Stable expression of Foxp3 is ensured by demethylation of CpG motifs in the
Forkhead box P3+ (Foxp3+) CD4+ regulatory T cells (Tregs) control a variety of immune responses in a Foxp3 dependent manner (Fontenot et al., 2003; Hori et al., 2003). During these processes, Tregs differentiate functionally and adapt themselves to diverse environmental stimuli (Campbell and Koch, 2011). For example, some Tregs co-express Foxp3 and other lineage or tissue specific transcription factors like T-bet, ROR-γt, Bcl6 and PPAR-γ to control the corresponding immune and non-immune responses (Burzyn et al., 2013; Chaudhry et al., 2009; Chung et al., 2011; Cipolletta et al., 2012; Koch et al., 2009; Linterman et al., 2011; Ohnmacht et al., 2015; Sefik et al., 2015; Wang et al., 2011). What is interesting is that some of the lineage specifying transcription factors expressed together with Foxp3 like T-bet, GATA3, ROR-γt and Bcl6 are induced by proinflammatory cytokines antagonizing Foxp3 expression. These reports suggest that unique mechanisms stabilizing the expression of Foxp3 should exist for Tregs to adapt to environmental changes and to preserve their identity at the same time (Sakaguchi et al., 2013).
Stable expression of Foxp3 is accompanied by epigenetic modulation of the CpG motifs within the evolutionarily conserved non-coding sequence 2 (CNS2) enhancer of the Foxp3 locus (Floess et al., 2007; Huehn and Beyer, 2015; Huehn et al., 2009; Kim and Leonard, 2007). Demethylation of the CpG motifs in the CNS2 enhancer enables critical transcription factors like Foxp3 and Runx1-Cbf-β complex to access the CNS2 enhancer region and the CNS2 enhancer itself to interact with the promoter through a loop structure, which, as a result, has central roles in stabilizing Foxp3 expression especially under inflammatory conditions (Feng et al., 2014; Li et al., 2014; Zheng et al., 2010). Consistent with the above reports, CNS2 is demethylated in Tregs expressing Foxp3 stably but methylated in activated CD4+ T cells or TGF-β-induced Tregs expressing Foxp3 transiently (Miyao et al., 2012; Polansky et al., 2008).
CNS2 demethylation occurs in the thymus or periphery through iterative DNA oxidation reactions done by Ten-Eleven-Translocation (Tet) family members (Sasidharan Nair et al., 2016; Toker et al., 2013; Yang et al., 2015; Yue et al., 2016) and is known to be maintained consistently once established (Miyao et al., 2012). Indeed, CNS2 demethylation, instead of Foxp3 expression, was reported to be used as a reliable method for counting Tregs in peripheral blood and solid tissue (Wieczorek et al., 2009). However, how CNS2 demethylation is maintained in Tregs has not been clearly elucidated yet. In this study, we addressed the question whether Tet proteins have a role in maintaining as well as generating CNS2 demethylation and found that Tet proteins recruited to the CNS2 locus by IL2 protect the demethylated CpG motifs from being re-methylated by the occupancy of the CNS2 locus and its demethylase activity resulting in the stable expression of Foxp3 in Tregs.
Wild-type (WT) C57BL/6 (B6) mice were purchased from Koatech (Pyeongtaek, Gyeongi-do, Korea). CD45.1 congenic (B6.SJL-
To sort naïve CD4+ cells and Treg cells, CD44, CD25 and YFP were used (naïve cells: CD4+CD8−CD44lowCD25−YFP+; Treg cells: CD4+CD8−CD25+YFP+). The post-sort purity for each cell type was usually > 95%. We purchased the following monoclonal antibodies from BD Biosciences, eBioscience (USA) or BioLegend (USA) for flow cytometry: R-phycoerythrin (PE)-, PerCP-Cy5.5-, Allophycocyanin (APC)- or Brilliant Violet 421 (BV421)-labeled anti-CD25 (clones PC61 and 7D4), PE-Cy7-, PerCP-Cy5.5- or APC- or BV421-labeled anti-CD4 (clones RM4-5 and GK1.5), Fluorescein isothiocyanate (FITC)-, PE- or PerCP-Cy5.5-labeled anti-CD44 (clone IM7), PerCP-Cy5.5- or APC-labeled anti-CD45.1 (clone A20), PE-, PE-Cy7-, PerCPCy5.5-or Brilliant Violet 510 (BV510)-labeled-anti-CD45.2 (clone 104), PE-labeled-anti-CD62L (clone MEL-14), PElabeled-anti-CD69 (clone H1.2F3), PerCP-Cy5.5- or APC- or APC-Cy7-labeled-anti-CD8 (clone 53-6.7), APC-labeled-anti-CD86 (clone GL1), PE-labeled-anti-CTLA4 (clone UC10-4F10-11), Alexa Fluor 488- or PE-labeled-anti-Foxp3 (clone FJK-16s), APC-labeled-anti-IFN-γ (clone XMG1.2), PE-labeled-IL17 (clone TC11-18H10.1) unconjugated anti-Myc (9E10, Santa Cruz Biotechnology, Dallas, Texas) and APC-labeled-donkey anti-mouse IgG (Jackson immunoresearch, West Grove, PA). Intracellular Foxp3, cytokines, myc-tagged proteins were stained using Foxp3 Staining Buffer set (eBioscience). For cytokine analysis, cells were cultured for 4 hours in the presence of PMA/ionomycin plus monensin (BD biosciences) before intracellular cytokine staining. Data were acquired through FACS Calibur or FACS Canto-II (BD Biosciences) and were analyzed with FlowJo software (Tree Star, USA) (Kim et al., 2015).
FACS-sorted cells were cultured in complete RPMI-1640 medium (WelGENE, Korea), supplemented with 10% FBS (WelGENE), penicillin, streptomycin (Sigma-Aldrich, USA), L-glutamine (2 mM; Life Technologies, USA), sodium pyruvate (2 mM; Sigma-Aldrich), non essential amino acid (0.1 mM; Sigma-Aldrich) and 2-ME (50 μM; Sigma-Aldrich). For CD3/CD28 stimulation, FACS-sorted cells were stimulated by plate-bound anti-CD3 (2C11, 1 μg/ml; eBioscience) plus CD28 (37.51, 1 μg/ml) in the presence or absence of recombinant murine IL2 (rIL2; Peprotech, Rocky Hill, NJ, USA), recombinant IL6 (rIL6; Peprotech), recombinant IL4 (rIL4; Peprotech) or recombinant IL12 (rIL12; Peprotech) for indicated periods.
The genomic DNA was extracted from the FACS-sorted live cells by using the Blood & Tissue Genomic DNA Extraction kit (Qiagen, USA). For isolation of DNA from cells fixed and stained with anti-Foxp3 mAb, we used the protocol described previously (Hansmann et al., 2010; Piper et al., 2014). Briefly, sorted cells (CD4+CD8−Foxp3+CD25+) were incubated with 300 μl lysis buffer (10 mM Tris-HCl, 100 mM NaCl, 50 mM EDTA, 0.5% SDS, 0.1 μg/ml proteinase K, and 20 μg/ml RNase A) for 24 hours at 60°C. Then DNA was extracted by phenol/chloroform/isoamyl alcohol solution (25:24:1) and precipitated overnight by ethanol. Extracted genomic DNAs were converted by the EZ DNA methylation gold kit (Zymo Research, USA). Anti-sense (Ohkura et al., 2012) strands of bisulfite-treated DNA were then subjected to PCR for amplification of CNS2 (12 CpG motifs were analyzed). The PCR products obtained were cloned into the pGemT-easy vector (Promega, USA) and 10–30 individual clones from each sample were sequenced with M13 reverse primer (GAAACAGCT ATGACCATG).
Nuclear lysate was sonicated to make small DNA fragments ranging from 100–500 base pairs and then incubated with anti-Tet2 (ab94580, Abcam, UK) or anti-Dnmt1 Ab (H-300, Santa Cruz Biotechnology) overnight at 4°C. Isotype-matched control Ab was used for the negative control. Immune complexes containing DNA fragments were precipitated using Dynabeads (Invitrogen, USA) or EZ-ChIP kit (Milipore, Germany). Relative enrichment of the target regions in the precipitated DNA fragments was analyzed by qPCR. The sequences of primers are as follows.
CNS2 forward, 5′-AAC CTT GGG CCC CTC TGG CA-3′
CNS2 reverse, 5′-GGC CGG ATG CAT TGG GCT TCA-3′
Foxp3 promoter forward, 5′-CTT CTG GGA GCC AGC CAT-3′
Foxp3 promoter reverse, 5′-GCT GTA CTC CCC CCA CAA ATT-3′
RNA was isolated from FACS-sorted cells using the RNeasy Mini kit (Qiagen) or Trizol (Life Technologies), and reverse-transcribed into cDNA using QuantiTect Reverse Transcription kit (Qiagen). Quantitative PCR (qPCR) reactions were performed on RotorGene 6000 system (Qiagen) using AccuPower GreenStar qPCR kit (Bioneer, Korea). All data were normalized to actin. Non-specific amplification was checked by the use of melting curve and agarose gel electorphoresis. The sequences of primers are as follows.
Plasmids transfection experiments were performed using FACS-sorted
Transfection of Tregs was performed using the Mouse T Cell Nucleofector Kit and the Nucleofector device (X-001 program) according to the kit directions (Lonza). Cells were transfected with 300 pmol SMART pool siRNAs (Dharmacon, USA) designed against mouse
FACS-sorted naïve CD4+ T cells labeled with cell trace violet (cell division dye, Life Technologies) were used as responders. Antigen presenting cells were prepared by depleting CD4+ and CD8+ cells from wild-type B6 splenocytes using flow cytometry. Responder cells (5 × 103) were cultured with antigen presenting cells (2 × 104) and soluble anti-CD3 mAb (0.3 μg/ml) in the absence or presence of various numbers of Treg cells for 3 days. The division of responder cells was assessed by dilution of cell trace violet.
FACS-sorted OT-II Tregs were injected intravenously into congenic WT B6 mice. The next day, the recipient mice were sub-cutaneously immunized in the left flank with 100 μg of OVA in CFA (Sigma-Aldrich) and injected intraperitoneally with sulfinpyrazone (10 μg/g body weight) or vehicle (DMSO) every day. In 7 days OT-II CD4+Foxp3+ Tregs of donor origin were FACS-sorted from the draining left axillary LNs and subjected to CNS2 demethylation study. In some cases, the recipient mice were treated with congenically marked OT-II naïve and Treg cells together with anti-IL2 mAb.
A two-tailed, unpaired, student’s
Previously, it was shown that Tet family members including Tet1, Tet2 and Tet3 have an essential role in demethylating the CpG motifs of the Foxp3 enhancer, CNS2, during the development of Tregs and contribute to the stable expression of Foxp3 (Yang et al., 2015; Yue et al., 2016). These reports led us to question how the established CNS2 demethylation is protected in mature Tregs where DNA methyltransferases (Dnmts) work actively (Wang et al., 2013) and whether Tet proteins might contribute by not only generating but also maintaining the CNS2 demethylation. To address these issues, the CNS2 methylation status of wild type (WT) and Tet2 deficient Tregs was investigated using the ovalbumin (OVA) immunization model. Tet2 deficient mice (referred to as
DNA methylation is known to be caused by Dnmts, which led us to check whether Dnmts are involved in the CNS2 re-methylation of
The finding that CNS2 demethylation was labile in
Next, we investigated the effect of CNS2 re-methylation on the Foxp3 stability and the suppressive function of the Tregs. Foxp3-expressing cells from Tregs treated with rIL6 shown in Fig. 2A were re-sorted and cultured with anti-CD3/CD28 plus rIL2, and the expression of Foxp3 was analyzed after 3 days. Foxp3 was maintained stably in all the Tregs except for the
IL2 is an important survival factor for Tregs (Malek, 2008), and media supplemented with rIL2 are commonly used to culture Tregs
Because IL2 could be produced by CD4+Foxp3− cells converted from Tregs and already available even without adding rIL2, we started to examine the potential roles of IL2 with a loss-of-function approach. To neutralize IL2, FACS-sorted WT or
Next, to verify the role of Dmnts
Subsequently, we investigated the role of IL2 on CNS2 demethylation with the OVA/CFA immunization model. FACS-sorted OT-II/WT Tregs were injected into congenically marked WT B6 mice, which were treated with anti-IL2 blocking mAbs plus sulfinpyrazone. IL2 neutralization treatment downregulated CD25 in the donor and host Tregs (
It was reported that IL2 is related to the binding of Tet2 to
Here, we showed that demethylation of the CpG motifs in the Foxp3 CNS2 region is determined by the competition between the DNA modifying enzymes Dnmts and Tets which have opposite functions. Dnmt1 was recruited to the CNS2 locus by the inflammatory cytokine IL6 (Fig. 5) and methylated the CpG motifs (Fig. 2). In contrast, the methylation activity of Dnmt1 was abrogated by Tet2 recruited to CNS2 by IL2 (Fig. 5). These findings suggest that IL2-Tet2 and IL6-Dnmt1 compete for the same binding sites in the CNS2 locus as STAT3 and STAT5 do in the IL17 locus (Yang et al., 2011) and Tet2 inhibits Dnmt1 through the occupation of CNS2. Furthermore, the enzymatic activity of Tet2 also contributed to CNS2 demethylation. While the demethylation status of CNS2 was restored completely in
In conclusion, our study showed that the methylation status of the Foxp3 CNS2 locus is dynamically regulated by a balance between two DNA modifying enzymes Dnmts and Tets and related cytokines, which influences the stability of Foxp3 expression in Tregs.