Analysis of FOXP3 DNA Methylation Patterns to Identify Functional FOXP3+ T-Cell Subpopulations

Human regulatory CD4⁺CD25⁺FOXP3⁺ T cells (Tregs) are involved in the suppression of immune responses and play important roles in the maintenance of self-tolerance and immune homeostasis. Abnormal Treg function may result in disease states of varying severity. As FOXP3-expressing Treg cells are phenotypically and functionally heterogeneous, the success of Treg therapies depends on the ability to reliably distinguish subpopulations of T cells bearing a Treg-like phenotype. Methylation of cytosines within CpG dinucleotides is an important epigenetic mechanism involved in regulation (and suppression) of gene expression. On the other hand, demethylation of regulatory DNA sequences, such as promoters and enhancers, is essential for initiation of gene transcription. This protocol shows that bisulfite sequencing (BS) distinguishes methylated and unmethylated cytosines within DNA and reveals the methylation status of individual CpGs in cells within each population, identifying functionally different FOXP3⁺ subpopulations.