acDC stimulation drives expansion of peptide-specific T cells. (A) Ex vivo detection of Flu HA_306-318–specific CD4⁺ T cells using Flu or control (Neg) peptide–loaded HLA-DR4 (DR*04:01) TMrs. (B) In vitro expansion of Flu HA_306-318–specific CD4⁺ T cells detected by TMrs. PBMCs were cultured without (first column) or with Flu HA_306-318 peptide (second and third columns) for 48 hours or 7 days with or without the acDC cocktail, as indicated. Flu-specific CD4⁺ T cells were identified with the corresponding HLA-DR4 TMrs (first and second columns), and background staining determined with control TMr (third column). (C-D) The same experiment was performed to detect Flu MP_58-66–specific CD8⁺ T cells using Flu or control peptide–loaded HLA-A2 (A*02:01) pentamers (PMrs). Numbers in the upper right corner of each dot plot indicate the percentage and absolute numbers of CD4⁺TMr⁺ or CD8⁺PMr⁺ cells. acDC maturation was induced with anti-CD40/IFN-α and dot plots are gated on CD14/CD19⁻ viable cells. Results are representative of 3 independent experiments.