Dendritic cells directly trigger NK cell functions: Cross-talk ... - Nature

© 1999 Nature America Inc. • http://medicine.nature.com

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Dendritic cells directly trigger NK cell functions: Cross-talk relevant in innate anti-tumor immune responses in vivo NADINE C. FERNANDEZ1, ANNE LOZIER1, CAROLINE FLAMENT1, PAOLA RICCIARDI-CASTAGNOLI2, DOMINIQUE BELLET1, MARK SUTER3, MICHEL PERRICAUDET4, THOMAS TURSZ, EUGENE MARASKOVSKY5,6 & LAURENCE ZITVOGEL1 Unité d’Immunologie, Département de Biologie Clinique and 4CNRS-UMR1582, Institut Gustave Roussy, Villejuif, France 2 University of Milano-Bicocca, Department of Biotechnology and Bioscience, Milan, Italy 3 University Institut for Virology, Zürich, Switzerland 5 Department of Immunobiology, Immunex Corporation, Seattle, Washington, 98101-2936, USA 6 Ludwig Oncology Unit, Austin and Repatriation Medical Center, Heidelberg, Australia Correspondence should be addressed to L.Z.; email: [email protected]


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Cytotoxic T lymphocytes and natural killer cells are essential effectors of anti-tumor immune responses in vivo. Dendritic cells (DC) ‘prime’ tumor antigen-specific cytotoxic T lymphocytes; thus, we investigated whether DC might also trigger the innate, NK cell-mediated anti-tumor immunity. In mice with MHC class I-negative tumors, adoptively transferred- or Flt3 ligand-expanded DC promoted NK cell-dependent anti-tumor effects. In vitro studies demonstrated a cellto-cell contact between DC and resting NK cells that resulted in a substantial increase in both NK cell cytolytic activity and IFN-γ production. Thus, DC are involved in the interaction between innate and adaptive immune responses.

Approaches to active cell-mediated immunotherapy of malignant diseases rely on the demonstration of autologous CD8+ cytotoxic T lymphocytes (CTL). They also include the activation of the innate arm of anti-tumor immunity; that is, natural killer (NK) or NKT effector cells. Dendritic cells (DC) are essential antigen-presenting cells that are sentinels of the immune system. Antigen-pulsed DC prime MHC class II- (refs. 1,2) and class I(refs. 3,4) restricted antigen-specific T cells in vivo. After being activated by inflammatory stimuli or ‘danger’ signals, they migrate to T cell-enriched areas of secondary lymphoid organs where they foster the egress of activated, antigen-specific T lymphocytes into the periphery as effectors5. DC might interact with effectors of innate immunity at various ports of entry. NKT cells are important effectors in tumor rejection6. Moreover, glycosylceramide-loaded DC are essential for inducing activation of NKT cells mediated by T-cell receptors7. Here we determined whether DC might be involved in nonMHC-restricted anti-tumor immunity by directly activating NK cells. Indeed, NK cells participate in the innate immune response against transformed cells in vivo8,9. Although expression of MHC class I molecules on antigen-presenting cells negatively regulates NK cell effector functions10,11, it is still unclear what initiates the activation of NK cells in vivo. DC-derived cytokines such as type I IFNs or IL-12 are essential in initiating the activation of NK cells in response to pathogens12,13. Our understanding of how DC and NK cells are functionally coordinated to provide resistance against tumor spreading remains incomplete. To investigate the role of DC in NK cell-dependent anti-tumor immune responses, we took advantage of the ability of Flt3 ligand (FL) to expand the number of DC (ref. 14) and to enhance the generation of NK cells in vivo15. First, we demonstrated that NK cells are unique effectors responsible for the FL-induced reNATURE MEDICINE • VOLUME 5 • NUMBER 4 • APRIL 1999

gression of MHC class I-negative tumors. We next investigated the mechanisms by which FL mediates NK cell-dependent tumoricidal activity in nude or Rag–/– mice. IL-12 and type I IFNs did not seem to be involved, whereas lymphoid-related DC were essential for FL-mediated NK cell-dependent anti-tumor effects. We formally demonstrated in vitro that DC are able to activate resting NK cells. DC-to-NK cell contact is necessary to trigger NK cell cytolytic activity and IFN-γ production. Finally, we showed that adoptive transfer of DC into the tumor site induces a significant NK cell-mediated tumor growth delay. Thus, DC initiate NK cell activation, resulting in innate anti-tumor immune responses in vivo. FL mediates NK cell-dependent tumor regression We used as a tumor model system the non-immunogenic mouse mesothelioma16 (AK7) that expresses low or undetectable levels of MHC class I and class II molecules in vitro (data not shown). Although FL showed no direct cytostatic or cytotoxic effect on AK7 cells in vitro (data not shown), systemic administration of FL in mice with AK7 tumors established for 20 days induced transient suppression of tumor growth, resulting in a significant growth delay (P < 0.05) in vivo. The FL-mediated anti-tumor effects observed in the B6-Rag–/– genetic background (Fig. 1a) and in nude mice (Fig. 3b) were similar to those achieved in immunocompetent mice (Fig. 1c), emphasizing that T cells were not required in the tumor growth delay. To investigate the role of NK cells in the FL-mediated antitumor effects, we used FL to treat B6-beige mice (which are deficient in NK cell functions) bearing established AK7 tumors. These mice had progressively growing mesotheliomas that were ultimately lethal despite FL administration (Fig. 1b). To further assess the involvement of NK cells in the FL-mediated tumorici405


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Fig. 1 FL induces NK cell-dependent anti-tumor effects in an MHC class Inegative mesothelioma. a and b, Tumor growth kinetics in B6-Rag–/– (a) and B6-beige mice (b) with established AK7 tumors injected daily for 20 days with FL or PBS. c, Co-administration of FL and depleting monoclonal antibody against NK1.1 abrogates FL-mediated anti-tumor effects. Immunocompetent B6-mice with AK7 tumors were injected daily with FL for 20 days. Depleting monoclonal antibody against NK1.1 or saline + 5%

mouse serum were administrated from day 8 to day 28 of FL treatment. Saline + 5% mouse serum did not significantly hamper FL efficacy in vivo. p, PBS; P, FL; G, FL + antibody against NK1.1. *, P < 0.05: significantly smaller tumors in mice treated with FL plus mouse serum compared with the mice depleted of NK1.1+ cells. Tumor sizes (mean ± s.e.m.) from the start of FL treatment are plotted. These experiments were done at least twice with identical results.

dal activity, we depleted immunocompetent B6 mice using monoclonal antibody against NK1.1. This depletion was sufficient to completely abrogate FL-mediated anti-tumor effects (Fig. 1c). AK7 had a similar tumor growth kinetics in beige mice, immunocompetent littermates and mice depleted of NK1.1+ cells. Thus, FL-induced effector cells are the only NK cells in this tumor model system. Transient complete tumor eradication could be achieved using a combination of FL with NK cell stimulatory factors (IL-12 or IL-15) (data not shown).

hibit the FL-induced NK cell-dependent anti-tumor effects (data not shown). Therefore, FL-expanded DC may be involved in these NK cell-mediated anti-tumor effects in which essential soluble mediators are not involved in vivo.

Type I IFNs and IL-12 are not involved Because type I IFNs and IL-12 are NK cell stimulatory cytokines9,17 secreted during infection or tumor development, we investigated the role of these soluble mediators in the FL-mediated NK cell-dependent anti-tumor effects. Despite slower AK7 growth kinetics in type I IFN receptor and Rag2 double-knockout mice (AR129), FL still promoted significant (P < 0.05) tumor growth delay (Fig. 2a). Although lymph node- or spleen-derived mononuclear cells collected from FL-treated mice did not secrete IL-12 in vitro (data not shown), we injected mice with neutralizing monoclonal antibody against p40 IL-12 from day 5 to day 17 of FL therapy. The FL-mediated anti-tumor effects were not hampered by the neutralization of endogenous IL-12 (Fig. 2b). Moreover, administration of the fusion chimeric protein CTLA4Ig or a blocking monoclonal antibody against B7.2 or a neutralizing monoclonal antibody against IFN-γ could not in-

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Fig. 2 Neither type I IFNs nor IL-12 are involved in FL-mediated anti-tumor effects. a, FL-mediated anti-tumor effects in type I IFN receptor and Rag2 double-knockout mice. AR129 mice with AK7 tumors were injected daily for 20 days with FL. p, PBS; P, FL. b, Coadministration of neutralizing monoclonal antibody against p40 IL-12 in vivo, in B6-nude mice, from day 5 to day 17 of FL treatment. p, PBS; P, FL + isotype-matched monoclonal antibody; G, FL + antibody against p40 IL-12. Tumor sizes (mean ± s.e.m.) after day 1 of FL treatment are plotted. These data were reproduced twice with similar results.

Involvement of the lymphoid-related DC subset The anti-tumor effects were considerable by day 10 of FL therapy, when splenomegaly and adenomegaly, related to the extramedullary hematopoiesis, were observed14 (data not shown). The kinetics of tumor growth returned to baseline 5–10 days after FL treatment ended, when the number of DC decreased. Thus, we next determined whether FL-expanded myeloid- or lymphoid-related DC were involved in the NK cell-dependent anti-tumor effects. We selectively depleted the lymphoid CD8α+ DC (ref. 18) using monoclonal antibody against mouse CD8α in B6-nude mice. This depleting monoclonal antibody was injected with FL from day 0 to day 28. The proportion of NK cells (DX5+) did not change after depletion with monoclonal antibody against CD8α, whereas the population co-expressing CD11c, I-Ab and CD8α was nearly undetectable in the depleted mice (Fig. 3a). The proportion of macrophages (F4/80+) and B cells (B220+) were not significantly modified, whereas the percentage of the subset CD11c+/I-Ab+ and CD8α– was slightly increased after depletion (data not shown). Thus, the depletion targeted only the lymphoid-related DC subset and not NK cells. Mice with AK7 tumors that were depleted with monoclonal antibody against CD8α re-



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ARTICLES Fig. 3 Role of the lymphoid-related DC subset. a, Distribution of DX5 and CD8a expression on CD11c and MHC class II-positive spleen cells (gated population R2) on splenocytes from B-6-nude mice treated for 10 days with either FL (thick line) or FL plus monoclonal antibody against CD8α (thin DX5 PE line). b, In vivo depletion studies using monoclonal antibody against CD8α or CD4. B6-nude mice with tumors were treated with PBS (p), FL alone (P), or with FL and monoclonal antibody against CD8α (G) or CD4 (g). *, P < 0.05: significantly smaller tumors in the undepleted mice treated with FL compared with the mice injected with monoclonal antibody against CD8α. Tumor sizes (mean ± s.e.m.) after day 1 of FL treatment are plotted. These data were reproduced twice with similar results.

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DC directly trigger NK cell functions in vitro We next determined whether mouse DC directly activate resting NK cells in vitro. We investigated the effects of bone marrowderived DC (BM-DC) or the D1 dendritic cell line19 on NK cell cytolytic activity and IFN-γ production. As described19, flow cytometry analyses showed a homogeneous population of cells expressing increased levels of MHC class II (I-Ab), CD86 and CD40 molecules after TNF-α stimulation compared with that expressed by unstimulated D1 cells. Loosely adherent BM-DC, cultured in complete medium containing granulocyte/monocyte colony-stimulating factor (GM-CSF) and IL-4, expressed high levels of CD11c, MHC class I and II, CD86 and CD40 molecules, and were negative for CD3 and B220 expression3. When allogeneic resting NK cells were co-cultured with untreated or TNF-α stimulated-D1 cells, up to 70% specific lysis of the resulting co-culture against YAC-1 cells was achieved at an effector:target ratio of 20:1 (Fig. 4a). Neither cell population cultured separately at similar concentrations yielded substantial


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YAC-1 cell lysis. Similarly, allogeneic (Fig. 4b) or syngeneic (Fig. 5d) NK cells were induced to kill YAC-1 cells when co-cultured with BM-DC derived from B6 or BALB/c mice. Freshly isolated NK cells showed at most 20% spontaneous YAC-1 cell lysis at an effector:target ratio of 100:1 (data not shown). Moreover, NK cells co-cultured with D1 cells acquired the ability to lyse not only MHC class I-negative tumor cell targets such as AK7 and MCA101, but also MHC class I-expressing tumor cells such as MCA205 (Fig. 4c). Nevertheless, substantial lysis of P815 mastocytoma cells could not be detected (Fig. 4c). Supernatants collected after 18 hours of NK/D1 co-cultures contained high levels of IFN-γ that decreased as a function of the number of stimulating D1 cells (Fig. 5a). IFN-γ was not detectable in the supernatants of D1 or NK cells cultured separately. Similar

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Fig. 4 DC directly stimulate NK cytolytic activity in vitro. Freshly isolated NK cells derived from SCID mice were co-cultured for 18h with immature D1 cells or D1 cells stimulated with TNF-α (a) or BM-DC derived from B6mice (GM-CSF/IL-4) (b) at a ratio of 0.5 NK cell to 1 DC. Viable lymphocytes were tested against YAC-1 cells in a chromium release assay. c, The cytolytic activity of viable lymphocytes against YAC-1, MCA205, AK7, NATURE MEDICINE • VOLUME 5 • NUMBER 4 • APRIL 1999

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sponded significantly less (P < 0.05) to FL treatment than their undepleted littermates or littermates injected with isotypematched monoclonal antibody against CD4 (Fig. 3b).

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MCA101 or P815 was assessed 40h after co-culture of resting NK cells with D1 cells stimulated with TNF-α. Results are expressed as a percentage of specific lysis at various effector:target ratios (a and b) or at a ratio of 25:1 (c) and represent the means of triplicate wells; s.e.m. were consistently less than 10% of means. Results are from one representative experiment of three to five. 407


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levels of IFN-γ were detected in supernatants of NK cell/BM-DC (GM-CSF/IL-4) co-cultures (data not shown). Mature dendritic cells secrete various cytokines20 that act in concert to activate NK cells. Type I IFNs, IL-1β and IL-12 were not found in supernatants of DC/NK cell co-cultures or in the D1 cell culture stimulated by TNF-α (data not shown). D1 cells were still capable of activating resting NK cells derived from type I IFN receptor and Rag2 double-knockout mice (AR129), as indicated by the detection of large amounts of IFN-γ in the supernatants of co-cultures (Fig. 5b). We cultured D1 and NK cells separately in transwells to determine the involvement of soluble factors in the in vitro NK cell triggering. DC-induced NK cell cytolytic activity (Fig. 5c) and IFN-γ secretion (data not shown) were detected only when NK cells were co-cultured in close contact with D1 cells but not when the cells were separated by a porous membrane, indicating that D1 cell-mediated NK cell activation requires intimate cell-to-cell contact. IL-15 might not be involved, as no IL15 bioactivity was found in the supernatant of DC/NK cell co-cultures (data not shown), and addition of exogenous IL-15 (50 ng/ml) was less effective for NK cell activation than the cellto-cell contact between DC and NK cell (Fig. 5c). Not all antigen-presenting cells activate NK cells Co-culturing resting NK cells with NK-sensitive target cells might trigger their cytolytic activity. We confirmed that BM-DC and D1 cells are not targets for resting NK cells, in contrast to YAC-1 cells (data not shown). Moreover, the co-culture of resting NK cells with YAC-1 or P815-B7.1 cells did not induce their cytotoxic activity (Fig. 5d). BM-DC cultured in GM-CSF alone21 and the adherent fraction of SCID mice-derived splenocytes, which consists mostly of macrophages (50% of F4/80- and CD11b-positive cells, as determined by FACS analysis), did not stimulate NK cells in vitro (Fig. 5d). 408

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Fig. 5 Cell contact requirement and specificity of the DC/NK cell interaction. a, IFN-γ secretion by DCactivated NK cells. Supernatants from immature D1 cells and allogeneic NK cells derived from BALB/c mice were cultured alone or together at various ratios were assayed for mIFN-γ, by ELISA. b, Type I IFN are not involved in the NK/DC crosstalk. AR129-derived NK cells and immature D1 cells were cultured alone or together. Supernantants were assayed for mIFN-γ, by ELISA. c, Cell contactdependent activation of NK cells by D1 cells. NK cells or D1 cells stimulated with TNF-α were cultured together in the same wells or separated by a porous membrane at a ratio of 0.5:1; rhIL-15 was added in the upper wells in parallel when NK cells were in the lower wells. Viable lymphocytes were tested against YAC-1 cells in a cytotoxicity assay. d, Neither targets nor other antigen-presenting cells are able to activate NK cells. NK cells were co-cultured with either BM-DC derived from BALB/c mice (GM-CSF/IL-4), YAC-1 cells, P815-B7.1 cells, BM-DC propagated with GM-CSF or macrophages derived from SCID mice. The lytic activity of the NK cells against YAC-1 cells was tested 18h later by chromium release assay. Results are expressed as a percentage of specific lysis at various effector:target ratios (c) or at a ratio of 50:1 (d) and represent the means of triplicate wells; s.e.m. were consistently less than 10% of means (c). Data represent one of three independent experiments achieving identical results.

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NK cell-dependent anti-tumor effects of DC transfer To assess the relevance of a direct cross-talk between DC and NK cells at the onset of innate anti-tumor immune responses, we injected 3 × 106 immature D1 cells twice a week for 3 weeks at the tumor sites of B6-nude mice with AK7 tumors established for 20 days. Fewer injected D1 cells or macrophages were not efficient in mediating anti-tumor effects (data not shown). Significant delays (P < 0.05) in tumor growth were achieved in mice treated with D1 cells compared with growth in mice injected with vehicle alone (Fig. 6a). These D1 cell-mediated anti-tumor effects were abrogated when NK1.1+ cells were depleted at the same time, using monoclonal antibody against NK1.1 (Fig. 6b). These results emphasize that DC-based immunotherapy may not only promote T cell-dependent anti-tumor immunity but also directly induce NK cell activation in vivo. Discussion These studies demonstrate direct activation of NK cell functions after DC/NK cell interactions in vitro and strongly support the idea that DC could initiate NK cell-mediated innate anti-tumor immune responses in vivo, indicating that DC are involved in the interaction between adaptive and non adaptive immunity. To our knowledge, very few studies have documented the ability of discrete cell types, such as B-EBV cell lines or in vitro-differentiated macrophages, to modulate NK cell activity22,23. Our results assign to DC a new role, as we show that these cells can trigger resting NK cell cytolytic activity against MHC class I-negative and -positive targets and IFN-γ secretion independently of exogenous cytokines. Here, we defined NK cells functionally as effector cells ‘licensed’ by DC to kill NK-sensitive tumor-cell targets. The possibility that murine DC might also represent effector cells has been eliminated, as murine DC do not express NK1.1 and are unNATURE MEDICINE • VOLUME 5 • NUMBER 4 • APRIL 1999


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able to kill NK-sensitive targets15. The co-culture of resting NK cells with B7- and CD40-expressing cells might result in the activation of their cytolytic activity24,25. However, we were able to exclude this possibility, because BM-DC and D1 cells were not targets for resting NK cells; NK cell targets such as YAC-1 or P815-B7.1 cells could not trigger NK cell cytolytic function; and other antigen-presenting cells such as macrophages or GM-CSF propagated BM-DC (expressing low levels of B7 and CD40 costimulatory molecules) could not activate resting NK cells. Moreover, the in vivo blockade of B7/CD28 interaction did not affect FL mediated-anti-tumor effects substantially, in accordance with previous observations26. Thus, our data show the unique capacity of DC to promote NK cell functions in vitro. The molecular mechanisms underlying DC/NK cell interaction remain to be elucidated. The stage of DC differentiation does not seem to be essential in triggering NK cell functions, as not only immature D1 cells or BM-DC (GM-CSF/IL-4) but also D1 cells stimulated by TNF-α (Fig. 4a) or LPS-treated BM-DC (GM-CSF/IL-4) (data not shown) were efficient in vitro. In vivo adoptive transfer of untreated D1 cells was efficient in promoting NK cell-dependent tumor growth retardation, although these D1 cells might mature in the tumor microenvironment. The DC lineage does not seem to discriminate, as in vivo studies indicated that lymphoid-related DC have an important role, whereas in vitro studies used myeloid-related DC. Abrogation of FL-mediated anti-tumor effects by selective depletion of CD8αexpressing DC was incomplete, indicating that both myeloidand lymphoid-related DC are involved in vivo. Alternative pathways of DC-induced NK cell activation may involve soluble mediators27,28. However, cell-to-cell contact between NK cells and DC is required in vitro; for this, type I IFNs and IL-12 are not essential. This does not preclude that other cytokines, such as IL-15 or IL-18, might be co-stimulatory in conjunction with the cellular interaction. However, so far only mature DC have been shown to secrete such NK cell stimulatory factors (ref. 29 and Scheicher, C. et al., Keystone Symposium on Cellular and Molecular Biology of Dendritic Cells, abstract 145, 1998). Our results indicate that the interaction between NK cells and DC involved membrane-associated determinants. It is generally accepted that the host immune status, particularly natural immune responses, is essential in controlling the dissemination and growth of metastatic tumors and therefore influences the prognosis of the tumor-bearing host. Few studies have reported a prominent role for NK cells on the growth of subcutaneous primary tumors in mice without using biological response modifiers. Some reports have suggested NK cells are involved in FL-mediated anti-tumor effects30,31. Our study has formally demonstrated that NK cells are the primary effectors responsible for the FL-mediated anti-tumor effects in a nonimNATURE MEDICINE • VOLUME 5 • NUMBER 4 • APRIL 1999

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Fig. 6 Adoptive transfer of D1 dendritic cells in B6-nude mice with AK7 tumors. a, Immature D1 cells (P) or saline vehicle (p) were injected twice each week for three weeks at the tumor site in B6-nude mice with AK7 tumors established for 20 days. l, control. *, P < 0.05: significantly smaller tumors in the mice injected with D1 cells compared with PBS-treated mice. b, Immature D1 cells were injected twice a week for three weeks at the tumor site in B6-nude mice with AK7 tumors established for 10 days, with co-administration of either depleting monoclonal antibody against NK1.1 (G) or saline + 5% mouse serum (P). l, control. *, P < 0.05: significantly smaller tumors in the mice treated with D1 cells compared with the mice depleted of NK1.1+ cells. Tumor sizes (mean ± s.e.m.) after day 1 of D1 cell inoculation are plotted. These experiments were done at least twice with identical results.

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munogenic, MHC class I-negative tumor model. An unusual lineage of lymphocytes, Vα14 NKT cells, is activated by DC pulsed with α-galactosylceramide7. Given that FL-mediated, NK cell-dependent anti-tumor effects were still observed in B6-Rag–/– mice and were IL-12-independent, a role for NKT cells or B lymphocytes in our model system is unlikely. The mechanism of NK celldependent AK7 killing is not clear. However, NK cells stimulated with DC in vitro became capable of lysing AK7. Moreover, administration of monoclonal antibody against mIFN-γ did not substantially hamper FL-mediated NK cell-dependent antitumor effects, precluding a prominent role for IFN-γ in AK7 tumor regression (data not shown). The substantial expansion of DC (ref. 14) and, to a lesser extent, NK cells15 in vivo after FL therapy allowed us to investigate a potential DC/NK cell interaction in vivo and its physiological relevance in the setting of tumor progression. In FL-treated animals, NK cell lytic activity in hematopoietic organs is enhanced15. Because mature NK cells do not express Flt3 (ref. 32), and FL has no effect on NK cell function 15, we could exclude the possibility of a direct activation of NK cells by FL therapy. We report here that NK cell-dependent anti-tumor immune responses are hampered considerably after depletion of the lymphoid-related DC subset. The idea of direct activation of NK cells by DC in vivo is strongly supported by our data showing, in an FL-free system, that intra-tumor inoculation of DC could significantly impair tumor progression in B6-nude mice in a NK cell-dependent manner. DC and NK cells might interact at the tumor site where they may be recruited and/or activated through chemokines and/or extracellular matrix components33,34. Innate immunity may control the development and the nature of adaptive immunity35. Tumor homologs of pathogen-associated molecular patterns may be recognized by pattern recognition receptors on DC that ‘license’ the DC to activate NK cells at the tumor site. The interactions between DC and NK cells in peripheral tissues may consequently result in tumor lysis, releasing apoptotic or necrotic bodies that will be uptaken, transported and presented by DC to T cells. DC, and therefore DC-based therapy, may modulate the interaction between innate and cognate immune responses. 409


ARTICLES Methods


Mice. Female C57BL/6 (B6), BALB/c, scid/scid (SCID), C57BL/6-bg/bg (B6beige), C57BL/6-B6-nude (B6-nude), C57BL/6 Rag2–/– (B6-Rag–/–) mice were purchased from the Centre d’élevage Janvier (Le Genest St Isle, France), the Centre d’élevage Iffa Credo (L’Arbresle, France), Harlan UK Limited (Oxon, UK), the Mollegaard Breeding & Research Centre A/S (Skensved, Denmark) and the Centre de Développement des Techniques Avancées du Centre National de la Recherche Scientifique (Orléans, France), respectively. 129Sv/Ev (H-2b) mice with deleted recombination activating gene 2 (ref. 36) were crossed with A129 mice37 to obtain homozygous AR129 (type I IFN receptor and Rag2 double-knockout). The altered genome of the newly bred mice was analyzed by PCR (refs. 36,37). AR129, SCID BALB/c, B6-beige and B6-nude mice were maintained in pathogenfree conditions. Cell lines. AK7, provided by A. Kane (Brown University, Providence, Rhode Island), is a murine mesothelioma cell line generated in B6 mice16. This cell line was maintained in DMEM supplemented with 10% FBS, 2 mM Lglutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. The D1 cell line is a splenic immature DC line of C57BL/6 background19; it was maintained in IMDM containing 10% endotoxin-free FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 5 × 10–5 M β-mercaptoethanol and supplemented with 30% R1-conditioned medium19. To induce maturation of D1 cells, recombinant mouse TNF-α (R&D Systems, Minneapolis, Minnesota) was added at a concentration of 10 ng/ml for 24 h. YAC-1 is an NK cell-sensitive Moloney virus-induced T-cell lymphoma of A/Sn background. P815 is a NK-resistant mastocytoma of DBA/2 background. P815B7.1 cells, a gift from P. Kourilsky (Institut Pasteur, Paris, France), were obtained by transfection of P1.HTR by the LL218 vector encoding murine B7.1. MCA205 and MCA101 are murine fibrosarcoma cell lines of B6 background, provided by M.T. Lotze (University of Pittsburgh, Pennsylvania). All cell culture media and reagents were obtained from Life Technologies. In vivo experimental settings. For FL treatment of tumor-bearing mice in vivo, mice were inoculated intradermally in the right flank with the minimal tumorigenic dose of AK7 (3 × 106 cells) tumor cells. Human FL derived from Chinese hamster ovary cells was provided by Immunex (Seattle, Washington). This cytokine was diluted in PBS at 100 µg/ml. AK7 tumors established for 20 days (about 20 mm2 in surface area: largest diameter × its perpendicular) were treated with one daily subcutaneous injection of either FL (10 µg) or vehicle for 20 consecutive days. For adoptive transfer of D1 cells in mice, B6-nude mice with AK7 tumors established for 20 days were injected twice each week at the tumor site with 3 × 106 immature D1 cells in PBS or vehicle alone for 3 weeks. For NK1.1+ cell depletion experiments, a similar protocol of D1 cell administration was used in B6-nude mice with AK7 tumors established for 10 days. Tumor growth was monitored twice each week and mice were killed when their tumors ulcerated. Tumor growth rates were determined by plotting the tumor size (mm2) versus time after day 1 of FL treatment or D1 cell inoculation. For in vivo antibody depletion experiments, monoclonal antibody against CD8α, clone YTS 191.1.2, monoclonal antibody against CD4, clone YTS 169.4.2.1 (both from S. Cobbold38) and monoclonal antibody against NK1.1, clone PK136 (HB-191; ATCC, Rockville, Maryland) were used. Hybridomas were grown in nude mice, and collected ascitis fluids were purified using classical procedures and were adjusted to 1 mg/ml in PBS. Depletion of CD8+ or CD4+ cells started at day 1 of FL treatment: 200 µg (per mouse) of monoclonal antibody was injected intraperitoneally for 3 consecutive days, then 300 µg was given every 3 days during the course of FL treatment. After FL treatment, the monoclonal antibodies were administrated twice, 4 days apart. Monoclonal antibody against NK1.1 (300 µg per mouse) was administrated intraperitoneally two days apart, from day 8 to day 28 of FL treatment, to immunocompetent B6 mice with tumors established for 20 days. For NK1.1+ cell depletion in D1 cell-treated mice, 300 µg (per mouse) of monoclonal antibody was injected intraperitoneally before and on the day of D1 cell inoculation. In NK cell depletion experiments, specific depletion greater than 95% was achieved. For endogenous mouse IL-12 neutralizing studies, purified monoclonal antibody against p40 mouse IL-12, clone C17-8, provided by G. Trinchieri (Wistar Institute, Philadelphia, Pennsylvania) was injected intraperitoneally at an individual dose of 300 µg 3 days apart, from day 5 to day 17 of FL therapy39. Isotype-matched mono410

clonal antibody or saline + 5% mouse serum (for NK1.1+ cell depletion) were injected as negative controls using the same protocols. All studies were done two to four times using individual treatment group of five mice. Flow cytometry analysis. Splenocytes from mice injected with FL or FL plus monoclonal antibody against CD8α were analyzed by FACS using a FACSCalibur (Becton Dickinson, Mountain View, California) on day 10 of FL treatment. Cells were collected after mechanical disruption of tissue and were stained using the following monoclonal antibodies: PE-labeled CD11c (PharMingen, San Diego, California), PE-labeled DX5 (NK cells) (PharMingen, San Diego, California), FITC-labeled anti-CD8α (Caltag, Burlingame, California) and biotin-conjugated anti-I-Ab (PharMingen, San Diego, California) followed by Tri-Color-conjugated Streptavidin (Caltag, Burlingame, California). We ensured that the FITC-labeled anti-CD8α staining was not blocked by the pre-incubation of cells with the YTS 169 monoclonal antibody used for in vivo depletion. Generation of bone marrow-derived DC (BM-DC). BM-DC were propagated from B6 or BALB/c mice BM progenitors for 5 days as described40 in complete medium (RPMI 1640 supplemented with 10% endotoxin-free FBS, 2 mM L-glutamine, 5 × 10–5 M β-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml streptomycin, essential amino acids and sodium pyruvate), with 1,000 IU/ml of rmGM-CSF alone or in combination with 1,000 IU/ml rmIL-4 (Schering-Plough, Kenilsworth, New Jersey). Co-culture assays. Spleens from AR129 or SCID BALB/c mice were dissociated in complete medium to yield single-cell suspensions. After erythrocyte lysis, cells were washed once and plated for 2 h at 37 °C. Adherent cells (called macrophages here) were collected using a cell scraper. The non-adherent cells, containing 30–40% NK cells, were resuspended at a concentration of 1.5 × 106/ml and plated in 96-well U-bottomed plates (0.1 ml/well). The percentage of NK cells was determined by flow cytometry analysis using PE-labeled DX5 monoclonal antibody (PharMingen, San Diego, California). Immature or TNF-α treated D1 cells, day 5 BM-DC (GM-CSF/ IL-4 or GM-CSF alone), YAC-1 cells, P815-B7.1 cells or macrophages were collected, washed, resuspended at a concentration of 1 × 106/ml and added to NK cells at different ratios in a final volume of 0.2 ml. Cells incubated individually were plated at a similar concentration. In some experiments, NK cells were cultured in 24-well flat-bottomed plates equipped with a transwell insert (Costar, Cambridge, Massachusetts). NK cells were co-cultured at a concentration of 2.5 × 106 cells/ml in 0.6 ml in the lower wells; D1 cells were added in 0.1 ml either together with the NK cells in the lower wells or by themselves in the upper wells of the transwell plates. For the latter, DC were separated from NK cells by 1 mm, but the soluble factors could diffuse freely through a microporous polycarbonate membrane (0.4 µm thick) between the upper and the lower wells. In some experiments, rhIL-15 (Immunex, Seattle, Washington) was added in the upper wells at 50 ng/ml in 0.1 ml for 18 h. Unless otherwise indicated, the NK:DC ratio was 0.5:1. In vitro assays. For mIFN-γ ELISA, the supernatants of the NK cells cultured with unstimulated D1 cells at different ratios were collected after18 h and assayed in commercial ELISA for mIFN-γ (Genzyme, Cambridge, Massachusetts). The detection limit of this assay was 5 pg/ml. For cytotoxicity assays, freshly extracted NK cells, cells from the NK/DC co-cultures or single cultures incubated for 18–40 h were collected. Viable trypan blueexcluded lymphocytes were counted and used as effector cells in chromium release assay using YAC-1, AK7, MCA205, MCA101, P815 cells as targets. The target cells were incubated for 1–2 h at 37 °C with Na51CrO4 and were plated at a concentration of 2 × 103 cells/well in 96-well V-bottomed microtiter plates after washing. Effectors and targets were mixed at various ratios and incubated for 4 h at 37 °C. Spontaneous release was determined from wells that contained labeled target cells alone, and maximum 51Cr release was determined by addition of 2% cetrimide (Sigma). Specific cytotoxicity was calculated as: percent 51Cr release = 100 x (cpm experimental–cpm spontaneous release)/(cpm maximum release–cpm spontaneous release). Statistical analyses of data. Fisher’s exact method was used to interpret the significance of differences between experimental groups (presented as NATURE MEDICINE • VOLUME 5 • NUMBER 4 • APRIL 1999


ARTICLES mean +/- s.e.m.). Significance at 95% confidence limits (asterisks in Figs.) are presented for individual experiments. Acknowledgments We are grateful to the staff of the Animal Facility of IGR. We thank C. Maliszewski, C. Bonnerot, S. Amigorena, F. Faure and E. Vivier for critical review of the manuscript. We are also indebted to M. Rescigno for contribution with the R1-conditioned medium and to P. Bousso for his input in flow cytometry analyses. E. Mottez is acknowledged for the generation of the P815-B7.1 cell line. This work was supported by the Association pour la Recherche sur le Cancer, the Ligue Nationale de Lutte contre le Cancer, the GEFLUC association, programme “Immunité Antitumorale”, the Institut Gustave Roussy, CRC IGR number 97.1, and by CNRS. NF was supported by the Institut de Formation Supérieure Biomédicale, the Fondation pour la Recherche Médicale and Rhône Poulenc RORER. AL was supported by CRC IGR number 97.1.


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