Targeted De-Methylation of the FOXP3-TSDR Is Sufficient to Induce Physiological FOXP3 Expression but Not a Functional Treg Phenotype

• Main Authors: Christopher Kressler, Gilles Gasparoni, Karl Nordström, Dania Hamo, Abdulrahman Salhab, Christoforos Dimitropoulos, Sascha Tierling, Petra Reinke, Hans-Dieter Volk, Jörn Walter, Alf Hamann, Julia K. Polansky
• Format: Article
• Language: English
• Published: Frontiers Media S.A. 2021-01-01
• Series: Frontiers in Immunology
• Subjects: T cell differentiation; regulatory T cells; epigenetic editing; adoptive T cell therapies; gene regulation; CRISPR-Cas9-based tool
• Online Access: https://www.frontiersin.org/articles/10.3389/fimmu.2020.609891/full

CD4+ regulatory T cells (Tregs) are key mediators of immunological tolerance and promising effector cells for immuno-suppressive adoptive cellular therapy to fight autoimmunity and chronic inflammation. Their functional stability is critical for their clinical utility and has been correlated to the demethylated state of the TSDR/CNS2 enhancer element in the Treg lineage transcription factor FOXP3. However, proof for a causal contribution of the TSDR de-methylation to FOXP3 stability and Treg induction is so far lacking. We here established a powerful transient-transfection CRISPR-Cas9-based epigenetic editing method for the selective de-methylation of the TSDR within the endogenous chromatin environment of a living cell. The induced de-methylated state was stable over weeks in clonal T cell proliferation cultures even after expression of the editing complex had ceased. Epigenetic editing of the TSDR resulted in FOXP3 expression, even in its physiological isoform distribution, proving a causal role for the de-methylated TSDR in FOXP3 regulation. However, successful FOXP3 induction was not associated with a switch towards a functional Treg phenotype, in contrast to what has been reported from FOXP3 overexpression approaches. Thus, TSDR de-methylation is required, but not sufficient for a stable Treg phenotype induction. Therefore, targeted demethylation of the TSDR may be a critical addition to published in vitro Treg induction protocols which so far lack FOXP3 stability.