Epigenetic analysis of immune cells is performed at Epiontis GmbH, a biotechnology company located in Berlin, Germany. The epigenetic measuring system consists of two quantitative PCR (qPCR) assays: FOXP3 qPCR for detection of Tregs and CD3 qPCR for detection of overall T-lymphocytes. DNA purified from peripheral blood is used for these analyses. The assay targets the Treg Specific Demethylation Region (TSDR) of the FOXP3 gene and the intragenic demethylated region between the gamma and delta region of CD3 gene (CD3G/CD3D) (Sehouli et al., Epigenetics 2011.6:2, 236-246).
The Immune Tolerance Network is shipping whole blood samples collected from several clinical trials to Epiontis. At the Epiontis laboratory, the percental share of overall T cells (CD3+ lymphocytes) and regulatory T cells (CD3+ CD4+ CD25++ T cells) in the samples are determined using epigenetic real time PCR based analyses. The assays applied target an epigenetic marker region in the CD3 gene and FOXP3 gene that is highly specific for overall T cells and regulatory T cells, respectively.