Quantitative DNA Methylation Analysis of FOXP3 as a New Method for Counting Regulatory T Cells in Peripheral Blood and Solid Tissue

Abstract

Regulatory T-cells (Treg) have been the focus of immunologic research due to their role in establishing tolerance for harmless antigens versus allowing immune responses against foes. Increased Treg frequencies measured by mRNA expression or protein synthesis of the Treg marker FOXP3 were found in various cancers, indicating that dysregulation of Treg levels contributes to tumor establishment. Furthermore, they constitute a key target of immunomodulatory therapies in cancer as well as transplantation settings. One core obstacle for understanding the role of Treg, thus far, is the inability of FOXP3 mRNA or protein detection methods to differentiate between Treg and activated T cells. These difficulties are aggravated by the technical demands of sample logistics and processing. Based on Treg-specific DNA demethylation within the FOXP3 locus, we present a novel method for monitoring Treg in human peripheral blood and solid tissues. We found that Treg numbers are significantly increased in the peripheral blood of patients with interleukin 2-treated melanoma and in formalin-fixed tissue from patients with lung and colon carcinomas. Conversely, we show that immunosuppressive therapy including therapeutic antibodies leads to a significant reduction of Treg from the peripheral blood of transplantation patients. In addition, Treg numbers are predictively elevated in the peripheral blood of patients with various solid tumors. Although our data generally correspond to data obtained with gene expression and protein-based methods, the results are less fluctuating and more specific to Treg. The assay presented here measures Treg robustly in blood and solid tissues regardless of conservation levels, promising fast screening of Treg in various clinical settings. [Cancer Res 2009;69(2):599-608]

In Vivo Maintenance of Human Regulatory T Cells during CD25 Blockade

“…Given the importance of IL-2 in driving FOXP3 expression and the modest decrease we observed in FOXP3 expression in Tregs during DAC HYP therapy, we sought to determine whether CD25 blockade effected TSDR methylation status. To perform this analysis, we measured epigenetically committed, stable Tregs using a quantitative PCR assay that enumerates Tregs based on the presence of a demethylated TSDR, termed TSDR + Tregs (25,33). In addition, total cells and CD3 + T cells were simultaneously quantified in the sample using epigenetic analysis of the GAPDH and CD3D/CD3G loci, respectively, and TSDR + Tregs were calculated as a percentage of total PBMCs or as a percentage of total CD3 + T cells.…”

Human FOXP3 and cancer

“…Recently, the group of Sakaguchi (Miyara et al, 2009) used CD45RA, a marker of naive T cells nonexpressed by activated and memory T cells, for the characterization and isolation of three types of circulating human CD4 þ T cells expressing Foxp3: CD45RA þ Foxp3 low resting Treg and CD45RA -Foxp3 high activated Treg, which were both suppressive in vitro, and CD45RA -Foxp3 low nonsuppressive T cells that were effector cells secreting high amounts of IL-2 and interferong (IFNg). Demethylation at a highly conserved region within the intronic enhancer of the FOXP3 gene was found to be a more valuable criterion than the expression of FOXP3 protein itself for discriminating Treg from activated T cells, notably in cancer patients (Wieczorek et al, 2009).…”

Allogeneic gene-modified tumor cells (RCC-26/IL-7/CD80) as a vaccine in patients with metastatic renal cell cancer: a clinical phase-I study

“…A minimum of 60 ng bisulfite-treated genomic DNA was used in a Realtime-PCR to quantify the FOXP3 TSDR as previously described. 51 Realtime-PCR was performed in a final reaction volume of 20 ml containing 10 ml FastStart Universal Probe Master (ROX) (Roche Diagnostics, Mannheim, Germany), 50 ng per ml Lamda DNA (New England Biolabs, Frankfurt, Germany), 5 pmol per ml methylation or non-methylation specific probe, 30 pmol per ml methylation-or non-methylation-specific primers and 60 ng bisulfite-treated DNA or respective amounts of plasmid standard. Primers and standards were provided by Epiontis GmbH (Berlin, Germany).…”

“…As PBMC samples were used, the obtained results were adjusted to the individual percentage of CD4 + T cells determined by flow cytometry. 51 The classification of changes in the percentage of TSDR+ CD4-T cells (increase vs decrease vs unchanged) is based on a comparison of the respective values pre-and post-vaccination as these parameters reflect the outcome of the whole vaccination period.…”