acDC stimulation reveals boosting of T-cell responses after Ag vaccination. (A) IFN-γ ELISPOT responses against MVA (0.1 PFUs/cell) were analyzed in frozen-thawed PBMCs from 21 healthy subjects before and after smallpox revaccination, using either nonamplified (diamonds) or acDC-amplified (circles) stimulation. The -fold changes in frequencies of MVA-specific IFN-γ SFCs before and after vaccinations are shown. Bars indicate the median and interquartile range of each distribution, whereas paired samples tested with or without the acDC cocktail are connected by lines. P = .019 for paired comparison between acDC and the “no cytokines” condition. (B) IFN-γ-, TNF-α-, and IL-2–producing CD4⁺ and CD8⁺ responses elicited by acDC stimulation in vitro with Flu vaccine or the HA and NP peptide pools analyzed by intracellular staining in frozen-thawed PBMCs from flu-vaccinated healthy subjects. The -fold changes in responses after vaccination are displayed, with each symbol representing a single individual and bars showing median values for each distribution. Dotted lines identify the 0.7- to 1.3-fold interval (ie, no -fold change). (C) acDC-amplified IFN-γ ELISPOT responses against DP4-restricted MAGE-3_247-258 (circles) or pan-DR-restricted TTX_830-844 (diamonds) epitopes were analyzed in frozen-thawed PBMCs from 10 stage IIIb/IV melanoma patients before (open symbols) and after (filled symbols) vaccination with exosomes loaded with MAGE-3_247-258, either alone or in combination with TTX_830-844, as indicated. Background-subtracted frequencies of IFN-γ SFC/10⁶ PBMCs (means ± SEM of triplicate wells) are shown before and after vaccination. Counts < 3 SD above background were scored as nondetectable (gray-shaded area). *P < .04.