Suppression of cytotoxicity by OT-II T reg cells depends on the strength of the antigen stimulation of OT-I T cells in a cell coculture system. (A) Ratio of apoptotic cleaved caspase-3+ EG.7 and EL-4 cells after 6 h of cocultivation with activated OT-I T cells at different ratios in the absence of externally applied OVA257-264 (n = 6, respectively). (B) Representative flow cytometry analysis of cleaved caspase-3 levels in EG.7 cells cocultured for 6 h at a ratio of 1:1 with activated OT-I T cells in the absence (left panel) and the presence (right panel) of OT-II T reg cells at a ratio of 2:1 (cleaved caspase-3+ EG.7 cells: no OT-I T cells: 6.5 ± 1.3%; OT-I T cells: 18.9 ± 2.7%; OT-I T cells + OT-II T reg cells: 18.3 ± 3.4%; P = 0.864; n = 4). (C) OVA257-264 concentration dependence of the OT-II T reg and wild-type T reg cell-mediated reduction of cleaved caspase-3+ EL-4 cells induced by activated OT-I T cells (ratio OT-I to T reg: 2:1; ratio OT-I to EL-4: 1:1). At low OVA257-264 concentrations (0.001 to 1 nM) OT-II T reg cells and wild-type T reg cells caused significant reduction of activated OT-I T cell-induced cleaved caspase-3+ EL-4 cells (n = 6, respectively). At high OVA257-264 concentrations (10 to 1,000 nM) neither OT-II T reg cells nor wild-type T reg cells exerted any suppressive effect (n = 6, respectively). (D) OVA257-264 peptide concentration dependence of intracellular ATP levels (detected following cell lysis) as a measure of cell viability in EL-4 cells following 4 h of cocultivation with activated OT-I T cells (ratio 1:1) in the absence and presence of OT-II T reg cells (ratio 2:1). At low OVA257-264 concentrations (0.001 to 0.1 nM) significantly higher intracellular ATP levels in EL-4 cells (ratio 1:1) were detected in the presence of OT-II T reg cells as compared to their absence (OVA257-264 0.1 nM: P = 0.0271; n = 8). At high OVA concentrations (1 to 10 nM) OT-II T reg cells did not exert any suppressive effect (OVA257-264 10 nM: P = 0.734; n = 8). P values < 0.05 were considered significant (indicated as **).