Transferrin

To generate DCs, CD14⁺ monocytes were cultured (3-5 x 10⁶/well; 6-well plate; total volume, 6 ml; 37°C/5% CO₂) for 8 days in R9 medium (RPMI 1640, 100 µg/ml penicillin, 100 U/ml streptomycin, 25 µg/ml transferrin, 10% human AB serum [Innovative Research], 25 mM HEPES buffer, 2 mM L-glutamine) supplemented with GM-CSF and IL-4 (800 U/ml) [PeproTech Ltd]. To induce DC maturation, 25 ng/ml TNF-α [PeproTech Ltd] and 1 µg/ml lipopolysaccharide [Sigma-Aldrich Ltd, E.coli 0111:B4] were added on the penultimate day of culture. Naïve or memory CD3⁺ T-cells, with Tregs removed, were cultured (2.5 x 10⁶/well; 24 well plate; total volume, 2 ml) with autologous mature DCs (0.8 x 10⁵/well) and SMX-NO (50 µM) for 8 days. To specific wells, human targeted anti-PD-L1, anti-CTLA4, or anti-TIM-3 antibodies (Biolegend, London, UK; anti-PD-L1, 5 µg/ml; anti-CTLA4, 10 µg/ml; anti-TIM-3, 7.5 µg/ml) were added prior to the addition of SMX-NO and incubated for ≥ 30 min (37°C/5% CO₂). Dose ranging studies were performed around those directed by the supplier to ascertain optimal antibody concentrations. Alternatively, specific quantities of autologous CD25⁺ Tregs were added to individual wells. Experiments were repeated a minimum of three times.