Article Figures & Data
Figures
- FIGURE 1.
Cytokine suppression of B16F10 lung metastases requires DNAM-1. A, Groups of 5–20 B6 WT and DNAM-1−/− mice were challenged i.v. with 2 × 105 B16F10 tumor cells as described in Materials and Methods. Groups of mice received PBS or cytokine treatment as follows: 100,000 IU IL-2 i.p. on day −1 relative to tumor inoculation, 50 μg IL-21 i.p. on days −1, 0, 1 and 2, or 500 ng IL-12 i.p. on days −1 and 0. B, Some groups of B6 WT mice were treated with either cIg or anti-mouse DNAM-1 (250 μg i.p. on days −1, 0, and 7 after tumor inoculation). Lungs were harvested on day 14 and fixed in Bouin’s solution. Tumor colonies were counted under a dissecting microscope. Data are depicted as individual mice (each symbol) and mean tumor colonies ± SEM (cross bar) for each group. Statistical differences between treated WT and DNAM-1−/− mice or cIg-treated and anti–DNAM-1–treated WT mice are depicted by asterisks. Mann-Whitney U test, p < 0.05.
- FIGURE 2.
IL-2 mediated suppression of B16F10 lung metastases requires early NK cell and DNAM-1/CD155 function. A, Groups of 5 B6 WT and DNAM-1−/− mice were challenged i.v. with 2 × 105 B16F10 tumor cells. Groups of mice received PBS or 100,000 IU IL-2 i.p. on day −1 relative to tumor inoculation. Some groups of B6 WT or DNAM-1−/− mice received cIg or were depleted of CD8+ T lymphocytes or NK cells in vivo, by treatment with anti-CD8 mAb or anti-asGM1, respectively, on days −1, 0, and 7. B, Groups of B6 WT mice were treated with either cIg, anti-mouse DNAM-1 (250 μg i.p.), or anti-asGM1 (100 μg) early (on days −1, 0, and 7; d-1) or late (on days 3, 4, and 10; d3). C, Groups of 10 B6 WT and DNAM-1−/− mice were challenged i.v. with 2 × 105 B16F10 tumor cells. Groups of mice received PBS or 100,000 IU IL-2 i.p. on day −1. Some groups of B6 WT or DNAM-1−/− mice were treated with either cIg or anti-mouse CD155 (250 μg i.p.) on days −1, 0, and 7. Lungs were harvested on day 14 and fixed in Bouin’s solution. Tumor colonies were counted under a dissecting microscope. Data are depicted as individual mice (each symbol) and mean tumor colonies ± SEM (cross bar) for each group. Statistical differences between (A) subset depleted and control treated mice and (B, C) treated WT and DNAM-1−/− or anti–DNAM-1–treated or anti-CD155–treated WT mice are depicted by asterisks. Mann-Whitney U test, p < 0.05.
- FIGURE 3.
DNAM-1-mediated suppression correlates with perforin function. Groups of 5 B6 WT, B6 pfp−/−, B6 IFN-γ−/−, and B6 pfp−/− × IFN-γ−/− mice were challenged i.v. with 2 × 105 B16F10 tumor cells as described in Materials and Methods. Groups of mice received (A) PBS or (B) 100,000 IU IL-2 i.p. on day −1 relative to tumor inoculation. Some groups of B6 WT mice were treated with either cIg or anti-mouse DNAM-1 (250 μg i.p. on days −1, 0, and 7). Lungs were harvested on day 14 and fixed in Bouin’s solution. Tumor colonies were counted under a dissecting microscope. Data are depicted as individual mice (each symbol) and mean tumor colonies ± SEM (cross bar) for each group. Statistical differences between cIg-treated and anti–DNAM-1–treated mice are depicted by asterisks. Mann-Whitney U test, p < 0.05.
- FIGURE 4.
Expression of NK cell ligands on B16F10, B16, and B16 derivatives. NK cell ligand expression was measured by flow cytometry on B16F10, B16, and B16 derivatives derived from cell culture. Isotype staining is shown as filled histograms, and expression of the ligand of interest is shown as open histograms. Histograms are representative of two independent experiments.
- FIGURE 5.
NKG2D-mediated suppression of B16-Rae-1 does not require DNAM-1. Groups of 5 B6 WT and DNAM-1−/− mice were challenged i.v. with (A) 2 × 105 B16-Rae-1ε, (B) 1 × 105 B16, (C) 7.5 × 105 B16-Rae-1ε, and (D) 2 × 105 B16 tumor cells. A and B, Groups of B6 WT or DNAM-1−/− mice were treated with either anti-asGM1 (100 μg i.p. on days −1, 0, and 7 after tumor inoculation), cIg, anti-mouse DNAM-1 or anti-NKG2D (250 μg i.p. on days −1, 0, and 7). C and D, Groups of mice additionally received PBS or 100,000 IU IL-2 i.p. on day −1. Lungs were harvested on day 14 and fixed in Bouin’s solution. Tumor colonies were counted under a dissecting microscope. Data are depicted as individual mice (each symbol) and mean tumor colonies ± SEM (cross bar) for each group. Statistical differences between cIg-treated and anti–DNAM-1–treated WT mice or DNAM-1−/− mice and are depicted by asterisks. Mann-Whitney U test, p < 0.05.
- FIGURE 6.
Costimulator-mediated suppression of B16-CD70 or -CD80 by NK cells requires DNAM-1. Groups of 5 B6 WT mice were challenged i.v. with 2 × 105 (A) B16-CD70 or (B) B16-CD80 tumor cells. Groups of B6 WT mice were treated with either anti-asGM1 (100 μg i.p. on days −1, 0, and 7 after tumor inoculation), cIg, anti-mouse DNAM-1, anti-CD70, anti-CD80 or a combination (250 μg i.p. on days −1, 0, and 7). Some groups of mice additionally received PBS or 100,000 IU IL-2 i.p. on day −1. Lungs were harvested on day 14 and fixed in Bouin’s solution. Tumor colonies were counted under a dissecting microscope. Data are depicted as individual mice (each symbol) and mean tumor colonies ± SEM (cross bar) for each group. Statistical differences between cIg-treated and Ab-treated mice are depicted by asterisks. Mann-Whitney U test, p < 0.05.
- FIGURE 7.
B16-CD70 or -CD80 suppression requires host DNAM-1. Groups of 5 B6 WT or DNAM-1−/− mice were challenged i.v. with (A) 2 × 105 B16, (B) 7.5 × 105 B16-CD70 or (C) 7.5 × 105 B16-CD80 tumor cells. Groups of mice additionally received PBS or 100,000 IU IL-2 i.p. on day −1 relative to tumor inoculation. Lungs were harvested on day 14 and fixed in Bouin’s solution. Tumor colonies were counted under a dissecting microscope. Data are depicted as individual mice (each symbol) and mean tumor colonies ± SEM (cross bar) for each group. Statistical differences between WT and DNAM-1−/− mice are depicted by asterisks. Mann-Whitney U test, p < 0.05.
- FIGURE 8.
DNAM-1 is not required for NKG2D-mediated and ADCC. IL-2–activated NK cells from mice of the indicated genotypes were tested for ex vivo cytotoxicity against parental B16 targets and variants expressing different ligands in a standard 4-h 51Cr-release assay. IL-2 activated NK cell-mediated cytotoxicity of (A) B16 in the presence of cIg, anti–DNAM-1 or anti-CD155; (B) B16-Rae-1ε; (C) B16-CD70; and (D) B16-CD80. Splenocytes (E) or IL-2–activated NK cells (F) were also tested for ADCC against NP-labeled B16 targets in the presence (+) or absence (−) of anti-NP Ab at several E:T ratios as shown. Each data point is the mean ± SEM of two independent experiments done in triplicate for each effector/target ratio.
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