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Aplastic Anemia in MicePromotes Th1 Cell Responses MediatingPosttranslational Expression of T-bet and Ezh2 Regulates Transcriptional and

ZhangSun, Yanyun Zhang, Yajun Guo, Elizabeth Hexner and YiYongnian Liu, Izumi Mochizuki, Lijun Meng, Hongxing Qing Tong, Shan He, Fang Xie, Kazuhiro Mochizuki,

http://www.jimmunol.org/content/192/11/5012doi: 10.4049/jimmunol.1302943April 2014;

2014; 192:5012-5022; Prepublished online 23J Immunol 

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3.DCSupplementalhttp://www.jimmunol.org/content/suppl/2014/04/23/jimmunol.130294

Referenceshttp://www.jimmunol.org/content/192/11/5012.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2014 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Journal of Immunology

Ezh2 Regulates Transcriptional and PosttranslationalExpression of T-bet and Promotes Th1 Cell ResponsesMediating Aplastic Anemia in Mice

Qing Tong,*,† Shan He,†,‡ Fang Xie,† Kazuhiro Mochizuki,† Yongnian Liu,†,‡

Izumi Mochizuki,† Lijun Meng,‡,x Hongxing Sun,x Yanyun Zhang,x Yajun Guo,*

Elizabeth Hexner,{ and Yi Zhang†,‡

Acquired aplastic anemia (AA) is a potentially fatal bone marrow (BM) failure syndrome. IFN-g–producing Th1 CD4+ T cells

mediate the immune destruction of hematopoietic cells, and they are central to the pathogenesis. However, the molecular events

that control the development of BM-destructive Th1 cells remain largely unknown. Ezh2 is a chromatin-modifying enzyme that

regulates multiple cellular processes primarily by silencing gene expression. We recently reported that Ezh2 is crucial for

inflammatory T cell responses after allogeneic BM transplantation. To elucidate whether Ezh2 mediates pathogenic Th1 responses

in AA and the mechanism of Ezh2 action in regulating Th1 cells, we studied the effects of Ezh2 inhibition in CD4+ T cells using

a mouse model of human AA. Conditionally deleting Ezh2 in mature T cells dramatically reduced the production of BM-

destructive Th1 cells in vivo, decreased BM-infiltrating Th1 cells, and rescued mice from BM failure. Ezh2 inhibition resulted

in significant decrease in the expression of Tbx21 and Stat4, which encode transcription factors T-bet and STAT4, respectively.

Introduction of T-bet but not STAT4 into Ezh2-deficient T cells fully rescued their differentiation into Th1 cells mediating AA.

Ezh2 bound to the Tbx21 promoter in Th1 cells and directly activated Tbx21 transcription. Unexpectedly, Ezh2 was also required

to prevent proteasome-mediated degradation of T-bet protein in Th1 cells. Our results demonstrate that Ezh2 promotes the

generation of BM-destructive Th1 cells through a mechanism of transcriptional and posttranscriptional regulation of T-bet. These

results also highlight the therapeutic potential of Ezh2 inhibition in reducing AA and other autoimmune diseases. The Journal of

Immunology, 2014, 192: 5012–5022.

Acquired aplastic anemia (AA) in humans is a fatal dis-order characterized by bone marrow (BM) hypoplasiaand blood pancytopenia (1, 2). Clinical studies indicate

that in most cases, AA is a disease caused by immune-mediateddestruction of hematopoietic stem cells and hematopoietic pro-genitor cells (1, 2). A role for T cells in AAwas first suggested bytheir inhibition of hematopoietic cell colony formation in culturesin vitro (2). Furthermore, CD4+ T cell clones isolated from the

patients with AA have a potent ability to lyse autologous CD34+

hematopoietic cells and inhibit formation of hematopoietic cellcolonies (3). Accumulating evidence indicates that CD4+ Th1cells, which are characterized by production of high levels ofIFN-g, play important roles in mediating bone marrow failure(BMF) (2, 4–8). IFN-g displays potent effects on suppressinghematopoiesis in vitro (2, 3). Immunosuppressive therapy andallogeneic BM transplantation (BMT) have significantly im-proved the survival of severe AA. However, relapse still occursin ∼35% of patients with AAwhen immunosuppressive therapyis withdrawn (1, 2, 9). Furthermore, graft-versus-host diseaseremains a major barrier to the success of allogeneic BMT (10,11). Novel approaches are needed to improve the outcome oftreatments for AA.The transcription factor T-bet (encoded by Tbx21) is crucial for

Th1 cell differentiation (12–15). T-bet is induced by TCR sig-naling and strongly upregulated by activation of the STAT1 tran-scription factor (16). T-bet binds to several enhancers and promoterof the Ifng genes, activating its transcription (12, 16). T-bet alsopromotes expression of the IL-12 receptor b2 chain (IL12Rb2),resulting in greater IL-12 responsiveness and further elevatedproduction of IFN-g (16). In addition, T-bet prevents Th2 differ-entiation by inhibiting Gata3 (16). T-bet is upregulated in pe-ripheral blood T cells from patients with AA, and it is a usefulmarker for predicting the responsiveness of patients to immuno-suppressive therapy (17). Furthermore, experimental studies sug-gested that T cells lacking T-bet were defective in induction of AAin mice (6). These observations suggest that T-bet can be an at-tractive target for modulating Th1 cell–mediated AA. However,transcription factors are difficult drug targets (18). Thus, identifying

*International Joint Cancer Institute, Second Military Medical University, Shanghai200433, China; †Department of Internal Medicine, University of Michigan MedicalSchool, Ann Arbor, MI, 48109; ‡Department of Microbiology and Immunology, FelsInstitute for Cancer Research and Molecular Biology, Temple University, Philadelphia,PA 19140; xInstitute of Health Sciences, Shanghai Institutes for Biological SciencesChinese Academy of Sciences, Shanghai 200433, China; and {Department of Med-icine and Abramson Cancer Center, University of Pennsylvania Perelman School ofMedicine, Philadelphia, PA 19104

Received for publication November 1, 2013. Accepted for publication March 19,2014.

This work was supported by the American Cancer Society (to Y.Z.), the U.S. De-partment of Defense (to Y.Z.) and National Institutes of Health Grant R01-CA-172106-01 (to Y.Z.).

Q. T. and Y. Z. conceived and designed the project; Q.T., S.H., F.X., K.M., Y.L., I.M.,L.M., H.S., Y.Z., Y.G., E.H., and Y.Z. performed experiments and analyzed the data;Q. T. and Y. Z. wrote the manuscript.

Address correspondence and reprint requests to Dr. Yi Zhang, Department of Micro-biology and Immunology, Fels Institute for Cancer Research and Molecular Biology,Temple University, Philadelphia, PA 19140. E-mail address: yi.zhang@temple.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: AA, aplastic anemia; BM, bone marrow; BMF,bone marrow failure; BMT, bone marrow transplantation; ChIP, chromatin immuno-precipitation; LN, lymph node; TSS, transcription start site.

Copyright� 2014 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/14/$16.00

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the molecular pathways that control T-bet expression in Th1 cellscan lead to new strategies to control AA.Ezh2 is a histone methyltransferase that specifically catalyzes

trimethylation of histone H3 at lysine 27 (H3K27me3) (19). Ezh2forms polycomb repressive complex 2 together with other polycombgroup proteins Suz12 and Eed (19), which is crucial for maintainingthe cellular memory and transcriptional patterns primarily througha mechanism of silencing genes (20, 21). Several studies indicate animportant role of Ezh2 and H3K27me3 in multiple lineages of ef-fector T cells (22–25). Genome-wide mapping analysis revealed thatrepressive H3K27me3 marked genes associated with differentiationand maintenance of effector and memory T cells (26, 27). Recently,we demonstrated new and essential roles of Ezh2 in regulating in-flammatory T cell responses in mice after allogeneic BMT (28).Loss of Ezh2 led to impaired production of alloreactive T cells thatinduce damage to epithelial organs (28). However, whether Ezh2mediates pathogenic Th1 responses in AA and the mechanism ofEzh2 action in regulating Th1 cells remain unknown.Mouse models of human AA have been established successfully

(4, 29). Transfer of parent lymph node (LN) cells into hap-loidentical daughter recipients caused BM hypoplasia and bloodpancytopenia, typical features of clinical AA. These AA mousemodels have proved to be a unique approach studying patho-physiology of immune cell-mediated BMF (4, 6, 30, 31). In thisreport, we exploited the functional impact of Ezh2 on Th1 cellresponses in vitro and in vivo. Using genetic approaches and amouse model of human BMF, we identified a novel and criticalrole of Ezh2 in regulating Th1 cells mediating AA.

Materials and MethodsMice

C57BL/6 (B6, H-2b) and B6xDBA/2 F1 (BDF1, H-2b/d) mice were pur-chased from Taconic (Rockville, MD). Cd4-Cre mice were originally de-rived from the Jackson Laboratory. B6/129 mice with floxed alleles ofEzh2 (Ezh2fl/fl) (32) were crossed to Cd4-Cre mice, before backcrossingto the B6 background (.8 generations). Age-matched and sex-matchedcontrols were used. Experimental protocols were approved by the Uni-versity of Michigan’s Committee on Use and Care of Animals.

Abs and flow cytometry analysis

Abs used for immunofluorescence staining were purchased from eBioscience,BioLegend, or BD Biosciences. Flow cytometry analyses were performedusing FACScan and Canto cytometer (Becton Dickinson) as described (33).

Induction of AA

The mouse AA model was induced as described previously (30, 31). B6 LNcells (10 3 106/mouse) isolated from B6 mice were transplanted into ir-radiated BDF1 mice (6.5 Gy). Peripheral blood was collected from theheart and the lateral tail vein. Complete blood counts were performedusing a Hemavet 950 analyzer (Drew Scientific). BM cells were extractedfrom bilateral tibiae and femurs. Lymphocytes were isolated from inguinal,axillary, and lateral axillary LNs.

Histologic examination

The histologic examination was performed on paraffin-embedded femurpieces, fixed with 10% formalin, and colored with H&E. The histologicimage was photographed with a OlympusBX41 microscope (10/0.3 NAlens; original magnification 3100; digital DP70 camera).

Real time RT-PCR

Total RNA was extracted from the indicated CD4+ T cell subsets usingTRIzol (Invitrogen Life Technologies). cDNA was quantified through thequantitative real-time PCR technique. Real-time PCR was performed witha SYBR Green PCR mix on a Mastercycler realplex (Eppendorf). Ther-mocycler conditions included an initial holding at 95˚C for 2 min; this wasfollowed by a three-step PCR program as follows: 95˚C for 30 s, 55˚C for30 s, and 72˚C for 30 s for 45 cycles. Transcript abundance was calculatedusing the DDCt method (normalization with 18 s). The primer sequencesare listed in Supplemental Table I.

Cell culture

Splenic and LN CD4+ T cells were prepared by MACS purification withCD4 microbeads (Miltenyi Biotec), and the purity was usually 90–95%.Th1-skewing culture conditions were set up as previously described (34–36). In brief, CD44loCD4+ naive T cells (Tn) were cultured in the presenceof anti-CD3 (2 mg/ml; BioLegend) and anti-CD28 (2 mg/ml; BioLegend)Abs together with recombinant human IL-2 (10 ng/ml; R&D Systems),recombinant mouse IL-12 (5 ng/ml; R&D Systems) and BM-deriveddendritic cells at a ratio of 1:16. Cultured T cells were restimulated withanti-CD3 Ab (1 mg/ml; BioLegend) 5 h before intracellular staining.

Retroviral construction and T cell infection

The MigR1 retroviral vector system was described previously (35, 37).MigR1 vector was provided by Warren Pear (University of Pennsylvania),and MigR1 vector encoding T-bet or STAT4 was provided by Steve Reiner(Columbia University, New York, NY) and Takashi Usui (Kyoto Univer-sity, Kyoto, Japan). MigR1 virus was produced as described (38). Forretroviral infection, CD4+ T cells were prestimulated with anti-CD3 andanti-CD28 Abs for 24 h, and then the retrovirus supernatant was added inthe presence of 8 mg/ml polybrene (Sigma). Cells were spinoculated at3000 rpm, 32˚C for 3 h. The same retroviral infection procedure was re-peated 24 h later.

Western blot analysis

Western blot was performed as described (28). Cell lysates were examinedwith routine Western blotting. The blots were incubated with anti-Ezh2(612667; BD Biosciences), Stat4 (sc-486; Santa Cruz Biotechnology),T-bet (sc-21749; Santa Cruz Biotechnology), GATA-3 (558686; BD Bio-sciences), or actin (ab3280; Abcam) Abs, and subsequently incubated withHRP-conjugated anti-rabbit or mouse IgG (Vector Laboratories) in TBScontaining 5% nonfat dry milk and 0.05% Tween 20. The final reactionwas developed with a chemiluminescent system (Pierce).

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) assays were performed as describedby using EZ-Magna ChIP (17-10086; Millipore) (27, 39). Sonicatedextracts were precleared and incubated with Abs specific to Ezh2 (39901,active motif), H3K27me3 (PAb-069-050; Diagenode) or H3K4me3(9751S; Cell Signaling Technology) at 4˚C overnight on a 360˚C rotator.The immunoprecipitated DNA was quantitated by real-time quantitativePCR. The primer sequences are listed in Supplemental Table II.

ELISA

CD4+ Tn isolated from WT B6 and T-KO mice were stimulated with anti-CD3 (2 mg/ml; BioLegend) and anti-CD28 (2 mg/ml; BioLegend) Abstogether with recombinant human IL-2 (10 ng/ml; R&D Systems) andrecombinant mouse IL-12 (5 ng/ml; R&D Systems). On day 7, cells wererestimulated in 96-well plates with plate-bound anti CD3 Abs (1 mg/ml;BioLegend) for 5 h before collecting the supernatants. Each group con-tained an equal number of cells (1 3 106 cells/ml). The concentrations ofIFN-g and IL-4 were measured in triplicate using recombinant mouse IFN-gand IL-4 ELISA kits in accordance with the manufacturer’s instructions(BioLegend).

Luciferase reporter assay

The Tbx21 promoter region ranging from +0.3 to 21.0 kb of the tran-scription start site (TSS) was cloned to pGL3 luciferase reporter vector togenerate Tbx21-specific reporter (named pGL3-Tbx21 reporter). 3T3 cellswere cotransfected with pGL3-Tbx21 reporter plasmid and MigR1 viralplasmid encoding Ezh2 or empty MigR1 plasmid. The cells were harvested48 h after transfection and analyzed with the Dual Luciferase system(Promega).

Statistical analysis

Survival in different groups was compared using the log-rank test. Com-parison of two means was analyzed using the two-tailed unpaired Studentt test.

ResultsIn the absence of Ezh2, LN cells are defective in mediating AAin mice

We used a genetic approach to determine the role of Ezh2 in theregulation of T cell–mediated AA. Mice with floxed alleles of

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Ezh2 (Ezh2fl/fl) (32) were crossed to B6 mice expressing Crerecombinase under control of the CD4 promoter to generateT cell–specific Ezh2 conditional knockout B6 mice (namedT-KO). These T-KO mice were further backcrossed to B6 micefor 10 generations. The development of mature thymocytes andT cells in peripheral lymphoid tissues was normal in these T-KOmice, which is in agreement with previous observations (28, 32).To assess whether conditionally deleting Ezh2 in T cells affected

their ability to mediate AA, we transferred donor LN cells derivedfrom WT and T-KO B6 mice into irradiated (6.5 Gy) BDF1recipients. In this setting, transfer of donor LN cells causes severeBM destruction and blood pancytopenia in these haploidenticalrecipients, which closely reflects the pathogenesis of human AA(4, 6, 30). As expected, BDF1 mice receiving WT B6 LN cellsdeveloped BM hypoplasia and severe blood pancytopenia afterLN cell infusion compared with control mice receiving only totalbody irradiation, with all of them dying from the disease within12 d after transfer (Fig.1A). Histologic examination showed thedestruction of BM and lack of hematopoietic cell islands in theBM of these recipients (Fig.1B). In contrast, transfer of T-KO LNcells did not cause severe AA in these BDF1 recipients (Fig.1A,1B). As compared with control mice receiving total body irradi-ation, there was no significant reduction of BM cellularity andperipheral blood WBCs in these BDF1 mice receiving T-KO LNcells (Fig. 1C, 1D). Importantly, all T-KO LN cell recipientssurvived without clinical signs and histologic evidence of AA(Fig. 1A, 1B). Thus, T cells required Ezh2 to mediate AA.

Ezh2 is required for the development of Th1 cells inducing AAin mice

Previous studies have demonstrated that Th1 cells are crucial forinducing BMF in this model of experimental AA (2, 3). To examinethe effects of Ezh2 deficiency on Th1 cell development in vivo, weharvested donor T cells from the spleen, LN, and BM of thesemice 10 d after transfer of WT or T-KO LN cells. We found thatloss of Ezh2 led to a significant reduction in the percentage andnumber of Th1 cells in the spleen, LN, and BM (Fig. 2A, 2B).Real-time RT-PCR analysis showed that alloantigen-activatedT-KO T cells expressed dramatically lower levels of Ifng (5-fold),

Stat4 (2.5-fold), and Tbx21 (4-fold) transcripts than their WTcounterparts did (Fig.2C).In this BMF model, only a small percentage of IL-4–producing

CD4+ T cells occurred in the spleen, LN, and BM of BDF1 micereceiving WT LN cells (Fig. 2A, 2B), suggesting that the in-flammatory stimuli produced during the AA process predomi-nantly induces Th1 cell differentiation in vivo. Ezh2 deficiencydid not result in significant changes in the ability of CD4+ T cellsto produce IL-4 protein as assessed by flow cytometry (Fig. 2A).Furthermore, loss of Ezh2 had no significant effect on the ex-pression of Th2 cell genes (e.g., Il4, Il5, Il13, Stat6, and Gata3;Fig. 2C). There was a moderate reduction in Rorgt and an increasein Foxp3 (Fig. 2C). However, these moderate changes in genesexpression did not lead to the reduction in Il17 expression (Fig. 2C)and increase in regulatory T cells (Tregs; data not shown).Collectively, these results suggest that during the AA process,

Ezh2 is critically involved in regulating the development of Th1cells, but has little effect on Th2 or Th17 cells.Data from our previous studies and others indicate that Ezh2

deficiency can lead to impaired expansion and survival of activatedT cells (28, 40). It is possible that the inability of T-KO LN cellsto cause AA could result from decreased expansion and survivalof BM-destructive Th1 cells. To test this possibility, we trackedthe longitudinal proliferation and differentiation of WT and T-KO LN cells that were injected into sublethally irradiated BDF1recipients. Compared with WT LN cells, T-KO LN cells pro-duced significantly less in frequency of both CD4+ T cells andIFN-g–producing CD4+ T cells in the spleen, LN, and BM 6 and10 d after transfer (Fig. 3A, 3B). Because WT LN cells causedlethal AA in all BDF1 recipients by day 12 (data not shown), wewere prevented from further comparing the difference betweenWT and T-KO LN cells at later time points. However, in BDF1mice receiving T-KO LN cells, although there was a markedincrease in numbers of total donor CD4+ T cells in the spleen,LN and BM at day 43 after transfer compared with that at day 6(Fig. 3C), the frequency of IFN-g–producing CD4+ T cells wasnot increased in parallel (Fig. 3B). These data suggest that thelack of both differentiation and expansion of Th1 cells may beresponsible for the inability of T-KO T cells to mediate AA early

FIGURE 1. In the absence of Ezh2,

LN cells are defective in mediating AA

in mice. WT B6 LN (10 3 106) or

T-KO LN (10 3 106) cells were injected

into irradiated BDF1 mice (6.5Gy). (A)

The survival was monitored over time.

(B) Histologic analysis of BM from

each group 7 or 43 d after LN cell in-

fusion. (H&E staining; original magni-

fication 3100) (C) Total BM cellularity

was calculated assuming that bilateral

tibia and femurs contain 25% of total

marrow cells (mean 6 SD; n = 6–8

mice per group). (D) Peripheral blood

was collected 10 or 43 d after LN cell

infusion, and the cell number of RBCs,

WBCs, platelets, and neutrophils were

calculated (mean 6 SD; n = 6–8 mice

per group). Data are representative of

two independent experiments. *p ,0.05, **p , 0.01.

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during disease process, whereas impaired Th1 differentiation ofT-KO T cells could account for reduced AA during later stage.

Ezh2 promotes in vitro Th1 cell differentiation in culturesunder Th1-skewing conditions

To further examine the importance of Ezh2 in regulating Th1 celldifferentiation, we highly purified CD4+ Tn from WT and T-KOB6 mice and cultured them under Th1-skewing conditions withoutanti–IL-4 Ab. One day after activation in Th1-skewing cultures,both WT and T-KO CD4+ Tn produced barely detectable IFN-g asassessed by intracellular cytokine staining (Fig. 4A). Three daysafter culture, there were 3.7-fold less in frequency of IFN-g–

producing CD4+ T cells in the culture of T-KO CD4+ T cells thanthat of WT CD4+ T cells (Fig. 4A). This impaired ability of T-KOCD4+ T cells to produce IFN-g persisted throughout a period of7 d during culture (Fig. 4A). ELISA further confirmed that T-KOCD4+ T cells secreted ∼2.5-fold less IFN-g than WT CD4+ T cellsdid 7 d after culture (Fig. 4B), when Th1 cells fully developed (12,35). These data confirmed the observations in our precedingexperiments in vivo (Fig. 2) that Th1 cell differentiation wasimpaired in the absence of Ezh2.It has been shown that Ezh2 is associated with Vav1 protein (40),

which is a protein important for mediating proximal TCR sig-naling in T cell activation (41). To rule out the possibility that the

FIGURE 2. Loss of Ezh2 results in

selective impairment of Th1 cell devel-

opment in vivo during AA induction. WT

B6 LN (10 3 106) or T-KO LN (10 3106) were injected into irradiated BDF1

mice (6.5 Gy). Seven days after transfer,

spleen, LN, and BMs were isolated, and

donor-derived CD4+ T cells were an-

alyzed for the expression of IFN-g and

IL-4. (A) Dot plots and graphs show

the percentage and the mean fluores-

cence intensity (MFI) of donor CD4+

T cells producing cytokines. (B) Bar

graphs show the number of donor CD4+

IFN-g+ T cells. (C) Seven days after

transfer, donor-derived CD4+ T cells

were sorted for the analysis of gene ex-

pression. Data are representative of two

independent experiments, with each group

containing 4–6 mice. Error bars indicate

mean 6 SD. *p , 0.05, **p , 0.01,

***p , 0.001.

FIGURE 3. Tracking the longitudinal proliferation

and differentiation of transferred WT and T-KO LN

cells in vivo. WT B6 LN (10 3 106) or T-KO LN (10 3106) cells were injected into irradiated BDF1 mice

(6.5 Gy). Spleen, LN, and BM were isolated to measure

cytokine production in donor derived CD4+ T cells at

days 6, 10, 20, and 43 after transfer, respectively. (A)

Dot plots show the percentage of cytokine-producing

donor CD4+ T cells in the spleen. (B) Graphs show the

percentage of donor CD4+ T cells (left panel) and donor

IFN-g+CD4+ T cells (right panel). (C) Graphs show the

total number of donor CD4+ T cells. Data are repre-

sentative of two independent experiments, with each

group containing 4–6 mice. Error bars indicate mean 6SD. *p , 0.05, **p , 0.01.

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decreased induction of Th1 cells by Ezh2 inhibition might beassociated with impaired activation of T cells in cultures, weexamined the effects of Ezh2 deficiency on the activation anddivision of T-KO CD4+ T cells cultured under Th1-skewingconditions. We found that in the absence of Ezh2, CD4+ Tn werenormally activated as evidenced by the upregulation of T cell ac-tivation markers CD25, CD44, and CD69 (Fig. 4C). Furthermore,like WT CD4+ T cells, T-KO CD4+ T cells underwent extensive celldivision as evidenced by their dilution of fluorescence dye CFSE incultures (Fig. 4D). These data suggest that in the absence of Ezh2,CD4+ T cells can be normally activated to undergo cell division andexpansion upon stimulation with Th1-skewing conditions.Previous studies suggest that IL-4 can reduce Th1 cell differ-

entiation (35, 42). We found that in vitro activated T-KO T cellsproduced more IL-4–producing T cells in frequency than their WTcounterparts (Fig. 4A). It is possible that increased production ofIL-4 in T-KO T cells might account for their impaired Th1 celldifferentiation. To test this possibility, we added neutralizing anti–IL-4 Ab into the cultures. Indeed, blockade of IL-4 efficientlyinhibited the production of IL-4 by both WT and T-KO CD4+

T cells (Fig. 4E). Seven days after cultures, there was no markeddifference in frequency of IL-4–producing T cells between activatedWTand T-KO CD4+ T cells (Fig. 4E). In contrast, neutralizing IL-4in cultures did not improve the ability of T-KO CD4+ T cells toproduce IFN-g (Fig. 4E). Furthermore, we confirmed that both WTand T-KO CD4+ T cells derived from Th1-skewing cultures ex-pressed similar levels of GATA3 protein and mRNA (Fig. 4F). All

these results suggest that Ezh2 promotes Th1 cell differentiationthrough a mechanism independent of IL-4 and GATA3.

Ezh2 associates with Th1 gene loci

Development of Th1 cells involves a complex mechanism (15).Th1 cell differentiation is initiated by IFN-g upregulation of T-bet,which specifically activates Ifng transcription (15). IL-12 activa-tion of STAT4 is also important to promote Th1 cell differentiation(13–15). To understand the mechanism by which Ezh2 promotedthe development of Th1 cells, we first used ChIP assay to examinethe presence of Ezh2 and histone methylation markers on Th1gene loci (e.g., Ifng, Tbx21, and Stat4). CD4+ Tn were isolatedfrom normal WT B6 mice and cultured under Th1-skewing con-ditions for 7 d. Chromatin was prepared from both CD4+ Tn andTh1 cells. As expected, the amount of H3K27me3 was markedlyreduced at the promoter regions of Ifng, Tbx21, and Stat4 geneloci (Fig. 5A). In contrast, H3K4me3, which is a permissive his-tone methylation marker associated with gene activation (27, 43),was increased at the promoter region of these genes (Fig. 5A).These data are in agreement with previous observations thatH3K27me3 strongly marked the promoter, intergenic, and 39UTRregions of TBX21 in CD4+ Tn, whereas differentiated Th1 cellshad reduced H3K27me3 but increased H3K4me3 at the TBX21regulatory regions (22, 27).Interestingly, there was no significant reduction in the amount of

Ezh2 at both regulatory and promoter regions of these Th1 genes indifferentiated Th1 cells compared with CD4+ Tn, with moderately

FIGURE 4. Ezh2 promotes in vitro Th1 cell differentiation in cultures under Th1-skewing conditions. Naive CD4+ T cells isolated from WT B6 and

T-KO mice were stimulated with anti-CD3 and anti-CD28 Abs under Th1-skewing condition. Cells were collected at the indicated time for analysis. (A) Dot

plots (upper panel) and graphs (lower panel) show the fraction and MFI of IFN-g– or IL-4–producing cells. (B) ELISA assays show the level of IFN-g and

IL-4 in the culture medium at day 7 after restimulation with anti-CD3 Ab. Each group contained an equal number of T cells (1 3 106 cells/ml). (C) Naive

CD4+ T cells isolated from WT B6 or T-KO mice were prestained with CFSE and stimulated with anti-CD3 and anti-CD28 Abs. Two days after culture,

cells were collected for the analysis of T cell activation markers. (D) Histograms show the cell divisions at the indicated time after culture. (E) Naive CD4+

T cells isolated from WT B6 and T-KO mice were stimulated with anti-CD3 and anti-CD28 Abs under Th1-skewing condition together with anti-IL-4 Ab

(10 mg/ml) for 7 d. Dot plots (left panel) and graphs (right panel) show the fraction of IFN-g– or IL-4–producing cells. (F) Naive CD4+ T cells before (Tn)

and after cultured under Th1-skewing condition for 7 d (Th1) were harvested and lysed for Western blot analysis (upper panel). The relative expression

level of each protein was indicated under the band, which was determined by densitometry analysis. Seven days after culture, the indicated gene expression

was analyzed in WT and T-KO cells (lower panel). Data are representative of two independent experiments. Error bars indicate mean 6 SD. *p , 0.05,

**p , 0.01, ***p , 0.001.

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increased amount of Ezh2 at the promoter region of Stat4 (Fig.5B). Real-time RT-PCR showed that Ezh2 was positively correlatedwith expression of these Th1 genes (Fig. 5C). All CD4+ T cells pro-ducing IFN-g also expressed high levels of Ezh2 (Fig. 5D).To further confirm that Ezh2 associated with Th1 gene loci, we

prepared chromatin from WT and T-KO CD4+ T cells activatedunder Th1-skewing conditions for 7 d. ChIP analysis revealed asignificantly reduced amount of Ezh2 and H3K27me3 at the

promoter regions of these gene loci in T-KO Th1 cells comparedwith WT Th1 cells (Fig. 6A–C). Thus, despite the reduction ofH3K27me3 at the promoter regions of these Th1 genes, its cata-lyzing enzyme Ezh2 remains associated with these regions. No-tably, T-KO Th1 cells showed a higher amount of H3K4me3 at thepromoter regions of these Th1 gene loci than WT Th1 cells did(Fig. 6D). This suggests that loss of Ezh2 leads to the switch to apermissive histone methylation signature for Th1 genes, which is

FIGURE 5. Ezh2 associates with the promoter regions of type-1 gene loci. Freshly isolated WT CD4+ T cells (Tn) and WT CD4+ T cells after 7 d culture

in Th1-skewing conditions (Th1) were processed for ChIP using Abs specific for H3K27me3, H3K4me3, Ezh2, and control IgG. (A) The graphs show the

ChIP–quantitative PCR (qPCR) for H3K27me3 or H3K4me3 binding to the promoter region of Ifng, Tbx21, or Stat4. (B) Schematic representation of the

mouse Ifng, Tbx21, or Stat4 locus (upper panel). Open rectangles indicate the transcriptional start site. Closed triangles show the locations of real-time

qPCR primer pairs used in the ChIP assay, which are indicated in relative kb to the transcriptional start site. The graphs (lower panel) show the ChIP-qPCR

for Ezh2 binding, with a serious of primer pairs shown above covering the regulatory and promoter region of Ifng, Tbx21, or Stat4 locus. (C) Graphs show

the relative expression of indicated genes measured by real-time PCR in freshly isolated WT CD4+ T cells (Tn) and WT CD4+ T cells after 7 d culture in

Th1-skewing conditions (Th1). (D) Dot plots show the fraction of IFN-g– and Ezh2-expressing cells in freshly isolated WT CD4+ T cells (Tn) and WT

CD4+ T cells after 7 d culture in Th1-skewing conditions (Th1). Data are representative of three independent experiments. Error bars indicate mean 6 SD.

**p , 0.01, ***p , 0.001.

FIGURE 6. Ezh2 specifically binds to promoter regions of

Th1 type gene loci. WT and T-KO CD4+ T cells collected 7 d

after culture under Th1-skewing conditions and processed for

ChIP using Abs specific for Ezh2, H3K27me3, and H3K4me3.

(A) A schematic of the primer regions in the mouse Ifng, Tbx21,

or Stat4 locus. Open rectangles indicate the TSS. Closed tri-

angles show the locations of real-time PCR primer pairs used

for ChIP assay. (B–D) The graphs show the relative amount of

Ezh2, H3K27me3, H3K4me3, and IgG at the regions of Ifng,

Tbx21, or Stat4 locus.

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favorable for their activation. However, our results showed thatloss of Ezh2 impaired Th1 cell development (Fig. 2C).

Ezh2 regulates T-bet at both the transcriptional level andposttranslational level

To determine how Ezh2 regulated the expression of T-bet andSTAT4 in Th1 cells, we examined the expression of T-bet andSTAT4 mRNA in WT and T-KO CD4+ T cell 7 d after cultureunder Th1-skewing conditions. CD4+ Tn were assessed as con-trols. Seven days after culture under Th1-skewing conditions,T-KO CD4+ T cells expressed ∼1.8-fold less T-bet mRNA thantheir WT counterparts did (Fig. 6A). STAT4 mRNA was slightlydecreased in activated T-KO CD4+ T cells (Fig. 7A). In addition,Ezh2 deficiency had no effect on the expression of Th1-relatedIfngr1 and Il12rb2 genes (Fig. 7B), two critical signaling mole-cules upstream of T-bet and STAT4, respectively (15). These datatogether with our observations that Ezh2 was positively associatedwith Tbx21 gene (Figs. 5, 6) indicate that Ezh2 promotes theexpression of T-bet at the transcriptional level.To assess whether Ezh2 directly activated Tbx21 transcription,

we cotransfected 3T3 cells with pGL3-Tbx21 reporter and MigR1viral plasmid encoding Ezh2 or empty MigR1 plasmid. These 3T3cells were harvested 48 h after transfection and analyzed with theDual Luciferase system. Overexpression of Ezh2 resulted in mod-erate induction of Tbx21 reporter activity (Fig. 7C). This findingindicates that Ezh2 can directly activate Tbx21 transcription.We further verified whether loss of Ezh2 led to reduction of

STAT4 and T-bet protein in Th1 cells. In a comparison with ac-tivated WT CD4+ T cells, there was only minimal reduction ofSTAT4 protein in these activated T-KO CD4+ T cells (Fig. 7D).Most notably, T-KO CD4+ T cells showed ∼4-fold and 10-foldless T-bet protein at day 3 and day 7 after culture, respectively,than their WT counterparts did (Fig. 7D). This dramatic reduction

of T-bet protein in T-KO Th1 cells appeared not to be completelysupported by moderate reduction of T-bet mRNA in these cells.We reasoned that loss of Ezh2 might lead to increased degradationof T-bet protein in Th1 cells. To test this reasoning, we treated WTand T-KO Th1 cells with the proteasome inhibitor MG115 (44).The addition of the proteasome inhibitor MG-115 restored the ex-pression of T-bet protein, but not STAT4 protein, in Ezh2-deficientTh1 cells (Fig. 7E), suggesting that T-bet protein is more susceptiblethan STAT4 to proteasome-mediated degradation in Th1 cellslacking Ezh2. Altogether, Ezh2 promotes T-bet expression attranscriptional and posttranslational levels, largely with the latter.These results identify a novel and important role for Ezh2 toregulate Th1 cells.

Introduction of T-bet into T-KO CD4+ T cells fully rescues theirdifferentiation into Th1 cells

To determine whether the downregulation of T-bet caused theimpairment of Th1 cell development, we used MigR1 virus bicis-tronically encoding T-bet and GFP (named MigR1/T-bet) to infectT-KO CD4+ T cells cultured under Th1-skewing conditions. T-KOCD4+ T cells infected with MigR1 encoding STAT4 and GFP(named MigR1/STAT4) or GFP alone (named MigR1/GFP) wereassessed in parallel. Expression of GFP allowed us to track cellsexpressing T-bet or STAT4. WT CD4+ T cells were also infectedwith each of these viruses as controls.We found that T-KO GFP-positive (GFP+) CD4+ T cells that

were derived from cultures infected by MigR1/T-bet, whichoverexpressed T-bet, produced a similar percentage of IFN-g+

T cells and WT GFP+ CD4+ T cells derived from cultures infectedby either MigR1/T-bet or MigR1/GFP (Fig. 8A, 8B). Interestingly,compared with WT GFP+CD4+ T cells infected with MigR1/GFPor MigR1/STAT4, T-KO GFP+CD4+ T cells expressing STAT4had significantly lower frequency of IFN-g+ T cells (Fig. 8A, 8B).

FIGURE 7. Ezh2 regulates T-bet at both the transcriptional level and posttranslational level. (A) Graphs show the gene expression of Stat4 and Tbx21 in

freshly isolated WT or T-KO CD4+ T cells (Tn) and cells after 7 d culture in Th1-skewing conditions (Th1). (B) The graph shows the relative expression of

indicated genes measured by real-time PCR in WT or T-KO CD4+ T cells after 7 d culture in Th1-skewing conditions. (C) The schematic (left panel) shows

the construction of Tbx21 promoter region ranging from +0.3 to –1.0 kb of the TSS. 3T3 cells were cotransfected with pGL3-Tbx21 reporter plasmid and

an empty vector control or Ezh2 in combination. Luciferase reporter activity was normalized to the activity obtained for the cotransfected Renilla control.

The graph (right panel) represents the relative light units (RLU). (D) Western blot shows the expression of Ezh2, Stat4, and T-bet after 3 or 7 d of Th1-

skewing culture conditions. The relative expression level of each protein was indicated under the band, which was determined by densitometry analysis. (E)

Western blots show the expression of T-bet and STAT4 in freshly isolated WT or T-KO CD4+ T cells (Tn) and cells after 7 d culture in Th1-skewing

conditions (Th1) with or without the treatment of MG115 (2 mM) for 6 h. Data are representative of two independent experiments. Error bars indicate

mean 6 SD. **p , 0.01.

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Furthermore, T-KO CD4+ T cells expressing STAT4 contained∼40% less IFN-g+ T cells than T-KO CD4+ T cells expressingT-bet (Fig. 8A, 8B). These data suggest that viral expressionof T-bet fully rescues the ability of Ezh2-deficient CD4+ T cellsto differentiate into Th1 cells, whereas overexpression of STAT4only partially improves Th1 cell differentiation of activated T-KOT cells.To validate these observations, we highly purified GFP-positive

T-KO T cells from these cultures (Fig. 8C) and confirmed theoverexpression of T-bet and STAT4 in these T-KO CD4+ T cells,respectively, using real-time RT-PCR (Fig. 8D). Furthermore,overexpression of T-bet in T-KO T cells induced significantlymore Ifng transcripts than did overexpression of STAT4 (Fig. 8D).Phosphorylation of STAT4 is critical for IFN-g production andTh1 cell differentiation (13, 45). Western blot analysis showedthat MigR1/STAT4-infected T-KO T cells expressed 7.5-fold moreSTAT4 protein and 1.2-fold more phosphorylated STAT4 thantheir WT counterparts did (data not shown). Thus, the reductionof T-bet in activated T-KO CD4+ T cells is a major contributorto their impaired development of Th1 cells.We finally examined whether retroviral expression of T-bet could

rescue the ability of Ezh2-deficient T cells to induce AA. BothCD4+ and CD8+ T cells were highly purified from T-KO B6 miceand activated in vitro for 24 h, followed by infection with MigR1/T-bet, MigR1/STAT4, and MigR1/GFP, respectively. Thirty-sixhours later, these infected T cells were harvested, assessed usingflow cytometry for the expression of GFP, and transferred intosublethally irradiated BDF1 mice. Each mouse received unfrac-tionated T cells that contained similar numbers of GFP+ cells(0.83 106 CD4+ T cells plus 0.63 106 CD8+ T cells). BDF1 micereceiving T-KO T cells transduced by either T-bet or STAT4survived .24 d after transfer, whereas all BDF1 mice receivingWT T cells infected by control MigR1/GFP died of AA within14 d (Fig. 9A and data not shown). This suggests that transfer ofthis amount of T-KO T cells expressing T-bet or STAT4 didnot cause lethal AA. However, as compared with control T-KOT cells infected by MigR1/GFP, MigR1/T-bet–infected T-KOT cells caused marked reduction of BM cells and peripheralblood cells (i.e., platelets, RBCs, WBCs, and neutrophils) 24 d

after transfer (Fig. 9A, 9B). Furthermore, GFP+ MigR1/T-bet-infected KO T cells (which expressed T-bet) had ∼4-fold more infrequency of IFN-g–producing T cells than control GFP+ T-KOT cells infected by MigR1/GFP (Fig. 9D). In contrast, transferof MigR1/STAT4-infected T-KO T cells resulted in moderatedecrease of BM cells, but not blood pancytopenia (Fig. 9A, 9B).Notably, GFP+ MigR1/STAT4-infected T-KO T cells (whichexpressed STAT4) failed to produce high levels of IFN-g (Fig. 9C,9D). Thus, Ezh2 regulation of T-bet is important for productionof Th1 cells mediating AA.

DiscussionOur findings elucidate a new and critical role of Ezh2 in controllingpathogenic Th1 cell responses during AA process, which has notbeen previously identified. Ezh2 is absolutely required for CD4+

Th1 cells to mediate fatal AA. Ezh2 inhibition led to dramaticreduction of BM-destructive Th1 cells in vivo, decreasing BM-infiltrating Th1 cells, and protecting mice from BMF. Decreasedcapability of Ezh2-deficient T cells to differentiate into Th1 cellsresulted from reduced expression of T-bet mRNA and protein, asretroviral expression of T-bet in Ezh2-deficient CD4+ T cells fullyrescued their differentiation into Th1 cells. In contrast, ectopicexpression of STAT4 only partially restored the ability of Ezh2-deficient CD4+ T cells to produce IFN-g. Interestingly, althoughEzh2 is known to act primarily as a gene silencer, it promoted theexpression of T-bet gene in Th1 cells via a mechanism of directlyactivating Tbx21 gene promoter. Furthermore, Ezh2 was requiredto prevent proteasome-mediated degradation of T-bet protein inTh1 cells. This constellation of Ezh2 actions induced the optimalproduction of Th1 cells destructing BM cells, highlighting thetherapeutic potential of Ezh2 inhibition in controlling AA.Because Ezh2 specifically catalyzes the repressive maker

H3K27me3, a corollary belief is that Ezh2 may be required forrepressing cytokine gene expression (22, 46, 47). CD4+ Tn showedmoderate levels of H3K27me3 in the promoter regions of Ifngand Tbx21 gene loci (46, 48, 49, 27). Upon Th1 differentiation,H3K27me3 was reduced in the promoter regions of Th1 genes(e.g., Ifng, Tbx21, and Stat4), whereas H3K4me3 was upregulatedin these gene loci (27). We confirmed and further extended these

FIGURE 8. Overexpression of T-bet fully

rescues T-KO CD4+ T cells to differentiate

into Th1 cells. CD4+ Tn isolated from WT

B6 and T-KO mice were stimulated with

anti-CD3 and anti-CD28 Abs under Th1-

skewing conditions. One day after culture,

T cells were infected with retrovirus en-

coding GFP-STAT4, GFP-T-bet, or GFP-

control and then cultured for another 6 d.

(A) Dot plots show the fraction of IFN-g–

and GFP-expressing cells 7 d after culture.

(B) Bar graphs show the percent and mean

fluorescence intensity (MFI) of IFN-g+ cells

in GFP+ (left panel) and GFP2 (right panel)

cells after infection. (C) GFP+CD4+ T-KO

cells were sorted, and total RNAs were iso-

lated and subjected to real-time PCR analysis.

Dot plots show the purity of sorted GFP+ cells.

(D) Graphs show the relative expression of

indicated genes in sorted donor GFP+CD4+

T cells. Data are representative of two in-

dependent experiments. Error bars indicate

mean6 SD. *p, 0.05, **p, 0.01, ***p,0.001.

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observations that loss of Ezh2 caused the reduction of H3K27me3at the Ifng, Tbx21, and Stat4 gene loci in Th1 cells, while in-creasing the amount of H3K4me3 in these gene loci. However,this skewed balance of histone methylation signature toward thetranscription permission state at these Th1 gene loci did not leadto increased expression of these genes in Ezh2-deficient CD4+

T cells. In contrast, Ezh2 deficiency caused downregulation ofTbx21 mRNA. ChIP assay and reporter analysis verified that Ezh2possessed the ability to directly activate Tbx21 transcription. Thisresult is in sharp contrast to conventional thoughts that Ezh2 mightrepress the expression of genes associated with effector differen-tiation of Ag-driven T cells during immune response (22, 27, 46).Past studies have shown that Ezh2 and other polycomb proteinswere unconventionally associated with effector cytokine genes(e.g., Ifng and Il4) (25, 50). However, these studies did not reportwhether Ezh2 influenced the expression of Tbx21 and the devel-opment of Th1 cells in vivo during inflammatory responses (25,50). Our data demonstrate that Ezh2 acted as an activator for in-ducing optimal expression of Tbx21 during Th1 cell differentia-tion. These findings are in agreement with observations by othersthat Ezh2 can activate some critical transcription factors in cancercells in a context-dependent manner (19, 51).Intriguingly, we identified a previously unknown role for

Ezh2 in protecting proteasome-mediated degradation of T-betprotein in Th1 cells. Ezh2 deficiency led to increased degra-dation of T-bet protein in Th1 cells. Addition of the proteasomeinhibitor MG-115 restored the expression of T-bet protein inEzh2-deficient Th1 cells. This effect of Ezh2 on protecting T-betfrom proteasome-mediated degradation differs from the effectof Ezh2 on repressing gene transcription (19). T-bet is crucial

for controlling appropriate Th1 cell differentiation through re-versing and establishing new epigenetic states in T cells (52, 53).T-bet recruited the histone methyltransferase SET7/9 to catalyzepermissive H3K4me2 at the Ifng promoter in Th1 cells (52, 53).Notably, T-bet recruited Jumonji domain–containing protein his-tone demethylase 3 (Jmjd3) to remove H3K27me3 across the Ifnglocus (54). These data suggest that epigenetic regulation of Th1cell differentiation involves a complex and functional cooperationof histone methyltransferase, demethylase, and transcription fac-tors (13, 52). In the current study, we show that Ezh2 plays acritical role in promoting optimal Th1 cell responses via a mech-anism of posttranslational regulation of T-bet. This effect of Ezh2appeared to be independent of its activity of methylating histoneproteins. Data from several studies indicate that Ezh2 can directlymethylate signaling proteins to regulate gene transcription (40,55). For example, Ezh2 methylates STAT3 in cancer cells, leadingto enhanced STAT3 activity (55). Given the presence of highamounts of Ezh2 at the promoter regions of Ifng gene locus in Th1cells, we speculated that Ezh2 could play critical roles in stabi-lizing T-bet protein at this gene locus, thereby facilitating theT-bet regulation of Th1 cell differentiation. Formal experimentsare needed to address this point and to determine the mechanismswhereby Ezh2 protects T-bet from proteasome-mediated degra-dation in Th1 cells.Accumulating evidence indicates that T-bet can be an effective

target for the prevention and treatment of immune-mediated AA.Abnormal expression of T-BET has been shown in human patientswith AA (17). T cells from patients with AA who were refractoryto immunosuppressive treatment expressed high levels of T-BET.In contrast, patients with AAwho responded to the therapy showed

FIGURE 9. Introduction of T-bet to T-KO T cells rescues their ability to mediate AA in mice . CD4+ Tn and CD8+ Tn were isolated from WT and T-KO

mice and separately cultured in the presence of anti-CD3 Ab (2 mg/ml), anti-CD28 Ab (2 mg/ml) and IL-2 (10 ng/ml). Twenty-four hours later, T-KO T cell

subsets were infected with MigR1/T-bet, MigR1/STAT4, and MigR1/GFP, respectively. WT T cell subsets were infected with MigR1/GFP as controls.

Thirty-six hours after infection, these T cells were harvested and transferred into sublethally irradiated (6.5 Gy) BDF1 mice. Each mouse received

unfractionated T cells that contained similar numbers of GFP+ cells (0.83 106 CD4+ T cells + 0.63 106 CD8+ T cells). (A) Peripheral blood was collected

24 d after LN cell infusion and the cell numbers of RBCs, WBCs, platelets, and neutrophils were calculated (mean 6 SD; n = 6–8 mice per group). (B)

Total BM cellularity was calculated assuming that bilateral tibia and femurs contain 25% of total marrow cells (mean 6 SD; n = 6–8 mice per group). (C)

Twenty-four days after LN-cell infusion, donor CD4+ T cells were isolated from the spleen, LN, and BM to measure the production of IFN-g. The plots

show the percentage of donor CD4+ T cells in spleen, the percentage of GFP2 and GFP+ cells, and the fraction of IFN-g–producing cells in GFP2 and GFP+

cells. (D) Graphs show the percentage (upper panel) and the number (lower panel) of donor IFN-g+CD4+ T cells in spleen, LN, and BM. *p, 0.05, **p, 0.01,

***p , 0.001.

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low levels of T-BET in their circulating blood cells (17). Experi-mental studies further indicate that genetic inhibition of T-bet leadsto decreased induction of AA in mice (6); however, so far, there isno pharmacologic approach available to modulate T-bet. Further-more, transcription factors are difficult drug targets (18). Given thatEzh2 plays critical roles in the regulation of T-bet mRNA andprotein in Th1 cells, we proposed that targeting Ezh2 may proveadvantageous to modulate T-bet and its controlled pathogenic T cellinflammation.Aberrant expression of Ezh2 has been associated with many

types of malignant diseases such as prostate cancer, breast cancer,and lymphomas (19, 56, 57). Data from our previous studies in-dicate that Ezh2 is markedly increased in activated T cells (58,59). Inhibition of Ezh2 leads to the reduction of graft-versus-hostdisease in mice of allogeneic BMT (28). In these mouse models ofallogeneic BMT, infusion of donor BM cells fully rescues he-matopoiesis and thymopoiesis in lethally irradiated recipients (10,11), which prevents us from precisely assessing the impact ofT cells in mediating BMF. Our experiments using the AA mousemodel and in vitro cultures provide compelling evidence fora T cell–intrinsic contribution of Ezh2 to the expression of Th1-associated transcription factors and cytokines. Inhibition of Ezh2in T cells through conditional deletion suppressed Th1 cell dif-ferentiation and development of BMF. As several Ezh2-specificinhibitors have recently been discovered for experimental treat-ment of cancer (60–63), it will be important to test whether theseEzh2-specific inhibitors may control AA in experimental models.In summary, we have identified the critical role of Ezh2 in

regulating Th1 responses during AA and the mechanism by whichEzh2 promotes Th1 cell responses mediating BMF in mice. Ourfindings open new perspectives to study the importance of Ezh2in regulating the development of other lineages of CD4+ T cells(e.g., Th2, Th17, Th9, and regulatory T cells). Indeed, a recent studyhas demonstrated that Ezh2 is involved in repressing the develop-ment of Th9 cells (64). Furthermore, it will be important to investigatewhether deregulated Ezh2 expression and activity are associatedwith inflammatory disorders in humans, such as autoimmunediseases and chronic infections.

AcknowledgmentsWe thank Jinfang Zhu (Molecular and Cellular Immunoregulation Unit

Laboratory of Immunology, National Institute of Allergy and Infectious

Diseases, National Institutes of Health) and Cheong-hee Chang (Depart-

ment of Microbiology and Immunology, University of Michigan) for

thoughtful discussion and critical reading of the manuscript.

DisclosuresThe authors have no financial conflicts of interest.

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