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The Critical Role of IL-15 in the Antitumor Effects Mediated by the Combination Therapy Imatinib and IL-2¹

Grégoire Mignot^2,*,,, Evelyn Ullrich^2,*,,, Mathieu Bonmort^*,,, Cédric Ménard^*,,, Lionel Apetoh^*,,, Julien Taieb^*, Daniela Bosisio, Silvano Sozzani^||, Maria Ferrantini^#, Jürg Schmitz^**, Matthias Mack, Bernard Ryffel^¶, Silvia Bulfone-Paus^*, Laurence Zitvogel^*,,, and Nathalie Chaput^3,*,,

^* Institut National de la Santé et de la Recherche Médicale, Unité 805, Villejuif, France; Institut Gustave Roussy and Center of Clinical Investigations CBT507, Villejuif, France; University Paris-Sud, Le Kremlin-Bicêtre, France; ^¶ Centre National de la Recherche Scientifique, Immunologie et Embryologie Moléculaire 2815, Institut Transgénose, Orléans, France; ^|| Section of General Pathology and Immunology, University of Brescia, Brescia, Italy; ^# Department of Cell Biology and Neuroscience, Istituto Superiore di Sanita, Rome, Italy; ^** Miltenyi Biotec GmbH, Bergisch Gladbach, Germany; Universitätsklinikum Regensburg, Abteilung für Nephrologie, Regensburg; and ^* Department of Immunology and Cell Biology, Research Center Borstel, Borstel, Germany

	Abstract

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The synergistic antitumor effects of the combination therapy imatinib mesylate (IM) and IL-2 depended upon NK1.1- expressing cells and were associated with the accumulation of CD11c^intB220⁺NK1.1⁺ IFN-producing killer dendritic cells (IKDC) into tumor beds. In this study, we show that the antitumor efficacy of the combination therapy was compromised in IL-15 and IFN-type 1R loss-of-function mice. IL-15R was required for the proliferation of IKDC during IM plus IL-2 therapy. Trans-presentation of IL-15/IL-15R activated IKDC to express CCR2 and to respond to type 1 IFN by producing CCL2. Moreover, the antitumor effects of the combination therapy correlated with a CCL2-dependent recruitment of IKDC, but not B220^– NK cells, into tumor beds. Altogether, the IL-15-driven peripheral expansion and the CCL-2-dependent intratumoral chemoattraction of IKDC are two critical parameters dictating the antitumor efficacy of IM plus IL-2 in mice.

	Introduction

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Accumulating evidence indicates that the innate and adaptive immune systems make a crucial contribution to the antitumor effects of conventional chemotherapy and radiotherapy-based cancer treatments (1, 2). The molecular and cellular bases of the immunogenicity of cell death induced by cytotoxic agents are being progressively unraveled (3, 4, 5, 6). Along these lines, we highlighted the off-target immunological effects of a tyrosine kinase inhibitor imatinib mesylate (IM;⁴ STI571/Gleevec) (7, 8). The 2-phenylaminopyrimidine compound has been initially designed to specifically block the ATP binding site of BCR/ABL tyrosine kinase and found to inhibit the kinase activity of the three related kinases BCR-ABL, platelet-derived growth factor receptor, and KIT (8, 9). IM is a clinically approved drug commonly used for the treatment of gastrointestinal stromal tumors because it directly targets the pathogenomic c-kit mutation responsible for the deregulated proliferation of tumor precursors (10). However, IM could mediate antitumor effects using an alternate mode of action than the direct effect on tumoral c-kit mutations. Borg et al. (7) selected mouse tumor models that resisted the antiproliferative effects of IM in vitro, yet responded to long-term exposure to IM in vivo. They showed that IM acted on host dendritic cells (DC) to promote NK cell activation and NK cell-dependent antitumor effects in mice and humans (7). c-kit triggering was required for the DC-mediated NK cell activation induced by IM.

To potentiate the NK cell activation induced by c-kit tyrosine kinase inhibition in vivo, IM was combined with IL-2 (11). Flow cytometry studies of single-cell suspensions obtained from regressing lung metastases highlighted that the CD11c⁺B220⁺NK1.1⁺ cells were increased by 4-fold during the combination therapy with IM plus IL-2 compared with each treatment modality alone (IM or IL-2). The CD11c⁺B220⁺NK1.1⁺ cell population expressed other NK cell markers such as the integrin VLA-2 recognized by anti-CD49b/Dx5 mAb, CD122, NKG2D, CD11b, but failed to express CD3, CD4, CD8, CD25, PDCA-1, and costimulatory molecules (CD40, CD80, CD86). Because up to 50% of the CD11c⁺B220⁺NK1.1⁺ cells coexpressed I-A^b in tumors (11) and because they could stimulate naive OVA -specific TCR-transgenic T cells when isolated from lymph nodes (12) and produce IFN- in contact with tumor cells (11), these CD11c⁺ cells were named IFN-producing killer DC (IKDC).

In an attempt to delineate the mechanisms by which IKDC could proliferate and/or be recruited into tumor beds, we investigated the role of key regulatory cytokines and chemokines in the antitumor effects mediated by IM plus IL-2 and found the critical involvement of the type 1 IFN/IL-15/CCL2 axis in the proliferation and recruitment of IKDC into tumors.

	Materials and Methods

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Mice and cell lines

Female C57BL/6 wild-type (WT) mice were obtained from the Centre d’ Elevage Janvier and used at 6–10 wk of age. IFN-type 1R^–/– and CCL2/MCP-1^–/– mice backcrossed on a C57BL/6 background were provided by Centre d’Elevage d’Orléans. IL-15R^–/– and IL-15^–/– mice were backcrossed on a C57BL/6 6–8 times and maintained at the animal facility of EB (Research Center Borstel, Borstel, Germany). Animals were all maintained according to the Animal Experimental Ethics Committee Guidelines. B16F10 is a melanoma cell line syngeneic of C57BL/6 (provided by M. T. Lotze, University of Pittsburgh, Pittsburgh, PA) that was cultured in RPMI 1640 (Life Technologies 31870) with 10% heat-inactivated FBS enriched with 5% L-glutamine, non essential amino acids, sodium pyruvate, and antibiotics. MS5 feeder cell lines (provided by W. Vainchenker, Institut Gustave Roussy, Villejuif, France) were cultured in IMDM (Sigma 13390) containing 10% heat-inactivated FBS, 5% L-glutamine, sodium pyruvate, and antibiotics.

Tumor model

Lung metastases model. B16F10 cells (3 x 10⁵) were injected at day 0 into the tail vein and mice were sacrificed at day 11. Two hundred microliters of H₂O or 150 mg/kg IM (Gleevec; Novartis) was given orally in mice twice a day (bid) from days 0 to 10 after tumor inoculation alone or combined with other therapies. Control groups (H₂O plus PBS named PBS), IM alone (IM plus PBS named IM), IL-2 alone (H₂O plus IL-2 named IL-2), cyclophosphamide (CTX, Endoxan; Baxter) alone (H₂O plus CTX named CTX), CpG oligodeoxynucleotide (ODN) alone (H₂O plus CpG named CpG), and combination therapies IM plus CTX, IM plus CpG, or IM plus IL-2 were preformed as stated. rhIL-2 (1 x 10⁵ IU; Chiron) were injected bid by the i.p. route from days 7 to 10 or 100 mg/kg CTX was administered i.p. at day 0, while 10 µg of CpG ODN (5'-TGACTGTGAACGTTCGAGATGA, given by A. Carpentier (Paris, France), AP-HP Pitié Salpétrière) were injected s.c. from days 7 to 10. PBS was injected i.p. or s.c. in control groups.

Skin model. B16F10 cells (3 x 10⁵) were injected s.c., and mice were treated with combination therapy IM plus IL-2 as described above, but with two different doses of IL-2 (10⁵ or 3 x 10⁵ bid).

Depletion of plasmacytoid DC (pDC). Mice were depleted by injection of 300 µg of anti-PDCA1-depleting mAb (clone 120G8; Miltenyi Biotec) at day –3 before tumor inoculation and day 0, day +3, and day +9 after tumor inoculation. Effective depletion was monitored by FACS using the pDC-specific staining (CD3^–CD19^–NK1.1^–CD11c^intB220⁺Gr1⁺).

Abs and flow cytometry analyses

FACS analyses were performed using allophycocyanin-conjugated anti-CD11c mAb (HL3), PE-Cy7-conjugated anti-NK1.1 mAb (PK136), allophycocyanin-Cy7-conjugated anti-B220 mAb (RA3-6B2), FITC-conjugated anti-CD3 mAb (17A2), FITC-conjugated CD19 (1D3), and PE-conjugated Gr1. IKDC are defined as CD3^–CD19^–NK1.1⁺B220⁺CD11c^intGR1^–, NK as CD3^–CD19^–NK1.1⁺B220^–CD11c^+/–GR1^–, and pDC as CD3^–CD19^–NK1.1^–B220^–CD11c^intGR1⁺. Abs were purchased from BD Pharmingen or eBioscience. Anti-CCR2 mAb (MC21, rat IgG2a) has been provided by M. Mack (Universitätsklinikum Regensburg, Abteilung für Nephrologie, Regensburg, Germany) (13). Cells were preincubated with Fc block for 20 min (CD16/CD32, 2.4G2; BD Pharmingen, diluted in PBS with 2% mice serum and 2% FBS) and afterward stained for 20 min at 4°C with the different Abs at 1/200 (anti-CCR2 at 1/20). Alexa Fluor 488 goat anti-rat IgG has been used as secondary Ab at 1/400 (Invitrogen). Immediately before FACS analysis, 4',6-diamidino-2-phenylindole (Sigma-Aldrich) was added. FACS analysis was performed by an LSRII (BD Biosciences) using FACSDiva software (BD Biosciences), CellQuestPro software (BD Biosciences), or Flow Jo (Tree Star).

Sorting of NK and IKDC cells and in vitro expansion of IKDC15 is described in our unpublished data. Sorting of NK and IKDC was realized on a Mo-Flo instrument (DakoCytomation), CD3^–CD10^–C11c^intB220⁺NK1.1⁺ cells defined "IKDC" and CD3^–CD19^–CD11c^+/–B220^–NK1.1⁺ cells defined B220^–NK cells. In vitro expansion of IKDC15 was realized as follows: 10⁴ freshly cell sorted IKDC were cultured in the presence of murine stromal cells MS-5 in DMEM (GIBCO 41966) culture medium containing 4500 mg/l of glucose, 5% L-glutamin, pyruvate, and enriched with Abs, 10% BGS (Lot ANB 18298, HyClone), and 20 ng/ml rmIL-15/ml (R&D Systems). While B220^– NK cells could not proliferate ex vivo in similar conditions as IKDC, we could maintain NK cells at higher concentrations (5 x 10⁵/ml) for 7 days on MS-5 feeders and rIL-15 to allow fair comparisons with IKDC.

Cytokine profiling of IKDC and NK cells

In brief, 10⁵ freshly sorted NK cells and IKDC or IKDC15 (obtained at day 7 of ex vivo expansion) or NK and IKDC stimulated with recombinant murine IL-15 (20 ng/ml; R&D Systems) for 24 h in the presence of MS-5 were further incubated with recombinant human (rh) IL-2 (10⁵ IU/ml) or type 1 IFN at 2500 IU/ml (14) (provided by M. Ferrantini, Instituto di Sanita, Rome, Italy). These in vitro cultures were performed in 200 µl of complete medium in 96-round-bottom well plates. After 24–36 h, cell supernatants were collected and commercial LUMINEX kits, (Linco and BioSource International) were used to determine chemokine and cytokine profiles (used according to the manufacturer’s conditions).

In vivo proliferation assays

WT C57BL/6 and IL-15R^–/– mice were treated with IL-2 combined with IM according to a protocol already reported (11). Mice received an i.p. injection of 100 µg of BrdU/100 µl PBS) 1 day before sacrifice. Spleen cells were harvested and processed according to the manufacturer’s protocol (BrdU Flow Kit; BD Biosciences). Briefly, cells were stained for surface Ags, fixed, and permeabilized. DNase digestion followed by staining with anti-BrdU mAb was performed before flow cytometry analyses.

Statistical analyses

Aberrant values were excluded using Dixon’s test. Normality of distributions was assessed using the Shapiro-Wilk test. Normal distributions were compared by the Student t test; non-normal samplings were compared using the Mann-Whitney U test. ANOVAs were performed with the Kruskal-Wallis test. Values of p inferior to 0.05 were considered significant. All tests were done using Prism 5 software (GraphPad).

	Results

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c-kit tyrosine kinase inhibition synergized with NK adjuvants

The natural immunosurveillance against B16F10 melanoma developing into lung metastases is known to rely on innate NK cell effectors (15, 16). We previously showed that IM could induce NK1.1⁺ cell-dependent tumor regressions (7). To further improve the NK cell triggering effects of IM, we combined IM with various NK adjuvants such as TLR9 ligands (CpG ODN (17, 18)) or metronomic CTX suppressing the regulatory T cell-mediated NK cell inhibitory effects (19, 20, 21) or IL-2 (22, 23). We observed significant synergistic effects with CTX (Fig. 1A) with CpG ODN (Fig. 1B) or rhIL-2 (Fig. 1C) but the most remarkable antitumoral effects were achieved with the combination of IM + IL-2 (Fig. 1D). Similarly, the growth of B16F10 melanoma inoculated into the skin was also impaired by the combination therapy but higher dosages of IL-2 were required for a significant tumor growth delay (Fig. 1E). It is of note that all of these agents did not impact on B16F10 proliferation in vitro (data not shown). We already reported that depleting anti-NK1.1 mAb completely curtailed the tumoricidal activity induced by the combination of IM + IL-2 (11).

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Combining IM with NK cell adjuvants. Established B16F10 lung metastases model in C57BL/6 mice. B16F10 tumor cells (5 x 105) were inoculated in the tail vein at day 0. A, Mice were injected i.p. with PBS or low-dose CTX (100 mg/kg) and were fed with H2O or IM bid at 150 mg/kg per day from days 0 to 10. B, Mice were fed with H2O or IM as in A, but were treated with s.c. administration of PBS or CpG ODN (10 µg/day) from days 7 to 10. C, Mice were fed with H2O or IM as in A, but were treated bid with i.p. administration of PBS or 105 IU of rhIL-2 from days 7 to 10. D, Mice were fed and treated systemically with the above-mentioned immunotherapies (A–C) in the same experiment. Graphs represent the number of lung metastases at day 11. E, s.c. B16F10 tumor model in C57BL/6 mice. Same as in C but 3 x 105 B16F10 cells were inoculated in the skin of flanks and two doses of IL-2 (105 IU or 3 x 105 IU bid) were used. The tumor size was monitored twice a week. A representative experiment of two to five including five mice per group is depicted. Statistical analyses were performed using the Kruskal-Wallis test. Values of p inferior to 0.05, 0.01, or 0.001 are indicated with *, **, or ***, respectively.

Absolute requirement for IL-15 in the antitumor efficacy of IM + IL-2

IKDC were found in tumor beds and were necessary and sufficient to prevent tumor outgrowth after adoptive cell transfer in Rag^–/– x IL-2R^–/– hosts (11, 24, 25). Moreover, we just showed that IL-15/IL-15R dictates IKDC effector functions (our unpublished data). Therefore, we investigated the role of IL-15 in the efficacy of IM + IL-2 against B16F10 lung metastases by comparing the numbers of tumor foci in lung parenchyma of WT vs IL-15^–/– mice (devoid of IKDC). IL-15 was not only involved in the natural immunosurveillance against B16F10 establishment but also in the IM + IL-2-induced antitumor activity (Fig. 2A). Like recombinant murine IL-15, IM + IL-2 could promote the accumulation of IKDC in the spleen by 4-fold (Fig. 2B and our unpublished data) while the absolute numbers of B220^– NK and B220⁺ cells remained unchanged (Fig. 2B). Importantly, IM + IL-2 promoted the entry of cells into cycle but IKDC cell proliferation was more efficient than that of B220^–NK cells (Fig. 2C). The IM + IL-2-driven IKDC proliferation was not entirely abolished in IL-15R^–/– mice (Fig. 2C), presumably as a result of a direct effect of IL-2 (our unpublished data). Nevertheless, these data outline the involvement of IL-15/IL-15R in the proliferation of IKDC during IM + IL-2 treatment.

View larger version (11K): [in this window] [in a new window]   FIGURE 2. IL-15 is required for the proliferation of IKDC and the antitumor effects of IM + IL-2. A, Established B16F10 lung metastases model in C57BL/6 mice (WT) and IL-15 knockout (IL-15–/–) mice. Same experimental setting as in Fig. 1C, but injections were performed in WT vs IL-15–/– mice. Statistical analyses were performed using the Kruskal-Wallis; ***, p < 0.001. B, Naive C57BL/6 mice (WT) mice were treated with PBS and combined therapy IM + IL-2. Splenocytes were harvested at day 11 for the enumeration of IKDC (CD3–CD19–CD11cintB220+NK1.1+), B220– NK cells (CD3–CD19–B220–NK1.1+), and B220+ cells. C, Naive C57BL/6 mice (WT) or IL-15R–/– mice were treated with PBS and combined therapy IM + IL-2 and injected with BrdU before flow cytometry analysis of splenocytes according to protocols described in Materials and Methods. Dividing cells that incorporated BrdU are measured as a percentage among the IKDC (upper panel) or B220– NK cell subsets (lower panel). *, p < 0.05; ND, Not detected; ns, nonsignificant.

The role of pDC and IFN-type 1R signaling in tumor regression

Trans-presentation of IL-15 is a biological attribute of conventional DC (cDC) stimulated through IFN-type 1R (26, 27) and is essential for the cDC-mediated NK cell triggering during TLR activation in vivo. We addressed whether the combination of IM + IL-2 would also implicate IFN-type 1R signaling. IFN-type 1R played a major role in the tumor growth inhibition provoked through therapy combining IM + IL-2 (Fig. 3A). Since the main source of IFN- remains pDC (28), we addressed the role of pDC in the antitumor effects of the combination therapy. Depletion of pDC using the 120G8 mAb before tumor inoculation significantly curtailed the therapeutic efficacy of IM + IL-2 (Fig. 3B).

View larger version (10K): [in this window] [in a new window]   FIGURE 3. Role of type 1 IFN in IM + IL-2-mediated antitumor effects. A, Established B16F10 lung metastases model in C57BL/6 mice (WT) and type I IFN receptor-deficient mice. Same experimental setting as in Fig. 1C. B, Depletion of C57BL/6 mice using the anti-PDCA1 Ab. Same experimental setting as in Fig. 1C. A representative experiment of two to three including five mice per group is depicted. Statistical analyses were performed using the Kruskal-Wallis test. Values of p inferior to 0.05 or 0.01 confidence interval are indicated with * or **, respectively. ns, nonsignificant.

In summary, IL-15 and IFN-type 1 signaling are markedly involved in the therapeutic effectiveness of the c-kit tyrosine kinase inhibitor combined with IL-2.

The synergistic effects of IFN-type 1 and IL-15 for CCL2 production by IKDC

Because IL-15 and type 1 IFN are major players in IM + IL-2-mediated immunosurveillance against B16F10, we investigated their role in modulating the chemokine/chemokine receptor expression profile in freshly sorted IKDC before and after stimulation by IL-15 trans-presentation (so-called "IKDC15" (our unpublished data)). In resting conditions, IKDC cells responded weakly to type 1 IFN and IL-2 for CCL2 (MCP-1), CCL3 (MIP-1), CCL4 (MIP-1β), CCL5 (RANTES), CXCL10 (IFN--inducible protein 10), and vesicular endothelial growth factor (Fig. 4A). Following trans-stimulation with IL-15, IKDC strongly produced CCL2 (MCP-1) and factors involved in tissue remodeling and/or angiogenesis such as vesicular endothelial growth factor and CXCL1 (KC/GRO) in response to type 1 IFN or IL-2 (Fig. 4A, right panel). Moreover, following trans-stimulation with IL-15, IKDC enhanced their transcription of the inflammatory chemokine receptors CCR2 and CCR5 (Fig. 4B). Flow cytometry analyses confirmed the expression of CCR2 and CCR5 on the IKDC15 cell surface (Fig. 4C and data not shown). Importantly, IL-15/IL-15R-driven B220^– NK cells failed to express CCR2 (Fig. 4C).

View larger version (17K): [in this window] [in a new window]   FIGURE 4. IL-15/IL-15R stimulates IKDC to respond to type 1 IFN and IL-2 for CCL2 and CCR2 expression. A, Cytokine and chemokine release. Resting IKDC or IKDC15 (IL-15R/IL-15 expanded IKDC, as described in our unpublished data) were stimulated for 24 h with medium, type 1 IFN, or IL-2. Multiplex analyses of cytokine and chemokine release were performed in the culture supernatants. B, Quantitative RT-PCR were performed on freshly sorted IKDC and IKDC15 (at day 7 of expansion). C, FACS analyses of CCR2 expression were performed on freshly sorted IKDC or B220– NK cells or on IL-15/IL15-R-stimulated IKDC or NK (for 7 days). Dotted line, Isotype control; black line, anti-CCR2 Ab staining.

CCL2-dependent trafficking of IKDC into tumor beds

We next addressed the role of CCL2 in the accumulation of IKDC into B16F10 lung metastases. Because our in vitro data support the view that CCL2 production by IKDC is stimulated by the synergistic effects between IL-15/IL-15R and type 1 IFN (Fig. 4), we determined the impact of IM + IL-2 therapy on the proportion of NK1.1⁺ innate effectors (B220^–NK vs IKDC) accumulating in tumor beds in WT, IL-15^–/–, and CCL2^–/–. IM + IL-2 significantly increased (up to 10-fold) the numbers of IKDC in WT mice in a CCL2 and IL-15-dependent manner (Fig. 5A, left panel). In contrast, B220^–NK cell numbers barely increased in a CCL2-independent fashion (Fig. 5A, right panel). Accumulation of IKDC into lung metastases could result from IKDC recruitment and/or local proliferation. The ratio between spleen and tumor-infiltrating IKDC augmented (by up to 3-fold) during therapy in WT but not CCL2-deficient hosts, supporting the contention that both recruitment and proliferation occurred during IM + IL-2 (Fig. 5B).

View larger version (13K): [in this window] [in a new window]   FIGURE 5. CCL2-dependent IKDC homing into tumor tissues. B16F10 tumor cells (5 x 105) were inoculated in the tail vein at day 0 and mice were sacrificed at day 11. A, Enumeration of tumor-infiltrating IKDC and B220– NK cells during PBS or combined therapy with IM + IL-2 in C57BL/6 (WT), CCL2-deficient (CCL2–/–), and IL-15-deficient (IL-15–/–) mice after lung dissociations. B, As in A, but calculation of an IKDC ratio defined as: number of IKDC in the lung divided by number of IKDC in the spleen (no. of lung IKDC ÷ no. of spleen IKDC). These data have been obtained from seven animals per group. Statistical analyses were performed using Student’s t test, p inferior to 0.05 are indicated with *. ns, nonsignificant.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
These findings underscore the antitumoral efficacy of associating two NK cell adjuvants, one blocking the c-kit tyrosine kinase of cDC (7) and the IL-2, a major lymphokine-inducing proliferation and activation of T and NK cells (22). This study addresses the mechanisms by which IKDC, recently described as novel innate effectors involved in antitumor immunosurveillance, could accumulate in tumor beds and be associated with tumor regression (11). In this study, we unravel the key role of the IFN type 1R/IL-15/CCL2 axis in the antitumor effects mediated by IM + IL-2 and underscore the potential of IL-15 to license IKDC to proliferate, kill (our unpublished data) and respond to IFN type 1 (and IL-2) by producing CCL2 and expressing CCR2. Finally, we highlight the relevance of the IL-15-driven CCL2/CCR2 selective expression on IKDC for their preferential homing to tumor tissues.

Before the description of IKDC cells, the functional interaction between conventional DC and bona fide NK cells (29, 30) was described to play a key role in the interplay between innate and cognate immunity (31, 32). IL-15/IL-15R is crucial for the DC-mediated NK cell priming in vitro (26, 33) and in vivo (26). TLR ligands or agonistic anti-CD40 mAb could both induce the expression of IL-15R on DC in vivo (26). Moreover, IFN- has been identified as a key inducer of IL-15/IL-15R (26, 27, 34). The precise mechanism by which IM + IL-2 could induce IL-15/IL-15R in our model system remains unclear. Our unpublished data indicated that IM alone could not mediate antitumor efficacy in TRIF^–/– mice, indicating that IFN type 1 could be necessary in the immunostimulatory effects of IM. Supporting this contention, we now show that the combination of IM + IL-2 required IFN type IR signaling (Fig. 3A). A previous work reported that IKDC derived from BALB/c mice and stimulated through TLR9 ligation could secrete IFN- (12), but these data remain controversial (35). Since pDC constitute a main source of IFN- (28), at least during viral infection or TLR7 and TLR9-dependent insults, we could hypothesize that pDC may also be implicated in the efficacy of IM + IL-2. Depleting pDC resulted in an abrogation of the IM +IL-2-mediated antitumor effects in vivo (Fig. 3B).

We could postulate that IM + IL-2 might stimulate IFN- production by pDC in vivo, leading to the up-regulation of IL-15/IL-15R on cDC, thereby promoting the DC/NK cell cross-talk. Alternatively, a direct pDC-NK cell interaction could occur whereby IL-2 and/or IM could play a regulatory role. Indeed, we and others reported that IL-2 could trigger TRAIL expression on both pDC and NK cells during a pDC/NK cell cross-talk in vitro (36) and that IL-2-activated NK cells could stimulate IFN- and IL-6 production by pDC (37). Interestingly, IM could synergize with a concomitant viral stimulation to enhance IFN- production by pDC in leukemic patients (38). It remains to be determined whether IM could increase IFN- secretion by pDC when associated with IL-2 in our model. In these two above-mentioned hypotheses (cDC/NK or pDC/NK dialogs), bona fide NK cells could be activated either through trans-presentation of IL-15 and/or by IFN-, accounting for the efficacy of IM + IL-2. However, several lines of evidence argue against those hypotheses. First, a significant proliferation of NK cells could not be documented during IM + IL-2 therapy (Fig. 2), neither in a tumor-free (Fig. 2) nor in tumor-bearing mice (Ref. 11 and not shown). Second, IM + IL-2 could not only prevent the establishment of B16F10 lung metastases (that are notoriously NK cell dependent) but also the s.c. expansion of the same tumor (Fig. 1), supporting the notion that effectors other than NK cells might be involved. Third, in synergy with IL-15/IL-15R, IFN type 1 and IL-2 could directly stimulate B220^– NK cells for CCL3 (MIP-1) secretion (data not shown). However, CCR5 was not critical for the efficacy of IM + IL-2 (our unpublished data).

To date, it remains quite complex to directly ascribe the tumoricidal activity of IM + IL-2 to IKDC. Since adaptive transfer of IM + IL-2-activated IKDC into Rag^–/– x IL-2R^–/– mice could abrogate B16F10 growth while NK cells failed to do so (11), we proposed that IKDC could represent the most potent final effector leading to an efficient antitumor activity of IM + IL-2. Supporting this view, we demonstrate here that IKDC are significantly enriched compared with B220^– NK cells in spleens of mice treated with IM + IL-2 (Fig. 2B). Indeed, the IKDC:B220^– NK cell ratio was increased by 4-fold during therapy (0.2 vs 0.05, in IM + IL-2 and PBS-treated mice, respectively, p < 0.001). Moreover, we showed the crucial role of IL-15/IL-15 R in the proliferation and in the CCR2/CCL2-dependent trafficking of IKDC cells into tumor beds during the therapy (Figs. 2, B and C and 5A). Finally, in our unpublished data, we showed that IL-15Ra/IL-15 licensed IKDC (and not bona fide NK cells) with TRAIL-dependent killing, which was critical in the efficacy of IM + IL-2 (11). However, the final demonstration of the key role of IKDC in tumor immunosurveillance, as an effector or conceivably as a professional APC (12), will await further investigations and the delineation of specific markers assigned to IKDC and not to other subsets of NK cells (39).

Because our data suggested that the combination therapy with IM + IL-2 could be useful to boost the natural immunosurveillance against tumors sensitive to TRAIL-dependent apoptosis (11) and was more potent than other combination therapies (Fig. 1), we launched a phase I trial "IMAIL-2" at the Institut Gustave Roussy aimed at targeting IM-resistant or stabilized gastrointestinal sarcomas and TRAIL-sensitive cancers. It is conceivable that the monitoring of innate effectors in patients treated with high dosages of IL-2 combined with Gleevec might allow the identification of the human counterpart of mouse IKDC.

	Acknowledgments

 
We thank W. Vainchenker, A. Mackensen, A. Caignard, and B. Azzarone for discussing our data and H. Yagita, E. Tomasello, E. Vivier, and T. Walzer for providing reagents and mice. We thank Y. Lecluse, P. Rameau, and D. Métivier for cell sorting and G. Elain, N. Brunel, and B. Besson-Lescure for technical assistance and the animal facility of the Institut Gustave Roussy under the direction of P. Gonin.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
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 work was supported by European Union grants (ALLOSTEM, DC THERA), Association pour la Recherche sur le Cancer, and Ligue Nationale Contre le Cancer (équipes labelisées de G. Kroemer and L.Z.). G.M. was supported by the Association pour la Recherche sur le Cancer, E.U. received a fellowship from the Deutsche Forschungsgemeinschaft and from the Fondation pour la Recherche Médicale, and M.B. was supported by the Poste d’Accueil Institut National de la Santé et de la Recherche Médicale.

² G.M. and E.U. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Nathalie Chaput, Center of Clinical Investigations CBT507, Institut National de la Santé et de la Recherche Médicale Unité 805, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, France. E-mail address: nathalie.chaput{at}igr.fr

⁴ Abbreviations used in this paper: IM, imatinib mesylate; DC, dendritic cell; IKDC, IFN-producing killer DC; pDC, plasmacytoid DC; rh, recombinant human; bid, twice a day; CTX, cyclophosphamide; ODN, oligodeoxynucleotide; WT, wild type; cDC, conventional DC.

Received for publication October 2, 2007. Accepted for publication March 1, 2008.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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