Pulsing DCs with Ag prior to their maturation is required to prime CD4⁺ T cells but not to elicit a recall response.

(A) Experimental design. DCs were pulsed with Ag on day 3, 6, or 7 of their culture, either before or during TNF-α–induced maturation. DCs were then harvested on day 8, irradiated, and used as stimulator cells in a primary proliferation assay with autologous CD4⁺ T cells at a T/DC ratio of 1:10. (B) Proliferation to recall Ag. DCs loaded with TT at indicated times were used to stimulate autologous CD4⁺ T cells. When indicated, Ag was removed from the DC culture after 4 hours of uptake (rem. 4h). [³H]-thymidine incorporation was measured at day 6 of the assay. (C) Proliferation to neoAg. DCs were incubated with KLH at indicated times and used at day 8 to stimulate autologous CD4⁺ T cells in a primary assay. When indicated, Ag was removed from the DC culture after 4 hours, 1 day, or 2 days of Ag uptake (rem. 4h, rem. d4, rem. d5). [³H]-thymidine incorporation by CD4⁺ T cells was measured in triplicate at day 7 of the assay. These results are representative of 3 experiments with 3 different donors.