Cd112

To evaluate phenotype features, wtGISTc and CIK were labeled and acquired on a FACS Cyan (CyAN ADP, Beckman Coulter s.r.l., Cassina de’ Pecchi, Italy). Flow cytometry data were analyzed using Summit Software (Beckman Coulter s.r.l., Cassina de’ Pecchi, Italy). wtGISTc were stained with the following fluorescein isothiocyanate (FITC), phycoerythrin (PE)-, or allophycocyanin (APC)-conjugated mouse monoclonal antibodies (mAbs): MICA/B and ULBP1, ULBP2, 5, 6 and ULPB3 for the NKG2D ligands (Pharmingen, Milan, Italy), CD112 and CD155 (R&D Systems, Minneapolis, MN, USA) for DNAM ligands and PD-L1 and PD-L2 (Pharmingen, Milan, Italy). HLA-I and β2-microglobulin (in collaboration with Soldano Ferrone, clones TP25.99.8.4 [37 (link)] and L368 [38 (link)], respectively), and Human IFNα/β R1 and IFNγ R1/CD119 Antibody (R&D Systems, Minneapolis, MN) were detected on wtGISTc with the use of a secondary antibody (Goat Anti-Mouse Ig, Pharmigen, Milan, Italy). Conjugated anti-human monoclonal antibodies for CD3 (Pharmingen, Milan, Italy), CD8, CD56, NKG2D, CD62L, CD45RA, PD-1, TIM-3 (MACS Miltenyi Biotec, Bologna Italy), DNAM-1, LAG3 (BD Biosciences, Milan Italy), TIGIT (eBiosciences, San Diego, CA, USA), NKp30, NKp44 and NKp46 (MACS Miltenyi Biotec, Bologna, Italy) were used to characterize CIK.

Target cancer cells were seeded at density of 40,000 cells/mL in 96-well plate and the day after, were incubated with 20γ of NK exosomes. After 24 h of co-culture, cells were washed in PBS, incubated with anti-CD19 antibody (Thermo Fischer, Waltham, MA, USA) for 20 min and washed in PBS. Then, propidium iodide (Sigma Aldrich, Saint Luis, MO, USA) was added to identify dead cells.
For experiments with blocking antibodies to LFA-1-L and DNAM1-L, before incubation with NK exosomes, NALM-18 cells were incubated for 30 minutes at 37 °C with 10 µg/mL of blocking antibodies to CD54 (R&D Systems, Minneapolis, USA), CD155 (Genetex, Irvine, CA, USA), CD112 (R&D Systems, Minneapolis, MN, USA). For DNAM1 blocking experiments, NK exosomes were previously incubated with anti-DNAM1 blocking mAb for 30 minutes at 37 °C and then incubated with target cells for 24 h.
Analysis of caspase activation was performed by using Vybrant FAM Caspase-3 and -7Assay Kit (Thermo Scientific, Waltham, MA, USA) according to manufacturer recommendations.

The cell phenotype was assessed by fluorescence-activated cell sorting (FACS) by using staining with the monoclonal antibodies MICA, MICB, CD112, CD155, B7-H6 (R&D Systems, clone 875001), and EphA2 (R&D Systems, clone 371805). The tumor cell lines were harvested, washed with cold phosphate-buffered saline (PBS, Sigma) containing 2% FBS, and incubated for 30 min on ice with fluorescently labelled monoclonal antibodies. Gamma-delta T cells were identified in freshly isolated PBMCs labelled with CD3 (Thermo Fisher Scientific, clone SK7), Vδ1 TCR (Thermo Fisher Scientific, clone TS8.2), Vδ2 TCR (BD Pharmingen, clone B6) or Vδ2 TCR (Sony, clone B6). CD27 (BD Pharmingen, clone M-T271), and CD45RA (Exbio, clone MEM-56) were used for immunophenotyping. Samples were washed and acquired using FACSCanto^® (BD Biosciences) and data analyzed using FlowJo^® software (FlowJo, Ashland, OR, USA). Forward and side scatter gating were used to discriminate live cells from dead cells and γδ T cells were derived from SSC vs. FSC-gated bulk PBMCs with doublet exclusion (FSC-A vs. FCS-H). To determine the placement of the gates, appropriate fluorescence minus one (FMO) and unstained controls were used.