FOXP3 Panel

Forkhead-box protein P3 (Foxp3) is a major transcription factor that is necessary for induction of the immunosuppressive function of regulatory T cells (Tregs). It is considered to be the most specific marker for Tregs in tumors, with its expression leading to poor survival rates in some types of cancer and improved survival rates in other types. Studies have shown that epigenetic modifications contribute to the stable expression of FOXP3 and other molecules associated with Treg cell function. These modifications include demethylation of CpG sites in the conserved non-coding sequence 2 (CNS2), Treg specific demethylated region (TSDR), proximal promoter, and upstream enhancer regions of the gene. Understanding the epigenetic regulation of FOXP3 and associated genes is critical in developing new approaches to autoimmune disease and cancer treatment.

CTLA4 is an inhibitory receptor protein found on the surface of FOXP3+ Tregs. The interaction between CTLA4 and receptors on antigen-presenting cells is known to turn off the immune response. While FOXP3 and CTLA4 are usually demethylated and expressed in Tregs, hypermethylation of the CTLA4 promoter has been found to result in dysfunctional FOXP3+ Tregs and autoimmune diseases such as rheumatoid arthritis. Assays covering SNPs in the gene have also been used to study the expression of CTLA4 in T-cells.

PD-1, or programmed cell death protein 1, is a receptor on T-cells that causes T-cell death or inhibition when bound with its ligand PDL1 (CD274), or programmed cell death ligand 1. High PDL1 expression has been associated with the reduction of conventional T-cell populations and the induction of FOXP3-expressing Tregs, downregulating the immune response. Thus, PD-1 and PD-L1 inhibitors are useful as cancer immunotherapy treatments.

EpigenDx has designed and validated DNA methylation assays covering the promoter region of PDL1.

Helios (IKZF2) and Eos (IKZF4) are two members of the Ikaros transcription factor family that are demethylated and expressed along with FOXP3 in Tregs. While Eos interacts directly with FOXP3 to silence certain genes (encoding T effector cytokines for instance), Helios binds to the FOXP3 promoter to upregulate FOXP3 expression. Both Eos and Helios are important biomarkers, essential for the proper function of CD4+ FOXP3+ Tregs. EpigenDx has designed and validated DNA methylation assays covering the intronic, non-coding regions of these two genes.

Before FOXP3 was discovered as the primary biomarker for the identification of Tregs, early studies used IL2RA (CD25), or the alpha chain of the interleukin-2 receptor, along with CD4 as the primary identifying membrane protein on Treg cells. IL2-IL2R signals play an important role in the maintenance of Tregs in the periphery, allowing suppressive Tregs to respond appropriately to T helper cells secreting IL2. Because naturally arising Tregs constitutively express IL2RA, whereas T helper cells only express IL2 and IL2RA upon activation, IL2RA remains an important biomarker for the epigenetic differentiation of Tregs.

TNFRSF18, or GITR (glucocorticoid-induced TNF tumor-necrosis factor-related receptor), is another protein that is demethylated and expressed in high levels in Tregs and activated T cells. Because the engagement of GITR with anti-GITR antibodies can inhibit Treg function and deplete Treg population, drugs blocking GITR/TNFRSF18 have been proposed as cancer immunotherapy treatments and allergy medication.