For example, while costimulatory signals generated by the engagement of CD80/86 and CD28 is indispensable for tTreg development ( 14– 17), pTreg generation in the intestine was reportedly normal in CD28-deficient mice ( 18). However, specific contribution of costimulatory signaling in pTreg or iTreg development has been controversial. Previous studies have demonstrated that TCR signaling is an essential requirement for Treg-type DNA hypomethylation as well as the expression of Foxp3 and other Treg function-associated genes in developing tTregs ( 7, 11– 13). Key questions are, therefore, how nTregs acquire Treg-specific DNA hypomethylation in the course of their physiological development in the thymus and the periphery ( 10) and how Treg-specific DNA hypomethylation can be generated de novo in iTregs for their clinical use to suppress immune responses stably. In contrast with tTregs and pTregs, iTregs are unstable in the expression of Foxp3 and other Treg signature genes mainly because of incomplete epigenetic changes at Treg-DRs, and can be driven, under certain in vivo conditions, to differentiate into effector T cells ( 7). In particular, Treg-specific DNA demethylation at specific genomic regions (hereafter referred to as Treg-specific demethylated regions or Treg-DRs) of Treg signature genes such as Foxp3, Cd25, and Ctla4 contributes to continued high expression of these genes and, consequently, leads to robust and stable Treg phenotype and function ( 4– 10). The Treg-specific epigenetic changes reinforce the Treg lineage determination/stability and acquisition of stable immunosuppressive function.
Both tTregs and pTregs possess epigenetic signatures distinct from those of Tconvs (reviewed in ref. Foxp3 + Tregs phenotypically similar to tTregs or pTregs can also be generated in vitro (induced Tregs ) from Tconvs by antigen stimulation in the presence of TGF-β and IL-2 ( 3). While a majority of nTregs present in the immune system are thymus-derived (thymus-derived Tregs ), a proportion of Foxp3 + Tregs appear to be generated in the periphery (peripherally derived Tregs ) from conventional T cells (Tconvs) under certain conditions ( 2). Naturally occurring regulatory T cells (nTregs), which specifically express the transcription factor Foxp3, play indispensable roles in the maintenance of immunological self-tolerance and homeostasis ( 1).
Taken together, inhibition of the CD28-PKC-NF-κB signaling pathway in iTreg generation enables de novo acquisition of Treg-specific DNA hypomethylation at Treg signature genes and abundant production of functionally stable antigen-specific iTregs for therapeutic purposes. These iTregs indeed stably expressed Foxp3 after in vivo transfer and effectively suppressed antigen-specific immune responses. In addition, in vitro maintenance of such epigenome-installed iTregs with IL-2 alone, without additional TGF-β or antigenic stimulation, enabled their expansion and stabilization of Treg-specific DNA hypomethylation. Furthermore, CD28 signal deprivation significantly reduced c-Rel expression in iTregs and the specific genomic perturbation of a NF-κB binding motif at the Foxp3 CNS2 locus enhanced the locus-specific DNA hypomethylation even in CD28 signaling-intact iTregs. Among various cytokines and signal activators/inhibitors, TNF-α and PKC agonists inhibited the hypomethylation. The signal abrogation could induce Treg-type hypomethylation in memory/effector as well as naive Tconvs, while hindering Tconv differentiation into effector T cells. It was achieved, for example, by TCR/TGF-β/IL-2 stimulation of CD28-deficient Tconvs or CD28-intact Tconvs without anti-CD28 agonistic mAb or with CD80/CD86-blocked or -deficient antigen-presenting cells.
Here we show that deprivation of CD28 costimulatory signal at an early stage of iTreg generation is able to establish Treg-specific DNA hypomethylation at Treg signature genes. However, unlike Foxp3 + naturally occurring Tregs, such in vitro induced Tregs (iTregs) are functionally unstable mainly because of incomplete Treg-type epigenetic changes at Treg signature genes such as Foxp3. Foxp3-expressing regulatory T cells (Tregs) can be generated in vitro by antigenic stimulation of conventional T cells (Tconvs) in the presence of TGF-β and IL-2.
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