Cxcr1

FITC-conjugated mouse antihuman CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR1, CXCR3, CXCR4, and CXCR5 or unconjugated monoclonal mouse-antihuman CCR1, CCR2, and CXCR6, as well as PE-conjugated rat antihuman CCR8, PE-conjugated rat antihuman CCR10, and PE-conjugated rat IgG2b, were obtained from R&D Systems Europe Ltd. (Abingdon, UK). FITC-conjugated mouse antihuman CX₃CR1 was purchased from Medical and Biological Laboratories Co. Ltd. (Nagoya, Japan). FITC-conjugated monoclonal mouse antihuman CD3, PE-conjugated monoclonal mouse antihuman CD56, and FITC-conjugated goat anti-mouse were purchased from Becton-Dickinson (San Diego, CA, USA). FITC-conjugated mouse IgG, PE-conjugated mouse IgG, unconjugated mouse IgG, and unconjugated rat IgG were obtained from either Becton-Dickinson or from R&D Systems. Pertussis toxin (PTX), MMF, and DMF were obtained from Sigma-Aldrich (Saint Louis, MO, USA). CCL1, CCL27, CCL28, and CXCL10 were purchased from PeproTech (London, UK).

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.

The cellular proteins from cultured cells were extracted by lysing in lysis buffer (50 mM Tris-HCl pH 7.5, 125 mM NaCl, 5 mM EDTA and 0.1% Triton X-100) containing 1% protease inhibitor and 1% protein phosphatase inhibitor cocktail (Sigma-Aldrich). Western blot was performed following previously reported methods [11 (link)]. Briefly, proteins were electrophoresed in a 5–20% sodium dodecyl sulfate-polyacrylamide gradient gel. After electrophoresis, proteins were transferred to the PVDF membrane with an iBlot^® Gel Transfer Stack (Invitrogen). The membrane was blocked using 5% skim milk and then incubated overnight at 4 °C with a primary antibody. After washing, the membrane was incubated with a secondary antibody for 90 min at 25 °C. Chemiluminescence was excited by ImmunoStar^® Reagents (FUJIFILM Wako Pure Chemical) and captured by the ImageQuant^TM LAS 4000 mini (FUJIFILM).
The primary antibodies used were phosphorylated (p)-Stat3 Tyr705 (#9145, Cell Signaling Technology; CST, Beverly, MA, USA), total (t)-Stat3 (#4904, CST), pAkt Ser473 (#4060, CST), tAkt (#9272, CST), pp38 MAPK (#4511, CST), tp38 MAPK (#8690, CST), CXCR1 (#MAB330, R&D Systems), CXCR2 (#ab65968, Abcam, Cambridge, UK), and β-actin (#4970, CST). The signal intensities of bands were quantified using the ImageJ 1.53t.