Abstract
Understanding T cell function in vivo is of key importance for basic and translational immunology alike. To study T cells in vivo, we developed a new knock-in mouse line, which expresses a fusion protein of granzyme B, a key component of cytotoxic granules involved in T cell-mediated target cell-killing, and monomeric teal fluorescent protein from the endogenous Gzmb locus. Homozygous knock-ins, which are viable and fertile, have cytotoxic T lymphocytes with endogeneously fluorescent cytotoxic granules but wild-type-like killing capacity. Expression of the fluorescent fusion protein allows quantitative analyses of cytotoxic granule maturation, transport and fusion in vitro with super-resolution imaging techniques, and two-photon microscopy in living knock-ins enables the visualization of tissue rejection through individual target cell-killing events in vivo. Thus, the new mouse line is an ideal tool to study cytotoxic T lymphocyte biology and to optimize personalized immunotherapy in cancer treatment.
Highlights
Cytotoxic T lymphocytes (CTLs) are essential for the removal of foreign agents such as viruses or bacteria, and play a key role in modern personalized cancer immunotherapy (Porter et al, 2011; Sharma and Allison, 2015; Watanabe et al, 2018; Minutolo et al, 2019)
Endogenous fluorescent label for cytotoxic granules (CG) we chose granzyme B (GzmB) (Young et al, 1986; Masson and Tschopp, 1987; Krahenbuhl et al, 1988), which belongs to a family of serine proteases that induce apoptosis of target cells and which is present in CGs of natural killer cells and CD4+ and CD8+ T lymphocytes (Peters et al, 1991)
The expression levels of the fusion protein varied between different preparations (59.1%, 53.6% and 183.9% of wt level for GzmB) as expected, but we always observed a robust fluorescence without the requirement to change the intensity of the excitation lasers for the experiments shown in the following figures
Summary
Cytotoxic T lymphocytes (CTLs) are essential for the removal of foreign agents such as viruses or bacteria, and play a key role in modern personalized cancer immunotherapy (Porter et al, 2011; Sharma and Allison, 2015; Watanabe et al, 2018; Minutolo et al, 2019). A detailed mechanistic understanding of the major CTL function, that is the killing of cells through release of cytotoxic substances from cytotoxic granules (CGs) at the immune synapse (IS), is of utmost interest for basic and clinical science alike (Dustin and Long, 2010; Griffiths et al, 2010; Mukherjee et al, 2017; Xiong et al, 2018). Immunology and Inflammation eLife digest Cytotoxic, or killer, T cells are a key part of the immune system. They carry a lethal mixture of toxic chemicals, stored in packages called cytotoxic granules. Inside the body, their killing rate slows down Researchers think that this has something to do with how killer T cells interact with other immune cells, but the details remain unclear
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