CD8⁺iTregs mediate the protective effect of rapamycin against graft versus host disease in a humanized murine model

CD8⁺Tregs are important immunoregulatory cells that participate in immunopathological processes in many diseases. Rapamycin (Rapa) is a macrolide immunosuppressant that inhibits the mammalian target of rapamycin (mTOR) and has been shown to improve CD4⁺-induced Tregs (iTregs) generation. This study aimed to evaluate the role of Rapa in the generation and function of CD8⁺iTregs. Human CD8 + CD25-CD45RA + T cells were divided into two groups, one with Rapa and the other without Rapa, and both groups were cultured under Treg-induced conditions. Rapa significantly improved Foxp3 expression and the suppressive function of CD8⁺iTregs in vitro. Further studies showed that Rapa suppressed inflammatory cytokine expression and enhanced anti-inflammatory cytokine expression. Under inflammatory conditions in vitro, Rapa-CD8 + iTregs sustained Foxp3 and anti-inflammatory cytokine expression. An in-depth study showed that Rapa regulated CpG demethylation in the Foxp3 region and STAT1 and STAT3 phosphorylation in CD8⁺iTregs. Finally, we compared the regulatory ability of Rapa and all-trans retinoic acid, another reagent that stimulates CD4⁺ iTreg generation in vitro, which showed that Rapa, but not all-trans retinoic acid, improved CD8⁺ iTreg induction and suppressed CD4⁺T cell expansion in vitro and protected against graft-versus-host disease in a humanized murine model in vivo. These results strongly suggest that CD8⁺iTregs initiated by Rapa may represent a new therapeutic strategy for inflammatory and autoimmune diseases.