Priming Naive HLA-A2–EV10 Specific T Cells

Naive precursors specific for HLA-A2–EV10 were primed in vitro using an accelerated dendritic cell (DC) coculture protocol as described previously (11 (link), 16 (link), 17 ). Briefly, thawed PBMCs were resuspended at 5 × 10⁶ cells/well in AIM-V medium (Thermo Fisher Scientific) supplemented with Flt3 ligand (Flt3L; 50 ng/mL; R&D Systems) in the absence or presence of rosiglitazone (40 μM; Sigma-Aldrich) or IL-7 (20 ng/mL; R&D Systems). After 24 hr (day 1), the Melan-A peptide EV20 (1 μM) was added to the cultures, and DC maturation was induced using a standard cocktail of inflammatory cytokines, incorporating IL-1β (10 ng/mL), IL-7 (0.5 ng/mL), PGE2 (1 μM), and TNF (1,000 U/mL) (all from R&D Systems), or ssRNA40 (TLR8L; 0.5 μg/mL; InvivoGen). The cultures were supplemented on day 2 with FCS (10% v/v; Sigma-Aldrich). Medium was replaced every 3 days thereafter with fresh R+ containing FCS (10% v/v; Sigma-Aldrich). Antigen-specific CD8⁺ T cells were characterized via flow cytometry on day 10.