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 CD44^highCD8⁺ T Cells

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 CD44^lowCD8⁺ T cells or from previously activated CD44^highCD8⁺ T cells. The patterns of IFN-γ gene demethylation and mRNA expression were faithfully inherited in all clones derived from CD44^high cells, but variable in clones derived from CD44^low 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.