OT-1 T cell isolation and adoptive transfer.

Cell suspensions from the lymph nodes of the MHC class I–restricted, OVA-specific, TCR-transgenic OT-1 mice (OT-1 cells) (8 weeks to 4 months old) were harvested (see tissue digestion protocol) and adoptively transferred by tail vein injection (2 × 10⁶ cells per mouse) on the specified day after tumor inoculation. When specified, OT-1 cells were labeled before intravenous injection with 2 μM CFSE (CellTrace CFSE Cell Proliferation Kit, Invitrogen, Thermo Fisher Scientific) by incubating them for 20 minutes at 37°C according to the manufacturer’s instructions. This was followed by an immunization with OVA protein (50 μg) and poly(I:C) (50 μg) 1 hour later. Cell division accompanied by CFSE dilution was analyzed by flow cytometry 2 days later (detected in the FITC channel).