Analysis of Sirtuin 1 Expression Reveals a Molecular Explanation of IL-2–Mediated Reversal of T-Cell Tolerance

Analysis of sirtuin 1 expression reveals a molecular explanation of IL-2–mediated reversal of T-cell tolerance

Beixue Gao, Qingfei Kong, Kyeorda Kemp, Yuan-Si Zhao, and Deyu Fang1

Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611

Edited by Arthur Weiss, University of California, San Francisco, CA, and approved December 8, 2011 (received for review November 9, 2011) The type III histone deacetylase sirtuin 1 (Sirt1) is a suppressor of both ably allowing accelerated T-cell proliferation and differentiation. innate and adoptive immune responses. We have recently found that Because Sirt1 is required for maintaining and IL-2 reverses T-cell Sirt1 expression is highly induced in anergic T cells. However, the peripheral tolerance, our findings here provide a possible explana- transcriptional program to regulate Sirt1 expression in T cells remains tion about the IL-2–mediated switch from anergy to activation. uncharacterized. Here we report that the early responsive genes 2 and 3, which can be up-regulated by T-cell receptor-mediated activa- Results tion of nuclear factor of activated T-cell transcription factors and are Differential Sirt1 Expression in Mouse Primary T Cells. We have re- involved in peripheral T-cell tolerance, bind to the sirt1 promoter to cently reported that sirt1 gene transcription is induced by TCR- transcript sirt1 mRNA. In addition, the forkhead transcription factor, mediated anergic signaling, and this increased Sirt1 expression is FoxO3a, interacts with early responsive genes 2/3 on the sirt1 pro- required to maintain peripheral T-cell tolerance (9). To further moter to synergistically regulate Sirt1 expression. Interestingly, IL-2, analyze the transcriptional regulation of Sirt1 expression in T cells, a cytokine that can reverse T-cell anergy, suppresses sirt1 transcrip- we compared Sirt1 protein and mRNA expression levels in naïve, tion by sequestering FoxO3a to the cytoplasm through activating the activated, anergic, and differentiated effector T cells. The highest PI3K-AKT pathway. Expression of the constitutively active form of levels of Sirt1 protein expression were detected in anergic T cells – FoxO3a blocks IL-2 mediated reversal of T-cell tolerance by retaining generated in OT-II TCR transgenic mice treated with ovalbumen IMMUNOLOGY sirt1 fi expression. Our ndings here provide a molecular explanation (OVA)323–339 peptide, confirming the results of our previous study of IL-2–mediated reversion of T-cell anergy. (9). Stimulation of T cells with anti-CD3 plus anti-CD28 also dramatically induced Sirt1 expression. In contrast, Sirt1 protein T-cell anergic gene | FoxO3a sequestration expression levels in Th1, Th2, Th17, and regulatory T cells (Tregs) were significantly reduced compared with its expression in anergic hen self-reactive T cells recognize MHC/antigen complexes and activated T cells (Fig. 1A). A similar pattern of sirt1 mRNA + Wthrough their receptors (T-cell receptor, TCR) at peripheral expression in CD4 T cells was observed by real-time RT-PCR lymphoid organs, they will either be deleted by apoptosis or si- analysis (Fig. 1B). Therefore, Sirt1 expression is differentially lenced through a mechanism known as anergy (1, 2). T-cell anergy regulated during T-cell immune responses. is a critical mechanism to prevent autoimmunity in mammals and TCR recognition of autoreactive antigen in the absence of failure in this anergic induction process causes autoimmune dis- costimulation induces T-cell tolerance through activation of eases, such as type 1 diabetes, multiple sclerosis, and rheumatoid NFAT (12, 19). In fact TCR-stimulation alone with anti-CD3 arthritis (3–6). It is well accepted that T-cell anergy is induced and antibody induced sirt1 transcription in a dose-dependent manner. maintained by a group of suppressive proteins, such as Itch, Cbl-b, Addition of anti-CD28 antibody did not further enhance, and in early growth-response genes (EGR) 2 and 3, Grail, and Sirt1, up- contrast slightly inhibited sirt1 gene transcription (Fig. 1C). regulated by self-antigen/TCR ligation in the absence of cos- Treatment of T cells with the calcineurin inhibitor cyclosporine A timulated signaling mediated by the CD28 receptor (7–10). The (CsA), which suppresses its downstream transcription factor up-regulation of these suppressive genes requires the nuclear NFAT, inhibited sirt1 gene transcription in a dose-dependent factor of activated T cells (NFAT), which can be activated by the manner (Fig. 1D), suggesting that the TCR-NFAT pathway is in- TCR-mediated calcium/calcineurin pathway (11–13). volved in sirt1 transcription and that CD28-mediated signaling may We and others have recently demonstrated that the class III suppress sirt1 gene transcription in T cells. histone deacetylase Sirt1, which plays important roles in many aspects of biological functions, such as aging, metabolism, and Transcription Factors EGR2 and EGR3 Are Involved in sirt1 Gene apoptosis, inhibits T-cell immune response (9, 14–16). Sirt1 ex- Expression in T Cells. Analyzing the sirt1 promoter sequence did pression in T cells is regulated by TCR-mediated signaling. The not identify any conserved NFAT-binding sites within the −30 kp up-regulated Sirt1 protein antagonizes T-cell immune response by region of sirt1 promoter in mammals. Instead, four conserved suppressing the activations of NF-κB and activated protein 1 binding elements for the EGR family transcription factors were (AP-1) transcription factors, both of which are required for pro- identified, three of which locate within the −196 bp region, and the duction of IL-2, a cytokine that promotes T-cell proliferation (9, fourth one is in the first exon (Fig. 2A), implying that EGR family 16, 17). Genetic depletion of the sirt1 gene in mice leads to ele- transcription factors may regulate sirt1 transcription. To test this vated T-cell immune response and the development of a lupus-like theory, we subcloned the 1-kb region (−1,063 to +79), which is autoimmune syndrome (9, 15). In addition, Sirt1 is also found to suppress innate-immune responses through opposing NF-κB– mediated inflammatory cytokine production by macrophages (18). Author contributions: D.F. designed research; B.G., Q.K., K.K., and Y.-S.Z. performed re- In the present study we report that TCR-mediated sirt1 tran- search; B.G. and D.F. analyzed data; and K.K. and D.F. wrote the paper. scription is promoted through EGR2 and EGR3, as well as the The authors declare no conflict of interest. forkhead O family transcription factor 3a (FoxO3a). FoxO3a binds This article is a PNAS Direct Submission. to the sirt1 promoter through its interaction with EGR2/3 to synergis- 1To whom correspondence should be addressed. E-mail: [email protected]. tically promote sirt1 mRNA transcription. IL-2 sequesters FoxO3a to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. suppress sirt1 mRNA expression during T-cell activation, presum- 1073/pnas.1118462109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1118462109 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 Fig. 1. Analysis of Sirt1 expression in CD4+ T cells. (A and B) Mouse naïve CD4+ T cells were left unstimulated (naïve) or stimulated with anti-CD3 plus anti-CD28 for 24 h (activated). Th1, Th2, and Th17 cells were generated under each speciﬁc polarization condition for 5 d. CD4+CD25+ cells from 6- to 8-wk- old mice were isolated as Tregs. For induction of anergic T cells,

OT-II TCR transgenic mice were treated with OVA323–339 peptide. Seven days after treatment, CD4+ Tcellsinthelymph nodes from the treated mice were used as anergic T cells. (A) Cells were lysed with Nonidet P-40 lysis buffer and Sirt1 protein expression was analyzed by Western blotting (Up- per) using β-Actin as a loading control (Lower). (B) Total RNA was puriﬁed and the mRNA level of sirt1 was analyzed by real-time RT-PCR. (C and D) Naïve T cells were stimulated with different amounts of anti-CD3 in the absence or presence of 1 μg/mL of anti-CD28 for 24 h (C) or stimulated with anti-CD3 plus anti-CD28 (1 μg/mL each) with different doses of CsA for 24 h (D). Total RNA was extracted and sirt1 mRNA levels were determined by real-time RT-PCR. Error bars represent data from three independent experiments (mean ± SE).

often the optimal region carrying transcription factor-binding ac- coexpressed (Fig. 2D). Therefore, our data indicate that EGR tivity, into a pGL3 luciferase vector. In contrast to a basal-level family transcription factors EGR2 and EGR3 regulate sirt1 gene pGL3-luciferase activity, this 1-kb fragment mediated significant transcription. Interestingly, both egr2 and egr3 genes are down- luciferase activity (Fig. 2B). A serial deletion of this 1-kb region stream of the NFAT transcription factor in T cells (20, 21). It is identified that the −196 to +79-bp region, which contains all four likely that TCR-mediated NFAT activation promotes egr2 and egr3 EGR binding sites, carries the transcriptional activity. These mRNA transcription for sirt1 gene expression in T cells. To support results suggest that EGR family transcription factors may be in- this hypothesis, treatment of cells with the calcineurin inhibitor volved in sirt1 mRNA transcription. There are four members of CsA dose-dependently suppressed sirt1 luciferase activity (Fig. 2E). EGR family transcription factors; we then tested which of them To further confirm the involvement of EGR family transcription mediates sirt1 transcription. As shown in Fig. 2C, cotransfection of factors in sirt1 gene transcription, we used a ChIP assay and ana- EGR2 and EGR3 transcription factors dramatically enhanced lyzed whether EGR transcription factors bind to the sirt1 promoter sirt1-luciferase activity. In contrast, EGR1 and EGR4 coex- in T cells. The binding of EGR2 to the sirt1 promoter was detected pression showed a modest luciferase activity of sirt1 promoter (Fig. in activated T cells, and this binding was significantly increased in 2C). Mutation of the EGR-binding sites completely abolished the tolerized T cells isolated from OT-II TCR transgenic mice that sirt1 reporter activity, even when either EGR2 or EGR3 was were treated with OVA323–339 peptide. In contrast, only back-

Fig. 2. EGR2/3 regulates sirt1 transcription in T cells. (A)The−196 bp to +79 bp region of Sirt1 promoter sequence is shown. EGR-binding sites are indicated in red and the partial sequence of the ﬁrst exon of sirt1 gene is underlined. (B) HEK293 cells were transfected with control pGL4-luc or with each of the pGL4-sirt1-luc plasmids that carry −1kb,−0.5 kb, or −196 bp region of sirt1 promoter region, or with muta- tions of all four EGR-binding sites. Luciferase activity in the lysates of transfected cells were determined by a dual luciferase assay. Error bars represent data from three independent experiments. (C and D)pGL4-sirt1-luc that carries −196 bp region of the sirt1 promoter (C)oritsmutant on all four EGR-binding sites (D)werecotrans- fected without or with EGR1, EGR2, EGR3, and EGR4, or FoxO3a expression plasmids, as indicated. The luciferase activity in transfected cells was determined. (E) Cells were transfected with pGL4-sirt1-luc that carries −196 bp region of the sirt1 promoter and treatment with different doses of CsA for 24 h and sirt1-luciferase activities were analyzed. (F–H) Naïve, activated, and anergic T cells were generated as described in Fig. 1. The binding of EGR2 (F, Middle,andG) and NFAT (F, Top,andH)tothesirt1 promoter was determined with ChIP assay. Their binding to the il-2 promoter was analyzed as a control (F and H, light-gray bars).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1118462109 Gao et al. Downloaded by guest on September 23, 2021 Fig. 3. Regulation of sirt1 transcription by FoxO3a. (A) pGL4-sirt1-luc plasmid DNA was cotransfected with different combinations of EGR2 (E2), EGR3 (E3), and FoxO3a (F) expression plasmids, as indicated (50 ng each). The luciferase activities in transfected cells were determined. (B and C) HA- tagged FoxO3a expression plasmid DNA was cotransfected with EGR2 (B) or with EGR3 (C) into HEK293 cells. FoxO3a protein in the lysates of transfected cells was immunopreci- pitated with anti-HA antibody. The binding of EGR2 or EGR3 with FoxO3a was determined by coimmunoprecipitation and Western blotting. (D) Mouse primary T cells were stimulated with anti-CD3 plus anti-CD28 for 2 h. The interaction of FoxO3a with EGR2 and EGR3 was determined by coimmunoprecipitation with anti-FoxO3a antibody and Western blotting with anti-EGR2 (Top, Left) and EGR3 (Middle, Left) antibodies. The same membrane was reblotted with anti- FoxO3a (Bottom, Left). The protein expression levels of EGR2 (Top, Right), EGR3 (Middle, Right), and FoxO3a (Bottom, Right) in the whole-cell lysates (WCL) were determined by Western blotting as loading controls. (E and F) Naïve T cells were transfected with siRNA against EGR2 or EGR3 by nuclear infection. The cells were then stimulated with anti-CD3 plus anti-CD28 for 24 h. (E) The protein expression levels of EGR2 (Top), EGR3 (Middle), and FoxO3a (Bottom) were determined by Western blotting. (F) The binding of FoxO3a and EGR2 to sirt1 promoter was determined by ChIP assay. The error bars represent data from three independent experi-

ments (mean ± SE). Student t test was used for statistic IMMUNOLOGY analysis. (G) The sirt1 promoter-binding activities of FoxO3a in the activated and anergic T cells were analyzed by ChIP assay.

ground levels of EGR2 binding to the sirt1 promoter was detected antibodies in our hand worked for ChIP analysis. It has been in naïve CD4 T cells (Fig. 2 F and G). In contrast, NFAT binding to reported that EGR2, EGR3, and Sirt1 are up-regulated to main- the il-2 but not sirt1 promoter was detected in activated and anergic tain T-cell tolerance, and that EGR2 and EGR3 are downstream T cells (Fig. 2H). Unfortunately, we could not analyze sirt1 pro- of NFAT (10, 21). Our results here provide a possible link of EGR moter-binding activity of EGR3 because none of the anti-EGR3 family transcription factors to Sirt1 in T-cell anergy induction.

Fig. 4. IL-2 suppresses sirt1 transcription in T cells. (A and B) Naïve T cells were stimulated with anti-CD3 (A) or anti- CD3 plus anti-CD28 (B) for different amounts of time as indicated (hours). The mRNA levels of sirt1 and egr2 were determined by real-time PCR. (C) Naïve CD4+ T cells were stimulated with anti-CD3 plus anti-CD28 for 24 h. The cells were then cultured with IL-2 (10 ng/mL) or with anti– IL-2 neutralization antibody (10 μg/mL) for each indicated amount of time (hours). (D) Naïve T cells were stimulated with anti-CD3 plus anti-CD28 for 12 h followed by cultivation with 10 ng/mL of IL-2 or with PI3K inhibitor LY294002 or with rapamycin for an additional 6 h. Cy- toplasmic (C) and nuclear (N) fractionations were performed and the protein levels of FoxO3a and EGR2 were analyzed by Western blotting (A). The levels of phos- phorylated FoxO3a (p-FoxO3a) and AKT (p-AKT), as well as their total protein levels in the whole-cell lysates, were analyzed by Western blotting. (E and F) Anergic CD4+ T cells were isolated from OT-II TCR transgenic mice treated

with OVA323–339 peptide. Puriﬁed cells were cultured with anti-CD3 plus anti-CD28 or with IL-2 or PI3K inhibitor LY294002 or rapamycin for 12 h. The levels of phos- phorylated and total FoxO3a and AKT were analyzed by Western blotting (E). The mRNA levels of sirt1 and egr2 were determined by real-time PCR. Error bars represent data from three independent experiments (F).

Gao et al. PNAS Early Edition | 3of6 Downloaded by guest on September 23, 2021 − − Fig. 5. Expression of the constitutively active form of FoxO3a blocks IL-2 ability to reverse T-cell anergy. Naïve CD4 T cells from wild-type and Sirt1 / mice were puriﬁed and cultivated with anti-CD3 plus anti-CD28 for 24 h followed by infection with retrovirus that carry (A and B) GFP only (GFP) or GFP with the constitutively active form FoxO3a (FoxO3a/Act), or (C and D) with control shRNA or with the shRNA against FoxO3a. Three days after infection, GFP+ cells were sorted out, rested, and then treated with or without ionomycin for 16 h to induce tolerance. Cells were restimulated with anti-CD3 and anti-CD28 in the absence or presence of IL-2. (A and C) Sirt1 (Top) and FoxO3a (Middle) expression levels were determined by Western blotting using β-Actin as a control (Bottom). (B and D) The proliferation was determined by 3H-thymidine (3H-TdR) incorporation. Error bars represent data from three independent experiments.

FoxO3a Promotes Sirt1 Transcription Through Its Binding with EGR2/3 When T cells only received EGR2 or EGR3 siRNA, a modest in T Cells. It has been reported that FoxO3a is a critical tran- reduction of FoxO3a-binding to sirt1 promoter was detected. In scription factor for sirt1 expression in human cells through its contrast, the sirt1 promoter binding activity of FoxO3a was sig- binding with p53 (22). Therefore, we attempted to test whether nificantly inhibited in T cells when both EGR2 and EGR3 were FoxO3a is also involved in sirt1 transcription in mouse T cells. knocked-down (Fig. 3F). As expected, suppression of EGR2 ex- Expression of FoxO3a induced a modest sirt1-luciferase activity. pression inhibited its binding to the sirt1 promoter, but EGR3 Coexpression with EGR2/3 synergistically promoted sirt1 lucif- binding activity was unaffected (Fig. 3F). In addition, a significant erase activity (Fig. 3A). Mutation of the EGR-binding sites in the increase in FoxO3a binding to sirt1 promoter DNA in anergic sirt1 promoter completely abolished FoxO3a-mediated sirt1-lucif- T cells was detected compared with that in the activated T cells erase activity (Fig. 2D). In human cells, FoxO3a binds to the sirt1 (Fig. 3G). Collectively, our data suggest that FoxO3a interacts promoter via interaction with p53 as two p53 binding sites were with EGR2/3 to promote sirt1 mRNA transcription in T cells. identified in the promoter region of human sirt1 gene (22). How- ever, the conserved p53 binding sites do not exist at the mouse sirt1 IL-2 Suppresses sirt1 Transcription in T Cells. Sirt1 is highly expressed promoter (Fig. 2A). Therefore, we asked whether FoxO3a pro- in anergic T cells and during the early stage of T-cell activation, motes sirt1 gene transcription in T cells by binding to EGR2 and but it is down-regulated in activated and differentiated effector T EGR3 at the sirt1 promoter. Indeed, EGR2 and EGR3 proteins cells, suggesting that cytokine-mediated signaling might be in- were detected in the anti-HA immunoprecipitates from the lysates volved in regulation of Sirt1 expression. To test this theory we of HEK293 cells when HA-FoxO3a expression plasmid DNA was analyzed the dynamics of sirt1 mRNA transcription in mouse cotransfected, but not in the nontransfected controls, suggesting primary T cells. As shown in Fig. 4A, a significant increase in sirt1 FoxO3a interacts with both EGR2 and EGR3 in transiently mRNA levels was detected at 12 h after TCR alone or TCR plus transfected cells (Fig. 3 B and C). Their interaction was also CD28 stimulation, which reached its peak at 24 h. A rapid de- detected in mouse primary T cells upon anti-CD3 stimulation, but crease in sirt1 mRNA transcription was observed from 36 h in T anti-CD28 did not further enhance FoxO3a-interaction with cells when stimulated with both anti-CD3 and anti-CD28, and the EGR2 and EGR3, suggesting that FoxO3a may promote Sirt1 sirt1 mRNA levels dropped to a similar level to that in naïve T transcription through binding to EGR2/3 (Fig. 3D). cells at 2 d poststimulation. However, when T cells were stimu- If FoxO3a binds to the sirt1 promoter through EGR2/3, sup- lated with anti-CD3 alone, only a modest decline in sirt1 mRNA pression of EGR2 and EGR3 expression should inhibit FoxO3a- levels was observed, and this high level of sirt1 gene transcription binding to the sirt1 promoter. We then used a siRNA-mediated was maintained at day 4 after TCR stimulation. These results knockdown approach to test this hypothesis. As shown in Fig. 3E, indicate that TCR-mediated signaling alone is sufficient in sirt1 EGR2-specific siRNA inhibited 70–80% of EGR2 expression gene transcription and that CD28 costimulation induces a nega- without affecting EGR3 protein levels. Similarly, EGR3-specific tive factor to suppress sirt1 gene transcription. In contrast, a dra- siRNA knocked-down EGR3 but not EGR2 expression in T cells. matic increase in egr2 mRNA was detected within 3 h of TCR/

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1118462109 Gao et al. Downloaded by guest on September 23, 2021 Fig. 6. A proposed model for IL-2–mediated reversal of T-cell tolerance through modulating Sirt1 expression. (A) Schematic representation of the transcriptional regulation of sirt1 during T-cell anergy induction. TCR signaling activates NFAT through calcium/calmodulin/calcineurin pathway to induce egr2/3 expression. EGR2 and EGR3 bind to FoxO3a at sirt1 promoter to synergistically induce sirt1 gene transcription for inducing and maintaining T-cell tolerance. (B) Schematic representation of IL-2–mediated reversal of T-cell anergy. When anergic T cells receive IL-2 (may be also CD28), its binding to IL-2R activates PI3K/AKT. The activated AKT phosphorylates FoxO3a leading to sequestering FoxO3a to cytoplasm. Therefore, sirt1 gene expression is inhibited leading to breakdown of T-cell tolerance.

CD28 stimulation, which peaked at 6–9 h. The expression of egr2 expression. Therefore, suppression of sirt1 transcription appears mRNA was maintained at relatively high levels during a 4-d pe- to be an explanation for IL-2–mediated reversal of T-cell anergy. riod cultivation, although with a signiﬁcant decline at about a day It has been shown that the mammalian target of rapamycin after stimulation, and CD28 costimulation appears not to affect (mTOR) kinase inhibitor rapamycin, which inhibits both mTORC1 egr2 gene transcription (Fig. 4B). Because both egr2 and egr3 are and mTORC2 in T cells, blocks the ability of IL-2 to reverse anergy downstream genes of NFAT, this relative delay in sirt1 mRNA (24, 25). As FoxO3a phosphorylation and inhibition is also medi- transcription during the early stages of T-cell activation suggests ated by mTORC2, we then asked whether rapamycin is able to that sirt1 gene transcription depends on EGR2 and EGR3 protein block FoxO3a phosphorylation and the down-regulation of sirt1. expression in T cells. In fact, treatment of T cells with rapamycin inhibited FoxO3a phosphorylation and sirt1 down-regulation that is induced by IL-2

Because sirt1 transcription is inhibited in all lineages of effector IMMUNOLOGY cells tested, including Th1, Th2, Th17, and Treg (Fig. 1 A and B), in anergic T cells (Fig. 4E). As a consequence, rapamycin treatment and IL-2 is the common cytokine during their differentiation, we also partially blocked the ability of IL-2 to suppress sirt1 gene ex- asked whether IL-2 is involved in suppression of sirt1 mRNA pression (Fig. 4F). – transcription. To test this theory, we stimulated CD4+ naïve T cells Because IL-2 mediated suppression of FoxO3a transcriptional with anti-CD3 plus anti-CD28 for 24 h, followed by addition of activity is responsible for sirt1 gene down-regulation and reversal recombinant mouse IL-2 or anti-mouse IL-2 neutralization anti- of T-cell tolerance, we asked whether the constitutively active form body. The exogenous IL-2, which induced T-cell proliferation, of FoxO3a can maintain sirt1 expression in anergic T cells when significantly reduced sirt1 mRNA and protein expression (Fig. 4C). stimulated with IL-2. To this end, the constitutively active form of In contrast, neutralization of IL-2 that was produced by activated T FoxO3a with a triple mutation of the AKT phosphorylation sites cells upon TCR/CD28 stimuli maintained the high levels of sirt1 (TM-FoxO3a), which is not susceptible to inactivation by Akt phosphorylation (26), was delivered into CD4+ during expansion mRNA expression (Fig. 4C). These data indicate that IL-2 is phase and rested. Anergy was induced by ionomycin treatment. a negative factor in regulating sirt1 gene expression in T cells. Data in Fig. 5A show that IL-2 treatment failed to down-regulate IL-2 Inhibits sirt1 Gene Transcription Through Phosphorylation of Sirt1 protein expression in T cells that express the constitutively FoxO3a in T Cells. It has been reported that IL-2 sequesters active form of FoxO3a. As a consequence, this constitutively active FoxO3a from the nucleus to the cytoplasm to promote T-cell FoxO3a expression partially blocked the ability of IL-2 in reversal proliferation and survival (23). It is possible that IL-2 suppresses of T-cell tolerance. The constitutively active FoxO3a suppresses IL-2–mediated T-cell anergy reversal is through Sirt1, because its sirt1 transcription by sequestering FoxO3a to cytoplasm in T cells. − − expression in Sirt1 / T cells failed to maintain T-cell anergy (Fig. Subcellular fractionation experiments confirmed that IL-2 se- 5B). However, knockdown of FoxO3a expression in T cells results questered FoxO3a to the cytoplasm. This IL-2–mediated se- in reduced Sirt1 expression, even after ionomycin treatment (Fig. questration of FoxO3a is inhibited by pretreatment of T cells with 5C). As a consequence, FoxO3a knockdown leads to a partial the PI3K inhibitor LY294002. In contrast, the subcellular distri- breakdown of tolerance of wild-type T cells (Fig. 5D). Collectively, bution of EGR2 was affected neither by IL-2 nor by PI3K in- our findings indicate that IL-2–mediated suppression of FoxO3a hibitor LY294002 treatment (Fig. 4D). Therefore, IL-2 appears to transcriptional activation inhibits sirt1 gene transcription to re- sequester FoxO3a through the PI3K-AKT pathway. To support verse T-cell tolerance. this finding, we further detected an elevated phosphorylation of FoxO3a and AKT by IL-2 treatment of anergic T cells, and this Discussion – IL-2 mediated AKT phosphorylation was inhibited by LY294002 Peripheral tolerance of self-reactive T cells is induced by the re- (Fig. 4E). Exogenous IL-2 reverses T-cell tolerance, and our re- organization of MHC/antigen complexes through TCR in the ab- cent findings show that TCR-mediated Sirt1 expression contrib- sence of costimulatory signaling (1, 2). Tolerized or anergic T cells utes significantly to maintain T-cell anergy (9). Combined with express the high-affinity IL-2Ra (CD25), but do not produce IL-2, our present data, these findings suggest that IL-2 may reverse T- even after reobtaining a combined TCR/CD28 stimulation. Add- cell anergy by suppressing sirt1 transcription. To test this hy- ing exogenous IL-2 leads to the breakdown of this tolerance, pothesis, we isolated CD4+ T cells from OT-II mice pretreated a phenomenon that has been known for decades. The present with OVA323–339 peptide and cultured them in the absence or study provides a possible molecular explanation of IL-2-mediated presence of IL-2 for 12 h. As shown in Fig. 4F, IL-2 dramatically reversal of T-cell tolerance by suppressing Sirt1 expression (Fig. 6). inhibited sirt1 mRNA expression without affecting egr2 mRNA in This conclusion is supported by the following observations: (i)the tolerized T cells. Treatment with PI3K inhibitor LY partially TCR-NFAT-EGR2/3 pathway induces Sirt1 expression to main- blocked IL-2-mediated down-regulation of sirt1 mRNA tain T-cell tolerance; (ii) FoxO3a interacts with EGR2/3 to

Gao et al. PNAS Early Edition | 5of6 Downloaded by guest on September 23, 2021 synergistically promote sirt1 gene expression during T-cell anergy four conserved binding sites for EGR-family transcription factors. induction; (iii) IL-2 sequesters FoxO3a to suppress sirt1 gene ex- Further analysis as reported in this study indicates that FoxO3a pression leading to reversal of T-cell tolerance; (iv) the constitu- interacts with EGR2 and EGR3 to synergistically regulate sirt1 tively active form of FoxO3a partially blocks IL-2–mediated − − mRNA transcription in T cells. reversal of T-cell anergy of wild-type but not Sirt1 / T cells; and (v) knockdown of FoxO3a inhibits sirt1 expression, enhances T- Materials and Methods cell activation and partially breaks down T-cell tolerance. Isolating Mouse Naïve T Cells and in Vivo CD4+ T-Cell Anergy Induction. Primary Anergic T cells express CD25, the high-affinity IL-2 receptor, T cells were isolated from the lymph nodes and spleens of 6- to 8-wk-old mice. − but not IL-2 cytokines, as the transcription factors mediating IL-2 CD4+CD25 CD44lowCD62hi naïve T cells were purified using a naïve CD4+ T-cell expression, such as NFAT, NF-κB, and AP-1, are inhibited (27– isolation kit (Miltenyi Biotec). These cells were maintained in RPMI media 29). The Sirt1-mediated deacetylation of both AP-1 and NF-κB supplemented with 10% FBS, 100 U/mL penicillin, 200 μg/mL streptomycin, transcription factors is responsible, at least partially, for sup- and 0.25 μg/mL amphotericin, and stimulated with anti-CD3 plus anti-CD28 (5 pressing IL-2 expression in anergic T cells (9, 18, 30, 31). It is well μg/mL each). For in vivo T-cell anergy induction, OT-II mice were treated with μ established that exogenous IL-2, together with TCR and CD28 200 g OVA323–339 peptide by tail intravenous injection. Mice were killed 7 d + stimuli, can induce the proliferation of anergic T cells (32), but the after treatment, CD4 T cells were isolated, and the anergy induction was fi underlying molecular mechanism remains unknown. IL-2 sup- con rmed as previously described (6). presses FoxO3a transcriptional activity by sequestering FoxO3a from nuclear to cytoplasm in a PI3K-AKT–dependent manner (23, Transfection, Immunoprecipitation, and Western Blotting. Transfection, immunoprecipitation, and Western blotting to analyze protein–protein inter- 33). As FoxO3a is a transcription factor of sirt1,andsirt1-deficency actions and expressions were performed as previously reported (14). Detailed leads to the breakdown of T-cell tolerance, this sequestration methods are described in SI Materials and Methods. of FoxO3a is likely a molecular explanation of IL-2–mediated reversal of T-cell anergy. Chromatin Immunoprecipitation. ChIP assays were performed as previously The forkhead transcription factor FoxO3a has been found as reported (14, 35) and a detailed method is described in SI Materials a transcription factor for sirt1 gene expression in human cells (22). and Methods. Our present study also demonstrates that FoxO3a promotes sirt1 gene transcription in mouse T cells. In human cells, FoxO3a binds Luciferase Assay. Experiments were performed as previously described (36). to the sirt1 promoter through an interaction with p53, which has been found to regulate sirt1 gene transcription upon DNA damage ACKNOWLEDGMENTS. We thank Dr. Fang Feng (Northwestern University) (34). However, p53 binding sites do not exist in mouse sirt1 pro- for early responsive-gene expression plasmids. This study was supported by National Institutes of Health Grant R01AI079056 and “The Type I Diabetes moter region, implying that FoxO3a regulates sirt1 gene tran- Pathfinder Award” (DK083050) from the National Institute of Health (to scription in mouse T cells differently from that in human cells. D.F.); and National Institutes of Health Training Grant 5K12GM088020-02 Interestingly, we analyzed the sirt1 promoter sequence and found (to K.K.).

1. Saibil SD, Deenick EK, Ohashi PS (2007) The sound of silence: Modulating anergy in T 19. Macián F, et al. (2002) Transcriptional mechanisms underlying lymphocyte tolerance. lymphocytes. Curr Opin Immunol 19:658–664. Cell 109:719–731. 2. Fathman CG, Lineberry NB (2007) Molecular mechanisms of CD4+ T-cell anergy. Nat 20. Rengarajan J, et al. (2000) Sequential involvement of NFAT and Egr transcription Rev Immunol 7:599–609. factors in FasL regulation. Immunity 12:293–300. 3. Brumeanu TD, Bona CA, Casares S (2001) T-cell tolerance and autoimmune diabetes. 21. Xiao S, et al. (1999) FasL promoter activation by IL-2 through SP1 and NFAT but not Int Rev Immunol 20:301–331. Egr-2 and Egr-3. Eur J Immunol 29:3456–3465. 4. Khan S, Greenberg JD, Bhardwaj N (2009) Dendritic cells as targets for therapy in 22. Nemoto S, Fergusson MM, Finkel T (2004) Nutrient availability regulates SIRT1 rheumatoid arthritis. Nat Rev Rheumatol 5:566–571. through a forkhead-dependent pathway. Science 306:2105–2108. 5. Waldner H, Collins M, Kuchroo VK (2004) Activation of antigen-presenting cells by 23. Stahl M, et al. (2002) The forkhead transcription factor FoxO regulates transcription – microbial products breaks self tolerance and induces autoimmune disease. J Clin In- of p27Kip1 and Bim in response to IL-2. J Immunol 168:5024 5031. vest 113:990–997. 24. Delgoffe GM, et al. (2011) The kinase mTOR regulates the differentiation of helper T 6. Lee SM, et al. (2011) Prevention and treatment of diabetes with resveratrol in a non- cells through the selective activation of signaling by mTORC1 and mTORC2. Nat Im- – obese mouse model of type 1 diabetes. Diabetologia 54:1136–1146. munol 12:295 303. 7. Heissmeyer V, et al. (2004) Calcineurin imposes T cell unresponsiveness through tar- 25. Powell JD, Delgoffe GM (2010) The mammalian target of rapamycin: Linking T cell differentiation, function, and metabolism. Immunity 33:301–311. geted proteolysis of signaling proteins. Nat Immunol 5:255–265. 26. Skurk C, et al. (2004) The Akt-regulated forkhead transcription factor FOXO3a con- 8. Anandasabapathy N, et al. (2003) GRAIL: An E3 ubiquitin ligase that inhibits cytokine trols endothelial cell viability through modulation of the caspase-8 inhibitor FLIP. J gene transcription is expressed in anergic CD4+ T cells. Immunity 18:535–547. Biol Chem 279:1513–1525. 9. Zhang J, et al. (2009) The type III histone deacetylase Sirt1 is essential for maintenance 27. Mondino A, et al. (1996) Defective transcription of the IL-2 gene is associated with of T cell tolerance in mice. J Clin Invest 119:3048–3058. impaired expression of c-Fos, FosB, and JunB in anergic T helper 1 cells. J Immunol 10. Safford M, et al. (2005) Egr-2 and Egr-3 are negative regulators of T cell activation. 157:2048–2057. Nat Immunol 6:472–480. 28. Li W, Whaley CD, Mondino A, Mueller DL (1996) Blocked signal transduction to the 11. Baine I, Abe BT, Macian F (2009) Regulation of T-cell tolerance by calcium/NFAT sig- ERK and JNK protein kinases in anergic CD4+ T cells. Science 271:1272–1276. naling. Immunol Rev 231:225–240. 29. Sundstedt A, et al. (1996) In vivo anergized CD4+ T cells express perturbed AP-1 and 12. Soto-Nieves N, et al. (2009) Transcriptional complexes formed by NFAT dimers regu- NF-kappa B transcription factors. Proc Natl Acad Sci USA 93:979–984. – late the induction of T cell tolerance. J Exp Med 206:867 876. 30. Salminen A, Kaarniranta K (2009) NF-kappaB signaling in the aging process. J Clin 13. Fu G, et al. (2010) Phospholipase Cgamma1 is essential for T cell development, acti- Immunol 29:397–405. – vation, and tolerance. J Exp Med 207:309 318. 31. Yeung F, et al. (2004) Modulation of NF-kappaB-dependent transcription and cell 14. Kong S, et al. (2011) The type III histone deacetylase Sirt1 protein suppresses p300- survival by the SIRT1 deacetylase. EMBO J 23:2369–2380. mediated histone H3 lysine 56 acetylation at Bclaf1 promoter to inhibit T cell acti- 32. Nossal GJ (1993) Tolerance and ways to break it. Ann N Y Acad Sci 690:34–41. vation. J Biol Chem 286:16967–16975. 33. Riou C, et al. (2007) Convergence of TCR and cytokine signaling leads to FOXO3a 15. Sequeira J, et al. (2008) sirt1-null mice develop an autoimmune-like condition. Exp phosphorylation and drives the survival of CD4+ central memory T cells. J Exp Med Cell Res 314:3069–3074. 204:79–91. 16. Kwon HS, et al. (2008) Human immunodeﬁciency virus type 1 Tat protein inhibits the 34. Luo J, et al. (2001) Negative control of p53 by Sir2alpha promotes cell survival under SIRT1 deacetylase and induces T cell hyperactivation. Cell Host Microbe 3:158–167. stress. Cell 107:137–148. 17. Chen J, et al. (2005) SIRT1 protects against microglia-dependent amyloid-beta toxicity 35. Lee SM, Gao B, Fang D (2008) FoxP3 maintains Treg unresponsiveness by selectively through inhibiting NF-kappaB signaling. J Biol Chem 280:40364–40374. inhibiting the promoter DNA-binding activity of AP-1. Blood 111:3599–3606. 18. Schug TT, et al. (2010) Myeloid deletion of SIRT1 induces inﬂammatory signaling in 36. Chen A, et al. (2009) The HECT-type E3 ubiquitin ligase AIP2 inhibits activation-in- response to environmental stress. Mol Cell Biol 30:4712–4721. duced T-cell death by catalyzing EGR2 ubiquitination. Mol Cell Biol 29:5348–5356.

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