Tumoricidal Activity of Monocyte-Derived Dendritic Cells: Evidence for a Caspase-8-Dependent, Fas-Associated Death Domain-Independent Mechanism 1 Nathalie Vanderheyde,* Ezra Aksoy,* Zoulikha Amraoui,* Peter Vandenabeele, † Michel Goldman,* and Fabienne Willems 2 * Monocyte-derived dendritic cells (DC) were found to be cytotoxic for several tumor cell lines including Jurkat cells, which were killed through a calcium-independent pathway. K562 cells were resistant, excluding a NK cell-like activity. DC-mediated apoptosis did not involve classical death receptors because it was not reversed by blocking TNF/TNFR, CD95/CD95 ligand, or TNF-related apoptosis-inducing ligand/TNF-related apoptosis-inducing ligand receptor interactions. Fas-associated death domain-deficient, but not caspase-8 deficient, Jurkat cells were killed by DC. Indeed, caspase-8 cleavage was demonstrated in Jurkat cells cocultured with DC, and the use of specific caspase inhibitors confirmed that apoptosis triggered by DC was caspase-8 dependent. Further- more, the involvement of Bcl-2 family members in the control of DC-mediated apoptosis was demonstrated by Bid cleavage in Jurkat cells cocultured with DC and resistance of Jurkat cells overexpressing Bcl-2 to DC-mediated cytotoxicity. Overall, these data indicate that monocyte-derived DC exert a caspase-8-dependent, Fas associated death domain-independent tumoricidal activity, a finding that could be relevant to their therapeutic use in cancer. The Journal of Immunology, 2001, 167: 3565–3569. E ndowed with a large array of specialized properties, den- dritic cells (DC) 3 are an essential link between the innate and adaptative immune responses. Besides their potent Ag-presenting function, DC were more recently found to act as killer cells in several in vitro expriments. In mice, Su ¨ss and Short- man (1) have demonstrated that a subset of spleen DC expressing CD8 are able to kill CD4 + T lymphocytes through the expression of CD95 ligand (CD95L). A CD95L-mediated killing was also described in murine bone marrow-derived DC (2). Two different types of cytotoxicity were discovered in the rat splenic DC pop- ulation. Indeed, cultured splenic DC exhibit a Ca 2+ -dependent NK-like cytotoxicity, whereas a subset of freshly extracted splenic DC display an intrinsic killing property through a Ca 2+ -indepen- dent mechanism (3, 4). In human DC, TNF-related apoptosis-in- ducing ligand (TRAIL) was detected on the surface of IFN-- or IFN--stimulated CD11c + blood DC subset, which enables them to kill TRAIL-sensitive target cells (5). Type I IFNs, in combina- tion with GM-CSF, were also shown to promote monocyte differ- entiation into TRAIL-expressing DC (6). Functional TRAIL pro- duction was found in GM-CSF/IL-4 monocyte-derived DC after IFN- treatment or measles virus infection (7, 8). All these data suggest an important role for DC in the killing of tumor cells. Human DC generated from peripheral blood monocytes in GM- CSF and IL-4 constitute a promising source of vaccines in antitu- mor immunotherapy. Recently, several clinical trials are investi- gating the effect of monocyte-derived DC in induction of efficient cell-mediated immune response against cancer (9, 10). In this con- text, we were interested in analyzing the cytotoxic potential of human monocyte-derived DC against a range of tumor cell lines. Materials and Methods Culture medium and reagents Culture medium consisted of RPMI 1640 (BioWhittaker, Verviers, Bel- gium) supplemented with 2 mM L-glutamine, 20 g/ml gentamicin, 50 M 2-ME, 1% nonessential amino-acids, and 10% FCS (BioWhittaker). The rIL-4 was kindly provided by Schering-Plough (Kenilworth, NJ), and rGM-CSF was obtained from Novartis (Basel, Switzerland). Agonistic anti- human CD95 mAb (clone CH11) was purchased from Immunotech (Mar- seille, France), recombinant human TRAIL from R&D Systems (Abing- don, U.K.), neutralizing monoclonal anti-human TNF- from BioSource International (Nivelles, Belgium), antagonistic anti-human CD95 mAb (clone ZB4) from Immunotech, and recombinant human TRAILR2-Fc chi- mera from R&D Systems. The caspase inhibitors benzyloxycarbonyl-Val- Ala-Asp-fluoromethylketone (zVAD-fmk) and z-Ile-Glu-Thr-Asp-flu- oromethylketone (zIETD-fmk) were purchased from Enzyme Systems Products (Livermore, CA). EGTA and MgCl 2 were purchased from Sigma (Bornem, Belgique). LPS from Escherishia coli (0128:B12) was also pur- chased from Sigma. Recombinant human IFN- was purchased from Sher- ing-Plough, recombinant human IFN-1a was kindly provided by G. J. van Daal (Serono, Benelux, Den Haag, The Netherlands), and recombinant human IFN- was purchased from R&D Systems. Generation of monocyte-derived DC DC were generated from the adherent fraction of PBMC cultured for 7 days in GM-CSF (800 U/ml) and IL-4 (500 U/ml) as described by Romani et al. (11). As we have previously reported (12), the DC-enriched fraction ob- tained according to this protocol routinely contains 95% DC. In some experiments, DC were stimulated for 24 h with either LPS (1 g/ml), or *Laboratory of Experimental Immunology, Universite ´ Libre de Bruxelles, Brussels, Belgium; and † Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology, University of Ghent, Ghent, Belgium Received for publication June 1, 2001. Accepted for publication July 10, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported by the Centre de Recherche Interuniversitaire en Vacci- nologie sponsored by the re ´gion Wallonne and Glaxo SmithKline Beecham Biologi- cals (Belgium) and by the Po ˆ le d’Attraction Interuniversitaire, the Fonds voor Weten- schappelijk Onderzoek-Vlaanderen (Grant 3G000601), and EC-RTD Grant QLRT- 199-00739. N.V. was supported by the Fonds National de la Recherche Scientifique- Te ´le ´vie program (Belgium). 2 Address correspondence and reprint requests to Dr. Fabienne Willems, Laboratory of Experimental Immunology, Universite ´ Libre de Bruxelles, 808 Route de Lennik, B-1070 Brussels, Belgium. E-mail address: fwillems@ulb.ac.be 3 Abbreviations used in this paper: DC, dendritic cell; TRAIL, TNF-related apoptosis- inducing ligand; CD95L, CD95 ligand; FADD, Fas-associated death domain; zVAD- fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; zIETD-fmk, z-Ile-Glu- Thr-Asp-fluoromethylketone; DIOC 6 , 3,3'-dihexylocarbocyanine iodide; PI, propidium iodide;  m , mitochondrial transmembrane potential; RIP, receptor-inter- acting protein; tBid, 15-kDa truncated form of Bid. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00