Antigen targeted to early endosomes via CD40 is cross presented by both DC subsets with similar efficacy. (A) Anti-CD40 antibody intracellular trafficking. Day 1 BDCA1⁺ DCs (top) or BDCA3⁺ DCs (bottom) were fed with Alexa Fluor 488–labeled anti-CD40 antibody (green) continuously for 3 h at 37°C, washed, and allowed to adhere to coverslips. After fixation and permeabilization, the lysosomes or early endosomes were stained using anti-Lamp1 or anti-EEA1 (red), respectively. Plasma membrane was stained using anti–HLA-DR (blue) antibodies. Cells were then analyzed using confocal microscopy. Bars, 5 µm. Shown is one representative experiment of three. (B) Accumulation of anti-CD40 antibody. Day 1 DCs were fed with Alexa Fluor 488–labeled anti-CD40 antibody continuously for 4–6 h at 4°C or 37°C. Results were analyzed by flow cytometry. The 4°C MFI was subtracted from the 37°C MFI, and the resulting MFI was normalized to BDCA1⁺ DCs. Data shown are the mean MFI ± SD (n = 3 independent experiments). (C) Antigen cross presentation via CD40 in DC subsets. Day 1 isolated DCs from HLA-A*0201 donors were fed with anti-CD40, or control isotype antibody conjugated to Flu-M1 (aa 55–72) at 1 µg/ml 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, Flu-M1–specific 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 more than five. (D) As in C; anti-CD40 and control isotype antibodies were conjugated to CMV-pp65 (aa 488–508) at the indicated doses. Shown is one representative experiment of two.