ICOS-LICOS interaction initiates ICOS up-regulation of TGN1412-treated T cells. (A) Freshly isolated and PKH26-labeled human T cells (2 × 105) per 96-well were stimulated with 3 μg/mL soluble recombinant LICOS protein (rLICOS) or 4 μg/mL soluble recombinant CD40 protein (rCD40; left panel). Alternatively, 5 μg/mL rLICOS or rCD40 were coated on 96-wells and 2 × 105 PKH-labeled T cells were added (right panel). Subsequently, PKH26-labeled T cells were stimulated with 1 μg/mL TGN1412 (solid lines). Controls were left unstimulated (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 at least 3 independent T-cell donors. (B) Freshly isolated human T cells (2 × 105) per 96-well were stimulated with 1 μg/mL TGN1412 followed by cross-linking with 2 μg/mL anti-IgG monoclonal antibody or 5 μg/mL TGN1412 were coated on 96-wells and T cells were added subsequently (top panel dotted lines). HUVECs (8 × 104) were irradiated and 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 human T cells were added to HUVECs and stimulated with 1 μg/mL TGN1412. 5 μg/mL rLICOS or rCD40 were coated on 96-wells. Subsequently, T cells were added and stimulated with 1 μg/mL TGN1412 (bottom panel dotted lines). Controls were left unstained (gray-shaded curves) or were stained but not TGN1412-stimulated (solid lines). At day 5 of stimulation, T cells were harvested, stained with an anti-CD3 and anti-ICOS antibody, and ICOS expression on CD3+ T cells was measured by flow cytometric analysis. Data shown are representative for at least 3 independent T-cell donors. (C) Freshly isolated and PKH26-labeled human T cells (1 × 106) per 24-well were treated with 10 μg/mL anti-ICOS blocking antibody for 1 hour. Subsequently, these T cells were added to 24-wells coated with 5 μg/mL rLICOS protein and stimulated with 1 μg/mL TGN1412. Controls were left unstimulated (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. Error bars indicate standard deviations of data retrieved from experiments performed with T cells derived of 3 independent donors. The frequency of T-cell proliferation is shown as percentage of total T cells, normalized to the control (samples stimulated with TGN1412 but without blocking antibody). The statistical analysis was performed with SAS/STAT software (**P < .01).
