Ikaros-deficient DCs show enhanced stimulation of allogeneic T cells. (A) BMDCs and peritoneal macrophages from WT (n = 3 mice/group), Ik^−/− (n = 3 mice/group) and IkDN^+/− B6 (n = 3 mice/group) animals were used as stimulators in an MLR with T cells from either BALB/c (allogeneic) or C57BL/6 (syngeneic) mice and analyzed for T-cell proliferation with ³H-thymidine incorporation at 96 hours. Data are means ± SEM of quadruplicate cultures. Results are from 1 of 3 similar experiments with the DCs and 1 of 4 for the macrophages. (B) Supernatants from the cultures were collected at 72 hours and analyzed for IFN-γ and IL-17A by ELISA. Data are means ± SEM of quadruplicate cultures. Results are from 1 of 3 similar experiments. (C) Analysis of T-cell phenotype after cultures with WT or Ik^−/− DCs. CD4⁺ T cells were harvested after 96-hour culture with DCs and analyzed for expression of CD44, CD62L, CD25, CD69, and Foxp3 on CD4⁺ cells. Data are means ± SEM of quadruplicate cultures. Results are from 1 of 2 similar experiments. (D) CFSE-labeled splenic CD90⁺ T cells from BALB/c mice were cultured for 96 hours with B6 BMDCs from WT, Ik^−/−, or IkDN^+/− animals, harvested, and analyzed for CFSE and Annexin V on cells gated for CD3⁺. Data shown are representative of 1 of 3 similar experiments. (E) Ik^−/− with WT BMDCs were mixed at the indicated ratios and used as stimulators in an MLR against BALB/c T cells. Shown is T-cell proliferation with ³H-thymidine incorporation at 96 hours. Data are means ± SEM of triplicate cultures. Results are from 1 of 2 similar experiments. (F) Treg suppression assay. BALB/c CD4⁺CD25⁻ T cells were cultured with either WT or Ik^−/− BMDCs along with BALB/c CD4⁺CD25⁺ Tregs at the indicated ratios. Proliferation was assessed after 5-day cultures with ³H-thymidine for the last 16 hours. Data are means ± SEM of quadruplicate cultures. Results are from 1 of 2 similar experiments.