Human T cell glycosylation and implications on immune therapy for cancer

Glycosylation is an important post-translational modification, giving rise to a diverse and abundant repertoire of glycans on the cell surface, collectively known as the glycome. When focusing on immunity, glycans are indispensable in virtually all signaling and cell-cell interactions. More specifically, glycans have been shown to regulate key pathophysiological steps within T cell biology such as T cell development, thymocyte selection, T cell activity and signaling as well as T cell differentiation and proliferation. They are of major importance in determining the interaction of human T cells with tumor cells. In this review, we will describe the role of glycosylation of human T cells in more depth, elaborate on the importance of glycosylation in the interaction of human T cells with tumor cells and discuss the potential of cancer immunotherapies that are based on manipulating the glycome functions at the tumor immune interface

A guide to Mycobacterium mutagenesis

The genus Mycobacterium includes several pathogens that cause severe disease in humans, like Mycobacterium tuberculosis (M. tb), the infectious agent causing tuberculosis. Genetic tools to engineer mycobacterial genomes, in a targeted or random fashion, have provided opportunities to investigate M. tb infection and pathogenesis. Furthermore, they have allowed the identification and validation of potential targets for the diagnosis, prevention, and treatment of tuberculosis. This review describes the various methods that are available for the generation of mutants in Mycobacterium species, focusing specifically on tools for altering slow-growing mycobacteria from the M. tb complex. Among others, it incorporates the recent new molecular biological technologies (e.g. ORBIT) to rapidly and/or genome-wide comprehensively obtain targeted mutants in mycobacteria. As such, this review can be used as a guide to select the appropriate genetic tools to generate mycobacterial mutants of interest, which can be used as tools to aid understanding of M. tb infection or to help developing TB intervention strategies

Characterization of genome-wide ordered sequence-tagged Mycobacterium mutant libraries by Cartesian Pooling-Coordinate Sequencing

Reverse genetics research approaches require the availability of methods to rapidly generate specific mutants. Alternatively, where these methods are lacking, the construction of pre-characterized libraries of mutants can be extremely valuable. However, this can be complex, expensive and time consuming. Here, we describe a robust, easy to implement parallel sequencing-based method (Cartesian Pooling-Coordinate Sequencing or CP-CSeq) that reports both on the identity as well as on the location of sequence-tagged biological entities in well-plate archived clone collections. We demonstrate this approach using a transposon insertion mutant library of the Mycobacterium bovis BCG vaccine strain, providing the largest resource of mutants in any strain of the M. tuberculosis complex. The method is applicable to any entity for which sequence-tagged identification is possible

Protein synthesis persists during necrotic cell death

Cell death is an intrinsic part of metazoan development and mammalian immune regulation. Whereas the molecular events orchestrating apoptosis have been characterized extensively, little is known about the biochemistry of necrotic cell death. Here, we show that, in contrast to apoptosis, the induction of necrosis does not lead to the shut down of protein synthesis. The rapid drop in protein synthesis observed in apoptosis correlates with caspase-dependent breakdown of eukaryotic translation initiation factor (eIF) 4G, activation of the double-stranded RNA-activated protein kinase PKR, and phosphorylation of its substrate eIF2-α. In necrosis induced by tumor necrosis factor, double-stranded RNA, or viral infection, de novo protein synthesis persists and 28S ribosomal RNA fragmentation, eIF2-α phosphorylation, and proteolytic activation of PKR are absent. Collectively, these results show that, in contrast to apoptotic cells, necrotic dying cells retain the opportunity to synthesize proteins

Reference genome and comparative genome analysis for the WHO reference strain for Mycobacterium bovis BCG Danish, the present tuberculosis vaccine

Background: Mycobacterium bovis bacillus Calmette-Guerin (M. bovis BCG) is the only vaccine available against tuberculosis (TB). In an effort to standardize the vaccine production, three substrains, i.e. BCG Danish 1331, Tokyo 172-1 and Russia BCG-1 were established as the WHO reference strains. Both for BCG Tokyo 172-1 as Russia BCG-1, reference genomes exist, not for BCG Danish. In this study, we set out to determine the completely assembled genome sequence for BCG Danish and to establish a workflow for genome characterization of engineering-derived vaccine candidate strains.ResultsBy combining second (Illumina) and third (PacBio) generation sequencing in an integrated genome analysis workflow for BCG, we could construct the completely assembled genome sequence of BCG Danish 1331 (07/270) (and an engineered derivative that is studied as an improved vaccine candidate, a SapM KO), including the resolution of the analytically challenging long duplication regions. We report the presence of a DU1-like duplication in BCG Danish 1331, while this tandem duplication was previously thought to be exclusively restricted to BCG Pasteur. Furthermore, comparative genome analyses of publicly available data for BCG substrains showed the absence of a DU1 in certain BCG Pasteur substrains and the presence of a DU1-like duplication in some BCG China substrains. By integrating publicly available data, we provide an update to the genome features of the commonly used BCG strains. Conclusions: We demonstrate how this analysis workflow enables the resolution of genome duplications and of the genome of engineered derivatives of the BCG Danish vaccine strain. The BCG Danish WHO reference genome will serve as a reference for future engineered strains and the established workflow can be used to enhance BCG vaccine standardization

Fed-batch fermentation of GM-CSF-producing glycoengineered Pichia pastoris under controlled specific growth rate

<p>Abstract</p> <p>Background</p> <p>Yeast expression systems with altered N-glycosylation are now available to produce glycoproteins with homogenous, defined N-glycans. However, data on the behaviour of these strains in high cell density cultivation are scarce.</p> <p>Results</p> <p>Here, we report on cultivations under controlled specific growth rate of a GlycoSwitch-Man5 <it>Pichia pastoris </it>strain producing Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) at high levels (hundreds of milligrams per liter). We demonstrate that homogenous Man₅GlcNAc₂N-glycosylation of the secreted proteins is achieved at all specific growth rates tested.</p> <p>Conclusions</p> <p>Together, these data illustrate that the GlycoSwitch-Man5 <it>P. pastoris </it>is a robust production strain for homogenously N-glycosylated proteins.</p

Development of a counterselectable transposon to create markerless knockouts from an 18,432-clone ordered Mycobacterium bovis bacillus Calmette-Guérin mutant resource

Mutant resources are essential to improve our understanding of the biology of slow-growing mycobacteria, which include the causative agents of tuberculosis in various species, including humans. The generation of deletion mutants in slow-growing mycobacteria in a gene-by-gene approach in order to make genome-wide ordered mutant resources is still a laborious and costly approach, despite the recent development of improved methods. On the other hand, transposon mutagenesis in combination with Cartesian pooling-coordinate sequencing (CP-CSeq) allows the creation of large archived Mycobacterium transposon insertion libraries. However, such mutants contain selection marker genes with a risk of polar gene effects, which are undesired both for research and for use of these mutants as live attenuated vaccines. In this paper, a derivative of the Himar1 transposon is described which allows the generation of clean, markerless knockouts from archived transposon libraries. By incorporating FRT sites for FlpE/FRT-mediated recombination and I-SceI sites for ISceIM-based transposon removal, we enable two thoroughly experimentally validated possibilities to create unmarked mutants from such marked transposon mutants. The FRT approach is highly efficient but leaves an FRT scar in the genome, whereas the I-SceI-mediated approach can create mutants without any heterologous DNA in the genome. The combined use of CP-CSeq and this optimized transposon was applied in the BCG Danish 1331 vaccine strain (WHO reference 07/270), creating the largest ordered, characterized resource of mutants in a member of the Mycobacterium tuberculosis complex (18,432 clones, mutating 83% of the nonessential M. tuberculosis homologues), from which markerless knockouts can be easily generated

Necrosis, a well-orchestrated form of cell demise: Signalling cascades, important mediators and concomitant immune response

AbstractNecrosis has long been described as a consequence of physico-chemical stress and thus accidental and uncontrolled. Recently, it is becoming clear that necrotic cell death is as well controlled and programmed as caspase-dependent apoptosis, and that it may be an important cell death mode that is both pathologically and physiologically relevant. Necrotic cell death is not the result of one well-described signalling cascade but is the consequence of extensive crosstalk between several biochemical and molecular events at different cellular levels. Recent data indicate that serine/threonine kinase RIP1, which contains a death domain, may act as a central initiator. Calcium and reactive oxygen species (ROS) are main players during the propagation and execution phases of necrotic cell death, directly or indirectly provoking damage to proteins, lipids and DNA, which culminates in disruption of organelle and cell integrity. Necrotically dying cells initiate pro-inflammatory signalling cascades by actively releasing inflammatory cytokines and by spilling their contents when they lyse. Unravelling the signalling cascades contributing to necrotic cell death will permit us to develop tools to specifically interfere with necrosis at certain levels of signalling. Necrosis occurs in both physiological and pathophysiological processes, and is capable of killing tumour cells that have developed strategies to evade apoptosis. Thus detailed knowledge of necrosis may be exploited in therapeutic strategies

Caspase inhibitors promote alternative cell death pathways

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