Cd155

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.

SAS cells (2 × 10⁷) were cultured in serum‐free DMEM for 24 h before harvesting the conditioned medium (CM). The proteins from CM were quantified (150 μg), and a human cytokine array (R&D Systems) was performed according to the manufacturer's protocol.
Proteins from the tissue, cell lysates, or CM were separated via SDS/PAGE and transferred onto a PVDF membrane, which was then blocked in 5% nonfat milk for 1 h at room temperature and incubated with primary antibodies against C1GALT1, MHC I, GAPDH (Santa Cruz Biotechnology), PD‐L1, IL‐1α, MCP‐1, IL‐6, IL‐8, STAT3 and phospho‐STAT3 (Cell Signaling Technology), CD155 (R&D Systems) at 4 °C overnight. The complete list of antibodies was in Table S1. HRP‐conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) were used to detect the protein bands.
For the lectin pull‐down assay, 250 μg of total proteins from CM was incubated with Vicia villosa agglutinin (VVA)‐conjugated beads (Vector Laboratories, Burlingame, CA, USA) for 18 h at 4 °C with constant rotation. The washed beads were boiled at 95 °C for 10 min, and VVA binding proteins were evaluated via western blot analysis.

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.