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Transcutaneous immunization (TCI) is a novel vaccination strategy that utilizes skin-associated lymphatic tissue to induce immune responses. Employing T-cell epitopes and the TLR7 agonist imiquimod onto intact skin mounts strong primary, but limited memory CTL responses. To overcome this limitation, we developed a novel imiquimod-containing vaccination platform (IMI-Sol) rendering superior primary CD8 + and CD4 + T-cell responses. However, it has been unclear whether IMI-Sol per se is restricted in terms of memory formation and tumor protection. In our present work, we demonstrate that the combined administration of IMI-Sol and CD40 ligation unleashes fullblown specific T-cell responses in the priming and memory phase, strongly enhancing antitumor protection in mice. Interestingly, these effects were entirely CD4 + T cell independent, bypassing the necessity of helper T cells. Moreover, blockade of CD70 in vivo abrogated the boosting effect of CD40 ligation, indicating that the adjuvant effect of CD40 in TCI is mediated via CD70 on professional APCs. Furthermore, this work highlights the so far underappreciated importance of the CD70/CD27 interaction as a promising adjuvant target in TCI. Summing up, we demonstrate that the novel formulation IMI-Sol represents a powerful vaccination platform when applied in combination with sufficient adjuvant thereby overcoming current limitations of TCI., (© 2019 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The epidermal application of the Toll Like Receptor 7 agonist imiquimod and a T-cell peptide epitope (transcutaneous immunization, TCI) mediates systemic peptide-specific cytotoxic T-cell (CTL) responses and leads to tumor protection in a prophylactic tumor setting. However, it does not accomplish memory formation or permanent defiance of tumors in a therapeutic set-up. As a distinct immunologic approach, CTLA-4 blockade augments systemic immune responses and has shown long-lasting effects in preclinical experiments as well as in clinical trials., Objective: The study investigates the vaccination capacity of TCI in combination with the checkpoint inhibitor CTLA-4 in matters of primary response, memory formation and tumor protection and characterizes the role of regulatory T cells (Tregs)., Methods: After performing TCI with IMI-Sol (containing 5% Imiquimod) and the model epitope SIINFEKL, 6-8 week old C57BL/6 mice received anti-CTLA-4 antibody either s.c or i.p. The CTL responses and frequency of peptide specific CD8 + T-cells were then evaluated on day 8. To determine anti-tumor effects, a therapeutic tumor challenge with B16 OVA melanoma was performed., Results: The combination of s.c. anti-CTLA-4 antibody and TCI leads to an enhanced systemic cytotoxic response, to memory formation and allows significantly improved survival in a tumor setting with B16 OVA melanoma. Towards the mechanism, we show that in this vaccination protocol the CTLA-4 antibody acts mainly Treg-independent., Conclusion: We demonstrate that the combination of TCI with IMI-Sol and anti-CTLA-4 can confer potent immune responses and tumor-protection. These results might contribute to the development of advanced vaccination approaches targeting tumors or persistent infectious diseases., (Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Background: Transcutaneous immunization (TCI) is a novel vaccination strategy utilizing the skin associated lymphatic tissue to induce immune responses. TCI using a cytotoxic T lymphocyte (CTL) epitope and the Toll-like receptor 7 (TLR7) agonist imiquimod mounts strong CTL responses by activation and maturation of skin-derived dendritic cells (DCs) and their migration to lymph nodes. However, TCI based on the commercial formulation Aldara only induces transient CTL responses that needs further improvement for the induction of durable therapeutic immune responses., Objective: Therefore we aimed to develop a novel imiquimod solid nanoemulsion (IMI-Sol) for TCI with superior vaccination properties suited to induce high quality T cell responses for enhanced protection against infections., Methods: TCI was performed by applying a MHC class I or II restricted epitope along with IMI-Sol or Aldara (each containing 5% Imiquimod) on the shaved dorsum of C57BL/6, IL-1R, Myd88, Tlr7 or Ccr7 deficient mice. T cell responses as well as DC migration upon TCI were subsequently analyzed by flow cytometry. To determine in vivo efficacy of TCI induced immune responses, CTL responses and frequency of peptide specific T cells were evaluated on day 8 or 35 post vaccination and protection in a lymphocytic choriomeningitis virus (LCMV) infection model was assessed., Results: TCI with the imiquimod formulation IMI-Sol displayed equal skin penetration of imiquimod compared to Aldara, but elicited superior CD8 + as well as CD4 + T cell responses. The induction of T-cell responses induced by IMI-Sol TCI was dependent on the TLR7/MyD88 pathway and independent of IL-1R. IMI-Sol TCI activated skin-derived DCs in skin-draining lymph nodes more efficiently compared to Aldara leading to enhanced protection in a LCMV infection model., Conclusion: Our data demonstrate that IMI-Sol TCI can overcome current limitations of previous imiquimod based TCI approaches opening new perspectives for transcutaneous vaccination strategies and allowing the use of this enhanced cutaneous drug-delivery system to be tailored for the improved prevention and treatment of infectious diseases and cancers., (Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Background: Transcutaneous immunization (TCI) is a non-invasive vaccination strategy targeting the skin-associated lymphoid tissue. Topical application of the TLR7 agonist imiquimod as adjuvant in combination with peptide antigens activates the innate immune system and mounts cytotoxic T lymphocyte (CTL) responses., Objective: Based on the commercial 5% imiquimod-containing drug Aldara we aimed to develop an improved formulation with superior vaccination efficiencies. The primary target was the enhancement of mast cell activation as important key for the function of the innate immune system., Methods: We investigated the effects of 9-phenanthrol (9-phe) on the activation of mast cells in vitro and in vivo. For TCI, we applied 0.2% 9-phe in Aldara or Aldara alone as adjuvants in combination with the MHC class I - restricted peptide SIINFEKL. To monitor vaccination, mast cell degranulation, migration of DC and frequencies of epitope-specific CTL was assessed. In a transgenic tumor model, the efficiencies of prophylactic immunization against a tumor antigen were also monitored., Results: 9-phe induced degranulation of mast cells in vitro and upon topical application in vivo. A mixture of 0.2% 9-phe in Aldara showed superior results regarding the migration of DC and the expansion of antigen-specific CTL. Consequently, prophylactic immunization with 0.2% 9-phe in Aldara caused enhanced protection against tumor inoculation., Conclusion: Our data demonstrate that a simple modification of an adjuvant formulation can yield superior results in experimental vaccination protocols by boosting critical steps leading to the generation of an efficient CTL response., (Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Immunologic approaches to combat cancer aim at the induction of tumor-reactive immune responses to achieve long-term protection. In this context, we recently developed a transcutaneous immunization (TCI) method using the Toll-like receptor (TLR) 7 agonist imiquimod and a peptide epitope. Application onto intact skin induces potent cytotoxic T lymphocyte (CTL) responses and protection against transplanted tumors. The purpose of this study was to explore the effects of UV irradiation on imiquimod-based TCI. Here we show that skin exposure to low-dose UV light before TCI with imiquimod strongly boosts specific CTL responses leading to memory formation and enhanced tumor protection. Toward the mechanisms, we show that the activation of bone-marrow-derived dermal dendritic cells (DCs), but not Langerin-expressing DCs, is responsible for enhanced CTL activation. We describe an optimized TCI method that mediates enhanced CTL and antitumor responses by a DC- and TLR-dependent mechanism. These data may provide the basis for the future development of advanced vaccination protocols against tumors and persistent virus infections.