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1 Supporting Information Bellora et al /pnas SI Materials and Methods Cells. NK cells purified from peripheral blood mononuclear cells (PBMC) of healthy donors (Human NK Cell Isolation Kit; Miltenyi Biotec) were frozen and thawed 24 h before coculture experiments. To obtain short-term activated NK cells, resting NK cells were cultured overnight with 0.5 ng/ml ril-12 (PeproTech) and/or 100 ng/ml ril-18 (BioSource International). To obtain macrophages (M0), monocytes purified from PBMC (Human Monocyte Cell Isolation Kit II; Miltenyi Biotec) were cultured ( /ml) for 7 d in 24 Lumox Multiwell TC-Qualitaet plates (Greiner Bio One GmbH) with 100 ng/ml rm-csf (PeproTech). M1 and M2 polarization were obtained culturing M0 for 18 h with 100 ng/ml LPS from Escherichia coli (Sigma- Aldrich), 20 ng/ml rifn-γ (PeproTech) used alone or in combination, or 20 ng/ml ril-4 (PeproTech), respectively (1). For TLR stimulation, macrophages were incubated overnight with 100 ng/ml of LPS or Mycobacterium bovis (bacille Calmette Guérin, bacillus Calmette Guérin; Pasteur Merieux) at the 1:1 bacillus Calmette Guérin:macrophage ratio. To obtain idcs, monocytes were cultured 7 d with 50 ng/ml rgm-csf (Mielogen; Schering-Plough) and 20 ng/ml ril-4 (PeproTech). To generate mdcs, idcs were treated for 24 h with 1 μg/ml LPS (2). Antibodies. mabs produced in our laboratories: KL247 (IgM, anti- NKp46), F252 (IgM, anti-nkp30), KS38 (IgM, anti-nkp44) ON72 (IgG1, anti-nkg2d), F5 (IgM, anti-dnam-1), CER1 (IgM, anti- NKp80), MA 127 (IgG1, anti-ntba), MA344 (IgM, anti-2b4), C227 (IgG1, anti-cd69), MAR93 (IgG1, anti-cd25), A6136 and 6A4 (IgM and IgG1 anti-hla class I-A, -B, -C, and -E), M5A10 (IgG1, anti-pvr), L14 (IgG2a, anti-nectin-2), 7E22 (IgG1, anti- ICAM-1), CO202 (IgM, anti-cd48), 5B14 (IgM, anti-b7h3), DF305 (IgM, anti-lfa-3), ECM17/120 (IgM, anti-cd11a), DF206 (IgG1, anti-gpr56). The anti-hla-e mab (3D12, IgG1) was kindly provided by Daniel Geraghty (Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle). Anti-CD14 (Immunotech); anti-cd80-pe, anti-cd206-fitc (BD Bioscience); anti-cd68-fitc (Genway Biotech); anti-hil-18 (IgG1; Medical Biological Laboratories); anti-hil-15 (IgG1), anti-hil- 18Ra/IL-1 R5 (goat IgG), anti-hil-15rα (goat IgG), anti-hil-12 (goat IgG) (3), anti-ccr7 (IgG2a; R&D Systems); anti-icam-2 (IgG1), anti-icam-3 (IgG1) (Diaclone). All of the antibodies are of mouse origin unless otherwise specified. Statistical Analysis. Independent samples t test was used for evaluating quantitative variables. The test is a statistical technique used to analyze the mean comparison of two independent groups. The statistical level of significance was preset at Graphic representation and statistical analyses were performed using the PASW Statistic version 18.0 software (formerly SPSS Statistics, IBM). 1. Martinez FO, Gordon S, Locati M, Mantovani A (2006) Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: New molecules and patterns of gene expression. J Immunol 177: Pende D, et al. (2006) Expression of the DNAM-1 ligands, Nectin-2 (CD112) and poliovirus receptor (CD155), on dendritic cells: Relevance for natural killer-dendritic cell interaction. Blood 107: Lapaque N, Walzer T, Méresse S, Vivier E, Trowsdale J (2009) Interactions between human NK cells and macrophages in response to Salmonella infection. J Immunol 182: of6

2 Fig. S1. Macrophages (M0) were polarized toward M1 or M2 using LPS (alone or in concert with IFN-γ) or IL-4, respectively. Monocytes and macrophages were analyzed by flow cytometry. White profiles refer to cells incubated with the second reagent only. The mean fluorescence intensity (MFI) is indicated. During differentiation from monocytes, macrophages down-regulated CD14 expression. In addition, when treated with LPS alone or LPS plus IFN-γ, macrophages expressed markers typical of M1 polarization (CD80, CD68, and CCR7). Conversely, M2-polarizing macrophages up-regulated CD206 (mannose receptor). Similar to monocytes, macrophages express at the cell surface important adhesion molecules and various ligands for NK receptors and coreceptors such as DNAM-1 (CD226). An exception was represented by NTBA that was de novo expressed by macrophages. Because DCs do not express NTBA, this molecule may represent a good marker of monocytes differentiation toward macrophages. Finally, macrophages and DCs de novo expressed B7-H3, a member of the B7 family that represents a ligand of a still-undefined inhibitory NK receptor. 2of6

3 Fig. S2. M0 and M2 cells were cocultured with autologous resting NK cells in the presence of bacillus Calmette Guérin. NK cells were recovered and analyzed for cytolytic activity in a 4-h 51 Cr-release assay against K562 or autologous idcs at the indicated E:T ratios. Average of two independent experiments. SD is indicated. *P < Fig. S3. (A and B) Representative cytofluorimetric analyses of CD69, CD25, and CCR7 expression in NK cells cocultured with untreated, LPS-treated (A), or bacillus Calmette Guérin-treated (B) macrophages. The experiments were performed either in the presence of cell-to-cell contact or in transwell. (C) Representative cytofluorimetric analysis of GPR56 expression in NK cells cocultured with LPS-treated macrophages. White profiles refer to cells incubated with the second reagent only. The mean fluorescence intensity (MFI) and the percentage of positive cells (%) are indicated. 3of6

4 Fig. S4. (A) The cell culture supernatants of M0, M1, and M2 polarizing or polarized macrophages were analyzed by ELISA for the presence of the indicated cytokines. Average of nine (Upper) or four (Lower) independent experiments. SD is indicated. *P < 0.05, **P < 0.01, ***P < (B) Macrophages were analyzed by flow cytometry for the expression of IL-15 and IL-15 receptor. White profiles refer to cells incubated with the second reagent only. The mean fluorescence intensity (MFI) is indicated. 4of6

5 Fig. S5. Resting NK cells were cocultured with autologous LPS-treated M0 or M2 either in the absence or in the presence of neutralizing Abs for the indicated cytokines. NK cells were recovered and analyzed for CD69 surface expression (A) and cytolytic activity against K562 target cells (B). Average of six independent experiments. SD is indicated. Fig. S6. M0 and M2 cells were cocultured overnight with autologous resting NK cells in the presence of bacillus Calmette Guérin. The cell culture supernatants were analyzed by ELISA for the presence of IFN-γ. The experiments were performed either in the presence of cell-to-cell contact or in transwell. Average of two independent experiments. SD is indicated. *P < 0.05, **P < 0.01, ***P < of6

6 Fig. S7. (A) M0 were treated with LPS + IFN-γ, IFN-γ alone, or LPS alone. Cells were analyzed by flow cytometry for the expression of the indicated surface molecules. White profiles refer to cells incubated with the second reagent only. The mean fluorescence intensity (MFI) is indicated. (B) Untreated and IFNγ-treated M0 cells were maintained in culture for additional 18 h either in the absence or in the presence of LPS. The cell culture supernatants were analyzed by ELISA for the presence of IL-12 and IL-18. Average of three independent experiments. SD is indicated. *P < 0.05, **P < 0.01, ***P < of6