Ficoll pacque

Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-Pacque (GE Healthcare) density gradient centrifugation. Monocytes from the PBMC fraction were positively isolated using human CD14 Microbeads (Miltenyi Biotec) according to the manufacturer's instructions. Negatively isolated monocytes using the EasySep Human Monocyte Isolation Kit (STEMCELL Technologies) were used for transmission electron microscopy.

Treatment-naïve OAC donor PBMCs were isolated from whole blood using Ficoll-Pacque (GE healthcare) and density gradient centrifugation, expanded for 5 days (using above anti-CD3/28 and IL-2 expansion protocol) and cultured for an additional 24 h under nutrient deprivation ± normoxic/hypoxic conditions (as described above). The supernatant was harvested and stored at − 80 °C for later use.
OE33 cells were seeded at a density of 1 × 10⁴ cells/100 μl in cRPMI in a flat-bottomed 96-well plate and left to adhere overnight. The media was replaced with 100 μl of cRPMI or 100 μl of 1 in 2 diluted supernatant that was collected from OAC donor PBMCs that had been cultured under hypoxia ± nutrient deprivation and cultured for 24 h at 37 °C 5% CO₂.

Treatment-naïve OAC donor peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Pacque (GE healthcare, Chicago, IL, USA) density gradient centrifugation and expanded in RPMI 1640 medium (Gibco, Waltham, MA, USA) supplemented with 10% foetal bovine serum and penicillin–streptomycin (Gibco, Waltham, MA, USA) using plate-bound anti-CD3 (10 μg/mL, Biolegend, San Diego, CA, USA), anti-CD28 (10 μg/mL, Ancell, Bayport, MN, USA), and recombinant human IL-2 (10 IU/mL, Immunotools, Friesoythe, Germany) for 3 days. PBMCs were irradiated with a 1.8 Gy dose of irradiation on days 1 and 2, 24 h apart, or cells were non-irradiated (NIR). All irradiations were performed using an X-Strahl cabinet X-ray irradiator (RS225) (X-Strahl Ltd., Walsall, UK). For experiments that included treatment with antagonists, in the last 24 h of the T cell activation process, PBMCs were treated with 1 nM CCR1 antagonist J113863 (Axon MedChem, Reston, VA, USA), 80 μM of CCR5 antagonist Maraviroc (Axon MedChem, Reston, VA, USA), 245 nM CX₃CR1 antagonist AZD8798 (Axon MedChem, Reston, VA, USA), or vehicle control (0.01% DMSO). The concentration of the antagonists was selected based on recommendations from the company and prior published studies [24 (link),31 (link),32 (link),33 (link)].