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-β. 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 nai{dotless}̈ve 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 nai{dotless}̈ve CD4+ cells suboptimally activated with IL-2 and TGF-β enhanced and stabilized FOXP3 expression, and accelerated their maturation to protective regulatory T cells. AtRA, by itself, accelerated conversion of nai{dotless}̈ve to mature cells but did not induce FOXP3 or suppressive activity. The combination of atRA and TGF-β enabled CD4⁺CD45RA⁺ cells to express a phenotype and trafficking receptors similar to natural Tregs. AtRA/TGF-β-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-antimouse GVHD as well as expanded endogenous Tregs. However, treatment of endogenous Tregs with IL-1b 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.