Analysis of DC surface markers on live cells was performed using the following mAbs in immunofluorescence assays: PE-conjugated anti-CD86 (HA5.2B7, IgG2b), FITC-conjugated anti-CD80 (MAB104, IgG1), PE-conjugated anti-CD1a (BL6, IgG1), FITC-conjugated anti-CD83 (HB15A, IgG2b), FITC-conjugated anti-ICAM-1 (CD54), PE-conjugated anti-CD83 (HB15a, Beckman Coulter), FITC-conjugated anti-HLA-A,B,C (BD PharMingen), FITC-conjugated anti-HLA-DR (BD PharMingen), PE-conjugated anti-CD11c (BD PharMingen), FITC-conjugated anti-CD40, and FITC-conjugated anti-CD25 (both from Immunotech). Anti-CD14 (63D3, IgG1) was provided by D. Vercelli, (Hospital San Raffaele-Dipartimento di Biotecnologie, Milan, Italy). Anti HLA-DR (D1.12,IgG2a) was provided by G. Frumento, Istituto Nazionale Ricerca sul Canero, Genoa, Italy. To analyze the phenotype of NK cells, we used the following FITC-labeled monoclonal antibodies: anti-CD16, anti-CD2, anti-CD7, anti-CD8, anti-CD56, obtained from Beckman Coulter. Anti-NKp30 (A76, IgG1), anti-NKp46 (BAB281, IgG1), anti-NKp44 (Z231, IgG; provided by A. Moretta, University of Genoa, Genoa, Italy) and anti-CD161 (NKRP1A; 191B8, IgG2a; provided by A. Poggi and D. Pende, Istituto Nazionale Ricerca sul Canero) mAbs were also analyzed on NK cell surface. Anti-ULBP2 (M311, IgG1; provided by Immunex Corporation, Seattle, WA) and anti-CD48 (TU145, IgM; BD PharMingen) mAbs were employed in order to identify possible NKG2D and 2B4 ligands on DCs. Direct immunofluorescence procedure was performed by diluting fluorochrome-labeled mAb with 1 mg/ml human γ-globulin (human therapy grade from a commercial source), in order to block nonspecific Fc-receptor binding. Cells were then washed and the flow cytometric analysis was performed. Indirect immunofluorescence assays were performed as follows: cell nonspecific binding sites were saturated with human γ-globulin and then the relevant mAb was added and incubated for 30 min at 4°C. After extensive washings, FITC-conjugated isotype-specific goat anti–mouse antibodies (GAM; Southern Biotechnology Associates, Inc.) were added and incubated for 30 min at 4°C. Negative controls included directly labeled or unlabeled isotype-matched irrelevant mAbs. Cells were then washed and analyzed by flow cytometry.