Figure 7. Induction of mitochondrion-dependent cell death in DCs by T_reg cells.

(A) Wild type or DKO BMDCs with or without incubation with T_reg cells for 6 h were labeled with TMRE, followed by flow cytometry. Percentages of TMRE⁺ cells (mean ± SD): wild type, 89.4 ± 2.1 (no T_reg), 44.9 ± 5.0 (with T_reg), P=0.0001; DKO, 91.2 ± 1.5 (no T_reg), 83.2 ± 5.8 (with T_reg), P=0.081 (n=3). (B) Wild type or DKO BMDCs with or without incubation with T_reg cells for 6 h were lysed for Western blotting of caspase-8 (Casp8), Casp9, Casp3, Bax or Bak. (C) T_reg cells were incubated with wild type, Bak^−/−, Bax^−/−, Bim^−/− or DKO BMDCs, followed by analysis of killing of DCs as in Fig. 4. Bim^−/− or DKO versus WT DCs: *P<0.05, **P<0.01 (n=3). (D) T_reg cells were incubated with wild type BMDCs for 6 h in the presence of various antibodies of control IgG, followed by quantification of killing of DCs as in Fig. 6A. Antibody treatments versus IgG control: *P<0.05; **P<0.01 (n=3). (E) CD4⁺FoxP3⁺ T_reg cells with or without stimulation with anti-CD3/anti-CD28 Dynal beads plus IL-2 for 3 days were stained with PE-conjugated anti-LAG3 (solid line) or an isotype control (dashed line), followed by analyses by flow cytometry. (F) WT or DKO DCs were incubated with anti-I-A^b or control IgG for 6 h, followed by incubation with TMRE. The cells were analyzed by flow cytometry and the loss of TMRE⁺ cells were calculated. **P<0.01 (n=3).