Revealing the mechanism of regulatory T-cell generation in vivo by novel Fluorescent Timer reporter

Abstract

Abstract Foxp3+ regulatory T-cells (Treg) are considered to mainly differentiate in the thymus and protect against autoimmunity. However, the physiological significance of peripheral Treg differentiation is still unclear, due to a lack of methods to identify newly generated Treg in vivo. In addition, the mechanism of Treg differentiation in the thymus is still obscure, because of difficulties in defining temporal sequences of developmental events in vivo. Here we develop a ground-breaking technology to reveal the time-dependent mechanisms of Treg generation and maturation in vivo. Using Fluorescent Timer protein that spontaneously and irreversibly changes its emission spectrum, we establish a novel Foxp3Timer reporter, and and visualize the dynamics of Foxp3 transcription and thereby identify de novo Foxp3 expression in vivo. Here we show that antigenic recognition immediately induces de novo Foxp3 expression in activated effector T-cells in vivo. Surprisingly, most of Foxp3-expressors in inflamed tissue are not derived from the pre-existing Treg pool, but are newly generated upon antigen recognition. RNA-seq analysis reveals novel genomic mechanisms for peripheral Treg generation and identifies new markers for antigen-reactive Treg, by which we successfully manipulate Treg differentiation dynamics. Collectively, we show that Foxp3 constitutes a negative feedback mechanism for activated effector T-cells, which induces the resolution of inflammation. Our novel Timer technology is applicable to many outstanding problems in immunology.