TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function

Abstract

T helper cells that produce IL-17 (T(H)17 cells) promote autoimmunity in mice and have been implicated in the pathogenesis of human inflammatory diseases. At mucosal surfaces, T(H)17 cells are thought to protect the host from infection, whereas regulatory T (T(reg)) cells control immune responses and inflammation triggered by the resident microflora. Differentiation of both cell types requires transforming growth factor-beta (TGF-beta), but depends on distinct transcription factors: RORgammat (encoded by Rorc(gammat)) for T(H)17 cells and Foxp3 for T(reg) cells. How TGF-beta regulates the differentiation of T cells with opposing activities has been perplexing. Here we demonstrate that, together with pro-inflammatory cytokines, TGF-beta orchestrates T(H)17 cell differentiation in a concentration-dependent manner. At low concentrations, TGF-beta synergizes with interleukin (IL)-6 and IL-21 (refs 9-11) to promote IL-23 receptor (Il23r) expression, favouring T(H)17 cell differentiation. High concentrations of TGF-beta repress IL23r expression and favour Foxp3+ T(reg) cells. RORgammat and Foxp3 are co-expressed in naive CD4+ T cells exposed to TGF-beta and in a subset of T cells in the small intestinal lamina propria of the mouse. In vitro, TGF-beta-induced Foxp3 inhibits RORgammat function, at least in part through their interaction. Accordingly, lamina propria T cells that co-express both transcription factors produce less IL-17 (also known as IL-17a) than those that express RORgammat alone. IL-6, IL-21 and IL-23 relieve Foxp3-mediated inhibition of RORgammat, thereby promoting T(H)17 cell differentiation. Therefore, the decision of antigen-stimulated cells to differentiate into either T(H)17 or T(reg) cells depends on the cytokine-regulated balance of RORgammat and Foxp3.

Figure 1. Co-expression of Foxp3 and RORγt in vitro and in vivo

a, Naïve CD4⁺ T cells were stimulated with anti-TCR and 5 ng/ml TGF-β for 48 hours, stained with DAPI (blue nuclear stain), anti-RORγ (red) and anti-Foxp3 (green) mAbs. All panels are of the same section, with Foxp3 and RORγ channels only (bottom), overlay of Foxp3 and RORγ channels (top right), and overlay of all three channels (top left). Foxp3, RORγt, and double expressing (DP) cells are indicated with colored arrows. b, Analysis of Foxp3⁺RORγt⁺ cells from the small intestinal lamina propria (LP). CD4⁺GFP^int and CD4⁺GFP^- cells were sorted from LP of RORc(γt)^+/gfp and RORc(γt)^gfp/gfp mice and Foxp3 expression was examined by intracellular staining. Results are representative of three experiments. c, Expression of IL-17 in Foxp3⁺RORγt⁺ and Foxp3^-RORγt⁺ T cells from small intestine. Foxp3 and IL-17 expression was examined by intracellular staining of sorted TCRβ⁺CD4⁺GFP^int cells from LP of RORc(γt)^+/gfp mice.

Figure 2. TGF-β inhibits RORγt-directed IL-17 production by up-regulating Foxp3

a, Effect of TGF-β on IL-17 expression following transduction of RORγt. Naïve CD4⁺ T cells incubated with TGF-β from day -1 were transduced with control vector (MIG) or RORγt-IRES-GFP (RORγt) on day 0 (24 h after TCR activation) and IL-17 intracellular staining was performed on day 5. b, Inhibitory effect of TGF-β when included at different times relative to transduction of RORγt. Percentage of IL-17⁺ cells among RORγt/GFP⁺ cells is shown. c, Knockdown of TGFβ-induced Foxp3 expression with an shRNA against Foxp3 (LMP1066). Naïve CD4⁺ T cells were stimulated as in (a) and co-transduced on days 0 and 1 with retroviral constructs encoding RORγt and the specific shRNA vector (LMP1066) or control vector (LMP). After transduction, the cells were cultured with or without TGF-β, and Foxp3 expression was measured by intracellular staining on day 5. d, Restoration of RORγt-induced IL-17 expression upon knock-down of Foxp3. IL-17 expression was assessed in cells co-transduced as in (c) and gated for the level of GFP expression. Percentage of IL-17⁺ T cells in GFP^hi cell populations is shown. Results with additional shRNA vectors that failed to down-regulate Foxp3 expression were similar to those with the control LMP vector. Representative data from three experiments are shown.

Figure 3. Foxp3 interacts with RORγt and inhibits RORγt-directed IL-17 expression

a, Co-immunoprecipitation of Foxp3 and RORγt from extracts of co-transfected 293T cells with or without DNaseI or ethidium bromide. Cells were transfected with murine RORγt and Flag-tagged wild-type (Flag-Foxp3) or exon 2-deleted Foxp3 (Flag-Foxp3ΔEx2). Anti-Flag immunoprecipitates and total lysates were immunoblotted with anti-RORγt antibody and anti-Flag antibody. b, Cells were transfected with Flag-tagged human RORγt and full-length (FL) or the exon 2-deleted isoform of human FOXP3 (ΔEx2). Anti-Flag immunoprecipitates and total lysates were probed with anti-FOXP3 and anti-Flag antibodies. c, Naïve CD4⁺ T cells were co-transduced with retroviruses encoding RORγt (MIT vector, Thy1.1 reporter) and various murine Foxp3 constructs (MIG vector, GFP reporter). IL-17 expression was assessed on day 4 in cells gated for expression of both Thy1.1 and GFP. Representative data from at least three experiments are shown in each of the panels.

Figure 4. TGF-β concentration influences IL-23R expression and levels of IL-17 in response to Th17-inducing cytokines

a, Foxp3-mediated inhibition of IL-6/IL-21-induced IL-23R expression. Naïve CD4⁺ T cells were transduced with MIG, full length Foxp3, or Foxp3ΔEx2 viruses and were treated with the indicated cytokines. RNA was isolated from GFP⁺ cells at day 2. IL-23R expression was measured by real-time RT-PCR and was normalized to the actin level. Error bars represent standard deviations obtained using the standard curve method. b, Induction of IL-23R mRNA in response to cytokines. Naïve CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 throughout the culture period in the presence of the indicated combinations of cytokines. TGF-β was titrated into the cultures at the concentrations of 5 ng/ml, 2.5 ng/ml, 1 ng/ml, 200 pg/ml, 40 pg/ml, or 8 pg/ml. IL-23R mRNA expression was measured after 48 h by real-time RT-PCR and was normalized to the actin expression level. c and d, IL-23 enhancement of IL-17 expression at low concentrations of TGF-β. Percent IL-17⁺ cells at 96 hours of stimulation with the indicated cytokines is shown. Results in histogram format are shown in Supplemental Fig. 7. e, Induction of Foxp3 at different concentrations of TGF-β. Representative data from at least three experiments are shown for each set of panels.