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Background: 5-10 % of all hospital admissions in Europe are caused by adverse drug reactions (ADR), many of them are considered to be preventable. Oral anticoagulants and antiplatelets range among the most frequently ADR-associated drugs and have a high potential for particularly serious ADR. Dosing[for full text, please go to the a.m. URL], 22. Jahrestagung der Gesellschaft für Arzneimittelanwendungsforschung und Arzneimittelepidemiologie

In times of globalization and the resulting contact to ever-new pathogens, it becomes even more important to understand how exactly protective immunity is induced to be able to generate more potent vaccinations. CD8+ T cells are crucial for protection against intracellular pathogens. Until today, vaccines capable to induce long lasting CD8+ T cell responses are usually based on living intracellular organisms. However, live vaccines can be potentially harmful, especially for immunocompromised individuals. In order to improve the safety of vaccines, the goal of many laboratories is to dissect the components derived from live vaccines, which are responsible to effectively induce long lasting protective immunity. Our aim of this study was to use purified Listeria monocytogenes (L.m.)-derived antigens and to modulate the immune system to promote the development of preferentially CD8+ effector memory T cells (TEM), which have recently shown to be the major population mediating protective immunity against L.m. We chose the L.m.-derived protein LLO and the LLO91-99 peptide as targets, because LLO-specific T cell responses have been demonstrated to confer efficient protection in this model. Furthermore, strong LLO91-99- specific immune responses are induced, increasing the chances to analyze also more subtle changes within the induced cell populations. While immunization with peptide alone or in combination with adjuvant did not lead to measurable CD8+ immune response, the full protein LLO induced readily detectable antigen specific CD8+ T cell numbers. Thereby, the route of application played a crucial role, since LLO91-99-specific immune responses were much higher after local application of LLO and almost absent after systemic administration. Priming of LLO91-99- specific CD8+ T-cell responses upon immunization with the purified protein requires access of antigen to the MHC class I cross-presentation pathway. As TLR9 ligands like bacterial CpG DNA are believed to be able to enhance cross-presentation by DCs, adjuvant effect of CpG DNA were analyzed. Indeed, CpG functioned as a strong stimulatory component, since mice treated with LLO and CpG as adjuvant showed almost equal levels of LLO91-99-specific T cell immune response as compared to L.m. infected animals. Immunization with LLO alone induced only weak immune responses. The superior CD8+ T cell immunogenicity of full protein versus peptide vaccination could be due to the induction of additional antigen specific CD4+ T cell responses, which provide enhancing factors by T cell help. To test the CD4+ T helper dependency, we performed antibody mediated CD4+ T cell depletion. To our surprise, LLO91-99-specific CD8+ T cell frequencies were even increased in CD4+ T cells depleted mice. Since CD4+ lymphocytes consist besides conventional CD4+ T cells also of a substantial fraction of regulatory T cells (Treg cells, CD4+CD25+), we analyzed which of these populations account for the unexpected increase of LLO91-99-specific CD8+ T cell numbers. Using a CD25+ depleting antibody, we could show that the absence of CD25+ T cells led to enhanced LLO91-99-specific CD8+ T cell responses in combination with CpG as adjuvant, as good as after CD4+ T cell depletion, identifying CD25+ Treg cells as inhibitory cell population during T cell priming. Challenge experiments with a high dose of L.m. indicated that CD25+ T cell depleted mice were long term protected against L.m. infection, even more effectively than CD4+ T cell depleted mice. This result correlates with the observation that CD25+ depleted animals showed increased numbers of TEM cells. Experiments using MHC class II-/- mice further confirmed these findings: CD8+ T-cell responses in MHC class II-/- mice were not effected by anti-CD25+ depletion; in contrast, treatment with a CD40 stimulating antibody to provide signals mimicking CD4+ T cell help substantially enlarged antigen-specific CD8+ T cell responses. Taken together, these data demonstrate that both CD4+ lymphocyte subsets affect memory T cell generation, with Treg cells being capable to reduce not only the population size, also the memory T cell differentiation pattern is largely affected. To our knowledge, this is the first report linking regulatory T cell activity to memory T cell differentiation. To address the question whether other TLR ligands are also able to support cross-priming and enhance CD8+ T cell immunogenicity, we generated purified LLO protein covalently linked to V7 peptide. V7 is a Yersinia-derived oligopeptide and has been shown to confer signaling via TLR2. Interestingly, V7 also induces IL-10 secretion from macrophages, already indicating that the modulatory capabilities might differ from CpG. Indeed, CD8+ immune responses were diminished when compared to LLO treated animals. This phenotype could be only partially reverted by additional application of CpG, indicating that the V7 effect might not only be mediated on the level of the APC. One candidate cell population were again the Treg cells. We found that depletion of CD25+ T cells upon vaccination with LLO-V7 plus CpG resulted in increased frequencies, but not to the same extent as observed for LLO plus CpG treated mice. This finding indicates that in this setting Treg cells partly seem to negatively regulate CD8+ T cell responses. In conclusion, we show for the first time that the activity of TLR-ligands as CD8+ T cell priming adjuvant can be strongly influenced by naturally occurring Treg cells. In addition, Treg downmodulation in combination with TLR stimulation might represent a novel approach to specifically support differentiation of CD8+ T cells to TEM cells. These results provide important implications for the design of more effective T cell vaccines when immunizing with purified proteins. Aufgereinigte Erregerproteine induzieren normalerweise schlechte T-Zell-Immunogenität. Mit dieser Arbeit sollte untersucht werden, ob durch definierte Immunmodulation die Immunogenität gesteigert werden kann. Es konnte im Infektionsmodell mit Listeria monocytogenes gezeigt werden, dass mit Hilfe des TLR9 Liganden CpG die CD8+ T-Zell Antwort deutlich gesteigert werden kann, wohingegen der TLR2 Ligand V7 einen gegenläufigen Effekt zeigt. Diese Daten weisen darauf hin, dass die Art des TLR Liganden entscheidenten Einfluss auf die Immunogenität haben kann. Die Abhängigkeit von konventioneller CD4+ T-Zell Hilfe war in Gegenwart steigernder TLR-Adjuvazien überraschend gering, hingegen wurde die CD8+-spezifische T-Zell Antwort und Gedächnisentwicklung deutlich durch die Präsenz von regulatorischen T-Zellen beeinflusst. Mit diesen Ergebnissen lassen sich neue Impfstrategien postulieren, duch welche T-Zell-abhängige Immunität wesentlich gesteigert werden kann.