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Natural T regulatory cells (Tregs) are challenging to expand  ex vivo , and this has severely hindered  in vivo  evaluation of their therapeutic potential. All  trans  retinoic acid (ATRA) plays an important role in mediating immune homeostasis  in vivo , and we investigated whether ATRA could be used to promote the  ex vivo  expansion of Tregs purified from adult human peripheral blood. We found that ATRA helped maintain FOXP3 expression during the expansion process, but this effect was transient and serum-dependent. Furthermore, natural Tregs treated with rapamycin, but not with ATRA, suppressed cytokine production in co-cultured effector T cells. This suppressive activity correlated with the ability of expanded Tregs to induce FOXP3 expression in non-Treg cell populations. Examination of CD45RA+ and CD45RA− Treg subsets revealed that ATRA failed to maintain suppressive activity in either population, but interestingly, Tregs expanded in the presence of both rapamycin and ATRA displayed more suppressive activity and had a more favorable epigenetic status of the  FOXP3  gene than Tregs expanded in the presence of rapamycin only. We conclude that while the use of ATRA as a single agent to expand Tregs for human therapy is not warranted, its use in combination with rapamycin may have benefit.

Retinoic Acid and Rapamycin Differentially Affect and Synergistically Promote the Ex Vivo Expansion of Natural Human T Regulatory Cells