Figure 4. Susceptibility of DCs to killing by T_reg cells.

(A) Sorted CD4⁺FoxP3⁺ T_reg cells were cultured in vitro with anti-CD3- anti-CD28-coated Dynabeads in the presence of IL-2 for 0, 1, 2 or 3 days. BMDCs were labeled with CFSE and incubated with T_reg cells for 6 h. Killing of DCs by T_reg cells was determined as described in Materials and Methods. Day 3 versus day 0 at 0.3: and 1:1 T_reg: DC ratios: **P<0.01 (n=3). (B) CD4⁺FoxP3⁺ Treg cells were sorted and stimulated with anti-CD3- anti-CD28-coated Dynabeads in the presence of IL-2 for 3 days. Treg-mediated killing (n=5) of BMDCs, splenic DCs, or activated B or T cells were was determined as described in Materials and Methods. (C) CD4⁺FoxP3⁺ T_reg cells or CD4⁺FoxP3⁻T_eff cells sorted and expanded as in Fig. 3 were incubated with wild type or Bax^−/−Bak^−/− (DKO) BMDCs for 6 h, followed by staining with 7-AAD and analyses by flow cytometry. Loss of 7-AAD⁻ DCs was quantitated. WT versus DKO: **P<0.01 (n=3). (D) Killing of DCs by CD4⁺FoxP3⁺ T_reg cells as in (A) in the presence of anti-MHC-II or control IgG. **P<0.01 (n=3). (E) CD4⁺FoxP3⁺ T_reg cells or CD4⁺FoxP3⁻ T_eff cells were sorted from OT2/FoxP3^GFP mice and expanded with OVA_323–339 peptide-pulsed BMDCs for 3 days. OT2 T_reg or T_eff cells were then isolated by removing DCs with anti-CD11c-MACS beads (Miltenyi Biotec) and used for killing of BMDCs with or without pulsing with OVA_323–339 peptide. WT versus DKO: **P<0.01 (n=3).