Figure 4.
IL-6 blocks cDC1 development by activating the Zeb2 −165 kb enhancer. (A) Upper panel: Retroviral reporter construct: Zeb2 −165 kb enhancer (yellow), CMV minimal promoter (red arrow), and EGFP (green). Lower panel: Sort-purified CDPs were transduced with reporter constructs harboring WT or Δ1+2+3 enhancers and cultured for 2 d with 5% Flt3L with or without 25 ng/ml IL-6, and EGFP expression assessed in Thy1.1+ cells. Numbers represent the MFI of cells. LTR, long terminal repeat. (B) A bar-scatter graph of three independent experiments from A for EGFP expression (average MFI ± SD). ***P < 0.001 (Student’s t test). (C) Sort-purified CDPs from WT or Δ1+2+3 mice were cultured in vitro with Flt3L and the indicated concentrations of IL-6 for 4 d. Shown is FACS analysis for MerTK− B220− SiglecH− cells. Numbers indicate the percentage of cells in the indicated gates. Data shown are representative of two independent similar experiments. (D) Sort-purified CDPs from WT or Δ1+2+3 mice were cultured in vitro with Flt3L alone (blue) or with 25 ng/ml IL-6 (red) for 20 h and analyzed for intracellular C/EBPβ expression. IC indicates staining with isotype control antibody. Number indicates MFI. (E and F) WT and Δ1+2+3 mice were inoculated with EL4-empty or EL4-IL-6 tumors and analyzed after 14 d. Shown is FACS analysis of (E) splenocytes pre-gated as MerTK− B220− SiglecH− MHCII+ CD11c+ cells and (F) BM DC progenitors pre-gated as Lin− SiglecH− Flt3+ CD11c+ MHCIIlo-int BM cells for cKit and CD24 expression. (G and H) Sort-purified OT-I and OT-II (2.5 × 105 cells, each) were transferred i.v. into WT or Δ1+2+3 mice inoculated 14 d earlier with EL4 tumors. OVA-loaded splenocytes (5 × 105 cells/mouse, i.v.) were transferred i.v. after 3 h. (G) In vivo proliferation of OT-I (upper panel) and OT-II (lower panel) on day 3 after transfer. (H) Bar-scatter graphs from G for OT-I (upper) and OT-II (lower) of the indicated conditions (average % ± SD). Individual mice are shown as dots. *P < 0.05, **P < 0.01, ****P < 0.0001 (Student’s t test). Refer to the image caption for details.

IL-6 blocks cDC1 development by activating the Zeb2 −165 kb enhancer. (A) Upper panel: Retroviral reporter construct: Zeb2 −165 kb enhancer (yellow), CMV minimal promoter (red arrow), and EGFP (green). Lower panel: Sort-purified CDPs were transduced with reporter constructs harboring WT or Δ1+2+3 enhancers and cultured for 2 d with 5% Flt3L with or without 25 ng/ml IL-6, and EGFP expression assessed in Thy1.1+ cells. Numbers represent the MFI of cells. LTR, long terminal repeat. (B) A bar-scatter graph of three independent experiments from A for EGFP expression (average MFI ± SD). ***P < 0.001 (Student’s t test). (C) Sort-purified CDPs from WT or Δ1+2+3 mice were cultured in vitro with Flt3L and the indicated concentrations of IL-6 for 4 d. Shown is FACS analysis for MerTK− B220− SiglecH− cells. Numbers indicate the percentage of cells in the indicated gates. Data shown are representative of two independent similar experiments. (D) Sort-purified CDPs from WT or Δ1+2+3 mice were cultured in vitro with Flt3L alone (blue) or with 25 ng/ml IL-6 (red) for 20 h and analyzed for intracellular C/EBPβ expression. IC indicates staining with isotype control antibody. Number indicates MFI. (E and F) WT and Δ1+2+3 mice were inoculated with EL4-empty or EL4-IL-6 tumors and analyzed after 14 d. Shown is FACS analysis of (E) splenocytes pre-gated as MerTK− B220− SiglecH− MHCII+ CD11c+ cells and (F) BM DC progenitors pre-gated as Lin− SiglecH− Flt3+ CD11c+ MHCIIlo-int BM cells for cKit and CD24 expression. (G and H) Sort-purified OT-I and OT-II (2.5 × 105 cells, each) were transferred i.v. into WT or Δ1+2+3 mice inoculated 14 d earlier with EL4 tumors. OVA-loaded splenocytes (5 × 105 cells/mouse, i.v.) were transferred i.v. after 3 h. (G) In vivo proliferation of OT-I (upper panel) and OT-II (lower panel) on day 3 after transfer. (H) Bar-scatter graphs from G for OT-I (upper) and OT-II (lower) of the indicated conditions (average % ± SD). Individual mice are shown as dots. *P < 0.05, **P < 0.01, ****P < 0.0001 (Student’s t test).

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