Transfection of immature murine bone marrow-derived dendritic cells with the granulocyte-macrophage colony-stimulating factor gene potently enhances their in vivo antigen-presenting capacity.

Ag presentation by dendritic cells (DC) is crucial for induction of primary T cell-mediated immune responses in vivo. Because DC culture from blood or bone marrow-derived progenitors is now clinically applicable, this study investigated the effectiveness of in vitro-generated murine bone marrow-derived DC (Bm-DC) for in vivo immunization protocols. Previous studies demonstrated that GM-CSF is an essential growth and differentiation factor for DC in culture and that in vivo administration of GM-CSF augments primary immune responses, which renders GM-CSF an attractive candidate to further enhance the effectiveness of DC-based immunotherapy protocols. Therefore, immature Bm-DC were transiently transfected with the GM-CSF gene and tested for differentiation, migration, and Ag-presenting capacity in vitro and in vivo. In vitro, GM-CSF gene-transfected Bm-DC were largely unaltered with regard to MHC and costimulatory molecule expression as well as alloantigen or peptide Ag-presenting capacity. When used for in vivo immunizations, however, the Ag-presenting capacity of GM-CSF gene-transfected Bm-DC was greatly enhanced compared with mock-transfected or untransfected cells, as determined by their effectiveness to induce primary immune reactions against hapten, protein Ag, and tumor Ag, respectively. Increased effectiveness in vivo correlated with the better migratory capacity of GM-CSF gene-transfected Bm-DC. These results show that GM-CSF gene transfection significantly enhances the capacity of DC to induce primary immune responses in vivo, which might also improve DC-based vaccines currently under clinical investigation.

Generation of tumor immunity by bone marrow-derived dendritic cells correlates with dendritic cell maturation stage.

Bone marrow-derived dendritic cells (BmDC) are potent APC and can promote antitumor immunity in mice when pulsed with tumor Ag. This study aimed to define the culture conditions and maturation stages of BmDC that enable them to optimally function as APC in vivo. BmDC cultured under various conditions (granulocyte-macrophage CSF (GM-CSF) or GM-CSF plus IL-4 alone or in combination with Flt3 ligand, TNF-alpha, LPS, or CD40 ligand (CD40L)) were analyzed morphologically, phenotypically, and functionally and were tested for their ability to promote prophylactic and/or therapeutic antitumor immunity. Each of the culture conditions generated typical BmDC. Whereas cells cultured in GM-CSF alone were functionally immature, cells incubated with CD40L or LPS were mature BmDC, as evident by morphology, capacity to internalize Ag, migration into regional lymph nodes, IL-12 secretion, and alloantigen or peptide Ag presentation in vitro. The remaining cultures exhibited intermediate dendritic cell maturation. The in vivo Ag-presenting capacity of BmDC was compared with respect to induction of both protective tumor immunity and immunotherapy of established tumors, using the poorly immunogenic squamous cell carcinoma, KLN205. In correspondence to their maturation stage, BmDC cultured in the presence of CD40L exhibited the most potent immunostimulatory effects. In general, although not entirely, the capacity of BmDC to induce an antitumor immune response in vivo correlated to their degree of maturation. The present data support the clinical use of mature, rather than immature, tumor Ag-pulsed dendritic cells as cancer vaccines and identifies CD40L as a potent stimulus to enhance their in vivo Ag-presenting capacity.

Ultraviolet light-induced immune tolerance is mediated via the Fas/Fas-ligand system.

Hapten sensitization through UV-exposed skin induces tolerance that is mediated via the induction of hapten-specific T suppressor cells. However, the detailed mechanisms underlying tolerance induction remain unclear to date. We show here that the apoptosis-related surface Ag Fas (APO-1, CD95) and its ligand, Fas ligand (FasL) are critically involved, since Fas-deficient lpr mice and FasL-deficient gld mice do not develop UV-induced tolerance. Adoptive transfer experiments revealed that the mediation of tolerance does not require the expression of Fas or FasL by the T suppressor cells but does require the expression of both molecules by the cells of mice receiving the T suppressor cells. To identify the mechanisms involved, the effect of suppressor cells on Ag-presenting dendritic cells (DC) was studied. Coincubation of hapten-pulsed DC with T cells that were obtained from UV-tolerized mice resulted in an enhanced death rate of DC, and this cell death was dependent upon Fas expression. The addition of IL-12, which recently was found to break established tolerance in vivo, prevented DC death. Moreover, IL-12 did not only rescue DC from T suppressor cell-induced death but also from apoptosis induced by rFasL, suggesting that IL-12 may interfere with the Fas/FasL system. Together, these data indicate a crucial role for the Fas/FasL system in UV-induced tolerance, and suggest that UV-induced T suppressor cells may act by inducing the cell death of APCs via the Fas pathway. The ability of IL-12 to break established tolerance may be due to the prevention of DC death induced by T suppressor cells.