Inhibition of the JAK/STAT Signaling Pathway in Regulatory T Cells Reveals a Very Dynamic Regulation of Foxp3 Expression

Abstract

The IL-2/JAK3/STAT-5 signaling pathway is involved on the initiation and maintenance of the transcription factor Foxp3 in regulatory T cells (Treg) and has been associated with demethylation of the intronic Conserved Non Coding Sequence-2 (CNS2). However, the role of the JAK/STAT pathway in controlling Foxp3 in the short term has been poorly investigated. Using two different JAK/STAT pharmacological inhibitors, we observed a detectable loss of Foxp3 after 10 min. of treatment that affected 70% of the cells after one hour. Using cycloheximide, a general inhibitor of mRNA translation, we determined that Foxp3, but not CD25, has a high turnover in IL-2 stimulated Treg. This reduction was correlated with a rapid reduction of Foxp3 mRNA. This loss of Foxp3 was associated with a loss in STAT-5 binding to the CNS2, which however remains demethylated. Consequently, Foxp3 expression returns to normal level upon restoration of basal JAK/STAT signaling in vivo. Reduced expression of several genes defining Treg identity was also observed upon treatment. Thus, our results demonstrate that Foxp3 has a rapid turn over in Treg partly controlled at the transcriptional level by the JAK/STAT pathway.

Short‐term cytokine stimulation reveals regulatory T cells with down‐regulated Foxp3 expression in human peripheral blood

“…F, the level of Foxp3 expression in the cells which remained within the Treg gate of the same experiment started to decline at 8 h of incubation, and reached a plateau of about half the initial expression level by 48 h. In contrast, CD25 expression levels remained stable over this time frame (Fig. G), in agreement with the higher stability of the CD25 as compared to the Foxp3 protein previously reported for primary murine and human Treg cells .…”

“…We here present an alternative explanation for the low frequency of CD4 + CD25 + Foxp3 + cells among circulating CD4 + T cells, which is based on transient cytokine withdrawal during lymphocyte recirculation. Our proposal is based on the known requirement for STAT5 activating cytokines such as IL‐2 for the maintenance of Foxp3 expression in vitro and its dependence on continuous IL‐2 exposure in vivo , the short half‐life of the Foxp3 protein , and its dynamic regulation in mouse and human Treg cells as demonstrated by pharmacologic interruption of the STAT5 signaling pathway . Furthermore, we propose that while STAT5‐activating cytokines are provided in the tissue context by other immune and non‐immune cells, many Treg cells spend sufficient time in the blood stream to lose detectable levels of Foxp3.…”

Ionizing radiation modulates the phenotype and function of human CD4+ induced regulatory T cells

“…Confirmation of this mechanism warrants further investigation. Alternatively, binding of STAT5 to the T REGspecific demethylated region (TSDR) within the conserved noncoding sequence 2 (CNS2) has been shown to stabilize Foxp3 expression [66], and blockade of the JAK3/STAT5 signaling pathway has been demonstrated to downregulate Foxp3 expression in both human and murine T REG cells [67]. It is possible that radiation alters the expression of STAT5, however, we did not observe any change in phosphorylated STAT5 following radiation treatment (unpublished data) suggesting that radiation-induced regulation of Foxp3 may be independent of STAT5.…”

Regulatory T Cells in the Mosaic of Liver Transplantation Tolerance

“…One may say that this is as two step process, starting with the generation of CD25 hi, but FoxP3 − Tregs-precursors, followed by the induction of FoxP3 through cytokine/STAT5-dependant signals involving HDAC [74,78,79]. Blocking of JAK/STAT pathway downmodulates Foxp3 expression [80]. In addition, a group of studies indicate that the maintenance of Tregs suppressive function is dependent on the epigenetic regulation of foxp3 locus by the Polycomb repressive complex 2 (PRC2).…”