Limits...
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.

Show MeSH

Related in: MedlinePlus

Transient expression of FOXP3 and CD25 in stimulated CD4+CD25lo cells from male donors.Plots showing results from one representative donor out of four analyzed. (A) FACS analysis of CD4+CD25lo cells stimulated at day 0 with CD3/CD28 Dynabeads (proportion cell∶beads 1∶1) in presence of 180 U IL-2. Stimuli removed after 48 h and cells followed for an additional 2 weeks with regards to their expression of CD25 (left) and FOXP3 (middle). Left column demonstrating the relationship between CD25 (y-axis) and FOXP3 (x-axis) expression. (B) Expression of CD25 (grey filled bars) and FOXP3 (open bars) in stimulated cells as described above, displayed as mean fluorescent intensity (MFI).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2238816&req=5

pone-0001612-g005: Transient expression of FOXP3 and CD25 in stimulated CD4+CD25lo cells from male donors.Plots showing results from one representative donor out of four analyzed. (A) FACS analysis of CD4+CD25lo cells stimulated at day 0 with CD3/CD28 Dynabeads (proportion cell∶beads 1∶1) in presence of 180 U IL-2. Stimuli removed after 48 h and cells followed for an additional 2 weeks with regards to their expression of CD25 (left) and FOXP3 (middle). Left column demonstrating the relationship between CD25 (y-axis) and FOXP3 (x-axis) expression. (B) Expression of CD25 (grey filled bars) and FOXP3 (open bars) in stimulated cells as described above, displayed as mean fluorescent intensity (MFI).

Mentions: Previously, activated CD4+CD25lo cells have been shown to transiently upregulate FOXP3 [13], [14]. Therefore, we stimulated CD4+CD25lo T cells with anti-CD3/CD28 Dynabeads for 48 hours and monitored the expression of FOXP3 and CD25 up to day 16 of culture (Figure 5A). Mean fluorescent intensity (MFI) for FOXP3 and CD25 expression at each time point was calculated (Figure 5B). CD25 showed a faster response to TCR stimulation in comparison to FOXP3. Maximum MFI of FOXP3 was recorded day 5 of culture after which it decreased. However, the cells still displayed some residual FOXP3 expression at day 16. These results confirm that FOXP3 expression occurs in CD4+CD25lo T cells upon stimulation, and thus is not solely limited to the Treg population in humans.


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)

Transient expression of FOXP3 and CD25 in stimulated CD4+CD25lo cells from male donors.Plots showing results from one representative donor out of four analyzed. (A) FACS analysis of CD4+CD25lo cells stimulated at day 0 with CD3/CD28 Dynabeads (proportion cell∶beads 1∶1) in presence of 180 U IL-2. Stimuli removed after 48 h and cells followed for an additional 2 weeks with regards to their expression of CD25 (left) and FOXP3 (middle). Left column demonstrating the relationship between CD25 (y-axis) and FOXP3 (x-axis) expression. (B) Expression of CD25 (grey filled bars) and FOXP3 (open bars) in stimulated cells as described above, displayed as mean fluorescent intensity (MFI).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001612-g005: Transient expression of FOXP3 and CD25 in stimulated CD4+CD25lo cells from male donors.Plots showing results from one representative donor out of four analyzed. (A) FACS analysis of CD4+CD25lo cells stimulated at day 0 with CD3/CD28 Dynabeads (proportion cell∶beads 1∶1) in presence of 180 U IL-2. Stimuli removed after 48 h and cells followed for an additional 2 weeks with regards to their expression of CD25 (left) and FOXP3 (middle). Left column demonstrating the relationship between CD25 (y-axis) and FOXP3 (x-axis) expression. (B) Expression of CD25 (grey filled bars) and FOXP3 (open bars) in stimulated cells as described above, displayed as mean fluorescent intensity (MFI).
Mentions: Previously, activated CD4+CD25lo cells have been shown to transiently upregulate FOXP3 [13], [14]. Therefore, we stimulated CD4+CD25lo T cells with anti-CD3/CD28 Dynabeads for 48 hours and monitored the expression of FOXP3 and CD25 up to day 16 of culture (Figure 5A). Mean fluorescent intensity (MFI) for FOXP3 and CD25 expression at each time point was calculated (Figure 5B). CD25 showed a faster response to TCR stimulation in comparison to FOXP3. Maximum MFI of FOXP3 was recorded day 5 of culture after which it decreased. However, the cells still displayed some residual FOXP3 expression at day 16. These results confirm that FOXP3 expression occurs in CD4+CD25lo T cells upon stimulation, and thus is not solely limited to the Treg population in humans.

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