acDC stimulation supports Ag-specific T-cell expansion. (A) Schematic of the acDC ELISPOT and T-cell clone-generation procedure. acDCs were induced by adding GM-CSF and IL-4 along with protein Ag to CFSE-labeled PBMCs on day 0, followed on day 1 by TNF-α/PGE₂/IL-1β. On day 2, nonadherent cells were transferred into ELISPOT wells for 6 hours to quantify Ag-specific responses. Cells were subsequently recovered and recultured without further Ag or cytokines. On day 10-12, proliferating (CFSE^low) cells were single-cell-sorted, expanded through 3 stimulation cycles with anti-CD3 + IL-2/IL-4, and tested for Ag specificity at day 32. (B) Representative acDC IFN-γ ELISPOT after TTX or control stimulation for 48 hours. (C) Proliferation of CFSE-labeled PBMCs recovered from ELISPOT wells and cultured for another 8 days. A comparison of standard versus acDC-driven expansion is shown. The TTX-specific CFSE^low fraction was sorted and cloned. (D) Recall assay of one clone on TTX- and control-pulsed DCs followed by intracellular IFN-γ staining is shown.