Foxp3 150d

Twenty-four hours after the last exercise session, mice were anesthetized, and tumors were harvested, and samples were processed as previously described (3 (link)). Flow cytometry data were obtained using an LSRII flow cytometer (BD Biosciences) and/or Aurora (Cytek) and analyzed with FlowJo software. The double/aggregated cells were gated out using forward scatter area (FSC-A) versus forward scatter width (FSC-W) and side scatter area (SSC-A) versus side scatter width (SSC-W). Different fluorophores conjugated with the following mAb were used: CD45 (30-F11, Biolegend), TCRβ chain (H57-597, Biolegend), CD4 (RM4-5, Biolegend), FOXP3 (150D, Biolegend), CD8a (53-6.7, Biolegend), CD11c (N418, Biolegend), Gr1 (RB6-8C5, Biolegend), MHC-II (M5/114.15.2, BD Biosciences) CD11b (M1/70, Biolegend), B220 (RA3-6B2, Biolegend), F4/80 (BM8, Biolegend), IFNγ (XMG1.2, Biolegend), Granzyme B (GB11, Biolegend), Ki67 (16A8, Biolegend), CD62L (W18021D, Biolegend), CD44 (NIM-R8, Biolegend), CD69 (H1.2F3, Biolegend), CXCR3 (173, Biolegend), IL15Rα (6B4C88, Biolegend), IL6Rα (D7715A7, Biolegend), CD247 (Biolegend, 10F.9G2).

For peripheral blood mononuclear cell (PBMC)-fHASC cocultures, PBMCs (3 × 10⁵/well) were activated with 5 μg/ml anti-CD3 mAb (clone OKT3) and then cocultured for 7 days with fHASCs that had been pre-treated or not with 1000 U/ml IFN-γ for 24 hrs. In some experiments, the PBMCs were incubated with NVs isolated from 1 × 10⁶ fHASCs that had been cultured in vitro for 2 days. The expression of FOXP3 was evaluated by FACS. Briefly, cells were treated with rat anti-CD16/32 (2.4G2) for 30 min. at 4°C for blocking of Fc receptors before assaying on an LSRFortessa (BD BioSciences, Franklin Lakes, NJ, USA) flow cytometer and analysed by flowJo data analysis software. The following fluorochrome-conjugated mAbs were used, CD4 (RPA-T4) and FOXP3 (150D) (Biolegend). For assessment of any cell–cell contact requirement, a 6.5 mm Transwell system (Corning, Union City, CA, USA) with a 0.4 μm pore polycarbonate membrane insert was used to separate PBMCs (upper chamber) from fHASCs (lower chamber). fHASCs at different PBMC-to-HASC ratios were added to the lower chamber, either as such or after treatment with IFN-γ or transfection with IDO1 siRNA or negative control siRNA. After 24 hrs, medium was removed and replaced with fresh medium alone. PBMCs (3 × 10⁵) were added to the upper chamber, and recovered at specific times, as detailed in the relevant figure legends.