Nofluorescence staining using unconjugated mAbs, followed by secondary GAM-APC. In all experiments, cells were stained with propidium iodide (PI) (1 g/ml) in order to assess cell viability (always higher than 90 after the different treatments). Nonspecific fluorescence was assessed by using an isotypematched irrelevant mAb R D System, followed by the same secondary antibody. Fluorescence was analyzed using a FACSCalibur flow cytometer (BD Biosciences), and data were analyzed using FlowJo Flow Cytometric Data Analysis Software (Tree Star, Inc). The analysis of ligands expression on patient-derived plasma cells was performed by gating on the CD138+ and CD38+ PC population. NK cell-mediated cytotoxicity was evaluated using the lysosomal marker CD107a as previously described [69]. As source of effector cells, we used primary NK cells obtained from PBMCs isolated from healthy donors by Lymphoprep ycomed gradient centrifugation and then co-cultured for 10 days with irradiated (30 Gy) EpsteinBarr virus (EBV)-transformed B cell line RPMI 8866 at 37 in a humidified 5 CO2 atmosphere, as previouslyAbruzzese et al. Journal of Hematology Oncology (2016) 9:Page 15 ofdescribed [69]. On day 10, the cell population was routinely more than 90 CD56+CD16+CD3-, as assessed by immunofluorescence and flow cytometry analysis. When patient-derived plasma cells were used as targets (myeloma cells were selected using anti-CD138 magnetic beads from Miltenyi Biotec), autologous CD138- bone marrow cells were cultured for 2 days in TAPI-2MedChemExpress TAPI-2 complete medium, supplemented with 200 U/ml IL-2, and used as source of effector cells. Drug-treated MM cell lines or patient-derived plasma cells were washed twice in complete medium and incubated with NK cells at effector/target (E:T) ratio of 2.5:1, in a U-bottom PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28192408 96-well tissue culture plate in complete medium at 37 and 5 CO2 for 2 h. Thereafter, cells were washed with PBS and incubated with anti-CD107a/APC (or cIgG/APC) for 45 min at 4 . Cells were then stained with anti-CD3/FITC, anti-CD56/PE, and anti-CD16/PerCPCy5.5 to gate the CD3-CD56+ CD16+ NK cell population. In some experiments, cells were pre-treated for 20 min at room temperature with anti-NKG2D neutralizing mAbs or a control Ab (anti-CD56). Fluorescence was analyzed using a FACSCalibur flow cytometer (BD Biosciences), and data were analyzed using FlowJo Flow Cytometric Data Analysis Software (Tree Star).PlasmidsIRF4-DN, encoding a truncated form of the human IRF4, consisting of its N-terminal DNA binding domain (1-405), was generated by inserting the mutant IRF4 cDNA in the pcDNA3 expression vector [70]. The lentiviral copGFP vectors pMIRNA1/pre-miR-125b and the pMIRNA1/control vector were purchased from System Biosciences. For knocking down cMYC and BRD4, we used the following lentiviral vectors: pLKO.1-sh-cMYC (TRCN0000039642), pLKO.1-sh-BRD4 (TRCN0000382028), and the control vector pLKO non-targeting shRNA MISSIONTM (Sigma-Aldrich).DNA transfections, virus production, and in vitro transductionMICA-270 bp promoter in pGL3-basic luciferase vector (Promega Corp.) was generated as previously described [68]. To generate the MICA promoter deletions MICA/ 3.2 kb and MICA/1.2 kb, the appropriate deletion fragments (Kpn-I/HindIII) were generated by PCR according to standard methods from a human 3.2 kb-wild-typeMICA/GFP reporter vector (kindly provided by Dr. Skov, University of Copenhagen, Frederiksberg, Denmark) and cloned in pGL3-basic luciferase vector. The primers u.