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Toussaint, W.

Publications and source records attributed to Toussaint, W..

3 recordsLinked to original sources

Modeling epithelial and endothelial cell tropism for SARS-CoV-2 through restricted transgenic hACE2 expression in mice

Severe COVID-19 is frequently associated with vascular complications, raising ongoing debate about whether SARS-CoV-2 can directly infect endothelial cells and thereby contribute to disease pathogenesis. Although endothelial cells express angiotensin-converting enzyme 2 (ACE2), the in vivo relevance of endothelial-restricted viral tropism remains unclear. To directly assess the consequences of endothelial-restricted SARS-CoV-2 tropism in vivo, we generated a transgenic mouse model expressing human ACE2 under control of the endothelial-specific Cdh5 promoter (Cdh5-hACE2). Despite confirmed pulmonary endothelial expression and protein presence of hACE2, SARS-CoV-2 infection of Cdh5-hACE2 mice did not induce clinical illness, detectable viral replication, immune cell influx in the lung, or histopathological abnormalities in the lung or brain. These findings indicate that endothelial-restricted SARS-CoV-2 tropism alone is insufficient to drive productive infection and clinical disease in vivo, suggesting that endothelial involvement in COVID-19 likely arises in the context of broader cellular infection or systemic host responses rather than from primary endothelial infection.

immunology↗

TCR transgenic clone selection guided by immune receptor analysis and single cell RNA expression of polyclonal responders

Since the precursor frequency of naive T cells is extremely low, investigating the early steps of antigen-specific T cell activation is challenging. To overcome this detection problem, adoptive transfer of a cohort of T cells purified from T cell receptor (TCR) transgenic donors has been extensively used but is not readily available for emerging pathogens. Constructing TCR transgenic mice from T cell hybridomas is a labor-intensive and sometimes erratic process, since the best clones are selected based on antigen-induced CD69 upregulation or IL-2 production in vitro, and TCR chains are PCR-cloned into expression vectors. Here, we exploited the rapid advances in single cell sequencing and TCR repertoire analysis to select the best clones without hybridoma selection, and generated CORSET8 mice (CORona Spike Epitope specific CD8 T cell), carrying a TCR specific for the Spike protein of SARS-CoV-2. Implementing newly created DALI software for TCR repertoire analysis in single cell analysis enabled the rapid selection of the ideal responder CD8 T cell clone, based on antigen reactivity, proliferation and immunophenotype in vivo. Identified TCR sequences were inserted as synthetic DNA into an expression vector and transgenic CORSET8 donor mice were created. After immunization with Spike/CpG-motifs, mRNA vaccination or SARS-CoV2 infection, CORSET8 T cells strongly proliferated and showed signs of T cell activation. Thus, a combination of TCR repertoire analysis and scRNA immunophenotyping allowed rapid selection of antigen-specific TCR sequences that can be used to generate TCR transgenic mice.

immunology↗

Engraftment of allotransplantated tumour cells in adult rag2 mutant Xenopus tropicalis

Modelling human genetic diseases and cancer in lab animals has been greatly aided by the emergence of genetic engineering tools such as TALENs and CRISPR/Cas9. We have previously demonstrated the ease with which genetically engineered Xenopus models (GEXM) can be generated. This included the induction of autochthonous tumour formation by injection of early embryos with Cas9 recombinant protein loaded with sgRNAs targeting multiple tumour suppressor genes. What has been lacking so far is the possibility to propagate the induced cancers via transplantation. In this paper we describe the generation of a rag2-/- knock-out line in Xenopus tropicalis that is deficient in functional T- and B-cells. This line was validated by means of an allografting experiment with a primary tp53-/- donor tumour. In addition, we optimized available protocols for sub-lethal gamma irradiation of X. tropicalis froglets. Irradiated animals also allowed stable, albeit transient, engraftment of transplanted tp53-/- tumour cells. The novel X. tropicalis rag2-/- line and the irradiated wild type froglets will further expand the experimental toolbox in this diploid amphibian, and help to establish it as a versatile and relevant model for exploring human cancer.

cancer biology↗