Alloantigen-Induced Regulatory T Cells Generated in Presence of Vitamin C Display Enhanced Stability of Foxp3 Expression and Promote Skin Allograft Acceptance

Abstract

Regulatory T cells (Tregs) are critical for the maintenance of immune homeostasis and self-tolerance and can be therapeutically used for prevention of unwanted immune responses such as allotransplant rejection. Tregs are characterized by expression of the transcription factor Foxp3, and recent work suggests that epigenetic imprinting of Foxp3 and other Treg-specific epigenetic signatures genes is crucial for the stabilization of both Foxp3 expression and immunosuppressive properties within Tregs. Lately, vitamin C was reported to enhance the activity of enzymes of the ten-eleven translocation family, thereby fostering the demethylation of Foxp3 and other Treg-specific epigenetic signatures genes in developing Tregs. Here, we in vitro generated alloantigen-induced Foxp3+ Tregs (allo-iTregs) in presence of vitamin C. Although vitamin C hardly influenced the transcriptome of allo-iTregs as revealed by RNA-seq, those vitamin C-treated allo-iTregs showed a more pronounced demethylation of Foxp3 and other Treg-specific epigenetic signatures genes accompanied with an enhanced stability of Foxp3 expression. Accordingly, when being tested in vivo in an allogeneic skin transplantation model, vitamin C-treated allo-iTregs showed a superior suppressive capacity. Together, our results pave the way for the establishment of novel protocols for the in vitro generation of alloantigen-induced Foxp3+ Tregs for therapeutic use in transplantation medicine.

Vitamin C supports conversion of human γδ T cells into FOXP3-expressing regulatory cells by epigenetic regulation

“…For the first time, we could demonstrate that pVC induced the demethylation of FOXP3 TSDR in human γδ T cells, as revealed by analyzing sorted FOXP3 + and FOXP3 − cells from γδ T-cell cultures supplemented with TGF-β and pVC. These results are well in line with previous reports on the induction of FOXP3 TSDR demethylation and stability by VC in murine CD4 T cells 23,24,44 . Remarkably, however, such FOXP3 TSDR demethylation was only observed in FOXP3 + γδ T cells sorted from [TGF-β + pVC]-supplemented cultures and not in FOXP3 + sorted γδ T cells derived from cultures with TGF-β but without pVC ( Fig.…”

“…A superior immunosuppressive capacity of these cells was reported 59 . Interestingly, a previous study demonstrated that the addition of VC to alloantigen-specific αβ Treg cultures led to the generation of a stable Treg population (epigenetically akin to naturally occurring Treg) with enhanced ability to promote (2020) 10:6550 | https://doi.org/10.1038/s41598-020-63572-w www.nature.com/scientificreports www.nature.com/scientificreports/ skin allograft acceptance 44 . Although some studies have highlighted the potential involvement of regulatory γδ T cells in the pathogenesis of some autoimmune diseases [60][61][62] , others provided evidence that γδ T cells could exert regulatory functions resulting in the prevention of autoimmune disorders 63,64 .…”

Large-Scale Generation of Human Allospecific Induced Tregs With Functional Stability for Use in Immunotherapy in Transplantation

“…It is interesting to point out that some clones acquired total demethylation TSDR, so the refinement in their characterization could improve the isolation of stable iTregs with an established TSDR demethylation pattern over the course of the culture. In this context, recent reports have shown that is possible to increase the demethylation status of the TSDR region by adding vitamin C to iTreg cultures (56,57), which was reported to be an activator of TET enzymes, which, in turn, control the stability of FOXP3 (47).…”

The Impact of Dietary Components on Regulatory T Cells and Disease

“…Calcitriol, a vitamin D metabolite, enhanced the growth of Tregs (141,142). Vitamin C has been found to increase the generation of FOXP3 + iTregs on alloantigenspecific Treg induction cultures and to cause a pronounced TSDR demethylation, resulting in an elevated FOXP3 stability (143). Interestingly, vitamin C treatment may act in a distinct manner on tTreg and iTreg function.…”