Endothelial cells confer TGN1412-mediated cytokine release and early T-cell activation. (A) Freshly isolated human T cells (1 × 10⁶) per 24-well were left unstimulated, stimulated with 1 μg/mL TGN1412, or 1 μg/mL TGN1412 followed by cross-linking with 2 μg/mL anti-IgG monoclonal antibody (left panel). HUVECs (8 × 10⁴) per 24-well were irradiated and cultured unstimulated for 24 hours. Alternatively, HUVECs were stimulated with 200 U/mL TNF-α + 100 U/mL IFN-γ for 3 days. After washing HUVECs with PBS, 1 × 10⁶ freshly isolated human T cells were added to HUVECs and left unstimulated or stimulated with 1 μg/mL TGN1412 (right panel). Twenty-four hours after the indicated stimulations, cell-free supernatant was collected and analyzed for TNF-α, TNF-β, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, and IL-12 p70 by FlowCytomix Th1/Th2 11plex analysis. Data shown are from 5 to 10 independent T-cell donors. #, data points are in the saturation area of the assay. (B) HUVECs (8 × 10⁴) were irradiated and cultured either unstimulated or stimulated with 200 U/mL TNF-α + 100 U/mL IFN-γ for 3 days. After washing HUVECs with PBS, 1 × 10⁶ freshly isolated human T cells were added to HUVECs and stimulated with 1 μg/mL TGN1412. As controls, T cells were cocultivated with cytokine prestimulated HUVECs without TGN1412 (gray-shaded curves). Twenty-four or 48 hours after cocultivation, T cells were harvested and stained with an anti-CD69, anti-CD25 (both after 24 hours) or anti–Ki-67 antibody (after 48 hours). Data shown are representative for 4 or 2 different T-cell donors, respectively.