CD4⁺and CD8⁺regulatory T cells characterization in the rat using a unique transgenicFoxp3-EGFPmodel

AbstractBackgroundCD4+and CD8+regulatory T cells (Treg) in diverse species include different subsets from different origins. In all species, CD8+Treg have been poorly characterized. CD4+and CD8+Treg in rats have only partially been characterized and there is no rat model in which FOXP3+Treg are genetically tagged.ResultsWe generated a rat transgenic line using the CRISPR/Cas9 system in which EGFP was inserted in frame on the 3’ end of theFoxp3gene using a 2A self-cleaving peptide. EGFP was exclusively expressed by CD4+and CD8+T cells in similar proportion as observed with anti-FOXP3 antibodies. CD4+EGFP+Treg were 5-10 times more frequent than CD8+EGFP+Treg. CD4+and CD8+EGFP+Treg expressed both the CD25highCD127lowCD45RClow/-markers. The suppressive activity of CD4+and CD8+Treg was largely confined to EGFP+cells. RNAseq analyses showed similarities but also differences among CD4+and CD8+EGFP+cells and provided the first description of the natural FOXP3+CD8+Treg transcriptome. In vitro culture of CD4+and CD8+EGFP-cells with TGFbeta and IL-2 resulted in the induction of EGFP+Treg. Preferential expansion of CD4+and CD8+EGFP+Treg could be detected upon in vivo administration of a low dose of IL-2.ConclusionsThis new and uniqueFoxp3-EGFPrat line constitutes a useful model to identify and isolate viable natural and induced CD4+and CD8+Treg. Additionally, it allows to identify new molecules expressed in CD8+Treg that may allow to better define their phenotype and function not only in rats but also in other species.