Ex vivo restimulation of OT-I T cells

For intracellular cytokine staining, cells from the CLN or skin were isolated 60 hours or 8 to 11 days after adoptive transfer of OT-I T cells. Cells were cultured for 4 hours in RPMI 1640 plus 10% FBS containing OVA peptide (257 to 264) (1 μg/ml). Brefeldin A (10 mg/ml) was added after the first hour of culture. Then, cells were surface stained to identify OT-I (Lin⁻TCRβ⁺CD5⁺CD8⁺Vα2⁺Vβ5.1/2⁺CD45.1⁺CD45.2⁺) cells and subsequently stained intracellularly with anti–IFN-γ antibody.