Epigenetic Control of Foxp3, CAMTA1 and FUT7 Genes in Human Regulatory T Cells

Regulatory T cells (Treg) are essential for immune tolerance and play a key role in autoimmune diseases, allergy and cancer. Foxp3 (Forkhead Box Transcription factor), encoded on Xp11.23 chromosome, is critical for the development, function and lineage commitment of Tregs, and has been widely used as a Treg specific marker. However, human CD4+CD25+Foxp3+ T cells are heterogeneous and include resting and activated Tregs, as well as non-suppressive Treg-like cells. The ability to distinguish between these subtypes may be critical in the development of Treg-based cell therapies. CD15s (sialyl Lewis x) was identified as a highly specific marker of activated and most suppressive effector Treg (eTreg), and the use of both CD45RA and CD15s enables the isolation of several CD4+CD25+Foxp3+ T cell subtypes with diverse functional profiles.

Foxp3 is mainly regulated by epigenetic processes such as histone acetylation and methylation of CpG islands. Three regions on Foxp3 locus - enhancer, promoter and CNS 2 (also known as Treg Specific Demethylated Region, TSDR) contribute to Foxp3 expression via demethylation of their CpG islands and can be used as molecular markers that distinguish Treg from conventional CD4+ T lymphocytes (Tcon) and different Treg maturation stages.

In this study, we characterised the epigenetic profile of four populations of CD4+CD25+ T cells: CD45RA+ CD15s- Foxp3^low (naïve nTregs), CD45RA-15s-Foxp3^low (non-suppressive Treg-like cells), CD45RA-CD15s+Foxp3^high (eTregs) and Tcons, and CD34+ cells, all isolated from the peripheral blood of healthy male and female donors. Methylation patterns of Foxp3 locus enhancer, promoter, pre-TSDR (located upstream of TSDR) and TSDR were investigated together with those of calmodulin binding transcription activator (CAMTA 1), and fucosyltransferase 7 (FUT 7) promoters. CAMTA 1 was previously identified as a potential second molecular marker that could differentiate Treg from Tcon as this gene is encoded on chromosome 1 and is not affected by X-chromosome inactivation. FUT 7 promoter was chosen as FUT 7 mediates synthesis of CD15s expressed in both eTregs and CD34+ cells and, therefore, it was expected to be demethylated in these cell populations to allow for protein expression.

Genomic DNA, isolated from the 5 cell populations, was subjected to bisulphite conversion followed by PCR-amplification of each gene region with bisulphite DNA specific primers and cloning into pGEM-T vector. Up to 24 clones (20 sequences fully covering each amplicon) were analysed and methylation status of each CpG position within all 6 gene regions was determined. As these gene regions were amplified from the same DNA sample, the methylation differences reflect the average status of the cell population.

We demonstrate the intricate methylation patterns of FUT7 and CAMTA1 promoters and Foxp3 enhancer, promoter, pre-TSDR and TSDR for 10 male and female donors. Overall, Treg cell populations from donors of both genders displayed high demethylation levels in Foxp3 promoter and TSDR while only certain CpGs (4, 9, 11, 20-30 in Foxp3 enhancer and 1, 5, 7-9 in pre-TSDR) were more demethylated in Treg subtypes compared to Tcon. CD34+, CD45RA-CD15s- Foxp3^low and eTreg populations had high demethylation levels in CpGs 5-16 of FUT7 promoter (highest in CpGs 5, 8-10) compared to nTreg and Tcon, suggesting that this regulatory element can be used as an additional molecular marker to distinguish eTreg from nTreg and Tcon. CAMTA 1 promoter (especially CpGs 2 and 11) was more demethylated in CD34+ and Treg cell populations compared to Tcon, with CD45RA-CD15s- Foxp3^low cells displaying the highest demethylation levels. In conclusion, the findings in this study suggest that Treg-specific epigenetic changes are evident not only in several regions of Foxp3 locus, but also in CAMTA1 and FUT7 promoters. Interestingly, certain CpG positions in these regions are consistently more demethylated in Treg subtypes, which suggests their importance in epigenetic control, most probably as part of the recognition sequence for methylation-sensitive transcription factors.