CpG Methylation of the IFNG Gene as a Mechanism to Induce Immunosupression in Tumor-Infiltrating Lymphocytes

Abstract

The execution of appropriate gene expression patterns during immune responses is of eminent importance where CpG methylation has emerged as an essential mechanism for gene silencing. We have charted the methylation status of regulatory elements in the human IFNG gene encoding the signature cytokine of the Th1 response. Surprisingly, human naive CD4+ T lymphocytes displayed hypermethylation at the IFNG promoter region, which is in sharp contrast to the completely demethylated status of this region in mice. Th1 differentiation induced demethylation of the IFNG promoter and the upstream conserved nucleotide sequence 1 enhancer region, whereas Th2-differentiated lymphocytes remained hypermethylated. Furthermore, CD19+ B lymphocytes displayed hypomethylation at the IFNG promoter region with a similar pattern to Th1 effector cells. When investigating the methylation status among tumor-infiltrating CD4+ T lymphocytes from patients with colon cancer, we found that tumor-infiltrating lymphocytes cells are inappropriately hypermethylated, and thus not confined to the Th1 lineage. In contrast, CD4+ T cells from the tumor draining lymph node were significantly more demethylated than tumor-infiltrating lymphocytes. We conclude that there are obvious interspecies differences in the methylation status of the IFNG gene in naive CD4+ T lymphocytes, where Th1 commitment in human lymphocytes involves demethylation before IFNG expression. Finally, investigations of tumor-infiltrating lymphocytes and CD4+ cells from tumor draining lymph node demonstrate methylation of regulatory regions within key effector genes as an epigenetic mechanism of tumor-induced immunosupression.

Myeloid-Derived Suppressor Cells Attenuate TH1 Development through IL-6 Production to Promote Tumor Progression

“…In particular, IFN-g production by tumorspecific T cells is shown to be inhibited in tumor-bearing hosts (7,15,17). This is consistent with the epigenetic silencing of ifng gene in tumor-infiltrating CD4 þ effector T cells (24). Moreover, a positive correlation between the survival of patients with cancer and the mRNA expression of T-helper cell (T H 1)-associated markers such as IFN-g and T-box expressed in T cells (T-bet) in tumor tissues has been observed (25).…”

Profiling of CD4+ T Cells with Epigenetic Immune Lineage Analysis

“…Naive T cell differentiation into Th1 or Th2 effector cells was performed as described previously (13). Th17 differentiation was conducted in serumfree AIM-V medium (Life Technologies) supplemented with 5 ng/ml TGFb, 20 ng/ml IL-6, 10 ng/mL IL-1b, 10 ng/ml IL-23, 25 ng/ml IL-21, 25 U/ ml IL-2, 5 mg/ml anti-IFN-g, 1 mg/ml anti-CD28, and 3 mg/ml platebound anti-CD3.…”

“…Locus-specific PCR amplification and snapshot reaction DNA bisulfite conversion was performed as described previously (10,13). Locus-specific PCR primers (Cybergene, Stockholm, Sweden) were designed for the FOXP3, IFNG, and IL-17 loci (Table I).…”

DNA methylation in the tumor microenvironment

“…Thus, it is possible that DAC can reverse PTEN silencing and halt the progression of breast cancer by increasing PTEN expression. Janson et al (2008) found increased expression of IFN-γ in 5-Aza-treated tumorinfiltrating lymphocytes, suggesting that DNMT inhibitors may stimulate the immune response in the TME. Most studies have described the effect of DNMT inhibitors on stromal cells in vitro.…”

“…Janson et al (2008) demonstrated the different methylation status of the interferon-γ (IFN-γ) gene promoter and upstream enhancer in naive CD4 + T lymphocytes. However, the IFN-γ gene promoter and enhancer were demethylated in Th1 lymphocytes, where the expression of IFN-γ was induced.…”