Eos mediates Foxp3-dependent gene silencing in CD4+ regulatory T cells - PubMed (original) (raw)

. 2009 Aug 28;325(5944):1142-6.

doi: 10.1126/science.1176077. Epub 2009 Aug 20.

Hong Yu, Eric V Dang, Joseph Barbi, Xiaoyu Pan, Joseph F Grosso, Dinili Jinasena, Sudarshana M Sharma, Erin M McCadden, Derese Getnet, Charles G Drake, Jun O Liu, Michael C Ostrowski, Drew M Pardoll

Affiliations

Eos mediates Foxp3-dependent gene silencing in CD4+ regulatory T cells

Fan Pan et al. Science. 2009.

Abstract

CD4+ regulatory T cells (Tregs) maintain immunological self-tolerance and immune homeostasis by suppressing aberrant or excessive immune responses. The core genetic program of Tregs and their ability to suppress pathologic immune responses depends on the transcription factor Foxp3. Despite progress in understanding mechanisms of Foxp3-dependent gene activation, the molecular mechanism of Foxp3-dependent gene repression remains largely unknown. We identified Eos, a zinc-finger transcription factor of the Ikaros family, as a critical mediator of Foxp3-dependent gene silencing in Tregs. Eos interacts directly with Foxp3 and induces chromatin modifications that result in gene silencing in Tregs. Silencing of Eos in Tregs abrogates their ability to suppress immune responses and endows them with partial effector function, thus demonstrating the critical role that Eos plays in Treg programming.

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Figures

Fig. 1

Fig. 1. Eos is highly expressed in subpopulation of CD4+ T cells, and it physically interacts with Foxp3 in Treg cells

CD4+ T cells pooled from spleens and LN of C57BL/6 or GFP-Foxp3 mice were sorted into indicated subsets, and the expression of Eos was assessed by (A) real-time quantitative RT-PCR analysis using primers and internal fluorescent probes specific for Eos and 18s rRNA, and (B) Western blot analysis, respectively. (C) Endogenous interaction between Eos and Foxp3 in Treg cells. Treg cells were lysed, subjected to immunoprecipitation (IP) with anti-Foxp3 (left), anti-Eos (right) antibodies or control IgG. The immunoprecipitates were resolved on SDS-PAGE followed by Western blot analysis (WB) with anti-Eos and anti-Foxp3 antibodies as indicated, respectively. (D) In vitro interaction between Eos and Foxp3 was analyzed by GST pull-down assay. In vitro transcribed/translated [35S]-methionine-labelled Eos or Foxp3 was incubated with glutathione beads bound with bacterially expressed GST-Foxp3 or GST-Eos, respectively. GST alone was included as a negative control. After 30 mins incubation, the beads were washed 3 times with PBS and eluted with 5mM reduced glutamine. The eluted product was subjected to SDS-PAGE and autoradiograph. Input consisted of 10% of the [35S]-methionine-labelled proteins. (E) Coomassie blue staining. GST fusion protein purified from bacterially expressed GST-Foxp3 or GST-Eos were subjected to SDS-PAGE and stained with Coomassie blue. (F) Mapping of the Eos-interacting domain in Foxp3 with a mammalian two-hybrid system. The cDNA of Eos was fused to the VP16 AD in the pVP16 vector (Clontech), the cDNAs of different fragments of Foxp3 were fused to GAL4 DB in the pM vector. 293T cells were transfected with the indicated vectors together with pnull-RL (Renilla Luciferase, Promega) and pG5Luc, a luciferase reporter plasmid containing GAL4 promoter. After 24h, cells were harvested and luciferase activity was measured and normalized to Renilla luciferase activity. (G) Mapping of the Eos-interacting Domain in Foxp3 by coimmunoprecipitation. 293T cells were transfected with the indicated plasmids. After 24hr, cell extracts were immunoprecipitated with anti-DBD antibodies (Santa Cruz), the immune complexes were subjected to SDS-PAGE, and blotted with anti-Flag (top), anti-DBD (middle). The whole cell lysates were blotted with anti-Flag antibody (bottom). (H) The co-IP assay was applied to map the Eos-interaction domain in Foxp3. Numbers at top of each lane represent the peptide fragments of Foxp3 fused to the DBD epitope. (I) The co-IP assay was applied to map the Foxp3-interaction domain in Eos. Numbers at top of each lane represent the peptide fragments of Eos fused to the Myc epitope. The results are representative of one of three independent experiments.

Fig. 2

Fig. 2

Foxp3 recruits Eos/CtBP co-repressor complexes to the core promoter region of IL-2 and epigenetically modify the promoter activity in Treg cells. (A) Knockdown of Eos by lentivirus-mediated RNA interference in Tregs. CD4+CD25high Treg cells were sorted from Balb/c mice and transduced with indicated lentivirus. 72h post-transduction, the CD4+GFP+ cells were sorted out and subject to Western blot and blot with anti-Eos(top), anti-Foxp3 (middle), and anti-actin, respectively. The target sequence for si-Eos was listed in the supplementary table 1. (B) Knockdown of Eos could reverse Foxp3-mediated IL-2 expression in T cells. CD4+ T cell were transduced with a retroviral vector (carrying RFP as an internal marker) expressing nothing (EV), full length Foxp3 or Foxp3 with the 148–198 Eos binding fragment deleted (ΔFoxp3). Cells were then retransduced with either the control siRNA expressing lentiviral vector or lentiviral vector (carrying GFP as an internal marker) expressing Eos siRNA (targeting to the 3′ UTR of Eos) in the presence or absence of a retroviral vector (carrying Thy1.1+ as an internal marker) expressing mouse Eos gene with one base pair mutation of the siRNA targeted region. 72h post-transduction, the RFP+GFP+ or RFP+GFP+Thy1.1+ cells were sorted out, and re-stimulated with anti-CD3/CD8, IL-2 production was assessed by ELISA. (C) Endogenous interaction between Eos and CtBP1 in Treg cells. Treg cells were lysed, subjected to immunoprecipitation (IP) with anti-CtBP1 antibody or control IgG. The immunoprecipitates were resolved on SDS-PAGE followed by Western blot analysis (WB) with anti-Eos (top) and anti-CtBP1 (Santa Cruz, bottom) antibodies, respectively. (D) Effect of Eos knockdown on the association between Foxp3 and CtBP1 in Treg cells. Eos knockdown in Treg cells were carried out with the same protocol as the panel A and B. Cell lysates were prepared from sorted siRL or si-Eos introduced Treg cells, followed by immunoprecipitation with anti-Foxp3 (eBioscience) or control IgG, and then analyzed by Western blotting with anti-CtBP1 (top), anti-Foxp3 (middle) and anti-Eos (bottom), respectively. (E) Effect of knockdown of Eos on indicated histone modifications at the Foxp3-binding region of the IL-2 promoter in Treg cells. The indicated cell population was sorter purified, followed by CHIP analysis (see material and method section for detail). (F) Knockdown of CtBP1 expression by RNA interference in Tregs. Cell lysates were prepared from sorted siRL(control) or si-CtBP1 transduced Treg cells, followed by Western blotting with anti-CtBP1(top), and anti-actin (bottom), respectively. The targeted sequence for si-CtBP1 is listed in the supplementary table 1. (G) CtBP1 siRNA decreases CtBP complex occupancy and H3 Lys9 methylation at the IL-2 promoter in Treg cells. siRL or si-CtBP1 treated Treg cells were sorted out and subjected to CHIP analysis. (H) Effect of knocking down CtBP1 on histone H3 and H4 modifications in the Foxp3-binding region at the IL-2 promoter in Treg cells. si-CtBP1 Treg cells were prepared (the same as panel F and G), and quantitative ChIP analysis was performed to determine the modification of histone at the IL-2 promoter using real-time PCR. (I) Effects of knockdown of CtBP1 on the suppressive activity of Treg cells in vitro. GFP+RFP+ cells from DO11.10/RAG2−/− CD4+ cells transduced with MIGR1-Foxp3-RFP/pFup3.1-siRL-GFP (closed circle), MIGR1-Foxp3-RFP/pFUP3.1-si-CtBP1-GFP (closed rhombus) or MIGR1-RFP/pFUP3.1-siRL-GFP (open circle) were cultured with 2×104 freshly isolated DO11.10 CD4+ in the presence of OVA peptide and 4×104 APCs (irradiated CD3- cells). [3H]-thymidine incorporation for the last 8 hrs of cell culture was measured as an indicator of cell proliferation and was expressed as the mean ± s.d. for triplicate cultures. (J) Effect of Eos and CtBP1 knockdown on the DNA methylation status at the core region of the IL-2 promoter in Treg cells. The results shown are the means ± s.d. of three independent experiments. All primers for the CHIP analysis are listed in the supplementary table 1.

Fig. 3

Fig. 3

Gene expression differences between siEos versus siRL-transduced Treg cells. Shown are the genes of up- (A) and down- (B) regulated transcripts in siEos vs. siRL-transduced Treg cells. Naïve T cell and nTreg cell gene expression profiling was carried out in parallel. siRL transduced Treg cell gene expression signature was comparable with non-transduced nTreg. Foxp3 dependent up- or down- regulated genes were compared with the databases reported by Zheng et al [Nature. 445,936 (2007)]. Genes with expression increased or decreased by two-fold or more in Treg vs. naïve T cells were considered up-or down-regulated, respectively, by Foxp3. (C) Knockdown of Eos enhances IL-2 and IFN-γ gene expression in Treg cells. Si-Eos and siRL Treg cells were sorted out and subjected to q-RT PCR assay using indicated gene specific primers. The primer sequences were listed in the supplementary table 1. (D) Mean fluorescence intensity for various cell surface molecules on CD25+CD4+ cells transduced with pFUP3.1siRL-GFPor pFUP3.1-siEos-GFP (see methods section for the antibodies). (E, F) The association between Foxp3 and Tip60 or HDAC7 in Treg cells remains unchanged upon si-Eos treatment. Cell lysates were prepared from sorted siRL or si-Eos transduced Treg cells, followed by immunoprecipitation with (E) anti-HDAC7, (F) anti-Tip60 (Santa Cruz) or control IgG, then analyzed by Western blotting with anti-Foxp3. The whole cell lysates were blotted with (E) anti-HDAC7, (F) anti-Tip60, anti-Foxp3 and anti-Eos as indicated.

Fig. 4

Fig. 4

Effects of knockdown of Eos on the suppressive activity of Treg cells invitro and in vivo. (A) Knockdown of Eos by siRNA reduces the suppressive activity of Foxp3-transduced naïve T cells. Sorted GFP+RFP+ or GFP+RFP+Thy1.1+ T cells from DO11.10/RAG-2−/− CD4+ cells transduced with the indicated viruses were co-cultured with 2×104 freshly isolated and CFSE labeled DO11.10 CD4+ (Thy1.2+, ratio 1:1) in the presence of OVA peptide and 4×104 APCs (irradiated CD3- cells) for 80h, followed by surface marker, Foxp3 and Ki67 staining. To quantify both frequency and absolute cell number, all cells under each condition were collected. Cell proliferation was determined by CFSE dilution and upregulation of Ki67. The proliferation index was calculated: the absolute number of Ki67+ CFSE diluting Thy1.2+CD4+Foxp3- T responder cells (co-cultured with the indicated Treg cells at 1:1 ratio) were divided by the number of proliferating T responder alone. The results were expressed as the mean ± s.d. for triplicate cultures. (B) Percentage weight change after transfer of the indicated cells demonstrates that co-transfer of siEos transduced Treg cannot suppress weight loss induced by transfer of CD4+CD25− CD62Lhigh Teff into Rag2−/− mice. (C) Representative photomicrography of the distal colon of Rag2−/− mice following T cells transfer. CD4+CD25− CD62Lhigh cells isolated from WT BALB/c were able to induce colitis in BALB/c Rag2−/− recipients (top left). Co-transfer of WT CD4+CD25+ Treg cells (top right) or siRL-transduced Treg (bottom left) was able to prevent colitis. However, siEos-transduced Treg had no effect on colitis (bottom right). Data represent 9–12 mice per group in each panel. (D) Colonic histology scores of experimental mice. Colons were removed from mice 8 weeks after T cells reconstitution and fixed in 10% neutral buffered formalin. Five-micrometer paraffin-embedded sections were cut and stained with haematoxylin and eosin (H&E). Pathology of the colon was evaluated by routine microscopic examination, then scored blindly using a semi-quantitative scale of zero to five (see method section for detail). 9–12 mice were used in each group. (E–G) Analysis of spleen (E), mensenteric LN (F), and lamina propria LN (G) Teff (CD4+Foxp3−) and Treg (CD4+Foxp3+) cell numbers. Foxp3 expression was determined by intracellular staining (eBioscience). (H) Comparable Treg cells are found in Eos deficient and control Treg recipient mice. FACS analysis of spleen and draining mesenteric lymph nodes of mice 8 weeks post-adopt transfer T cells in the model of IBD. Sorted Thy1.1+ Treg cells transduced with siRL or siEos were cotransfered with or without CD4+CD25−CD62Lhi T cells marked as Thy1.1− into Rag2−/− mice. 8 weeks post adoptive transfer, pooled spleen and lymph nodes were stained with APC-CD4 together with FITC-Foxp3 or Thy1.1. Data shown are a representative staining of three independent experiments. (I) Immunohistochemistry (IHC) staining of CD3+ or Foxp3+ T cells in colon tissue. IHC staining of paraffin sections of colon was performed by a peroxidase technique, using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame,CA) and diaminobenzidine substrate kit (Vector). CD3+ T cells were visualized using goat anti-CD3 polyclonal antibodies (Santa Cruz, top panel), and Foxp3+ cells were visualized with rat anti-Foxp3 clone FJK-16 antibody (eBioscience, bottom panel). Haematoxylin serves as a counterstaining (magnification, x 20). Substitution of irrelevant antibodies of the same isotype was used as a control (not shown). The IHC analysis was performed on five mice per group.

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References

    1. Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Cell. 2008 May 30;133:775. - PubMed
    1. Maloy KJ, Powrie F. Nat Immunol. 2001 Sep;2:816. - PubMed
    1. Shevach EM. Annu Rev Immunol. 2000;18:423. - PubMed
    1. Wu Y, et al. Cell. 2006 Jul 28;126:375. - PubMed
    1. Marson A, et al. Nature. 2007 Feb 22;445:931. - PMC - PubMed

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