Human ab serum

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.

CD14⁺ cells were cultured in medium (RPMI-1640, 100μg/ml penicillin, 100U/ml streptomycin, 25μg/ml transferrin, 10% human AB serum [Innovative Research; MI, USA], 25mM HEPES buffer, and 2mM L-glutamine) supplemented with GM-CSF and IL-4 (800U/ml; 37°C/5% CO₂) for 7 days to generate dendritic cells. On the penultimate day, 25ng/ml TNFα and 1μg/ml LPS were added as maturation factors. Dendritic cell phenotype (CD1a,CD11a,CD11c,CD14,CD40,CD80,CD83,CD86,CD274,MHC class II) was measured by flow cytometry.
Mature dendritic cells were plated (0.8×10⁵ per well) and cultured with naive CD3+ T cells (2.5×10⁶ per well; 24 well plated total volume 1.5ml) and SMX-NO (50μM) or flucloxacillin (2mM) for 8 days_. Anti-PD-L1 and/or PD-L2 antibodies (Biolegend UK Ltd, London, UK; 10μg/ml) were added to certain wells. Both antibodies have previously been shown to block the receptors (24 (link)). Antibody concentrations were optimized in dose-ranging studies around the concentration suggested by the supplier. Where indicated, TGF-β (5ng/ml), IL-1β (10ng/ml) and IL-23 (20ng/ml) or TNFα (50ng/ml) and IL-6 (20ng/ml) were added to the cultures to induce the differentiation of Th17 and Th22 cells, respectively. All experiments were performed at least three times using cells from different blood donors with no previous history of sulfonamide/flucloxacillin exposure.