From stability to dynamics: understanding molecular mechanisms of regulatory T cells through Foxp3 transcriptional dynamics

Abstract

Studies on regulatory T cells (T_reg ) have focused on thymic T_reg as a stable lineage of immunosuppressive T cells, the differentiation of which is controlled by the transcription factor forkhead box protein 3 (Foxp3). This lineage perspective, however, may constrain hypotheses regarding the role of Foxp3 and T_reg in vivo, particularly in clinical settings and immunotherapy development. In this review, we synthesize a new perspective on the role of Foxp3 as a dynamically expressed gene, and thereby revisit the molecular mechanisms for the transcriptional regulation of Foxp3. In particular, we introduce a recent advancement in the study of Foxp3-mediated T cell regulation through the development of the Timer of cell kinetics and activity (Tocky) system, and show that the investigation of Foxp3 transcriptional dynamics can reveal temporal changes in the differentiation and function of T_reg in vivo. We highlight the role of Foxp3 as a gene downstream of T cell receptor (TCR) signalling and show that temporally persistent TCR signals initiate Foxp3 transcription in self-reactive thymocytes. In addition, we feature the autoregulatory transcriptional circuit for the Foxp3 gene as a mechanism for consolidating T_reg differentiation and activating their suppressive functions. Furthermore, we explore the potential mechanisms behind the dynamic regulation of epigenetic modifications and chromatin architecture for Foxp3 transcription. Lastly, we discuss the clinical relevance of temporal changes in the differentiation and activation of T_reg .

Keywords: Foxp3; Nr4a3; Time of cell kinetics and activity (Tocky); regulatory T cells (Treg); transcriptional autoregulatory circuit.

Figure 1

Comparison of tools to investigate forkhead box protein 3 (Foxp3)‐expressing T cells in vivo. (a) Most Foxp3 reporter mice use stable fluorescent proteins (FP), such as enhanced green fluorescent protein (EGFP), the half‐life of which is > 56 h. (b) Foxp3 fate mappers such as Foxp3^GFPCre:Rosa26^RFP allow the identification of regulatory T cells (T_regs) with Foxp3 expression and ex‐T_regs that lost Foxp3 expression. Notably, both GFP and red fluorescent protein (RFP) are stable FPs. (c) Foxp3–Tocky uses fluorescent timer, the emission spectrum of which changes spontaneously and irreversibly from blue to red fluorescence. The half‐life of blue fluorescence is ~4 h, and thus reports the ‘real‐time’ activity of Foxp3 transcription. In contrast, the half‐life of red fluorescence is ~120 h and thus reports the history of Foxp3 transcription. The Tocky system combines blue and red fluorescence data and identifies characteristic transcriptional dynamics including new, persistent and arrested (inactive).

Figure 2

Activation versus consolidation and tuning of Foxp3 transcription. We propose to classify Foxp3 transcriptional regulation into two major mechanisms. (a) Activation of Foxp3 transcription is regulated mainly by T cell receptor (TCR) signals and enhanced by interleukin (IL)‐2, transforming growth factor (TGF)‐β and retinoic acid (RA). This may lead to thymic regulatory T cell (T_reg) selection and peripheral T_reg differentiation. In addition, tonic TCR signals through self‐reactive TCRs may use this mechanism to regulate homeostatic Foxp3 transcription. (b) Consolidation and tuning of Foxp3 transcription. The maintenance of Foxp3 transcription requires CNS2 of the Foxp3 gene, which may provide a platform for Foxp3–Runx1/CBF‐β complex to form the autoregulatory transcriptional circuit (autoregulatory loop) for the Foxp3 gene. The activity of this loop can be affected by IL‐2 signalling via phosphorylated signal tranducer and activator of transcription‐5 (STAT)‐5. This mechanism may lead to temporally persistent Foxp3 transcription, which promotes effector T_reg differentiation, and the dynamic regulation of epigenetic modifications during T_reg differentiation.