Characterization of Protective Human CD4+CD25+ FOXP3+ Regulatory T Cells Generated with IL-2, TGF-β and Retinoic Acid

Background: Protective CD4+CD25+ regulatory T cells bearing the Forkhead Foxp3 transcription factor can now be divided into three subsets: Endogenous thymus-derived cells, those induced in the periphery, and another subset induced ex-vivo with pharmacological amounts of IL-2 and TGF-beta. Unfortunately, endogenous CD4+CD25+ regulatory T cells are unstable and can be converted to effector cells by pro-inflammatory cytokines. Although protective Foxp3+CD4+ CD25+ cells resistant to proinflammatory cytokines have been generated in mice, in humans this result has been elusive. Our objective, therefore, was to induce human naive CD4+ cells to become stable, functional CD25+ Foxp3+ regulatory cells that were also resistant to the inhibitory effects of proinflammatory cytokines. Methodology/Principal Findings: The addition of the vitamin A metabolite, all-trans retinoic acid (atRA) to human naive CD4+ cells suboptimally activated with IL-2 and TGF-beta enhanced and stabilized FOXP3 expression, and accelerated their maturation to protective regulatory T cells. AtRA, by itself, accelerated conversion of naive to mature cells but did not induce FOXP3 or suppressive activity. The combination of atRA and TGF-beta enabled CD4+ CD45RA+ cells to express a phenotype and trafficking receptors similar to natural Tregs. AtRA/TGF-beta-induced CD4+ regs were anergic and low producers of IL-2. They had potent in vitro suppressive activity and protected immunodeficient mice from a human-anti-mouse GVHD as well as expanded endogenous Tregs. However, treatment of endogenous Tregs with IL-1 beta and IL-6 decreased FOXP3 expression and diminished their protective effects in vivo while atRA-induced iTregs were resistant to these inhibitory effects. Conclusions/Significance: We have developed a methodology that induces human CD4(+) cells to rapidly become stable, fully functional suppressor cells that are also resistant to proinflammatory cytokines. This methodology offers a practical novel strategy to treat human autoimmune diseases and prevent allograft rejection without the use of agents that kill cells or interfere with signaling pathways.