Galectin-9-CD44 interaction enhances stability and function of adaptive regulatory T cells - PubMed (original) (raw)

Galectin-9-CD44 interaction enhances stability and function of adaptive regulatory T cells

Chuan Wu et al. Immunity. 2014.

Abstract

The β-galactoside-binding protein galectin-9 is critical in regulating the immune response, but the mechanism by which it functions remains unclear. We have demonstrated that galectin-9 is highly expressed by induced regulatory T cells (iTreg) and was crucial for the generation and function of iTreg cells, but not natural regulatory T (nTreg) cells. Galectin-9 expression within iTreg cells was driven by the transcription factor Smad3, forming a feed-forward loop, which further promoted Foxp3 expression. Galectin-9 increased iTreg cell stability and function by directly binding to its receptor CD44, which formed a complex with transforming growth factor-β (TGF-β) receptor I (TGF-βRI), and activated Smad3. Galectin-9 signaling was further found to regulate iTreg cell induction by dominantly acting through the CNS1 region of the Foxp3 locus. Our data suggest that exogenous galectin-9, in addition to being an effector molecule for Treg cells, acts synergistically with TGF-β to enforce iTreg cell differentiation and maintenance.

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Figures

Figure 1

Figure 1. Galectin-9 promotes Foxp3 expression during iTreg cell differentiation

(A) The percentage of Foxp3+ Treg cells in mesenteric LN (mLN) and lamina propria (LP) was determined by flow cytometry; (B) Flow cytometry analysis of CD4+ T cells from the LP of WT or Lgals9−/− mice; (C) Activated naïve CD4+ T cells were stimulated with TGF-β and/or recombinant galectin-9. The frequency of Foxp3+ cells was determined by flow cytometry; CD45.1+ WT and CD45.2+ Lgals9−/− naïve T cells were cultured (D) separately or (E) together in the presence of TGF-β. The frequency of Foxp3+ cells was then determined by flow cytometry; OT-II Rag2−/− and OT-II Rag2−/−Lgals9−/− CD45.2+CD4+ T cells were transferred into congenic CD45.1 (F) Lgals9−/− or (G) WT recipient mice, followed by administration of OVA in the drinking water for 5 days. CD45.2+Foxp3+ iTreg cells from Peyer’s patches (PP) and LP of Lgals9−/− recipient mice were determined by flow cytometry. Data are representative of three independent experiments with n≥4 mice each group.

Figure 2

Figure 2. Smad3 regulates galectin-9 during iTreg cell differentiation

(A) Naïve CD4+CD44loCD62L+CD25− T cells from WT mice were differentiated into different T cell subsets. Quantitative real-time PCR analysis of Lgals9 mRNA in different subsets is presented relative to the expression of GAPDH mRNA; (B) Protein expression of galectin-9 was determined in different T cell subsets by flow cytometry; (C) The binding of Smad3 to the Lgals9 promoter in Th0 and iTreg cells was assayed by ChIP-PCR. Six horizontal bars represent the locations of Smad3 binding sites on Lgals9 locus detected by real-time PCR; (D–E) Luciferase assay using a Lgals9 promoter-driven reporter in EL4 LAF cells transfected with a control or Smad3-expressing vector under the indicated conditions. Promoter activity was determined 24 h after the addition of stimuli; (F) mRNA and (G) protein expression levels of Foxp3 and galectin-9 within WT and Smad3−/− iTreg cells; Data are representative of three independent experiments with n ≥ 4 mice each group (A–B, F–G) or are pooled from three independent experiments (C–E). *P< 0.05 and **P< 0.01 (Student’s _t_-test, error bars, SD).

Figure 3

Figure 3. Lgals9−/− iTreg cells have reduced suppressive activity

(A) Proliferation of WT naïve CD4+ T cells in the presence of anti-CD3 and anti-CD28 and GFP+ (Foxp3+) iTreg cells from Foxp3GFP and Foxp3GFPLgals9−/− mice. Data shown are presented as mean [3H]-thymidine incorporation; (B) The expression of indicated co-stimulatory molecules on Foxp3GFP and Foxp3GFPLgals9−/− CD4+GFP+ iTreg cells was determined by flow cytometry; (C) Body weight of Rag2−/−Lgals9−/− mice transferred with WT CD4+CD25−CD45RBhi T cells with or without WT or Lgals9−/− Foxp3+ (GFP) iTreg cells; (D) Hematoxylin and eosin staining of colon samples from the different groups as in (C) (original magnification, ×20); (E) Flow cytometry of IL-17 and IFN-γ secretion by CD4+ T cells isolated in spleen and LP from indicated groups as in (C) 10 weeks after colitis induction. The data are representative of three independent experiments with n ≥ 4 mice each group.* P< 0.05 (Student’s _t_-test, error bars, SD).

Figure 4

Figure 4. Galectin-9 enhances iTreg cell stability

(A) Purified GFP+ iTreg from Foxp3GFP and Foxp3GFPLgals9−/− mice were i.v. transferred into Rag2−/−Lgals9−/− recipient mice. 2 and 5 days after transfer, frequency of GFP+ cells were determined by flow cytometry; (B) IL-17 and IFN-γ production in the GFP− fraction in (A) were determined by flow cytometry; (C) Body weight of Rag2−/−Lgals9−/− mice transferred i.p. with CD4+CD25−CD45RBhiRFP−YFP− T cells from WT or Lgals9−/− FM mice; (D) Flow cytometry of IL-17 and IFN-γ, YFP and RFP by CD4+ T cells isolated in LP from indicated groups as in (C) 10 weeks after colitis induction. The data are representative of three independent experiments with n ≥ 5 mice each group. *P < 0.05, **P< 0.01 (Student’s _t_-test, error bars, SD).

Figure 5

Figure 5. CD44 mediates galectin-9 signaling during iTreg differentiation

(A) Confocal images of WT or Lgals9−/− Th0 and iTreg cells stained with anti-TGF-βRI and anti-CD44 antibodies; (B) Immunoprecipitation (with control IgG, anti-TGF-βRI or anti-CD44) of lysates of WT and Cd44−/− iTreg cells, followed by immunoblot analysis with the indicated antibodies; (C) TGF-βRI and CD44 association was visualized in iTreg cells with an in situ proximity ligation assay. Punctate staining (red) indicates a TGF-βRI-CD44 interaction as detected by the assay; (D) Anti-CD3 and anti-CD28 activated WT and Cd44−/− naïve CD4+ T cells were stimulated with TGF-β or recombinant galectin-9 and TGF-βRI kinase activity was determined. Data are representative of two independent experiments (A–C) or are pooled from three independent experiments (D) with n≥4 mice each group.* P< 0.05 (Student’s _t_-test, error bars, SD).

Figure 6

Figure 6. CNS1 region is essential for galectin-9 signaling within iTreg but not nTreg cells

(A) The binding of Smad3 and AcH4 to the Foxp3 CNS1 region from sorted GFP+T cells of Figure S6C from Foxp3GFP and Foxp3GFPLgals9−/− iTreg cells was determined by ChIP-PCR; (B) The binding of AcH4 to the Foxp3 CNS1 region from activated naïve T cells as in Figure S6D was determined by ChIP-PCR; (C) EL4 LAF cells were transfected with a Foxp3 promoter construct with or without the CNS1 region and stimulated with anti-CD3 and anti-CD28 antibodies in the presence of the indicated stimulations. Luciferase activity was measured 48 hours later. Data are representative of two independent experiments; (D) Anti-CD3 and anti-CD28 activated naïve CD4+ T cells from WT or Foxp3_Δ_CNS1 mice were stimulated with different combinations of TGF-β and recombinant galectin-9 and the frequency of Foxp3+ cells was determined by flow cytometry; (E) Chimeric mice were generated by transferring WT or Foxp3_Δ_CNS1 BM into WT or Lgals9−/− host mice. 10 weeks after reconstitution, the frequency of Foxp3+Nrp1lo iTreg cells or Foxp3+Nrp1hi nTreg cells in PP and LP was determined by flow cytometry. Data are pooled from three independent experiments (A–C) or are representative of two independent experiments (D–E) with n≥4 mice each group. *P < 0.05 and **P < 0.01 (Student’s _t_-test, error bars, SD).

Figure 7

Figure 7. Galectin-9-CD44 interaction regulates iTreg differentiation and function

(A) Activated WT naïve CD4+ T cells were stimulated with combinations of anti-CD44 antibody and recombinant galectin-9 with or without TGF-β for 3 days. The frequency of Foxp3+ cells was determined by flow cytometry; (B) Anti-CD3 and anit-CD28 activated naïve CD4+ T cells from WT or Cd44−/− mice were stimulated with different combinations of TGF-β and recombinant galectin-9 and the frequency of Foxp3+ cells was determined by flow cytometry; (C) Heat map displaying nanostring data, fold change of selected gene subsets in the experimental settings: Cd44−/− versus Lgals9−/− versus WT iTreg cells; (D) Proliferation of naïve CD4+ T cells in the presence of anti-CD3 and anti-CD28 and GFP+ (Foxp3+) iTreg cells from WT or Cd44−/− mice. Data shown are presented as mean [3H]-thymidine incorporation (cpm ± SEM, performed in triplicate); (E) Body weight of Rag2−/− mice transferred with WT CD4+CD25−CD45RBhi T cells with or without WT or Cd44−/− Foxp3+ (GFP) iTreg cells. Data are representative of three independent experiments with n ≥ 5 mice each group. *P < 0.05 (Student’s _t_-test, error bars, SD).

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