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Bone Marrow-Derived IFN-Producing Killer Dendritic Cells Account for the Tumoricidal Activity of Unpulsed Dendritic Cells¹

Nourredine Himoudi^*, Stephen Nabarro^*, Jo Buddle, Ayad Eddaoudi, Adrian J. Thrasher and John Anderson^2,*

^* Unit of Molecular Haematology and Cancer Biology and Unit of Molecular Immunology, Institute of Child Health, London, United Kingdom

	Abstract

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The CD11c^intB220⁺NK1.1⁺CD49⁺ subset of cells has recently been described as IFN-producing killer dendritic cells (IKDC), which share phenotypic and functional properties of dendritic cells and NK cells. Herein we show that bone marrow-derived murine dendritic cell preparations contain abundant CD11c^intB220⁺NK1.1⁺CD49⁺ cells, the removal of which results in loss of tumoricidal activity of unpulsed dendritic cells in vivo. Moreover, following s.c. injection, as few as 5 x 10³ highly pure bone marrow-derived IKDC cells are capable of shrinking small contralateral syngeneic tumors in C57BL/6 mice, but not in immunodeficient mice, implying the obligate involvement of host effector cells in tumor rejection. Our data suggest that bone marrow-derived IKDC represent a population that has powerful tumoricidal activity in vivo.

	Introduction

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Dendritic cells (DC)³ are pivotal to the induction of innate and adaptive immune responses. In vivo DC are attracted to sites of infection or inflammation where they can take up Ag. Subsequent maturation of DC results in the up-regulation of costimulatory molecules and MHC class II and their migration to sentinel lymph nodes where they present Ags to T cells and can stimulate innate responses through secretion of inflammatory cytokines. For example, NK cells and DC can exchange bidrectional activating signals in a positive feedback, referred to as cross-talk (1, 2). Both myeloid DC and plasmacytoid DC promote cytoxicity and cytokine production by NK cells, while activated NK cells can reciprocate by providing immunoregulatory helper function to DC, stimulating them to produce proinflammatory cytokines that stimulate cytotoxic lymphocyte (CTL) and Th1 responses.

It has long been recognized that adoptive transfer of DC has the potential to inhibit cancer growth through stimulation of both innate and adaptive immune responses (3, 4, 5, 6). Innate responses induced by DC can result, for example, from the stimulation of tumor-reactive NK cells. Adaptive anticancer immune responses result from the presentation of tumor Ags by DC. In adoptive transfer studies stimulation of adaptive immunity has generally resulted from the priming of DC ex vivo with tumor Ags (7, 8), although unprimed DC have shown effective tumoricidal activity in animal models following direct intratumoral injection (9, 10, 11). In tumor models involving injection of DC at distant sites, unpulsed DC usually show low or no antitumor activity compared with Ag-pulsed DC (12, 13, 14, 15, 16). However, some studies have shown tumoricidal activity of unpulsed DC injected at sites distant to a tumor, attributed, at least in part, to stimulation of innate NK responses (4, 17, 18, 19).

Heightened interest of the role of DC in cross-talk of innate and adaptive responses follows the recent description of IFN--producing killer dendritic cells (IKDC), which were reported to show both NK and Ag presenting cell surface phenotype and function (20, 21, 22). IKDC were originally described as having intermediate expression of CD11c coupled with high expression of B220, NK1.1, and CD49b, and absence of Gr-1, to distinguish them from conventional NK cells (NK1.1⁺CD49b⁺CD11c^–B220^–) and plasmacytoid DC (pDC; NK1.1^–CD49b^–Gr-1⁺B220⁺) (20, 21). However, the distinction between IKDC, DC, and NK cells has been challenged in recent reports that have described the presence of CD11c and B220 on conventional NK cells, and failed to demonstrate presentation of Ag by IKDC, leading to the suggestion that IKDC represent an activated form of NK cells (23, 24, 25).

Herein we provide further evidence for the antitumor properties of cells with the IKDC phenotype. We show that the IKDC can be readily isolated in large numbers from conventional bone marrow DC preparations, but that remarkably low numbers of bone marrow-derived IKDC administered by s.c. injection are required for effective adoptive transfer immunotherapy of distant tumors. Both innate NK response and adaptive T cell responses are observed in mice undergoing tumor rejection following administration of unpulsed DC.

	Materials and Methods

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Mice and cell lines

Studies were conducted on 8-wk-old C57BL/6 or immunodeficient RAG^–/–/common -chain^–/–/complement C5^–/– mice. Tumor cells suspended in 150 µl PBS were injected s.c. into the flanks of groups of at least six mice, and DC subtypes were injected s.c. at the contralateral flank. Tumor size was monitored every 2 days using calipers, and mice were killed when tumors reached a maximum diameter of 1.2 cm. Tumors were mechanically disaggregated before staining for flow cytometric analysis, or fixed in 10% formalin for frozen sections. 76-9 and 76-9-P3F (76-9:C23) have been previously described (26). B16F10 melanoma and LL/2 lung cancer cell lines, which are syngeneic to C57BL/6, and NK-sensitive YAC-1 cells, were from the American Type Culture Collection. Mouse experiments were performed in accordance with a U.K. Home Office-approved project license held by one of the authors (J.A.).

Abs and flow cytometry analyses

All the Abs used in this study are listed in Table I. Stained cells were analyzed using CyAn ADP flow cytometer (Dako) using Summit 4.3 software.

Recombinant murine GM-CSF (800 IU/ml) was added to freshly isolated bone marrow cells of C57BL/6 mice, and the cells were cultured for 7 days at 37°C in 5% CO₂. On day 3, nonadherent cells were discarded and fresh RPMI 1640 containing 10% FCS, 800 IU/ml recombinant murine (rm)GM-CSF and 1000 IU/ml rmIL-4 (PeproTech) was added. On day 6, the nonadherent cells were replated. On day 7, nonadherent immature DC were harvested, resuspended in fresh media, and matured with 10 µg/ml keyhole limpet hemocyanin (Calbiochem) and prostaglandin E₂ (1 µg/ml, Cambridge Laboratories). In some experiments DC were matured in the presence of 500 ng LPS, with 800 IU/ml rmGM-CSF and 1000 IU/ml rmIL-4 for 24 h as indicated in the figure legends. CD11c^intB220⁺CD49⁺NK1.1⁺ cells were generated from bulk DC activated by LPS in two different ways. First (see Fig. 4, A and B), bone marrow-derived DC preparations were stained for CD11c, NK1.1, and B220, and the CD11c^intNK1.1⁺B220⁺ population was gated for FACS sorting. To generate larger numbers for adoptive transfer experiments (see Figs. 4C and 5), bone marrow-derived DC were first enriched for CD49b⁺ cells by a magnetic separation using a CD49b-specific mAb (Miltenyi Biotec) according to the manufacturer’s protocol. CD49b⁺ cells were then stained with mAb against NK1.1, CD11c, and B220, and gated on the intermediate level of expression of CD11c before isolating the double-positive cells for B220 and NK1.1 by FACS sorting (EPICS Altra; Beckman Coulter and software Expo32). Expression of MHC class II and NKG2D was assessed using appropriate conjugated mAb. Purified NK cells (CD11c^–B220^–CD49b⁺NK1.1⁺) and CD11c^intB220⁺CD49b⁺NK1.1⁺ cells were then used for functional experiments. The purity of cell separation was >90%.

Cytotoxicity, proliferation and IFN- release assays

CTL analysis was performed as previously described (27) with minor modifications. Briefly, spleen cells or sorted DC or NK cell subsets were used as effectors to test specific cytolytic activity against tumor targets in a standard 4-h ⁵¹Cr-release assay. In blocking experiments, effector cells were incubated in the presence of specific neutralizing Ab for 2 h (72 h for anti-TRAIL Ab). Spontaneous and maximum release were determined from wells containing either medium alone or lysis buffer (0.5% Triton X-100). The percentage specific cytotoxicity was calculated by the formula: (release in assay – spontaneous release)/(maximum release – spontaneous release) x 100. For each E:T ratio, the results are expressed as the mean of triplicates. IFN--releasing cells were quantified by cytokine-specific ELISPOT as previously described (27). Briefly, effector cells were cocultured with target cells for 72 h in the presence of 10 IU/ml of IL-2. The background level was measured in wells containing effector cells in medium only. The results are shown as the mean value obtained for triplicate wells. We assessed T cell proliferation by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt) assay according to the manufacturer’s instructions (Promega).

Statistical analyses

We compared groups using ANOVA followed by multiple comparisons of means. Nonparametric statistical methods were used when the variables studied were not normally distributed. Wilcoxon two-sample rank-sum test was used to compare the values of continuous variables between two groups. Paired comparisons were made using a Wilcoxon paired test. p-values were determined and considered significant when p < 0.05.

	Results

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Unpulsed DC are capable of shrinking small established tumors

In initial experiments designed to assess tumor lysate-pulsed DC vaccination in alveolar rhabdomyosarcoma, we made use of murine 76-9 methylcolanthrene-induced rhabdomyosarcoma cells stably transfected with the PAX3-FKHR oncogene, referred to as 76-9:C23 cells (26). To our surprise, subcutaneously administered unpulsed bone marrow-derived DC were as effective as tumor-lysate-pulsed DC in inhibiting outgrowth of subsequently administered tumor (Fig. 1A). Furthermore, unpulsed DC had a similar effect on the growth of syngeneic B16F10 melanoma and LL/2 lung cancer cells (Fig. 1B). Unpulsed DC could also inhibit growth of small established tumors (Fig. 1C), and splenocytes from DC-vaccinated tumor-bearing mice secreted large amounts of IFN- following addition of tumor cells in vitro (Table II). Furthermore, these splenocytes demonstrated a high degree of cytotoxicty against 76-9:C23 cells, and the cytotoxicity was abrogated by antagonistic anti-CD3, anti-CD8, or anti-NKG2D Abs, suggesting that unpulsed DC induced both CTL- and NK-mediated cytotoxicity (Fig. 1D). This was confirmed by analysis of tumor-infiltrating lymphocytes, which showed a large increase in NK cells and CTL (but not NKT cells) as well as other inflammatory cells in unpulsed DC-treated mice (Table III). In beige mice lacking functional NK cells, 76-9:C23 tumor growth was more rapid, but unpulsed DC prepared from wild-type mice still inhibited tumor growth (Fig. 1E), indicating that 76-9:C23 are NK targets but that host NK cells are not essential for tumor inhibition mediated by unpulsed DC. Furthermore, the role of T cells in beige mice was suggested by the high number of activated tumor-infiltrating CD4 and CD8 T cells compared with untreated mice (Fig. 1F).

View larger version (32K): [in this window] [in a new window]   FIGURE 1. Unpulsed DC inhibition of 76-9:C23 tumors is mediated by NK and T cells. In all experiments C57BL/6 mice were injected s.c. with 5 x 106 76-9:C23 tumor cells on one flank and 1 x 106 unpulsed or 76-9:C23 lysate-pulsed bone marrow-derived DC on the contralateral flank in accordance with the protocol schema. Eight mice per group; small palpable tumors were already established in protocol 3 at the time of the second DC vaccination. Tumor volume results are the means ± SEM. A, DC were pulsed or not with 76-9:C23 tumor lysate in protocol 2. B, C57BL/6 mice received two injections with 1 x 106 unpulsed DC followed by a challenge at the contralateral flank with 76-9:C23 or LL/2 or B16 tumor cell lines (protocol 2). A control group of mice receiving only tumor cells was left untreated (protocol 1). C, Unpulsed DC could also inhibit growth of small established 76-9:C23 tumors in protocol 3. D, Splenocytes from unpulsed DC-treated 76-9:C23 tumor-bearing mice (protocol 2) kill 76-9:C23 target cells in standard 51Cr-release assay. Results are the mean of specific killing from six mice per group (±SEM). NK cell blocking used an anti-NKG2D-blocking Ab. Isotype control Abs had no signigicant blocking activity (not shown). E, C57BL/6 beige or wild-type mice were treated with unpulsed DC prepared from wild-type mice and challenged with 76-9:C23 tumor cells (protocol 2). Tumor volume results are the mean of measurements from eight mice per group (±SEM). F, Flow cytometric analysis of tumor-infiltrating lymphocytes from NK-deficient C57BL/6 beige mice. Results are expressed as percentage of cells within the viable lymphocyte gate. Tumors from eight treated mice were analyzed for tumor-infiltrating lymphocytes, and the mean of relative number of total viable cells ± SEM are plotted.

View this table: [in this window] [in a new window]   Table II. Unpulsed DC vaccine induces IFN--secreting cells in tumor- or non-tumor-bearing micea

To confirm that unpulsed DC had induced an adaptive T cell-mediated immune response, we investigated whether the activated T cells were specific for the tumor implanted in the immunized mice. Purified FACS-sorted T cells and NK cells, but not NKT cells, from unpulsed DC-treated 76-9:C23 tumor-bearing mice showed markedly enhanced in vitro cytotoxicity against 76-9:C23 targets (Fig. 2A), but the T cells did not show significant killing of B16 or LL2 targets (Fig. 2B). Moreover, in an MTS assay these purified T cells showed enhanced proliferation response following addition of tumor lysate from 76-9:C23 but not from LL2 or B16 tumors (Fig. 2C). This proliferation correlated with increased IFN- secretion following addition of 76-9:C23 tumor lysate to splenocytes from tumor-bearing unpulsed DC-treated mice (Fig. 3A). T cells and NK cells were also purified from spleens of non-tumor-bearing DC-treated mice, and here the T cells showed no killing of 76-9:C23 cells, although NK cells had been similarly activated (Fig. 3B). Therefore, a mixed NK and adaptive T cell-mediated immune response occurs following unpulsed DC treatment, and the adaptive T cell component is dependent on the presence of tumor at a distant s.c. site.

View larger version (24K): [in this window] [in a new window]   FIGURE 2. Unpulsed DC cause a specific T cell immune response dependent on the presence of tumor at a distant site. Splenocytes were prepared from unpulsed DC-treated mice 2 wk after 76-9:C23 tumor challenge. CD3 T cells (CD3+ NK1.1–), NK cells (CD3–NK1.1+), and NKT cells (CD3+NK1.1+) were FACS sorted. In these experiments, DC were matured with prostaglandin E2 and keyhole limpet hemocyanin. A, Sorted cells were stimulated for 5 days with bone marrow-derived DC pulsed with 76-9:C23 lysate in the presence of 10 U/ml of murine IL-2, and then incubated with 51Cr-labeled 76-9:C23 target cells. B, CD3 T cells were stimulated as described in A and incubated with 51Cr-labeled LL/2 or B16 or 76-9:C23 tumor cell lines in a 4-h 51Cr-release assay at E:T ratio of 50:1. C, Sorted CD3 T cells were stimulated with DC pulsed with tumor lysate prepared from LL/2 or B16 or 76-9 cell lines for 5 days in the presence of 10 U/ml of murine IL-2, and the proliferation of cells was analyzed by MTS assay. Each assay point was done in triplicate, and the result is representative of mean data ± SEM obtained with six mice per group.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. T cell tumoricidal effect following DC vaccination is dependent on presence of tumor in vivo. A, Bone marrow-derived DC and splenocytes were prepared from mice that had been treated with two DC injections and challenged with 76-9:C23 tumor cells, or from naive mice, or from mice challenged only with tumor cells. CD3 T cells and NK cells and NKT cells were FACS sorted. DC were pulsed with 76-9:C23 tumor lysate for 2 h, washed, and mixed with T cells, NK cells, or NKT cells and then incubated in presence of 10 U/ml of murine IL-2 with 76-9:C23 target cells for 72 h in an ELISPOT IFN--release assay. B, CD3 T or NK cells were FACS sorted from non-tumor-bearing mice treated with DC or from naive mice, and stimulated as described in Fig. 2A and incubated with 51Cr-labeled 76-9 or YAC-1 target cell lines in a 4-h 51Cr-release assay in the presence or absence of anti-NKG2D-blocking Ab or its isotype control. Each assay point was done in triplicate and the result is representative of mean data obtained with six mice per group.

We hypothesized that the recently described IKDC (20, 21) might account for the apparent ability of unpulsed DC to trigger regression of tumor implanted at a distant site. We first demonstrated the presence of cells with the IKDC phenotype in the bulk bone marrow DC preparation that had been used in the DC vaccinations, and refer to these hereafter as bone marrow-derived IKDC (BM-IKDC: CD11c^intB220⁺NK1.1⁺, Fig. 4A). These cells represented between 2 and 10% of the viable cells in DC preparations depending on the method of enrichment (Table IV). As previously described (20, 21), FACS-sorted BM-IKDC stained brightly for the NK marker CD49b but low for MHC class II, and did not express Gr-1 (Fig. 4B).

View larger version (32K): [in this window] [in a new window]   FIGURE 4. CD11cintB220+NK1.1+ cells are necessary and sufficient for the tumoricidal effect of unpulsed DC vaccination in tumor-bearing mice. A, Bone marrow-derived DC were separated by flow cytometry after staining with FITC-conjugated B220-specific, PE-conjugated CD11c-specific, and allophycocyanin-conjugated-specific NK1.1 Abs. We gated on CD11cint cells and FACS sorted B220+ and NK1.1+ double-positive cells. B, Sorted cells were further analyzed for their purity and for the expression of CD49b, NK1.1, MHC class II (I-A/I-E) and Gr-1 using PE-Cy7-conjugated-specific Abs. Isotype controls are open histograms. C, Left panel, 76-9:C23 tumor-bearing C56BL/6 mice were left untreated or treated, as specified in protocol 3, with 1 x 106 sorted negative fraction (shown in A) or unsorted total DC, or negative fraction supplemented with 5% or 10% of CD11cintB220+NK1.1+CD49b+. Animals were culled when tumor size reached 1.2-cm diameter. Right panel, Tumor-bearing mice were left untreated or treated as per protocol 3 with 105, 104, or 5 x 103 of FACS-sorted CD11cintB220+NK1.1+CD49b+ cells. One representative out of two experiments including six mice per group is shown.

In vitro killing capacity of BM-IKDC

We next investigated whether the BM-IKDC in the bulk bone marrow DC culture shared the same in vivo killing activity as the IKDC described previously (20, 21). We hypothesized that the activating receptor NKG2D might be essential for killing because it was consistently present in the previously published IKDC, and it was present in the CD11c^intB220⁺NK1.1⁺ cells that we purified from bone marrow DC (Fig. 5A). NKG2D ligands RAE1 and H60 were present on 76-9:C23 and LL2 targets but not on B16. Moreover, IFN- release by BM-IKDC following addition of these targets correlated with the presence of the cognate ligands and was blocked by anti-NKG2D blocking Abs (Fig. 5B). Unlike plasmacytoid and conventional DC (CD11c⁺B220⁺NK1.1^– and CD11c⁺B220^–NK1.1^– negative fraction cells, respectively), purified BM-IKDC were cytotoxic for 76-9:C23, LL2, B16, and YAC1 targets, and cytotoxicty was partially blocked by NKG2D Abs only in the cell lines expressing the cognate ligands. In agreement with previous findings, cytotoxicity against all the targets was inhibited by TRAIL-blocking Ab (Fig. 5C). Therefore, BM-IKDC present in bulk bone marrow DC cultures share cytotoxicity properties with the previously described IKDC. To determine whether the inherent cytotoxic function of the BM-IKDC could account for tumor regression, we repeated the tumor growth assays using immunodeficient tumor-bearing host mice lacking functional NK and T cells. Here the BM-IKDC had no measurable effect on tumor growth, although parallel tumor growth assays in C57BL/6 mice showed the same growth inhibition as previously observed (Fig. 5D). Therefore, BM-IKDC are dependent on the presence of host effector cells for tumor regression following injection at a distant site.

View larger version (29K): [in this window] [in a new window]   FIGURE 5. In vitro killing capacity of BM-IKDC. A, 76-9:C23, LL/2, and B16 cell lines stained for the surface expression NKG2D ligands, RAE1, and H-60. B, FACS-sorted CD11cintB220+NK1.1+CD49b cells were incubated with 76-9:C23 or LL/2 or B16 in the presence or absence of an anti-NKG2D-specific monoclonal neutralizing Ab or its isotype control for 48 h in an ELISPOT IFN--release assay at an E:T ratio of 10:1. C, FACS-sorted CD11cintB220+NK1.1+CD49b+ killed 76-9, LL/2, B16, and YAC-1. Cytotoxicity toward 76-9:C23 and LL/2 was blocked by an NKG2D-specific mAb, whereas the NKG2D blocking effect on B16 cells is moderate. Cytotoxicity of C57BL/6 CD11cintB220+NK1.1+ toward 76-9, LL/2, and B16 was blocked by an anti-TRAIL-specific mAb. YAC-1 target cells were used as a positive control for NK killing, and negative fraction DC were effector cells in a negative control. D, FACS-sorted CD11cintB220+NK1.1+CD49b+ cells were subcutaneously injected in the contralateral flank of mice bearing 10 days of established tumors. A control group of tumor-bearing mice was left untreated. Tumor volume is shown for individual mice.

	Discussion

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Remarkably, removal of CD11c^intB220⁺NK1.1⁺CD49⁺ IKDC cells from the DC bulk culture caused virtually complete loss of the tumoricidal effect. This was unlikely to result from artifact caused by FACS sorting because add-back of the sorted BM-IKDC fraction was capable of restoring killing function to a level greater than that which had been induced by unsorted DC, and remarkably few (5 x 10³) BM-IKDC were capable of inducing antitumor response. The BM-IKDC population is shown to have TRAIL- and NKG2D-dependent NK-type cytotoxicity in vitro. Ab-blocking experiments on splenocytes from unpulsed DC-treated mice show induction of cytotoxic activity of both CD8-positive T cells and NK1.1-positive cells. Moreover, in the tumor regression we have observed induced by purified BM-IKDC, there is infiltration of tumors by activated T cells and accumulation of Ag-specific activated T cells in spleen, showing in vivo Ag presentation to have occurred. However, it is not clear whether the host or the adoptively transferred DC (or both) are responsible for the presentation. One model is that direct cytotoxicity by IKDC allows for recruitment of host DC, which take up tumor Ag and stimulate host Ag-specific T cells or NK cells. An alternate model is that IKDC perform both killing and presentation of Ags from the killed tumor cells. These different models need to be formally assessed, for example in animals lacking host Ag presentation function. Moreover, very high degrees of purity of FACS-sorted IKDC are needed to exclude Ag presentation by contaminating conventional DC for both in vitro and in vivo assessments of IKDC Ag presentation function. Similarly, experiments using unpulsed DC and BM-IKDC from perforin-deficient hosts would be one way to delineate whether direct target cell killing is an esssntial component of the tumoricidal effect. Our data showing absence of tumoricidal effect of IKDC in hosts lacking T cells and NK cells suggests that the BM-IKDC-mediated direct tumor cytotoxicity observed in vitro is insufficient to inhibit tumor growth in vivo following administration of BM-IKDC at a distant site.

A surprising finding is that as well as regression of established tumors, the unpulsed DC also inhibited outgrowth of subsequently challenged tumor (protocol 2 in Fig. 1) by stimulating the mixed T cell and NK cell response. This suggests that the adoptively transferred DC survive in vivo until the time of the tumor challenge 1 wk later and then are able to stimulate an mixed adaptive and innate immune response on encounter with tumor. This contention will need formal testing in experiments with marked DC, both conventional DC and IKDC.

Zitvogel and coworkers have previously demonstrated the capacity of IKDC derived from spleens of mice treated with IL2 and imatinib to induce the regression of B16 tumors in immunodeficient mice following intratumoral injection (20). In common with our findings, in the absence of host immune effector cells there was no effect on growth of a contralateral tumor. This suggests that in vivo cytotoxic effects of IKDC on tumor growth require direct inoculation into tumor. Alternatively, the lack of BM-IKDC effect on tumor growth in immunodeficient mice in our hands could be due to different migratory or functional NK activity of BM-IKDC compared with splenic IKDC from IL-2- and imatinib-treated mice.

Therefore, we have identified BM-IKDC as the primary cell type involved in the initiation of tumor growth inhibition induced by unpulsed bone marrow-derived DC adoptive transfer in a murine cancer model. However, some controversy has arisen regarding the identity and function of the IKDC population, which shares the same phenotype as the CD11c^intB220⁺NK1.1⁺CD49⁺ population described in this study (23, 24, 25, 28). We have elected to call these cells BM-IKDC but stress that our data, as far as they go, do not exclude the possibility that the BM-IKDC may represent activated and highly potent NK cells capable of inducing regression of established tumor through induction of secondary immune responses, but without having Ag presentation capacity in their own right. It would be important in future experiments to perform a direct comparison in vivo of the tumor inhibitory properties of BM-IKDC and conventional NK cells. The lack of tumor regression in hosts lacking functional B cells, T cells, or NK cells confirms that BM-IKDC, although cytotoxic, rely on cells of the host immune system for the majority of their tumoricidal activity in vivo. Conventional NK cells are also known to activate host APC and so this direct comparison would provide interesting data on relative tumoricidal potency.

The relationship of IKDC to NK cells and DC has been questioned in terms of ontogeny as well as function (23, 24, 25). For example, both NK and IKDC depend primarily on IL-15 and common -chain signaling for development (23, 25) and both lack expression of PU.1 (23). Analysis of published gene expression profiles suggests that splenic IKDC share a transcriptional signature with NK cells but have relatively little overlap with DC subsets (29). However, it has recently been shown that IKDC arise from progenitors of the lymphocytic lineage distinct from pDC or NK progenitors (28). It will be important to determine whether BM-IKDC within bulk DC preparations also appear most closely derived from NK cells and whether BM-IKDC from IL-15 knockout mice have similar tumoricidal activity.

Further work is needed to elucidate the mechanism of tumor regression induced by BM-IKDC. We have unequivocally demonstrated cytotoxic activity of these cells in vitro; however, the ability to present Ag in vitro and in vivo needs formal demonstration, especially as others have failed to demonstrate any Ag presentation function in IKDC derived from splenocytes (23). Moreover, the ability of as few as 5 x 10³ purified BM-IKDC to inhibit growth of established tumors in immunocompetent mice suggests a remarkable potency in terms of induction of antitumor immunity, as well as obvious potential clinical applications. It will be of great interest to determine whether an analogous population exists in human bone marrow- or monocyte-derived DC populations and to assess their Ag presentation and NK functions.

	Disclosures

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The authors have no financial conflicts 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 research grants from SPARKS (Sport Aiding Medical Research in Kids), Cancer Research UK, and RICC (Research into Childhood Cancer).

² Address correspondence and reprint requests to Dr. John Anderson, Unit of Molecular Haematology and Cancer Biology, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, United Kingdom. E-mail address: j.anderson{at}ich.ucl.ac.uk

³ Abbreviations used in this paper: DC, dendritic cell; BM-IKDC, bone marrow-derived IKDC; CTL, cytotoxic lymphocyte; IKDC, IFN-producing killer dendritic cells; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt; pDC, plasmacytoid DC; rm, recombinant murine.

Received for publication April 1, 2008. Accepted for publication August 21, 2008.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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