Tobias Warger

Cutting Edge: Priming of CTL by Transcutaneous Peptide Immunization with Imiquimod

CTL are important in combating cancer and viruses. Therefore, triggering the complete potential of CTL effector functions by new vaccination strategies will not only improve prophylaxis of tumor or virus-related diseases, but also open opportunities for effective therapeutic immunizations. Using transcutaneous immunization, we show that epicutaneous (e.c.)4 application of an ointment containing a CTL epitope and the TLR7 ligand imiquimod is highly effective in activating T cells in mice using TCR-transgenic CTL or in wild-type mice. Transcutaneous immunization-activated CTL mount a full-blown immune response against the target epitope characterized by proliferation, cytolytic activity, and the production of IFN-γ that is completely restricted to the epitope used for vaccination. Our results obtained by simple e.c. application of an ointment, without further skin irritating procedures, provide the basis for the development of new, easy to use vaccines against cancer or virus-associated diseases.

Interaction of TLR2 and TLR4 Ligands with the N-terminal Domain of Gp96 Amplifies Innate and Adaptive Immune Responses

Activation of dendritic cells by ligands for Toll-like receptors (TLR) is a crucial event in the initiation of innate and adaptive immune responses. Several classes of TLR ligands have been identified that interact with distinct members of the TLR-family. TLR4 ligands include lipopolysaccharide derived from different Gram-negative bacteria and viral proteins. Recent reports have demonstrated the TLR-mediated activation of dendritic cells by heat shock proteins (HSPs). However, doubts were raised as to what extent this effect was due to lipopolysaccharide contaminations of the HSP preparations. We re-examined this phenomenon using Gp96 or its N-terminal domain, nominally endotoxin-free (<0.5 enzyme units/mg). As described previously, innate immune cells are activated by Gp96 at high concentrations (≥50 μg/ml) but not at lower concentrations. However, preincubation of low amounts of Gp96 with TLR2 and TLR4 ligands at concentrations unable to activate dendritic cells by themselves results in the production of high levels of proinflammatory cytokines, up-regulation of activation markers, and amplification of T cell activation. Our results provide significant new insights into the mechanism of HSP-mediated dendritic cell activation and present a new function of HSPs in the amplification of dendritic cell activation by bacterial products and induction of adaptive immune responses.

Glycoprotein 96-activated dendritic cells induce a CD8-biased T cell response.

Abstract Heat shock proteins (Hsps) are able to induce protective immune responses against pathogens and tumors after injection into immunocompetent hosts. The activation of components of the adaptive immune system, including cytotoxic T lymphocytes specific for pathogen- or tumor-derived peptides, is crucial for the establishment of immunoprotection. Hsps acquire these peptides during intracellular protein degradation and when released during necrotic cell death, facilitate their uptake and Minor Histocompatibility Complex (MHC)-restricted representation by professional antigen-presenting cells (APCs). In addition, the interaction of Hsps with APCs, including the Endoplasmatic Reticulum (ER)-resident chaperone glycoprotein 96 (Gp96), induces the maturation of these cells by Toll-like receptor (TLR)– mediated signaling events. We now provide evidence that in contrast to lipopolysaccharides (LPS)-mediated dendritic cell (DC) maturation, the interaction of Gp96 with DCs leads to the preferential expansion of antigen-specific CD8-positive T cells in vitro and in vivo. This CD8 preference induced by mouse and human DCs did not correlate with enhanced levels of interleukin-12 secretion. Thus, despite the fact that both LPS and Gp96 activate DCs in a TLR4-dependent manner, the experiments of this study clearly demonstrate qualitative differences in the outcome of this maturation process, which preferentially favors the expansion of CD8-positive T cells.

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.

UV Exposure Boosts Transcutaneous Immunization and Improves Tumor Immunity: Cytotoxic T-Cell Priming through the Skin

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.

Precursor frequency can compensate for lower TCR expression in T cell competition during priming in vivo

The factors controlling clonal dominance of cytotoxic T lymphocyte (CTL) responses are currently not well understood. To study the functional impact of the strength of the interaction of a T cell with an antigen‐presenting cell in this context, we established a new mouse model comprised of two T cell receptor (TCR)‐transgenic strains expressing the identical TCR in differing amounts, hence providing two CTL clones with different avidities but identical specificity and affinity. Utilizing this new model, we show that upon antigen challenge higher‐avidity CTL expand at the expense of moderate‐avidity CTL in vivo if present in equal numbers. Beyond this, moderate‐avidity T cells can also contribute to a CTL response when present in excess. These results suggest that in addition to a proposed affinity/avidity threshold, the precursor frequency is important in defining clonal dominance. A new model in which TCR density and precursor frequency define the outcome of a CTL response is discussed.