NDLI: Quantification of Regulatory T Cells in Septic Patients by Real-Time PCR–Based Methylation Assay and Flow Cytometry

Content Provider	PubMed Central
Author	Roman, Tatura Michael, Zeschnigk Michael, Adamzik Michael, Probst-kepper Buer, Jan Kehrmann, Jan
Editor	Heimesaat, Markus M.
Copyright Year	2012
Abstract	During sepsis, a relative increase of regulatory T (Treg) cells has been reported. Its persistence is associated with lymphocyte anergy, immunoparalysis and a poor prognosis. Currently, an exact quantification of human Treg cells based on protein expression of marker molecules is ambiguous, as these molecules are expressed also by activated non-regulatory T cells. Furthermore, no firm criteria for flow cytometer gate settings exist so far. Recently, a specific DNA methylation pattern within FOXP3-TSDR has been reported that allows distinguishing Treg and non-regulatory T cells, independent of their activation status. Using this epigenetic marker, we established a single-tube real-time PCR based methylation assay (QAMA) for relative quantification of Treg cells. Validation was performed on defined ratios of methylated and unmethylated target sequence and on mixtures of Treg and non-regulatory T cells. DNA-methylation was measured in CD4+ T cells isolated from blood samples of 30 septic patients and 30 healthy subjects and compared with results of Treg cell quantification by flow cytometry based on CD4+ CD25hiCD127low measurement. In septic patients both methods showed an increased ratio of Treg cells to all CD4+ T cells. In healthy individuals, the results obtained by both methods were clearly positively correlated. However, the correlation between both methods in septic patients was only weak. We showed that quantification of Treg cells by QAMA detects CD4+ T cells with unmethylated FOXP3-TSDR, hidden in the CD25med/low fraction of flow cytometry. Given that unmethylated FOXP3-TSDR is the most specific feature of Treg cells to date, our assay precisely quantifies Treg cells, as it additionally detects those committed Treg cells, hidden in the CD25med/low fraction of CD4+ cells. Furthermore, QAMA is a reliable method, which is easier to standardize among laboratories and can thus improve reproducibility of Treg cell quantification.
Related Links	http://dx.doi.org/10.1371/journal.pone.0049962
Starting Page	49962
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	11
Volume Number	7
Language	English
Publisher	Public Library of Science
Publisher Date	2012-11-01
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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