Abstract

Dendritic cells (DCs) are characterized by their unique capacity for primary T cell activation, providing the opportunity of DC-based cancer vaccination protocols. Recently, we defined a novel major subset of human blood DCs by using the monoclonal antibody M-DC8 which recognizes a carbohydrate modification of P selectin glycoprotein ligand-1 (PSGL-1) selectively expressed on these cells (

Immunity
2002
;
17
:
289
-301
). In addition to a marked capacity to activate tumor-reactive cytotoxic T cells M-DC8+ DCs efficiently mediate antibody-dependent cytotoxicity (
Blood
;
2002
;
100
:
1502
-1504
). In the present study, we analyzed the capacity of M-DC8+ DCs to kill tumor cells in the absence of antibodies and to enhance the tumor-directed cytotoxicity of NK cells. To determine whether M-DC8+ DCs exhibit tumoricidal activity, DCs were isolated at high purity (>93%) from the blood of healthy donors by immunomagnetic separation. These cells were cultured for 6 h in the presence or absence of 200 U/ml interferon (IFN)-gamma. Subsequently, DCs were coincubated with four chromium-labeled tumor cell lines and two normal cell lines for 18 h. Whereas unstimulated DCs demonstrated only moderate tumor-directed cytotoxicity (specific lysis: 7–13%), IFN-gamma-stimulated M-DC8+ DCs displayed potent killing of each of these tumor cell lines (specific lysis: 27–35%). Only a marginal cytotoxic effect was seen when normal human cells such as lung fibroblasts or endothelial cells were used as targets. When evaluating the cytotoxic effector mechanisms FACS analysis and ELISA assays revealed that IFN-gamma-stimulated M-DC8+ DCs secreted a high amount of tumor necrosis factor (TNF)-alpha induced by direct cell-to-cell contact with the different tumor cell lines. This effect was already observed after 3 h of cocultivation. Interestingly, no significant induction of TNF-alpha was detected during contact of M-DC8+ DCs with the normal human cell lines. These results suggest that tumor-associated surface molecules are important for the observed increase of TNF-alpha production in M-DC8+ DCs. Inhibition experiments with neutralizing antibodies clearly demonstrated that tumor cell-induced TNF-alpha play an important role in tumor-directed cytotoxicity mediated by M-DC8+ DCs. To investigate whether M-DC8+ DCs enhance the tumoricidal activity of NK cells freshly isolated DCs were cultured for 6 h in the presence or absence of IFN-gamma. Thereafter, DCs were coincubated with highly enriched (>90%) NK cells. The cytotoxic potential of the stimulated NK cells was tested towards various tumor cell lines in a 4 h chromium release assay. We observed a two- to threefold increase of NK cell-mediated cytotoxicity towards all analyzed tumor cell lines by IFN-gamma-stimulated M-DC8+ DCs. In addition, transwell experiments demonstrated that this triggering effect was mainly dependent on cell-to-cell contact. In conclusion, our data provide evidence that a major subpopulation of circulating human blood DCs exhibits efficient tumoricidal activity and clearly enhances NK cell-mediated tumor-directed cytotoxicity. The capacity of DCs to induce tumor-specific T cell responses and to kill tumor cells either directly or by activating NK cells points to the pivotal role of DCs in triggering the innate and adaptive immune response against tumors.

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