Tumor Immunization and Checkpoint Blockade

Mice were immunized intramuscularly into both tibialis anterior muscles with 100 µg/mouse DNA or transplanted subcutaneously into the left/right flank with 2.5×10⁵ tumor cells (in 100 µL PBS). For cell-based immunizations, tumor cells were pretreated with recombinant mouse IFN-γ (20 ng/mL, cat. no. 554587, BD Biosciences, Heidelberg, Germany) for 16–20 hours, washed and cultured for indicated times before gamma irradiation (30 Gy). Where indicated, anti-PD-1 (cat. no. BP0146; Bio X Cell), anti-CD8 (cat. no. BE0117; Bio X Cell), anti-CD4 (cat. no. BE0119; Bio X Cell) and rat IgG2a or IgG2b isotype control antibodies (cat. no. BP0089, BE0090; Bio X Cell) were injected intraperitoneally (100 µg/mouse). Tumor growth was monitored by regular palpation with calipers. Mice were sacrificed when the tumor diameter reached 1 cm. Determination of antigen-specific CD8 T-cell frequencies by flow cytometry (FCM) was carried out as described previously.30 (link)