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FOXP3 promoter demethylation reveals the committed Treg population in humans.

Janson PC, Winerdal ME, Marits P, Thörn M, Ohlsson R, Winqvist O - PLoS ONE (2008)

Bottom Line: We show that a partial demethylation occurs in long-term cultures after activation, whereas the addition of TGF-beta and/or IL-10 does not induce any additional change in methylation level.Presently, FOXP3 is used to identify Tregs in several human diseases and there are future implications for adoptive Treg transfer in immunotherapy.In these settings there is a need to distinguish true Tregs from transiently FOXP3(+) activated T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Clinical Allergy Research Unit, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

Background: Naturally occurring thymus derived regulatory T cells (Tregs) are central in the maintenance of self-tolerance. The transcription factor FOXP3 is crucial for the suppressive activity of Tregs and is considered the most specific marker for this population. However, human non regulatory T cells upregulate FOXP3 transiently upon activation which calls for other means to identify the Treg population. Since epigenetic mechanisms are involved in the establishment of stable gene expression patterns during cell differentiation, we hypothesized that the methylation profile of the FOXP3 promoter would allow the distinction of truly committed Tregs.

Methodology/principal findings: Human CD4(+)CD25(hi) Tregs displayed a demethylated FOXP3 promoter (1.4%+/-0.95% SEM methylated) in contrast to CD4(+)CD25(lo) T cells which were partially methylated (27.9%+/-7.1%). Furthermore, stimulated CD4(+)CD25(lo) T cells transiently expressed FOXP3 but remained partially methylated, suggesting promoter methylation as a mechanism for regulation of stable FOXP3 expression and Treg commitment. In addition, transient FOXP3 expressing cells exhibited suppressive abilities that correlate to the methylation status of the FOXP3 promoter. As an alternative to bisulphite sequencing, we present a restriction enzyme based screening method for the identification of committed Tregs and apply this method to evaluate the effect of various culturing conditions. We show that a partial demethylation occurs in long-term cultures after activation, whereas the addition of TGF-beta and/or IL-10 does not induce any additional change in methylation level.

Conclusions/significance: The unique FOXP3 promoter methylation profile in Tregs suggests that a demethylated pattern is a prerequisite for stable FOXP3 expression and suppressive phenotype. Presently, FOXP3 is used to identify Tregs in several human diseases and there are future implications for adoptive Treg transfer in immunotherapy. In these settings there is a need to distinguish true Tregs from transiently FOXP3(+) activated T cells. The screening method we present allows this distinction and enables the identification of cells suitable for in vitro expansions and clinical use.

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FOXP3 expression in sorted cell populations and confirmation of Treg function.(A) The sorted CD4+CD25lo, CD4+CD25hi and CD19+ cells were analyzed by flow cytometry. (B) FOXP3 expression in the sorted populations as determined by intracellular flow cytometry. Plots showing results from one representative donor out of four analyzed, except for plot showing FOXP3 expression in CD19+ cells where one single donor was analyzed (C) Sorted CD4+CD25+ cells suppress the proliferation of CD4+CD25− cells. CD4+CD25− cells were activated with CD3 and CD28 antibodies together with CD4− feeder cells and increasing numbers of CD4+CD25+ cells in triplicate samples. Proliferation was measured as incorporation of [3H]Thymidine for 18 hours, here illustrated as counts per minute (cpm) on the y-axis. The CD4+CD25+ to CD25− cell ratio is displayed on the x-axis. Squares indicate coculture of CD25− and CD25+ cells. Triangles indicate control samples with only CD4+CD25− cells. (D) FOXP3 mRNA expression of sorted cell populations. FOXP3 mRNA was measured by real-time PCR in FACS sorted CD4+CD25hi, CD4+CD25lo and CD19 cells. Data was normalized to the expression in CD4+CD25lo cells using the 2−ΔΔCt method and RPII as housekeeping gene. Data represent mean of triplicate samples from one single donor.
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pone-0001612-g001: FOXP3 expression in sorted cell populations and confirmation of Treg function.(A) The sorted CD4+CD25lo, CD4+CD25hi and CD19+ cells were analyzed by flow cytometry. (B) FOXP3 expression in the sorted populations as determined by intracellular flow cytometry. Plots showing results from one representative donor out of four analyzed, except for plot showing FOXP3 expression in CD19+ cells where one single donor was analyzed (C) Sorted CD4+CD25+ cells suppress the proliferation of CD4+CD25− cells. CD4+CD25− cells were activated with CD3 and CD28 antibodies together with CD4− feeder cells and increasing numbers of CD4+CD25+ cells in triplicate samples. Proliferation was measured as incorporation of [3H]Thymidine for 18 hours, here illustrated as counts per minute (cpm) on the y-axis. The CD4+CD25+ to CD25− cell ratio is displayed on the x-axis. Squares indicate coculture of CD25− and CD25+ cells. Triangles indicate control samples with only CD4+CD25− cells. (D) FOXP3 mRNA expression of sorted cell populations. FOXP3 mRNA was measured by real-time PCR in FACS sorted CD4+CD25hi, CD4+CD25lo and CD19 cells. Data was normalized to the expression in CD4+CD25lo cells using the 2−ΔΔCt method and RPII as housekeeping gene. Data represent mean of triplicate samples from one single donor.

Mentions: PBMC from healthy blood donors were recovered from buffy coats. Subsequently, CD4+ T cells were isolated and analyzed with respect to expression of CD4, CD25 and FOXP3 (Figure 1A–B). In agreement with previous results [30], FOXP3 expression was prominent in the CD4+CD25hi T cell population, with very few CD4+CD25lo/int cells being FOXP3+. As a control the CD19+ B cell population was found to be FOXP3− (Figure 1A–B). After cell sorting, cell populations were >95% pure as demonstrated with FACS (Figure 1A).


FOXP3 promoter demethylation reveals the committed Treg population in humans.

Janson PC, Winerdal ME, Marits P, Thörn M, Ohlsson R, Winqvist O - PLoS ONE (2008)

FOXP3 expression in sorted cell populations and confirmation of Treg function.(A) The sorted CD4+CD25lo, CD4+CD25hi and CD19+ cells were analyzed by flow cytometry. (B) FOXP3 expression in the sorted populations as determined by intracellular flow cytometry. Plots showing results from one representative donor out of four analyzed, except for plot showing FOXP3 expression in CD19+ cells where one single donor was analyzed (C) Sorted CD4+CD25+ cells suppress the proliferation of CD4+CD25− cells. CD4+CD25− cells were activated with CD3 and CD28 antibodies together with CD4− feeder cells and increasing numbers of CD4+CD25+ cells in triplicate samples. Proliferation was measured as incorporation of [3H]Thymidine for 18 hours, here illustrated as counts per minute (cpm) on the y-axis. The CD4+CD25+ to CD25− cell ratio is displayed on the x-axis. Squares indicate coculture of CD25− and CD25+ cells. Triangles indicate control samples with only CD4+CD25− cells. (D) FOXP3 mRNA expression of sorted cell populations. FOXP3 mRNA was measured by real-time PCR in FACS sorted CD4+CD25hi, CD4+CD25lo and CD19 cells. Data was normalized to the expression in CD4+CD25lo cells using the 2−ΔΔCt method and RPII as housekeeping gene. Data represent mean of triplicate samples from one single donor.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2238816&req=5

pone-0001612-g001: FOXP3 expression in sorted cell populations and confirmation of Treg function.(A) The sorted CD4+CD25lo, CD4+CD25hi and CD19+ cells were analyzed by flow cytometry. (B) FOXP3 expression in the sorted populations as determined by intracellular flow cytometry. Plots showing results from one representative donor out of four analyzed, except for plot showing FOXP3 expression in CD19+ cells where one single donor was analyzed (C) Sorted CD4+CD25+ cells suppress the proliferation of CD4+CD25− cells. CD4+CD25− cells were activated with CD3 and CD28 antibodies together with CD4− feeder cells and increasing numbers of CD4+CD25+ cells in triplicate samples. Proliferation was measured as incorporation of [3H]Thymidine for 18 hours, here illustrated as counts per minute (cpm) on the y-axis. The CD4+CD25+ to CD25− cell ratio is displayed on the x-axis. Squares indicate coculture of CD25− and CD25+ cells. Triangles indicate control samples with only CD4+CD25− cells. (D) FOXP3 mRNA expression of sorted cell populations. FOXP3 mRNA was measured by real-time PCR in FACS sorted CD4+CD25hi, CD4+CD25lo and CD19 cells. Data was normalized to the expression in CD4+CD25lo cells using the 2−ΔΔCt method and RPII as housekeeping gene. Data represent mean of triplicate samples from one single donor.
Mentions: PBMC from healthy blood donors were recovered from buffy coats. Subsequently, CD4+ T cells were isolated and analyzed with respect to expression of CD4, CD25 and FOXP3 (Figure 1A–B). In agreement with previous results [30], FOXP3 expression was prominent in the CD4+CD25hi T cell population, with very few CD4+CD25lo/int cells being FOXP3+. As a control the CD19+ B cell population was found to be FOXP3− (Figure 1A–B). After cell sorting, cell populations were >95% pure as demonstrated with FACS (Figure 1A).

Bottom Line: We show that a partial demethylation occurs in long-term cultures after activation, whereas the addition of TGF-beta and/or IL-10 does not induce any additional change in methylation level.Presently, FOXP3 is used to identify Tregs in several human diseases and there are future implications for adoptive Treg transfer in immunotherapy.In these settings there is a need to distinguish true Tregs from transiently FOXP3(+) activated T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Clinical Allergy Research Unit, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

Background: Naturally occurring thymus derived regulatory T cells (Tregs) are central in the maintenance of self-tolerance. The transcription factor FOXP3 is crucial for the suppressive activity of Tregs and is considered the most specific marker for this population. However, human non regulatory T cells upregulate FOXP3 transiently upon activation which calls for other means to identify the Treg population. Since epigenetic mechanisms are involved in the establishment of stable gene expression patterns during cell differentiation, we hypothesized that the methylation profile of the FOXP3 promoter would allow the distinction of truly committed Tregs.

Methodology/principal findings: Human CD4(+)CD25(hi) Tregs displayed a demethylated FOXP3 promoter (1.4%+/-0.95% SEM methylated) in contrast to CD4(+)CD25(lo) T cells which were partially methylated (27.9%+/-7.1%). Furthermore, stimulated CD4(+)CD25(lo) T cells transiently expressed FOXP3 but remained partially methylated, suggesting promoter methylation as a mechanism for regulation of stable FOXP3 expression and Treg commitment. In addition, transient FOXP3 expressing cells exhibited suppressive abilities that correlate to the methylation status of the FOXP3 promoter. As an alternative to bisulphite sequencing, we present a restriction enzyme based screening method for the identification of committed Tregs and apply this method to evaluate the effect of various culturing conditions. We show that a partial demethylation occurs in long-term cultures after activation, whereas the addition of TGF-beta and/or IL-10 does not induce any additional change in methylation level.

Conclusions/significance: The unique FOXP3 promoter methylation profile in Tregs suggests that a demethylated pattern is a prerequisite for stable FOXP3 expression and suppressive phenotype. Presently, FOXP3 is used to identify Tregs in several human diseases and there are future implications for adoptive Treg transfer in immunotherapy. In these settings there is a need to distinguish true Tregs from transiently FOXP3(+) activated T cells. The screening method we present allows this distinction and enables the identification of cells suitable for in vitro expansions and clinical use.

Show MeSH
Related in: MedlinePlus