Eine neue Helfer-Funktion von T Helfer 1 Zellen bei der Induktion von CD8⁺ T Zellen durch sinusoidale Endothelzellen der Leber

Die sinusoidalen Endothelzellen der Leber (LSEC) nehmen kontinuierlich zirkulierende Antigene auf und kreuzpräsentieren diese CD8+ und CD4+ T Zellen. Unter homöostatischen Bedingungen leiten LSEC die Differenzierung von naiven CD8+ T Zellen zu Leber-induzierten Gedächtnis T Zellen ein. Dem geht ein sehr früher, jedoch vorübergehender Gewinn zytotoxischer Effektorfunktionen voraus, was durch IL 6 Trans-Signalübertragung induziert wird. In der vorliegenden Arbeit wurde untersucht, ob Th1 Zellen die Induktion von CD8+ T Zellen durch LSEC in Abwesenheit von Inflammation verstärken können. Es zeigte sich, dass Th1 Zellen LSEC-spezifisch die Expression von Granzym B und CD25 der CD8+ T Zellen steigern konnten. Voraussetzung dafür war die Antigen-spezifische Interaktion der Endothelzellen mit den Th1 Zellen, was zu der Aktivierung beider Zellpopulationen führte. Die beobachtete Helfer-Funktion war Zytokin-vermittelt und bedurfte keiner konventionellen kostimulatorischen Signale. Th1 Zellen steigerten die Granzym B- und CD25-Expression der CD8+ T Zellen vermutlich durch Sekretion von IL-2. Die Induktion der spezifischen Granzym-B-Hochregulation war jedoch abhängig von LSEC-vermittelten Stimuli und konnte nicht durch Signale von Th1 Zellen eingeleitet werden. LSEC erlangten nach Aktivierung die Fähigkeit, die Expression von Granzym B Zytokin-vermittelt zu induzieren. Dies ergänzt den bereits beschriebenen Mechanismus der IL 6 Trans-Signalübertragung mit Hilfe des membranständigen IL-6/IL-6R-Komplexes auf LSEC. Die Analyse der beobachteten Helfer-Funktion lieferte weiterhin Belege für die essenzielle Bedeutung von IL-2 für die durch IL-6 Trans-Signalübertragung induzierte Expression von Granzym B. Im Rahmen der vorliegenden Arbeit konnte somit eine neue Helfer-Funktion von Th1 Zellen bei der Induktion von CD8+ T Zellen durch LSEC beschrieben werden. Es ist durchaus denkbar, dass Th1 Zellen auch in vivo unter homöostatischen Bedingungen die Differenzierung von Pathogen-spezifischen CD8+ T Zellen in der Leber unterstützen können. Dies kann möglicherweise die Einleitung von Immunantworten durch CD8+ T Zellen gegen zirkulierende Antigene verbessern und so die effiziente Eliminierung von viralen Infektionen und Tumoren ermöglichen.Liver sinusoidal endothelial cells (LSEC) constantly take up circulating antigens and cross-present these to CD8+ and CD4+ T cells. Under homeostatic conditions this cross-presentation process induces differentiation of naive CD8+ T cells to liver-primed memory T cells. This is preceded by an early, but temporary acquisition of cytotoxic effector function induced by IL-6 trans-Signaling. The aim of this study was to analyze whether Th1 cells are able to enhance the induction of granzyme B and surface marker expression of CD8+ T cells by LSEC in the absence of inflammation. We demonstrate that Th1 cells help increase the expression of granzyme B and CD25 specifically in LSEC-primed CD8+ T cells. The antigen-specific interaction of LSEC and Th1 cells was a prerequisite for this effect and led to the mutual activation of both cell types. The observed helper function was cytokine mediated and independent of conventional costimulatory signals. Th1 cells presumably increased the granzyme B and CD25 expression of CD8+ T cells by secretion of IL 2. However, the specific induction of granzyme B depended on LSEC-delivered stimuli and could not be induced by Th1 signals alone. Upon activation, LSEC gained the ability to induce the expression of granzyme B in a cytokine-mediated manner. This finding complements the already described mechanism of IL 6 trans-Signaling via the membrane-bound IL 6/IL 6R-complex on LSEC. Furthermore, analysis of the observed helper function provided evidence for an essential role for IL-2 in the IL-6 trans-Signaling-mediated expression of granzyme B. Therefore, a new helper function of Th1 cells during priming of CD8+ T cells by LSEC is described in this study. It is conceivable that Th1 cells can support the differentiation of pathogen-specific CD8+ T cells in the liver also in vivo under homeostatic conditions. This could possibly improve the induction of CD8+ T cell-mediated immune responses towards circulating antigens, allowing for efficient elimination of viral infections and tumors

Virulence-associated protein A from Rhodococcus equi is an intercompartmental pH-neutralising virulence factor

Professional phagocytic cells such as macrophages are a central part of innate immune defence. They ingest microorganisms into membrane-bound compartments (phagosomes), which acidify and eventually fuse with lysosomes, exposing their contents to a microbicidal environment. Gram-positive Rhodococcus equi can cause pneumonia in young foals and in immunocompromised humans. The possession of a virulence plasmid allows them to subvert host defence mechanisms and to multiply in macrophages. Here, we show that the plasmid-encoded and secreted virulence-associated protein A (VapA) participates in exclusion of the proton-pumping vacuolar-ATPase complex from phagosomes and causes membrane permeabilisation, thus contributing to a pH-neutral phagosome lumen. Using fluorescence and electron microscopy, we show that VapA is also transferred from phagosomes to lysosomes where it permeabilises the limiting membranes for small ions such as protons. This permeabilisation process is different from that of known membrane pore formers as revealed by experiments with artificial lipid bilayers. We demonstrate that, at 24 hr of infection, virulent R. equi is contained in a vacuole, which is enriched in lysosome material, yet possesses a pH of 7.2 whereas phagosomes containing a vapA deletion mutant have a pH of 5.8 and those with virulence plasmid-less sister strains have a pH of 5.2. Experimentally neutralising the macrophage endocytic system allows avirulent R. equi to multiply. This observation is mirrored in the fact that virulent and avirulent R. equi multiply well in extracts of purified lysosomes at pH 7.2 but not at pH 5.1. Together these data indicate that the major function of VapA is to generate a pH-neutral and hence growth-promoting intracellular niche. VapA represents a new type of Gram-positive virulence factor by trafficking from one subcellular compartment to another, affecting membrane permeability, excluding proton-pumping ATPase, and consequently disarming host defences