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

TGF-b and Retinoic Acid. PLoS ONE 5(12): e15150. doi:10.1371/journal.pone.0015150 + + Characterization of Protective Human CD4 CD25 + FOXP3 Regulatory T Cells Generated with IL-2, TGF-b and Retinoic Acid Ling Lu 0 Xiaohui Zhou 0 Julie Wang 0 Song Guo Zheng 0 David A. Horwitz 0 Derya Unutmaz, New York University, United States of America 0 1 Division of Rheumatology, Department of Medicine, Keck School of Medicine at University of Southern California, Los Angeles, California, United States of America, 2 Department of Liver Transplantation, First Affiliated Hospital of Nanjing Medical University , Nanjing , China , 3 Immune Tolerance Center Shanghai East Hospital, Tonji University of Medicine , Shanghai , China 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-b. 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 nave 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 nave CD4+ cells suboptimally activated with IL-2 and TGF-b enhanced and stabilized FOXP3 expression, and accelerated their maturation to protective regulatory T cells. AtRA, by itself, accelerated conversion of nave to mature cells but did not induce FOXP3 or suppressive activity. The combination of atRA and TGF-b enabled CD4+CD45RA+ cells to express a phenotype and trafficking receptors similar to natural Tregs. AtRA/TGF-b-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. - CD4+ regulatory T cells (Tregs) bearing the Forkhead Box P3 (Foxp3) transcription factor are required to maintain immunologic homeostasis and prevent autoimmunity [1,2]. Mutations of the Foxp3 gene result in immune dysregulation and multiorgan autoimmunity [3]. Both CD4+ cells and CD8+ cells can express Foxp3 [4,5], but the former have received the most attention. Because abnormalities in the numbers and function of Tregs can lead to autoimmunity, allergy and graft rejection, manipulation of these cells to correct these defects offers a novel treatment strategy [6]. Endogenous CD4+Foxp3+ cells can be divided into thymusderived, natural regulatory T cells (nTregs) which constitutively express high levels of CD25, the IL-2 receptor alpha chain and those induced in the periphery from CD4+CD252Foxp32 precursors by a TGF-b dependent mechanism (iTregs). In mice and humans these two subsets have been indistinguishable phenotypically until recently [7], and may have separate or synergistic roles in vivo [8,9]. In humans CD4+FOXP3+ Tregs express high levels of CD25 and low levels of CD127, the IL-7 receptor alpha chain [10]. In addition to endogenous Foxp3+ Tregs, substantial evidence exists that the combination of IL-2 and TGF-b can induce nave CD4+CD252 cells to become FOXP3+ iTregs in both mice and humans. In mice, suboptimal polyclonal TCR stimulation of nave CD4+ cells with IL-2 and TGF-b can induce iTregs that have protective effects in autoimmune diabetes [11], experimental autoimmune encephalitis[12] and myasthenia gravis [13]. Because of decreased numbers and/or function of FOXP3+ Tregs in human autoimmune diseases [14], the transfer of iTregs generated ex-vivo could be therapeutic to subjects with these diseases. In humans CD4+CD252 cells activated by either superantigens or alloantigens with IL-2 and TGF-b developed potent in vitro suppressive activity [15,16], and these alloantigen-induced FOXP3+ iTregs could also induce other CD4+CD252 cells to become TGF-b dependent suppressor cells [17]. One group recently also reported that polyclonal TCR stimulation of nave CD4+ cells with TGF-b could result in FOXP3+ suppressor cells [18]. However, the generation of fully functional polyclonal human FOXP3+ iTregs ex vivo is controversial. First, TCR activation without TGF-b can induce naive CD4+ cells to transiently express FOXP3 [19]. Secondly, although we and others have observed that TGF-b can greatly increase FOXP3 expression and stability, after one week in vitro suppressive activity of these human CD4+ cells was not greater than control cells [20,21]. Moreover, unlike nTregs which are anergic in response to TCR stimulation, these human CD4+ cells primed with TGF-b produced IL-2 and proliferated robustly following re-stimulation. Interestingly, however, repeated stimulation of TGF-b primed CD4+ cells did result in anergy, membrane-expression of TGF-b, and in vitro suppressive activity similar to that described with nTregs [20,22]. We concluded that human TGF-b primed CD4+ cells one week after culture were partially differentiated cells and required a much longer time to mature than similar mouse Foxp3+ iTregs [21]. Thus, agents that accelerate cell differentiation might be useful for a more rapid generation of human iTregs ex-vivo. Retinoic acid (RA), a vitamin A derivative, has an important role in the development of various organs including the immune system. RA metabolites strongly contribute to the maintenance of immunologic tolerance. All-trans retinoic acid (atRA), an active metabolite of retinoic acid, markedly enhances TGF-b-induced Foxp3 expression and stability in mice [23], and the expansion of these iTregs by either direct cytokine-dependent [24] or cytokine independent mechanisms [25]. In human CD4+ cells, atRA has been reported to induce histone acetylation at the FOXP3 gene promoter and expression of the FOXP3 protein [26]. Recently, atRA has been shown to enhance the stability and expansion of TGF-b induced iTreg and endogenous nTreg cells [27]. Here we have extensively characterized the phenotype and functional properties of iTregs induced by TGF-b a (...truncated)