FOXP3 is the master transcription factor of CD4+CD25+FOXP3+ T-regulatory (Treg) cells, and demethylation of the Treg-specific demethylation region (TSDR) of FOXP3 is considered a specific marker of stable, functionally competent thymic Tregs, allowing their discrimination from activated CD4+CD25+FOXP3+ T-effector cells. Assessment of TSDR demethylation in PBMC has thereby come to be regarded as a gold standard for calculating numbers of “true” Tregs. However, data regarding use of FOXP3 TSDR demethylation in PBMC were derived from healthy donor cells, i.e. mostly resting cells isolated from non-inflammatory conditions. Methods: We assessed FOXP3 TSDR demethylation in Tregs isolated from liver and kidney allograft recipients, as well as evaluating their number and phenotype (CD4, CD25, CD127, CTLA4, FOXP3), and assessed any correlations with the numbers of “true” Tregs calculated using FOXP3 TSDR demethylation in PBMC from the same samples. Results: Pediatric recipients had stable liver (n=53, 26 M) or kidney (n=9, 6 M) allografts, receiving monotherapy with CNI (39) or RAPA (9), and 12 patients had CNI (8) or RAPA (4) plus MMF, Aza or steroids. 8 adult patients (n=8, 6 M) were from a follow-up study with serial sampling at day of liver Tx, then 0.5, 3 and 12 months, with similar levels of steroids and Aza, but a different dosage of CNI. In purified Tregs, FOXP3 TSDR demethylation correlated with their expression of CTLA4 and with the absence of CD127, and with suppressive function (r=0.545, p=0.007 for autologous responder CD4+ cells, r=0.456, p=0.019 and r=0.423, p=0.031 for healthy donor CD4+ and CD8+responders, respectively). However, TSDR demethylation in PBMC in clinical samples had no correlations with numbers of Tregs, evaluated by any phenotype: CD4+CD25+, CD4+CD25+FOXP3+ or even as CD4+CD25+TSDR-demethylated cells. Using healthy donor PBMC, we also found that demethylation of FOXP3 can be an active and dynamic process, sensitive to cell activation and cytokine exposure. Conclusions: TSDR demethylation is an important feature of human Tregs, but unfortunately cannot be used to calculate their number in PBMC samples. Our data identify hitherto unrecognized and important limitations to its diagnostic and/or prognostic use when using unfractionated cells in clinical settings.

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