10 research outputs found
Die Bedeutung regulatorischer T-Zellen für den Verlauf einer Sepsis
Regulatorische T Zellen sind an der Entstehung verschiedener Autoimmun- und Infektionskrankheiten beteiligt. Die Bedeutung dieser Zellen für die Sepsis ist jedoch noch unklar und sollte im Rahmen dieser Arbeit weitergehend analysiert werden. Hierfür wurde ein assay etabliert, der spezifische Unterschiede in der DNA-Methylierung nutzt, um stabile Treg-Zellen im Menschen und der Maus zuverlässig mit Hilfe einer methylierungsempfindlichen real-time PCR zu quantifizieren. Der Vorteil dieses assays liegt in der Unabhängigkeit vom Aktivierungsstatus der Zellen, der gerade bei Erkrankungen wie der Sepsis eine bedeutende Rolle spielt. Durch Anwendung des assays bei septischen Patienten konnte der durchflusszytometrisch bestimmte relative Anstieg von Treg-Zellen bestätigt werden. Im Mausmodell wurde die Bedeutung von Treg-Zellen differenziell für die Früh- und Spätphase der Sepsis untersucht. Hier konnte gezeigt werden, dass Treg-Zellen in der Frühphase die Hyperinflammation begrenzen und dass ein Mangel an Treg-Zellen mit einem schwereren Krankheitsverlauf und einer erhöhten Letalität einhergeht. Andererseits konnte während der Spätphase der Sepsis kein Einfluss dieser Zellen auf eine Sekundärinfektion mit P. aeruginosa festgestellt werden. Die Möglichkeit einer Depletion von Treg-Zellen während der Spätphase war jedoch in den verwendeten Mäusen nur partiell möglich, was die Aussagekraft der Versuche für die Bedeutung dieser Zellen in der Spätphase der Sepsis einschränkt.
Zusätzlich wurden in dieser Arbeit T-Zellen mit den beiden demethylierenden Substanzen 5-Aza-2-deoxycytidin (5-Aza-dC), das in der Therapie hämato-onkologischer Erkrankungen zur Anwendung kommt und Epigallocatechingallat aus dem grünen Tee kultiviert, um über eine Demethylierung der FOXP3-TSDR Treg-Zellen zu induzieren. Beide Substanzen induzierten eine globale DNA-Hypomethylierung, jedoch nur 5-Aza-dC war in der Lage, eine relevante Demethylierung der FOXP3-TSDR in humanen CD4+CD25- T-Lymphozyten herbeizuführen. Ebenso wurde nur durch 5-Aza-dC eine gesteigerte Genexpression von Treg-spezifischen Genen erreicht. Jedoch konnte auch 5-Aza-dC keine voll funktionstüchtigen Treg-Zellen induzieren, da die Zellen keine suppressiven Eigenschaften aufwiesen und neben FOXP3 auch verstärkt für Th1- und Th17-Zellen typische Master-Transkriptionsfaktoren und Zytokine exprimierten, was ihre therapeutische Anwendungen im Sinne zukünftiger T-Zell-Therapien ausschließt
Impact of 5-aza-2'-deoxycytidine and epigallocatechin-3-gallate for induction of human regulatory T cells.
The epigenetic regulation of transcription factor genes is critical for T-cell lineage specification. A specific methylation pattern within a conserved region of the lineage specifying transcription factor gene FOXP3, the Treg-specific demethylated region (TSDR), is restricted to regulatory T (Treg) cells and is required for stable expression of FOXP3 and suppressive function. We analysed the impact of hypomethylating agents 5-aza-2'-deoxycytidine and epigallocatechin-3-gallate on human CD4(+) Â CD25(-) T cells for generating demethylation within FOXP3-TSDR and inducing functional Treg cells. Gene expression, including lineage-specifying transcription factors of the major T-cell lineages and their leading cytokines, functional properties and global transcriptome changes were analysed. The FOXP3-TSDR methylation pattern was determined by using deep amplicon bisulphite sequencing. 5-aza-2'-deoxycytidine induced FOXP3-TSDR hypomethylation and expression of the Treg-cell-specific genes FOXP3 and LRRC32. Proliferation of 5-aza-2'-deoxycytidine-treated cells was reduced, but the cells did not show suppressive function. Hypomethylation was not restricted to FOXP3-TSDR and expression of master transcription factors and leading cytokines of T helper type 1 and type 17 cells were induced. Epigallocatechin-3-gallate induced global DNA hypomethylation to a lesser extent than 5-aza-2'-deoxycitidine, but no relevant hypomethylation within FOXP3-TSDR or expression of Treg-cell-specific genes. Neither of the DNA methyltransferase inhibitors induced fully functional human Treg cells. 5-aza-2'-deoxycitidine-treated cells resembled Treg cells, but they did not suppress proliferation of responder cells, which is an essential capability to be used for Treg cell transfer therapy. Using a recently developed targeted demethylation technology might be a more promising approach for the generation of functional Treg cells
Quantification of Regulatory T Cells in Septic Patients by Real-Time PCR–Based Methylation Assay and Flow Cytometry
<div><p>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 <em>FOXP3-TSDR</em> 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<sup>+</sup> 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<sup>+</sup> CD25<sup>hi</sup>CD127<sup>low</sup> measurement. In septic patients both methods showed an increased ratio of Treg cells to all CD4<sup>+</sup> 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<sup>+</sup> T cells with unmethylated <em>FOXP3-TSDR</em>, hidden in the CD25<sup>med/low</sup> fraction of flow cytometry. Given that unmethylated <em>FOXP3-TSDR</em> 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<sup>med/low</sup> fraction of CD4<sup>+</sup> cells. Furthermore, QAMA is a reliable method, which is easier to standardize among laboratories and can thus improve reproducibility of Treg cell quantification.</p> </div
Characteristics of the septic patients enrolled in the study.
<p>SAPS = Simplified Acute Physiology Score.</p
Correlation of qRT-PCR and flow cytometry.
<p>Correlation of ratio of Treg cells as quantified by <i>FOXP3-TSDR</i> QAMA qRT-PCR and as quantified by flow cytometry shows a clear positive correlation in healthy subjects (<i>r</i> = 0.60) and a weak positive correlation in septic patients (<i>r</i> = 0.37) FACS = fluorescence-activated cell sorting qRT-PCR = quantitative real-time polymerase chain reaction.</p
<i>FOXP3-TSDR</i> standard curve.
<p>The difference of both cT values (cT methylated probe – cT unmethylated probe) is determined and the methylation ratio of each sample deduced from a standard curve running along with each assay. ΔcT = difference in cycle-threshold values.</p
Illustration of the CD25 and CD4 gate settings used to sort various CD4<sup>+</sup> populations.
<p>CD4<sup>+</sup> CD25<sup>med</sup> cells showed an unmethylated ratio of 4.4% (SD ±0.17%); CD4<sup>+</sup> CD25<sup>−</sup> cells, of 0.8% (SD ±0.60%); and CD4<sup>+</sup> CD25<sup>hi</sup>CD127<sup>low</sup> cells of 99.8% (SD ±0.24%)with <i>FOXP3-TSDR</i> QAMA. Results were calculated of four experiments from four healthy men.</p
Schematic illustration of <i>FOXP3</i> Treg-specific demethylated region (<i>TSDR</i>) quantitative analysis of methylated alleles (QAMA) assay.
<p><i>FOXP3-TSDR</i>-QAMA is a single tube quantitative real-time PCR. Bisulfite-treated target sequence is amplified with a single primer set irrespective of its methylation status. Two different labelled internal MGB Taqman® probes bind specifically to the methylated or unmethylated target sequence and are cleaved by the 5′nuclease activity of <i>Taq</i> DNA polymerase. The amount of fluorescence dyes VIC and FAM released during PCR is directly proportional to the amount of PCR product generated from the methylated or unmethylated allele. FAM probe = 6-carboxyfluorescein met probe = probe specific for methylated target unmet probe = probe specific for unmethylated target VIC probe = 4,7,2′-trichloro-7′-phenyl-6-carboxyfluorescein.</p
Characteristics of healthy subjects enrolled in the study.
<p>Characteristics of healthy subjects enrolled in the study.</p
Quantification of Treg cells in septic patients and healthy individuals. A
<p>Relative quantification of Treg cells by <i>FOXP3</i> Treg-specific demethylated region (<i>TSDR</i>) real-time quantitative polymerase chain reaction (quantitative analysis of methylated alleles, QAMA) <b>B</b> Relative quantification of Treg cells (characterized as CD4<sup>+</sup> CD25<sup>hi</sup>CD127<sup>low</sup>) by flow cytometry <b>C</b> Representative quantification of one septic patient and healthy subject by flow cytometry. Setting of the CD25 and CD127 flow cytometer gates are shown.</p