Cell lines and cultures

The human cell lines H929, RPMI-8226, MM.1S, MM.1R, and HEK293T cells were purchased from the American Type Culture Collection (ATCC), while the human cell lines U266 and OPM-2 were purchased from the Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures (DSMZ). Human cell lines KMS-11 and KMS-11 BTZ were kindly provided by Dr. Arianna Giacomini, University of Brescia, Italy. All MM cell lines (H929, RPMI-8226, MM.1S, MM.1R, U266, OPM-2, KMS-11, and KMS-11 BTZ) were cultured in RPMI-1640 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) medium containing 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific), 2 µM/L glutamine, 10,000 U/mL penicillin G, and 10,000 μg/mL streptomycin (Gibco, Thermo Fisher Scientific) and maintained at 37 °C with 5% CO2. HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Thermo Fisher Scientific), containing 10% FBS, 2 µM/L glutamine, 10,000 U/mL penicillin G, and 10,000 μg/mL streptomycin and maintained at 37 °C with 5% CO2. Cells were used within 2–3 months after thawing (24–40 passages at maximum). Cell lines were also regularly tested for mycoplasma contamination using the MycoAlert mycoplasma detection kit (Lonza, Basel, Switzerland).

MM patient samples

Bone marrow (BM) aspirates were obtained under Institutional Review Board (IRB)-approved protocols (2010C0126 and 2021C0218) after informed consent in accordance with the Declaration of Helsinki was obtained from all subjects. MM cells were separated by Ficoll-Paque (Cytiva, Cat. No. 17144002) suspension, followed by CD138 positive selection, using EasySep™ Human CD138 Positive Selection Kit II (RosetteSep™, STEMCELL Technologies, Cat. No. 17877). Purity was confirmed by CD138-V450 (BD Biosciences, San Jose, CA, USA, Cat. No. 562098, RRID:AB_10894011) and CD38-PE (BD Biosciences, Cat. No. 555460, Clone HIT2, RRID:AB_395853) staining. Peripheral blood (PB) samples were separated by density gradient centrifugation over Ficoll-Paque, washed with centrifugation with room-temperature phosphate-buffered saline (PBS, Gibco), and stained with specific flow cytometry fluorescent antibodies.

NK cell isolation by RosetteSep™ immunodensity cell separation method

Apheresis cones were obtained from Versiti, Inc. After dilution of full apheresis blood in a 50 mL tube with PBS, 500 µL of RosetteSep™ Human NK Cell Enrichment Cocktail (STEMCELL Technologies, Cat. No. 15065) were added. Blood was incubated for 20 min at room temperature on a rocker. 15 mL of Ficoll solution was added to a new tube and topped with 35 mL of blood. The sample was centrifuged at 1800 rpm for 30 min at room temperature. Buffy layers were collected, and cells were washed twice in PBS (1300 rpm for 5 min). Ammonium chloride solution (STEMCELL Technologies, Cat. No. 07850) for RBC lysis was added to the pellet and incubated at room temperature for 5 min. Cells were washed in PBS, counted, and stained with specific flow cytometry fluorescent antibodies.

RNA extraction and quantitative real-time PCR analysis

RNA for quantitative real-time PCR was extracted using TRIzol method (Invitrogen, Life Technologies, Carlsbad, CA, USA) or ReliaPrep™ RNA Miniprep System (Promega, Madison, WI, USA, Cat. No. Z6011); cDNA was synthesized using the ProtoScript® II First Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA). Quantitative real-time PCR analysis was performed using SYBR GREEN PCR Master Mix protocol (Applied Biosystems, CA, USA, Cat. No. 4309155). Quantitative real-time PCR analysis was performed on a ViiA 7 Real-Time PCR System (Applied Biosystems, CA, USA). Data were analyzed using the ΔΔ Ct method. Gene-specific primers are reported in the Supplementary Methods. GAPDH was used for normalization.

Western blot analysis

MM cells were harvested and lysed using RIPA lysis buffer (Cell signaling, Cat. No. 9806), with addition of 1 mM PMSF (Cell signaling, Cat. No. 8553). Laemmli sample buffer (BIO-RAD, Cat. No. 1610747) was added and samples were boiled at 95 °C. Cell lysates were subjected to SDS–PAGE, transferred to nitrocellulose membranes, and immunoblotted with the specific antibodies reported in the Supplementary Methods. All antibodies were diluted to a concentration of 1:1000 and prepared in milk 5% diluted in TBS (BIO-RAD, Cat. No. 1706435) with Tween 20 (BIO-RAD, Cat. No. 1706531).

Gain-of- and loss-of-function experiments in MM cell lines

For gain-of-function experiments, U266 cells were transiently transfected using Nucleofector 4D Unit X, Kit SF, program DY-100 (Lonza, Amaxa Biosystems, Köln, Germany). Viruses were produced by co-transfection of specific plasmids and packaging vectors (psPAX2, Addgene Cat. No. 12260, RRID:Addgene_12260 and pMD2.G, Addgene Cat. No. 12259, RRID:Addgene_12259) into HEK293T cells. H929 and OPM-2 cells were infected with lentiviral particles using polybrene followed by spinoculation of suspended cells. Stably infected cells were selected using 0.5–1 μg/mL of puromycin (InvivoGen, Cat. No. ant-pr-1). After transfection or infection, MM cells were subjected to mRNA analysis, western blotting, and flow cytometry analyses.

Flow cytometry analysis

MM cell lines, MM patient-derived samples, and PB samples were washed with room-temperature PBS and then incubated with specific antibodies for 20 min. Cells were then washed with PBS and acquired on Attune NxT Flow cytometry machine using Attune NxT Software v 3.1 (Thermo Fisher Scientific). At least 50,000 events were acquired. The following antibodies were used: HLA-E-PE (BioLegend, San Diego, CA, USA, Cat. No. 342604), CD56-APC (BD Biosciences, Cat. No. 555518, RRID:AB_398601), CD56-PE (Miltenyi Biotec, Cat. No. 170-081-014, Clone REA196), CD138-V450 (BD Biosciences, Cat. No. 562098, RRID:AB_10894011), and CD38-PE (BD Biosciences, Cat. No. 555460, Clone HIT2, RRID:AB_395853). For staining of NK cell populations in the peripheral blood, the following antibodies were used: CD3-VioBlue (Miltenyi Biotec, Cat. No. 170-081-046, Clone BW264/56), CD16-FITC (BD Biosciences, Cat. No. 555406, Clone 3G8), CD56-APC Vio770 (Miltenyi Biotec, Cat. No. 130-114-548, Clone REA196), CD94-PE (BD Biosciences, Cat. No. 555889, Clone HP-3D9, RRID:AB_396201), NKG2A/CD159a-APC (Miltenyi Biotec, Cat. No. 130-113-563, Clone REA110). Post-acquisition analyses including t-SNE analysis were performed using FlowJo Software (BD Biosciences).

Co-culture experiments of NK cells with MM cells

MM cells were modified by gain-of- or loss-of-function approaches or treated with different compounds (day 1) for 48 h in a six-well plate. On day 2, apheresis cones from healthy donors were shipped overnight in dry ice from Versiti, Inc. On day 3, NK cells were isolated by RosetteSep™ immunodensity cell separation method as described above. Both MM and NK cells were counted and plated at an effector to target (E:T) ratio of 5:1 or 10:1 in V-shaped 96-well plates. After 4 h, the cells were stained with CD138-V450 (BD Biosciences, Cat. No. 562098, RRID:AB_10894011), CD56-PE (Miltenyi Biotec, Cat. No. 170-081-014, Clone REA196), CD16-FITC (BD Biosciences, Cat. No. 555406, Clone 3G8), and SYTOX™ Red Dead Cell Stain (Thermo Fisher Scientific, Cat. No. S34859) and acquired on Attune NxT Flow cytometry machine. MM cells without NK cells were plated, stained, and acquired in the same conditions. The percentage of lysis was calculated normalizing to the average of n = 3 treated cells without NK cells.

Analysis of RNA-sequencing or gene-expression profiling from multiple myeloma datasets

The RNA-sequencing data were downloaded from the MMRF CoMMpass database, with data generated as part of the Multiple Myeloma Research Foundation Personalized Medicine Initiatives (https://research.themmrf.org and www.themmrf.org). mRNA expression data were collected from: GSE4452, a collection of 12 healthy donors and 65 newly diagnosed MM patients; GSE5900, a collection of 22 cases of healthy donors, 44 cases of MGUS, and 12 cases of SMM; and GSE8546, a collection of relapsed patients with MM prior to (n = 36) and after (n = 19) lenalidomide administration. For GSE8546, 16 samples were paired and used for the analysis. All three datasets were analyzed by Affymetrix microarray (HG-U133 Plus 2.0 array for GSE4452 and GSE5900; HG U95Av2 array for GSE8546). For evaluation of the relationship between HLA-E, STAT1, IRF1, and IRF9, with CREB1 or CD56, median value of expression was used as threshold to define two groups. Gene sets of Canonical Pathways (C2_CP_BIOCARTA, C2_CP_KEGG, C2_CP_PID, C2_CP_REACTOME, C2_CP_WIKIPATHWAYS) or gene ontology (C5_GOBP) were used to perform enrichment analysis at http://www.broad.mit.edu/gsea [12, 13]. Two-sided p values < 0.05 were considered statistically significant. Statistical analyses were performed using GraphPad software (GraphPad Prism, RRID:SCR_002798).