Epigenetic Analysis Demonstrates that Natural Treg Only Infiltrate the Cardiac Allograft During an Acute Rejection Episode

Abstract

Introduction: FOXP3+ regulatory T cells (Treg) infiltrate the cardiac allograft after transplantation; however, it is unknown whether these infiltrating cells are derived from the thymus (nTreg) or are induced by antigen in the periphery (iTreg). Using demethylation of the FOXP3 gene as a marker for nTreg, we investigated the origin of infiltrating cells in heart transplant biopsies during both immunological quiescence and acute rejection. Methods: The proportion of demethylated FOXP3 DNA was determined with a quantitative PCR based method with a sensitivity limit of 0.06%. In total, 66 EMB (endomyocardial biopsies) were analyzed of 17 heart transplantation patients who experienced at least 1 rejection episode requiring anti-rejection therapy (ISHLT rejection grade 2R; rejectors) within the first 3 months after transplantation and 9 patients who remained free from rejection during this period (non-rejectors). EMB were taken before transplantation (time-zero biopsies; n=24), before and during acute rejection (AR) for the rejectors (n=28) and within the first 3 months for the non-rejectors (n=14). Results: In time-zero biopsies, no evidence for nTreg was detected. However, after transplantation the proportion of nTreg marker significantly increased in the EMB where cellular infiltrates were present. Grade 1R EMB (n=19) displayed a proportion of 0.22% nTreg and grade 2R EMB (n=16) 0.52% nTreg (p< 0.0001 versus time-zero). Within the grade 1R EMB, including 9 EMB from rejectors and 10 EMB from non-rejectors, the proportional increase in nTreg marker was predominantly observed in the rejectors (0.51% versus 0.05% for non-rejectors; p< 0.01). In all EMB from non-rejectors (0R and 1R) no evidence for accumulation in nTreg marker was observed. Conclusion: In conclusion, rejectors display a higher proportion of nTreg marker after heart transplantation, already before histologically proven AR occurs. This indicates that nTreg home in the graft during alloreactivity where they dampen rather than prevent the rejection process.