Eomes pe cy7

PBMCs, isolated as described above, were resuspended to 1×10⁶ cells/ml in PBS. The cells were surface-stained with CD3-BV786, CD38-BUV737, HLA-DR-PE, CCR7-BV421, CD45RA-AF700, CD71-APC-H7 (BD), CD4-APC-Fire750, CD8-BV510 (BioLegend) for 30 min in the dark at 4°C, followed by fixation and permeabilization. After permeabilization, cells were stained with ki67-FITC, Granzyme B-AF700, T-bet-BV421, BAX-FITC, Bcl2-PE (BD Biosciences), Eomes-PE-Cy7 (Ebioscience), perforin-APC (BioLegend) antibodies for 30 min in the dark at room temperature. Following staining, cells were washed and acquired on an LSRFortessa.

PBMCs were stimulated with anti-CD3/CD28 (5 µg/mL, Ebioscience) for 5 h in the presence of anti-CD107a BV421 (BioLegend) and Golgiplug (BD Biosciences). CD107a expression was measured as a marker of degranulation on CD8⁺ T cells after stimulation. The cells were surface-stained with CD3-BV786, CD4-APC-Fire750, CD8-BV421, CD244-PE-D594, CD160-AF488, and intracellularly stained with TNF-α-BV711, IL-2-BV650 (BioLegend), or IFN-γ-AF700 (Ebioscience) antibodies. For Ki67, perforin, Granzyme B, T-bet, or Eomes staining, PBMCs were surface-stained with CD3-BV786, CD4-APC-Fire750, CD8-BV421, CD244-PE-D594, CD160-AF488, and intracellularly stained with Granzyme B-AF700, T-bet-BV421 (BD Biosciences), Ki67-BV711, perforin-APC (BioLegend), or Eomes-PE-CY7 (Ebioscience) antibodies. A fixable viability dye eFluor^® 506 (Ebioscience) was used to label dead cells.

The production of proangiogenic factors by NK cells was studied in 8 HCC patients (paired LINK and TINK) after overnight stimulation with IL-12 and IL-18 (5 ng/mL) in the presence of 10 µg/mL of brefeldin A (BFA). Surface staining with anti-CD3-BUV805, anti-CD56-BUV805, and anti-CD49a-BV510 (BD Bioscience, Franklin Lakes, NJ, USA) was performed. Cells were then fixed and permeabilized with Fixation Permeabilization Concentrate and Diluent and Permeabilization Buffer (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions and stained with Eomes-PE-Cy7 (Invitrogen), Angiopoietin 1 (ANGPT1)-Alexa Fluor750 (Bioss Antibodies, Boston, MA, USA), CXCL10/IP-10-Alexa Fluor 700 (R&D System, Minneapolis, MN, USA), Osteopontin-eFluor660 (Thermo Fisher Scientific, Waltham, MA, USA), IL-8-PerCp-eFluor710 (Thermo Fisher Scientific, Waltham, MA, USA), MMP-9-Alexa Fluor 488 (Abcam, Cambridge, UK), Placental growth Factor (PlGF)-Alexa Fluor594 (Bioss Antibodies, Boston, MA, USA), VEGF-PE (R&D system, Minneapolis, MN, USA), and Angiogenin-Alexa Fluor405 (Novus Biotechne, R&D System, Minneapolis, MN, USA). Analysis was performed on an 18-fluorescence flow cytometer (FACS Fortessa, Becton Dickinson (BD) Immunocytometry System, CA, USA). Data are expressed as the percentage of cytokine-positive cells in the CD49a+Eomes+ NK cell subset.

CD8⁺ T‐cells were isolated using the MagCellect Mouse CD8⁺ T Cell Isolation Kit (R&D Systems, Minneapolis, MN, USA) from the spleens of naive C57BL/6 and PD‐1 KO mice. Purified CD8⁺ T‐cells were placed into culture and stimulated with anti‐CD3 (2 µg/ml) Ab for 1 h prior to transfer onto mixed glial cell culture. CD8⁺ T‐cells were added at a 10:1 CD8: glial cell ratio. Neutralization of PD‐1 and its ligand was performed by treating glial cells with anti‐PD‐1 (J43 clone; eBiosciences, San Diego, CA, USA), anti‐PD‐L1 (M1H5 clone; eBiosciences), anti‐PD‐L2 (TY25 clone; eBiosciences), or IgG2a for 2 h prior to the addition of anti‐CD3‐activated CD8⁺ T‐cells. Cells were collected 48 h after the addition of T‐cells and stained for 15–20 min at 4°C for surface markers anti‐CD45‐PE‐Cy5, anti‐KLRG1‐PE‐Cy7, anti‐CD103‐FITC (clone 2E7), anti‐CD127‐APC, anti‐CD69‐e‐F 450, (eBioscience, San Diego CA), and anti‐CD8‐BV‐510 from (Biolegend). For intracellular staining, cells were stained for 30 min with anti‐IFN‐γ ef450, T‐bet‐PE (eBioscience), and EOMES‐PE‐Cy7 (Invitrogen, Carlsbad, CA). Control isotype Abs were used to assess nonspecific Ab binding.