Functional properties of LN DC subsets. DC subsets from LNs (black symbols) or blood (blue symbols) were purified and cultured with allogeneic naive CD4⁺ T cells for 6 d before T cell restimulation. (A–C) Symbols represent cells purified from the same donor (for LNs, n = 4 for macrophages and n = 7–11 for DCs; for blood, n = 5). Black closed symbols represent experiments with CD1a⁺ DCs alone, and other symbols represent experiments with pooled CD1a⁺ DCs and LCs. Mean is shown. (A) T cell proliferation was assessed by calculating fold expansion (ratio of the number of T cells at the end of the culture divided by the number of T cells plated at the start of the culture). (B and C) Cytokine concentration was measured in culture supernatant by ELISA or cytometric bead array. (D) Cultured T cells were permeabilized and stained for GATA-3, T-bet, and RORγt. Representative plots of five independent experiments and mean ± SD are shown. (E) Purified LN or blood DCs were incubated with (+) or without (−) MelanA long or short peptide and cultured with antigen-specific CD8⁺ T cell clones. Secretion of IFN-γ was assessed as a measure of T cell activation. Symbols represent DCs purified from the same donor (n = 3; except for CD206⁺ DCs n = 2). CD1a⁺ DCs and LCs were pooled. Mean is shown.