RESUMEN
Regulatory T cells (Treg) are negative regulators of the immune response; however, it is poorly understood whether and how Foxp3 transcription is induced and regulated in the periphery during T-cell responses. Using Foxp3-Timer of cell kinetics and activity (Tocky) mice, which report real-time Foxp3 expression, we show that the flux of new Foxp3 expressors and the rate of Foxp3 transcription are increased during inflammation. These persistent dynamics of Foxp3 transcription determine the effector Treg programme and are dependent on a Foxp3 autoregulatory transcriptional circuit. Persistent Foxp3 transcriptional activity controls the expression of coinhibitory molecules, including CTLA-4 and effector Treg signature genes. Using RNA-seq, we identify two groups of surface proteins based on their relationship to the temporal dynamics of Foxp3 transcription, and we show proof of principle for the manipulation of Foxp3 dynamics by immunotherapy: new Foxp3 flux is promoted by anti-TNFRII antibody, and high-frequency Foxp3 expressors are targeted by anti-OX40 antibody. Collectively, our study dissects time-dependent mechanisms behind Foxp3-driven T-cell regulation and establishes the Foxp3-Tocky system as a tool to investigate the mechanisms behind T-cell immunotherapies.
Asunto(s)
Factores de Transcripción Forkhead/inmunología , Linfocitos T Reguladores/inmunología , Transcripción Genética/inmunología , Animales , Anticuerpos/farmacología , Antígeno CTLA-4/genética , Antígeno CTLA-4/inmunología , Factores de Transcripción Forkhead/genética , Ratones , Ratones Transgénicos , Receptores OX40/antagonistas & inhibidores , Receptores OX40/genética , Receptores OX40/inmunología , Receptores Tipo II del Factor de Necrosis Tumoral/antagonistas & inhibidores , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral/inmunología , Linfocitos T Reguladores/citología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genéticaRESUMEN
Understanding the mechanisms of cellular differentiation is challenging because differentiation is initiated by signaling pathways that drive temporally dynamic processes, which are difficult to analyze in vivo. We establish a new tool, Timer of cell kinetics and activity (Tocky; or toki [time in Japanese]). Tocky uses the fluorescent Timer protein, which spontaneously shifts its emission spectrum from blue to red, in combination with computer algorithms to reveal the dynamics of differentiation in vivo. Using a transcriptional target of T cell receptor (TCR) signaling, we establish Nr4a3-Tocky to follow downstream effects of TCR signaling. Nr4a3-Tocky reveals the temporal sequence of events during regulatory T cell (Treg) differentiation and shows that persistent TCR signals occur during Treg generation. Remarkably, antigen-specific T cells at the site of autoimmune inflammation also show persistent TCR signaling. In addition, by generating Foxp3-Tocky, we reveal the in vivo dynamics of demethylation of the Foxp3 gene. Thus, Tocky is a tool for cell biologists to address previously inaccessible questions by directly revealing dynamic processes in vivo.