Differential contribution of canonical and noncanonical NF-κB signaling in DCs to naive T-cell proliferation and apoptosis induction. (A) Blockade of the canonical NF-κB pathway inhibits the capacity of DCs to induce proliferation in naive T cells. Immature DCs were incubated with NBD/MUT peptides and matured for 2 days with LPS or CD40L. Subsequently, the cells were thoroughly washed and loaded with Staphylococcus aureus enterotoxin B (SEB; 10 pg/mL), and different concentrations of DCs were used to stimulate naive CD4⁺ Th cells. The proliferative response was determined at day 5 of coculture by [³H]TdR incorporation. Data are presented as mean cpm plus or minus SD of triplicate cultures. Results are representative of 3 independent experiments. (B) Blockade of noncanonical NF-κB signaling does not change the capacity of DCs to induce T-cell proliferation. Immature DCs were treated with siRNA, incubated with NBD/MUT peptides, and matured for 2 days with CD40L. Subsequently, the cells were thoroughly washed, loaded with SEB, and used to stimulate naive CD4⁺ Th cells. The proliferative response was determined at day 5 of coculture by [³H]TdR incorporation. Data are presented as mean cpm plus or minus SD of triplicate cultures. Results are representative of 3 independent experiments (*P < .01). (C) Blockade of noncanonical NF-κB signaling does not alter apoptosis induction in naive CD4⁺ T cells by CD40L-NBD DCs. Monocyte-derived DCs were treated with siRNA, incubated with NBD/MUT peptides and stimulated with CD40L. After 48 hours, DCs were washed extensively, loaded with SEB, and cocultured with naive CD4⁺ T cells for 24 hours. Subsequently, T-cell apoptosis was assessed by annexin V/propidium iodine (PI) staining and evaluated by flow cytometry. Results represent mean plus or minus SEM from 3 independent experiments.