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

Abstract

Th17-derived Th1 lymphocytes, termed nonclassic, differ from classic Th1 cells because of the presence of retinoic acid orphan receptor (ROR)C2 and the surface expression of CD161 and CCR6. We demonstrate in this article that nonclassic Th1 cells, like Th17 cells, have a marked RORC2 and IL17A demethylation, whereas classic Th1 cells exhibit a complete methylation of these genes. The analysis of RORC2 DNA methylation in the CD4+CD161+ and CD4+CD161− naive Th subsets from umbilical cord blood surprisingly revealed comparable hypermethylation levels. PCR analysis at the single-cell level revealed that RORC2 mRNA was expressed by none of the CD4+CD161− and present only in a minority of CD4+CD161+ naive Th cells. These findings provide two important novel observations on the physiology of human Th17 cells: 1) they confirm at the epigenetic level the origin of nonclassic Th1 cells from Th17 cells, also identifying in the RORC2 and IL17A methylation status a novel tool for their distinction from classic Th1 cells, and 2) they demonstrate that RORC2-expressing cells are only a minority in the subset of CD4+CD161+ naive Th cells, which are known to contain all Th17 cell precursors.

Do Memory CD4 T Cells Keep Their Cell-Type Programming: Plasticity versus Fate Commitment?

“…These cells may derive directly from naïve T cells in a RORgt-dependent fashion or from T H 17 cells that convert to T H 1 Ã under the influence of IL-12, TNF-a, and/or IL-1b. The latter possibility is consistent with the finding that in T H 1 Ã cells, like in T H 17 cells, RORC2 and IL17A are demethylated (Mazzoni et al 2015).…”

CCR6+ Th cells in the cerebrospinal fluid of persons with multiple sclerosis are dominated by pathogenic non-classic Th1 cells and GM-CSF-only-secreting Th cells

“…This protocol has been widely used to reveal the cytokine production capabilities of T cells, including studies on MS (Mazzoni et al, 2015). Cells were incubated with antibodies specific for surface markers for 20 min at 4 °C before washing.…”

“…Potentially pathogenic CCR6 + Th subsets that do not secrete IL-17A includes non-classic or non-conventional Th1 cells, a population originally characterised by the expression of CD161 and/or CCR6 (Acosta-Rodriguez et al, 2007, Becattini et al, 2015, Maggi et al, 2010, Maggi et al, 2012). Epigenetic analysis of these cells showed that they most likely originate from Th17 cells, but have lost their ability to produce IL-17A (Mazzoni et al, 2015), with studies in juvenile idiopathic arthritis demonstrating their presence in both the blood and synovial fluid. In this study we explored the association of CCR6 with the pathogenic cytokines IL-17, IFNγ and GM-CSF in CD4 + T cells and in particular their association with MS. We demonstrate that the dominant CSF CD4 + T cell subset is not composed of IL-17-secreting T cells, but non-classic/non-conventional Th1 cells as well as GM-CSF-only-secreting Th cells.…”

Disease Manifestation and Inflammatory Activity as Modulators of Th17/Treg Balance and RORC/FoxP3 Methylation in Systemic Sclerosis

“…A second explanation for the trend towards higher RORγτ expression in CD4+CCR6+ T cells and intracellular IL-17 production despite hypermethylation at RORC regions in dcSSc is that in our assay, DNA from total PBMCs due to small sample volumes of dcSSc patients and HC was taken to analyze methylation levels at RORC1 , RORC2 , FOXP3 , STAT3, and STAT5A regions, but not from isolated T-cell subsets. Recently, it has been shown that RORC2 is differentially methylated in classic CD4+CD161-CCR6-IFNγ+ Th1 cells, and transiently in Th17/Th1 (CD4+CD161+CCR6+IL-17A+IFNγ+) cells and nonclassic CD4+CD161+CCR6+IFNγ+ Th1 cells [57]. Thus, hypermethylation at RORC regions may result rather from other Th cell subpopulations, such as Th1 or Th2, than from the relatively small peripheral Th17 subpopulation.…”