Nkp44 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).