeleton in cytoplasmatic extensions of nerve growth cones. Its differential expression in maturing LC coincides with the formation of numerous dendritic projections in LC. Their formation was inhibited by incubation of LC with fascin antisense oligonucleotides during cultivation. Therefore, we conclude that fascin is necessary for the formation of the dendritic processes of maturing Langerhans cells and may thus influence T cell-LC interaction.