The Mechanism And Possibility Of Application Of TGF-β Induced Regulatory T Cells

AIM: To clarify the possibility and mechanism of TGF-βsuperfamily members BMPs affect the development of Foxp3⁺ Treg cells in vitro and in vivo.METHODS: Na?ve CD4⁺ T cells were separated from the spleen cells from wild type and transgenic mice. These cells were underwent TCR stimulation, with IL-2 (20 U/ml) for 3days. TGF-β(2 ng/ml), BMP-2 (10 ng/ml), or BMP-4 (10 ng/ml) was added in some culture. Cell was intracellular stained with Foxp3. Some conditions were added Noggin, P38 inhibitor, JNK inhibitor, or ERK inhibitor. The effect of phosphorylation Smad by BMP was analyzed by Weston Blot. In vivo, BMP was i.p. injected into the mouse. The percentage of CD4⁺CD25⁺Foxp3⁺T cells in the blood, lymph nodes and spleen was analyzed by FACS at day 12. Spleen CD4⁺CD25⁺ T cells was separated and assayed for assessment of the mixed lymphocyte reaction RESULT: BMP-2 and BMP-4, typical representatives of the BMPs, are unable to induce non-regulatory T cells to become Foxp3⁺ regulatory T cells. Neutralization studies with Noggin have revealed that BMP-2/4 and the BMP receptor signaling pathway is not required for TGF-βto induce naive CD4⁺CD25- cells to express Foxp3. However, BMP-2/4 and TGF-βhave a synergistic effect on the induction of Foxp3⁺ regulatory T cells. BMP-2/4 affects non-Smad signaling molecules including phosphorylated ERK and JNK, which could subsequently promote the differentiation of Foxp3⁺ regulatory T cells induced by TGF-β.CONCLUTION: TGF-βis a key signaling factor for Foxp3⁺ regulatory T cell development. BMP-2 and BMP-4 significantly increased the ability of TGF-βto promote the generation of Foxp3⁺ iTreg cells, and this synergistic effect was also dependent upon TGF-βreceptor signaling, as well as ERK and/or JNK MAPK pathways.AIM: To observation that Smad and non-Smad signaling pathways differentially affect the Th17 and Foxp3⁺ Treg cell differentiation.METHODS: Na?ve CD4⁺ T cells were separated from the spleen cells from wild type and transgenic mice. Under induction of Treg, cells were underwent TCR stimulation, with IL-2 (20 U/ml) for 3days. TGF-β(2 ng/ml), P38 inhibitor, JNK inhibitor, or ERK inhibitor, or atRA (10 nM) was added in some culture. The percentage of Foxp3⁺ in T cells was analyzed by FACS following the injection of TsA. T cells were isolated from spleen, LN and blood in EAE mice at day 18 after immunization, intracellular IL-17 expression was stained and analyzed by FACS.RESULT: TGF-βcan induce regulatory cells from Smad2 or Smad gene knockout mice. The MAKP JNK, and ERK inhibitor can down regulate the ability of Treg induction by TGF-β. AtRA also can favorate the Treg and inhibitor the Th17 cells in Smad3 knockout mice.CONCLUTION: Neither Smad2 nor Smad3 deficiency abrogates TGF-β-dependent iTreg induction by a deacetylase inhibitor Trichostatin A (TsA) in vivo although loss of Smad2 or Smad3 partially reduces iTreg induction in vitro. Similarly, Smad2 and Smad3 have a redundant role in development of Th17 in vitro and in experimental autoimmune encephalomyelitis (EAE). In addition, ERK and/or JNK pathways were shown to be involved in regulating iTregs, while the p38 pathway predominately modulates Th17 and EAE induction.AIM: The possibility to induce human regulatory cells with stable suppressor function both in vitro and in vivo.METHODS: Na?ve CD4⁺ T cells were separated from health donor PBMC. These cells were stimulated with anti-CD3CD28-beads. IL-2, TGF-β, and RAPA were added to some culture. The cells were harvested and stained the phenotypes of nature regulatory cells. Membran bound TGF-βwas stained after restimulation for 72 hours. The suppress function of different condition cells was analyzed by the dilution of CFSE of T cells. Human peripheral blood mononuclear cells (huPBMCs) with or whithout different condition cells were injected into RAG2^-/-γc^-/- mice. The weight and engraft human T cells of these mice were monitored.RESULT: Combine RAPA with TGF-βnot only enhanced FOXP3 expression by CD4⁺ cells, but also appeared more resistant to apoptosis compared to others. The combination of RAPA and TGF-βenabled CD4⁺ cells to express a phenotype and trafficking receptors similar to natural Tregs, and no significant cytotoxicity was observed. CD4RAPA were anergic and had potent in vitro suppressive activity. When transfer human peripheral blood mononuclear cells (huPBMCs) with CD4_RAPA into RAG2^-/-γc^-/- mice, CD4RAPA can decrease human cell engraftment and extend survival.CONCLUTION: Stimulation of human CD4+T cells in the presence of RAPA results in a highly increased suppressor function is TGF-βdependent, these cells also has stable in vivo suppressor function as compared with that of CD4+ T cells stimulated in the absence of the drug.