Abstract
This protocol was developed to generate chimeric mice in which T lymphocytes could be stratified by age on the basis of congenic marker expression. The conditioning drug busulfan is used to ablate host haematopoietic stem cells while leaving the peripheral immune system intact. Busulfan treatment is followed by bone marrow transplantation (BMT), with T-cell depleted donor bone marrow bearing a different congenic marker (CD45.2) to that of the host mouse (CD45.1). New cell production post-BMT can thus be tracked by measuring the fraction of CD45.2+ cells over time within a population of interest (Hogan et al., 2015; Gossel et al., 2017).
Keywords: T cells, T cell homeostasis, Bone marrow chimeras, Busulfan, Temporal fate mapping
Background
Bone marrow chimeras are a valuable tool for studying immune system development and function. Typically, chimeras are generated by irradiation of host mice followed by transplantation with donor bone marrow. Irradiation causes considerable damage to the haematopoietic system, and full immune reconstitution is delayed by weeks to months post-transplant (Fry and Mackall, 2005). The resulting period of lymphopenia drives spontaneous proliferation of naïve T cells and the acquisition of a memory-like phenotype (Goldrath et al., 2004). To avoid the perturbation of immune homeostasis caused by irradiation, we turned our attention to the conditioning drug busulfan. Busulfan is an alkylating agent that is toxic to haematopoietic stem cells (HSC) but does not deplete circulating lymphocytes (Westerhof et al., 2000; Hsieh et al., 2007). Following busulfan conditioning and bone marrow transplantation (BMT), chimeras are indistinguishable from untreated controls: they have normal numbers of naïve and memory CD4 and CD8 T cells, and these cells express normal levels of the proliferation marker Ki67 (Hogan et al., 2015; Gossel et al., 2017). Therefore, busulfan conditioning allows induction of chimerism while preserving peripheral immune homeostasis. By using donor bone marrow bearing a congenic marker, it is possible to distinguish host- or donor-origin cells by flow cytometry. While HSC replacement is never absolute, emergence of donor origin cells can be used as an accurate proxy for de novo haematopoetic development since BMT, thereby allowing cells within a population to be stratified by age on the basis of congenic marker expression.
Materials and Reagents
Equipment
Procedure
Note: The steps in this procedure are summarised in Figure 1. All animal procedures were performed in accordance with UK Home Office regulations. Figure 1. Generation of busulfan chimeric mice. This schematic summarises the principal steps of this protocol for the generation of busulfan chimeric mice. Bone marrow cells from CD45.2+ donor mice are labeled with biotinylated antibodies to CD3 and TCRb, followed by labelling with streptavidin-coated magnetic beads. Labelled cells are immobilised using a magnet, allowing the unbound fraction to be collected. T cell depleted bone marrow cells are then injected into busulfan-conditioned CD45.1+ host mice.
Data analysis
Notes
Recipes
Acknowledgments
This protocol was developed for two published studies (Hogan et al., 2015; Gossel et al., 2017), both supported by the National Institutes of Health (R01 AI093870) and the Medical Research Council (MC-PC-13055). The authors declare no conflicts of interest or competing interests.
References
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