All work conducted on animals were cared for following the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals under a protocol approved by the NIH Clinical Center Animal Care and Use Committee. Female C57BL/6 mice (6 to 8 weeks) were purchased from the Jackson Laboratory. For cancer immunotherapy studies, C57BL/6 mice were subcutaneously injected with 0.1 × 106 MC38 cells on the right shoulder. On day 9, C57BL/6 mice were immunized with different formulations containing antigen peptides (20 μg per mouse), CpG (2 nmol per mouse), and R848 (8 μg per mouse) in 100-μl volume by subcutaneous injection at the tail base on days 9, 15, and 21. For the combinatorial immunotherapy, anti-mouse PD-1 (200 μg per mouse) antibodies were administered intraperitoneally on days 9, 12, 15, 18, and 21. Tumor growth was monitored every 3 days, and the tumor volume throughout this study was calculated by caliper measurement using the following equation: tumor volume = length × width2/2. Animals were euthanized when the maximum tumor diameter reached 1.8 cm or more. For prophylaxis studies, C57BL/6 mice (6 to 8 weeks) were immunized with tumor vaccines on days 0, 7, and 14. Vaccinated mice were challenged 6 days later after last immunization by inoculation with 0.3 × 106 MC38 cells on the right shoulder per mouse. Tumor growth was monitored as indicated above. Mice were euthanized if the tumor diameter exceeded 1.8 cm.

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