In vitro and in vivo cross-presentation of cell-associated, soluble, and viral antigens in Batf3-deficient mice. (A) Ly5.2⁺CD45RA⁻ CD11c⁺Sirpα⁺ (top panels), CD11c⁺Sirpα⁻CD103⁻ (CD8α⁺ equivalent; middle panels) and CD11c⁺Sirpα⁻CD103⁺ DCs (bottom panels) were purified from bone marrow cultures of Id2^gfp/gfp(Ly5.2⁺)→Ly5.1 or Id2^gfp/gfpBatf3^−/−(Ly5.2⁺)→Ly5.1 chimeric mice that had been allowed to reconstitute for 10 weeks. Bone marrow was stimulated with Flt3L for 8 days with the addition of GM-CSF on day 6. FACS purified populations of DCs were cocultured with soluble OVA together with either CD8⁺ OT-I cells labeled with CellTracker Violet (CTV). Numbers represent the percentage of cells that have proliferated in response to exogenous antigen. Data are representative of bone marrow cells derived from 2 separate cohorts of Id2^gfp/gfp(Ly5.2⁺)→Ly5.1 or Id2^gfp/gfpBatf3^−/−(Ly5.2⁺)→Ly5.1 chimeric mice and each DC subset analyses was performed in duplicate or triplicate within each experiment. (B) In vivo cross-presentation of soluble OVA (top panels). OVA-coated irradiated bm-1 splenocytes or soluble OVA was injected into recipient wild-type or Batf3^−/− mice containing 1.5 × 10⁶ CTV-labeled Ly5.1⁺ OT-I cells. After 3 days, the spleen was isolated and analyzed for proliferation by loss of CTV from Ly5.1⁺Vα2⁺CD8⁺ T cells. (C) Cross-presentation of cell-associated OVA in GVHD. Bm1.ActmOVA mice were irradiated and transplanted with 5 × 10⁶ BM from either Id2-GFP or Id2 × Batf3^−/− bone marrow, and 2 × 10³ CD3⁺ T cells from Ly5.1 congenic mice. CSFE-labeled Ly5.1⁺ OT-I cells were adoptively transferred into mice on day 10 after transplantation and proliferation in the OVA-specific Vα2⁺ CD8⁺ T-cell population analyzed after 3 days. Data show representative proliferation profiles (left panels) and the mean number ± SD of K^b-OVA Vα2⁺ CD8⁺ T cells recovered per spleen (n = 8). NS, not significant. (D) Responses to HSV-1 virus infection. 1.5 × 10⁶ CTV labeled Ly5.1⁺ HSV-1–specific (glycoprotein B, gB) CD8α⁺ T cells were adoptively transferred into wild-type or Batf3^−/− mice 1 day before subcutaneous foot pad inoculation with HSV-1. After 3 days, the spleen was isolated and analyzed for proliferation by loss of CTV from Ly5.1⁺Vα2⁺CD8⁺ T cells. Data are representative of 3 experiments with 2 to 4 mice in each group for each experiment.