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PBMC were isolated from non-cancer controls by density gradient centrifugation and seeded at a density of 1 × 10⁶ cells/ml RPMI supplemented with 10% FBS and 1% pen/strep. Cells were treated with ACM or TCM from non-obese or obese OAC patients for 2 or 24 h. Cells were stained with CD56-FITC-Viobright, NKG2A-APC (Miltenyi Biotec), CD3-APC-Cy7, CD71-PE-Cy7, CD36-PerCP-Cy5.5, NKp30-BV421, NKp46-PE-Cy7, NKG2D-PE-Cy5, PD-1-PE-Cy7, TIGIT-PE-Cy5, CD69-BV510, TRAIL-APC and FasL-BV421 (BioLegend). NK cells were quantified as CD56⁺CD3⁻ cells within the lymphocyte gate. Cells were acquired using the CANTO II (BD Biosciences) flow cytometer and analysed using FlowJo software (BD Biosciences).

Naïve T cells were purified from cryopreserved peripheral blood mononuclear cells (PBMCs) using the Naïve Pan T Cell Isolation Kit (Miltenyi Biotec). After purification, isolated naïve T cells were >99% CCR7⁺ CD45A⁺ as determined by flow cytometry with CD3‐APC‐Cy7 (Tonbo), CCR7‐PE‐Cy7 (Miltenyi Biotec), and CD45RA‐APC (Biolegend). Naïve T cells were stained with Cell Proliferation Dye eFluor‐670 (eBioscience) as recommended by the manufacturer. Purified mucosal CD1a⁺ or CD14⁺ cells (5 × 10³ cells) were plated with naïve T cells (7.5 × 10⁴ cells) (1:15 ratio) in round‐bottom 96‐well plates, in Xvivo 15 media (Invitrogen) supplemented with 10% human AB serum (Valley Biomedical). After 6 days in culture, proliferation of T cells was assessed by flow cytometry after staining with zombie yellow dye (Biolegend) and CD3‐APC‐Cy7, CD8‐FITC (Tonbo), CD4‐PE, CD103‐PE‐Cy7 (eBiosciences), and CD11c‐PerCp‐Cy5.5 (Biolegend). Naïve T cells alone were used as a negative control. For some experiments, TGFβ receptor 1 blocker, SB431542 (10 μmol/L, Tocris Cookson Inc) (Ochiel, Ochsenbauer, et al., 2010), was added to the cultures at the beginning of each experiment.