Targeting CD40 and MR leads to superior cross presentation compared with targeting DEC205. (A) Schematic for the generation of antibody-peptide conjugates. On average, our peptide conjugates had 1 peptide per antibody. (B) Matched immature or (C) mature HLA-A*0201⁺ DCs were incubated with various concentrations of anti–CD40-M1, anti–MR-M1, or anti–DEC205-M1 for 4-6 hours, washed, and cocultured with autologous CFSE-labeled CD8⁺ T cells for 8-10 days in the presence of LPS and IL-2. “Immature” or “mature” refers to the activation state of the DCs before antigen uptake and coculture. MuIso-M1 is the isotype for anti–CD40-M1, and huIso-M1 is the isotype for anti–MR-M1 and anti–DEC205-M1. Graphs depict frequencies of total CFSE^lo and Influenza M1 (58-66) specific CD8⁺ T cells. One representative experiment of 6 to 8 independent donors (immature samples) or 3 to 4 independent donors (mature samples) is shown. Targeting via 1 μg/mL of anti-CD40 and MR was judged superior to targeting via anti-DEC205 across 6 to 8 independent donors with the use of a paired t test for immature DCs (CD40 vs DEC205: MoDC, P = .0270, BDCA1⁺ DC, P = .0337; MR vs DEC205: MoDC, P = .0264; BDCA1⁺ DC was not statistically significant possibly because of variable surface MR expression). (D) Influenza M1 (58-66) nonextended peptide control for antigen presentation assay. (E top panel) As in panel B, DCs were incubated with anti–CD40-pp65 or anti–DEC205-pp65 conjugates. (Bottom panel) CMV pp65 (495-503) nonextended peptide control. (F) DCs were incubated with 1 μg/mL antibody-M1 conjugates or 25 ng/mL M1 peptide with or without 0.1μM epoxomicin or DMSO for 4-6 hours, followed by washing and coculture with autologous CFSE-labeled CD8⁺ T cells. “No ag” shows the background when DCs and CD8⁺ T cells are cocultured in the absence of antigen. Graphs depict frequencies of total CFSE^lo and influenza M1 (58-66) specific CD8⁺ T cells. One representative experiment from ≥ 3 independent donors is shown.