Antigen-Specific T Cell Activation by DCs

For DC transfer experiments, splenic DCs were isolated as described above from mice subcutaneously injected with 1 × 10⁶ B16 melanoma cells that constitutively secrete FMS-like tyrosine kinase 3 ligand (Flt3L)^{58 (link)} 10 days prior to harvest. Cells were resuspended at 10⁷ cells/ml and incubated with 10 μM OVA^323–339, LCMV-GP^61–80, OVA^257–264, or LCMV^276–286 peptides (Anaspec) in RPMI + 10% FBS, for 30 min at 37 °C. For cell labelling, CFSE or CTV (ThermoFisher Scientific) was added to a final concentration of 2 μM during the last 5 or 20 minutes of incubation, respectively. Cells were washed three times in RPMI + 10% FBS and resuspended at 2 × 10⁷ cells/ml in PBS supplemented with 0.4 μg ml⁻¹ LPS (Sigma-Aldrich). DCs were injected (5 × 10⁵ cells in 25 μl) subcutaneously into the hind footpads. For CD4⁺ T cell and CD8⁺ T cell transfer experiments, T cells isolated as described above were injected intravenously in 100 μl PBS per mouse.