CD4+ regulatory T cells control TH17 responses in a Stat3-dependent manner

Abstract

Distinct classes of protective immunity are guided by activation of STAT transcription factor family members in response to environmental cues. CD4+ regulatory T cells (T(regs)) suppress excessive immune responses, and their deficiency results in a lethal, multi-organ autoimmune syndrome characterized by T helper 1 (TH1) and T helper 2 (TH2) CD4+ T cell-dominated lesions. Here we show that pathogenic TH17 responses in mice are also restrained by T(regs). This suppression was lost upon T(reg)-specific ablation of Stat3, a transcription factor critical for TH17 differentiation, and resulted in the development of a fatal intestinal inflammation. These findings suggest that T(regs) adapt to their environment by engaging distinct effector response-specific suppression modalities upon activation of STAT proteins that direct the corresponding class of the immune response.

Fig. 1

Phosphorylated Stat3 interacts with Foxp3 and its expression in Treg cells is required for suppression of fatal colitis. (A) Foxp3 immunoprecipitation from nuclear lysates of sorted CD4⁺Foxp3⁻ and CD4⁺Foxp3⁺ cells followed by western blot analysis for the indicated proteins. The input lanes represent 3–5% of cell equivalents used for Foxp3 immunoprecipitation. (B) Spleen and lymph node cellularity. ** P<0.001; * P<0.01. (C) Examples of colon thickening. (D) Weight loss over time (n = 7). (E) IBD scores derived from histopathologic evaluation of colon and cecum from 8–9 week-old mice. Formalin fixed sections were H&E stained prior to examination (n = 4/group). (F) Representative H&E stained colon sections from 8–9 week old mice (original magnification, 20x).

Fig. 2

Stat3 ablation in Treg cells does not affect their numbers, yet leads to CD4⁺ T cell activation and selective increase in Th17 responses. (A) CD4⁺ and CD8⁺ T cell numbers in the spleen and lymph nodes. ** P<0.001; * P<0.01. (B) Flow cytometric analysis of CD44 and CD62L expression on CD4⁺ Foxp3⁻ T cells in 6–8 wk-old mice. A representative of five independent experiments is shown. (C) Frequency and numbers of CD4⁺Foxp3⁺ T. ** P<0.001; * P<0.01. (D) Cytokine production by splenic CD4⁺Foxp3⁻ T cells as determined by flow cytometric analysis. A representative of four independent experiments is shown.

Fig. 3

Increased IL-17 production triggers IBD development in Foxp3^CreStat3^fl/fl mice. (A) Total numbers of PP cells, IEL and LPL as well as CD4⁺, CD8⁺, CD11b⁺ cell numbers in the IEL population in 8 wk-old mice. (B) Frequencies of indicated cytokine secreting CD4⁺ and CD8⁺ T cells in PP, IEL and LPL populations in 7–8 week old mice. (C) Frequencies of indicated cytokine producing CD4⁺ and CD8⁺ T cells in PP, IEL and LPL in 3–4 wk-old mice. (D) Flow cytometric analysis of cytokine production by splenic CD4⁺Foxp3⁻ T cells in Foxp3^CreStat3^fl/fl mice treated with isotype-matched IgG or IL-17 neutralizing antibody. A representative of three independent experiments is shown. (E) Weight loss and IBD scores and (F) representative H&E stained colon sections from 7–8 wk-old Foxp3^CreStat3^fl/fl mice treated with isotype-matched IgG control or neutralizing IL-17 antibody (original magnification, 10x).

Fig. 4

Stat3-deficient Treg cells efficiently suppress in vitro T cell proliferation, but fail to suppress Th17 cell differentiation and colitis. (A) Stat3-sufficient and -deficient Treg cells from 5–6 wk-old mice suppress in vitro proliferative response of CD4⁺Foxp3⁻ T cells (Teff). A representative of three independent experiments is shown. (B) Frequencies and numbers of splenic Treg cells, CD4⁺IL-17⁺Foxp3⁻, CD4⁺IFN-γ⁺Foxp3⁻, CD4⁺IL-4⁺Foxp3⁻ T cells within the indicated donor-derived population 4 weeks after co-transfer of either Foxp3^CreStat3^fl/wt or Foxp3^CreStat3^fl/fl Treg cells with Foxp3⁻ Teff cells into Rag2^−/− recipients. (C) Representative H&E stained sections of cecum, skin, and salivary gland tissues collected from recipient mice 4 wk after adoptive T cell transfer (original magnification, 20x).

Fig. 5

Stat3-dependent gene expression in Treg cells. (A, B) Expression pattern of Stat3dependent genes potentially contributing to Treg suppressor function. The data represent average of two independent microarray experiments. qPCR analysis of relative expression of indicated genes in YFP-Cre⁺ Treg cells from indicated mice. The results represent mean and standard deviation of relative expression values for indicated genes over Hprt in two independent experiments using three replicates each. (C) Flow cytometric analysis of CCR6, IL-1R and IL-6R expression on Tregs from indicated mice. (D) ELISA and western blot analysis of amounts of TGF-β1, IL-6, and Ebi3 in supernatants of Stat3-sufficient and -deficient Tregs cultured in presence of IL-2. (E) Ratio of Foxp3⁺YFP⁺ to Foxp3⁺YFP⁻ Tregs in spleen, IEL and LPL populations of indicated mice. (F) CD4⁺Foxp3⁻ were stimulated with anti-CD3 in the presence of culture supernatants derived from Tregs isolated from indicated mice and the frequency of CD4⁺IL-17⁺ cells was assessed by flow cytometry. (G) qPCR analysis of Foxp3- and Stat3-bound chromatin isolated from wild-type Tregs using primer set corresponding to the promoter region of the indicated genes. The housekeeping gene Gmpr was used as a specificity control.