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Eos Mediates Foxp3-Dependent Gene Silencing in CD4+ Regulatory T Cells

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Science  28 Aug 2009:
Vol. 325, Issue 5944, pp. 1142-1146
DOI: 10.1126/science.1176077

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.

Naturally occurring CD4+CD25+ regulatory T cells (Tregs), which specifically express the transcription factor Foxp3, are essential for the suppression of pathological immune responses to both self and foreign antigens (13). Although it has been shown that Foxp3 interacts with a number of transcription and chromatin-modifying regulators (47), the molecular mechanism by which Foxp3 mediates selective gene silencing in Tregs is still largely unknown. For example, the silencing of cytokine genes such as those that express interleukin-2 (IL-2) and interferon-γ (IFN-γ) is critical in assuring that Tregs suppress rather than facilitate immune responses.

To gain insight into the molecular mechanisms of Treg function, we conducted comparative gene chip analysis on antigen-specific CD4+ T cells under tolerizing versus activating conditions (8). One of the genes that exhibited substantially higher expression in tolerizing/Treg conditions relative to activating conditions was Eos, which expresses a zinc-finger transcription factor that belongs to the Ikaros family. We confirmed the result by real-time quantitative reverse transcription–polymerase chain reaction (qRT-PCR) and Western blot analysis. Eos was highly expressed in the CD4+CD25+ and CD4+Foxp3+ Treg population (Fig. 1, A and B).

Fig. 1

Eos is highly expressed in Tregs and physically interacts with Foxp3. CD4+ T cells pooled from spleens and lymph nodes (LNs) of C57BL/6 or green fluorescent protein (GFP)-Foxp3 mice were sorted into indicated subsets, and the expression of Eos was assessed by (A) real-time qRT-PCR analysis, and (B) Western blot analysis. (C and D) Endogenous interaction between Eos and Foxp3 in Tregs. Treg cell lysates were subjected to immunoprecipitation (IP) with antibodies to Foxp3 (C), antibodies to Eos (D), or control immunoglobulin G (IgG) and then subjected to Western blot (WB) analysis with antibodies to Eos and Foxp3, as indicated. (E) In vitro interaction between Eos and Foxp3 was analyzed by GST pull-down assay. Data represent three or more independent experiments. Vertical bars indicate SEM.

Given that both Eos and Foxp3 are highly expressed in Tregs and that Eos exhibits transcriptional repression activity in certain tumor cell lines (9), we speculated that Eos might interact with Foxp3 and mediate its gene-silencing activity. To test this hypothesis, we carried out coimmunoprecipitation between Eos and Foxp3 ectopically expressed in Jurkat T cells (a transformed human T cell line), which lack endogenous Foxp3. Eos was found to coimmunoprecipitate with Foxp3 (fig. S1A). Endogenous Foxp3 and Eos also coimmunoprecipitated in lysates prepared from natural Tregs (Fig. 1, C and D). Similar experiments in cell lines and natural Tregs revealed that two other Ikaros family members (Ikaros and Helios) did not interact with Foxp3 (figs. S1, B and C, and S2). To verify that the interaction between Eos and Foxp3 is direct, we used affinity-purified recombinant glutathione S-transferase (GST)–Eos fusion protein (fig. S1D) to bind [35S]-labeled Foxp3 that was produced by in vitro transcription and translation. GST-Eos, but not GST, bound to [35S]-Foxp3, which suggests a direct interaction between the two proteins in vitro (Fig. 1E). Next, we determined the minimal Eos-interacting domain in Foxp3 using coimmunoprecipitation. Human embryonic kidney (HEK) 293T cells were cotransfected with constructs encoding various truncated forms of Eos and Foxp3. This approach led to the identification of a 51-residue (amino acids 148 to 198) fragment of Foxp3 that is necessary and sufficient to bind to the C-terminal region (amino acids 281 to 586) of Eos (fig. S3).

In Tregs, the IL-2 locus is silenced, and Foxp3 has been shown to be responsible for the suppression of IL-2 expression in both T cell lines and primary T cells (4). We thus investigated whether Eos was involved in IL-2 suppression by Foxp3 in T cells by using small interfering RNA (siRNA) to inhibit (knockdown) Eos (si-Eos) in primary CD4+ T cells (10) (Fig. 2A) that were then transduced with a biscistronic retroviral vector expressing full-length Foxp3 or a deletion of the 51-amino acid fragment that is required for Eos interaction (ΔFoxp3). As expected, the expression of the full-length Foxp3 almost completely blocked IL-2 production (Fig. 2B), and Eos protein expression remained unchanged upon overexpression of Foxp3 (fig. S4). ΔFoxp3 completely abrogated the ability of Foxp3 to repress IL-2 production. Knockdown of Eos reversed Foxp3-mediated suppression of IL-2 production (Fig. 2B), which could be rescued by retransduction with a retroviral vector expressing Eos with one base pair mutation at the siRNA-targeted region. Together, these results indicate that Foxp3-mediated IL-2 gene suppression requires Eos and the Eos-interacting domain of Foxp3.

Fig. 2

Foxp3 recruits Eos-CtBP co-repressor complexes to the core promoter region of IL-2 and epigenetically modifies the promoter activity in Tregs. (A) Knockdown of Eos by lentivirus-mediated RNAi in Tregs. (B) Knockdown of Eos reverses Foxp3-mediated IL-2 expression in T cells. (C) Endogenous interaction between Eos and CtBP1 in Tregs. (D) Effect of Eos knockdown on the association between Foxp3 and CtBP1 in Tregs. (E) Effect of knockdown of Eos on indicated histone modifications at the Foxp3-binding region of the IL-2 promoter in Tregs. (F) Effect of knocking down CtBP1 on histone H3 and H4 modifications in the Foxp3-binding region at the IL-2 promoter in Tregs. (G) Effect of Eos and CtBP1 knockdown on the DNA methylation status at the core region of the IL-2 promoter in Tregs. All primers for the ChIP analysis are listed in table S2. Data represent three or more independent experiments. Error bars indicate SEM.

C-terminal binding protein–1 (CtBP1) has been implicated as a key repressor of Eos-mediated gene expression (9, 1113); thus, we hypothesized that Eos might serve as a co-repressor in Tregs to recruit CtBP1to Foxp3. To test this hypothesis, we determined the association between Eos and CtBP1 and assessed IL-2 promoter epigenetic status on Eos knockdown in Tregs. Eos indeed interacted with CtBP1 in Tregs (Fig. 2C). Moreover, both Eos and CtBP1 coimmunoprecipitated with Foxp3, whereas knockdown of either Eos or ΔFoxp3 [which is still capable of associating with NFAT (nuclear factor of activated T cells) or AML1/Runx1 (acute myeloid leukemia 1/runt related transcription factor 1) while not binding Eos (fig. S5)] abrogated the incorporation of CtBP1 into Foxp3-suppressive complexes (Fig. 2D). These data suggest that CtBP1 is part of the Foxp3-Eos suppression complex.

It has been reported that CtBP1 suppresses transcription by affecting histone methylation and acetylation (11). To determine whether this was the case in Tregs, we applied chromatin immunoprecipitation (ChIP) analysis to detect the histone modifications at the IL-2 promoter region in Tregs by examining histone 3 lysine 4 (H3K4) and lysine 9 (H3K9) methylation, as well as histone 3 and histone 4 acetylation (H3Ac, H4Ac). In vitro–primed effector CD4+ cells, which transcribe the IL-2 gene more rapidly than naïve cells do after activation (14), exhibited a 10- to 40-fold increase in histone trimethylation (Me3H3K4) and acetylation (H3Ac and H4Ac), but a concurrent decrease in histone methylation (MeH3K9) at the IL-2 promoter (Fig. 2E). In contrast, Tregs exhibited little or no change in histone methylation and acetylation at the IL-2 promoter compared to that of naïve T cells. Upon knockdown of Eos, histone trimethylation (Me3H3K4) and acetylation (H3Ac and H4Ac) were significantly increased at the IL-2 promoter (Fig. 2E) but not at the CD3ε promoter that does not contain a Foxp3 binding site (fig. S6). We attribute the changes in epigenetic modifications of histones at the IL-2 promoter to abrogation of the Foxp3 interaction with CtBP1 in si-Eos Tregs (Fig. 2D), because Foxp3 itself still binds to the IL-2 promoter even after si-Eos knockdown (fig. S7). CtBP1 associates with a co-repressor complex including EuHMT(G9a), which is responsible for H3K9 methylation in other cell types (15, 16). We thus used RNA interference (RNAi) to determine whether CtBP1 is associated with the methylation of H3K9 at the IL-2 promoter in Tregs. Upon knockdown of CtBP1 by RNAi (fig. S8A), we observed a marked decrease in CtBP1 and EuHMT occupancy at the IL-2 promoter, whereas there was no significant change to Foxp3-Eos occupancy. CtBP1 knockdown was accompanied by a significant decrease in H3K9 methylation (MeK9, fig. S8B). In addition, we observed that histone H3 and H4 acetylation was enhanced upon si-CtBP1 treatment (Fig. 2F). At the functional level, knockdown of CtBP1 in Tregs abrogated their suppressive activity (fig. S9). Similar changes in epigenetic histone modifications were also observed at the IFN-γ promoter in Tregs (fig. S10).

Another epigenetic modification that regulates IL-2 gene expression is methylation of genomic DNA at CpG dinucleotides in the promoter. We thus determined whether knockdown of Eos influenced the extent of DNA methylation at the IL-2 locus. Given the documented negative correlation between methylation status of the –68 CpG dinucleotide in the IL-2 promoter region and IL-2 transcription (17, 18), we examined the DNA methylation status of the CpG islands flanking the –68 region within the IL-2 promoter using a methylation-sensitive PCR assay in Tregs. Knockdown of Eos or CtBP1 led to demethylated CpG dinucleotides at the IL-2 promoter upon CD3 and CD28 treatment (Fig. 2G). Taken together, these findings lend further support to the notion that Eos or CtBP represses the IL-2 promoter through epigenetic modification mechanisms in Tregs.

We next sought to determine whether Eos plays a broader role in Foxp3-mediated Treg gene silencing without affecting gene activation. Using the Foxp3-dependent gene set from natural Tregs defined by Rudensky and colleagues (19) as a basis for comparison, we analyzed changes in global gene expression patterns from Eos-siRNA versus control Renilla luciferase (RL)-siRNA–transduced Tregs. The majority (>70%) of the si-Eos–affected genes were Foxp3-dependent (fig. S11). Among them, 90% of the genes known to be suppressed by Foxp3 were no longer down-regulated upon Eos knockdown in Tregs (Fig. 3A). In marked contrast, >95% of known Foxp3-dependent up-regulated genes (19) were unaltered by Eos knockdown (Fig. 3B), a subset of which was further verified by qRT-PCR (Fig. 3C) and cell-surface staining (table S1).

Fig. 3

si-Eos could reverse most Foxp3-mediated suppression genes in Tregs. Shown are the genes of up-regulated (A) and down-regulated (B) transcripts in si-Eos versus si-RL–transduced Tregs. Naïve T cell and naïve Treg gene expression profiling was carried out in parallel. (C) Knockdown of Eos enhances IL-2 and IFN-γ gene expression but does not affect Foxp3, CTLA-4, or GITR gene expression in Tregs. si-Eos and si-RL Tregs were sorted out and subjected to qRT-PCR assay with the indicated gene-specific primers. The results shown are the means ± SEM of three independent experiments.

The selective effect of Eos knockdown on Foxp3-repressed genes prompted us to study the specific role of Eos in Treg function. Sorted Tregs, which were transduced with the same si-Eos lentivirus as above, were subjected to an in vitro suppression assay based on carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution (20). Although both fresh wild-type Tregs and si-RL–transduced Tregs could suppress the proliferation of naïve T cells, Tregs with Eos knockdown were not suppressive but could be rescued by a functional human Eos construct (hEos) lacking the siRNA-targeted region (fig. S12). To further confirm that Eos is involved specifically in Foxp3-mediated suppression (21), we used recombination-activating gene (RAG)–deficient (Rag2−/−) DO11.10 CD4+ T cells, which express ovalbumin (OVA) peptide-specific transgenic T cell receptors. Transgenic CD4+ T cells transduced with MIGR1-Foxp3-RFP suppressed the proliferation of freshly prepared nontransduced transgenic T cells upon stimulation with specific OVA peptides, whereas those transduced with the control MIGR1-RFP or MIGR1-∆Foxp3-RFP mutant (148– to 198–amino acid deletion that is unable to interact with Eos) did not (Fig. 4A). siRNA knockdown of Eos reduced the suppressive activity of Foxp3-expressing antigen-specific CD4+ T cells, which could be rescued by retransduction with RV-Eos or chimeric construct ΔFoxp3/hEos (Fig. 4A). Moreover, si-Eos treatment did not block suppressive activity conferred by a chimeric Foxp3/hEos gene (Fig. 4A). Foxp3 protein abundance remained unchanged upon si-Eos treatment (fig. S13). Together, these results suggest that Eos is required for Foxp3-mediated suppressive activity of Tregs.

Fig. 4

Effects of knockdown of Eos on the suppressive activity of Tregs in vitro and in vivo. (A) Knockdown of Eos by siRNA reduces the suppressive activity of Foxp3-transduced naïve T cells. The results are representative of one of three independent experiments. (B) Percentage weight change after transfer of the indicated cells demonstrates that cotransfer of si-Eos–transduced Tregs cannot suppress weight loss induced by transfer of CD4+CD25 CD62Lhigh Teff cells into Rag2/ mice. Nine mice were used in each group, and the results shown are the means ± SD of three independent experiments. (C) Representative photomicrography of the distal colon of Rag2/ mice after T cell transfer. (D) Colonic histology scores of experimental mice. (E to G) Analysis of spleen (E), mesenteric LNs (F), and lamina propria LN (G) Teff (CD4+Foxp3) and Treg (CD4+Foxp3+) cell numbers. Eight to 10 mice were used in each group, and the results shown are the means ± SD of three independent experiments. (H) Immunohistochemistry (IHC) staining of CD3+ or Foxp3+ T cells in colon tissue. Hematoxylin serves as a counterstain (magnification 20×). The IHC analysis was performed on five mice per group.

Finally, we determined the role of Eos in the function of Tregs in an in vivo colitis model, where severe T helper type 1 (TH1)–mediated inflammation within the colon is induced after the transfer of naïve CD4+CD25CD62Lhigh T cells in the absence of Tregs into Rag2−/− recipient mice (22). The cotransfer of CD4+CD25+ Tregs is sufficient to prevent the development of disease (23). Disease progression was monitored by weight loss, and colitis was assessed by histological analysis of colon tissue (24). Wild-type or si-RL–modified Tregs effectively suppressed the development of disease as judged by both body weight loss (Fig. 4B) and histological analysis of tissue isolated from the colon (Fig. 4, C and D). Transfer of si-Eos Tregs in the presence of CD4+CD25CD62Lhigh T effector (Teff) cells, however, failed to prevent the disease development (Fig. 4, B to D). In vivo tracking of transferred cells demonstrated that the colitis that developed in mice receiving CD4+CD25CD62Lhigh T cells and si-Eos–transduced Tregs was not due to impaired accumulation or homing of Foxp3 Tregs to the mesenteric lymph nodes or spleen (Fig. 4, E to H). Given that Eos knockdown de-represses effector cytokine genes in Tregs, we asked whether Tregs developed effector function in the absence of Eos. Indeed, transfer of a higher dose of pure si-Eos Tregs (1 × 106) into Rag2−/− mice produced colitis (figs. S14 to S16), which was less severe than that caused by transfer of the same number of Teff cells. At a lower dose of transferred cells (2 × 105), Teff cells still induced colitis, whereas si-Eos Tregs did not (fig. S15). Thus, Eos knockdown alone partially converts Treg into functional Teff cells, consistent with our finding that immune-suppressive functions associated with Foxp3-activated genes in Tregs are unaffected by Eos knockdown.

Collectively, our findings demonstrate that Foxp3 suppresses gene expression through its interaction with Eos and define Eos as a key missing link in this process. These results also raise the possibility that the interaction between Foxp3 and Eos, along with its associated co-repressor complexes, might be a potential therapeutic target for controlling physiological and pathological immune responses. This may be particularly true in the setting of cancer and chronic infections, where excessive Treg activity seems to play an important role. Further dissection of the subset of genes silenced by Eos-Foxp3 complexes will likely lead to the identification of downstream molecules involved in the regulation of Treg function.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1176077/DC1

Materials and Methods

Figs. S1 to S16

Tables S1 and S2

References

References and Notes

  1. We thank A. Rudensky, Y. Shi, M. Crossley, S. Smale, A. Friedman, H. Levisky, C. V. Dang, J. D. Powell, and S. L. Topalian for reagents and helpful suggestions. We are grateful to G. Zhou, K. J. Rhee, and members of the Pardoll laboratory for helpful discussions. The array data sets are deposited at Gene Expression Omnibus (accession number GSE17166). This work was supported by grants from NIH and the Melanoma Research Alliance, the Janey Fund and Seraph Foundation, and gifts from Bill and Betty Topecer and Dorothy Needle.
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