Specific stabilization of the 4F7 molecule on dendritic cells by contact allergens.

Our laboratory has recently developed the monoclonal antibody 4F7 which recognizes a molecule on dendritic cells in the dermis of mice that is upregulated after application of contact allergens in vivo. Furthermore, this antibody detects an antigen on dendritic cells in spleen, lymph nodes and colon. In order to study the influence of contact allergens on the surface expression of the 4F7 molecules on dendritic cells, FACScan analysis of splenic dendritic cells was carried out after in vitro application of contact allergens. Freshly isolated splenic dendritic cells were found to be positive for 4F7, 33D1, N418 (CD11c) and MHC class II. After overnight culture the expression of the dendritic…

CD83+ autologous human dendritic cells transfected with tumorpeptide CDNA: A potential tool for gene-immunotherapy?

Multiple levels of MHC class I down-regulation by ras oncogenes.

A number of tumours and oncogene transformed cells displayed reduced MHC class I surface expression which seemed to enable their escape from immune surveillance. To test whether oncogenic activation is directly involved in suppressing MHC class I expression, a model of inducible oncogene expression was chosen. Mouse fibroblasts transfected with different oncogenes expressed under the control of the dexamethasone-inducible MMTV promoter were analysed in the presence and absence of hormone for the mRNA and protein expression of MHC class I molecules as well as the respective oncogenes. Immunofluorescence analyses demonstrated an inverse association of MHC class I and oncogene expression after…

Direct evidence that FK506 inhibition of FcεRI-mediated exocytosis from RBL mast cells involves calcineurin

eRI-mediated exocytosis of preformed mediators from mast cells and basophils (e.g. histamine, serotonin, beta-hexosaminidase) is sensitive to the immunosuppressants cyclosporin A and FK506 (IC 50 200 and 4 n M , respectively) but not rapamycin. The mechanism of inhibition does not appear to involve tyrosine phosphorylation, hydrolysis of inositol phosphates or calcium flux. Here we report experiments using a molecular approach to assess the role of calcineurin, a serine/threonine phosphatase thought to be the primary pharmacological target of these drugs. Calcineurin’s activity requires association of its catalytic (A) subunit with an intrinsic regulatory (B) subunit. We hypothesized that c…

CD83+ human dendritic cells transfected with tumor peptide cDNA by electroporation induce specific T-cell responses: A potential tool for gene immunotherapy.

Dendritic cells (DC) are the most potent immunostimulatory cells, with the capacity to induce primary T-cell responses. Functional autologous DC can be generated from fetal calf serum-free peripheral blood mononuclear cells in the presence of interleukin-4 and granulocyte-macrophage colony-stimulating factor and are stimulated with a defined cytokine cocktail for terminal maturation. We were able to establish a nonviral transfection protocol for these DC by electroporation. Using enhanced green fluorescent protein as a reporter gene, we achieved transfection efficiencies of up to 10%. FACScan analyses revealed a stable phenotype, and the expression of major histocompatibility complex class …

Down-regulation of the MHC class I antigen-processing machinery after oncogenic transformation of murine fibroblasts

Malignant transformation is often associated with genetic alterations providing tumor cells with mechanisms for escape from immune surveillance. Human and murine tumors of various origin as well as in vitro models of viral and oncogenic transformation express reduced levels of major histocompatibility complex (MHC) class I antigens resulting in decreased sensitivity to MHC class I-restricted cytotoxic T lymphocyte (CTL)-mediated lysis. We here investigate whether the suppressed MHC class I surface expression of ras-transformed fibroblasts is due to dysregulation of the genes of the antigen-processing machinery, the peptide transporters TAP-1 and TAP-2 and the proteasome subunits LMP-2 and L…

Epidermal Cells Enhance Interleukin 4 and Immunoglobulin E Production After Stimulation with Protein Allergen

Exposure to certain allergens via epithelial tissues is the primary route for tile induction of immunoglobulin E–dependent allergies of the immediate type associated with atopic diseases. In order to address the question whether and how epithelial cells might contribute to the induction or increase of T H2 -dependent IgE production, we performed co-culture experiments of syngeneic epidermal cells and cells from the associated lymphoid tissue or spleen (responder cells) of BALB/c mice primed with ovalbumin in vivo . In the presence of ovalbumin in vitro , immunoglobulin E but not immunoglobulin G 2a production was significantly enhanced by the addition of epidermal cells, and separation of e…