Regulatory T-cell suppressor program co-opts transcription factor IRF4 to control T(H)2 responses - PubMed (original) (raw)

Regulatory T-cell suppressor program co-opts transcription factor IRF4 to control T(H)2 responses

Ye Zheng et al. Nature. 2009.

Abstract

In the course of infection or autoimmunity, particular transcription factors orchestrate the differentiation of T(H)1, T(H)2 or T(H)17 effector cells, the responses of which are limited by a distinct lineage of suppressive regulatory T cells (T(reg)). T(reg) cell differentiation and function are guided by the transcription factor Foxp3, and their deficiency due to mutations in Foxp3 results in aggressive fatal autoimmune disease associated with sharply augmented T(H)1 and T(H)2 cytokine production. Recent studies suggested that Foxp3 regulates the bulk of the Foxp3-dependent transcriptional program indirectly through a set of transcriptional regulators serving as direct Foxp3 targets. Here we show that in mouse T(reg) cells, high amounts of interferon regulatory factor-4 (IRF4), a transcription factor essential for T(H)2 effector cell differentiation, is dependent on Foxp3 expression. We proposed that IRF4 expression endows T(reg) cells with the ability to suppress T(H)2 responses. Indeed, ablation of a conditional Irf4 allele in T(reg) cells resulted in selective dysregulation of T(H)2 responses, IL4-dependent immunoglobulin isotype production, and tissue lesions with pronounced plasma cell infiltration, in contrast to the mononuclear-cell-dominated pathology typical of mice lacking T(reg) cells. Our results indicate that T(reg) cells use components of the transcriptional machinery, promoting a particular type of effector CD4(+) T cell differentiation, to efficiently restrain the corresponding type of the immune response.

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Figures

Figure 1

Figure 1. Ablation of IRF4 in Treg cells results in autoimmune lymphoproliferative disease

a, _Foxp3Cre_-mediated deletion of Irf4 is restricted to Treg cells. Spleen and lymph node CD4+ T cells from _Irf4fl/− Foxp3Cre mice were FACS-sorted into GFP+ and GFP− cells containing a recombined and unrecombined Irf4fl allele, respectively, and stained for Foxp3. The post-sorting purity of GFP+ and GFP− population was greater than 97%. b, Intracellular staining of IRF4 in splenic and thymic CD4+ Foxp3− (red) and CD4+ Foxp3+ (blue) cells and splenic B220− CD138+ (green) plasma cells (positive control) in Irf4fl/+ Foxp3Cre (top panels) and Irf4fl/flFoxp3Cre mice (bottom panels). c, Splenomegaly and lymphadenopathy in Irf4fl/flFoxp3Cre mice. d, Spleen and lymph node cellularity in Irf4fl/+ Foxp3Cre (filled circles) and Irf4fl/flFoxp3Cre (open circles) mice. e, Histopathology induced after IRF4 ablation in Treg cells. Representative haematoxylin and eosin (H&E)-stained tissue sections from 8-week-old Irf4+/_− Foxp3Cre and Irf4fl/-Foxp3Cre mice and diphtheria-toxin-treated Foxp3DTR mice. Note pronounced infiltrates in the lung, pancreas and stomach of _Irf4fl/_− Foxp3Cre mice and severe lesions in tissues from diphtheria-toxin-treated Foxp3DTR mice (n = 3–5). Original magnification (c, e), ×10.

Figure 2

Figure 2. Increased numbers and activation of CD4+ T cells in mice harbouring IRF4-deficient Tregcells

a, CD4+ and CD8+ T cell numbers in the spleen (sp) and lymph nodes (ln) of Irf4fl/flFoxp3Cre (open circles) mice and Irf4fl/+ Foxp3Cre (filled circles) littermate control mice. NS, not significant. b, Flow cytometric analysis of CD44, CD62L and CD69 expression on CD4+ T cells in 8-week-old Irf4fl/flFoxp3Cre mice and Irf4fl/+ Foxp3Cre littermates. A representative of three independent experiments is shown. c, d, Increased Foxp3+ Treg cell subset in Irf4fl/flFoxp3Cre mice. Flow cytometric analyses of spleen and lymph node cells from Irf4fl/+ Foxp3Cre (filled circles) and Irf4fl/flFoxp3Cre (open circles) mice. A representative of three independent experiments is shown.

Figure 3

Figure 3. IRF4 deficiency in Treg cells results in a selective failure to control TH2 responses

a, b, Flow cytometric analysis of cytokine production by splenic CD4+ T cells from Irf4fl/+ Foxp3Cre mice and Irf4fl/flFoxp3Cre littermates. Splenocytes were stimulated with CD3 (5 μg ml−1) and CD28 (5 μg ml-1) antibodies in the presence of Golgi-Plug (1 μg ml−1) for 5 h before staining for CD4, CD8 and the indicated cytokines. A representative of three independent experiments is shown.

Figure 4

Figure 4. Increased serum IgG1 and IgE concentration, germinal centre formation, and plasma cell tissue infiltration caused by IRF4 deficiency in Treg cells

a, Analysis of immunoglobulin isotype amounts in sera of 8-week-old Irf4fl/flFoxp3Cre mice and Irf4fl/+ Foxp3Cre littermates, and of 3–4-week-old _Foxp3_− and Foxp3+ littermates. b, Immunofluorescent staining of germinal centre B cells (GL7+, green), follicular B cells (IgD+, red), and CD4+ T cells (blue) in spleens of Irf4fl/flFoxp3Cre mice and Irf4fl/+ Foxp3Cre littermates. Original magnification, ×20. c, Histological sections of H&E-stained pancreas from 8-week-old _Irf4fl_− Foxp3Cre mice and Treg-deficient diphtheria-toxin-treated Foxp3DTR mice. The _Irf4fl/_− Foxp3Cre pancreas is infiltrated primarily by plasma cells (arrows), that is, distinct round cells containing an eccentric nucleus with a cartwheel chromatin appearance and perinuclear clearing (inset; original magnification, ×60). In contrast, the pancreatic infiltrates of diphtheria-toxin-treated Foxp3DTR mice contained principally macrophages (arrows), that is, large cells with abundant eosinophilic cytoplasm, reniform to oval nuclei, and indistinct cell borders (inset; original magnification, ×60). Original magnification for both panels, ×40. Representative sections are shown.

Figure 5

Figure 5. IRF4 interacts with Foxp3 and diminished expression of a subset of suppressor effector and TH2 specific genes in IRF4-deficient Treg cells

a, IRF4-sufficient (Irf4+ TR, red) and -deficient (_Irf4_− TR, blue) Treg cells from 5-week-old Irf4+/+ Foxp3Cre mice and Irf4fl/flFoxp3Cre littermates suppress in vitro proliferative response of Foxp3− CD4+ T cells (TE) from B6 mice. A representative of two independent experiments is shown. b, Flow cytometric analysis of CD25, CTLA4 and ICOS expression by IRF4-sufficent (red) and -deficient (blue) Treg cells. c, Numbers of IRF4-independent genes that were up- (red bars) or downregulated (green bars), respectively, in Treg cells compared to naive CD25− Foxp3− CD4+ T cells. Open bars represent genes in which expression was changed by twofold or more in the absence of IRF4. d, Decreased expression of genes with a presumed role in Treg suppressor function in IRF4-deficient compared to IRF4-sufficient Treg cells. The data (c, d) represent average of two independent microarray experiments (exp) performed using YFP-Cre+ Treg cells FACS-purified from healthy _Irf4fl/_− Foxp3Cre/wt and Irf4+/− Foxp3Cre littermates. e, qPCR analysis of relative expression of genes shown in d in Irf4+ Treg (black) and _Irf4_− Treg (red). f, The decreased expression of TH2-specific or functionally important genes in IRF4-deficient in comparison to IRF4-sufficient Treg cells (two independent microarray experiments as above). g, h, qPCR analysis of relative expression of the TH2-specific gene set in Irf4+ Treg (black) and _Irf4_− Treg (red) (g), and in in vitro differentiated TH1 (black) and TH2 cells (red) (h). Data in e, g and h represent mean and s.d. of the expression of genes relative to Hprt1 in two independent experiments using three replicates each. i, Both IRF4 and Foxp3 bind to the promoter region of the Icos gene. qPCR analysis of Foxp3- and IRF4-bound chromatin isolated from wild-type Treg cells using primer set corresponding to the Icos promoter region. IgG ChIP and qPCR using primers corresponding to the promoter region of Gmpr was used as a specificity controls. j, Western blot (WB) analysis of IRF4 in nuclear lysates of wild-type Foxp3+ Treg cells and total Foxp3− CD4+ T cells (control) (lanes 1 and 2), and in Foxp3 complexes immunoprecipitated (IP) from the nuclear lysates using Foxp3 antibody. Transcription factor p65 was a negative control. IRF4 signal in control nuclear lysates is due to the presence of activated IRF4+ CD25+ Foxp3− T cells.

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