Induction of Treg-type DNA hypomethylation and stable Treg function in in vitro- induced Treg cells by CD28 signal deprivation

Naturally occurring regulatory T cells (Treg) stably exert their suppressive function in vivo through stable expression of the master transcription factor Foxp3 and Treg-type CpG DNA demethylation. Meanwhile, in vitro induced Treg cells generated by conventional methods do not establish such Treg-type DNA demethylation so that they exhibit instability of Foxp3 expression and pathogenicity in vivo. Until now, the molecular mechanism that establishes stably functional Foxp3+ Treg cells with Treg-type demethylation in vitro has been largely unknown. Here, we found that CD28 co-stimulation constrains the establishment of Treg-type DNA demethylation during iTreg generation via PKC-NF-kB signaling pathway. iTreg cells generated without CD28 stimulation are more stable and suppressive in vitro and in vivo, which is sufficient to suppress allergic contact hyper sensitivity response. This provides a molecular-based evidence for the formation of lineage identity of Treg cells, which might accelerate development of safe and efficient iTreg cell therapy.