Imaging of cytotoxic antiviral immunity while considering the 3R principle of animal research

Abstract

TCR CD8 T cells are obtained repetitively from the blood samples of single donors. One thousand transferred TCR CD8 T cells get activated, are functional, and proliferate. Several adoptive cell transfers from the same donor show reproducible results. One thousand transferred cells take part in the FV immune response without modifying it. Use of fluorescent transfer cells allows in vivo imaging and single cell tracking.

PD-1 knockout on cytotoxic primary murine CD8+ T cells improves their motility in retrovirus infected mice

“…DEREG mice were originally provided by Tim Sparwasser and bred at the University of Duisburg-Essen and the University Hospital Essen under pathogen-restricted conditions. For donor mice we used the previously described TCR-Lck-tdTom mice, which show a T cell specific expression of tdTomato (tdTom) as well as a FV-specific TCR directed against the GagL 85-93 -epitope on more than 90% of all CD8 + T cells ( 17 , 18 ).…”

“…Blood draw from donor mice, CD8 T cell isolation and preparation for cell transfer was carried out as previously described ( 12 , 18 ). For transfer of PDCD1 targeted and control CD8+ cells, gene editing was performed as described below prior to cell transfer.…”

“…Hence, we performed a CRISPR/Cas9 mediated knockout (KO) of the PD-1 coding gene PDCD1 on naïve primary CD8+ T cells that are specific for an immunodominant FV epitope ( 17 ). We used our established two-photon intravital bone marrow imaging model ( 12 , 18 ) to evaluate the impact of cell-selective PD-1 knockout on CTL motility in the bone morrow of living FV-infected mice. Bone morrow was chosen because it is an organ of massive viral replication during acute FV-infection ( 19 ).…”

From chemotherapy to biological therapy: A review of novel concepts to reduce the side effects of systemic cancer treatment (Review)

“…Intravital two-photon microscopy also allowed single cell visualization and tracking of a physiological antiviral CD8+ T-cell response. A low dose of 1,000 antigen-specific TCR transgenic naïve T-cells was revealed to be sufficient to transfer the response into virus-infected recipients, where the cells expanded strongly in the spleen and bone marrow within 10 days post-infection ( 32 ).…”

Embryo-Engineered Nonhuman Primate Models: Progress and Gap to Translational Medicine

“…However, some principles have been introduced internationally to help solve such problems, such as the implementation of the 3Rs principle. 3Rs include the use of unconscious experimental materials to replace live animals (replacement), reduce the number of experimental animals in the experiment (reduction), and improve animal welfare (refinement) in the whole process of the experiment [84][85][86][87][88][89]. At present, this principle has also become a recognized global animal scientific standard in animal ethics.…”