Epigenetically modifying the Foxp3 locus for generation of stable antigen-specific Tregs as cellular therapeutics

Abstract

Foxp3+ regulatory T cells (Tregs) are potent immunoregulatory cells, prompting strong interests in manipulating them for therapeutic purposes. However, significant challenges remain, including their heterogeneity and functional instability. Here we focused on the inducible Tregs (iTregs) and studied whether the Foxp3 locus can be epigenetically edited ex vivo to produce stable therapeutic iTregs. Under iTreg‐inducing condition where activated CD4+ T effector cells were converted to Foxp3+ Tregs, we tested approximately 30 compounds and identified 3 chromatin‐modifying chemical compounds (3C) consisting of sodium butyrate (a broad histone deacetylase inhibitor), UNC0646 (a histone methyltransferase inhibitor), and vitamin C (a TET dioxygenase co‐activator), that together produced complete demethylation at the conserved noncoding sequence 2 (CNS2) region of Foxp3 locus. We found that iTregs induced in the presence of 3C (3C‐iTregs) are stable, even after exposure to inflammatory cytokines. They expressed high levels of Foxp3 and exhibited potent suppressive activities both in vitro and in vivo. We showed that in models of autoimmunity and transplant rejection, adoptive transfer of antigen‐specific 3C‐iTregs prevented the induction of experimental autoimmune encephalitis and enabled long‐term skin allograft survival. Our data demonstrate that the Foxp3 locus can be epigenetically edited ex vivo to generate stable therapeutic iTregs.

Regulatory T Cells-Related Genes Are under DNA Methylation Influence

“…This is because T effectors may acquire transient Foxp3 expression upon activation [63]. Considering the Tregs populations, nTregs acquire stable and profound TSDR demethylation, while iTregs remain almost completely methylated, which makes them less stable during cell culture [64]. In addition, Tregs that are generated in vivo have similar, albeit lower TSDR demethylation compared to nTregs [65].…”

“…The research conducted on mice has shown a beneficial effect of stimulated iTregs on the prevention of GvHD or allograft rejection [86,87]. More complex research on iTregs, including the cooperation of three epigenetic modifiers, including vitamin C, has disclosed a deep CNS2 demethylation in stimulated cells with high stability and usefulness in therapeutic trials [64].…”

IL-4 inhibits regulatory T cells differentiation by HDAC9-mediated epigenetic regulation

“…Independent of the known anticancer effects of HDACi, such as proapoptotic activity or cell cycle arrest induction 18 , the anti-inflammatory functions of these agents have recently aroused interest 19 . Previous researches have showed that epigenetic regulation play roles in Treg stability 20 ; for example, the HDACi trichostatin A (TSA) prevented the differentiation of human Foxp3 + Tregs into IL-17 producing cells 21 . Among the multiple HDACs Treg expresses, HDAC9 was proved to play an important role in regulating Foxp3-dependent suppression 22 .…”

Suppressive effects of vitamin C-treated induced-regulatory T cells on heart allograft rejection under vitamin C-deficient or –sufficient conditions

“…Previous studies showed that V-iTregs prolonged skin allograft survival to a greater extent than did C-iTregs, which is consistent with their better in vitro suppressive activity. However, these studies had induced V-iTregs with vitamin C together with vitamin A or sodium butyrate [19,27]. On the other hand, systemic administration of vitamin C alone did not improve skin allograft survival in vitamin C-sufficient RAG KO mice, although vitamin C increased generation of iTregs and Foxp3 levels [25].…”

“…A limitation associated with the current study was that we did not combine vitamin C with other agents, such as vitamin A and histone modifiers, for stabilization of Foxp3 in iTregs to boost the beneficial effects of vitamin C on iTregs [16,27,29,30]. P2X7 receptor blockade suppressed effector T cells or induced Tregs, thereby suppressing ischemia-reperfusion injury and allograft rejection [31,32].…”