Nkg2d pe

Leukocytes were defined as CD45pos, and the different cell subtypes were defined as CD3pos (T cells), CD19pos (B cells) CD14pos (monocytes) and Cd56pos (NK cells). Cell viability was assessed by propidium iodide staining. Anti-isotype controls (Exbio, Praha, CZ) were performed.
1 × 10⁵ eNK cells were labeled with fluorophore-conjugated antibodies: CD3-PE-Cy7, CD16-PE, CD56-APC, NKG2D-PE, NKG2C-PE, NKG2A-PE, NKp30-PE, NKp44-PE, NKp46-PE, KIR2DL2/3-PE, KIR2DL1-PE (BD, Italy), KIR2DL4-PE (R&D Systems, Italy); CXCR1, CXCR2, CXCR3, CX3CR1, CXCR4, CCR1, CCR2, CCR3, CCR5, and CCR7 (R&D Systems, Italy). eNK cells were gated as CD56pos CD3neg. eNK cells activation status was determined by CD107a staining (R&D Systems, Italy), as previously reported (Rizzo et al., 2016 (link)).
5 × 10⁵ epithelial cells were stained specific Ab HLA-I (HLA-A,-B,-C)-PE (BD Biosciences, Italy), HLA-E (clone MEM-E/08, Exbio, Praha, CZ) or HLA-DR (BD Biosciences, Italy) and matched isotype controls.
The NKG2D-ligands were detected on epithelial cells by binding of NKG2D-Fc chimera (R&D Systems, Italy) and indirect labeling with the secondary Ab FITC-coupled mouse anti-human IgG1 (Abcam, Cambridge, United Kingdom).
Data were analyzed using FACS CantoII flow cytometer (BD, Milan, Italy) and FlowJo LLC analysis software (Ashland, OR, United States). Ten thousand events were acquired.

Peripheral blood mononuclear cells (PBMCs) were isolated from the fresh blood of patients using Ficoll density gradients as described previously [16 (link)]. Isolated PBMCs were stained for surface markers, fixed, permeabilised with IntraPreReagent (Beckman Coulter, Fullerton, CA) and further stained with antibodies directed against intracellular markers. Leukocytes were stimulated with Leukocyte Activation Cocktail (BD Bioscience, USA) at 37 °C for 4 h prior to intracellular staining using the manufacturer’s staining protocol. Anti-human mAbs against CD3-PE-CF594, CD56-FITC, NKG2D-PE, NKp46-PE-CY7, NKp-30-APC, NKp44-PE, NKG2A-APC, CD69-PE-CY7, PD1-Pacific blue, Tim-3-APC, perforin-APC, Granzyme B-BV421, IFN-γ-PE, and TNF-α-PE with corresponding isotype-matched controls were purchased from BD Biosciences (San Jose, CA, USA). Data were acquired on a Gallios instrument (Beckman Coulter, Brea, CA, USA) and analysed using FlowJo software (Flow jo, LCC, USA).

This study utilized frozen PBMC that were previously collected using the Ficoll-Paque method and stored in liquid nitrogen
[24 (link)]. PBMC were thawed in a water-bath at 37°C for one minute and thereafter, washed and re-suspended in RPMI media containing 10% v/v fetal calf serum before surface staining. PBMC viability was evaluated using trypan blue dye and viable cells were counted under microscopy. Minimum cell viability was 80%. Cells were rested for four hours, and subsequently stained with the following anti-human monoclonal antibodies; CD14/19 FITC CD3 Amcyan, CD8 APC-Cy7, CD16 PerCP Cy5.5, CD56 PE Cy7, NKG2D PE and NKp46 APC (BD Biosciences, San Jose CA). At least 50,000 events in the CD3-negative gate were collected. Gating was standardized and set using fluorescence minus one control (FMOs) for CD14/19, CD16, CD56, NKG2D and NKp46. NK cells were identified as CD3^-negative, CD14^-/CD19^- and NK cell subsets were identified by co-expression of CD56 and CD16 on NK cells; CD56^bri (CD56⁺⁺CD16^-), CD56^INTERMEDIATE (CD56⁺CD16^-), CD56^dim (CD56⁺CD16⁺) and CD56^neg (CD56^-CD16⁺); see Figure 
2. Expression of NK receptors NKG2D and NKp46 was defined by the percentage of NKG2D + and NKp46+ NK cell subsets (Figure 
3).

The following antibodies were used for flow cytometry (all anti-human): CD16-PE (BD Biosciences, 560995, clone 3G8), CD16-APC (BD Biosciences, 561248, clone 3G8) NKG2D-PE (BD Biosciences, 557940, clone 1D11), NKp44-PE (BD Biosciences, 558563, clone p44-8), NKp46-PE (BD Biosciences, 557991, clone 9E2), TRAIL-PE (BD Biosciences, 565499, clone YM366), FAS ligand-PE (BD Biosciences, 56426, clone NOK-1), NKG2A-PE (Beckman Coulter, IM3291U, clone Z199), CD158a,h (KIR2DL1, KIR2DS1)-PE (Beckman Coulter, A09778, clone EB6B), CD158b1/b2,j (KIR2DL2, KIR2DL3, KIR2DS2)-PE (Beckman Coulter, IM2278U, clone GL183), CD158e1(KIR3DL1)-BV421 (BioLegend, 312713, clone DX9), CD158a(KIR2DL1)-APC (Miltenyi, 130-120-584, clone REA284), CD158b1/b2,j (KIR2DL2, KIR2DL3, KIR2DS2)-PE-Cy5.5 (Beckman Coulter, A66900, clone GL183), CD158a,h(KIR2DL, KIR2DS1)-PE-Cy7 (Beckman Coulter, A66899, clone EB6B), CD158b2(KIR2DL3)-PE, (R&D systems, FAB2014P, clone 180701), CD155-PE (BioLegend, 337609, Clone SKII.4), HLA E-PE (BioLegend, 342603, Clone 3D12) MICA/MICB-PE (BD Biosciences, 558352, Clone 6D4), CD112-PE (BD Biosciences, 551057, Clone R2.525), ULBP2/5/6-PE (R&D Systems, FAB1298P, Clone 16590).

Cell surface and intracytoplasmic marker expression were detected using a BD CantoII flow cytometer. For extracellular staining, anti-human CD56-FITC (eBiosciences, MA1-19129), NKG2D-PE (BD, 561,815), MICA/B-PE (Biolegend, 320,906), ULBP1-FITC (Invitrogen, MA5-38655), CD107a-APC (BD, 641,581), and TIM-3-PE-cy7 (Biolegend, 345,013) were used for extracellular staining. For intracellular staining, CAR-NK cells were fixed and permeabilized using a BD Cytofix/Cytoperm kit (BD Biosciences, Franklin Lakes, USA). following the manufacturer’s protocol, and anti-human IFN-γ-eFluor 450 (eBiosciences, 85-48-7319-42) was used for intracellular staining. Stained samples were acquired on a BD CantoII flow cytometry and analyzed with FlowJo software (Tree Star, Ashland, USA).