Endothelial cells confer TGN1412-mediated T-cell proliferation. (A) Freshly isolated and PKH26-labeled human T cells (2 × 10⁵) per 96-well were stimulated with 1 μg/mL TGN1412 (solid line left panel) followed by cross-linking with 2 μg/mL anti-IgG mAb (solid line middle panel). Five μg/mL TGN1412 was coated on 96-wells and PKH26-labeled T cells were added (solid line right panel). Controls were left untreated (no TGN1412; gray-shaded curves). At day 5 of stimulation, PKH26-labeled T cells were harvested, stained with an anti-CD3 antibody, and T-cell proliferative responses were measured by flow cytometric analysis. Data shown are representative for experiments using T cells of at least 20 independent donors. (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 and PKH26-labeled human T cells were added to HUVECs and stimulated with 1 μg/mL TGN1412. As controls, T cells were left untreated or were cocultivated with cytokine prestimulated HUVECs without TGN1412 (gray-shaded curves). At day 5 of cocultivation, PKH26-labeled T cells were harvested, stained with an anti-CD3 antibody, and T-cell proliferative responses were measured by flow cytometric analysis. Error bars indicate standard deviations from data obtained with T cells of 30 independent donors. The frequency of T-cell proliferation is shown as percentage of total T cells. The statistical analysis was performed with SAS/STAT Version 9.3 software (SAS System for Windows; ***P < .001).