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, 193 (6), 661-70

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Contributes to Interferon Gamma-Dependent Natural Killer Cell Protection From Tumor Metastasis

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Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Contributes to Interferon Gamma-Dependent Natural Killer Cell Protection From Tumor Metastasis

M J Smyth et al. J Exp Med.

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expressed by in vitro activated natural killer (NK) cells, but the relevance of this observation to the biological function of NK cells has been unclear. Herein, we have demonstrated the in vivo induction of mouse TRAIL expression on various tissue NK cells and correlated NK cell activation with TRAIL-mediated antimetastatic function in vivo. Expression of TRAIL was only constitutive on a subset of liver NK cells, and innate NK cell control of Renca carcinoma hepatic metastases in the liver was partially TRAIL dependent. Administration of therapeutic doses of interleukin (IL)-12, a powerful inducer of interferon (IFN)-gamma production by NK cells and NKT cells, upregulated TRAIL expression on liver, spleen, and lung NK cells, and IL-12 suppressed metastases in both liver and lung in a TRAIL-dependent fashion. By contrast, alpha-galactosylceramide (alpha-GalCer), a powerful inducer of NKT cell IFN-gamma and IL-4 secretion, suppressed both liver and lung metastases but only stimulated NK cell TRAIL-mediated function in the liver. TRAIL expression was not detected on NK cells from IFN-gamma-deficient mice and TRAIL-mediated antimetastatic effects of IL-12 and alpha-GalCer were strictly IFN-gamma dependent. These results indicated that TRAIL induction on NK cells plays a critical role in IFN-gamma-mediated antimetastatic effects of IL-12 and alpha-GalCer.

Figures

Figure 1
Figure 1
IL-12 and α-GalCer induce TRAIL expression on NK cells. MNCs from the liver, spleen, lung, and peripheral blood (PBMC) were isolated from PBS or vehicle-treated (control) BALB/c mice or IL-12 (500 U intraperitoneally on days −5, −4, −3, −2, and −1), α-GalCer (α-GC; 2 μg intraperitoneally on days –8, −4, and 0), or IL-2 (100,000 U intraperitoneally on days −5, −4, −3, −2, and −1)–treated BALB/c mice at 24 h after the treatment. These cells were stained with biotin-conjugated anti-TRAIL mAb followed by PE-conjugated streptavidin, FITC-conjugated anti-DX5 mAb, and Cy-Chrome–conjugated anti-CD3 mAb. (A) DX5 (y-axis) vs. CD3 (x-axis) staining profiles of MNCs from each organ after the indicated treatments. Percentages of DX5+CD3 cells and DX5+CD3+ cells are indicated at the top left and top right, respectively. Two- to threefold more cells were present in the livers and spleens of IL-12–, α-Gal Cer–, or IL-2–treated mice. (B) Expression of TRAIL was analyzed on electronically gated CD3DX-5+ (NK) cells, CD3+DX-5 (T) cells, CD3+DX-5+ T cells, and CD3DX-5 (DN) cells by flow cytometry. Bold lines indicate staining with anti-TRAIL mAb and plain lines indicate staining with isotype-matched control IgG2a. These analyses (A and B) have been performed on more than three occasions and these profiles are representative.
Figure 1
Figure 1
IL-12 and α-GalCer induce TRAIL expression on NK cells. MNCs from the liver, spleen, lung, and peripheral blood (PBMC) were isolated from PBS or vehicle-treated (control) BALB/c mice or IL-12 (500 U intraperitoneally on days −5, −4, −3, −2, and −1), α-GalCer (α-GC; 2 μg intraperitoneally on days –8, −4, and 0), or IL-2 (100,000 U intraperitoneally on days −5, −4, −3, −2, and −1)–treated BALB/c mice at 24 h after the treatment. These cells were stained with biotin-conjugated anti-TRAIL mAb followed by PE-conjugated streptavidin, FITC-conjugated anti-DX5 mAb, and Cy-Chrome–conjugated anti-CD3 mAb. (A) DX5 (y-axis) vs. CD3 (x-axis) staining profiles of MNCs from each organ after the indicated treatments. Percentages of DX5+CD3 cells and DX5+CD3+ cells are indicated at the top left and top right, respectively. Two- to threefold more cells were present in the livers and spleens of IL-12–, α-Gal Cer–, or IL-2–treated mice. (B) Expression of TRAIL was analyzed on electronically gated CD3DX-5+ (NK) cells, CD3+DX-5 (T) cells, CD3+DX-5+ T cells, and CD3DX-5 (DN) cells by flow cytometry. Bold lines indicate staining with anti-TRAIL mAb and plain lines indicate staining with isotype-matched control IgG2a. These analyses (A and B) have been performed on more than three occasions and these profiles are representative.
Figure 2
Figure 2
IL-12 and α-GalCer induce TRAIL-mediated cytotoxicity. Liver, spleen, and lung MNCs were isolated (day 0) from PBS or vehicle-treated (control) BALB/c mice or IL-12 (500 U intraperitoneally on days −5, −4, −3, −2, and −1), α-GalCer (α-GC; 2 μg intraperitoneally on days −8, −4, and 0), or IL-2 (100,000 U intraperitoneally on days −5, −4, −3, −2, and −1)–treated BALB/c mice at 24 h after the treatment. Their cytotoxic activities were tested against TRAIL-sensitive Renca tumor cells in the presence or absence of 50 nM CMA, 10 μg/ml of anti-TRAIL mAb, or 10 μg/ml of control rat IgG2a mAb (control IgG) by 8 h 51Cr-release assay at several effector/target ratios (100:1 spleen MNCs, 50:1 liver and lung MNCs, shown). Data are represented as the mean ± SD of triplicate samples. Similar results were obtained in three independent experiments. *P < 0.05, compared with control IgG by two sample t tests.
Figure 3
Figure 3
The antimetastatic activity of IL-12– or α-GalCer–activated liver NK cells involves TRAIL effector function. Groups of 5 to 20 WT mice were inoculated intrasplenically with (A) between 1 × 104 and 3 × 105 Renca tumor cells or (B) 3 × 105 Renca tumor cells on day 0. As indicated, some groups of mice were treated with one or more of: 500 U IL-12 intraperitoneally on days 3 through 7; 100,000 U IL-2 intraperitoneally on days 3 through 7; 2 μg α-GalCer (α-GC) intraperitoneally on days 0, 4, and 8; 20 μg anti-asGM1 on days −1, 0, and 7; and 0.25 mg anti-TRAIL mAb or control IgG intraperitoneally on days 0, 1, and 7. The livers were removed from mice on day 14, and the metastatic nodules quantified. Data are recorded as the mean ± SE, with the significance of anti-TRAIL mAb-treated mice compared with untreated or control IgG-treated mice as defined by Mann Whitney U test, *P < 0.05, **P < 0.01. IL-12, α-GalCer, and IL-2 were also statistically effective alone compared with no treatment in the livers of WT mice (P < 0.01). There was no effect of rabbit IgG (control for anti-asGM1) on the efficacy of α-GalCer, IL-12, or IL-2 (data not shown).
Figure 4
Figure 4
IL-12 stimulates TRAIL-mediated antimetastatic activity in the lung. Groups of 5 to 20 WT mice were inoculated intravenously with (A) between 104 and 105 Renca tumor cells or (B) 105 Renca tumor cells on day 0. As indicated, some groups of mice were treated with one or more of: 500 U IL-12 intraperitoneally on days 3 through 7; 100,000 U IL-2 intraperitoneally on days 3 through 7; 2 μg α-GalCer (α-GC) intraperitoneally on days 0, 4, and 8; 20 μg anti-asGM1 on days −1, 0, and 7; and 0.25 mg anti-TRAIL mAb or control IgG intraperitoneally on days 0, 1, and 7. The lungs were removed from mice on day 14, and the metastatic nodules quantified. Data are recorded as the mean ± SE, with the significance of anti-TRAIL mAb-treated mice compared with untreated or control IgG-treated mice as defined by Mann Whitney U test; *P < 0.05, **P < 0.01. IL-12, α-GalCer, and IL-2 were also statistically effective alone compared with no treatment in the livers of WT mice (P < 0.01). There was no effect of rabbit IgG (control for anti-asGM1) on the efficacy of α-GalCer, IL-12, or IL-2 (data not shown).
Figure 5
Figure 5
Key role of IFN-γ in TRAIL-mediated antimetastatic effect of IL-12. Groups of 5 to 15 IFN-γ−/− or pfp−/− mice were inoculated (A) intrasplenically with 3 × 105 Renca tumor cells or (B) intravenously with 1 × 105 Renca tumor cells on day 0. As indicated, some groups of mice were treated with: 500 U IL-12 intraperitoneally on days 3 through 7 and/or 0.25 mg anti-TRAIL mAb or control IgG intraperitoneally on days 0, 1, and 7. The (A) livers and (B) lungs were removed from mice on day 14, and the metastatic nodules quantified. Data are recorded as the mean ± SE, with the significance of anti-TRAIL mAb-treated mice compared with untreated or control IgG-treated mice as defined by *P < 0.05, **P < 0.01, Mann-Whitney U test. IL-12 was statistically effective alone compared with no treatment in the livers and lungs of both pfp−/− and IFN-γ−/− mice (P < 0.01). Control IgG was without effect in the experiment shown in B (data not shown).
Figure 6
Figure 6
Role of IFN-γ and TRAIL in antimetastatic effect of α-GalCer. Groups of five IFN-γ−/− or pfp−/− mice were inoculated (A) intrasplenically with 3 × 105 Renca tumor cells or (B) intravenously with 1 × 105 Renca tumor cells on day 0. As indicated, some groups of mice were treated with: 2 μg α-GalCer (α-GC) intraperitoneally on days 0, 4, and 8 and/or 0.25 mg anti-TRAIL mAb on days 0, 1, and 7. The (A) livers and (B) lungs were removed from mice on day 14, and the metastatic nodules quantified. Data are recorded as the mean ± SE, with the signi-ficance of anti-TRAIL mAb-treated mice compared with mice treated with α-GalCer alone as defined by *P < 0.05, **P < 0.01, Mann-Whitney U test. Control IgG was without effect in pfp−/− or IFN-γ−/− mice (data not shown). α-GalCer was statistically effective alone compared with no treatment in the livers and lungs of pfp−/− mice (P < 0.01).
Figure 7
Figure 7
Correlation between IFN-γ activity and NK cell TRAIL expression. (A) Serum was collected from untreated (▪) or anti-asGM1–treated BALB/c WT mice (•) or BALB/c CD1d−/− mice (▴) as indicated, and mouse IFN-γ measured by a specific ELISA. (B) Sera were also taken 24 h after treatment as per the Renca tumor therapy regime (above) from IL-12– (black bars), α-GalCer– (white bars), or IL-2 (striped bars)–treated mice. For A and B, data represent the mean ± SE (ng/ml) of duplicate samples from three different mice. The serum of all untreated mice contained <50 pg/ml of IFN-γ. (C) MNCs from the liver and spleen were isolated from PBS- or vehicle-treated (control) BALB/c IFN-γ−/− mice at 24 h after the treatment with IL-12 (500 U intraperitoneally on days −5, −4, −3, −2, and −1) or α-GalCer (α-GC; 2 μg intraperitoneally on days −8, −4, and 0), or at 3 h after the treatment with IFN-γ (50,000 U intraperitoneally). These cells were stained with biotin-conjugated anti-TRAIL mAb followed by PE-conjugated streptavidin, FITC-conjugated anti-DX5 mAb, and Cy-Chrome–conjugated anti-CD3 mAb. Expression of TRAIL was analyzed on electronically gated CD3DX-5+ (NK) cells by flow cytometry. Bold lines indicate staining with anti-TRAIL mAb and plain lines indicate staining with isotype-matched control IgG2a.
Figure 7
Figure 7
Correlation between IFN-γ activity and NK cell TRAIL expression. (A) Serum was collected from untreated (▪) or anti-asGM1–treated BALB/c WT mice (•) or BALB/c CD1d−/− mice (▴) as indicated, and mouse IFN-γ measured by a specific ELISA. (B) Sera were also taken 24 h after treatment as per the Renca tumor therapy regime (above) from IL-12– (black bars), α-GalCer– (white bars), or IL-2 (striped bars)–treated mice. For A and B, data represent the mean ± SE (ng/ml) of duplicate samples from three different mice. The serum of all untreated mice contained <50 pg/ml of IFN-γ. (C) MNCs from the liver and spleen were isolated from PBS- or vehicle-treated (control) BALB/c IFN-γ−/− mice at 24 h after the treatment with IL-12 (500 U intraperitoneally on days −5, −4, −3, −2, and −1) or α-GalCer (α-GC; 2 μg intraperitoneally on days −8, −4, and 0), or at 3 h after the treatment with IFN-γ (50,000 U intraperitoneally). These cells were stained with biotin-conjugated anti-TRAIL mAb followed by PE-conjugated streptavidin, FITC-conjugated anti-DX5 mAb, and Cy-Chrome–conjugated anti-CD3 mAb. Expression of TRAIL was analyzed on electronically gated CD3DX-5+ (NK) cells by flow cytometry. Bold lines indicate staining with anti-TRAIL mAb and plain lines indicate staining with isotype-matched control IgG2a.
Figure 7
Figure 7
Correlation between IFN-γ activity and NK cell TRAIL expression. (A) Serum was collected from untreated (▪) or anti-asGM1–treated BALB/c WT mice (•) or BALB/c CD1d−/− mice (▴) as indicated, and mouse IFN-γ measured by a specific ELISA. (B) Sera were also taken 24 h after treatment as per the Renca tumor therapy regime (above) from IL-12– (black bars), α-GalCer– (white bars), or IL-2 (striped bars)–treated mice. For A and B, data represent the mean ± SE (ng/ml) of duplicate samples from three different mice. The serum of all untreated mice contained <50 pg/ml of IFN-γ. (C) MNCs from the liver and spleen were isolated from PBS- or vehicle-treated (control) BALB/c IFN-γ−/− mice at 24 h after the treatment with IL-12 (500 U intraperitoneally on days −5, −4, −3, −2, and −1) or α-GalCer (α-GC; 2 μg intraperitoneally on days −8, −4, and 0), or at 3 h after the treatment with IFN-γ (50,000 U intraperitoneally). These cells were stained with biotin-conjugated anti-TRAIL mAb followed by PE-conjugated streptavidin, FITC-conjugated anti-DX5 mAb, and Cy-Chrome–conjugated anti-CD3 mAb. Expression of TRAIL was analyzed on electronically gated CD3DX-5+ (NK) cells by flow cytometry. Bold lines indicate staining with anti-TRAIL mAb and plain lines indicate staining with isotype-matched control IgG2a.

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