ICOS-LICOS interaction facilitates TGN1412-induced T-cell proliferation. (A) HUVECs were irradiated and cultured either unstimulated or stimulated with 200 U/mL TNF-α + 100 U/mL IFN-γ for 3 days in 24-wells or transwell inserts. After washing HUVECs with PBS, 1.9 × 106 freshly isolated and PKH26-labeled human T cells per 24-well or 1 × 106 per transwell insert were added and stimulated with 1 μg/mL TGN1412 (solid lines). 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. As controls, T cells were cocultivated with unstimulated or cytokine prestimulated HUVECs without the addition of TGN1412 (gray-shaded curves). The frequency of T-cell proliferation is shown as percentage of total T cells. Data shown are representative for at least 4 independent T-cell donors. (B) Unstimulated (solid lines) or with 200 U/mL TNF-α + 100 U/mL IFN-γ for 3 days stimulated HUVECs (2 × 105; dotted lines) were stained with fluorochrome-conjugated anti–MHC I, anti–MHC II, anti-CD40, anti-LICOS, anti-CD80 or anti-CD86 antibodies and analyzed by flow cytometry. Controls were left unstained (gray-shaded curves). Data shown are representative for at least 9 independent HUVEC donors. (C) HUVECs (8 × 104) were irradiated and stimulated with 200 U/mL TNF-α + 100 U/mL IFN-γ for 3 days. After washing with PBS, HUVECs MHC I, MHC II, or costimulatory molecules were blocked specifically with anti–MHC I, anti–MHC II, anti-CD40, anti-LICOS, anti-CD80, or anti-CD86 antibodies for 1 hour. Freshly isolated and PKH26-labeled human T cells (1 × 106) were added to HUVECs and stimulated with 1 μg/mL TGN1412 (dotted lines). As controls, T cells were cocultivated with cytokine prestimulated HUVECs without TGN1412 (gray-shaded curves) or with TGN1412 but without blocking antibodies (solid lines). 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 of data retrieved from experiments performed with T cells derived of at least 8 independent donors. The frequency of T-cell proliferation is shown as percentage of total T cells, normalized to the control (samples without blocking antibody). The statistical analysis was performed with SAS/STAT Version 9.3 software (SAS System for Windows; ***P < .001).
