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The Journal of Immunology, 2006, 176: 4113-4124.
Copyright © 2006 by The American Association of Immunologists

Immature NK Cells Suppress Dendritic Cell Functions during the Development of Leukemia in a Mouse Model

Kazumi Ebata*, Yukihiro Shimizu1,*, Yasuhiro Nakayama*, Masami Minemura*, Jun Murakami*, Tsutomu Kato*, Satoshi Yasumura*, Terumi Takahara*, Toshiro Sugiyama* and Shigeru Saito{dagger}

* The Third Department of Internal Medicine, Faculty of Medicine, and {dagger} Department of Obstetrics and Gynecology, Faculty of Medicine, University of Toyama, Toyama, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
To analyze the mechanisms by which cancer cells escape from hosts’ immune surveillance, we investigated the changes in immune status during the progression of leukemia induced by injecting mice with WEHI-3B cells. In the bone marrow (BM) of leukemic mice, only DX5+CD3– cells were continuously increased, despite the progression of leukemia. In addition, DX5+CD3– cells were rapidly increased in peripheral blood (PB) 20 days after inoculation. We also found that myeloid dendritic cells (DCs) expressing low levels of I-Ad and having low allo-T cell stimulatory activity were markedly increased in PB and spleen. The increase in DX5+ cells in BM was thought to be induced by soluble factors from leukemic cells. DX5+ cells from leukemic mice were CD3–, B220–, Gr-1–, CD14–, CD94–, Ly-49C/F–, asialo GM1+, CD25+, CD122+, Thy-1bright, and c-kitdim and showed low killing activity against YAC-1 cells, suggesting that those DX5+ cells were immature NK cells. NK cells from leukemic PB down-regulated the expression of I-Ad on DCs, an effect mediated by TGF-beta. Moreover, these NK cells significantly suppressed the allo-T cell stimulatory activity of DCs, an effect requiring cell-to-cell contact between NK cells and DCs and thought to involve CD25. Importantly, NK cells from leukemic PB inhibited generation of autotumor-specific CTL induced by DCs in primary MLR or by DC immunization. In conclusion, we identified circulating immature NK cells with immunosuppressive activities. These cells may be important for understanding the involvement of the host immune system during the development of leukemia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Malignant neoplasms are generally fatal diseases if they are diagnosed at advanced stages. Many tumors express tumor-associated Ag, enabling the recognition of cancer cells by cells of the host’s immune system (1). Although tumor-associated Ag-specific T cells have been detected in the circulation of patients with cancer (2), cancer cells usually continue proliferating by escaping from the host’s immune attack (3).

Among the many types of immune cells involved in anticancer immunity are dendritic cells (DCs)2 and NK cells. In patients with malignant diseases, however, DCs and/or NK cells have been reported to be quantitatively and/or functionally defective (4, 5, 6, 7). For example, in patients with breast or pancreatic cancer, the numbers of circulating DCs were reduced, and their allo-T cell stimulatory ability was impaired (4, 5). As DCs are potent APCs, which can activate both innate and acquired immunity (8, 9), functional impairment of DCs may be one of the mechanisms by which a tumor can escape from the control of the immune system.

NK cells can kill various cancer cells that express little or no MHC Ags on their surface (10, 11). Cells from hematological tumors are often more sensitive to NK-mediated killing than are cells from solid tumors (12), suggesting that NK cells are especially important in the control of hematological malignancies.

The kinetics of functional changes in DCs and NK cells during the development of hematological malignancies, however, have not been analyzed. Moreover, although NK cells have been reported to regulate immune responses in autoimmune diseases (13, 14, 15, 16), it is not known whether they possess similar functions in malignant diseases.

We have established a leukemia model in BALB/c mice by i.p. injection of the monocyte-derived leukemic cell line, WEHI-3B (17, 18). In this model, only DX5+ cells in bone marrow (BM) were continuously increased during the progression of leukemia, despite a rapid increase in leukemia cells in BM and peripheral blood (PB). We also found that DX5+ cells in PB were increased 24 days after inoculation. We therefore characterized these DX5+ cells and analyzed their functional properties by focusing on their effects on the expression of phenotypic markers and functions of DCs, a population that was also markedly increased during tumor progression in this model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Six- to 8-wk-old female BALB/c (H-2d) and C57BL/6 (H-2Kb) mice were purchased from Japan SLC. All animal protocols were approved by the University of Toyama Committee on Animal Welfare.

Cell lines

WEHI-3B cells (H-2d, mouse myelomonocytic leukemia cell line, IFO50296) were purchased from Health Science Research Resources Bank (Osaka, Japan) and cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. YAC-1 cells were purchased from RIKEN BioResource Center (Ibaraki, Japan).

Establishment of a leukemia model in BALB/c mice

WEHI-3B cells were i.p. injected into BALB/c mice (1 x 104/mouse) to establish a leukemia model, as reported previously with minor modifications (18).

Preparation of mononuclear cells from PB, spleen, and BM

Mononuclear cells were isolated from PB by density gradient centrifugation (specific gravity, 1.090). Spleen cells were isolated by mechanical disruption and pipetting, followed by filtration through nylon mesh (pore size, 70 µm; BD Biosciences). BM cells were obtained from bilateral tibiae and femurs by gentle pipetting, followed by filtration through nylon mesh.

Flow cytometry

For the analysis of surface markers, cells were suspended in PBS containing 0.1% NaN3 and 1% FBS, and 2 x 105 cells/tube were incubated with anti-CD16/32 Ab (eBioscience) for Fc receptor blocking, followed by incubation with specific Ab for 20 min at 4°C. Among the anti-mouse Abs used were FITC-conjugated anti-MHC class II (I-Ad; Cedarlane Laboratories), anti-CD3 (Immunotech), anti-MHC class I (H-2Ld), anti-DX5, anti-CD45R (B220), Ly-6G (Gr-1), and anti-Ly-49C/F (eBioscience); PE-conjugated anti-DX5 (Southern Biotechnology Associates), anti-CD11c, anti-CD14, anti-CD90.2 (Thy-1.2; BD Biosciences), anti-CD25, anti-CD94, anti-CD117 (c-kit), and anti-CD122 (eBioscience); biotin-conjugated anti-CD11b and anti-B220 (BD Biosciences); and purified anti-asialo GM1 (rabbit; Wako Pure Chemical). After incubation with biotin-conjugated Abs and purified anti-asialo GM1 Ab, the cells were incubated with allophycocyanin-conjugated streptavidin (Biomedia) and FITC-conjugated goat IgG F(ab')2 fraction to rabbit IgG (ICN Pharmaceuticals), respectively. Isotype controls were used for the determination of negative cells. The stained cells were washed twice with washing buffer, fixed in a 2% paraformaldehyde solution and analyzed on a FACSCalibur flow cytometer (BD Biosciences).

DCs were defined as I-Ad+CD11c+ cells and were divided into two subsets, myeloid (CD11b+) and plasmacytoid (B220+) DCs.

Cell separation by magnetic bead-conjugated Abs

CD11c+ and DX5+ cells were separated using Abs conjugated to microbeads. In brief, mononuclear cells from PB, spleen, or BM were suspended in 2% FBS/PBS and incubated with anti-CD16/CD32 (eBioscience) for FcR blocking. The cells were then incubated with anti-CD11c or anti-DX5 Ab-conjugated MACS microbeads (Miltenyi Biotec) for 20 min at 4°C, and each cell population was separated by MACS separation columns MS (Miltenyi Biotec), according to the manufacturer’s instructions. The purity of each cell population was >98%. The cells were stained with May-Grünwald-Giemsa and their morphology was examined under a light microscope.

Allo-T cell stimulatory activity of splenic DCs in leukemic mice

To examine the function of splenic DCs in leukemic mice, CD11c+ cells, isolated from the spleens of normal and leukemic mice using anti-CD11c-conjugated MACS microbeads, were seeded in 96-well flat-bottom wells at 1 x 104 cells/100 µl. To each well was added 2 x 105 allo-T cells, which had been separated (in 100 µl) from the spleens of C57BL/6 mice by a nylon wool-column method. The mixed cultures were maintained at 37°C, 5% CO2 for 5 days, with the addition of 1 µCi of [3H]thymidine (Amersham Biosciences) to each well 18 h before harvesting. After harvesting the cells on filter paper (Skatron), responder cell proliferation was determined by measuring the radioactivity in a beta counter (Beckman LS3801; Beckman Coulter).

Cytotoxic activity of freshly isolated or IL-2-stimulated DX5+ cells

DX5+ cells isolated from PB and BM and grown in the presence or absence of IL-2 were examined for NK activity against YAC-1 cells. YAC-1 cells were labeled with 200 µCi of 51Cr-sodium chromate (Amersham Biosciences) for 60 min, with gentle mixing every 20 min, and washed three times. A total of 1 x 103 51Cr-labeled target cells were seeded in each well of a 96-well V-bottom plate. DX5+ cells from PB or BM or the same cells grown in the presence of 10 or 100 U/ml IL-2 (PeproTech) were added at three different E:T ratios (25, 12, and 6). The plates were centrifuged at 1200 rpm for 5 min and incubated for 4 h at 37°C in 5% CO2. Medium (100 µl) was collected from each well and added to 1.5 ml of liquid scintillation fluid (ACS II; Amersham Biosciences), and the radioactivity was measured in a beta counter. The percentage of cytotoxicity in each well was calculated as ((experimental cpm) – (spontaneous release mean cpm)/(maximal mean cpm) – (spontaneous release mean cpm)) x 100.

Coculture of WEHI-3B and BM cells

To analyze the mechanism by which DX5+ cells increase in BM during the progression of leukemia, we cocultured 1 x 102, 1 x 103, or 1 x 104 WEHI-3B cells and 4 x 106/ml freshly isolated BM cells in double-chambered 24-well plates with permeable inserts, Transwells (pore size 0.4 µm, Corning), or in a mixed condition. Half of the culture medium was changed on days 3 and 5. After 7 days in culture, the cells were harvested, and the percentages of DX5-, CD3-, CD11b-, CD25-, CD122-, B220-, and Gr-1-positive cells were analyzed by single histogram flow cytometry. YAC-1 cells (H-2d) were used as negative controls. For the analysis of cells in mixed cultures, only BM cells were gated by dot plot with side and forward scatter.

Expansion of DCs from BM

DCs were expanded from BM of normal mice as reported previously with minor modifications (19). Cells (2 x 106/well) were seeded in a collagen type I-coated 24-well plate (IWAKI Glass) and cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 x 10–2 mM 2-ME. DCs were expanded using 10 ng/ml recombinant mouse GM-CSF (R&D Systems) and 10 ng/ml recombinant mouse IL-4 (PeproTech), changing half the medium every other day. On day 7 of culture, loosely attached cells were collected by gentle pipetting and used as BM-derived DCs.

Effect of DX5+ cells from normal and leukemic mice on the proliferation and functions of normal BM-derived DCs

On day 2 of BM cell cultures with GM-CSF and IL-4, 12.5% of that number of MACS-purified DX5+ cells were added. To determine the necessity for cell-to-cell contact, DX5+ cells and DCs were also cocultured in Transwells (Corning), with DCs in the lower chambers and DX5+ cells in the upper chambers. DC proliferation was assayed by seeding DCs (5 x 104/well), harvested on day 5 of culture, in 96-well flat-bottom plates and adding 1 µCi of [3H]thymidine to each well 18 h before harvest. After 24 h in culture, the cells were harvested on filter papers, and the radioactivity was counted in a beta counter. To determine the immunostimulatory or immunosuppressive properties of DCs cocultured with DX5+ cells, the level of expression of I-Ad, the allo-T cell stimulatory effect, and the production of TGF-beta1 were measured. To examine the allo-T cell stimulatory effect of DCs, with or without coculture with DX5+ cells, allo-T cells were separated from the spleens of C57BL/6 mice by a nylon wool-column method. DCs were incubated in complete medium containing 30 µg/ml mitomycin C (MMC; MP Biomedicals) for 30 min at 37°C to inhibit cell proliferation, washed three times with PBS containing 2% FBS, and seeded at 1 x 104 cells/100 µl in 96-well flat-bottom plates. Finally, 2 x 105/100 µl allo-T cells was added to each well, and responder cell proliferation was determined as described earlier.

Role of TGF-beta in the effect of DX5+ cells from leukemic mice on normal BM-derived DCs

To examine the role of TGF-beta in the effect of DX5+ cells on normal BM-derived DCs, 10 µg/ml anti-TGF-beta neutralizing Ab (mouse IgG1, 1D11.16.8), produced by hybridoma cells (HB 9849; American Type Culture Collection), were added to cultures of normal BM-derived DCs with or without DX5+ cells. The expression of I-Ad on DCs was analyzed by flow cytometry. Mouse IgG1 (10 µg/ml) was used as an isotype control. To test the effect of IFN-{gamma} on I-Ad expression on DCs, recombinant murine IFN-{gamma} (5–100 U/ml; PeproTech) was added to some DC cultures.

Role of CD25 in the function of DX5+ cells

To examine the role of CD25 in the functions of DX5+ cells, 10 µg/ml anti-CD25 Ab (PC61, azide-free, rat IgG1; BD Biosciences) or rat IgG1 as an isotype control was added together with DX5+ cells to the DC cultures to block the IL-2R {alpha}-chain. The level of expression of I-Ad and the allo-T cell stimulatory activity of DCs and the production of TGF-beta1 from those cell populations were analyzed as earlier described.

Induction of autotumor-specific CTL by normal BM-derived DCs expanded in the presence or absence of PBDX5+ cells from leukemic mice

The ability to induce autologous tumor-specific CTL by DCs expanded with or without leukemic PBDX5+ cells was first examined by primary MLR (20). Splenic T cells (5 x 106/well), isolated by a nylon-wool column method from normal mice, were cocultured with MMC-treated (30 µg/ml for 30 min) DCs (2.5 x 105/well), expanded in the presence or absence of leukemic PBDX5+ cells in 24-well plates for 7 days. IL-2 (10 U/ml) was added to all cultures, and MMC-treated (100 µg/ml for 30 min) WEHI-3B cells (2.5 x 105) were added to half of total wells. CD8+ cells were isolated by MACS after in vitro culture and examined for cytotoxic activity against WEHI-3B and YAC-1 cells at three E:T ratios as previously discussed. To confirm that cytotoxicity was MHC class I-dependent, the labeled target cells were incubated with anti-H-2Ld Ab (mouse IgG2a; eBioscience) or isotype control mouse IgG2a (1–10 µg/ml) for 15 min at 4°C and used for cytotoxicity assay at an E:T ratio of 25:1.

Induction of autotumor-specific CTL in vivo after immunization with DCs expanded in the presence or absence of leukemic PBDX5+ cells

We injected DCs, expanded in the presence or absence of leukemic PBDX5+ cells, into normal mice with or without i.v. preinjection of WEHI-3B cells (1 x 106/mice) 1 day before DC injection. Spleen cells were harvested 1 wk after DC injection by mechanical disruption, and T cells were isolated by a nylon wool-column method to avoid contamination with WEHI-3B cells. Cells (2 x 106) were cultured in 24-well plates with MMC-treated (100 µg/ml for 30 min) WEHI-3B cells (1 x 105) and MMC-treated (30 µg/ml for 30 min) spleen cells (2 x 106) from normal mice in the presence of IL-2 (10 U/ml) for 7 days as reported previously with minor modifications (19). CD8+ cells were isolated by MACS and tested for their cytotoxic activity against WEHI-3B and YAC-1 cells at three E:T ratios, as previously described. Again, to confirm that cytotoxicity was MHC class I-dependent, the labeled target cells were incubated with anti-H-2Ld Ab or isotype control mouse IgG2a (1–10 µg/ml) for 15 min at 4°C and used for cytotoxicity assay at an E:T ratio of 25:1.

Measurement of cytokine concentrations in culture medium

The concentrations of TGF-beta1 and IFN-{gamma} in culture media were measured using ELISA kits obtained from TECHNE and BioSource International, respectively. Latent and active forms of TGF-beta1 were separately measured according to the kit instructions.

Semiquantitative RT-PCR

Total RNA was isolated using an RNeasy Mini kit (Qiagen). First-strand cDNA was reverse transcribed from 1 µg of total RNA using 1 µg of oligo(dT)12–18 primer and a SuperScript First-Strand Synthesis System (Invitrogen Life Technologies). The cDNA was amplified using AmpliTaq Gold DNA Polymerase (Applied Biosystems), and specific primers for beta-actin, forward, 5'-GTG GGC CGC TCT AGG CAC CAA-3', and reverse 5'-CTC TTT GAT GTC ACG CAC GAT TTC-3' (21); and TGF-beta1 (Maxim Biotech) and IFN-{gamma}, forward, 5'-TGC ATC TTG GCT TTG CAG CTC TTC CTCATG GC-3', and reverse 5'-TGG ACC TGT GGG TTG TTG ACC TCA AAC TTG GC-3' (21). The amplification profile consisted of an initial denaturation at 94°C for 10 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1.5 min. The PCR products were electrophoresed on 2% agarose gels and visualized by staining with ethidium bromide.

Statistical analysis

Results are expressed as mean ± SD. Statistical analysis was performed using the Mann-Whitney U test, and statistical significance was set at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Phenotypes of WEHI-3B cells

Flow cytometric analysis of WEHI-3B cells isolated from leukemic mice showed that they were CD11c–, DX5–, CD11b+, MHC class I+, and MHC class II (I-Ad)–.

Changes in leukemic cell numbers after i.p. inoculation of WEHI-3B cells

Following i.p. injection of 1 x 104 WEHI-3B cells per BALB/c mouse, we measured the number of white blood cells on days 0, 10, 20, 24, 27, and 30. Circulating cell numbers rapidly increased after 24 days, with the mean ± SD cell numbers being 1.76 ± 1.13 x 105/µl on day 30 (data not shown).

Changes in PB and BM cell composition after WEHI-3B inoculation

Using flow cytometry, we analyzed the cell compositions in PB and BM on days 0, 10, 20, 24, 27, and 30 after WEHI-3B inoculation, with the composition on day 0 representing data from untreated normal mice. During progression of leukemia, both I-Ad+CD11c+ DCs and DX5+CD3– cells showed marked increases in PB, although most cell types were increased. The numbers of circulating DCs were rapidly increased by day 20 (Fig. 1A), with most being I-Ad+CD11c+CD11b+ myeloid DCs (Fig. 1B). The expression level of I-Ad in DCs from PB and spleen of leukemic mice was significantly lower than levels found in normal mice (Fig. 2A). Moreover, splenic CD11c+ cells of leukemic mice showed significantly lower allo-T cell stimulatory activity than cells from normal mice (Fig. 2B).


Figure 1
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  		FIGURE 1. Analysis of cell compositions in PB at time points after WEHI-3B inoculation. A, Changes in total circulating DC numbers after inoculation of WEHI-3B cells. B, Changes in the numbers of myeloid (CD11b+) and plasmacytoid (B220+) DCs in the circulation after inoculation of WEHI-3B cells. Data represent the mean ± SD from five mice. *, p < 0.05.

 

Figure 2
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  		FIGURE 2. Expression level of I-Ad and allo-T cell stimulatory activity of DCs from normal and leukemic mice. A, Mean fluorescence intensity of I-Ad in DCs, as determined by flow cytometry. Each point represents the mean ± SD from five mice. B, Allo-T cell stimulatory activity of splenic CD11c+ cells, as measured by [3H]thymidine incorporation. Data represent the mean ± SD of triplicate assays. *, p < 0.0001; **, p < 0.05.

 
Similarly, circulating DX5+CD3– cells were abruptly increased 24 days after inoculation (Fig. 3A), although the percentages of the cell population were unchanged (data not shown). The numbers (Fig. 3B) and percentages of DX5+CD3– cells were gradually increased in BM, although leukemic cells also increased in BM during leukemia progression. On day 30, nonleukemic cells in the BM were predominantly DX5+ cells. Most DX5+ cells from PB and BM in normal and leukemic mice were CD3–, as determined by two-color flow cytometry (Fig. 4A). Further phenotypic analysis of isolated DX5+ cells showed that they were negative for B220, Gr-1, and CD14 (data not shown). Morphologically, the majority of DX5+ cells from PB and BM of normal and leukemic mice were large agranular or granular lymphocytes (Fig. 4, B and C), with morphology distinct from that of WEHI-3B cells (Fig. 4D). These results indicate that both myeloid DCs with low immunogenic phenotype and DX5+ NK cells were increased in PB and BM during the progression of leukemia.


Figure 3
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  		FIGURE 3. Changes in the numbers of circulating (A) and BM (B) DX5+CD3– cells after inoculation of WEHI-3B cells. Data represent the mean ± SD from five mice. *, p < 0.05.

 

Figure 4
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  		FIGURE 4. Phenotypic and morphologic characteristics of DX5+ cells in PB and BM from normal and leukemic mice. A, Two-color staining for CD3 and DX5 in mononuclear cells from PB and BM of normal and leukemic mice performed on day 30 after WEHI-3B inoculation. B, Morphological analysis of DX5+ cells isolated from PB and BM of normal and leukemic mice. Cells with large granules ({cjs2113}) are represented. lym, lymphocytes; seg, segmented granulocytes; mono, monocytes. Representative morphology of circulating DX5+ cells (C) and WEHI-3B cells (D) from leukemic mice.

 
Phenotypic characteristics of DX5+ cells from PB and BM

When we compared the phenotypes of DX5+ cells from PB and BM in normal and leukemic mice by flow cytometry (Fig. 5), we found that DX5+ cells from both PB and BM in normal mice expressed CD94, Ly49C/F, asialo GM1, and CD122, and were CD25–Thy-1.2dimc-kit–. In contrast, most DX5+ cells from PB and BM of leukemic mice expressed lower levels of CD94 and Ly49C/F, but expressed CD25 and were asialo GM1+Thy-1.2brightc-kitdim.


Figure 5
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  		FIGURE 5. Phenotypic characteristics of isolated DX5+ cells. Expression of CD94, Ly-49C/F, asialo GM1, CD25, CD122, Thy-1.2, and c-kit was assayed on DX5+ cells from PB and BM of normal and leukemic mice. The cells were isolated using an anti-DX5 Ab conjugated to magnetic beads.

 
Changes in the numbers of circulating DX5+CD25+ cells and percentages of CD94+ cells in circulating DX5+ cells after inoculation of WEHI-3B cells

As most DX5+ cells from leukemic mice also expressed CD25, we analyzed the changes in number of circulating DX5+CD25+ cells after WEHI-3B inoculation by flow cytometry. Untreated normal mice had 1.90 ± 0.60% of DX5+CD25+ cells in mononuclear cells from PB and their absolute numbers were 141 ± 43.3/µl. We observed an increase in those cells on day 10 and a rapid increase after day 27, with 3.89 ± 0.35 x 104 cells/µl on day 30 (Fig. 6A). In contrast, the percentage of circulating DX5+ cells that were CD94+ gradually decreased during the progression of leukemia (Fig. 6B).


Figure 6
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  		FIGURE 6. Analysis of the changes in circulating DX5+CD25+ cells and CD94+ cells after WEHI-3B inoculation. A, Change in the number of circulating DX5+CD25+ cells after inoculation of WEHI-3B cells. B, Change in the percentage of CD94+ cells in circulating DX5+ cells after inoculation of WEHI-3B cells. Data represent the mean ± SD from three mice. *, p < 0.05.

 
NK activity of DX5+ cells from PB and BM

We found that DX5+ cells from PB and BM of normal, but not leukemic, mice had NK activity (Fig. 7). When DX5+ cells from normal and leukemic mice were cultured in the presence of IL-2, however, both showed dose-dependent cytotoxicity against YAC-1 cells. Importantly, IL-2-activated DX5+ cells from leukemic and normal mice showed comparable levels of NK activity.


Figure 7
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  		FIGURE 7. Cytotoxic activity against YAC-1 cells of freshly isolated and IL-2-stimulated (10 U/ml or 100 U/ml) DX5+ cells from PB and BM of normal and leukemic mice, as determined by 4-h 51Cr release assay. Data represent the mean ± SD of triplicate assays.

 
Coculture of WEHI-3B and BM cells

To analyze the mechanism responsible for the selective increase in DX5+ cells in BM, we cocultured WEHI-3B and BM cells using Transwells, in which the two cell populations were not in direct contact, or using mixed culture, in which the two cell types were in direct contact. After coculture using Transwells for 7 days, the percentages of DX5+ and CD25+ cells were significantly increased (Fig. 8). We observed similar increases in DX5+ and CD25+ cells when the cells were cultured using mixed cultures (data not shown). In contrast, there was no increase in the cells positive for CD3, CD11b, CD122, B220, or Gr-1 in both cultures (data not shown). The percentages of both cell types was similar in both culture systems, indicating that the increase in DX5+ cells in PB and BM in our mice leukemia model may be due to soluble factors produced by WEHI-3B cells. In contrast, when we cocultured YAC-1 and BM cells, there was no effect on the percentages of DX5+ cells and CD25+ cells (Fig. 8), indicating that the selective increase in both cell types was due specifically to WEHI-3B cells.


Figure 8
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  		FIGURE 8. Percentages of DX5+ and CD25+ cells after culture of BM cells with different numbers of WEHI-3B or YAC-1 cells in Transwells for 7 days. Representative results of three experiments.

 
Effect of DX5+ cells from normal and leukemic mice on the proliferation of BM-derived DCs

When we added DX5+ cells isolated from PB or BM of normal and leukemic mice to cultures of BM-derived DCs, and determined the proliferation of the latter by [3H]thymidine incorporation, we found that none of the DX5+ cell populations had any effect on the proliferation of BM-derived DCs (data not shown).

Effect of DX5+ cells from normal and leukemic mice on the expression levels of I-Ad on DCs

We also assayed the level of expression of I-Ad on DCs after culture with or without DX5+ or DX5– cells from PB or BM of normal and leukemic mice. Using flow cytometry (Fig. 9) and both two-color staining with CD11c and single histograms of CD11c+ cells, we found that the level of I-Ad expression on DCs was markedly reduced only when DX5+ cells from leukemic PB were added to the DC cultures (Fig. 9, E–G). Moreover, this effect was observed even when the DX5+ cells were added in Transwells (Fig. 9G), indicating that soluble factors produced by the DX5+ cells were responsible for the immunosuppressive effect. We also found that blocking CD25 with a specific Ab had no effect on the reduction of I-Ad expression induced by DX5+ cells from leukemic PB (Fig. 9F).


Figure 9
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  		FIGURE 9. Expression of I-Ad on normal BM-derived DCs cultured in the absence (A) or presence of 12.5% DX5+ (B–G) or DX5– (H) cells isolated from various origins. Cells were cocultured in mixed conditions (B, D–F, and H) or in Transwells (C and G), and the percentages of I-Ad+ cells were analyzed by two-color staining with Abs to CD11c and I-Ad or as a single histogram of CD11c+ cells. Where indicated, the cultures included 10 µg/ml of a blocking Ab against CD25. Representative results are shown from five experiments.

 
To analyze the mechanism of this immunosuppression, we measured TGF-beta1 in the culture media of normal BM-derived DCs cocultured with or without DX5+ or DX5– cells (Fig. 10A). The concentrations of TGF-beta1, most of it in latent form, were significantly increased when DX5+ cells from PB of leukemic mice were added, either in mixed culture or in Transwells. In addition, the increase in TGF-beta1 concentration was not affected by blocking CD25 with a specific Ab (Fig. 10A). These results indicated that direct contact between NK cells from leukemic mice and activated DCs is not necessary, and CD25 is not involved, in the production of TGF-beta1.


Figure 10
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  		FIGURE 10. Analysis of the mechanism responsible for decreased I-Ad expression induced by PBDX5+ cells from leukemic mice. A, Concentrations of TGF-beta1 in the media of normal BM-derived DCs cultured with or without leukemic PBDX5+ cells in mixed cultures or in Transwells, and in the medium of leukemic PBDX5+ cells cultured alone (§) on day 6. Where indicated, the cultures included 10 µg/ml of a blocking Ab against CD25. TGF-beta1 concentration in the medium of DCs alone was subtracted from each value, except for that marked (§). Data represent the mean ± SD of three experiments. B, RT-PCR analysis of TGF-beta1 mRNA of DX5+ and DX5– cells separated from cocultures of DCs and DX5+ cells using an anti-DX5 Ab conjugated to magnetic beads. C, Expression of I-Ad on normal BM-derived DCs cultured alone or in the presence of 12.5% DX5+ from PB of leukemic mice, 12.5% DX5+ from PB of leukemic mice, and anti-TGF-beta neutralizing Ab (10 µg/ml) or mouse IgG1 (10 µg/ml). The percentages of I-Ad+ cells were analyzed by two-color staining with Abs to CD11c and I-Ad or single histogram of CD11c+ cells. Representative results of two independent experiments are shown. D, Concentrations of TGF-beta1 in the media of normal BM-derived DCs cultured with normal PBDX5+ cells in mixed cultures or in Transwells, and in the medium of normal PBDX5+ cells cultured alone (§). TGF-beta1 concentration in the medium of DCs alone was subtracted from each value, except where marked (§). Data represent the mean ± SD of three experiments. E, Concentrations of IFN-{gamma} in the media of normal BM-derived DCs cultured with leukemic or normal PBDX5+ cells on day 6 of DC culture. F, Expression of I-Ad on normal BM-derived DCs cultured in the presence of 12.5% leukemic PBDX5+ cells and murine rIFN-{gamma} (5 U/ml), added 24 h before harvest. The percentages of I-Ad+ cells were analyzed by two-color staining with Abs to CD11c and I-Ad or single histogram of CD11c+ cells. Representative results of two independent experiments are shown. nor, normal; leu, leukemic. *, p < 0.05.

 
When we assayed the origin of this TGF-beta1 by semiquantitative RT-PCR after separation of the DX5+ cells from the cultures, we found that both DX5+ cells and DX5– DCs showed increased expression of mRNA encoding TGF-beta1 (Fig. 10B). Addition of anti-TGF-beta neutralizing Ab, but not isotype control mouse IgG1, to the culture significantly reversed the suppressive effect on I-Ad expression of DCs, indicating that the immunosuppressive activity of DX5+ cells from leukemic PB was mediated mostly by TGF-beta (Fig. 10C). However, the concentrations of TGF-beta1 in cultures of normal PBDX5+ cells, alone or cocultured in Transwells with DCs, were even higher than those of leukemic PBDX5+ cells alone or cocultured with DCs (Fig. 10D), suggesting that another mechanism is involved in the DX5+ cell regulation of I-Ad expression on DCs. Because IFN-{gamma} is a potent stimulator of I-Ad expression, we then measured IFN-{gamma} concentrations in the media of cocultures of DCs and DX5+ cells. We found that the IFN-{gamma} concentrations in cocultures of DCs and PBDX5+ cells from normal mice were significantly higher than those in cocultures of DCs and PBDX5+ cells from leukemic mice (Fig. 10E). These results were reflected in the expression of mRNA encoding IFN-{gamma} by these DX5+ cell populations, as assessed by semiquantitative RT-PCR (Fig. 10E). We also found that the addition of a low concentration (5 U/ml for 24 h) of murine rIFN-{gamma} to DC cultures reversed the suppression of I-Ad expression induced by leukemic PBDX5+ cells (Fig. 10F). This result suggests that the absence of immunosuppressive activity in PBDX5+ cells from normal mice, despite their high production of TGF-beta1, may be due, at least in part, to the concomitant production of immunostimulatory cytokines such as IFN-{gamma}.

Effect of DX5+ cells from normal and leukemic mice on allo-T cell stimulatory activity of normal BM-derived DCs

To determine the effect of DX5+ cells on the Ag-presenting function of DCs, DX5+ cells isolated from PB or BM of normal and leukemic mice were added to cultures of BM-derived DCs, and the latter were harvested on day 7 and cocultured for 5 days with allo-T cells. We found that DCs cocultured with DX5+ cells from leukemic PB induced significantly lower proliferation of allo-T cells than did DX5– cells from the same mice (Fig. 11A). In contrast, DX5+ cells from PB and BM of normal mice or from BM of leukemic mice did not show this immunosuppressive effect. Furthermore, the effect induced by DX5+ cells from leukemic PB was not observed when the assay was performed in Transwells, indicating a requirement for cell-to-cell contact. Blocking of CD25, however, partially restored the ability of inhibition of DX5+ cells from leukemic PB to stimulate allo-T cells (Fig. 11B).


Figure 11
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  		FIGURE 11. A, Allo-T cell stimulatory activity of normal BM-derived DCs cocultured with or without DX5+ cells from various origins, as indicated. B, Effect of anti-CD25 blocking Ab on the allo-T cell stimulatory ability of normal BM-derived DCs. Rat IgG1 was used as an isotype control. Data represent the mean ± SD of three experiments. *, p < 0.05.

 
Effect of PBDX5+ cells from leukemic mice on the ability of autotumor-specific CTL induction of normal BM-derived DCs

To examine the role of PBDX5+ cells from leukemic mice in inhibiting acquired immune responses against autologous tumor cells, CTL induced by DCs expanded in the presence of PBDX5+ cells from leukemic mice were compared with those expanded in the absence of PBDX5+ cells in primary MLR of splenic T cells with or without MMC-treated WEHI-3B cells. We found that autotumor-specific CTL activity induced by DCs expanded in the presence of PBDX5+ and WEHI-3B cells was significantly lower than that of DCs expanded alone, indicating that immature DX5+ NK cells suppress the generation of autotumor-specific CTL responses in primary mixed culture by inhibiting DC functions (Fig. 12A). Although splenic CD8+ T cells showed cytotoxicity against YAC-1 cells, addition of anti-MHC class I (H-2Ld) Ab completely abolished their cytotoxicity only against WEHI-3B cells, indicating that the autotumor-specific cytotoxic activity is MHC class I-dependent. When we assayed CTL activity induced by immunization with DCs expanded in the presence or absence of PBDX5+ cells from leukemic mice, with or without preinjection of WEHI-3B cells, we found that immunization with DCs expanded in the absence of leukemic PBDX5+ cells induced autotumor-specific CTL activity only when WEHI-3B cells were preinjected (Fig. 12B). In contrast, immunization with DCs expanded in the presence of leukemic PBDX5+ cells, with or without preinjection of WEHI-3B cells, failed to induce autotumor-specific CTL activity. Similar to CTL induction in vitro, addition of anti-MHC class I Ab again completely suppressed their cytotoxicity against WEHI-3B cells but not against YAC-1 cells. These data indicate that immature DX5+ NK cells can also suppress the generation of autotumor-specific CTL response in vivo through inhibition of DC functions.


Figure 12
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  		FIGURE 12. Immature DX5+ NK cells suppress the ability of DCs to induce autotumor-specific CTL. A, Autotumor-specific CTL activity induced by primary MLR. Splenic T cells from normal mice were cocultured with (filled) or without (open) MMC-treated WEHI-3B cells and with normal BM-derived DCs expanded in the presence (triangle) or absence (circle) of leukemic PBDX5+ cells for 7 days with 10 U/ml IL-2. CD8+ cells were isolated by MACS, and cytotoxic activity against WEHI-3B and YAC-1 cells was examined by 4-h 51Cr release assay. In addition, labeled target cells were incubated with anti-H-2Ld Ab ({blacksquare}) or mouse IgG2a as an isotype control ({square}) and used for cytotoxicity assay at an E:T ratio of 25:1. Representative data from four independent experiments is shown and represent the mean ± SD of triplicate assays. B, Autotumor-specific CTL activity induced after immunization with DCs expanded with (triangle) or without (circle) leukemic PBDX5+ cells. DCs were injected into normal mice with (filled) or without (open) preinjection of WEHI-3B cells. Splenic T cells harvested 1 wk after DC injection were cultured with MMC-treated WEHI-3B cells and MMC-treated normal spleen cells in the presence of IL-2 (10 U/ml) for 7 days. CD8+ cells were isolated and examined for cytotoxic activity against WEHI-3B cells and YAC-1 cells. In addition, labeled target cells were incubated with anti-H-2Ld Ab ({blacksquare}) or mouse IgG2a as an isotype control ({square}) and used for cytotoxicity assay at an E:T ratio of 25:1. Representative data from two independent experiments is shown. Data represent the mean ± SD of triplicate assays. Ms, mouse.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
NK cells specifically kill virus-infected or transformed cells (22), and they are especially effective at killing cells with low MHC class I expression (10, 11). The molecular mechanism by which MHC class I expression can lower the susceptibility to NK cell killing has now been elucidated (11, 23, 24). NK cells specialize in killing non-self cells, an activity controlled by various cytokines, including IL-2 and IL-12 (12, 25). In this study, we have shown that, in our mouse leukemia model, there was a marked increase in the numbers of circulating myeloid DCs and DX5+ NK cells as leukemia progressed, indicating that these immunocompetent cells are not effective for controlling tumor cell proliferation or for killing tumor cells. Both circulating and splenic myeloid DCs expressed low levels of surface I-Ad, suggesting that these DCs are not immunostimulatory. In addition, splenic DCs from leukemic mice showed significantly lower allo-T cell stimulatory activity than did cells from normal mice. Although the mechanism by which myeloid DCs were increased during the progression of leukemia is unclear, the ability of NK cells to kill immature DCs (26, 27) suggests that an increase in the number of immature NK cells with low killing activity may lead to the overproduction of DCs in this model.

DX5+ cells are a heterogeneous population, and subsets of CD8+ T cells (28) and immature B cells (29) have been reported to express DX5. To confirm that our DX5+ cells are actually NK cells, we assayed their surface markers. We found that most DX5+ cells from both normal and leukemic mice were negative for markers of any other lineage, but were positive for asialo GM1. Moreover, a significant proportion of DX5+ cells from normal mice were positive for CD94, Ly-49C/F (inhibitory NK receptors), and CD122, with the former two being mature NK cell markers (30, 31, 32). In contrast, most DX5+ cells from leukemic mice were positive for CD25 and CD122, but not for CD94 and Ly-49C/F, indicating that these cells have lost their mature NK markers, while acquiring CD25 expression. Importantly, DX5+ cells from leukemic mice were Thy-1brightc-kitdim, suggesting that they were immature NK cells. Freshly isolated NK cells from leukemic mice were also shown to have low killing activity against YAC-1 cells. The diminished NK activity of this population may be associated with their low surface expression of CD94 (31). When cultured in the presence of IL-2, however, these cells were able to kill YAC-1 cells, indicating that they have the potential for killing tumor cells after cytokine activation. Their surface expression of CD25 is similar to that of regulatory CD4+ T cells, and IL-2 has been shown to play an essential role in the immunosuppressive function of the latter (33, 34, 35).

Although DCs have been reported to activate NK cells (36, 37, 38), and activated NK cells have been reported to kill DCs (26), there have been no reports showing that NK cells can suppress DC function without killing them or affecting their growth. We have shown that NK cells from leukemic PB could not kill YAC-1 cells, but could suppress I-Ad expression on activated DCs in a manner independent of CD25. TGF-beta1 concentration was increased when NK cells and DCs were cocultured, both in mixed cultures and in Transwells, and addition of anti-TGF-beta neutralizing Ab to the culture reversed the suppressed expression of I-Ad on DCs. These observations confirm that this activity was mediated mostly by TGF-beta (39, 40). Culture medium of PBDX5+ cells from normal mice contained higher concentrations of TGF-beta1 than those from leukemic mice, although normal PBDX5+ cells had no immunosuppressive activity on DCs. Although this inconsistency needs to be further analyzed, the concomitant production of immunostimulatory cytokines such as IFN-{gamma} may reverse the TGF-beta-induced inhibition of I-Ad expression on DCs.

We also found that NK cells markedly inhibited the allo-T cell stimulatory activity of normal BM-derived DCs. This effect was not due primarily to decreased I-Ad expression, because DCs cocultured with NK cells in Transwells had low surface expression I-Ad, while their allo-T cell stimulatory activity was similar to that of DCs expanded alone. This suppression requires direct cell-to-cell contact, as well as the involvement of CD25. More importantly, we have also shown that autotumor-specific CTL induction was significantly decreased, both in primary MLR with DCs and by DC immunization, when normal BM-derived DCs that were expanded in the presence of leukemic PBDX5+ cells were used as stimulants. This autotumor-specific cytotoxic activity was mediated by CD8+ CTLs, as shown by our use of purified CD8+ cells as effectors. Although splenic cells had cytotoxicity toward YAC-1 cells, this activity was not inhibited by addition of anti-MHC class I (H-2Ld) Ab, indicating that the killing mechanism or cytotoxic cell population against WEHI-3B cells was distinct from that against YAC-1 cells (41). Thus, overall, our results indicate that immature NK cells, expanded during the progression of leukemia, have immunoregulatory rather than killing function.

In this study, we have shown that immunosuppressive immature NK cells were induced by soluble factors produced by leukemic cells, suggesting that this may constitute a mechanism by which leukemic cells escape from the host’s immune responses. These results indicate that NK cells do not exist solely to fight cancer cells, but may be used by cancer cells to suppress DC function, leading to the inhibition of acquired immune responses against cancer cells.

Because injection of P815 or YAC-1 cells into BALB/c mice did not increase the number of immature NK cells (data not shown), it is not clear whether the phenomenon we described occurs in other types of malignancies. However, the percentage of CD56+ cells in the BM of two of three patients with acute monocytic leukemia was higher than the percentage found in normal BM or in the BM of patients with other types of leukemia (data not shown). Together with our observations on WEHI-3B myelomonocytic leukemia cells, this finding suggests that this phenomenon may be specific to malignancies involving monocytic cells. Further studies are required to clarify this hypothesis.

In the late 1980s, Thy-1+ BM cells were reported to have immunoregulatory functions in BM transplantation (42, 43), but these cells have not been further characterized, and their phenotypic markers have not been determined. During early pregnancy in humans and rodents, c-kit+CD25+CD122+CD16–CD56bright NK cells and Thy-1+NK1.1+ asislo GM1+ granulated metrial gland cells, respectively, were reported to accumulate in the uterine deciduas (44, 45, 46). These uterine NK cells produce TGF-beta1 and are thought to have immunoregulatory functions. The findings we present suggest that immature Thy-1+ NK cells with immunosuppressive activities may be a population common to these cells.

In conclusion, we have identified circulating immature NK cells with immunosuppressive activity in a mouse leukemia model. We found that these cells suppressed DC functions by down-regulating the expression of I-Ad or by inhibiting allo-T cell stimulatory activity without affecting their growth. This immunosuppressive activity may have been due to the enhanced production of TGF-beta by DCs and NK cells after their interaction, and is thought to involve CD25. Moreover, these NK cells were shown to suppress the ability of DCs to induce autotumor-specific CTL. This phenomenon is important for understanding the involvement of host immunity during the development of leukemia.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
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 Address correspondence and reprint requests to Dr. Yukihiro Shimizu, The Third Department of Internal Medicine, Faculty of Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan. E-mail address: yukihiro-tym{at}umin.ac.jp Back

2 Abbreviations used in this paper: DC, dendritic cell; BM, bone marrow; MMC, mitomycin C; PB, peripheral blood. Back

Received for publication February 25, 2005. Accepted for publication January 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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