Transcutaneous immunization with imiquimod is amplified by CD40 ligation and results in sustained cytotoxic T-lymphocyte activation and tumor protection.

Transcutaneous immunization (TCI) using ligands of Toll-like receptors (TLRs) and cytotoxic T-lymphocyte (CTL) epitopes lead to the induction of potent T-cell responses. To characterize the efficacy of TCI-mediated CTL activation, we monitored the frequency and functional activity of specific CTL induced with TCI using the ovalbumin-derived epitope SIINFEKL composed in creme containing the synthetic TLR7 ligand R-837. We found that the frequency and activity decayed rapidly 10 d post-TCI. Consistently, no significant memory T-cell formation was detectable. In a prophylactic vaccination setting, TCI was protective against a lethal challenge with ovalbumin expressing EG.7 thymoma cells when the tumor cells were inoculated 5 d later. However, only a delay of tumor growth was observed when the tumor challenge was performed 55 d after immunization. Conversely, a single combined treatment with TCI and an agonist anti-CD40 (FGK-45) monoclonal antibody greatly enhanced the primary response, with up to 30% of peptide-specific CTL and the effective induction of memory cells. Consequently, mice treated with TCI/anti-CD40 were completely protected against a lethal tumor challenge with EG.7 tumor cells after 55 d. In this article, we demonstrate that transcutaneous immunization approaches using TLR ligands deliver sufficient amounts of antigen to mediate durable protection against tumors if adequate costimulation is provided. These results may contribute to the development of advanced vaccination protocols against malignancies and persistent virus infections.