Methylation of FOXP3 TSDR Underlies the Impaired Suppressive Function of Tregs from Long-term Belatacept-Treated Kidney Transplant Patients

Abstract

Regulatory T cells (Tregs) are considered key players in the prevention of allograft rejection in transplanted patients. Belatacept (BLT) is an effective alternative to calcineurin inhibitors that appears to preserve graft survival and function; however, the impact of this drug in the homeostasis of Tregs in transplanted patients remains controversial. Here, we analyzed the phenotype, function, and the epigenetic status of the Treg-specific demethylated region (TSDR) in FOXP3 of circulating Tregs from long-term kidney transplant patients under BLT or Cyclosporine A treatment. We found a significant reduction in the proportion of CD4+CD25hiCD127lo/−FOXP3+ T cells in all patients compared to healthy individual (controls). Interestingly, only BLT-treated patients displayed an enrichment of the CD45RA+ “naïve” Tregs, while the expression of Helios, a marker used to identify stable FOXP3+ thymic Tregs remained unaffected. Functional analysis demonstrated that Tregs from transplanted patients displayed a significant reduction in their suppressive capacity compared to Tregs from controls, which is associated with decreased levels of FOXP3 and CD25. Analysis of the methylation status of the FOXP3 gene showed that BLT treatment results in methylation of CpG islands within the TSDR, which could be associated with the impaired Treg suppression function. Our data indicate that analysis of circulating Tregs cannot be used as a marker for assessing tolerance toward the allograft in long-term kidney transplant patients. Trial registration number IM103008.

End‐stage renal disease, dialysis, kidney transplantation and their impact on CD4+ T‐cell differentiation

“…Recent studies also confirmed that neither frequencies nor the suppressive function of Treg cells were impaired in ESRD patients before and after the initiation of dialysis . However, similar to our studies, Treg frequencies and Treg function were shown to be reduced in long‐term kidney transplant patients . Our findings indicate that, similar to aging or pregnancy, renal insufficiency may provoke diminished thymic output of RTE Treg/Tresp cells and therefore accelerated peripheral differentiation of these cells.…”

Involvement of Tumor Necrosis Factor Receptor Type II in FoxP3 Stability and as a Marker of Treg Cells Specifically Expanded by Anti–Tumor Necrosis Factor Treatments in Rheumatoid Arthritis

“…We further showed that TNF induced CD25 expression in Treg cells and also increased IL‐2–induced CD25 and STAT5 phosphorylation in Treg cells. Phosphorylated STAT5 binds to demethylated CNS2, which contains the TSDR and thus may stabilize Foxp3 expression, leading to the high suppressive activity of Treg cells . On another note, the STAT5/STAT3 balance controls the fate of CD4+ T cells.…”

CTLA4Ig Improves Murine iTreg Induction via TGFβ and Suppressor Function In Vitro

“…Whereas some groups reported no short- or long-term effects on Treg numbers and function when compared to treatment with CNIs [ 46 , 47 ], others reported a decrease in Treg and FoxP3 mRNA levels [ 48 ]. The only conclusion from clinical experience with CD28 blockade via CTLA4Ig, which could be agreed on, was the fact that induction of tolerance with CTLA4Ig and current concomitant regimens was unlikely [ 13 , 49 ]. In mouse models, on the other hand, CTLA4Ig treatment seems to be able to favor regulatory mechanisms in order to induce an operational tolerant state.…”

“…Here, we have shown that CTLA4Ig does not negatively impact Treg conversion via TGF β in vitro , which in our opinion is of major relevance as it mimics the generation of allospecific pTregs in the periphery. Clinical data and murine studies suggest that in long-term kidney transplant patients, indirect allospecific T cells mainly contribute to late graft rejection [ 13 , 57 , 58 ]. As tTregs and pTregs are generally believed to represent distinct TCR repertoires, several reports have suggested a division of labor between those subsets [ 59 ].…”

“…Therefore, Tregs would be promising candidates for the intentional induction of transplantation tolerance or as part of calcineurin inhibitor- (CNI-) sparing immunosuppressive regimens, as chronic use of CNIs—still being the backbone of current immunosuppressive regimens—is associated with substantial side effects, including profound nephrotoxicity. Furthermore, previous studies already demonstrated that CNIs inhibit Treg function and have markedly negative effects on Tregs [ 12 ], including decreased FoxP3 expression and demethylation status and subsequent impaired suppressive capacity [ 13 ].…”