FIGURE 4.

Understanding the role of transcription factors Irf8, Batf3, and Id2 in CD8α⁺ DC development. (A) DC9 cells were transduced with Mig-control-IRES-hCD8t or Mig-Irf8-IRES-hCD8t retrovirus. At 72 h posttransduction, cells were purified using anti-human CD8 MicroBeads, and the Id2 levels were compared by Western blot analysis. (B) DC9 cells were transduced with MSCV-control-puro or MSCV-Irf8-puro retrovirus, and 48-h puromycin–selected populations were analyzed for the expression of Batf family members (Batf, Batf2, and Batf3). (C) DC9 cells were transduced with retrovirus expressing Irf8, Batf3, or Id2 individually and coexpressed with each other and selected for 48 h with puromycin. Selected populations were treated with CpG (1826; 1 μg/ml) for 24 h and analyzed by flow cytometry. Expression of Id2 or Batf3 alone or coexpressed together did not induce CD8α surface marker. When coexpressed with Irf8, Id2 or Batf3 showed a synergistic effect on the expression of surface markers CD8α and MHCII. Data are representative of three independent experiments. (D) DC9 cells were transduced with retroviruses expressing Irf8, Batf3, or Id2 individually and coexpressed with each other and selected for 48 h with puromycin. Selected populations were treated with CpG (1826; 1 μg/ml) for 24 h and analyzed by flow cytometry. DC maturation markers CD40 and CD80 also showed a synergistic increase when Id2 or Batf3 was coexpressed with Irf8. Data are representative of three independent experiments. (E) DC9 cells were transduced with retroviruses expressing transcription factors and selected for 48 h with puromycin. SiglecH, pDC-specific surface marker was downregulated by Id2 and Batf3 alone or by their coexpression. Expression of Irf8 rescues downregulation by Id2 and Batf3 and increases SiglecH on the cell surface. Data are representative of three independent experiments.