Anti-CD40 does not enhance cross presentation of MR or DEC205. (A) We added 1 μg/mL anti-CD40 (S2C6), anti-MR (B11), anti-DEC205 (3G9), isotype (iso), or 200 ng/mL LPS to MoDCs overnight. After overnight culture, supernatants were harvested, and DCs were labeled for surface markers that are normally up-regulated after maturation, as indicated. One representative of 3 independent donors is shown. (B) Supernatants from panel A were analyzed for cytokine production with the use of Luminex technology. IL-12p70 levels were insignificant, except for LPS controls. (C-D) MoDCs or (E) BDCA1⁺ DCs were incubated with 1 μg/mL antibody-M1 peptide conjugates alone or with antibody-M1 conjugates plus 1 μg/mL unconjugated CD40 (S2C6) antibody or 1 μg/mL isotype (muIso) together 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. (F) BDCA1⁺ DCs were incubated with 1 μg/mL DEC205–NY-ESO peptide conjugates alone or with DEC205–NY-ESO peptide conjugates plus 1 μg/mL unconjugated CD40 (S2C6) antibody or 1 μg/mL isotype (muIso) together for 1.5 hours, followed by washing and coculture with an NY-ESO–specific CD4⁺ T-cell clone. Graphs depict percentage of IFNγ⁺ CD4⁺ T cells. One representative of 2 independent DC donors is shown.