Decreased DNA methyltransferase levels contribute to abnormal gene expression in “senescent” CD4+CD28− T cells
Introduction
Chronic stimulation of CD4+ T cells results in the development of a “senescent” CD28− subset in vitro, in chronic inflammatory diseases like rheumatoid arthritis (RA), and with aging [1], [2], [3]. CD4+CD28− T cells aberrantly express genes not normally expressed by CD4+CD28+ T cells, such as members of the killer cell immunoglobulin-like receptor (KIR) gene family and perforin [4], [5], normally expressed by NK cells [6], and overexpress interferon–γ (IFNγ) [7], LFA-1 [8] and CD70 [9]. The CD4+CD28− T cells acquire cytolytic activity, lysing endothelial and other cells through mechanisms involving the aberrantly expressed KIR molecules and self class I MHC molecules, and have been isolated from ruptured atherosclerotic plaques of people dying from myocardial infarctions, implicating these cytotoxic and inflammatory functions in acute coronary events [4], [6]. The mechanisms causing aberrant overexpression of genes like KIR, perforin and CD70 in CD4+CD28− T cells are incompletely understood.
Our group and others have found that some of the genes aberrantly overexpressed in CD4+CD28− T cells, including KIR, perforin, IFNγ, LFA-1 and CD70, are suppressed in CD4+ T cells from young healthy people by DNA methylation, and that demethylation of their promoters is sufficient to increase their transcription in T cells [10], [11], [12], [13]. Total genomic T cell DNA is demethylated in RA and aging, although specific subsets have not been studied [14], [15]. Levels of T cell DNA methyltransferase 1 and 3a (Dnmt1 and Dnmt3a), which maintain DNA methylation patterns during mitosis and DNA repair [16], also decrease with aging [17], although again subsets have not been studied, the significance of the decreases has not been established, and the relative roles of Dnmt1 and Dnmt3a in maintaining T cell DNA methylation patterns during mitosis have not been determined.
We hypothesized that the aberrant expression of methylation sensitive genes in the CD4+CD28− T cell subset is due to demethylation of crucial promoter sequences, caused by decreased levels of Dnmt1 and/or Dnmt3a. We therefore compared expression as well as methylation status of crucial regulatory regions of the CD70, perforin and KIR2DL4 promoters in CD4+CD28+ and CD4+CD28− T cells, and compared the results with levels of Dnmt1 and Dnmt3a in these subsets. We also used siRNA “knockdowns” to determine the consequences of decreased Dnmt1 and Dnmt3a levels on the expression and methylation status of these genes in T cells.
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Subjects
Patients with RA were recruited from the outpatient rheumatology clinics at the University of Michigan. Older subjects (ages 50–85) were recruited from the Human Subjects Core of the University of Michigan Claude D. Pepper Older Americans Independence Center. Young healthy controls (ages 18–30) were recruited by advertising. All RA patients fulfilled the American College of Rheumatology criteria for the diagnosis of RA [18]. This protocol was approved by the University of Michigan Institutional
Methylation sensitive gene overexpression in CD4+CD28− T cells
Initial studies confirmed overexpression of the methylation sensitive genes CD70, perforin and KIR2DL4 on CD4+CD28− T cells using flow cytometry. Figures 1a–d show representative histograms of CD70, perforin and KIR2DL4 in CD4+CD28+ and CD4+CD28− T cells from a patient with RA. Figure 1B shows the mean + SD of 3 independent experiments similarly comparing CD70, perforin and KIR2DL4 levels on CD4+CD28+ and CD4+CD28− subsets from 3 RA patients. Expression of all 3 proteins is significantly higher
Discussion
T cells undergoing replicative stress in RA, aging and chronic infections [1], [2], [3], [24] become senescent. Characteristic changes include altered secretion of a variety of molecules including proteases, cytokines and growth factors, telomere shortening with eventual proliferative arrest, and resistance to apoptosis [25], [26]. The mechanisms causing senescence are multiple, and include oxidative damage [27], [28], decreased clearance of aberrant proteins [29], and changes in DNA including
Acknowledgments
The authors thank Ms. Cindy Bourke for her expert secretarial assistance. This work was supported by PHS grants AG25877, AR42525, ES015214, the University of Michigan Pepper Center grant AG024824, and a Merit grant from the Veterans Administration.
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