Induction and regulation of KC apoptosis in KC–T cell cocultures. (a) Coculture of primary human KCs and stimulated autologous CD45RO⁺ T cells. After 3 days of coculture, cells were stained with annexin V and subjected to flow cytometry. With the indicated stimuli, the number of apoptotic KCs increases and the cells show an increase in annexin V binding (open histograms). Filled histograms show live KCs. The percentage of apoptotic KCs is indicated at upper right. Panel 1: KC apoptosis induced by coculture with CD45RO⁺ T cells stimulated with anti-CD2, anti-CD3, and anti-CD28 mAb’s. Panel 2: Isotype control mAb. Panel 3: Blocking anti–IFN-γ receptor mAb (1 μg/mL). Panel 4: Fas-Fc protein (1 μg/mL). Panel 5: The caspase inhibitor Z-VAD-FMK (50 μM). Panel 6: Z-VAD-FMK (100 μM). (b) Coculture of primary human KCs and stimulated autologous CD45RO⁺ T cells in Transwell plates. After 3 days in coculture, permeabilized KCs were stained with propidium iodide and subjected to flow cytometry. Filled histograms demonstrate control KCs containing diploid DNA. The percentage of apoptotic KCs with hypodiploid DNA (open histograms) is shown in the upper right corner. Panel 1: Primary human KCs alone were cultured in the lower well. Panel 2: KCs were pretreated with 10 ng/mL IFN-γ for 24 hours, and KC apoptosis was determined 3 days after 1 μg/mL activating anti-FasR mAb was added. Panel 3: KC apoptosis induced by Transwell coculture with stimulated CD45RO⁺ T cells. Panel 4: Inhibition of KC apoptosis induced by IFN-γ and anti-FasR mAb, with 1 μg/mL ZB4 blocking mAb. Panels 5 and 6: Inhibition of KC apoptosis induced by Transwell coculture with stimulated CD45RO⁺ T cells, with 1 μg/mL Fas-Fc protein (panel 5), and with 10 μg/mL neutralizing anti–IFN-γ mAb (panel 6). Results in a and b are representative of three experiments.