Loss of methylation at the IFNG promoter and CNS‐1 is associated with the development of functional IFN‐γ memory in human CD4⁺T lymphocytes

Abstract

Cytokine memory for IFN-γ production by effector/memory Th1 cells plays a key role in both protective and pathological immune responses. To understand the epigenetic mechanism determining the ontogeny of effector/memory Th1 cells characterized by stable effector functions, we identified a T-cell-specific methylation pattern at the IFNG promoter and CNS-1 in ex vivo effector/memory Th1 cells, and investigated methylation dynamics of these regions during the development of effector/memory Th1 cells. During Th1 differentiation, demethylation occurred at both the promoter and CNS-1 regions of IFNG as early as 16 h, and this process was independent of cell proliferation and DNA synthesis. Using an IFN-γ capture assay, we found early IFN-γ-producing cells from 2-day differentiating cultures acquired "permissive" levels of demethylation and developed into effector/memory Th1 cells undergoing progressive demethylation at the IFNG promoter and CNS-1 when induced by IL-12. Methylation levels of these regions in effector/memory Th1 cells of peripheral blood from rheumatoid arthritis patients correlated inversely with reduced frequencies of IFN-γ-producers, coincident with recruitment of effector/memory Th1 cells to the site of inflammation. Thus, after termination of TCR stimulation, IL-12 signaling potentiates the stable functional IFN-γ memory in effector/memory Th1 cells characterized by hypomethylation at the IFNG promoter and CNS-1.Keywords: Demethylation r Human IFNG gene r IFN-γ cytokine memory r Promoter and CNS-1 r Th1 cell differentiation Additional supporting information may be found in the online version of this article at the publisher's web-site 794Jun Dong et al. Eur. J. Immunol. 2013. 43: 793-804 Introduction A unique feature of adaptive immunity is the generation of effector/memory T cells after primary activation that control pathogens and mediate effective protection during secondary challenges [1]. Depending on the stimuli and cytokine environment experienced during activation, naive T cells make decisions to proliferate and differentiate into Th1, Th2, or Th17 lineages [2]. When challenged by intracellular pathogens, naïve CD4 + T cells can adopt a proinflammatory cell fate and differentiate into effector and memory Th1 cells, which are characterized by the production of the signature cytokine IFN-γ. Th1 cells play a key role in intracellular pathogen killing and exert proinflammatory effects in organspecific autoimmune diseases [3]. During primary activation, the expression of the Ifng/IFNG gene in naïve CD4 + T cells is initiated by TCR signaling, in conjunction with instructive IL-12 signaling through lineage-specific transcription factors (such as T-bet and the IL-12-responsive transcription factor STAT4, respectively) [4][5][6]. After termination of antigen stimulation, IL-12 also controls Ifng reactivation in effector/memory Th1 cells through STAT4 and T-bet [7]. During secondary activation, TCR signaling in the absence of the original instructive signal is sufficient to trigger rapid Ifng/I...

Demethylation of the RORC2 and IL17A in Human CD4+ T Lymphocytes Defines Th17 Origin of Nonclassic Th1 Cells

“…As shown in Fig. 1B, two ROI in the IFNG gene locus were selected, the promoter and CNS-1 (located 4.2 kb upstream of the transcriptional start site) (19). The analysis of DNA methylation status of the two ROI by bisulfite pyrosequencing showed that whereas Th17/Th1, nonclassic Th1, and classic Th1 cells had a marked demethylation, Th17 cells and naive CD4 + Th cells were characterized by a complete methylation (Fig.…”

“…DNA methylation of the ROI was assessed by pyrosequencing of bisulfite-specific PCR products using standard procedures (Varionostic, Ulm, Germany), as described previously by Dong et al (19). Briefly, 40 ng bisulfite-converted DNA was PCR amplified for the ROI of the IL17A, IL17F, IFNG, RORC2, and TBX21 genes using bisulfite-DNA-specific primers (Table I) in a PCR using the following program: 95˚C for 3 min; 50 cycles of 95˚C for 35 s, annealing temperature (Table I) for 35 s, and 72˚C for 40 s; and a final extension at 72˚C for 10 min.…”

“…In this study, we examined the methylation patterns of regions of interest (ROI), identified by sequence homology analysis between mice and humans, of the human genes RORC2 (17), TBX21, IL17A (17), IL17F (18), and IFNG (19) in either human T cell clones or circulating freshly isolated CD4 + T cells, isolated from both peripheral blood (PB) of healthy subjects and synovial fluid (SF) of juvenile idiopathic arthritis (JIA) patients, characterized by the following phenotypes: Th17, Th17/Th1, nonclassic (Th17-derived), and classic Th1. We further analyzed the methylation changes of the above-mentioned ROI in Th17 clones during the in vitro polarization of Th17 into the nonclassic Th1 phenotype.…”

Adoptively transferred natural killer cells maintain long-term antitumor activity by epigenetic imprinting and CD4+T cell help

“…40 Although the underlying mechanism is not completely understood, it was shown that IL-12 activates STAT4 that could further limit recruitment of DNA methyltransferase 41 leading to DNA demethylation. In addition, NK cells preactivated with IL-12/15/18 showed increased STAT5 phosphorylation in response to low doses of IL-2.…”

Immunological memories of the bone marrow

“…Ever since the original classification of circulating memory CD4 T cells into Th1 and Th2 cells,82 functional compartmentalization of antigen‐experienced T cells has become a favorite exercise of immunologists 83, 84, 85. We have shown that GATA3 mediates the epigenetic imprinting of the interleukin‐4 ( Il4 ) gene,86, 87 and how imprinting of the interferon‐γ ( Ifng ) gene is induced by STAT4 88, 89. Most importantly, we and others have shown that imprinting of cytokine genes in activated naive T cells but not in memory T cells requires progression through the S phase of the first cell division.…”