DEC205-targeted cross presentation is rescued by the addition of protease and acidification inhibitors. Alexa 488 (4 μg/mL) covalently conjugated (A) anti-DEC205 (3G9), (B) anti-CD40 (S2C6), or (C) anti-MR (B11) antibodies were internalized by MoDCs for 1 hour at 37°C. Cells were washed extensively and chased for indicated times with or without leupeptin and NH₄Cl (inh), followed by fixation and analysis by flow cytometry. Graphs depict the mean MFI ± SEM of 3 independent donors. MFI differences in the absence or presence of inhibitors were assessed with the paired t test, and statistically significant differences were depicted, *P < .05. (D) Immature or (E) mature BDCA1⁺ DCs were incubated with 1 μg/mL DEC205-M1 conjugates with or without indicated inhibitors for 4-6 hours, followed by washing and coculture with autologous CFSE-labeled CD8⁺ T cells. Graphs depict frequencies of CFSE^lo, influenza M1 (58-66) specific CD8⁺ T cells. One representative of 3 independent donors is shown (D) and 1 representative of 2 independent donors is shown (E). (F) Immature BDCA1⁺ DCs were incubated with 1 μg/mL CD40-M1 conjugates with or without indicated inhibitors for 4-6 hours, followed by washing and coculture with autologous CFSE-labeled CD8⁺ T cells. Graphs depict frequencies of total CFSE^lo and Influenza M1 (58-66) specific CD8⁺ T cells. One representative of 3 independent donors is shown. (G) BDCA1⁺ DCs were incubated with 1 μg/mL DEC205-pp65 conjugates with or without indicated inhibitors for 4-6 hours, followed by washing and coculture with autologous CFSE-labeled CD8⁺ T cells. Graphs depict frequencies of CFSE^lo, CMV pp65 (495-503) specific CD8⁺ T cells. One representative of 2 independent donors is shown. (H) CMV pp65 (495-503) nonextended peptide control (25 ng/mL).