Technology Description

Comparison with flow cytometry; dried blood spot analysis

• Baron U et al. Epigenetic immune cell counting in human blood samples for immunodiagnostics. Sci Transl Med. 2018 Aug 1;10(452).
• Nettenstrom L et al. An optimized multi-parameter flow cytometry protocol for human T regulatory cell analysis on fresh and viably frozen cells, correlation with epigenetic analysis, and comparison of cord and adult blood. J Immunol Methods. 2013 Jan 31;387(1-2):81-8.
• Liu J et al. T regulatory cells in cord blood–FOXP3 demethylation as reliable quantitative marker. PLoS One. 2010 Oct 12;5(10).
• Ramirez et al. Epigenetic immune cell quantification for diagnostic evaluation and monitoring of patients with inborn errors of immunity and secondary immune deficiencies. Clinical Immunology 260, 2024

DNA (de)methylation markers for detection of specific immune cell subtypes

• Pink M et al. Imprinting of Skin/Inflammation Homing in CD4+ T Cells Is Controlled by DNA Methylation within the Fucosyltransferase 7 Gene. J Immunol. 2016 Oct 15;197(8):3406-3414.
• Steinfelder S et al. Epigenetic modification of the human CCR6 gene is associated with stable CCR6 expression in T cells. 2011 Mar 10;117(10):2839-46.
• Wieczorek G et al. Quantitative DNA methylation analysis of FOXP3 as a new method for counting regulatory T cells in peripheral blood and solid tissue. Cancer Res. 2009 Jan 15;69(2):599-608.
• Polansky JK et al. DNA methylation controls Foxp3 gene expression. Eur J Immunol. 2008 Jun;38(6):1654-63.
• Baron U et al. DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3(+) conventional T cells. Eur J Immunol. 2007 Sep;37(9):2378-89.
• Floess S et al. Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol. 2007 Feb;5(2):e38

Applications

Newborns/Infants

• Schoenaker MHD et al. Epigenetic Immune Cell Counting to Analyze Potential Biomarkers in Preterm Infants: A Proof of Principle in Necrotizing Enterocolitis. Int J Mol Sci. 2023;24(3):2372.
• Blom M et al. Second Tier Testing to Reduce the Number of Non-actionable Secondary Findings and False-Positive Referrals in Newborn Screening for Severe Combined Immunodeficiency. J Clin Immunol. 2021 Nov;41(8):1762-1773.
• Blom M et al. Future Perspectives of Newborn Screening for Inborn Errors of Immunity. Int J Neonatal Screen. 2021 Nov 2;7(4):74.

Immune regulatory disorders

• Wyatt RC et al. FOXP3 TSDR Measurement Could Assist Variant Classification and Diagnosis of IPEX Syndrome. J Clin Immunol. 2023;43(3):662-669.
• Narula M et al. Epigenetic and immunological indicators of IPEX disease in subjects with FOXP3 gene mutation. J Allergy Clin Immunol. 2023;151(1):233-246.e10.
• Cepika AM et al. Tregopathies: Monogenic diseases resulting in regulatory T-cell deficiency. J Allergy Clin Immunol. 2018 Dec;142(6):1679-1695.
• Goudy K et al. Human IL2RA null mutation mediates immunodeficiency with lymphoproliferation and autoimmunity. Clin Immunol. 2013 Mar;146(3):248-61.
• Barzaghi F et al. Demethylation analysis of the FOXP3 locus shows quantitative defects of regulatory T cells in IPEX-like syndrome. J Autoimmun. 2012 Feb;38(1):49-58.
• Passerini L et al. Forkhead box protein 3 (FOXP3) mutations lead to increased TH17 cell numbers and regulatory T-cell instability. J Allergy Clin Immunol. 2011 Dec;128(6):1376-1379.e1.
• Di Nunzio S et al. Wild-type FOXP3 is selectively active in CD4+CD25(hi) regulatory T cells of healthy female carriers of different FOXP3 mutations. 2009 Nov 5;114(19):4138-41.

Solid Tumors

• Le Cornet C et al. Circulating Immune Cell Composition and Cancer Risk: A Prospective Study Using Epigenetic Cell Count Measures. Cancer Res. 2020 May 1;80(9):1885-1892.
• Barth SD et al. Treg-Mediated Immune Tolerance and the Risk of Solid Cancers: Findings From EPIC-Heidelberg. J Natl Cancer Inst. 2015 Aug 22;107(11).
• Thomas AA et al. Regulatory T cells are not a strong predictor of survival for patients with glioblastoma. Neuro Oncol. 2015 Jun;17(6):801-9.
• Türbachova I et al. The cellular ratio of immune tolerance (immunoCRIT) is a definite marker for aggressiveness of solid tumors and may explain tumor dissemination patterns. 2013 Nov;8(11):1226-35.
• Sehouli J et al. Epigenetic quantification of tumor-infiltrating T-lymphocytes. 2011 Feb;6(2):236-46.
• Loddenkemper C et al. Regulatory (FOXP3+) T cells as target for immune therapy of cervical intraepithelial neoplasia and cervical cancer. Cancer Sci. 2009 Jun;100(6):1112-7.

Cardiovascular Disease

• Barth SD et al. The Ratio of Regulatory (FOXP3+) to Total (CD3+) T Cells Determined by Epigenetic Cell Counting and Cardiovascular Disease Risk: A Prospective Case-cohort Study in Non-diabetics. 2016 Sep;11:151-156.

Allergy, inflammation, autoimmunity

• Blokland SLM et al. Epigenetically quantified immune cells in salivary glands of Sjögren’s syndrome patients: a novel tool that detects robust correlations of T follicular helper cells with immunopathology. Rheumatology (Oxford). 2020 Feb 1;59(2):335-343.
• Burska AN et al. Quantifying circulating Th17 cells by qPCR: potential as diagnostic biomarker for rheumatoid arthritis. Rheumatology (Oxford). 2019 Nov 1;58(11):2015-2024.
• Winter M et al. The benzene metabolite 1,4-benzoquinone reduces regulatory T-cell function: A potential mechanism for tobacco smoke-associated atopic dermatitis. J Allergy Clin Immunol. 2017 Aug;140(2):603-605.
• Herberth G et al. Prenatal phthalate exposure associates with low regulatory T-cell numbers and atopic dermatitis in early childhood: Results from the LINA mother-child study. J Allergy Clin Immunol. 2017 Apr;139(4):1376-1379.e8.
• Roesner LM et al. Foxp3(+) regulatory T cells are expanded in severe atopic dermatitis patients. 2015 Dec;70(12):1656-60.
• Rodríguez-Perea AL et al. Statins increase the frequency of circulating CD4+ FOXP3+ regulatory T cells in healthy individuals. J Immunol Res. 2015;2015:762506.
• Singh A et al. Th17/Treg ratio derived using DNA methylation analysis is associated with the late phase asthmatic response. Allergy Asthma Clin Immunol. 2014 Jun 24;10(1):32.
• Sebode M et al. Reduced FOXP3(+) regulatory T cells in patients with primary sclerosing cholangitis are associated with IL2RA gene polymorphisms. J Hepatol. 2014 May;60(5):1010-6.
• Herberth G et al. Maternal and cord blood miR-223 expression associates with prenatal tobacco smoke exposure and low regulatory T-cell numbers. J Allergy Clin Immunol. 2014 Feb;133(2):543-50.
• Weisse K et al. Maternal and newborn vitamin D status and its impact on food allergy development in the German LINA cohort study. 2013 Feb;68(2):220-8.
• Peiseler M et al. FOXP3+ regulatory T cells in autoimmune hepatitis are fully functional and not reduced in frequency. J Hepatol. 2012 Jul;57(1):125-32.
• Hinz D et al. Cord blood Tregs with stable FOXP3 expression are influenced by prenatal environment and associated with atopic dermatitis at the age of one year. 2012 Mar;67(3):380-9.
• Ferraro A et al. Expansion of Th17 cells and functional defects in T regulatory cells are key features of the pancreatic lymph nodes in patients with type 1 diabetes. 2011 Nov;60(11):2903-13.
• McClymont SA et al. Plasticity of human regulatory T cells in healthy subjects and patients with type 1 diabetes. J Immunol. 2011 Apr 1;186(7):3918-26.
• Schaub B et al. Maternal farm exposure modulates neonatal immune mechanisms through regulatory T cells. J Allergy Clin Immunol. 2009 Apr;123(4):774-82.e5.

Autoimmune hepatitis

• Taubert R et al. Intrahepatic regulatory T cells in autoimmune hepatitis are associated with treatment response and depleted with current therapies. J Hepatol. 2014 Nov;61(5):1106-14.

Tumor vaccination

• Pohla H et al. High immune response rates and decreased frequencies of regulatory T cells in metastatic renal cell carcinoma patients after tumor cell vaccination. Mol Med. 2013 Feb 8;18:1499-508.
• Schwarzer A et al. Regulatory T-cells and associated pathways in metastatic renal cell carcinoma (mRCC) patients undergoing DC-vaccination and cytokine-therapy. PLoS One. 2012;7(10):e46600.

Hematopoietic stem cell transplantation

• Sprangers B et al. Origin of Enriched Regulatory T Cells in Patients Receiving Combined Kidney-Bone Marrow Transplantation to Induce Transplantation Tolerance. Am J Transplant. 2017 Aug;17(8):2020-2032.
• Delemarre EM et al. Autologous stem cell transplantation aids autoimmune patients by functional renewal and TCR diversification of regulatory T cells. 2016 Jan 7;127(1):91-101.
• Peccatori J et al. Sirolimus-based graft-versus-host disease prophylaxis promotes the in vivo expansion of regulatory T cells and permits peripheral blood stem cell transplantation from haploidentical donors. 2015 Feb;29(2):396-405.

Solid organ transplantation

• Taubert R et al. Enrichment of regulatory T cells in acutely rejected human liver allografts. Am J Transplant. 2012 Dec;12(12):3425-36.
• Stauch D et al. Induction of bona fide regulatory T cells after liver transplantation – the potential influence of polyclonal antithymocyte globulin. Transpl Int. 2012 Mar;25(3):302-13.
• Bestard O et al. Intragraft regulatory T cells in protocol biopsies retain foxp3 demethylation and are protective biomarkers for kidney graft outcome. Am J Transplant. 2011 Oct;11(10):2162-72.

Immunosuppressive therapy (adoptive transfer of Treg)

• Wang H et al. Human LAP(+) GARP(+) FOXP3(+) regulatory T cells attenuate xenogeneic graft versus host disease. 2019 Apr 12;9(8):2315-2324.
• Velaga S et al. Clinical-grade regulatory T cells: Comparative analysis of large-scale expansion conditions. Exp Hematol. 2017 Jan;45:27-35.e1.
• Bégin P et al. Human in vitro induced T regulatory cells and memory T cells share common demethylation of specific FOXP3 promoter region. Clin Transl Allergy. 2015 Oct 20;5:35.
• Noyan F et al. Isolation of human antigen-specific regulatory T cells with high suppressive function. Eur J Immunol. 2014 Sep;44(9):2592-602.
• Ukena SN et al. Human regulatory T cells of G-CSF mobilized allogeneic stem cell donors qualify for clinical application. PLoS One. 2012;7(12):e51644.
• Hoffmann P et al. Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation. Eur J Immunol. 2009 Apr;39(4):1088-97.

Quality control in regenerative medicine

• Baron U et al. DNA methylation analysis as a tool for cell typing. 2006 Jan-Mar;1(1):55-60.
• Rapko S et al. DNA methylation analysis as novel tool for quality control in regenerative medicine. Tissue Eng. 2007 Sep;13(9):2271-80.

Data Analysis

Schildknecht K et al. Simultaneous statistical inference for epigenetic data. PLoS One. 2015 May 12;10(5):e0125587.