Nkp44

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.

NK cells were stained with antibodies against surface molecules for 30 min a 4 °C, washed and then analyzed by LX-Cytoflex or Cytoflex S flow-cytometers (Beckman) and FlowJo software (BD Biosciences, Franklin Lakes, NJ, USA) was used for data analysis. For exosome analysis, 5 µg of exosomes were coated to 5 µL of aldehyde/sulphate latex beads (Invitrogen, Carlsbad, CA, USA) and following 15 min incubation, exosome-coupled beads were diluted with 1 mL in PBS and incubated overnight at 4 °C. The day after, samples were washed twice and incubated with conjugated antibodies for 30 min at 4 °C. After 3 washings, they were analyzed by flow-cytometry. For detection of cytotoxic proteins or inner proteins inside exosomes (Granzyme A-B, Perforin, IFN-γ and PD-1), samples were fixed in 4% paraformaldehyde and permeabilized in Triton 0.1% before proceeding with incubation at 37 °C with conjugated antibodies.
Antibodies used were CD3, CD81, CD63, NKp44, CD69, PD1, and CD54 (Miltenyi, Bergish Galdbach, Germany), CD56, CD16, Granzyme A and B (Beckton Dickinson, Franklin Lakes, USA), NKp46, IFN-γ (Thermo Fischer, Waltham, USA), NKG2D, DNAM1, Perforin, CD115, CD112 (Biolegend, San Diego, CA, USA), NKp30 (Beckman Coulter, Brea, CA, USA), CD45, CD14, and CD19 (Beckman Coulter).