Epigenetic Analysis of the TSDR of FOXP3 Demonstrates That Natural Treg Infiltrate the Cardiac Allograft Already before an Acute Rejection Episode

Purpose Two subtypes of FOXP3+ regulatory T cells (Treg) have been identified. Natural (n) Treg originate in the thymus, while antigen specific induced (i) Treg are induced in the periphery by antigen exposure and cytokines. Their role in regulating allogeneic responses in immunosuppressed heart transplant patients is unknown. Therefore we investigated the origin of graft infiltrated FOXP3+ Treg and examined their immunosuppressive capacities by studying FOXP3 characteristics during acute rejection (AR) and immunological quiescence. Methods and Materials In total, 91 endomyocardial biopsies (EMB) were analyzed of 42 patients, 28 patients experienced at least 1 rejection episode requiring anti-rejection therapy (ISHLT rejection grade 2R; rejectors) and 14 patients remained free from rejection (non-rejectors). The percentage of demethylated TSDR (Treg Specific Demethylated Region) in the FOXP3 gene represents the percentage of nTreg. FOXP3 mRNA levels represent the total regulatory T cell population. Results In time-zero biopsies, no evidence for nTreg was detected. In grade 1R EMB of non-rejectors no significant accumulation of nTreg was observed. However, in rejectors, in all grade 1R EMB (taken before AR) and 2R EMB (AR) nTreg were detected with a significant higher percentage compared to EMB of non-rejectors (p≤0.001). FOXP3 mRNA levels significantly increased after transplantation with the highest expression in grade 2R EMB. Remarkably, no significant difference was observed in the FOXP3 mRNA levels in 1R EMB of non-rejectors compared to 1R EMB of rejectors (p=0.32), while the latter had significant evidence for a higher percentage nTreg (p=0.001), suggesting more iTreg in the non-rejectors. Conclusions These data show that nTreg infiltrate the cardiac allograft already before histologically proven AR, indicating that nTreg are unable to prevent the rejection process. The characteristics of the FOXP3 gene in EMB of non-rejectors support a role for antigen specific iTreg in the prevention of rejection.