Treatment of B16F10 melanoma–bearing mice

The dorsal skin of C57Bl6 mice was shaved, and B16F10 or B16F10-OVA cells (10⁵) were implanted in the right dorsal flank on day 0. After 5 (when all tumors were visible), 7, and 9 days, mice were i.d. injected with 150, 50, or 12.5 μg of anti-mouse CTLA-4 (clone 9H10 or UC10-4F10-11; BioXCell) and/or rat anti-mouse PD-1 (clone RMP1-14; BioXCell) i.t., i.d. in the forelimb, or i.p. in 30 μl of saline. In abscopal tumor immunotherapy experiments, 10⁵ B16F10 cells were injected i.d. on the right dorsal skin of the mouse on day 0 and on the left dorsal skin on day 2. On days 5, 7, and 9, mice were injected with 150 μg of aCTLA-4 (clone 9H10) and aPD-1 (i.d. or i.p.) in saline. For immune cell phenotyping, mice were euthanized on day 12 and tissues were harvested. In vaccination studies, at 4 and 10 days, CpG (3 μg) and OVA (10 μg) were i.d. administered in 30 μl of saline in each limb. On days 5, 8, 11, and 14, mice received 150 μg of aCTLA-4 and aPD-1 mAb in 30 μl of saline either i.t., i.d. in the forelimb, or i.p. In studies evaluating the effects of sustained mAb release, Pluronic F127 (Sigma-Aldrich) was dissolved at 25 weight % in cold phosphate-buffered saline (PBS). Before injection, 25 μg of aCTLA-4 (clone 9H10) and aPD-1 mAb (5 μl) was mixed with 25 μl of the gel solution or PBS and i.d. injected once into one forelimb on day 5.