Biology subjects

Plets, E.

Publications and source records attributed to Plets, E..

6 recordsLinked to original sources

bioRxiv2026

Addition of DMSO to T cell cultures skews differentiation towards a memory phenotype.

Dimethyl sulfoxide (DMSO) is a polar aprotic organic solvent that is widely used in biological applications. It is routinely applied as a cryoprotectant for long-term cell freezing as well as to dissolve peptides or drugs for immune cell functional assays. We report on a remarkable impact of low concentrations of DMSO on in vitro expansion of the total CD3+ pool isolated from human PBMCs as well as on the purified CD8+ T cell fraction thereof in the presence of different cytokine combinations typically used for therapeutic T cell expansion. Characterizing survival, proliferation, activation, exhaustion and differentiation, we demonstrate that DMSO at low concentrations substantially skews the differentiation of T cells towards a memory phenotype in a dose-dependent way. This is a desirable outcome for the field of adoptive T cell therapies for cancer, where it has been established that T cells with a memory phenotype exert superior anti-cancer immune responses.

immunology↗

bioRxiv2023

N-glycosylation engineering in chimeric antigen receptor T cells enhances anti-tumor activity

Chimeric antigen receptor (CAR) T cell therapy has had limited success in solid tumors, requiring novel enhancement strategies. Modifying the glycocalyx of CAR T cells is unexplored; we report on genome-editing of the MGAT5 gene to abolish human CAR T N-glycan poly-LacNAc modifications. This boosted tumor control in carcinoma and lymphoma models, for donors of whom the non-engineered CAR T cells largely failed in tumor control. More blood-circulating MGAT5 KO CD70 nanoCAR T cells were found, exhibiting potent tumor cell-killing activity ex vivo, while non-glycoengineered CAR T cells faltered. MGAT5 KO CD70 nanoCAR T cells also mediated durable anti-tumor immunity, improving control of secondary carcinoma challenge months later. Single-cell transcriptomics revealed increased mitotic activity and type I interferon signaling, indicating sustained intratumoral activation. The glyco-engineered cells had unaltered antigen sensitivity and dependence on T cell growth factors, preserving key safety features. MGAT5 KO is readily compatible with clinical manufacturing, representing a promising approach to enhance CAR T cell therapy.

immunology↗

bioRxiv2021

Exploration of synergistic action of cell wall-degrading enzymes against Mycobacterium tuberculosis

SynopsisO_ST_ABSBackgroundC_ST_ABSThe major global health threat tuberculosis is caused by Mycobacterium tuberculosis (Mtb). Mtb has a complex cell envelope - a partially covalently linked composite of polysaccharides, peptidoglycan and lipids, including a mycolic acid layer - which conveys pathogenicity but also protects against antibiotics. Given previous successes in treating gram-positive and -negative infections with cell wall degrading enzymes, we investigated such approach for Mtb. Objectives(i) Development of an Mtb microtiter growth inhibition assay that allows undisturbed cell envelope formation, to overcome the invalidation of results by typical clumped Mtb-growth in surfactant-free assays. (ii) Exploring anti-Mtb potency of cell wall layer-degrading enzymes. (iii) Investigation of the concerted action of several such enzymes. MethodsWe inserted a bacterial luciferase-operon in an auxotrophic Mtb strain to develop a microtiter assay that allows proper evaluation of cell wall degrading anti-Mtb enzymes. We assessed growth-inhibition by enzymes (recombinant mycobacteriophage mycolic acid esterase (LysB), fungal -amylase and human and chicken egg white lysozymes) and combinations thereof, in presence or absence of biopharmaceutically acceptable surfactant. ResultsOur biosafety level-2 assay identified both LysB and lysozymes as potent Mtb-inhibitors, but only in presence of surfactant. Moreover, most potent disruption of the mycolic acid hydrophobic barrier was obtained by the highly synergistic combination of LysB, -amylase and polysorbate 80. ConclusionsSynergistically acting cell wall degrading enzymes are potently inhibiting Mtb - which sets the scene for the design of specifically tailored antimycobacterial (fusion) enzymes. Airway delivery of protein therapeutics has already been established and should be studied in animal models for active TB.

microbiology↗

bioRxiv2019

Use of a counterselectable transposon to create markerless knockouts from a 18,432-clone ordered M. bovis BCG 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 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 is 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 M. tb complex (18,432 clones, mutating 83% of the non-essential M. tb homologues), from which clean knockouts can be generated.\n\nImportanceWhile speeding up research for many fields of biology (e.g. yeast, plant, and C. elegans), genome-wide ordered mutant collections are still elusive in mycobacterial research. We developed methods to generate such resources in a time- and cost-effective manner, and developed a newly engineered transposon from which unmarked mutants can be efficiently generated. Our library in the WHO reference vaccine strain of M. bovis BCG Danish targets 83% of all non-essential genes and was made publicly available via the BCCM/ITM Mycobacteria Collection. This resource will speed up Mycobacterium research (e.g. drug resistance research, vaccine development) and paves the way to similar genome-wide mutant collections in other strains of the M. tb complex. The stretch to a full collection of mutants in all non-essential genes is now much shorter, with just 17% remaining genes to be targeted using gene-by-gene approaches, for which highly effective methods have recently also been described.

microbiology↗

bioRxiv2019

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

Mycobacterium bovis bacillus Calmette-Guerin (M. bovis BCG) is the only vaccine available against tuberculosis (TB). This study reports on an integrated genome analysis workflow for BCG, resulting in the completely assembled genome sequence of BCG Danish 1331 (07/270), one of the WHO reference strains for BCG vaccines. We demonstrate how this analysis workflow enables the resolution of genome duplications and of the genome of engineered derivatives of this vaccine strain.

microbiology↗

bioRxiv2018

SapM mutation to improve the BCG vaccine: genomic, transcriptomic and preclinical safety characterization

The Mycobacterium bovis Bacille Calmette Guerin (BCG) vaccine shows variable efficacy in protection against adult tuberculosis (TB). Earlier, we have described a BCG mutant vaccine with a transposon insertion in the gene coding for the secreted acid phosphatase SapM, which led to enhanced long-term survival of vaccinated mice challenged with TB infection. To facilitate development of this mutation as part of a future improved live attenuated TB vaccine, we have now characterized the genome and transcriptome of this sapM::Tn mutant versus parental BCG Pasteur. Furthermore, we show that the sapM::Tn mutant had an equal low pathogenicity as WT BCG upon intravenous administration to immunocompromised SCID mice, passing this important safety test. Subsequently, we investigated the clearance of this improved vaccine strain following vaccination and found a more effective innate immune control over the sapM::Tn vaccine bacteria as compared to WT BCG. This leads to a fast contraction of IFN{gamma} producing Th1 and Tc1 cells after sapM::Tn BCG vaccination. These findings corroborate that a live attenuated vaccine that affords improved long-term survival upon TB infection can be obtained by a mutation that further attenuates BCG. These findings suggest that an analysis of the effectiveness of innate immune control of the vaccine bacteria could be instructive also for other live attenuated TB vaccines that are currently under development, and encourage further studies of SapM mutation as a strategy in developing a more protective live attenuated TB vaccine.

microbiology↗