While murine studies showed increased numbers of FOXP3+ Tregs in tolerant allograft recipients (15
), the induction of CD25 and FOXP3 upon activation of human Teffs (16
) has led to far more variable clinical data. Thus, Treg numbers were increased in tolerant recipients (17
), whereas others found no differences (19
). Similarly, Treg numbers were decreased in patients with acute (21
) or chronic (19
) rejection in some Centers but not others (25
). Despite an ever-expanding list of markers to identify “true” human Tregs, including CD127−/low and CD49d−, CD39+, CTLA4+, GARP+, CD120+, LAP+, CD27+ etc., none have proven appropriately Treg-specific using patient samples. E.g. in healthy donors CD4+CD25+CD127−/low gated cells are 90–95% FOXP3+, whereas that subset is enriched by FOXP3− cells in rheumatoid arthritis (27
). In Tx recipients, we found variable FOXP3 expression by CD4+CD25+ cells, many FOXP3+ cells outside the CD25++CD127− gate, and contamination of that gate by FOXP3− cells. Others noted similar findings in renal Tx patients (28
). Thus, currently flow cytometry alone cannot satisfactorily enumerate Treg cells post-Tx, and needs to be accompanied by assessment of Treg function.
Treg suppression assays have pitfalls, since isolation of CD4+CD25+ (or CD4+CD25+CD127−) cells from Tx recipient results in a mix of suppressive (Tregs) and activated (Teffs) cells. We are unaware of any literature assessing at least FOXP3 (or better, FOXP3, CD127 and CTLA4) expression within isolated cells and corresponding assessment of Treg suppressive function post-Tx. Moreover, using autologous cells as responders in Treg assays can be misleading, since immunosuppression can decrease their proliferative capacity. Such use may explain why Tregs from patients with rejection had impaired suppressive function in some studies (24
) but not others (23
). We therefore assessed CD25, CD127, CTLA4 and FOXP3 expression in isolated Tregs, and performed suppression assays under highly standardized conditions, with Tregs as the only variable. We found Treg function post-Tx correlated with CTLA4 and absence of CD127 expression, but not with FOXP3, despite its role as a master regulator in Tregs (2
). This likely reflects how activated human T cells can induce FOXP3 without acquiring a Treg phenotype or suppressive function, and is consistent with dominance of CTLA4 over FOXP3 for human Treg suppressive function (11
The epigenetic status of FOXP3 is useful given the TSDR is demethylated only in natural Tregs with stable FOXP3 expression (13
). We found that TSDR-demethylation indeed correlated with a “true” Treg CD127−CTLA4+ phenotype and suppressive function, and described a new way to evaluate Treg subsets within isolated Tregs: the ratio of FOXP3+ cells to TSDR-demethylated cells (FOXP3/TSDR). Almost all Tregs from CNI patients had FOXP3/TSDR ratios <1, showing FOXP3 gene transcription was impaired, while TSDR FOXP3 demethylation was preserved. Conversely, most Tregs in rapamycin-treated patients had FOXP3/TSDR ratios >1, showing the Treg subset was enriched by suppressive, CTLA4+FOXP3+ cells with a methylated-TSDR, and indicative of iTregs (). Rapamycin stimulates iTreg conversion through inhibition of the mTOR pathway and enhancing TGF-β production by conventional T cells (2
Assessment of TSDR-demethylation is proposed as way to determine Treg numbers in PBMC or tissue samples (13
), but requires at least two conditions. First, every TDSR-demethylated cell should have normal FOXP3 transcription and hence, FOXP3 protein. Second, cells with methylated-TSDR should be unable to produce FOXP3 or, if FOXP3 is produced, the cells should not be suppressive. This concept does not adequately account for iTregs. TSDR-demethylation in PBMC post-Tx showed no correlations with Treg numbers, calculated as CD4+CD25+, CD4+CD25+FOXP3+ or even as CD4+CD25+TSDR-demethylated cells. We are unaware of any previous comparison of Treg numbers calculated using TSDR-demethylation in PBMC and TSDR-demethylation in Tregs, and note that TSDR-demethylation in PBMC varies with activation.
Our study design has several points to note. We excluded patients undergoing acute or chronic rejection, or those graft failure, allowing us to evaluate Tregs from patients with stable graft function and receiving conventional immunosuppression. However, patients with impaired grafts may provide additional insights. Second, we excluded patients with conditions affecting Treg number or function, such as cancer, autoimmune diseases, hepatitis and serious infections, to minimize their possibly confounding effects. Lastly, our study had a small size and cross-sectional design. Despite this, our composite approach showed that even in patients with stable graft function, CNI use may adversely affect Tregs. Serial studies including collection of samples preand post-Tx will likely produce key additional insights, and are now underway in our lab.