Antigen targeted to early endosomes via CD11c is cross presented by both DC subsets with similar efficacy. (A) Anti-CD11c antibody intracellular trafficking. Day 1 BDCA1⁺ DCs were fed with Alexa Fluor 488–labeled anti-CD11c antibody (green) continuously for 3 h, washed, and allowed to adhere to coverslips. After fixation and permeabilization, the early endosomes and plasma membrane were stained using EEA1 (red) and anti–HLA-DR (blue) antibodies, respectively. Cells were then analyzed using confocal microscopy. Bar, 5 µm. Shown is one representative experiment of three. (B) Antigen cross presentation via CD11c in DC subsets. Day 1 isolated DCs from HLA-A*0201 donors were fed with anti-CD11c or control isotype antibody conjugated to Flu-M1 (aa 55–72) at indicated doses for 4 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8⁺ T cells in the presence of IL-2 and TLR7/8 L. 8–10 d later, CD8⁺ T cell expansion was evaluated by gating on CFSE^low cells positive for Flu-M1 (aa 58–66) pentamer. Shown is one representative experiment of three.