(A) NCR ligand expression was measured by flow cytometry on paired LN and skin (SK) melanoma cell lines obtained from metastases of patients Mel1 and Mel16. Staining with NKp30-Fc, NKp44-Fc, and NKp46-Fc (filled histograms) was compared with staining with irrelevant CD5-Fc (open histograms). (B) Quantification and statistical analysis of NCR ligand expression on 6 unpaired LN (white bars) and skin (black bars) metastatic cell lines from 7 patients measured as fluorescence ratio calculated as MFI (mean + SD) of NKp44-Fc and NKp46-Fc staining over MFI of negative control (CD5-Fc staining). *P < 0.05, ***P < 0.0005. (C) DNAM-1 and NKG2D ligand expression (filled histograms) measured by flow cytometry on melanoma cells obtained from different anatomical sites of patients Mel1 and Mel16. Negative controls were stained with FITC-conjugated anti-mouse Ig xenoantibodies (open histograms). Histograms are representative of 2–5 independent experiments. AS, ascite metastasis; PL, pleural metastasis; PR, primary skin melanoma.

(A) Expression of the indicated ligands (filled histograms) relative to background staining determined by isotype-matched controls (open histograms) on single-cell suspensions prepared from tumor lesions freshly explanted from RET transgenic mice. CD45 expression was used to discriminate tumor infiltrating lymphocytes (CD45⁺) from the melanoma cells (CD45^–). Histograms are representative of 4 individual mice that displayed similar stages of disease progression as judged by tumor size. (B) Expression of the indicated ligands (filled histograms) relative to background staining determined by isotype-matched controls (open histograms) on melanoma cell lines. Histograms are representative of 2–5 independent experiments.

(A) Allogeneic IL-2–activated NK cells from healthy donors were incubated at a 20:1 E:T ratio in the presence of the indicated combinations of the blocking monoclonal antibody, with tumor cells derived from paired melanoma cell lines of LN and skin metastases obtained from patient Mel16 and subjected to cytotoxicity assays. Cells incubated with control (Ctrl) IgG and control IgM antibodies were used as isotype controls. Data are mean + SD of 6 independent experiments, of which 4 included a control sample in which no monoclonal antibody was added (data not shown) and 2 included control IgG or IgM monoclonal antibody. (B) IL-2–activated WT and Ncr1^–/– NK cells were co-incubated with the indicated melanoma cells at a 20:1 E:T ratio in the presence of control IgG or anti–DNAM-1 monoclonal antibody and subjected to cytotoxicity assays. Data are the mean + SEM of 3 WT and 4 Ncr1^–/– mice and are pooled from 3 independent experiments. **P < 0.01 relative to WT cells in the presence of control IgG in that group.

(A and B) Resting allogeneic NK cells from healthy donors were co-incubated with paired melanoma cell lines from LN and skin metastases obtained from patients Mel14 and Mel16 and subjected to cytotoxicity assays. Data are mean ± SEM of results from 6 experiments. *P < 0.05, **P < 0.005 by Student’s t test. (C and D) Autologous NK cells were co-incubated with paired melanoma cell lines from LN and skin metastases obtained from patients Mel14 and Mel16 and subjected to cytotoxicity assays. Data are from 1 experiment performed. (E) Resting allogeneic NK cells from healthy donors were co-incubated with unpaired melanoma cell lines from LN (open symbols) and skin (filled symbols) metastases obtained from different patients and subjected to cytotoxicity assays. Data are representative of 1 of 4 experiments. (F) Resting allogeneic NK cells from healthy donors were co-incubated with melanoma cell lines from LN metastases, primary uveal melanoma (PRU), and uveal melanoma liver metastases (LMs) and subjected to cytotoxicity assays. Data are representative of 1 of 3 experiments.

(A) Cells (1 × 10⁴) were injected s.c. into control IgG–treated or NK1.1-depleted syngeneic B6 mice. Tumor growth was monitored daily until mice were killed at day 14. Data are mean ± SEM of 5 mice per group. ***P < 0.01 NK1.1 compared with control IgG at each time point. (B) RET cells (3 × 10⁴) were injected i.v. into B6, Rag2^–/–, and Rag2^–/–Il2rg^–/– mice. Lung metastases were counted 14 days later. Results are representative of 3 independent experiments. Data are mean + SEM of 4 mice per group. **P < 0.001 for Rag2^–/–Il2rg^–/– compared with B6. (C) CFSE-labeled RET cells (2 × 10⁵) were injected i.p. into B6, Rag2^–/–, and Rag2^–/–Il2rg^–/– mice, and the number of residual RET cells in the peritoneal cavity were counted following lavage 48 hours later. Results are representative of 4 independent experiments. Data are mean + SEM of 5 mice per group. *P < 0.05 for Rag2^–/–Il2rg^–/– compared with B6.

(A) CFSE-labeled RET cells (2 × 10⁵) were injected i.p. into control IgG– and DNAM-1–treated or NK1.1-depleted B6 mice, and the number of residual RET cells were counted 48 hours later. Representative plots of peritoneal lavage CFSE gating are shown. (B) Quantification of residual cells. Data are mean + SEM of 8–10 mice per group and are pooled from 2 experiments. *P < 0.05 compared with control. (C) NK cells were expanded for 5 days in IL-2, sorted into DNAM-1⁺ and DNAM-1^–, recultured for 48 hours, and tested for cytotoxicity at the indicated E:T ratio. Results are representative of 2–3 experiments. (D) NK cells from WT or Ncr1^–/– mice were expanded, sorted, recultured as described in C, and injected i.p. into Rag2^–/–Il2rg^–/– mice (2.5 × 10⁵/mouse) immediately after i.p. injection of 5 × 10⁵ CFSE-labeled RET cells. Residual RET cells were counted following lavage 48 hours later. Results are individual mice from 3 independent experiments. The bar represents the mean of 8–9 mice per group. (E and F) RET cells (10⁵) were injected i.v. into control IgG, anti–DNAM-1–treated or anti-NK1.1–depleted WT or Ncr1^–/– mice (E). (F) RET cells (2 × 10⁵) were injected i.v. into WT, Ncr1^–/–, or NK1.1-depleted WT mice. Lung metastases were counted 14 days later. Results are from individual mice, and the bar represents the mean of 4–5 (E) and 6–11 (F) mice per group. **P < 0.01.

(A) NOD-SCID mice (3 mice/group) were engrafted with melanoma cell lines (mel) (2 × 10⁶ cells/mouse) from LN, liver (OMM1), pleura, and skin in the presence (filled symbols) or absence (open symbols) of NK cells adoptively transferred from allogeneic healthy donors (1.5 × 10⁶ cells/mouse) on day 10 following the administration of melanoma cells. Mice were then monitored for survival up to day 70. (B) The same procedure was followed as described in A, except that fewer NK cells (5 × 10⁵ NK cells/mouse) were adoptively transferred from allogeneic healthy donors on day 10 following the administration of melanoma cells. Mice were monitored for survival up to day 70. The experiment was repeated twice with similar results.

(A) The early stages of melanoma cells are opposed by only rare resident or infiltrating NK cells, and these melanoma cells eventually metastasize to the draining LN. Although ligands for both DNAM-1 and NCRs are expressed, along with low expression of MHC class I molecules, targeting the DNAM-1 pathway might be an effective immunotherapeutic strategy at this stage, for example, by infusing autologous or allogeneic DNAM-1⁺ NK cells. (B) Once in the LN, the melanoma cells might undergo a weak and inefficient selection by the low cytotoxic CD56^bright NK cells. (C) Hematogenous spread of melanoma cells might undergo a stronger selection by the highly cytotoxic CD56^dim blood NK cells, resulting in an editing out of the melanoma cells expressing NCR ligands. (D) The melanoma cells not expressing NCR ligands escape NK cells and metastasize to visceral organs. Immunotherapeutic intervention at later stages of the disease based on the use of strong cytotoxic NK cells could be envisaged by either deliberately recruiting autologous NK cells from the patient’s pool into the LN or by adoptively transferring allogeneic NK cells from healthy donors in an attempt to halt the metastatic progression to visceral organs. This oversimplified view of melanoma progression is placed in the context of NK cell recognition and does not take into account the interactions of melanoma cells with other immune cells.