Megacd40l

The human monocytic leukemia cell line THP-1 (kindly provided by Prof. J. Dziadek, Polish Academy of Sciences, Lodz, Poland) was cultured at 5 × 10⁵–1 × 10⁶ cells/mL in RPMI 1640 medium, supplemented with 10% FBS, 2 mM l-glutamine, 0.168 mM penicillin, 0.172mM streptomycin, 0.05 mM β-mercaptoethanol, and 1mM sodium pyruvate in a humidified incubator at 37 °C and 5% CO₂ (Binder GmbH, Tuttlingen, Germany). Primary human monocytes were isolated from the blood of healthy donors by a magnetic cell separation procedure using anti-CD14 antibodies (MACS system, Miltenyi Biotech, Bergisch Gladbach, Germany). The MACS system was also used to isolate T cells with the pan T cell isolation kit (MACS system, Miltenyi Biotech). The differentiation of primary monocytes or THP-1 cells into dendritic cells (DCs) was achieved by culturing the cells in the presence of recombinant human IL-4 (100 ng/mL) and GM-CSF (100 ng/mL) for three to five days. For the stimulation of cytokine production, cells were incubated at a density of 5 × 10⁵ cells per mL in serum-free medium (AIM V, Gibco–Thermo Fisher Scientific, Life Technologies Polska, Warsaw, Poland) for 24 h with LPS (Lipopolysaccharide (E. coli 0111:B4), 1 µg/mL; Sigma-Aldrich, St Louis, MO, USA) and CD40 ligand (100 ng/mL, soluble, human, recombinant Mega CD40L, Enzo Life Sciences, Lausen, Switzerland).

The following cytokines and antibodies were used for in vitro stimulation of HDBEC: hIFNγ (Biosite), MegaCD40L (Enzo lifesciences), hIFNγ neutralizing antibody (BD biosciences) and the isotype control for the hIFNγ-neutralizing antibody (BD biosciences). All cytokines and antibodies used in in vitro HDBEC stimulations were diluted in endothelial cell starvation medium (basal EMV2 plus 1% FBS). HDBEC were pre-starved for 2 hours in starvation medium prior to stimulation.