Early induced CD8α⁺ DCs exhibit hallmarks of classic CD8α⁺ DCs. (A) Ly5.2⁺CD11c⁺CD45RA⁻ CD8α⁺, CD4⁺ and CD8α⁻CD4⁻ DN DCs derived from Id2^gfp/gfp (Ly5.2⁺)→Ly5.1 or Id2^gfp/gfpBatf3^−/− (Ly5.2⁺)→Ly5.1 chimeric mice 3 weeks after transplantation of bone marrow were purified by FACS sorting from spleen. DCs (1.25 × 10⁴) were cocultured with OVA-coated bm1 splenocytes and 5 × 10³ CTV CD8⁺ OT-I or CD4⁺ OT-II T cells. After 60 hours, the level of proliferation induced in cultures were analyzed by staining cells for Vα2⁺ within the CD4⁺ and CD8⁺ populations. Data are representative profiles of 2 experiments with similar results. (B) Expression of CD8α, CD24, and Clec9A surface molecules in splenic CD8α⁺ DCs. Ly5.2⁺ CD11c⁺ splenic DCs from bone marrow chimeric mice as described in panel A. Numbers indicate the mean fluorescence intensity of each marker within the PI⁻CD45⁻CD11c⁺CD8α⁺ DC subset. (C) Quantitative PCR analyses of Xcr1, Tlr3, and Batf3 expression by CD8α⁺ DCs 3 weeks after bone marrow transplantation. FACS purified CD8α⁺ DCs isolated from Id2^gfp/gfp (Ly5.2⁺) or Id2^gfp/gfpBatf3^−/− (Ly5.2⁺)→Ly5.1 recipients and analyzed for expression of Tlr3. (D) Analysis of CX₃CR1 and CCR2 expression on splenic DC subset 3 weeks after reconstitution of Ly5.1 recipients with Ly5.1 (WT), Cx₃cr1-GFP, or Ccr2-DTR-CFP bone marrow. Dotted line shows expression on Ly6C^high monocytes. (A-D) Data are representative of at least 2 experiments for each condition and error bars represent mean ± SD.