IFNγ+ Treg in-vivo and in-vitro represent both activated nTreg and peripherally induced aTreg and remain phenotypically stable in-vitro after removal of the stimulus.
BACKGROUND
IFNγ-producing CD4+CD25+Foxp3+CD127- Treg represent the first line of Treg during an immune response. In the present study we determined whether IFNγ+ Treg in-vivo and in-vitro are Helios-positive representing activated natural (nTreg) or Helios-negative representing adaptive Treg (aTreg) and whether they originate from CD4+CD25+ and/or CD4+CD25- PBL. Furtheron, we investigated whether they are inducible by recombinant IFNγ (rIFNγ) as a single stimulus, decrease in-vitro after elimination of the stimulus, and have a demethylated Foxp3 Treg-specific demethylated region (TSDR) which is associated with stable Foxp3 expression.


METHODS
Subsets of IFNγ+ Treg were determined in peripheral blood of healthy controls using eight-color flow cytometry and were further investigated in-vitro. Foxp3 TSDR methylation status was determined using bisulphite polymerase chain reaction (PCR) and high resolution melt (HRM) analysis.


RESULTS
Nearly all Treg in the peripheral blood were Helios+IFNγ- (1.9 ± 1.1/μl) and only few were Helios+IFNγ+ or Helios-IFNγ+ Treg (both 0.1 ± 0.1/μl). Enriched IFNγ+ Treg subsets showed in part strong Foxp3 TSDR demethylation. In-vitro, rIFNγ was unable to induce Treg. CD4+CD25+ enriched PBL stimulated with PMA/Ionomycin in the presence of rIFNγ were rather resistant to the effect of rIFNγ, in contrast to CD4+CD25- enriched PBL which showed increasing total Treg with Helios+ Treg switching from IFNγ- to IFNγ+ and increasing Helios-IFNγ+ Treg. The data indicate that rIFNγ, in combination with a polyclonal stimulus, activates nTreg and induces aTreg. When phorbol 12-myristate 13-acetate (PMA)/Ionomycin was washed out from the cell culture after 6 h stimulation, Treg induction continued for at least 96 h of cell culture, contradicting the hypothesis that removal of the stimulus results in significant decrease of IFNγ- and IFNγ+ CD4+CD25+Foxp3+CD127- Treg due to loss of Foxp3 expression.


CONCLUSIONS
IFNγ+Helios- aTreg as well as IFNγ+Helios+ nTreg are detectable in the blood of healthy individuals, show in part strong Foxp3 TSDR demethylation and are inducible in-vitro. The present data provide further insight concerning the in-vivo and in-vitro characteristics of IFNγ+ Treg and help to understand their role in immunoregulation. Alloantigen-specific demethylated IFNγ+Helios+ nTreg might represent a suitable marker for monitoring graft-specific immunosuppression in renal transplant recipients.
Department of Transplantation-Immunology, Institute of Immunology, University Hospital Heidelberg, Im Neuenheimer Feld 305, 69120, Heidelberg, Germany. Volker.Daniel@med.uni-heidelberg.de.
Volker
V
Daniel
Department of Transplantation-Immunology, Institute of Immunology, University Hospital Heidelberg, Im Neuenheimer Feld 305, 69120, Heidelberg, Germany. Karina.Trojan@med.uni-heidelberg.de.
Karina
K
Trojan
Department of Transplantation-Immunology, Institute of Immunology, University Hospital Heidelberg, Im Neuenheimer Feld 305, 69120, Heidelberg, Germany. Martina.Adamek@med.uni-heidelberg.de.
Martina
M
Adamek
Department of Transplantation-Immunology, Institute of Immunology, University Hospital Heidelberg, Im Neuenheimer Feld 305, 69120, Heidelberg, Germany. Gerhard.Opelz@med.uni-heidelberg.de.
Gerhard
G
Opelz
D016022:Case-Control Studies; D002478:Cells, Cultured; D019175:DNA Methylation / Q000187:drug effects; D051858:Forkhead Transcription Factors / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008213:Lymphocyte Activation / Q000187:drug effects / Q000276:immunology*; D018655:Lymphocyte Count; D016131:Lymphocyte Subsets / Q000187:drug effects / Q000276:immunology; D010641:Phenotype; D011994:Recombinant Proteins / Q000494:pharmacology; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects / Q000276:immunology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D011994:Recombinant Proteins; D015759:Ionomycin; D007371:Interferon-gamma; D013755:Tetradecanoylphorbol Acetate