title: Quantification of regulatory T cells in septic patients by real-time PCR-based methylation assay and flow cytometry.
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(+) CD25(hi)CD127(low) 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 CD25(med/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 CD25(med/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.
authors.affiliation: Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.
authors.forename: Roman
authors.initials: R
authors.lastname: Tatura
authors.forename: Michael
authors.initials: M
authors.lastname: Zeschnigk
authors.forename: Michael
authors.initials: M
authors.lastname: Adamzik
authors.forename: Michael
authors.initials: M
authors.lastname: Probst-Kepper
authors.forename: Jan
authors.initials: J
authors.lastname: Buer
authors.forename: Jan
authors.initials: J
authors.lastname: Kehrmann
mesh_terms: D000328:Adult; D000368:Aged; D015704:CD4 Antigens / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D016022:Case-Control Studies; D019175:DNA Methylation; D005260:Female; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism; D053699:Interleukin-7 Receptor alpha Subunit / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction; D018805:Sepsis / Q000235:genetics* / Q000276:immunology*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology* / Q000378:metabolism*; D055815:Young Adult
chemical_list: D015704:CD4 Antigens; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D053645:Interleukin-2 Receptor alpha Subunit; D053699:Interleukin-7 Receptor alpha Subunit