BDCA3⁺ DCs and BDCA1⁺ DCs have a similar ability to present endogenous antigen on MHCI. (A) Presentation of preprocessed peptide by DCs. Day 1 DCs from HLA-A*0201 donors were incubated with Flu-M1 (aa 58–66) or HIV-p17 (aa 77–85, negative control) peptide at 25 ng/ml for 3 h at 37°C. The cells were then washed and cultured with autologous CFSE-labeled CD8⁺ T cells at indicated DC/T cell ratio in the presence of TLR7/8 L. 8–10 d later, Flu-M1–specific CD8⁺ T cell expansion was evaluated by gating on CFSE^low cells positive for Flu-M1 (aa 58–66) pentamer. Shown data are normalized to BDCA1⁺ DC/T cells at a 1:30 ratio and the mean ± SD (n = 4 independent experiments) is depicted. (B) EGFP fluorescence intensity in DC subsets. DCs from HLA-A*0201 donors were transfected directly after isolation with a plasmid encoding for Flu-M1 (aa 55–72)–EGFP fusion protein. DCs were cultured overnight in the presence or absence of TLR7/8 L. 16 h after transfection, DCs were analyzed by flow cytometry for the expression of EGFP. Shown histograms are DCs gated on live EGFP⁺ cells. Shown is one representative experiment of three. (C) Presentation of endogenous antigen by DCs. Flu-M1 (aa 55–72)-EGFP–transfected DCs were cultured overnight in the presence or absence of TLR7/8 L and then cultured with autologous CFSE-labeled CD8⁺ T cells at indicated DC/T cell ratios, normalized to percentage of EGFP⁺ DCs. 8–10 d later, Flu-M1–specific CD8⁺ T cell expansion was evaluated as in A. Shown is one representative experiment of three.