The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 2389-2396
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dong, J.
Right arrow Articles by Thiel, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dong, J.
Right arrow Articles by Thiel, A.

IL-10 Is Excluded from the Functional Cytokine Memory of Human CD4+ Memory T Lymphocytes1

Jun Dong2,3,*, Claudia Ivascu2,{ddagger}, Hyun-Dong Chang{dagger}, Peihua Wu*,§, Roberta Angeli, Laura Maggi, Florian Eckhardt{ddagger}, Lars Tykocinski{dagger}, Carolina Haefliger{ddagger}, Beate Möwes*, Jochen Sieper§, Andreas Radbruch{dagger}, Francesco Annunziato and Andreas Thiel3,*

* Clinical Immunology Group and {dagger} Cell Biology Group, Deutsches Rheuma-Forschungszentrum Berlin, Berlin, Germany; {ddagger} EPIGENOMICS, Berlin, Germany; § Department of Rheumatology, Charité Campus Mitte, Berlin, Germany; and Center of Research, Transfer, High Education MCIDNENT, University of Florence, Firenze, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Epigenetic modifications, including DNA methylation, profoundly influence gene expression of CD4+ Th-specific cells thereby shaping memory Th cell function. We demonstrate here a correlation between a lacking fixed potential of human memory Th cells to re-express the immunoregulatory cytokine gene IL10 and its DNA methylation status. Memory Th cells secreting IL-10 or IFN-{gamma} were directly isolated ex vivo from peripheral blood of healthy volunteers, and the DNA methylation status of IL10 and IFNG was assessed. Limited difference in methylation was found for the IL10 gene locus in IL-10-secreting Th cells, as compared with Th cells not secreting IL-10 isolated directly ex vivo or from in vitro-established human Th1 and Th2 clones. In contrast, in IFN-{gamma}+ memory Th cells the promoter of the IFNG gene was hypomethylated, as compared with IFN-{gamma}-nonsecreting memory Th cells. In accordance with the lack of epigenetic memory, almost 90% of ex vivo-isolated IL-10-secreting Th cells lacked a functional memory for IL-10 re-expression after restimulation. Our data indicate that IL10 does not become epigenetically marked in human memory Th cells unlike effector cytokine genes such as IFNG. The exclusion of IL-10, but not effector cytokines, from the functional memory of human CD4+ T lymphocytes ex vivo may reflect the need for appropriate regulation of IL-10 secretion, due to its potent immunoregulatory potential.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immune reactions are tightly controlled to avoid excessive activation in the course of pathogen-specific immune responses and to suppress the activation of autoreactive lymphocytes. Different subsets of regulatory T cells represent pivotal players of immune regulation in the immune system (1). Populations of regulatory T cells identified include naturally occurring CD4+CD25+ T regulatory cells (2), Ag-induced T regulatory type 1 cells (Tr1)4 (3), and Th3 cells (4), which all display certain abilities to produce the immunoregulatory cytokine IL-10. IL-10 exerts its inhibitory action on macrophages and dendritic cells, thereby regulating effector cell activation, but has also stimulatory effects on B and T cells (5). Il10-deficient mice develop severe chronic enterocolitis (6) while tissue- or cell-specific overexpression of Il10 leads to impaired immune responses (7, 8, 9, 10). Tr1 cells secret high levels of IL-10 and low levels of IFN-{gamma} and IL-2 upon activation, but display their regulatory function in an IL-10-dependent manner (11, 12, 13, 14). IL-10 production by CD4+CD25+ regulatory T cells has been reported in different in vivo (15, 16) and in vitro (17) experimental systems. Thus, IL10 gene expression by regulatory T cell subsets is an essential factor for effective regulation of immune responses.

The immune system needs to act rapidly and specifically to pathogens but also has to control autoimmunity. These actions are partially mediated by effector and regulatory cytokines produced by memory Th cells that memorize the expression of cytokine genes after primary activations triggered by TCR signaling and instructive costimulatory signals, and re-express memorized cytokine genes within a few hours upon restimulation via TCR, in the absence of original instructive costimulatory signals (18). Control of effector cytokines expression via networks of transcription factors and epigenetic regulation has been well documented. Epigenetic modifications such as changes in DNA methylation (primarily at the C5 position of cytosine in CpG dinucleotides), histone modifications, and chromatin rearrangement within the nucleus influence the accessibility of transcription factors to their DNA binding sites. These associated molecular changes are heritable and provide a basis for memory of gene expression (19, 20, 21, 22). Indeed, these mechanisms have been documented for Th1 and Th2 cells in the transcriptional regulation of the Ifng/IFNG and Il4 clustered genes (23, 24, 25, 26, 27, 28, 29, 30). However, epigenetic regulation of IL10, especially by DNA methylation, has remained poorly understood.

The expression of Il10 is under the control of several transcription factors such as Stat3 (31), Sp1 (32) and Sp3 (33), NF-{kappa}B (34), Smad-4 (35), c-Maf (36), and Jun proteins (37). Recent data obtained in mouse on Il10 gene accessibility according to DNase I hypersensitivity have suggested chromatin remodeling of the Il10 gene locus. However, some DNase I hypersensitive sites (HSS) were described as both enhancing and silencing Il10 gene expression (34, 38, 39).

To gain insight into the epigenetic regulation of the IL10 gene in comparison to the IFNG gene in human Th cells, we characterized IL-10+IFN-{gamma}–, IL-10+IFN-{gamma}+, IL-10–IFN-{gamma}+, and IL-10–IFN-{gamma}– Th cell subsets isolated directly ex vivo from peripheral blood of healthy adults. Using a bisulfite-modified DNA sequencing approach, we performed semiquantitative assessments of the DNA methylation pattern of the entire IL10 gene locus spanning 9.1 kb upstream and 9 kb downstream of the transcriptional start site that encompasses 88 selected CpGs. We demonstrate the lack of a specific DNA methylation pattern of the IL10 gene in IL-10-secreting Th cells isolated from ex vivo- and in vitro-established Ag-specific human Th1 and Th2 clones. However, we show an unambiguous hypomethylation of the IFNG gene promoter in different IFN-{gamma}-producing Th cell subsets. Thus, contrary to the effector cytokine gene IFNG, expression of the immunoregulatory cytokine gene IL10 is not mainly regulated by DNA methylation. Moreover, IL-10-producing Th cells lack a memory for IL-10 re-expression in vitro. In conclusion, IL-10 is excluded from the functional cytokine memory in human Th cells, preventing the generation of memory Th cells with an inherited program to secrete IL-10, possibly to ensure a limited effect of IL-10 in down-regulation of adaptive pathogen-specific immunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Media and reagents

The media used were RPMI 1640 supplemented with 1% glutamax, 100 U/ml penicillin, 100 µg/ml streptomycin (Invitrogen Life Technologies), and 10% human AB serum (PAA). PMA (P; 5 ng/ml; Sigma-Aldrich) and 1 µg/ml ionomycin (I; Sigma-Aldrich) were used for stimulation. rIL-7 and rIL-15, each 10 µg/ml (R&D Systems), and 5–10 µM 5-azacytidine (Sigma-Aldrich) were applied to in vitro cell cultures. Brefeldin A (5 µg/ml; Sigma-Aldrich) was used to block cytokine secretion. The following Abs were used for FACS analysis: anti-CD69 FITC, anti-IFN-{gamma} PerCP Cy5.5, anti-IL-10 allophycocyanin, anti-CD45RO (allophycocyanin), anti-CD45RA (allophycocyanin), -CCR7 (FITC; BD Biosciences), and anti-CD4 Cy5 (TT1, mouse IgG1, house conjugate).

Sample collection and preparation

Buffy coats from healthy adult anonymous donors were obtained in accordance with local ethical committee approval. PBMCs were isolated from each buffy coat by density gradient sedimentation using Ficoll-Hypaque (Sigma-Aldrich). Cells were washed twice with PBS before CD4+ T cell separation.

Purification, sorting of CD4+ T lymphocytes

CD4+ T cells were purified from PBMCs by MACS using CD4 microbeads (Miltenyi Biotec). The purity of the sorted population was 95–99%, as determined by FACSCalibur using CellQuest software (BD Biosciences).

Isolation of Th cell subsets secreting IL-10 or IFN-{gamma} or both

Human Th1 and Th2 clones, highly purified CD4+ T lymphocytes or CD4+ T lymphocytes labeled with CD45RO or CD45RA and CCR7 were stimulated with P/I, followed by detection and isolation of IL-10-secreting and IFN-{gamma}- and/or IL-10-secreting Th cell subsets using single or double cytometric cytokine secretion assays (Miltenyi Biotec) and a FACSDiva (BD Biosciences). In brief, cells were activated at 1 x 107 cells/ml. After 4 h of stimulation, cells were washed with ice-cold buffer (PBS with 0.5% BSA and 2 mM EDTA) and labeled with IL-10- or IFN-{gamma}-specific or premixed (1:1) IFN-{gamma}- and IL-10-specific capture matrix in cold medium for 5 min on ice. Subsequently, the labeled cells were diluted in prewarmed medium (≤105 cells/ml) and subjected to a 45-min cytokine secretion period at 37°C under slow continuous rotation. The cytokine secretion was stopped by filling up the tube with cold buffer and subsequent incubation on ice for 15 min. Cells were harvested and surface stained with specific detection Abs for IL-10 (allophycocyanin or PE) or IFN-{gamma} (PE) or equal amounts of specific detection Abs for IL-10 (allophycocyanin or PE) and IFN-{gamma} (PE or FITC). IL-10+ and IL-10–, or IFN-{gamma}+ and IFN-{gamma}–, or IFN-{gamma}+IL-10–, IFN-{gamma}+IL-10+, IFN-{gamma}–IL-10+, and IFN-{gamma}–IL-10– Th cell subsets were purified (>95%) by cell sorting.

Cytometric bead array (CBA) assay

Supernatants from 48- or 72-h cultures of P/I- stimulated Th cell subpopulations were analyzed using the human Th1/Th2 cytokine CBA kit (BD Biosciences), which allows the simultaneous detection and quantification of soluble IL-2, IL-4, IL-5, IL-10, TNF-{alpha}, and IFN-{gamma} in a single sample. In brief, a mixture of 10 µl of each of the six different bead suspensions specific for each cytokine (resolved in FL3 channel) was incubated with 50 µl of sample and 50 µl of PE-conjugated detection Ab (resolved in FL2 channel) for 3 h. Following acquisition of sample data by a FACSCalibur, the results were analyzed using the BD Biosciences CBA analysis software.

In vitro expansion and cytokine profiling of Th cell subsets

Sorted Th cell subsets were seeded in a 96-well plate (2 x 104 cells/well). Cytokines were added twice during the expansion. The cytokine profile was assessed at day 7, after 6 h of P/I stimulation (the last 4 h with brefeldin A) and by intracellular cytokine staining for IFN-{gamma} and/or IL-10 and FACS analysis.

Real-time quantitative PCR analysis

Total RNA from the different Th cell subsets was extracted using the Absolutely RNA Microprep Kit (Stratagene) and reverse transcribed with TaqMan reverse transcription reagents (Roche Applied Biosystems) according to the manufacturer’s recommendations. cDNA was analyzed for the expression of IL10, IFNG, GATA3, Tbet, Sp1, and ubiquitin (UBCH5B) by real-time PCR (primer sequences available upon request) using a LightCycler FastStart DNA Master SYBR GreenI Kit and a LightCycler (Roche Applied Science). Quantification of target gene expression was calibrated according to the values relative to the expression of ubiquitin gene.

Bisulphite treatment, PCR amplification, and semiquantitative DNA sequencing

Different Th cells were isolated from buffy coats of 30 healthy donors as described above. High molecular weight genomic DNA from each purified cell subset was isolated using a QIAamp DNA minikit (Qiagen) as recommended by the manufacturer. Three pools for each subset were analyzed. Each pool consisted of equal amounts of DNA from 10 age- and sex-matched (5 male and 5 female) donors. The average age of the donors are 39.1, 44.7, and 40.0 years for pools 1, 2, and 3, respectively. The DNA methylation status of pooled DNA was analyzed by direct bisulfite-modified DNA sequencing. The bisulfite treatment was performed as described earlier (40). Briefly, heat-denatured genomic DNA was embedded in 2% low-melting agarose (SeaPlaque; Biozym) and incubated for 4 h at 50°C in sodium disulfite solution (Merck) and then washed with Tris-EDTA buffer. Subsequently, PCR fragments were generated using the bisulfite-treated DNA as template. Primers were designed corresponding to the bisulfite-modified DNA sequence (primer sequences available upon request). PCR products were sequenced from both orientations with the dye terminator chemistry (Applied Biosystems V3.1.) using the PCR primers. The sequencing reaction was performed with an annealing temperature at 55°C and extension at 60°C for 24 cycles. Methylation levels were calculated using the Applied Biosystems trace files and previously described software (41, 42).

Chromatin immunoprecipitation assay (ChIP)

Sorted IL-10+ and IL-10– Th cells were fixed with 1% formaldehyde for 10 min at room temperature. The fixation was stopped with 0.125 M glycine. The chromatin was sheared to 200–1000 bp in length by sonication with five pulses of 10 s at 30% power (Bandelin). The chromatin was incubated with Ab directed against hyperacetylated histone H3 or lysine 4 trimethylated histone H3 (Upstate Biotechnology) overnight, followed by incubation with protein A-MicroBeads (Miltenyi Biotec) for 2 h. Washing steps were performed on µ columns (Miltenyi Biotec) with high salt, low salt, LiCl, and Tris-EDTA buffer sequentially. Chromatin precipitate was eluted with 1% SDS and 0.1 M NaHCO3. Cross-links were reversed by incubation at 65°C for 4 h in the presence of 0.2 M NaCl, and the DNA was purified with NucleoSpin Extract II (Macherey-Nagel). The amount of immunoprecipitated DNA was determined by real-time PCR with LightCycler (Roche Applied Science) using FASTStart SYBR Green Master (Roche Applied Science).The relative amount of DNA was calculated with 2(crossing point input – crossing point input).

Statistics

The two-tailed Welch t test was performed using GraphPad Prism 4.00. Values of p ≤ 0.05 were regarded as significant. Data are presented as mean of replicates of relative mRNA expression from the same donor.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Distinct cytokine profiles of IL-10+IFN-{gamma}–, IL-10+IFN-{gamma}+, and IL-10–IFN-{gamma}+ and IL-10–IFN-{gamma}– Th cell subsets

In our initial experiments, we observed that after both polyclonal stimulation with P/I or staphylococcal enterotoxin B and Ag-specific stimulation with CMV-derived Ags, human IL-10+ Th cells can be delineated into a subset of only IL-10-secreting cells and a subset of Tr1-like IL-10- and IFN-{gamma}-secreting cells (data not shown). To isolate ex vivo different human Th cell subsets secreting IL-10 or IFN-{gamma} or both, we used a new technology, a double cytokine secretion assay for IFN-{gamma} and IL-10. After stimulation of CD4+ T cells with P/I, IL-10+IFN-{gamma}– (1–5%), IL-10+IFN-{gamma}+ (0.6–3%), IL-10–IFN-{gamma}+ (5–15%), and IL-10–IFN-{gamma}– (80–90%), Th cell subsets were induced (Fig. 1A) and sorted to a purity exceeding 95% in all experiments (Fig. 1B). To confirm the supposed cytokine secretion pattern of the isolated Th cell subsets, purified cells were cultured for 48 or 72 h and supernatants were analyzed for various cytokines. IL-10+IFN-{gamma}– Th cells produced only IL-10 but no IFN-{gamma}, while IL-10–IFN-{gamma}+ Th cells produced only IFN-{gamma}, but no IL-10. As expected, Tr1-like IL-10+IFN-{gamma}+ Th cells secreted both cytokines, whereas IL-10–IFN-{gamma}– Th cells secreted neither (Fig. 1C).


Figure 1
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 1. Isolation and characterization of human IL-10+IFN-{gamma}–, IL-10+IFN-{gamma}+, and IL-10–IFN-{gamma}+ and IL-10–IFN-{gamma}– Th cell subsets. A, Induction and analysis of CD4+ Th cell subsets. CD4+ T cells were stimulated with P/I followed by IL-10/IFN-{gamma} secretion assay. The following Th cell subsets were identified: IL-10+IFN-{gamma}–, IL-10+IFN-{gamma}+, and IL-10–IFN-{gamma}+, and IL-10–IFN-{gamma}–. B, Purity analysis of CD4+ Th cell subsets. The induced Th cell subsets were sorted by a FACSDiva. A small fraction of each sorted subset was reanalyzed for purity on a FACSCalibur. Similar detection and purification results were obtained in 46 independent experiments. C, Distinct cytokine profiles of CD4+ Th cell subsets. Equal amounts of cells from each subset were cultured for 48 or 72 h. Supernatants were assessed for IL-2, IL-4, IL-5, IL-10, TNF-{alpha}, and IFN-{gamma} (from top to bottom) by CBA assay and results were analyzed on a FACSCalibur. Data shown are representative of five separate experiments.

 
To further phenotypically analyze ex vivo IL-10-secreting Th cells, an IL-10 secretion assay was performed with CD45RO or CD45RA and CCR7-labeled CD4+ cells. As characterized by expression of the CD45A and CD45RO isoforms and homing receptor CCR7, IL-10-secreting Th cells resembled Ag-experienced CD45RA–CCR7+ central memory Th cells (43) (Fig. 2).


Figure 2
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 2. Phenotypic analysis of primary IL-10-secreting Th cells. A, IL-10-secreting Th cells are enriched in CD45RO+ memory compartment. IL-10 secretion assay was performed with CD45RO-labeled CD4+ Th cells before P/I stimulation. B, Ex vivo IL-10-secreting Th cells resemble Ag-experienced CD45RA–CCR7+ central memory cells. IL-10 secretion assay was performed with CD45RA- and CCR7-labeled CD4+ Th cells before P/I stimulation. The data shown are representative of three independent experiments.

 
mRNA expression in human IL-10-secreting Th cell subsets

We next performed quantitative real-time PCR analysis to assess mRNA expression of IL10 and IFNG and to evaluate expression levels of transcription factors that have been associated with the regulation of IL10 and IFNG such as Sp1 (32), FoxP3 (44) and GATA3 (45, 46, 47), and Tbet (29). Abundant amounts of IL10 mRNA were obtained only in IL-10-secreting subsets, IL-10+IFN-{gamma}–, IL-10+IFN-{gamma}+ Th cells. Notably, GATA3 mRNA expression was significantly higher in IL-10+IFN-{gamma}– Th cells (p < 0.05) in comparison to IL-10+IFN-{gamma}+ Th cells. Sp1 and FoxP3 were not differentially expressed (data not shown). As expected, IFNG and higher Tbet mRNA expression levels were restricted to IFN-{gamma}-secreting Th cells, except for one sample with lower expression in the IL-10+IFN-{gamma}+ Th cells in comparison to the other four donors (Fig. 3). Together, these data demonstrate that IL10 gene expression in human Th cells is regulated at the level of transcription.


Figure 3
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 3. mRNA expression by IL-10+IFN-{gamma}–, IL-10+IFN-{gamma}+, IL-10–IFN-{gamma}+, and IL-10–IFN-{gamma}– Th cell subsets. IL10, IFNG, and transcription factors GATA3 and Tbet mRNA levels in ex vivo-differentiated CD4+ Th cell subsets from a same donor (as described in Fig. 1, A and B) were quantified by real-time PCR and normalized to human E2 ubiquitin-conjugating enzyme (UBCH5B) mRNA levels. The data shown are derived from five independent experiments.

 
Epigenetic status of the IL10 gene

We next assessed the level of DNA methylation of the entire IL10 gene by DNA methylation in CD4+ Th cell subsets purified according to IL-10 and/or IFN-{gamma} secretion. Fig. 4A illustrates a schematic map of the 18.1-kb IL10 gene locus spanning 9.1 kb upstream and 9 kb downstream of the transcriptional start site. Thirteen regions of interest (ROI) were analyzed, encompassing 88 CpGs. ROI were selected preferentially in conserved sequences (man and mouse), with priority given to known regulatory regions (promoter, HSS) and CpG density. There are no CpG islands within this locus as predicted by a cpgplot program (http://www.ebi.ac.uk/emboss/cpgplot/). Due to the low frequency of IL-10+ Th cells and to increase uniformity of all measurements, the same amounts of DNA of each Th cell subset (Fig. 1, A and B) from peripheral blood samples of 30 healthy donors were pooled and subjected to bisulfite conversion, PCR amplification, and DNA sequencing.


Figure 4
View larger version (43K):
[in this window]
[in a new window]

  		FIGURE 4. Genomic organization and quantitative DNA methylation and histone modification analyses of the human IL10 gene. A, Genomic organization of the human IL10 gene and alignment of human and mouse IL10 gene loci with DNA sequence identity >50% over at least 100 bp are shown in the histogram plot (59 ). The selected ROI are labeled below the gene locus. B, The DNA methylation status of three pooled DNA populations of each IL-10+IFN-{gamma}–, IL-10+IFN-{gamma}+, IL-10–IFN-{gamma}+ and IL-10–IFN-{gamma}– Th cell subsets and IL-10+ and IL-10– cells from Th1 and Th2 clones (C) are shown in rows. DNA methylation levels were determined by signal proportions between C and T peaks in colors shown in columns. Both peaks represent the average signal of all DNA copies generated during the PCR. Each rectangle in the grid represents a distinct CpG site on the designated ROI. ChIP assay assessing H3Ac (D) and H3K4me3 (E) at indicated ROI in ex vivo-purified IL-10+ and IL-10– Th cells. Immunoprecipitated DNA was quantified by real-time PCR and normalized to input. The data shown are from two of three independent experiments.

 
Strikingly, no methylation pattern specific for IL-10-secreting Th cells could be demonstrated as compared with IL-10-nonsecreting Th cells. Two CpGs of ROI 1, located 9.1 kb upstream of the transcriptional start site, showed low levels of methylation in both IL-10+ and IL-10– Th cells. CpGs of ROI 2 and 3 (5.5 and 5 kb upstream) and ROI 12 (3' end) were almost fully methylated in all cell subsets. CpGs of ROI 4 (2.5 kb upstream) displayed alternating patterns of methylation, without an explicit preference assignable to a certain Th cell subset. Two CpGs in ROI 5 (proximal promoter) were slightly demethylated in IL-10+ vs IL-10– Th cells. There was no similar methylation pattern observed for adjacent CpGs. In contrast to the IL10 loci conservation, the two CpGs in ROI 5 were not evolutionary conserved between man and mouse. Some CpG sites of ROI 6 (intron I), 7 (intron I and exon II), 8 (exon III), 9 (intron IV), 10 (intron IV and exon V), 11 (3' untranslated region), and 13 (3' end) showed low levels of methylation throughout all Th cell subsets analyzed (Fig. 4B). Similar results were obtained for IL-10-secreting Th cells isolated from in vitro-established human Th1 and Th2 clones (Fig. 4C). Further analysis on histone modifications by histone 3 acetylation (H3Ac; Fig. 4D) and histone 3 lysine 4 trimethylation (H3K4me3; Fig. 4E), which closely correlate with transcriptional activity (48), showed that H3Ac and H3K4me3 associated with activation of IL10 not only in ROI 5, but also in ROI 1, 2, 4, and 13 to a lesser extent. Taken together, these data indicate that limited differential demethylation of only two CpGs at the proximal promoter correlated with IL10 gene expression. However, there is no methylation pattern correlating with IL10 gene expression in any other selected ROI of the IL10 locus, regardless of whether evolutionary conserved coding or noncoding regions were analyzed.

Hypomethylation pattern of the IFNG gene promoter in IL-10+IFN-{gamma}+ and IL-10–IFN-{gamma}+ Th cell subsets

For comparison, we investigated the methylation status of the IFNG gene focusing on the promoter region in these Th cell populations. IFN-{gamma}+ Th cell subsets (IL-10+IFN-{gamma}+ and IL-10–IFN-{gamma}+ cells) were hypomethylated at the IFNG locus compared with IFN-{gamma}-nonsecreting Th cell subsets (IL-10+IFN-{gamma}– and IL-10–IFN-{gamma}– cells; Fig. 5). Particularly, CpG sites at positions –295, –186, –54, +122, and +128 relative to the start of transcription were hypomethylated specifically in IFN-{gamma}+ Th cells. Our data emphasize the strong correlation between expression of the IFNG gene and hypomethylation of its promoter.


Figure 5
View larger version (27K):
[in this window]
[in a new window]

  		FIGURE 5. Correlation between hypomethylation and IFN-{gamma} expression in the IFNG gene promoter, exon I, and intron I in human IFN-{gamma}-secreting vs nonsecreting Th cells. A, Genomic organization of the human IFNG gene and alignment of human and mouse IFNG gene loci with DNA sequence identity >50% over at least 100 bp are shown in the histogram plot. The selected ROI are labeled below the gene locus. Overlapping ROI 1, 2, and 3 in the proximal promoter, exon 1, and intron 1 are depicted below the gene locus. ROI 1 consists of CpG sites of –295 and –186; ROI 2, –186, –54 and +122; and ROI 3, –54, +122 and +128 relative to the transcriptional start site. B, Cell types tested and method of CpG methylation quantification are the same as in Fig. 4B. These hypomethylation and hypermethylation patterns were reproducible in IFN-{gamma}+ and IFN-{gamma}– Th cells isolated from at least four individual donors, respectively.

 
Limited IL-10 re-expression in ex vivo-expanded IL-10+ Th cell subsets

To determine a possible functional relevance of the lack of epigenetic memory of IL10, we analyzed the stability of IL-10 re-expression in comparison to IFN-{gamma} re-expression in ex vivo-isolated IL-10+ and IFN-{gamma}+ Th cell subsets following short-term in vitro cultures. One week after isolation and expansion under neutral conditions with rIL-7 plus rIL-15 (rIL-7/15), re-expression of IL-10 and IFN-{gamma} was assessed after restimulation with P/I. Little induction of IFN-{gamma} was observed in cultured IL-10+IFN-{gamma}– and IL-10+ Th cell subsets, most likely due to a small fraction of pre-Th1 cells (49) in response to homeostatic cytokines rIL-7/15 (Fig. 6A). Strikingly, from both sorted IL-10+IFN-{gamma}– (Fig. 6A) and IL-10+IFN-{gamma}+ (Fig. 6B) Th cells, only ~10% re-expressed IL-10. In contrast, in both IFN-{gamma}-secreting subsets, IL-10+IFN-{gamma}+ (Fig. 6B) and IL-10–IFN-{gamma}+ (Fig. 6C) Th cells, >90% of the cells maintained IFN-{gamma} expression. To address whether stable IL-10 re-expression is restricted to a special subset of Th cells, IL-10+IFN-{gamma}– Th cells were reisolated following 1 wk of culture of IL-10+IFN-{gamma}– Th cells. After 1 wk of further expansion and stimulation of these reisolated IL-10+IFN-{gamma}– cells, again only ~10% of cells re-expressed IL-10 (Fig. 6A). Thus, 99% of ex vivo-isolated human IL-10+ Th cells were unable to maintain IL-10 expression upon secondary restimulation, implicating that the immunoregulatory cytokine IL-10 is excluded from the functional cytokine memory of human memory Th cells compared with the effector cytokine IFN-{gamma}.


Figure 6
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 6. Attenuation of IL-10 re-expression. Ex vivo IL-10+IFN-{gamma}– (A), IL-10+IFN-{gamma}+ (B), and IL-10–IFN-{gamma}+ (C) Th cell subsets were sorted as described in Fig. 1. Purified Th cell subsets were expanded under neutral (rIL-7 plus rIL-15) conditions. After 1 wk, a second-round cytokine secretion assay for IL-10 and IFN-{gamma} was performed to reisolate IL-10+IFN-{gamma}– cells of first-round sorted IL-10+IFN-{gamma}– cells. In parallel, re-expression of IL-10 and IFN-{gamma} was analyzed by intracellular staining (i.c.) following 6 h of P/I restimulation for all of the subsets. Similar results were obtained in three independent experiments. D, Kinetics of IL10 and GATA3 expression. IL-10+ and IL-10– Th cell subsets were reisolated after 1 wk of culture of IL-10+ cells and subjected to real-time PCR analysis. Normalization of IL10 and GATA3 expression was the same as in Fig. 3. One of two independent experiments is shown. E, DNA methylation status of the promoter region of the IL10 gene (ROI 5) in secondary (2°) IL-10+ and IL-10– cell subsets. Method of CpG methylation quantification is the same as described in Fig. 4B. The data shown are representative of four independent experiments.

 
Transcriptional regulation of secondary IL-10-producing Th cells

To understand the molecular mechanisms underlying the inefficiency of functional IL-10 memory, we next performed quantitative real-time PCR analysis to examine mRNA expression of IL10 and GATA3 by short-term cultured cells. IL-10+ and IL-10– cells were reisolated after 1 wk of culture of IL-10+ Th cells. As expected and similar to the results observed in primary Th cell subsets (Fig. 3), IL10 mRNA expression was restricted to IL-10-secreting Th cells (Fig. 6D). Also, a higher level of GATA3 was detected only in IL-10-secreting Th cells (Fig. 6D). The kinetics of IL10 and GATA3 expression suggest that IL10 gene expression is regulated at the level of transcription and GATA-3 is important with regard to the sustained IL10 gene expression. To exclude the possibility that the lack of IL-10 re-expression could be due to reversible alterations in DNA methylation status, we compared the level of DNA methylation of ROI 3 (upstream), 5 (promoter), and 9 (intron IV) in reisolated IL-10+ vs IL-10– Th cell subsets. The detected methylation patterns of secondary IL-10+ and IL-10– cells resembled those of primary Th cell subsets (Figs. 4B and 6E, and data not shown).

5-Azacytidine not only induces IL-10 expression in IL-10– cells but also increases IL-10 re-expression in cultured IL-10+ cells

We next investigated whether IL10 gene expression can be in principle regulated by DNA methylation. To this end, 5-azacytidine was applied to IL-10+ and IL-10– and IFN-{gamma}+ and IFN-{gamma}– Th cell cultures. 5-Azacytidine is an analog of cytidine that inhibits DNA methyltransferases (Dnmts) during replication, thereby changing DNA methylation status and the expression of genes silenced by methylation (50). 5-Azacytidine treatment led to not only a 4.5-fold induction of IL-10 production by primary IL-10– cells, but also a 2.5-fold increase of IL-10 re-expression in primary IL-10+ cells, compared with cells cultured in the absence of 5-azacytidine, respectively (Fig. 7). In agreement with previous finding (51), 5-azacytidine treatment resulted in a 4-fold induction of IFN-{gamma} production in primary IFN-{gamma}– cells. As expected, this treatment did not increase the IFN-{gamma} production in primary IFN-{gamma}+ cells (Fig. 7). These data suggest that like IFNG gene expression can be regulated by DNA methylation, IL10 gene expression can be in principle regulated by DNA methylation as well.


Figure 7
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 7. Dnmts inhibitor 5-azacytidine treatment of primary IL-10-secreting and nonsecreting Th cells vs IFN-{gamma}-secreting and nonsecreting Th cells. IL-10+ and IL-10–, IFN-{gamma}+ and IFN-{gamma}– Th cell subsets were highly purified following IL-10 and IFN-{gamma} secretion, respectively. rIL-7/15 or rIL-7/15 plus 5-azacytidine were applied to cultures at days 0 and 2. At day 7, re-expression of cytokine was assessed by P/I stimulation for 6 h in the presence of brefeldin A for the last 4 h. The data shown are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Epigenetic modification of effector cytokine genes such as Ifng/IFNG and Il4 have been demonstrated in Th cells by changes in DNA methylation, histone de/acetylation, and rearrangement of the chromatin within the nucleus. The "poised" state apparent in effector/memory Th cells is thought to allow for rapid secretion of effector cytokines when rechallenged by invading pathogens (52, 53, 54). In this study, we show that things are different for the immunoregulatory cytokine IL-10. IL-10-secreting memory Th cells isolated ex vivo did not display a specific DNA methylation pattern as compared with Th cells not secreting IL-10. In contrast, hypomethylation of the IFNG gene promoter strongly correlated with IFN-{gamma} expression in memory Th cells. In accordance with the lack of methylation memory, the majority of ex vivo-isolated IL-10-secreting Th cells lack a functional memory for IL-10 re-expression after restimulation. The unique role of IL-10 with its broad immunoregulatory functions might be a major reason for these differences in expression and regulation. So far, the regulation of IL10 gene expression, particularly in human Th cells representing a major source of IL-10 in the course of adaptive immune responses had remained poorly understood.

Unlike memory Th cell subsets producing effecter cytokines such as IFN-{gamma} and IL-4, efficient protocols for the generation of IL-10-secreting cells from naive Th cells have not been established yet or results remain controversial. Moreover, in vivo-generated human IL-10-secreting Th cells are usually rare and cannot be assessed according to characteristic surface markers. Using a new technology, we isolated human Th cell subsets secreting IL-10 or IFN-{gamma}, or both, directly ex vivo from peripheral blood of healthy donors after short-term polyclonal stimulation. To evaluate epigenetic mechanisms that underlie IL10 gene expression, we provide here the first assessments of DNA methylation status of the IL10 gene, encompassing 88 selected CpGs, and the IFNG gene promoter, encompassing 5 CpGs. Our results offer initial evidence that in contrast to the expression of the effector cytokine IFNG, the expression of immunoregulatory cytokine IL10 is not mainly regulated by DNA methylation. Concomitantly, ex vivo IL-10-secreting Th cells lack a functional cytokine memory for IL-10 re-expression after short-term in vitro expansion, as compared with the epigenetically marked IFNG in IFN-{gamma}-secreting Th cells.

Demethylation of cytosine in CpG dinucleotides in regulatory regions often correlates with other epigenetic modifications such as increased nuclease sensitivity. DNase I HSS, are believed to reflect the "open" chromatin configuration (55). With respect to the regulation of Il10 gene expression, such DNase I HSS have been described in macrophages, dendritic cells, and Th1 and Th2 clones (34, 38, 56). One HSS described as Th2 specific and constitutive coexists with DNA methylation in the promoter region (38) that is similar to our ROI 5. At the level of histone modifications, the relative hyper-H3Ac and H3K4me3 do not completely match the DNA methylation status. It seems that a slight coincidence between H3Ac and H3K4me3 and DNA methylation only exists in the ROI 5 (Fig. 4, D and E), suggesting an intricate interplay between different layers of chromatin modifications given by histone acetylation and methylation and DNA methylation that contribute to the regulation of IL10 gene expression.

Of note, GATA-3 (47) that has been implicated in the regulation of Il10 gene expression (45, 46, 57) was significantly up-regulated in IL-10+IFN-{gamma}– vs IL-10+IFN-{gamma}+ Th cells. Furthermore, GATA3 remained up-regulated in reisolated IL-10+ Th cells in comparison to IL-10– Th cells following 1-wk culture of primary IL-10+ Th cells (Fig. 6D), confirming the importance of GATA-3 with regard to IL10 gene expression. Conversely, Sp1 is ubiquitously expressed (58) and we did not find a correlation between Sp1 and IL10 gene expression (data not shown). However, we currently cannot exclude the possibility that the limited difference in DNA methylation is conferring transcriptional specificity for undefined transcription factors in human Th cells.

In accordance with the lack of epigenetic memory of IL10 by DNA methylation, human IL-10-secreting Th cells displayed a limited memory for IL-10 re-expression after short-term in vitro culture. In contrast, IFN-{gamma}-secreting Th cell subsets were characterized by a specific epigenetic memory for IFNG gene expression. However, when treated with DNA Dnmts inhibitor 5-azacytidine, IL-10 expression was significantly augmented in both ex vivo IL-10+ and IL-10– Th cells after 1 wk of culture to a similar extent as augmented IFN-{gamma} production in treated ex vivo IFN-{gamma}-nonproducers (Fig. 7). Although this shows that in principle expression of the IL10 gene can be subject to regulation by demethylation, the IL10 gene in IL-10+ Th cells directly isolated ex vivo (Fig. 4B) in in vitro-established IL-10+ Th cell clones (Fig. 4C) and in IL-10+ Th cells reisolated from in vitro cultures initiated with IL-10+ Th cells (Fig. 6E) is not imprinted by demethylation, in contrast to the IFNG gene (Fig. 5B). Apparently, the expression of the major immunoregulatory cytokine IL10 in human memory Th cells is regulated in a completely different manner compared with an effector cytokine such as IFNG. IL10 gene expression might be determined by a variable mixture of signals rather than by a fixed heritable program acquired during Th cell differentiation as seen for effector cytokines. An epigenetic memory for IL10 gene expression in memory Th cells generated in immune responses specific for exogenous Ags could be dangerous, because it could allow for an immediate and undesired suppression of an immune response.

In summary, in contrast to effector cytokines such as IFN-{gamma}, IL-10 is excluded from the functional cytokine memory of human memory Th cells. Our results indicate that cytokine expression in memory Th cells is differentially regulated for the major immunoregulatory cytokine IL-10 and the effector cytokine IFN-{gamma}, most likely to ensure efficient pathogen-specific recall immune responses.


   Acknowledgments
 
We thank Drs. J. L. Grogan, M. Bros, and J. Li for valuable comments and S. Cottrell and F. Hatam for critical reading of this manuscript. We are grateful to K. Raba, T. Kaiser, L. Reiners-Schramm, R. Cortese, and J. Dietrich for their expert technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Deutsche Forschungsgemeinschaft (TH 806/1-1, SFB421 TP B6, SFB 650 TP 11, and BMBF 312102). Back

2 J.D. and C.I. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Andreas Thiel and Dr. Jun Dong, Clinical Immunology, Deutsches Rheuma-Forschungszentrum Berlin, Charitéplatz 1, Berlin, Germany. E-mail addresses: thiel@drfz.de and dong{at}drfz.de Back

4 Abbreviations used in this paper: Tr1, T regulatory type 1 cell; P/I, PMA/ionomycin; HSS, hypersensitivity site; ChIP, chromatin immunoprecipitation; H3Ac, histone 3 acetylation; H3K4me3, histone 3 lysine 4 trimethylation; ROI, regions of interest; Dnmt, DNA methyltransferase. Back

Received for publication December 12, 2006. Accepted for publication May 16, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Bluestone, J. A., A. K. Abbas. 2003. Natural versus adaptive regulatory T cells. Nat. Rev. Immunol. 3: 253-257. [Medline]
  2. Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, M. Toda. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor {alpha}-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155: 1151-1164. [Abstract]
  3. Groux, H., A. O’Garra, M. Bigler, M. Rouleau, S. Antonenko, J. E. de Vries, M. G. Roncarolo. 1997. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389: 737-742. [Medline]
  4. Chen, Y., V. K. Kuchroo, J. Inobe, D. A. Hafler, H. L. Weiner. 1994. Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 265: 1237-1240. [Abstract/Free Full Text]
  5. Moore, K. W., R. de Waal Malefyt, R. L. Coffman, A. O’Garra. 2001. Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 19: 683-765. [Medline]
  6. uhn, R., J. Lohler, D. Rennick, K. Rajewsky, W. Muller. 1993. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75: 263-274. [Medline]
  7. Ding, W., S. Beissert, L. Deng, E. Miranda, C. Cassetty, K. Seiffert, K. L. Campton, Z. Yan, G. F. Murphy, J. A. Bluestone, R. D. Granstein. 2003. Altered cutaneous immune parameters in transgenic mice overexpressing viral IL-10 in the epidermis. J. Clin. Invest. 111: 1923-1931. [Medline]
  8. Lang, R., R. L. Rutschman, D. R. Greaves, P. J. Murray. 2002. Autocrine deactivation of macrophages in transgenic mice constitutively overexpressing IL-10 under control of the human CD68 promoter. J. Immunol. 168: 3402-3411. [Abstract/Free Full Text]
  9. Murray, P. J., L. Wang, C. Onufryk, R. I. Tepper, R. A. Young. 1997. T cell-derived IL-10 antagonizes macrophage function in mycobacterial infection. J. Immunol. 158: 315-321. [Abstract]
  10. Pinderski, L. J., M. P. Fischbein, G. Subbanagounder, M. C. Fishbein, N. Kubo, H. Cheroutre, L. K. Curtiss, J. A. Berliner, W. A. Boisvert. 2002. Overexpression of interleukin-10 by activated T lymphocytes inhibits atherosclerosis in LDL receptor-deficient mice by altering lymphocyte and macrophage phenotypes. Circ. Res. 90: 1064-1071. [Abstract/Free Full Text]
  11. Asseman, C., S. Mauze, M. W. Leach, R. L. Coffman, F. Powrie. 1999. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J. Exp. Med. 190: 995-1004. [Abstract/Free Full Text]
  12. Barrat, F. J., D. J. Cua, A. Boonstra, D. F. Richards, C. Crain, H. F. Savelkoul, R. de Waal-Malefyt, R. L. Coffman, C. M. Hawrylowicz, A. O’Garra. 2002. In vitro generation of interleukin 10-producing regulatory CD4+ T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2-inducing cytokines. J. Exp. Med. 195: 603-616. [Abstract/Free Full Text]
  13. Jonuleit, H., E. Schmitt, G. Schuler, J. Knop, A. H. Enk. 2000. Induction of interleukin 10-producing, nonproliferating CD4+ T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J. Exp. Med. 192: 1213-1222. [Abstract/Free Full Text]
  14. Kemper, C., A. C. Chan, J. M. Green, K. A. Brett, K. M. Murphy, J. P. Atkinson. 2003. Activation of human CD4+ cells with CD3 and CD46 induces a T-regulatory cell 1 phenotype. Nature 421: 388-392. [Medline]
  15. Belkaid, Y., C. A. Piccirillo, S. Mendez, E. M. Shevach, D. L. Sacks. 2002. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420: 502-507. [Medline]
  16. Hara, M., C. I. Kingsley, M. Niimi, S. Read, S. E. Turvey, A. R. Bushell, P. J. Morris, F. Powrie, K. J. Wood. 2001. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. J. Immunol. 166: 3789-3796. [Abstract/Free Full Text]
  17. de la Rosa, M., S. Rutz, H. Dorninger, A. Scheffold. 2004. Interleukin-2 is essential for CD4+CD25+ regulatory T cell function. Eur. J. Immunol. 34: 2480[Medline]
  18. Lohning, M., A. Richter, A. Radbruch. 2002. Cytokine memory of T helper lymphocytes. Adv. Immunol. 80: 115-181. [Medline]
  19. Grogan, J. L., M. Mohrs, B. Harmon, D. A. Lacy, J. W. Sedat, R. M. Locksley. 2001. Early transcription and silencing of cytokine genes underlie polarization of T helper cell subsets. Immunity 14: 205-215. [Medline]
  20. Jaenisch, R., A. Bird. 2003. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 33: ((Suppl.)):245[Medline]
  21. Reiner, S. L.. 2005. Epigenetic control in the immune response. Hum. Mol. Genet. 14: R41-R46. [Abstract/Free Full Text]
  22. Wilson, C. B., K. W. Makar, M. Shnyreva, D. R. Fitzpatrick. 2005. DNA methylation and the expanding epigenetics of T cell lineage commitment. Semin. Immunol. 17: 105-119. [Medline]
  23. Agarwal, S., A. Rao. 1998. Modulation of chromatin structure regulates cytokine gene expression during T cell differentiation. Immunity 9: 765-775. [Medline]
  24. Avni, O., D. Lee, F. Macian, S. J. Szabo, L. H. Glimcher, A. Rao. 2002. Th cell differentiation is accompanied by dynamic changes in histone acetylation of cytokine genes. Nat. Immunol. 3: 643-651. [Medline]
  25. Fields, P. E., G. R. Lee, S. T. Kim, V. V. Bartsevich, R. A. Flavell. 2004. Th2-specific chromatin remodeling and enhancer activity in the Th2 cytokine locus control region. Immunity 21: 865-876. [Medline]
  26. Grogan, J. L., Z. E. Wang, S. Stanley, B. Harmon, G. G. Loots, E. M. Rubin, R. M. Locksley. 2003. Basal chromatin modification at the IL-4 gene in helper T cells. J. Immunol. 171: 6672-6679. [Abstract/Free Full Text]
  27. Lee, D. U., O. Avni, L. Chen, A. Rao. 2004. A distal enhancer in the interferon-{gamma} (IFN-{gamma}) locus revealed by genome sequence comparison. J. Biol. Chem. 279: 4802-4810. [Abstract/Free Full Text]
  28. Martins, G. A., A. S. Hutchins, S. L. Reiner. 2005. Transcriptional activators of helper T cell fate are required for establishment but not maintenance of signature cytokine expression. J. Immunol. 175: 5981-5985. [Abstract/Free Full Text]
  29. Szabo, S. J., S. T. Kim, G. L. Costa, X. Zhang, C. G. Fathman, L. H. Glimcher. 2000. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100: 655-669. [Medline]
  30. Young, H. A., P. Ghosh, J. Ye, J. Lederer, A. Lichtman, J. R. Gerard, L. Penix, C. B. Wilson, A. J. Melvin, M. E. McGurn, et al 1994. Differentiation of the T helper phenotypes by analysis of the methylation state of the IFN-{gamma} gene. J. Immunol. 153: 3603-3610. [Abstract]
  31. Ziegler-Heitbrock, L., M. Lotzerich, A. Schaefer, T. Werner, M. Frankenberger, E. Benkhart. 2003. IFN-{alpha} induces the human IL-10 gene by recruiting both IFN regulatory factor 1 and Stat3. J. Immunol. 171: 285-290. [Abstract/Free Full Text]
  32. Brightbill, H. D., S. E. Plevy, R. L. Modlin, S. T. Smale. 2000. A prominent role for Sp1 during lipopolysaccharide-mediated induction of the IL-10 promoter in macrophages. J. Immunol. 164: 1940-1951. [Abstract/Free Full Text]
  33. one, M., M. J. Powell, Y. Tone, S. A. Thompson, H. Waldmann. 2000. IL-10 gene expression is controlled by the transcription factors Sp1 and Sp3. J. Immunol. 165: 286-291. [Abstract/Free Full Text]
  34. Saraiva, M., J. R. Christensen, A. V. Tsytsykova, A. E. Goldfeld, S. C. Ley, D. Kioussis, A. O’Garra. 2005. Identification of a macrophage-specific chromatin signature in the IL-10 locus. J. Immunol. 175: 1041-1046. [Abstract/Free Full Text]
  35. Kitani, A., I. Fuss, K. Nakamura, F. Kumaki, T. Usui, W. Strober. 2003. Transforming growth factor (TGF)-beta1-producing regulatory T cells induce Smad-mediated interleukin 10 secretion that facilitates coordinated immunoregulatory activity and amelioration of TGF-beta1-mediated fibrosis. J. Exp. Med. 198: 1179-1188. [Abstract/Free Full Text]
  36. Cao, S., J. Liu, L. Song, X. Ma. 2005. The protooncogene c-Maf is an essential transcription factor for IL-10 gene expression in macrophages. J. Immunol. 174: 3484-3492. [Abstract/Free Full Text]
  37. Wang, Z. Y., H. Sato, S. Kusam, S. Sehra, L. M. Toney, A. L. Dent. 2005. Regulation of IL-10 gene expression in Th2 cells by Jun proteins. J. Immunol. 174: 2098-2105. [Abstract/Free Full Text]
  38. Im, S. H., A. Hueber, S. Monticelli, K. H. Kang, A. Rao. 2004. Chromatin-level regulation of the IL10 gene in T cells. J. Biol. Chem. 279: 46818-46825. [Abstract/Free Full Text]
  39. Jones, E. A., R. A. Flavell. 2005. Distal enhancer elements transcribe intergenic RNA in the IL-10 family gene cluster. J. Immunol. 175: 7437-7446. [Abstract/Free Full Text]
  40. Olek, A., J. Oswald, J. Walter. 1996. A modified and improved method for bisulphite based cytosine methylation analysis. Nucleic Acids Res. 24: 5064-5066. [Abstract/Free Full Text]
  41. Lewin, J., A. O. Schmitt, P. Adorjan, T. Hildmann, C. Piepenbrock. 2004. Quantitative DNA methylation analysis based on four-dye trace data from direct sequencing of PCR amplificates. Bioinformatics 20: 3005-3012. [Abstract/Free Full Text]
  42. Rakyan, V. K., T. Hildmann, K. L. Novik, J. Lewin, J. Tost, A. V. Cox, T. D. Andrews, K. L. Howe, T. Otto, A. Olek, et al 2004. DNA methylation profiling of the human major histocompatibility complex: a pilot study for the human epigenome project. PLoS Biol. 2: E405[Medline]
  43. Sallusto, F., D. Lenig, R. Forster, M. Lipp, A. Lanzavecchia. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401: 708-712. [Medline]
  44. Hori, S., T. Nomura, S. Sakaguchi. 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science 299: 1057-1061. [Abstract/Free Full Text]
  45. Pai, S. Y., M. L. Truitt, I. C. Ho. 2004. GATA-3 deficiency abrogates the development and maintenance of T helper type 2 cells. Proc. Natl. Acad. Sci. USA 101: 1993-1998. [Abstract/Free Full Text]
  46. Shoemaker, J., M. Saraiva, A. O’Garra. 2006. GATA-3 directly remodels the IL-10 locus independently of IL-4 in CD4+ T cells. J. Immunol. 176: 3470-3479. [Abstract/Free Full Text]
  47. Zheng, W., R. A. Flavell. 1997. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89: 587-596. [Medline]
  48. Shahbazian, M. D., M. Grunstein. 2007. Functions of site-specific histone acetylation and deacetylation. Annu. Rev. Biochem. 76: 26.1-26.26.
  49. Rivino, L., M. Messi, D. Jarrossay, A. Lanzavecchia, F. Sallusto, J. Geginat. 2004. Chemokine receptor expression identifies pre-T helper (Th)1, pre-Th2, and nonpolarized cells among human CD4+ central memory T cells. J. Exp. Med. 200: 725-735. [Abstract/Free Full Text]
  50. Christman, J. K.. 2002. 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 21: 5483-5495. [Medline]
  51. Fitzpatrick, D. R., K. M. Shirley, L. E. McDonald, H. Bielefeldt-Ohmann, G. F. Kay, A. Kelso. 1998. Distinct methylation of the interferon {gamma} (IFN-{gamma}) and interleukin 3 (IL-3) genes in newly activated primary CD8+ T lymphocytes: regional IFN-{gamma} promoter demethylation and mRNA expression are heritable in CD44highCD8+ T cells. J. Exp. Med. 188: 103-117. [Abstract/Free Full Text]
  52. Ansel, K. M., I. Djuretic, B. Tanasa, A. Rao. 2006. Regulation of Th2 differentiation and Il4 locus accessibility. Annu. Rev. Immunol. 24: 607-656. [Medline]
  53. Grogan, J. L., R. M. Locksley. 2002. T helper cell differentiation: on again, off again. Curr. Opin. Immunol. 14: 366-372. [Medline]
  54. Lee, G. R., S. T. Kim, C. G. Spilianakis, P. E. Fields, R. A. Flavell. 2006. T helper cell differentiation: regulation by cis elements and epigenetics. Immunity 24: 369-379. [Medline]
  55. Gross, D. S., W. T. Garrard. 1988. Nuclease hypersensitive sites in chromatin. Annu. Rev. Biochem. 57: 159-197. [Medline]
  56. Kang, K. H., S. H. Im. 2005. Differential regulation of the IL-10 gene in Th1 and Th2 T cells. Ann. NY Acad. Sci. 1050: 97-107. [Medline]
  57. Chang, H. D., C. Helbig, L. Tykocinski, S. Kreher, J. Koeck, U. Niesner, A. Radbruch. 2007. Expression of IL-10 in Th memory lymphocytes is conditional on IL-12 or IL-4, unless the IL-10 gene is imprinted by GATA-3. Eur. J. Immunol. 37: 807-817. [Medline]
  58. Li, L., S. He, J. M. Sun, J. R. Davie. 2004. Gene regulation by Sp1 and Sp3. Biochem. Cell Biol. 82: 460-471. [Medline]
  59. Bray, N., I. Dubchak, L. Pachter. 2003. AVID: A global alignment program. Genome Res. 13: 97-102. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
K. K. McKinstry, T. M. Strutt, A. Buck, J. D. Curtis, J. P. Dibble, G. Huston, M. Tighe, H. Hamada, S. Sell, R. W. Dutton, et al.
IL-10 Deficiency Unleashes an Influenza-Specific Th17 Response and Enhances Survival against High-Dose Challenge
J. Immunol., June 15, 2009; 182(12): 7353 - 7363.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
K. Tsuji-Takayama, M. Suzuki, M. Yamamoto, A. Harashima, A. Okochi, T. Otani, T. Inoue, A. Sugimoto, T. Toraya, M. Takeuchi, et al.
The Production of IL-10 by Human Regulatory T Cells Is Enhanced by IL-2 through a STAT5-Responsive Intronic Enhancer in the IL-10 Locus
J. Immunol., September 15, 2008; 181(6): 3897 - 3905.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dong, J.
Right arrow Articles by Thiel, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dong, J.
Right arrow Articles by Thiel, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS