Fc-FcR interaction is dispensable for endothelial cell-conferred TGN1412-mediated T-cell proliferation. (A) Untreated HUVECs (2 × 105; solid lines) or HUVECs treated with 200 U/mL TNF-α + 100 U/mL IFN-γ for 3 days (dotted lines) were stained with fluorochrome-conjugated anti-FcγRI, II or III antibody and analyzed by flow cytometry. Controls were left unstained (gray-shaded curves). Data shown are representative for experiments using HUVECs of 8 independent donors. (B) 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' FcγRs were blocked specifically with anti-FcγRI, anti-FcγRII, anti-FcγRIII antibodies, or with Polyglobin 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 from at least 7 independent T-cell donors. The frequency of T-cell proliferation is shown as percentage of total T cells, normalized to the control (samples without blocking antibody). (C) Freshly isolated and PKH26-labeled human T cells (2 × 105) per 96-well were cocultivated with 2 × 105 freshly isolated and irradiated human B cells or 1 × 106 freshly isolated and PKH26-labeled human T cells per 24-well were cocultivated with prestimulated HUVECs as described in Figure 1B. Subsequently, T cells were stimulated with 1 μg/mL control TGN1412* (see “Methods”; solid line) or 1 μg/mL deglycosylated TGN1412 (dotted lines). 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 cytometry. Data shown are representative for at least 6 independent T-cell donors.
