Tanja Breinig

Differential cell reaction upon Toll-like receptor 4 and 9 activation in human alveolar and lung interstitial macrophages

BackgroundInvestigations on pulmonary macrophages (MΦ) mostly focus on alveolar MΦ (AM) as a well-defined cell population. Characteristics of MΦ in the interstitium, referred to as lung interstitial MΦ (IM), are rather ill-defined. In this study we therefore aimed to elucidate differences between AM and IM obtained from human lung tissue.MethodsHuman AM and IM were isolated from human non-tumor lung tissue from patients undergoing lung resection. Cell morphology was visualized using either light, electron or confocal microscopy. Phagocytic activity was analyzed by flow cytometry as well as confocal microscopy. Surface marker expression was measured by flow cytometry. Toll-like receptor (TLR) expression patterns as well as cytokine expression upon TLR4 or TLR9 stimulation were assessed by real time RT-PCR and cytokine protein production was measured using a fluorescent bead-based immunoassay.ResultsIM were found to be smaller and morphologically more heterogeneous than AM, whereas phagocytic activity was similar in both cell types. HLA-DR expression was markedly higher in IM compared to AM. Although analysis of TLR expression profiles revealed no differences between the two cell populations, AM and IM clearly varied in cell reaction upon activation. Both MΦ populations were markedly activated by LPS as well as DNA isolated from attenuated mycobacterial strains (M. bovis H37Ra and BCG). Whereas AM expressed higher amounts of inflammatory cytokines upon activation, IM were more efficient in producing immunoregulatory cytokines, such as IL10, IL1ra, and IL6.ConclusionAM appear to be more effective as a non-specific first line of defence against inhaled pathogens, whereas IM show a more pronounced regulatory function. These dissimilarities should be taken into consideration in future studies on the role of human lung MΦ in the inflammatory response.

Saccharomyces cerevisiae: a versatile eukaryotic system in virology

The yeast Saccharomyces cerevisiae is a well-established model system for understanding fundamental cellular processes relevant to higher eukaryotic organisms. Less known is its value for virus research, an area in which Saccharomyces cerevisiae has proven to be very fruitful as well. The present review will discuss the main achievements of yeast-based studies in basic and applied virus research. These include the analysis of the function of individual proteins from important pathogenic viruses, the elucidation of key processes in viral replication through the development of systems that allow the replication of higher eukayotic viruses in yeast, and the use of yeast in antiviral drug development and vaccine production.

Uptake of various yeast genera by antigen-presenting cells and influence of subcellular antigen localization on the activation of ovalbumin-specific CD8 T lymphocytes

Yeasts of the genus Saccharomyces expressing recombinant antigens are currently evaluated as candidate T cell vaccines. Here, we compared the interaction kinetics between four biotechnologically relevant yeast genera (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis and Pichia pastoris) and human dendritic cells as well as the involvement of Dectin-1 and mannose receptor in phagocytosis. Further, we analyzed the activation capacity of recombinant yeasts expressing ovalbumin (OVA) either intracellular, extracellular or surface-displayed by OVA-specific CD8 T lymphocytes. We found that the kinetic patterns of yeast uptake by phagocytic cells varied between the tested yeast genera and that both genus and subcellular OVA antigen localization influenced the strength of T cell activation. In particular, in S. cerevisiae, a secreted antigen was less effectively delivered than its cytosolic variant, whereas most efficient antigen delivery with P. pastoris was obtained by cell surface bound antigen. Our data indicate that protein secretion might not be an effective delivery pathway in yeast.

T cell activation on a single-cell level in dielectrophoresis-based microfluidic devices.

The gentle and careful in vitro processing of live cells is essential in order to make them available to future therapeutic applications. We present a protocol for the activation of single-T cells based on the contact formation with individual anti-CD3/anti-CD28 presenting microbeads in a lab-on-chip environment. The chips consist of microfluidic channels and microelectrodes for performing dielectrophoretic manipulation employing a.c. electric fields. The dielectrophoretic guiding elements allow the assembly of cell-bead pairs while avoiding ill-defined physical contacts with their environment. After overnight cultivation of the manipulated cells, 77% of the bead-associated T cells expressed the activation marker molecule CD69. Physiological stress on the cells was shown to be mainly due to the single-cell cultivation and not to the manipulation in the chips. The same approach could be useful for the in vitro regulation of stem cell differentiation.

Exploiting the Yeast L-A Viral Capsid for the In Vivo Assembly of Chimeric VLPs as Platform in Vaccine Development and Foreign Protein Expression

A novel expression system based on engineered variants of the yeast (Saccharomyces cerevisiae) dsRNA virus L-A was developed allowing the in vivo assembly of chimeric virus-like particles (VLPs) as a unique platform for a wide range of applications. We show that polypeptides fused to the viral capsid protein Gag self-assemble into isometric VLP chimeras carrying their cargo inside the capsid, thereby not only effectively preventing proteolytic degradation in the host cell cytosol, but also allowing the expression of a per se cytotoxic protein. Carboxyterminal extension of Gag by T cell epitopes from human cytomegalovirus pp65 resulted in the formation of hybrid VLPs that strongly activated antigen-specific CD8+ memory T cells ex vivo. Besides being a carrier for polypeptides inducing antigen-specific immune responses in vivo, VLP chimeras were also shown to be effective in the expression and purification of (i) a heterologous model protein (GFP), (ii) a per se toxic protein (K28 α-subunit), and (iii) a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A). Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production.

Yeast-based protein delivery to mammalian phagocytic cells is increased by coexpression of bacterial listeriolysin

Yeast-mediated protein delivery to mammalian antigen-presenting cells is a powerful approach for inducing cell-mediated immune responses. We show that coexpression of the pore-forming protein listeriolysin O from Listeria monocytogenes leads to improved translocation of a proteinaceous antigen and subsequent activation of specific T lymphocytes. As the resulting yeast carrier is self-attenuated and killed after antigen delivery without exhibiting any toxic effect on antigen-presenting cells, this novel carrier system suggests itself as promising approach for the development of yeast-based live vaccines.

Heat treatment improves antigen-specific T cell activation after protein delivery by several but not all yeast genera.

A central prerequisite in using yeast as antigen carrier in vaccination is its efficient interaction with cellular components of the innate immune system, mainly mediated by cell surface structures. Here, we investigated the distribution of major yeast cell wall components such as mannan, β-glucan and chitin of four different and likewise biotechnologically relevant yeasts (Saccharomyces, Pichia, Kluyveromyces and Schizosaccharomyces) and analyzed the influence of heat-treatment on β-1,3-glucan exposure at the outer yeast cell surface as well as the amount of yeast induced reactive oxygen species (ROS) production by antigen presenting cells (APC) in human blood. We found that yeasts significantly differ in the distribution of their cell wall components and that heat-treatment affected both, cell wall composition and yeast-induced ROS production by human APCs. We further show that heat-treatment modulates the activation of antigen specific memory T cells after yeast-mediated protein delivery in different ways and thus provide additional support of using yeast as vehicle for the development of novel T cell vaccines.

mRNA Delivery to Human Dendritic Cells by Recombinant Yeast and Activation of Antigen-Specific Memory T Cells

The import of functional nucleic acids like messenger RNA into mammalian cells has proven to be a powerful tool in cell biology and several delivery systems have been described. However, as targeting of particular cell types is a major challenge and RNA vaccination represents a promising means for the induction of cellular immune responses, there is a need for novel delivery systems that permit the introduction of functional messenger RNA to the cytosol of immune cells. Here, we describe a delivery system based on the yeast Saccharomyces cerevisiae that allows the delivery of functional messenger RNA to mammalian antigen-presenting cells such as human dendritic cells. Further, we present a method to prove antigen processing and presentation by stimulation of human autologous T lymphocytes.

Human yeast-specific CD8 T lymphocytes show a nonclassical effector molecule profile.

Pathogenic yeast and fungi represent a major group of human pathogens. The consequences of infections are diverse and range from local, clinically uncomplicated mycosis of the skin to systemic, life-threatening sepsis. Despite extensive MHC class I-restricted frequencies of yeast-specific CD8 T lymphocytes in healthy individuals and the essential role of the cell-mediated immunity in controlling infections, the characteristics and defense mechanisms of antifungal effector cells are still unclear. Here, we describe the direct analysis of yeast-specific CD8 T lymphocytes in whole blood from healthy individuals. They show a unique, nonclassical phenotype expressing granulysin and granzyme K in lytic granules instead of the major effector molecules perforin and granzyme B. After stimulation in whole blood, yeast-specific CD8 T cells degranulated and, upon cultivation in the presence of IL-2, their granula were refilled with granulysin rather than with perforin and granzyme B. Moreover, yeast-specific stimulation through dendritic cells but not by yeast cells alone led to degranulation of the effector cells. As granulysin is the only effector molecule in lytic granules known to have antifungal properties, our data suggest yeast-specific CD8 T cells to be a nonclassical effector population whose antimicrobial effector machinery seems to be tailor-made for the efficient elimination of fungi as pathogens.