Selective loss of CD8α⁺ DCs in Nfil3^−/− mice and impaired Flt3-dependent development of CD8α⁺ DCs in the absence of NFIL3. (A) Nfil3 is highly expressed in DC subsets. Quantitative real-time RT-PCR analysis of Nfil3 mRNA in purified cells (NKs and DCs) or in vitro stimulated cells (B and T cells) was performed. Data show the mean and SD from 3 experiments. (B) Splenocytes from Nfil3^+/+ or Nfil3^−/− mice were stained for CD11c and PDCA-1 to identify pDCs and cDCs (left) and for CD11c, CD8, CD4, DEC-205, SIRP-α, and CD24 to detect CD8α⁺ DCs and CD8α ⁻ DCs (middle) or to detect CD8α⁺ DC precursors in CD8α⁻CD4⁻ DCs (right). Data are representative of 5 experiments with similar results. (C) Thy1.2⁻ thymocytes from Nfil3^+/+ or Nfil3^−/− mice were stained for CD11c and PDCA-1 to identify pDCs and cDCs (left) and for CD11c, CD8, DEC-205, SIRP-α, and CD24 to detect CD8α⁺ DCs (right). Data are representative of 5 experiments with similar results. (D) Number of CD8α⁺DEC-205⁺ cDCs in spleen and thymus from Nfil3^+/+ (○) and Nfil3^−/− (●) mice. Cell numbers were calculated on the basis of analyses by flow cytometry (n = 5; *P < .001, **P < .0001). (E) Impaired IL-12 production by splenic DCs in response to polyinosinic:polycytidylic acid [Poly (I:C)] in Nfil3^−/− mice. CD11c^hi cDCs were enriched by a MACS system with anti-Thy1.2 and anti-B220 beads for depletion of T cells, B cells, and pDCs, followed by anti-CD11c beads for isolation of cDCs. Enriched cDCs were unstimulated or stimulated with CpG (0.5 μg/mL), lipopolysaccharide (LPS; 100 μg/mL), or Poly (I:C) (100 μg/mL) for 4 hours. One hour after stimulation, brefeldin A was added to the culture. After stimulation, cells were stained for CD11c and CD45RA, followed by intracellular staining for IL-12. Data are representative of 4 experiments with similar results. (F) Developmental defect of Nfil3^−/− BM progenitor cells to CD8α⁺-equivalent cDCs. BM cells from Nfil3^+/+ or Nfil3^−/− mice were cultured with FL (50 ng/mL) for 9 days and stained for CD11c, CD45RA, SIRP-α, and CD24. Data are representative of 3 experiments with similar results. (G) FL-DCs from Nfil3^+/+ or Nfil3^−/− mice were stimulated with Poly (I:C) (100 μg/mL) for 4 hours and stained for IL-12. Data are representative of 3 experiments with similar results. (H) Lack of FL-dependent development of CD8α⁺ cDC in vivo. Nfil3^−/− and Nfil3^+/+ mice were injected with B16-FL cells subcutaneously, and spleens were harvested 8 days after injection. Splenocytes were analyzed for CD11c⁺ (left), and CD8α⁺ cDCs (right) by flow cytometry. Data are representative of 3 independent experiments. (I) Equal numbers of BM progenitor cells between Nfil3^−/− and Nfil3^+/+ mice. BM cells from Nfil3^−/− and Nfil3^+/+ mice were stained for LSK (Lin⁻c-kit^hiSca-1⁺CD127⁻), CLPs (common lymphoid progenitors; Lin⁻c-kit^intSca-1^intCD127⁺), CMP/GMP/MEPs (myeloid progenitors; Lin⁻c-kit^hiSca-1⁻CD127⁻), CDPs (common DC progenitors; Lin⁻Flt3⁺c-kit^loCD127^-CD115⁺), and pre-cDCs (Lin⁻CD11c⁺MHCII^-Flt3⁺SIRP-α^lo), and cell numbers of each population per 1 × 10⁶ BM cells were calculated. Each dot represents an individual mouse, and gray lines indicate mean cell number. (J) Relative expression of Batf3, Irf8, Id2, Irf4, and Nfil3 genes in Nfil3^−/− and Nfil3^+/+ BM pre-cDCs was determined by quantitative real-time RT-PCR. Mean expression of RNA in wild-type cells was set as 1. The mean and SD of 2 independent experiments are shown. (K) Rescue of CD8α⁺-equivalent cDCs by BATF3 and NFIL3 transduction. Nfil3^−/− BM cells stimulated with FL were transduced with vector or Batf3- or Nfil3-carrying viruses and cultured for 9 days, then stained for CD11c, CD45RA, SIRP-α, and CD24. Virus-infected cells (green fluorescent protein–positive cells) were analyzed. Data are representative of 3 experiments with similar results.