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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: 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.The screening method we present allows this distinction and enables the identification of cells suitable for in vitro expansions and clinical use.

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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Related in: MedlinePlus

Activation results in a partial demethylation at long-term follow-up.CD25 and FOXP3 expression was monitored in CD4+CD25lo cells during activation with (A) CD3/CD28 dynabeads as described in Figure 5 or repeated stimulation with anti-CD3 (5 µg/mL) and anti-CD28 (1 µg/mL) antibodies every 7 days. CD4+CD25hi Tregs were also monitored as they were stimulated continuously with CD3/CD28 dynabeads. (B) CD4+CD25lo cells stimulated with high (10 µg/mL), medium (5 µg/mL) or low (0.5 µg/mL) anti-CD3 antibody together with 1 µg/mL anti-CD28 antibody. (C) CD4+CD25lo cells stimulated with anti-CD3 (5 µg/mL) and anti-CD28 (1 µg/mL) antibodies together with TGF-β (5 ng/mL), TGF-β (5 ng/mL) and IL-10 (10 ng/mL) or IL-10 only (10 ng/mL). Stimuli were removed on day 2 after stimulation and FACS analysis performed on days 0, 2, 5, 7, 10, 14 and 21. Data represent mean values from three separate donors±SEM, except for expanded Tregs where data represent mean values from two separate donors. Methylation status of the −77 reporter position was monitored during conditions described above (D–F). Evaluation of methylation status was performed on DNA from one donor at days 0, 7, 14 and 21 for each separate population. Methylation of expanded Tregs at day 7, 12 and 16 represent mean values from two separate donors.
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pone-0001612-g007: Activation results in a partial demethylation at long-term follow-up.CD25 and FOXP3 expression was monitored in CD4+CD25lo cells during activation with (A) CD3/CD28 dynabeads as described in Figure 5 or repeated stimulation with anti-CD3 (5 µg/mL) and anti-CD28 (1 µg/mL) antibodies every 7 days. CD4+CD25hi Tregs were also monitored as they were stimulated continuously with CD3/CD28 dynabeads. (B) CD4+CD25lo cells stimulated with high (10 µg/mL), medium (5 µg/mL) or low (0.5 µg/mL) anti-CD3 antibody together with 1 µg/mL anti-CD28 antibody. (C) CD4+CD25lo cells stimulated with anti-CD3 (5 µg/mL) and anti-CD28 (1 µg/mL) antibodies together with TGF-β (5 ng/mL), TGF-β (5 ng/mL) and IL-10 (10 ng/mL) or IL-10 only (10 ng/mL). Stimuli were removed on day 2 after stimulation and FACS analysis performed on days 0, 2, 5, 7, 10, 14 and 21. Data represent mean values from three separate donors±SEM, except for expanded Tregs where data represent mean values from two separate donors. Methylation status of the −77 reporter position was monitored during conditions described above (D–F). Evaluation of methylation status was performed on DNA from one donor at days 0, 7, 14 and 21 for each separate population. Methylation of expanded Tregs at day 7, 12 and 16 represent mean values from two separate donors.

Mentions: CD4+CD25hi cells isolated from peripheral blood are CD45RO+ and CD45Rblo. They also have short telomere length consistent with memory T cells that have undergone several rounds of replication [31]. These observations together with the finding of antigen-specific Treg cells suggest that Tregs are generated as a result of time, after one or repeated antigen encounters. In order to investigate whether long-term culture after stimulus or repeated stimuli over time could affect the methylation pattern and/or FOXP3 expression in CD4+CD25lo cells we followed the expression of CD25 and FOXP3 with FACS while monitoring the methylation status with the COBRA based method (figure 7A and D). Stimulation with CD3/CD28 Dynabeads or repeated stimulations with anti-CD3 and and-CD28 antibodies induced a transient expression of CD25 and FOXP3 and a gradual demethylation, similar to what was previously observed (Figure 6A and B). It is also worth noting that the Treg population remained FOXP3hi throughout the stimulation.


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)

Activation results in a partial demethylation at long-term follow-up.CD25 and FOXP3 expression was monitored in CD4+CD25lo cells during activation with (A) CD3/CD28 dynabeads as described in Figure 5 or repeated stimulation with anti-CD3 (5 µg/mL) and anti-CD28 (1 µg/mL) antibodies every 7 days. CD4+CD25hi Tregs were also monitored as they were stimulated continuously with CD3/CD28 dynabeads. (B) CD4+CD25lo cells stimulated with high (10 µg/mL), medium (5 µg/mL) or low (0.5 µg/mL) anti-CD3 antibody together with 1 µg/mL anti-CD28 antibody. (C) CD4+CD25lo cells stimulated with anti-CD3 (5 µg/mL) and anti-CD28 (1 µg/mL) antibodies together with TGF-β (5 ng/mL), TGF-β (5 ng/mL) and IL-10 (10 ng/mL) or IL-10 only (10 ng/mL). Stimuli were removed on day 2 after stimulation and FACS analysis performed on days 0, 2, 5, 7, 10, 14 and 21. Data represent mean values from three separate donors±SEM, except for expanded Tregs where data represent mean values from two separate donors. Methylation status of the −77 reporter position was monitored during conditions described above (D–F). Evaluation of methylation status was performed on DNA from one donor at days 0, 7, 14 and 21 for each separate population. Methylation of expanded Tregs at day 7, 12 and 16 represent mean values from two separate donors.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2238816&req=5

pone-0001612-g007: Activation results in a partial demethylation at long-term follow-up.CD25 and FOXP3 expression was monitored in CD4+CD25lo cells during activation with (A) CD3/CD28 dynabeads as described in Figure 5 or repeated stimulation with anti-CD3 (5 µg/mL) and anti-CD28 (1 µg/mL) antibodies every 7 days. CD4+CD25hi Tregs were also monitored as they were stimulated continuously with CD3/CD28 dynabeads. (B) CD4+CD25lo cells stimulated with high (10 µg/mL), medium (5 µg/mL) or low (0.5 µg/mL) anti-CD3 antibody together with 1 µg/mL anti-CD28 antibody. (C) CD4+CD25lo cells stimulated with anti-CD3 (5 µg/mL) and anti-CD28 (1 µg/mL) antibodies together with TGF-β (5 ng/mL), TGF-β (5 ng/mL) and IL-10 (10 ng/mL) or IL-10 only (10 ng/mL). Stimuli were removed on day 2 after stimulation and FACS analysis performed on days 0, 2, 5, 7, 10, 14 and 21. Data represent mean values from three separate donors±SEM, except for expanded Tregs where data represent mean values from two separate donors. Methylation status of the −77 reporter position was monitored during conditions described above (D–F). Evaluation of methylation status was performed on DNA from one donor at days 0, 7, 14 and 21 for each separate population. Methylation of expanded Tregs at day 7, 12 and 16 represent mean values from two separate donors.
Mentions: CD4+CD25hi cells isolated from peripheral blood are CD45RO+ and CD45Rblo. They also have short telomere length consistent with memory T cells that have undergone several rounds of replication [31]. These observations together with the finding of antigen-specific Treg cells suggest that Tregs are generated as a result of time, after one or repeated antigen encounters. In order to investigate whether long-term culture after stimulus or repeated stimuli over time could affect the methylation pattern and/or FOXP3 expression in CD4+CD25lo cells we followed the expression of CD25 and FOXP3 with FACS while monitoring the methylation status with the COBRA based method (figure 7A and D). Stimulation with CD3/CD28 Dynabeads or repeated stimulations with anti-CD3 and and-CD28 antibodies induced a transient expression of CD25 and FOXP3 and a gradual demethylation, similar to what was previously observed (Figure 6A and B). It is also worth noting that the Treg population remained FOXP3hi throughout the stimulation.

Bottom Line: 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.The screening method we present allows this distinction and enables the identification of cells suitable for in vitro expansions and clinical use.

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