Impact of 5-aza-2′-deoxycytidine and epigallocatechin-3-gallate for induction of human regulatory T cells

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The epigenetic regulation of transcription factor genes is critical for T-cell lineage specification. A specific methylation pattern within a conserved region of the lineage specifying transcription factor geneFOXP3, theTreg-specific demethylated region(TSDR), is restricted to regulatory T (Treg) cells and is required for stable expression of FOXP3 and suppressive function. We analysed the impact of hypomethylating agents 5-aza-2′-deoxycytidine and epigallocatechin-3-gallate on human CD4+ CD25− T cells for generating demethylation withinFOXP3-TSDRand inducing functional Treg cells. Gene expression, including lineage-specifying transcription factors of the major T-cell lineages and their leading cytokines, functional properties and global transcriptome changes were analysed. TheFOXP3-TSDRmethylation pattern was determined by using deep amplicon bisulphite sequencing. 5-aza-2′-deoxycytidine inducedFOXP3-TSDRhypomethylation and expression of the Treg-cell-specific genesFOXP3andLRRC32. Proliferation of 5-aza-2′-deoxycytidine-treated cells was reduced, but the cells did not show suppressive function. Hypomethylation was not restricted toFOXP3-TSDRand expression of master transcription factors and leading cytokines of T helper type 1 and type 17 cells were induced. Epigallocatechin-3-gallate induced global DNA hypomethylation to a lesser extent than 5-aza-2′-deoxycitidine, but no relevant hypomethylation withinFOXP3-TSDRor expression of Treg-cell-specific genes. Neither of the DNA methyltransferase inhibitors induced fully functional human Treg cells. 5-aza-2′-deoxycitidine-treated cells resembled Treg cells, but they did not suppress proliferation of responder cells, which is an essential capability to be used for Treg cell transfer therapy. Using a recently developed targeted demethylation technology might be a more promising approach for the generation of functional Treg cells.

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