Different and synergistic effect of 5-Azacytidine and Rapamycin on ex vivo expansion of natural human T Regulatory cells

Natural T regulatory cells (Treg) are challenging to expand ex vivo, and this has severely hindered in vivo evaluation of their therapeutic potential. 5-Azacytidine (5-azaC) and Rapamycin (RAPA) are immunosuppressive drugs that promote selectively the expansion of CD4+CD25highFoxp3+ regulatory T cells. We investigated whether 5-azaC and RAPA could be used together to promote the ex vivo expansion of Tregs purified from adult human peripheral blood.
: We found that 5-azaC helped maintain FOXP3 expression during the expansion process probably by promoting the conversion of T conventional (Tconv) in Treg, instead Rapa induces selectively apoptosis in Tconv cells and expansion in Treg. Addition of 5-azaC to RAPA treated cultures improved gene expression of FOXP3, CD25, STAT5 and TGF-B resulted in enhanced Treg expansion and suppressive activity. Also Rapa and 5-AzaC combination sustain Bcl-2 protein expression in Treg conferring resistance to apoptosis process. 5-azaC may have utility in ex vivo expansion of human Tregs, not as a single agent, but in combination with RAPA. These data may considerably accelerate the development of immunotherapeutic approaches for the treatment of autoimmune disease or posttransplant alloreactions by the adoptive transfer of nTreg cells.