Journal Article

FOXP3 Promoter Demethylation Reveals the Committed Treg Population in Humans

Janson, Peter C. J., Marits, Per, Winerdal, Malin E., Winqvist, Ola, Thörn, Magnus, Ohlsson, Rolf

2008

Overview

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. 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. 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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Antigens / B cells / Binding sites / Biologi / Biology / Blood & organ donations / CD25 antigen / CD4 antigen / Cell activation / Cell Culture Techniques / Cell differentiation / Cytokines / Dehydrogenases / Demethylation / DNA Methylation / Epigenetic inheritance / Epigenetics / Flow cytometry / Forkhead Transcription Factors - genetics / Foxp3 protein / Gene expression / Gene Expression Regulation / Genes / Genetics and Genomics/Epigenetics / Genetics and Genomics/Genetics of the Immune System / Genotype & phenotype / Humans / Immunological tolerance / Immunology/Genetics of the Immune System / Immunology/Immunomodulation / Immunoregulation / Immunotherapy / Interleukin 10 / Lymphocytes / Lymphocytes T / MEDICIN / MEDICINE / Methylation / Molecular Biology/DNA Methylation / NATURAL SCIENCES / NATURVETENSKAP / Polymerase chain reaction / Population / Promoter Regions, Genetic / Screening / Self Tolerance / T cell receptors / T cells / T-Lymphocytes, Regulatory - chemistry / T-Lymphocytes, Regulatory - cytology / T-Lymphocytes, Regulatory - immunology / Thymus / Transcription factors / Transforming growth factors