Figure 6

Apoptosis of ESb-MP cells is mediated by perforin and granzyme B. (A) Shows perforin expression in Vβ6⁺ T cells. Vβ6⁺ T cells were stimulated with vSAG7⁻ Eb or vSAG7⁺ ESb-MP cells and APCs for 4 days. Then unstimulated (control) or stimulated Vβ6⁺ T cells were stained with an anti perforin mAb. Cells were counterstained with hemalaun. Membrane staining indicates positively labelled cells. Black dots are magnetic beads which were used for the purification and could not be removed after 4 days of stimulation. Magnification × 400. (B) ESb-MP cells were cultured either alone (ESb-MP) or with stimulated Vβ6⁺ T cells at an effector : target ratio of 10 : 1. Separate groups of effector cells were treated either with 10 nM Concanamycin A (Concan.A), 20 μM granzyme B inhibitor (Z-AAD-CMK) or with a combination of both inhibitors (Vβ6+Concan.A+GranzB.Inh.) for 15 min before coincubation with target cells for 4 h. Incubation of ESb-MP cells alone with Concanamycin A and granzyme B inhibitor revealed no significant difference in comparison to the ESb-MP control. Apoptosis was determined by PI incorporation and FACScan analysis. The mean±standard deviation from three independent experiments are shown.