Distinct Methylation of the Interferon γ (IFN-γ) and Interleukin 3 (IL-3) Genes in Newly Activated Primary CD8+ T Lymphocytes: Regional IFN-γ Promoter Demethylation and mRNA Expression Are Heritable in CD44highCD8+ T Cells

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Abstract
Differential genomic DNA methylation has the potential to influence the development of T cell cytokine production profiles. Therefore, we have conducted a clonal analysis of interferon (IFN)-γ and interleukin (IL)-3 gene methylation and messenger (m)RNA expression in primary CD8+ T cells during the early stages of activation, growth, and cytokine expression. Despite similar distributions and densities of CpG methylation sites, the IFN-γ and IL-3 promoters exhibited differential demethylation in the same T cell clone, and heterogeneity between clones. Methylation patterns and mRNA levels were correlated for both genes, but demethylation of the IFN-γ promoter was widespread across >300 basepairs in clones expressing high levels of IFN-γ mRNA, whereas demethylation of the IL-3 promoter was confined to specific CpG sites in the same clones. Conversely, the majority of clones expressing low or undetectable levels of IFN-γ mRNA exhibited symmetrical methylation of four to six of the IFN-γ promoter CpG sites. Genomic DNA methylation also has the potential to influence the maintenance or stability of T cell cytokine production profiles. Therefore, we also tested the heritability of IFN-γ gene methylation and mRNA expression in families of clones derived from resting CD44lowCD8+ T cells or from previously activated CD44highCD8+ T cells. The patterns of IFN-γ gene demethylation and mRNA expression were faithfully inherited in all clones derived from CD44high cells, but variable in clones derived from CD44low cells. Overall, these findings suggest that differential genomic DNA methylation, including differences among cytokine genes, among individual T cells, and among T cells with different activation histories, is an important feature of cytokine gene expression in primary T cells.