Figure 6. Dendritic cells present antigen in injection sites throughout the primary immune response.

Single cell preparations from the ears of B6 mice injected with either EαRFP/IFA in both ears or OVA/IFA in both ears were enriched as indicated. The events in shown (A) and (B) represent viable cells, in (C-D, F-G) CD11c+ cells, and in (E) CD11c⁺, CD11b^high cells. (A) CD11c staining versus forward light scatter of anti-MHCII magnetic bead-enriched cells from EαRFP/IFA injection sites of B6 mice 24 hours or 12 days after injection. Numbers indicate the percentage of cells in the CD11c⁺ gate. (B) CD11c versus CD11b staining (left panel) of anti-CD11c magnetic bead enriched cells from EαRFP/IFA injection sites of B6 mice 12 days after injection. F4/80 staining (right panel, thick line) is shown for the CD11c⁺, CD11b^high cells from the left panel. The shaded histogram shows staining of the same population with an isotype control antibody. (C) CD11b staining versus Langerin expression in anti-CD11c magnetic bead enriched cells from EαRFP/IFA injection sites of control B6 mice (left panel) or Langerin-GFP transgenic mice (right panel) 12 days after injection. Dermal Langerin⁺ cells were identified in the left gate and epidermal Langerhans cells in the right gate in the panel from Langerin-GFP transgenic mice. Numbers indicate the percentage of cells in the indicated gates. (D) B220 versus CD11b staining for anti-CD11c magnetic bead enriched cells from lymph nodes (left panel) or EαRFP/IFA injection sites (right panel) of B6 mice 12 days after injection. Plasmacytoid DC were identified as B220⁺, CD11b⁻ cells based on the gate from the lymph nodes (left panel). A population of B220⁻, CD11b⁻ cells was also identified with this staining combination. (E) Y-Ae staining (left panel) and antigen uptake (right panel) in the CD11c⁺ CD11b^high cells (dermal DCs) in (B) in Y-Ae magnetic bead-enriched populations from OVA/IFA (shaded) or EαRFP/IFA (thick line) injection sites of B6 mice 12 days after injection. (F) Y-Ae staining (left panel) and antigen uptake (right panel) in the Langerin⁺, CD11b⁻ (dermal Langerin⁺ DCs) and (G) Langerin⁺, CD11b⁺ (epidermal Langerhans cells) in (C) in anti-CD11c magnetic bead-enriched populations from OVA/IFA (shaded) or EαRFP/IFA (thick line) injection sites of Langerin-eGFP mice 12 days after injection. (H) Y-Ae staining in the B220⁺, CD11b⁻ (plasmacytoid DCs) and B220⁻, CD11b⁻ cells in (D) in anti-CD11c magnetic bead-enriched populations from OVA/IFA (shaded) or EαRFP/IFA (thick line) injection sites of B6 mice 12 days after injection. Data are representative of two to five independent experiments.