Antigen delivered to early endosomes via CD40 is more efficiently cross presented by all DC subsets than antigen delivered to lysosomes via DEC205. (A) Antibody accumulation. Day 1 DCs were fed with Alexa Fluor 488–labeled anti-DEC205 or anti-CD40 antibody continuously for 3–4 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 resultant MFI was normalized to BDCA1⁺ DCs fed with anti-DEC205. MFI was also normalized for the number of fluorophores per antibody. Data shown are the mean MFI ± SD (n = 5 independent experiments). (B and C) Antigen cross presentation via CD40 and DEC205 in DC subsets. Day 1 isolated DCs from HLA-A*0201 donors were fed with anti-DEC205, anti-CD40, or control isotype antibodies 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, Flu-M1–specific CD8⁺ T cells were detected by staining with Flu-M1 (aa 58–66) pentamer. T cell proliferation was measured by CFSE dilution. The graphs shown in B show frequency of CFSE^low, Flu-M1 (aa 58–66) pentamer-positive CD8⁺ T cells. In C, FACS plots of CD8⁺ T cells showing CFSE dilution in response to antigen presentation and numbers indicate frequency of CD8⁺ T cells in each quadrant. Shown is one representative experiment of three.