Quantitative DNA methylation analysis of FOXP3 as a new method for counting regulatory T cells in peripheral blood and solid tissue.
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.
Wieczorek
Georg
G
Epiontis GmbH, Berlin,Germany.
Asemissen
Anne
A
Model
Fabian
F
Turbachova
Ivana
I
Floess
Stefan
S
Liebenberg
Volker
V
Baron
Udo
U
Stauch
Diana
D
Kotsch
Katja
K
Pratschke
Johann
J
Hamann
Alf
A
Loddenkemper
Christoph
C
Stein
Harald
H
Volk
Hans Dieter
HD
Hoffmüller
Ulrich
U
Grützkau
Andreas
A
Mustea
Alexander
A
Huehn
Jochen
J
Scheibenbogen
Carmen
C
Olek
Sven
S
D000328:Adult; D000368:Aged; D019175:DNA Methylation; D005260:Female; D051858:Forkhead Transcription Factors / Q000096:biosynthesis / Q000097:blood / Q000235:genetics*; D006801:Humans; D007376:Interleukin-2 / Q000627:therapeutic use; D008297:Male; D008875:Middle Aged; D009369:Neoplasms / Q000097:blood / Q000188:drug therapy / Q000276:immunology*; D016133:Polymerase Chain Reaction / Q000379:methods; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000276:immunology*; D014181:Transplantation Immunology / Q000235:genetics
C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D007376:Interleukin-2