Protein/peptide Ags and different stimulation periods trigger different T-cell responses. (A) IFN-γ ELISPOT performed on PBMCs cultured with or without the acDC cocktail. PBMCs from a HLA-A2⁺ (A*02:01) and -DR4⁺ (DR*04:01) subject were stimulated with BSA control or no Ag, HLA-A2–restricted Flu MP_58-66 peptide, DR4-restricted Flu HA_306-318 peptide, or TTX, as indicated. Peptides were added after the first 24 hours of culture, whereas TTX was introduced ab initio. (B) TTX-specific IFN-γ ELISPOT responses in acDC stimulations performed on PBMCs magnetically depleted of CD45RA⁺ or CD45RO⁺ cells or left undepleted. Results are expressed as relative IFN-γ responses normalized to undepleted PBMCs. (C) IFN-γ and IL-10 ELISPOT responses to TTX and KLH (background-subtracted) on acDC-stimulated PBMCs labeled with CFSE. (D) CFSE-labeled PBMCs were recovered from the assay wells of panel C and cultured for an additional 10 days in the absence of further stimuli and cytokines. CFSE proliferation of CD4⁺ and CD8⁺ T cells to different Ags is shown. Results in panels A-D were obtained after maturing acDCs with TNF-α/PGE₂/IL-1β and are representative of 3 independent experiments, with the exception of panel B, in which the means ± SEM of 3 separate experiments are shown.