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

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

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

International audienceThe 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

Rapid turn over of the Foxp3 protein and mRNA in Treg.

<p><b>(a)</b> MFI of Foxp3 on GFP⁺ Treg treated with proteasome inhibitors MG-132 or Epoxomycine (Epox) in presence of IL-2 and AG for 2 hrs. Cells were pretreated during 2 hrs with the proteasome inhibitors before adding AG. <b>(b)</b> Foxp3, USP7 and STUB1 mRNA were quantified using the TaqMan RT-PCR assay normalized to vehicle control conditions 45 minutes after AG-treatment in expanded CD4⁺GFP⁺ cells by the 2^-ΔΔCt method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153682#sec009" target="_blank">method</a> section). This result originate from a single experiment performed in triplicates. <b>(c)</b> Foxp3 mRNA was assessed by quantitative RT-PCR normalized to vehicle control conditions at the indicated times after AG-treatment in expanded CD4⁺GFP⁺ cells by the 2^-ΔΔCt method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153682#sec009" target="_blank">method</a> section). The experiment was repeated 3 times. Each dot represent the mean ± SDEV of results obtained in 3 independent experiments. Two outliers were removed from the calculation <b>(d)</b> MFI of Foxp3 and CD25 in cycloheximide-treated sorted activated CD4⁺GFP⁺ cells relative to the vehicle control at the indicated times. Results are compiled from 3 independent experiments.</p

Loss of Foxp3 is not due to remethylation of the CNS2 and is reversible.

<p><b>(a)</b> STAT5 binding on a putative binding site in the TSDR enhancer of the <i>foxp3</i> gene was assessed by chromatin immunoprecipitation with control IgG or anti-STAT-5 antibodies. Quantification of binding was performed as described in Methods section. These data are from one representative experiment out of two. (<b>b</b>) Methylation status of the <i>Foxp3</i> TSDR was determined by bisulphite sequencing. Expanded CD4⁺GFP⁺ T cells were treated with IL-2 and ZM (IL-2+ZM) for 4 (4 hrs) or 6 (6 hrs) hours. Freshly purified CD4⁺GFP⁺ (GFP⁺), CD4⁺GFP^- (GFP^-), or untreated expanded cells (IL-2) were used as controls. These data are representative of two independent experiments. Each line indicate the position in the Foxp3 locus (CpG), according to Floess et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153682#pone.0153682.ref011" target="_blank">11</a>]. A shaded scale of grey illustrates the frequency of each methylated CpG. <b>(C)</b> For <i>in vivo</i> analysis, expanded CD4⁺GFP⁺ cells treated or not with AG for two hours were injected into congenic C57BL/6 mice. Animals were euthanized at the indicated times after transfer. The MFI of Foxp3 was normalized to vehicle-treated control cells.</p

Blockade of JAK/STAT signaling pathway leads to down modulation of Foxp3 in Treg.

<p>(<b>a</b>) CD25-enriched T cells were cultured for one hour in complete medium alone (Med.), with IL-2 (IL-2), or IL-2 supplemented with ZM-39923 (IL-2+ZM) or AG-490 (IL-2+AG). Profiles shown are gated in CD4⁺ cells and are representative of 4 independent experiments. (<b>b</b>) Frequencies of pSTAT5⁺ cells among CD4⁺ cells cultured for one hour with IL-2 alone (IL-2) or in presence of IL-2 and the indicated JAK inhibitors (ZM, AG). Results are compiled from 4 independent experiments. <b>(c)</b> In vitro expanded CD4⁺GFP⁺ Treg were treated with IL-2 and with either 100ug/mL of AG490 (IL-2+AG) or vehicle control (absolute ethanol; EtOH) for 2 hrs (IL-2). Proteins were extracted in lysis buffer and blotted followed by anti-Foxp3 and anti-actin staining. Intensity values were normalized to the actin band in each blot. The specificity of the Foxp3 staining is attested by the absence of the Foxp3 band in extracts from splenocytes of a scurfy (genetically deficient for Foxp3) mouse. <b>(d)</b> Human CD4⁺CD25⁺ cells were enriched from PBMC of healthy donors by magnetic sorting and treated with IL-2 (600 IU/mL) with or without ZM (50 μM) for 1h. MFI of CD25 and FOXP3 in human CD4⁺CD3⁺ cells after treatment with IL-2 alone (IL-2) or IL-2 in presence of ZM (IL-2+ZM). The MFI of Foxp3 and CD25 shown were normalized by the MFI of control cultures. These data are compiled from 3 independent experiments. Statistical significance was tested using Student t-test (***p<0.001, **p<0.01, *p<0.05).</p

Reduced Foxp3 by JAK/STAT inhibitors is independent of cell death.

<p><b>(a)</b> CD4⁺GFP⁺ T cells sorted from Foxp3-GFP transgenic mice were stimulated with anti-CD3/CD28 beads for 8 days and treated with vehicle and IL-2 (IL-2), or IL-2 supplemented with ZM (IL-2+ZM) or AG (IL-2+AG) JAK inhibitors for two hours. Cells were then stained with a dye allowing exclusion of dead cells by flow cytometry compatible with Foxp3 intracellular staining. Profiles are representative of 6 and 7 independent experiments for AG and ZM inhibitors, respectively. <b>(b)</b> Foxp3 and Bcl-2 expression in gated CD4⁺CD25⁺ cells from expanded CD4⁺GFP⁺ cells after 2 hour treatment with vehicle control and IL-2 (IL-2) or ZM in presence of IL-2 (IL-2+ZM). Similar results were observed in 3 independent experiments. <b>(c)</b> Median fluorescence intensity (MFI) of CD25 and Foxp3 in dead cell–excluded subset, represented as percentages of the MFI relative to Foxp3 staining in vehicle control condition (control) after two hours treatment with the indicated inhibitors. The results are compiled from 6 (AG) and 7 (ZM) independent experiments. <b>(d)</b> Freshly isolated or <b>(e)</b> anti-CD3/CD28 activated CD4⁺GFP⁺ cells from Foxp3-eGFP reporter mice were treated for the indicated times with either ZM or AG inhibitors. MFI of CD25 and Foxp3 are relative to the MFI of vehicle control conditions. These data are compiled from 3 independent experiments. Statistical significance was tested using Student t-test (***p<0.001, **p<0.01, *p<0.05).</p

CMIP is a negative regulator of T cell signaling

International audienceUpon interaction with cognate antigen, T cells integrate different extra and intracellular signals, involving basal and induced protein-protein interactions as well as binding of proteins to lipids, which can lead to either cell activation or inhibition. Here we show that selective T-cell expression of CMIP, a new adaptor protein, by targeted transgenesis drive