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Keywords:

  • DNA methylation;
  • Epigenetic modifications;
  • Treg marker

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

The transcription factor FOXP3 is critical for development and function of regulatory T cells (Treg). Their number and functioning appears to be crucial in the prevention of autoimmunity and allergy, but also to be a negative prognostic marker for various solid tumors. Although expression of the transcription factor FOXP3 currently constitutes the best-known marker for Treg, in humans, transient expression is also observed in activated non-Treg. Extending our recent findings for the murine foxp3 locus, we observed epigenetic modification of several regions in the human FOXP3 locus exclusively occurring in Treg. Importantly, activated conventional CD4+ T cells and TGF-β-treated cells displayed no FOXP3 DNA demethylation despite expression of FOXP3, whereas subsets of Treg stable even upon extended in vitro expansion remained demethylated. To investigate whether a whole set of genes might be epigenetically imprinted in the Treg lineage, we conducted a genome-wide differential methylation hybridization analysis. Several genes were found displaying differential methylation between Treg and conventional T cells, but none beside FOXP3 turned out to be entirely specific to Treg when tested on a broad panel of cells and tissues. We conclude that FOXP3 DNA demethylation constitutes the most reliable criterion for natural Treg available at present.

Abbreviations:
Amp:

amplicon

MS-SNuPE:

methylation-sensitive single-nucleotide primer extension

TSDR:

Treg-specific demethylated region

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Regulatory T cells (Treg) play a pivotal role in the maintenance of self tolerance within the immune system 1. In healthy individuals Treg balance the required aggressive immune response against foes with the required tolerance towards self constituents or harmless antigens. Reduced numbers or decreased activity of Treg has been reported in several autoimmune diseases 28. In contrast, patients diagnosed with solid tumors often display an elevated number of Treg within the peripheral blood or an accumulation at the tumor site itself 912, and these cells might be involved in tumor progression 1317. The significance of Treg in tumor biology is additionally stressed by their prognostic value in various cancer types, e.g., breast and ovarian cancers 13, 14, 18. To further validate the role of Treg in those diseases and, ultimately, to use such information in disease diagnostics and/or outcome prediction, unique and specific markers for their identification and quantification are mandatory.

Initially, Treg were described as CD4+ T cells constitutively expressing high levels of CD25 1. While the marker CD25 was important to detect and characterize this cell type, its specificity was always known to be limited, since this molecule is induced in conventional T cells following stimulation. Major progress was achieved when expression of the forkhead box transcription factor Foxp3 was shown to be a master transcription factor critically involved in Treg development and function 19. In the murine immune system, specificity of Foxp3 expression in Treg was confirmed using transgenic animals harboring Foxp3-promoter-driven reporter genes 2022. High levels of FOXP3 expression were also found in human natural CD25highCD4+ Treg 2325. However, in the human system, FOXP3 is not expressed exclusively in Treg 26 since it has also been found in non-Treg, most notably in conventional CD4+ T cells upon activation 23, 2732. Thus, FOXP3 expression does not allow the unambiguous characterization of Treg in humans.

Since FOXP3 expression in activated conventional T cells is only transient and disappears after a number of days 29, 31, we hypothesize that constitutive expression of FOXP3 in naturally occurring human Treg requires regulation on an epigenetic level, a finding we have recently described in the murine system 33. DNA methylation constitutes a biologically and chemically stable epigenetic modification, resulting in long-term gene expression changes 3437. Thus, we here asked whether differential methylation of the FOXP3 locus might be less susceptible to short-term fluctuation of gene expression, and, therefore, be superior to determination of mRNA or protein expression for the unambiguous identification of FOXP3+ Treg. In addition, we performed a genome-wide screen to analyze the specificity of epigenetic imprints in the FOXP3 locus and to search for further cell-type-specific methylation patterns in Treg. Our analysis identified an array of differentially methylated gene regions between FOXP3+CD25highCD4+ Treg and naive CD45RA+CD25CD4+ T cells. However, only demethylation at the FOXP3 locus was found to be restricted to Treg when tested against all major peripheral blood cell types and a selection of non-blood cells. Most importantly, FOXP3 demethylation was only observed in natural Treg, but not in activated conventional T cells transiently expressing FOXP3, and was also preferentially associated with Treg displaying stable FOXP3 expression upon in vitro expansion. These data indicate that epigenetic modifications, especially in the FOXP3 locus, might serve as valuable markers for the identification of cells with stable Treg phenotype and function.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Methylation differences in the FOXP3 gene between Treg and naive T cells

Based on homology analyses and our previous findings in the murine system 33 we designed several amplicons covering candidate regions for differential DNA methylation of the human FOXP3 gene including putative regulatory elements. The DNA methylation status was tested in FACS-sorted CD25highCD45RACD4+ Treg and CD25CD45RA+CD4+ naive T cells derived from peripheral blood of male donors.

Intracellular FOXP3 staining of FACS-sorted subsets revealed specific FOXP3 expression only in CD25highCD4+ Treg (Fig. 1A), in accordance with findings from other groups 2325. After bisulfite sequencing, CpGs were found to be methylated in both cell types in the 3′ region of the gene (Amps 1 and 2 in Fig. 1B and Supporting Information Table I). Upstream of exon-1, CpGs in both cell types were demethylated up to the 5′ end of a CpG island (Amps 9 and 10). At the 5′ end of the FOXP3 gene, methylation was observed only in naive CD4+ T cells, while tending towards demethylation in Treg (Amps 10 and 11). Striking differences in the methylation pattern were observed within the gene body (Amps 3–8), in particular in a region highly conserved between mammals (Amp 5), for which we recently have proposed the name TSDR (Treg-specific demethylated region) 33.

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Figure 1. Demethylation of the FOXP3 gene in Foxp3+CD25highCD4+ Treg. (A) MACS-sorted CD4+ T cells isolated from peripheral blood were stained for CD25 and CD45RA and sorted into CD25highCD45RA Treg and CD25CD45RA+ naive T cells by FACS. Flow cytometry analysis shows staining of cells before FACS sorting (left panel) as well as the sort purity and FOXP3 expression in sorted CD25CD45RA+ naive T cells and CD25highCD45RA Treg (middle and right panels, respectively). Numbers display frequency of cells within indicated populations. Staining of cells from one representative donor is depicted. (B) Schematic overview of the FOXP3 gene and positioning of amplicons designed for methylation analysis (upper panel). The lower panel depicts merged CpG methylation rates measured by bisulfite sequencing from donors 1–3; each box represents methylation rate of a single CpG according to the color code (yellow = 0% methylation, blue = 100% methylation).

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Since FOXP3 is encoded on the X-chromosome and, in females, epigenetic random X-inactivation affects approximately 85% of X-linked genes 38, we tested whether sex-dependent effects affected the methylation status at the TSDR. This analysis was performed both by bisulfite sequencing of TSDR and by methylation-sensitive single-nucleotide primer extension (MS-SNuPE) of a single CpG motif. As expected, FOXP3 TSDR was fully methylated in naive T cells derived from both male and female donors (Fig. 2 and Supporting Information Table II). In Treg derived from the male donor, FOXP3 TSDR was fully demethylated, whereas Treg derived from the female donor displayed hemimethylation, likely reflecting epigenetic silencing of one allele of the X-chromosome. Thus, when deducting this assumed effect of X-inactivation, relative demethylation of FOXP3 TSDR measured by MS-SNuPE approximately corresponded to the percentage of FOXP3+ cells among CD25highCD4+ Treg as determined by flow cytometry.

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Figure 2. Methylation pattern of the evolutionarily conserved region in Foxp3+CD25highCD4+ Treg. Left panel: DNA methylation pattern of amplicon 5 (Amp5) covering the TSDR from donors 4 and 5. Right panel: With the same donor samples, a single CpG motif within Amp5 (red arrow) was analyzed by MS-SNuPE. For MS-SNuPE analysis blue peaks correspond to a methylated cytosine and green peaks correspond to unmethylated cytosine. Original data are shown in the electropherogram (x-axis: fragment size in bases; y-axis: signal intensities in relative light units). Numbers display the percentage of unmethylated cytosines. Two representative donors from 6 analyzed samples (n=3 female; n=3 male) are depicted.

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Search for further methylation markers for Treg

To analyze the specificity of epigenetic imprints in the FOXP3 locus and to search for further cell-type-specific methylation patterns in Treg, we performed a genome-wide differential methylation hybridization according to Huang et al. 39 as a genome-wide approach to compare the methylation status between CD25high CD45RACD4+ Treg and CD25CD45RA+CD4+ naive T cells. Methylation differences of 12 selected candidate regions were independently confirmed for 5 samples of FACS-sorted Treg and naive T cells by bisulfite sequencing (Fig. 3 and Supporting Information Table III). For all selected candidate regions, including MRIP (myosin phosphatase-Rho interacting protein), NP_001008745.1, APOBEC3H (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3H), PSL1 (presenilin-like protein 1), TNIP3 (TNFAIP3-interacting protein 3), ZBTB32 (zinc finger and BTB domain-containing protein 32), AMZ1 (archaemetzincin-1), TCF7 (transcription factor 7), NP_689963.2 (hypothetical protein FLJ23834), EZH1 (enhancer of zeste homolog 1), Amp864 (no corresponding gene locus), CAMTA1 (calmodulin-binding transcription activator 1) and FOXP3 as a positive control, differences in the methylation status between Treg and naive T cells were confirmed to be donor independent, and, accordingly, a property of the cell type. Thus, these candidate regions potentially might be used as markers for the identification and quantification of Treg.

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Figure 3. Methylation profiling of candidate genes in naive T cells and Treg. DNA methylation patterns of 14 differential methylation hybridization (DMH)-derived candidate genes were analyzed in CD25CD45RA+ naive T cells and CD25high CD45RA Treg isolated from five independent donors (6–10) by bisulfite sequencing. In the matrix, cell types and donors are arranged in the columns. Analyzed genes and their chromosomal localization are indicated at the right side. Genes are separated by red lines, with each row representing a single CpG site. CpG methylation levels are color coded according to the color scale ranging from yellow (0% methylation) to blue (100% methylation). At the bottom of the matrix the methylation profile of the FOXP3 TSDR was analyzed in the same five independent donors.

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Testing selective marker specificity

To prove the specificity of the selected markers, we sorted the major leukocyte populations, including granulocytes (CD15+), monocytes (CD14+), NK cells (CD56+), naive B cells (CD19+IgD+CD27), memory B cells (CD19+IgDCD27+/–), memory CD4+ T cells (CD3+CD4+CD45RACD27+/–), naive CD8+ T cells (CD3+CD8+CD45RA+CD27+) and memory CD8+ T cells (CD3+CD8+CD45RACD27+/–), from peripheral blood of healthy donors (Fig. 4A) and analyzed the methylation status of the candidate genes and of FOXP3 TSDR by performing bisulfite sequencing (Fig. 4B and Supporting Information Table IV). Out of 13 tested gene regions, 11 share their methylation status between two or more cell types. Therefore, they are not suitable for the unambiguous identification of Treg. Only two gene regions, i.e., FOXP3 and CAMTA1 are entirely specific to Treg. FOXP3 TSDR is fully methylated in all cell types, and completely demethylated only in Treg (Fig. 4B). The same holds true for CAMTA1, showing only weak demethylation in some CpG motifs in all memory cell populations (memory Th cell, memory CTL and memory B cell). Extending our analysis to selected tissues (adipose and skin), non-leukocyte cell populations (chondrocytes, keratinocytes, melanocytes, mesenchymal stem cells and osteoblasts) and immortalized cell lines (HCC1937 and HOSE), we demonstrated full methylation of FOXP3 TSDR for all samples analyzed, while CAMTA1 showed different degrees of methylation (Fig. 4C and Supporting Information Table V). Viewed as a whole, the methylation status of both FOXP3 TSDR and CAMTA1 gene region can be used to discriminate Treg from major leukocyte populations of the peripheral blood with demethylated FOXP3 TSDR even promising to be an exclusive Treg marker throughout all human cell types.

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Figure 4. Analysis of Treg-specific methylation markers in blood cell subtypes and non-hematopoietic tissues. (A) Summary of the purification scheme for the various peripheral blood leukocyte subtypes. (B) DNA methylation patterns of the 12 differential methylation hybridization (DMH)-derived candidate genes and FOXP3 were analyzed in indicated leukocyte subtypes sorted from pooled peripheral blood of five healthy donors. CD25highCD45RA Treg served as control. In the matrix, each column represents one cell type. Analyzed genes are indicated at the right side and separated by red lines, with each row representing a single CpG site. DNA methylation was measured by means of bisulfite sequencing. CpG methylation levels are color coded according to the color scale ranging from yellow (0% methylation) to blue (100% methylation). (C) DNA methylation patterns of FOXP3 TSDR and CAMTA1 gene region measured in various non-hematopoietic tissues. Two samples were analyzed from each: adipose tissue, chondrocytes, keratinocytes, melanocytes, osteoblasts, skin tissue. One sample of the human breast cancer cell line HCC1937, the human ovarian surface epithelial cell line HOSE and mesenchymal stem cells (MSC) were also analyzed. DNA methylation was measured and depicted as described in (B).

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Methylation status of FOXP3 TSDR in recently activated conventional FOXP3+ CD4+ T cells

Transient FOXP3 expression in the absence of regulatory function has recently been reported for activated conventional CD4+ T cells 27, 29. To determine whether demethylation of FOXP3 TSDR would occur in such transiently FOXP3 expressing CD4+ T cells, we stimulated FACS-sorted naive CD25CD45RA+CD4+ T cells for varying times followed by flow cytometry analysis of FOXP3 expression and MS-SNuPE-analysis of the methylation status of FOXP3 TSDR.

The starting population of FACS-sorted naive CD25CD45RA+CD4+ T cells in general contained less then 0.2% FOXP3+ cells. Upon stimulation, the cells uniformly up-regulated CD25 and a significant fraction expressed high levels of FOXP3 (Fig. 5A), confirming recently published data 2729, 31, 32. When we analyzed the methylation status of the FOXP3 TSDR in these activated FOXP3+ CD4+ T cells by MS-SNuPE-analysis (Fig. 5B and Supporting Information Table VI) and bisulfite sequencing (data not shown), we observed almost complete methylation of the selected CpG motif despite high FOXP3 expression. Importantly, when we analyzed total CD4+ T cells, which contained roughly 10% FOXP3+CD25highCD4+ Treg, these natural Treg were detected by MS-SNuPE-analysis of the FOXP3 TSDR (12.7% demethylated CpG motifs) (Fig. 5A and B). Similar results were observed for the methylation status of the CAMTA1 gene region, showing complete methylation of the selected CpG motif in recently activated T cells, whereas 14.0% demethylated CpG motif was measured in total CD4+ T cells (Fig. 5B and Supporting Information Table VI). Thus, analysis of the methylation status of both FOXP3 TSDR and CAMTA1 gene region allows discrimination between natural FOXP3+CD25highCD4+ Treg and conventional, non-regulatory CD4+ T cells only transiently expressing FOXP3 upon activation.

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Figure 5. Methylation status of FOXP3 TSDR and CAMTA1 gene region in activated conventional CD4+ T cells. CD4+ T cells from a male donor were sorted into naive CD25CD45RA+CD4+ T cells by FACS and stimulated in vitro for 4 days. (A) Flow cytometry analysis of FOXP3 and CD25 from MACS-sorted total CD4+ T cells (left panel), FACS-sorted naive CD25CD45RA+CD4+ T cells (middle panel) and stimulated naive CD4+ T cells (right panel) for CD25 and FOXP3 are shown. Numbers indicate the frequency of FOXP3+ cells among total CD4+ T cells and FACS-sorted naive CD4+ T cells (left and middle panel, respectively) as well as FOXP3high cells among stimulated naive CD4+ T cells (right panel). (B) MS-SNuPE analysis of a single CpG motif within FOXP3 TSDR (upper row) and CAMTA1 gene region (lower row) in total CD4+ T cells (left panel), FACS-sorted naive CD25CD45RA+CD4+ T cells (middle panel) and stimulated naive CD4+ T cells. Blue peaks correspond to a methylated cytosine and green peaks correspond to unmethylated cytosine. Original data are shown in the electropherogram (x-axis: fragment size in bases; y-axis: signal intensities in relative light units). Numbers display the percentage of unmethylated cytosines. One representative out of four independent experiments is depicted.

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Methylation status of FOXP3 TSDR in TGF-β-induced Treg

Recently, it has been reported that human naive CD4+ T cells upon stimulation in the presence of TGF-β acquire both FOXP3 expression and suppressive capacity 40. To investigate whether these culture conditions would lead to an epigenetic modification of the FOXP3 locus, we stimulated FACS-sorted naive CD25CD45RA+CD4+ T cells for various times in the presence of TGF-β and analyzed both FOXP3 expression and methylation status of FOXP3 TSDR in the stimulated cells. Stimulation of naive CD4+ T cells in the presence of TGF-β led to the induction of a significant fraction of FOXP3+ cells (Fig. 6A). The fraction of FOXP3+ cells was higher when compared to CD4+ T cells stimulated in the absence of exogenous TGF-β (Fig. 5A). Nevertheless, not even the presence of exogenous TGF-β resulted in an epigenetic modification of the FOXP3 locus, since the FOXP3 TSDR showed almost complete methylation (Fig. 6B and Supporting Information Table VII). Again, the same holds true for CAMTA1 gene region that also showed nearly complete methylation. Thus, activation of naive human CD4+ T cells in the presence of TGF-β did not lead to a permanent change of the methylation status of the FOXP3 TSDR and CAMTA1 gene region.

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Figure 6. Methylation status of FOXP3 and CAMTA1 gene regions in TGF-β-induced Treg. CD4+ T cells from the same male donor as depicted in Fig. 5 were sorted into naive CD25CD45RA+CD4+ T cells by FACS and stimulated in vitro for 4 days in the presence of TGF-β (0.5 ng/mL). (A) Flow cytometry analysis shows staining for CD25 and FOXP3. Numbers indicate frequency of FOXP3high cells. (B) MS-SNuPE analysis of a single CpG motif within FOXP3 TSDR (middle panel) and CAMTA1 gene region (right panel). Blue peaks correspond to a methylated cytosine and green peaks correspond to unmethylated cytosine. Original data are shown in the electropherogram (x-axis: fragment size in bases; y-axis: signal intensities in relative light units). Numbers display percentage of unmethylated cytosines. One representative out of five independent experiments is depicted.

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Methylation status of FOXP3 TSDR in ex vivo expanded Treg

To determine whether the demethylation of FOXP3 TSDR observed in natural Treg is influenced by repetitive stimulation, we analyzed the DNA methylation status in expanded Treg. For these experiments naive CD25highCD45RA+CD4+ natural Treg were isolated from peripheral blood of healthy donors since we have recently shown that these cells give rise to a homogeneous and highly suppressive Treg population upon in vitro expansion 41. Conventional CD25CD4+ T cells isolated from the same donors served as controls. Sorted cells were expanded for 11 days followed by a 3-day resting phase in the presence of IL-2. At this time, expanded CD25highCD45RA+CD4+ Treg still showed a high frequency of FOXP3+ cells, whereas expanded conventional CD25CD4+ T cells had already partially lost the transient FOXP3 expression (Fig. 7A and unpublished data). As expected, expanded conventional cells, when analyzed by bisulfite sequencing, displayed complete methylation of FOXP3 TSDR after this extended culture period (Fig. 7B and Supporting Information Table VIII). In contrast, expanded CD25highCD45RA+CD4+ Treg still showed complete demethylation of FOXP3 TSDR. Interestingly, cell lines derived from CD25highCD45RACD4+ Treg, which we have previously shown to contain a substantial fraction of T cells with non-Treg phenotype and function 41, displayed only incomplete demethylation of FOXP3 TSDR. Comparable results were obtained for CAMTA1 gene region (Fig. 7B). Taken together, these data confirm that not even extensive in vitro stimulation and expansion results in an epigenetic modification of the FOXP3 TSDR and CAMTA1 gene region in conventional T cells, whereas the demethylated status of both gene regions is maintained in natural Treg and identifies them in mixed cell populations.

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Figure 7. Methylation status of FOXP3 and CAMTA1 gene regions in expanded Treg. CD25CD4+ T cells, CD25high CD45RA+CD4+ Treg and CD25highCD45RACD4+ Treg were isolated from leukapheresis products from healthy volunteers and expanded in vitro by stimulation with anti-CD3 and anti-CD28 antibodies for 11 days, followed by cultivation for further 3 days without stimulation. (A) Flow cytometry analysis shows staining of in vitro expanded CD25CD4+ T cells (left panel), stimulated CD25highCD45RA+CD4+ Treg (middle panel) and stimulated CD25highCD45RACD4+ Treg (right panel) for CD25 and FOXP3. Numbers indicate frequency of FOXP3high cells. (B) Methylation pattern of the FOXP3 TSDR (left) and CAMTA1 gene region (right) in expanded T cell populations. The amplicons are subdivided by vertical lines each representing an individual CpG motif. The methylation status of each motif is color coded according to the degree of methylation at that site. The color code ranges from yellow (0% methylation) to blue (100% methylation) according to the color scale. One representative out of four independent experiments is depicted.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Lineage decisions in the differentiation of cells require stable imprinting of gene expression patterns, which then are passed to daughter cells in a heritable manner. Epigenetic modification, notably DNA demethylation, and the resulting chromatin remodeling appear to be mechanisms crucial for fixation of gene expression patterns 3437. Chromatin remodeling upon epigenetic modification is thought to determine the accessibility of genes by transcriptional activators or repressors. Methylated sequences are equivalent to silent genes and opening of the locus is linked with demethylation. We here show that the human FOXP3 gene coding for a master transcription factor of Treg is subject to epigenetic modification upon differentiation into natural Treg, a stable T cell lineage produced in the thymus 1.

The forkhead box transcription factor FOXP3 is the most accepted and widely used molecular marker for Treg identification and characterization 19. As Treg are pivotal for the induction and maintenance of post-thymic immune tolerance, they are considered to be of high relevance in various areas of medical research. The modulation of Treg cell numbers or function seems promising for the treatment of diseases associated with imbalances in immune tolerance, including autoimmune diseases and solid tumors. Currently, however, the analysis of Treg especially in humans is severely hampered by the lack of specificity of all known Treg markers including FOXP3 26.

The aim of the current approach was to exploit the alleged property of DNA demethylation to be associated only with imprinted gene expression. We conducted a methylation analysis that included both a candidate gene approach with FOXP3 as a marker known to be relevant in Treg biology as well as an unbiased genome-wide approach to identify novel epigenetic markers. Extending our previously published murine data 33, we observed not only a selective demethylation of the FOXP3 TSDR for freshly isolated Treg when compared to naive T cells, but also of additional up- and downstream regions of the FOXP3 gene, indicating that specific demethylation is not restricted to the TSDR. Further studies investigating the role and relevance of these surrounding regions are required.

In the current study, demethylation of FOXP3 TSDR turned out to be only associated with naturally occurring Treg constitutively expressing FOXP3, thereby providing an exclusive and the most specific marker for natural Treg known so far. This exclusivity – but not its superior specificity when compared to FOXP3 or CD25 expression – might be challenged when novel data indicating that FOXP3 mRNA is expressed in healthy but not in diseased mammary gland epithelial cells 42 is paralleled in the methylation status. No demethylation was observed in activated conventional CD4+ T cells or in TGF-β-induced Treg, which both displayed high levels of FOXP3 expression as had been described before 27, 29, 30, 32, 40. Thus, our current data on the epigenetic status of the FOXP3 gene provide clear evidence that opening of the locus as reflected by DNA demethylation is not a mere correlative of gene expression. Instead, both the data of the current study and the preceding results from the murine system suggest that demethylation of the TSDR is restricted to conditions of stable imprinting of lineage properties, indicating evolutionary conservation not only on a genetic, but also on an epigenetic level. As shown previously, induction of Foxp3 in the presence of TGF-β, which is associated with only partial demethylation of the TSDR, was unstable and disappeared in a large fraction of cells upon restimulation 33. Together with the finding in the human system that FOXP3 expression in activated conventional T cells is only transient and disappears after a number of days 29, 31, 32, these data suggest that demethylation of the TSDR is only associated with the development of stable Foxp3-expressing natural Treg.

Whether transient expression of FOXP3 in human T cells is associated with transient suppressive effects is currently disputed. While Walker et al. 23 and Pillai et al. 31 reported that naive CD4+ T cells not only acquired FOXP3 expression, but also transient suppressive capacity after stimulation via the TCR, others have observed FOXP3 expression without Treg development 29, 32, suggesting that FOXP3 expression does not always directly correlate with suppressive capabilities. In the study performed by Wang and colleagues 30 only part of the activated cells displayed suppressive function. Interestingly, those activated CD4+ T cells that did not acquire suppressive capacity showed only transient FOXP3 expression, whereas activated CD4+ T cells harboring suppressive activity also showed a more stable FOXP3+ phenotype and maintained high FOXP3 levels during the resting phase. It is tempting to speculate that demethylation of the FOXP3 gene would be observable only in this subfraction of cells.

The genome-wide approach carried out in the current study revealed a number of donor-independent, differentially methylated candidates when freshly isolated Treg were compared to naive T cells. However, when all candidate regions were tested against an array of sorted peripheral blood cell types, it was not surprising that the analysis yielded hardly any true cell-type identifiers. Instead, as was to be expected using only two cell types for the original screening (Treg and naive T cells), most markers discriminated only groups of cell types with methylation in one group and demethylation in the other group. The only two markers specific for Treg throughout the comparisons with all peripheral blood leukocyte populations were FOXP3 and CAMTA1. CAMTA1 is a member of a recently characterized protein family designated as calmodulin-binding transcription activators 43. It is involved in the development and progression of neuroblastoma 4446. A role for CAMTA1 in T cell development and function has not been described before.

In the light of recent findings that Treg are of prognostic value in various cancer types 1315, 17 and that their presence in tumor tissues suppresses anti-tumor immune response, as most recently shown in colorectal cancer 16, the ability to specifically detect this cell type in both peripheral blood and tumor tissue moves into the focus of developing diagnostic, prognostic and predictive cancer markers.

For all these applications precise determination of Treg numbers is a prerequisite. However, with both FOXP3 and CD25 expression being not truly specific and subjected to short-term fluctuations, DNA methylation analysis of the genes proposed in this study may provide a more specific marker for this cell type, not blurred by varying levels of T cell activation. This is despite some limitations encountered with the method, such as its inadequacy for single-cell analysis and the inability to determine the exact localization of cells within a tissue. For quantification in peripheral blood, the percentage of both FOXP3 TSDR and CAMTA1 demethylation signals directly translates into the number of natural Treg. For quantification in tissues, as recently suggested by Curiel et al. 13 for prognostic applications in tumors, only FOXP3 TSDR demethylation is suitable for the measurement, since we observed CAMTA1 demethylation also in various non-blood tissues.

In conclusion, our data suggest that genome-wide methylation analysis is a promising approach to identify novel epigenetic markers to differentiate cellular components of the immune system complementing the currently almost exclusively used surface marker system. As a more immediate consequence from this study, the option to detect “true” natural FOXP3+ Treg in the presence of activated FOXP3+ conventional T cells constitutes a significant advantage amending the measurement of FOXP3 expression. The CAMTA1 and, more so, FOXP3 TSDR methylation status shows a previously unknown specificity for human natural Treg. Thus, this system provides a new avenue to prognosis and diagnosis by exploiting Treg numbers in disease settings.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Cells, antibodies and sorting reagents

Buffy coats (DRK Blutspendedienst, Berlin, Germany) and peripheral blood samples were obtained from healthy donors after informed consent in accordance with local ethical committee approval. PBMC were separated with a Ficoll-Hypaque gradient (Sigma-Aldrich). Chondrocytes, osteoblasts, keratinocytes, melanocytes and mesenchymal stem cells (MSC) were obtained from Promocell, Cambrex, Cell Applications or Cascade Biologics. Adipose and skin tissues were obtained from Asterand and HCC1937 cells from LGC Promochem GmbH. All antibodies for cell surface staining were from BD-Pharmingen or Caltag Laboratories, and the PE anti-human FOXP3 staining set was from eBioscience. All microbeads were purchased from Miltenyi Biotec.

Cell sorting of CD25highCD4+ Treg subsets and of conventional CD25CD4+ T cells

CD4+ T cells were isolated from buffy-coat-derived PBMC using anti-CD4 microbeads and the AutoMACS magnetic separation system (Miltenyi Biotec). MACS-sorted CD4+ T cells were stained using anti-CD45RA-FITC and anti-CD25-allophycocyanin. Cells were sorted into CD25highCD45RA Treg and CD25CD45RA+ naive T cells by FACS (Aria, BD-Bioscience). The purity of the sorted population was 95–99%, as determined by flow cytometry. For the expansion of natural Treg, CD45RA+ and CD45RA CD25highCD4+ Treg were isolated from leukapheresis products as described previously 41. Conventional CD25CD4+ T cells were taken as controls.

Cell sorting of major peripheral blood leukocyte populations

Peripheral blood samples of five healthy donors (30 mL each) were pooled and fractionated by a combined MACS/FACS sorting strategy into eight different leukocyte populations: granulocytes (CD15+), monocytes (CD14+), NK cells (CD56+), naive B cells (CD19+IgD+CD27), memory B cells (CD19+IgDCD27+/–), memory CD4+ T cells (CD3+CD4+CD45RACD27+/–), naive CD8+ T cells (CD3+CD8+CD45RA+CD27+) and memory CD8+ T cells (CD3+CD8+CD45RACD27+/–). In detail, erythrocytes were lysed at 4°C with EL-buffer (Qiagen) according to manufacturer's instructions. After lysis has been completed, leukocytes were washed twice with 50 mL PBS containing 5 mM EDTA (Sigma). Leukocytes were quantified by a CASY® cell counter (Schaerfe System) and incubated with an appropriate amount of anti-CD15 microbeads. Separation was done by an automated separation system (autoMACS, Miltenyi Biotec). The fraction of CD15-depleted cells was divided in two portions and prepared for two independent four-channel high-speed FACS-sorts (DIVA, BD-Biosciences). For the first sort separating monocytes, NK cells, naive and memory B lymphocytes, cells were stained for 15 min at 10°C with an antibody cocktail containing anti-CD3-Alexa405, anti-CD56-PE-Cy7, anti-CD14-FITC, anti-CD19-APC-Cy7, anti-IgD-PE and anti-CD27-FITC mAb. For the second sort of memory CD4+ T cells and naive and memory CD8+ T cells, cells were stained with an antibody cocktail containing anti-CD3-Alexa405, anti-CD4-APC-Cy7, anti-CD8-PE-Cy7 (Caltag Laboratories), anti-CD27-PE and anti-CD45RA-FITC mAb. DAPI (diamidophenylindole, Molecular Probes) was added to discriminate dead cells during FACS analysis. Purities of cells sorted were >97% as determined by flow cytometry and viabilities were always >99%.

Flow cytometry

Cytometric analysis was performed as previously described 47 using a LSRII (BD Biosciences) and the CellQuestTM software. Dead cells were excluded by DAPI staining. Intracellular FOXP3 staining was performed with the PE anti-human Foxp3 staining set (eBioscience) according to the manufacturers instructions.

In vitro stimulation of naive CD4+ T cells

FACS-sorted naive CD4+ T cells (CD25CD45RA+) were stimulated for 3–7 days using anti-CD3/anti-CD28-coated beads (Dynal) according to the manufacturers instructions. In part of the cultures, TGF-β (R&D) was added at 0.5 ng/mL. Cell culture was performed in RPMI medium (Gibco) supplemented with 10% FCS (Sigma). On indicated time points, stimulated cells were harvested and aliquots were taken for both the analysis of FOXP3 expression by flow cytometry and for the analysis of the methylation status of the FOXP3 and CAMTA1 genes.

In vitro expansion of Treg

Isolated CD45RA+ and CD45RA CD25highCD4+ Treg as well as conventional CD25CD4+ T cells as controls were expanded in vitro as described previously 41. In brief, cells were stimulated for 11 days via anti-CD3 and anti-CD28 in the presence of high-dose IL-2, followed by a 3-day resting phase in medium with IL-2.

DNA preparation, bisulfite conversion, PCR and sequencing

Genomic DNA was isolated using the DNeasy tissue kit (Qiagen) following the protocol for cultured animal cells. Bisulfite treatment of genomic DNA was performed as previously described 48. PCR was performed in a final volume of 25 µL containing 1× PCR Buffer, 1 U Taq DNA polymerase (Qiagen), 200 µM dNTP, 12.5 pmol each of forward and reverse primers, and 7 ng bisulfite-treated genomic DNA at 95°C for 15 min, and 40 cycles of 95°C for 1 min, 55°C for 45 s and 72°C for 1 min, and a final extension step of 10 min at 72°C. PCR products were purified using ExoSAP-IT (USB Corp.) and sequenced applying the PCR primers and the ABI Big Dye Terminator v1.1-chemistry (Applied Biosystems) followed by capillary electrophoresis on an ABI 3100 genetic analyzer. AB1 files were interpreted using ESME 49.

MS-SNuPE

MS-SNuPE was performed using the ABI-Prism-Snapshot kit (Applied Biosystems). Substrates were PCR products produced from bisulfite-converted genomic DNA using primers for Amp5 and CAMTA1 (“p” and “o”). The assay utilizes internal extension primer(s) annealing immediately 5′ of the relevant nucleotide. In presence of labeled ddNTP, the primer is extended by a single nucleotide: Extension primers: FOXP3: 5′-CCCAACAAACAATACAAAAAACC-3′; CAMTA1: 5′-CTAAAAAAATCTCTAATATACAATTAATACC-3′. Capillary electrophoretic analysis was performed by using ABI 3100 Genetic Analyzer and GeneMapper software (v3.5).

Differential methylation hybridization

Discovery was performed with two samples of FACS-sorted CD25highCD45RA Treg and CD25CD45RA+ naive T cells from matched donors. In addition, one comparison was done with Treg and naive T cells sorted from a pool of five donors. Amplicon preparation for methylation analysis was performed as previously described 39. Briefly, 2 µg genomic DNA was restricted with MseI. Digests were ligated with 0.5 nmol PCR linkers H-24/H-12 (H-24, 5′-AGGCAACTGTGCTATCCGAGGGAT and H-12, 5′-TAATCCCTCGGA). DNA was then digested with the methylation-sensitive endonucleases, HpaII and BstUI. Linker-PCR was performed with digests serving as templates. The amplified products were used for fluorescent labeling and subsequent chip hybridization on a custom-made microarray and resulting data were processed to convert spot intensities into quantitative methylation signal profiles. Experiments were performed as described by Huang et al. 39 with minor modifications. Gene regions that showed cell type-specific differing spot intensities in all three matched donor comparisons were considered for further analysis. Differences between cell types were verified in 12 gene regions using bisulfite sequencing as a different methodological approach in one of the matched donor pairs.

Oligonucleotides

Sequences in Table 1 are given in 5′ to 3′ direction. Primers were used for bisulfite-specific PCR and sequence reactions. Strand specificity and orientation: primers “p” and “o” produce amplicons based on the +1 strand, “r” and “q” on the –1 strand. Primers “p” and “r” indicated forward, primers “o" and “q” denote reverse orientation.

Table 1. Primers used for bisulfite-specific PCR and sequence reactions
FOXP3 (ENSG00000049768):
Amp1:p-GTTATTTGTGGAGTTTTATGGG
o-CCCCACTACTTACCTCTCTACA
Amp2:r-AAAACCCTCCTATCTACCTCC
q-AGGGTATGTGTTTGGTTATTGT
Amp3:r-AAACCTCACTTCTTAATCCCTA
q-TTGGGATGGTTTTAAGTGTTAT
Amp4:r-AACCCTCAAACCTAACTCATAC
q-GGAGGTGATAGTAAAGAAAGGA
Amp5:p-TGTTTGGGGGTAGAGGATTT
o-TATCACCCCACCTAAACCAA
Amp6:r-AAATCCTAAAATCTCAAAACCA
q-GGTGATGATGGAGGTATGTTA
Amp7:p-TAGAGATGGTAATAGGGGGAG
o-CCAACCTCACAAAAACTAAACT
Amp8:p-GTGAGGTTGGGTTTTATATTGT
o-TATCCCTATCTCTCAACCAATC
Amp9:r-TCCTAATTCACACACCAAAATA
q-AAGGTTAAAAGGAGATTAAGAGG
Amp10:r-AATTTTACCTAATCCCCACATT
q-GGTTGTTGGTTTAGAAAGTGTT
Amp11:p-AGGAGTAGGAGATTTTATTTTGG
o-TTCAACTACCTAACCTCAACCT
PSL1 (ENSG00000005206):r-TTAACCCAAATACTCCCAAAC
q-GAGGGGTTTTAGAAGAAAAATTA
EZH1 (ENSG00000108799):p-TTTGGGATGAGATAAAGTGATT
o-TTTAACAACTCCAAACACAAAA
Amp864 (no Ensembl-ID):p-TTAAGTTGGGGAAATGTTTATT
o-AACAACCCTAAATCTACTACCCT
TCF7 (ENSG00000081059):r-TCTAACACAAAACATCCAACA
q-TAGGGTAGTGGTTTGGGAA
AMZ1 (ENSG00000174945):r-TCTTCCCAAAATAACTTTCTCT
q-TTAGAAGGAAGGAGGGTTTAGT
TNIP3 (ENSG00000050730):r-TAAAACTCACAAACCCCTAAAT
q-TGGAGAGTTGAGAATAGAATGA
ZBTB32 (ENSG00000011590):p-TTTTGAGGATGTGTTTTAGAAGT
o-CTTATCCAAAATCATAACAAATAAAC
APOBEC3H (ENSG00000100298):r-TCCCTTCATCTTTAATTTTCC
q-AGAGGGGTAGAAGGAGTATTTT
CAMTA1 (ENSG00000171735):p-TTTATGGTGGTTTTTATGAGAAT
o-AAACCTAACTCCTTCTCCTCTC
NP_689963 (ENSG00000128536):p-TAAGATGAATATAAGGTAGAGTTGGT
o-TTTCTCTCAACTACACAAAAACA
MRIP (ENSG00000133030):r-CCCACCTAATAAACCACTTAAT
q-GGGTATAGGGGATTTAGAATTG
NP001008745 (ENSG00000204934):r-CTCTTTCAATAACTCCTTCTCC
q-TAGGGTTTGTGTTTAGGTTGA

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

We thank Alexander Hellwag (Epiontis), Michael Weber (Epiontis), and Kristina Hartwig (VDI-VDE) for their support. This work was supported by the Wilhelm Sander Foundation (J.H. and A.H.), by the Deutsche Krebshilfe (H. E.), the Bundesministerium für Bildung und Forschung der Bundesrepublik Deutschland (NGFN-II, A.H.) and by the Deutsche Forschungsgemeinschaft (SFB633 and SFB650, J.H. and A.H.). Conflict of interest: U.B., G.W. and S.O. have declared a financial interest in a company whose potential product was studied in the present work.

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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2040/2007/37594_s.pdf or from the author.

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