*Purpose: Foxp3 acetylation is essential to T_REG stability and function. Acetylation protects Foxp3 from degradation, and acetylated Foxp3 translocates to the nucleus to activate the immunosuppressive transcriptional program. Current methods to measure Foxp3 acetylation (proximity-ligation assays) are inadequate as they are amplification-based and thus semiquantitative, can only assess a limited number of cells, and underperform when combined with flow cytometry.

*Methods: We developed a FRET-based method to measure Foxp3 acetylation in primary cells using flow cytometry. We utilized an anti-Foxp3 antibody labeled with Alexa Fluor (AF) 488 as the donor and an anti-acetylated lysines antibody labeled with AF555 as the acceptor (Fig. 1A), and calculated the single-cell FRET efficiency distribution from flow cytometry measurements to reduce the variability between samples and increase the signal-to-noise ratio. We utilized this technique to test the effect on T_REG of three molecules (NU9056, MG149, TH1834) that target the acetyltransferase TIP60.

*Results: Our results indicate that these molecules, previously described as TIP60 inhibitors, enhance in vitro murine and human T_REG inductions (Fig. 1B) through the increase of Foxp3 acetylation levels in Foxp3⁺ cells (Fig. 1C).

*Conclusions: Our FRET-based quantitative technique is perfectly adapted to monitor T_REG Foxp3 acetylation status in peripheral blood mononuclear cells (PBMC) from organ transplant recipients and could provide clinically-relevant information regarding their function.

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