Published April 23, 2014, doi:10.4049/jimmunol.1302943 The Journal of Immunology

Ezh2 Regulates Transcriptional and Posttranslational Expression of T-bet and Promotes Th1 Cell Responses Mediating 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: 000–000.

cquired aplastic anemia (AA) in humans is a fatal dis- patients with AA have a potent ability to lyse autologous CD34+ order characterized by bone marrow (BM) hypoplasia hematopoietic cells and inhibit formation of hematopoietic cell and blood pancytopenia (1, 2). Clinical studies indicate colonies (3). Accumulating evidence indicates that CD4+ Th1

by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. A that in most cases, AA is a disease caused by immune-mediated cells, which are characterized by production of high levels of destruction of hematopoietic stem cells and hematopoietic pro- IFN-g, play important roles in mediating bone marrow failure genitor cells (1, 2). A role for T cells in AA was first suggested by (BMF) (2, 4–8). IFN-g displays potent effects on suppressing their inhibition of hematopoietic cell colony formation in cultures hematopoiesis in vitro (2, 3). Immunosuppressive therapy and in vitro (2). Furthermore, CD4+ T cell clones isolated from the allogeneic BM transplantation (BMT) have significantly im- proved the survival of severe AA. However, relapse still occurs ∼ *International Joint Cancer Institute, Second Military Medical University, Shanghai in 35% of patients with AA when immunosuppressive therapy 200433, China; †Department of Internal Medicine, University of Michigan Medical is withdrawn (1, 2, 9). Furthermore, graft-versus-host disease ‡ School, Ann Arbor, MI, 48109; Department of Microbiology and Immunology, Fels remains a major barrier to the success of allogeneic BMT (10,

http://classic.jimmunol.org Institute for Cancer Research and Molecular Biology, Temple University, Philadelphia, PA 19140; xInstitute of Health Sciences, Shanghai Institutes for Biological Sciences 11). Novel approaches are needed to improve the outcome of Chinese Academy of Sciences, Shanghai 200433, China; and {Department of Med- treatments for AA. icine and Abramson Cancer Center, University of Pennsylvania Perelman School of The transcription factor T-bet (encoded by Tbx21) is crucial for Medicine, Philadelphia, PA 19104 Th1 cell differentiation (12–15). T-bet is induced by TCR sig- Received for publication November 1, 2013. Accepted for publication March 19, 2014. naling and strongly upregulated by activation of the STAT1 tran- This work was supported by the American Cancer Society (to Y.Z.), the U.S. De- scription factor (16). T-bet binds to several enhancers and promoter Downloaded from partment of Defense (to Y.Z.) and National Institutes of Health Grant R01-CA- of the Ifng genes, activating its transcription (12, 16). T-bet also 172106-01 (to Y.Z.). promotes expression of the IL-12 receptor b2 chain (IL12Rb2), Q. T. and Y. Z. conceived and designed the project; Q.T., S.H., F.X., K.M., Y.L., I.M., resulting in greater IL-12 responsiveness and further elevated 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. production of IFN-g (16). In addition, T-bet prevents Th2 differ- entiation by inhibiting Gata3 (16). T-bet is upregulated in pe- Address correspondence and reprint requests to Dr. Yi Zhang, Department of Micro- biology and Immunology, Fels Institute for Cancer Research and Molecular Biology, ripheral blood T cells from patients with AA, and it is a useful Temple University, Philadelphia, PA 19140. E-mail address: [email protected] marker for predicting the responsiveness of patients to immuno- The online version of this article contains supplemental material. suppressive therapy (17). Furthermore, experimental studies sug- Abbreviations used in this article: AA, aplastic anemia; BM, bone marrow; BMF, gested that T cells lacking T-bet were defective in induction of AA bone marrow failure; BMT, bone marrow transplantation; ChIP, chromatin immuno- in mice (6). These observations suggest that T-bet can be an at- precipitation; LN, lymph node; TSS, transcription start site. tractive target for modulating Th1 cell–mediated AA. However, Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 transcription factors are difficult drug targets (18). Thus, identifying

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302943 2 EPIGENETIC REGULATION OF Th1 CELLS MEDIATING APLASTIC ANEMIA

the molecular pathways that control T-bet expression in Th1 cells Cell culture can lead to new strategies to control AA. Splenic and LN CD4+ T cells were prepared by MACS purification with Ezh2 is a histone methyltransferase that specifically catalyzes CD4 microbeads (Miltenyi Biotec), and the purity was usually 90–95%. trimethylation of histone H3 at lysine 27 (H3K27me3) (19). Ezh2 Th1-skewing culture conditions were set up as previously described (34– lo + forms polycomb repressive complex 2 together with other polycomb 36). In brief, CD44 CD4 naive T cells (Tn) were cultured in the presence group proteins Suz12 and Eed (19), which is crucial for maintaining of 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), the cellular memory and transcriptional patterns primarily through recombinant mouse IL-12 (5 ng/ml; R&D Systems) and BM-derived a mechanism of silencing genes (20, 21). Several studies indicate an dendritic cells at a ratio of 1:16. Cultured T cells were restimulated with important role of Ezh2 and H3K27me3 in multiple lineages of ef- anti-CD3 Ab (1 mg/ml; BioLegend) 5 h before intracellular staining. fector T cells (22–25). Genome-wide mapping analysis revealed that Retroviral construction and T cell infection repressive H3K27me3 marked genes associated with differentiation and maintenance of effector and memory T cells (26, 27). Recently, The MigR1 retroviral vector system was described previously (35, 37). MigR1 vector was provided by Warren Pear (University of Pennsylvania), we demonstrated new and essential roles of Ezh2 in regulating in- and MigR1 vector encoding T-bet or STAT4 was provided by Steve Reiner flammatory T cell responses in mice after allogeneic BMT (28). (Columbia University, New York, NY) and Takashi Usui (Kyoto Univer- Loss of Ezh2 led to impaired production of alloreactive T cells that sity, Kyoto, Japan). MigR1 virus was produced as described (38). For + induce damage to epithelial organs (28). However, whether Ezh2 retroviral infection, CD4 T cells were prestimulated with anti-CD3 and mediates pathogenic Th1 responses in AA and the mechanism of anti-CD28 Abs for 24 h, and then the retrovirus supernatant was added in the presence of 8 mg/ml polybrene (Sigma). Cells were spinoculated at Ezh2 action in regulating Th1 cells remain unknown. 3000 rpm, 32˚C for 3 h. The same retroviral infection procedure was re- Mouse models of human AA have been established successfully peated 24 h later. (4, 29). Transfer of parent lymph node (LN) cells into hap- Western blot analysis loidentical daughter recipients caused BM hypoplasia and blood pancytopenia, typical features of clinical AA. These AA mouse Western blot was performed as described (28). Cell lysates were examined models have proved to be a unique approach studying patho- with routine Western blotting. The blots were incubated with anti-Ezh2 (612667; BD Biosciences), Stat4 (sc-486; Santa Cruz Biotechnology), physiology of immune cell-mediated BMF (4, 6, 30, 31). In this T-bet (sc-21749; Santa Cruz Biotechnology), GATA-3 (558686; BD Bio- report, we exploited the functional impact of Ezh2 on Th1 cell sciences), or actin (ab3280; Abcam) Abs, and subsequently incubated with responses in vitro and in vivo. Using genetic approaches and a HRP-conjugated anti-rabbit or mouse IgG (Vector Laboratories) in TBS mouse model of human BMF, we identified a novel and critical containing 5% nonfat dry milk and 0.05% Tween 20. The final reaction was developed with a chemiluminescent system (Pierce). role of Ezh2 in regulating Th1 cells mediating AA. Chromatin immunoprecipitation assay Materials and Methods Chromatin immunoprecipitation (ChIP) assays were performed as described Mice by using EZ-Magna ChIP (17-10086; Millipore) (27, 39). Sonicated extracts were precleared and incubated with Abs specific to Ezh2 (39901, b b/d C57BL/6 (B6, H-2 ) and B6xDBA/2 F1 (BDF1, H-2 ) mice were pur- active motif), H3K27me3 (PAb-069-050; Diagenode) or H3K4me3 chased from Taconic (Rockville, MD). Cd4-Cre mice were originally de- (9751S; Cell Signaling Technology) at 4˚C overnight on a 360˚C rotator. rived from the Jackson Laboratory. B6/129 mice with floxed alleles of The immunoprecipitated DNA was quantitated by real-time quantitative fl/fl Ezh2 (Ezh2 ) (32) were crossed to Cd4-Cre mice, before backcrossing PCR. The primer sequences are listed in Supplemental Table II. by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. to the B6 background (.8 generations). Age-matched and sex-matched controls were used. Experimental protocols were approved by the Uni- ELISA versity of Michigan’s Committee on Use and Care of Animals. CD4+ Tn isolated from WT B6 and T-KO mice were stimulated with anti- Abs and flow cytometry analysis 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) and Abs used for immunofluorescence staining were purchased from eBioscience, recombinant mouse IL-12 (5 ng/ml; R&D Systems). On day 7, cells were BioLegend, or BD Biosciences. Flow cytometry analyses were performed restimulated in 96-well plates with plate-bound anti CD3 Abs (1 mg/ml; using FACScan and Canto cytometer (Becton Dickinson) as described (33). BioLegend) for 5 h before collecting the supernatants. Each group con- 3 6 Induction of AA tained an equal number of cells (1 10 cells/ml). The concentrations of IFN-g and IL-4 were measured in triplicate using recombinant mouse IFN-g http://classic.jimmunol.org The mouse AA model was induced as described previously (30, 31). B6 LN and IL-4 ELISA kits in accordance with the manufacturer’s instructions cells (10 3 106/mouse) isolated from B6 mice were transplanted into ir- (BioLegend). radiated BDF1 mice (6.5 Gy). Peripheral blood was collected from the heart and the lateral tail vein. Complete blood counts were performed Luciferase reporter assay using a Hemavet 950 analyzer (Drew Scientific). BM cells were extracted The Tbx21 promoter region ranging from +0.3 to 21.0 kb of the tran- from bilateral tibiae and femurs. Lymphocytes were isolated from inguinal, scription start site (TSS) was cloned to pGL3 luciferase reporter vector to axillary, and lateral axillary LNs. generate Tbx21-specific reporter (named pGL3-Tbx21 reporter). 3T3 cells

Downloaded from Histologic examination were cotransfected with pGL3-Tbx21 reporter plasmid and MigR1 viral plasmid encoding Ezh2 or empty MigR1 plasmid. The cells were harvested The histologic examination was performed on paraffin-embedded femur 48 h after transfection and analyzed with the Dual Luciferase system pieces, fixed with 10% formalin, and colored with H&E. The histologic (Promega). image was photographed with a OlympusBX41 microscope (10/0.3 NA lens; original magnification 3100; digital DP70 camera). Statistical analysis Real time RT-PCR Survival in different groups was compared using the log-rank test. Com- parison of two means was analyzed using the two-tailed unpaired Student Total RNA was extracted from the indicated CD4+ T cell subsets using t test. TRIzol (Invitrogen Life Technologies). cDNA was quantified through the quantitative real-time PCR technique. Real-time PCR was performed with a SYBR Green PCR mix on a Mastercycler realplex (Eppendorf). Ther- Results mocycler conditions included an initial holding at 95˚C for 2 min; this was In the absence of Ezh2, LN cells are defective in mediating AA followed by a three-step PCR program as follows: 95˚C for 30 s, 55˚C for in mice 30 s, and 72˚C for 30 s for 45 cycles. Transcript abundance was calculated using the DDCt method (normalization with 18 s). The primer sequences We used a genetic approach to determine the role of Ezh2 in the are listed in Supplemental Table I. regulation of T cell–mediated AA. Mice with floxed alleles of The Journal of Immunology 3

Ezh2 (Ezh2fl/fl) (32) were crossed to B6 mice expressing Cre Stat4 (2.5-fold), and Tbx21 (4-fold) transcripts than their WT recombinase under control of the CD4 promoter to generate counterparts did (Fig.2C). T cell–specific Ezh2 conditional knockout B6 mice (named In this BMF model, only a small percentage of IL-4–producing T-KO). These T-KO mice were further backcrossed to B6 mice CD4+ T cells occurred in the spleen, LN, and BM of BDF1 mice for 10 generations. The development of mature thymocytes and receiving WT LN cells (Fig. 2A, 2B), suggesting that the in- T cells in peripheral lymphoid tissues was normal in these T-KO flammatory stimuli produced during the AA process predomi- mice, which is in agreement with previous observations (28, 32). nantly induces Th1 cell differentiation in vivo. Ezh2 deficiency To assess whether conditionally deleting Ezh2 in T cells affected did not result in significant changes in the ability of CD4+ T cells their ability to mediate AA, we transferred donor LN cells derived to produce IL-4 protein as assessed by flow cytometry (Fig. 2A). from WT and T-KO B6 mice into irradiated (6.5 Gy) BDF1 Furthermore, loss of Ezh2 had no significant effect on the ex- recipients. In this setting, transfer of donor LN cells causes severe pression of Th2 cell genes (e.g., Il4, Il5, Il13, Stat6, and Gata3; BM destruction and blood pancytopenia in these haploidentical Fig. 2C). There was a moderate reduction in Rorgt and an increase recipients, which closely reflects the pathogenesis of human AA in Foxp3 (Fig. 2C). However, these moderate changes in genes (4, 6, 30). As expected, BDF1 mice receiving WT B6 LN cells expression did not lead to the reduction in Il17 expression (Fig. 2C) developed BM hypoplasia and severe blood pancytopenia after and increase in regulatory T cells (Tregs; data not shown). LN cell infusion compared with control mice receiving only total Collectively, these results suggest that during the AA process, body irradiation, with all of them dying from the disease within Ezh2 is critically involved in regulating the development of Th1 12 d after transfer (Fig.1A). Histologic examination showed the cells, but has little effect on Th2 or Th17 cells. destruction of BM and lack of hematopoietic cell islands in the Data from our previous studies and others indicate that Ezh2 BM of these recipients (Fig.1B). In contrast, transfer of T-KO LN deficiency can lead to impaired expansion and survival of activated cells did not cause severe AA in these BDF1 recipients (Fig.1A, T cells (28, 40). It is possible that the inability of T-KO LN cells 1B). As compared with control mice receiving total body irradi- to cause AA could result from decreased expansion and survival ation, there was no significant reduction of BM cellularity and of BM-destructive Th1 cells. To test this possibility, we tracked peripheral blood WBCs in these BDF1 mice receiving T-KO LN the longitudinal proliferation and differentiation of WT and T- cells (Fig. 1C, 1D). Importantly, all T-KO LN cell recipients KO LN cells that were injected into sublethally irradiated BDF1 survived without clinical signs and histologic evidence of AA recipients. Compared with WT LN cells, T-KO LN cells pro- (Fig. 1A, 1B). Thus, T cells required Ezh2 to mediate AA. duced significantly less in frequency of both CD4+ T cells and IFN-g–producing CD4+ T cells in the spleen, LN, and BM 6 and Ezh2 is required for the development of Th1 cells inducing AA 10 d after transfer (Fig. 3A, 3B). Because WT LN cells caused in mice lethal AA in all BDF1 recipients by day 12 (data not shown), we Previous studies have demonstrated that Th1 cells are crucial for were prevented from further comparing the difference between inducing BMF in this model of experimental AA (2, 3). To examine WT and T-KO LN cells at later time points. However, in BDF1 the effects of Ezh2 deficiency on Th1 cell development in vivo, we mice receiving T-KO LN cells, although there was a marked harvested donor T cells from the spleen, LN, and BM of these increase in numbers of total donor CD4+ T cells in the spleen, mice 10 d after transfer of WT or T-KO LN cells. We found that LN and BM at day 43 after transfer compared with that at day 6 by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. loss of Ezh2 led to a significant reduction in the percentage and (Fig. 3C), the frequency of IFN-g–producing CD4+ T cells was number of Th1 cells in the spleen, LN, and BM (Fig. 2A, 2B). not increased in parallel (Fig. 3B). These data suggest that the Real-time RT-PCR analysis showed that alloantigen-activated lack of both differentiation and expansion of Th1 cells may be T-KO T cells expressed dramatically lower levels of Ifng (5-fold), responsible for the inability of T-KO T cells to mediate AA early

FIGURE 1. In the absence of Ezh2, http://classic.jimmunol.org 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- Downloaded from 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. 4 EPIGENETIC REGULATION OF Th1 CELLS MEDIATING APLASTIC ANEMIA

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)orT-KOLN(103 106) 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+ Tcells.(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.

during disease process, whereas impaired Th1 differentiation of producing CD4+ T cells in the culture of T-KO CD4+ T cells than T-KO T cells could account for reduced AA during later stage. that of WT CD4+ T cells (Fig. 4A). This impaired ability of T-KO CD4+ T cells to produce IFN-g persisted throughout a period of Ezh2 promotes in vitro Th1 cell differentiation in cultures 7 d during culture (Fig. 4A). ELISA further confirmed that T-KO under Th1-skewing conditions CD4+ T cells secreted ∼2.5-fold less IFN-g than WT CD4+ T cells To further examine the importance of Ezh2 in regulating Th1 cell did 7 d after culture (Fig. 4B), when Th1 cells fully developed (12, differentiation, we highly purified CD4+ Tn from WT and T-KO 35). These data confirmed the observations in our preceding B6 mice and cultured them under Th1-skewing conditions without experiments in vivo (Fig. 2) that Th1 cell differentiation was anti–IL-4 Ab. One day after activation in Th1-skewing cultures, impaired in the absence of Ezh2. + by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. both WT and T-KO CD4 Tn produced barely detectable IFN-g as It has been shown that Ezh2 is associated with Vav1 protein (40), assessed by intracellular cytokine staining (Fig. 4A). Three days which is a protein important for mediating proximal TCR sig- after culture, there were 3.7-fold less in frequency of IFN-g– naling in T cell activation (41). To rule out the possibility that the http://classic.jimmunol.org

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 3 106) 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 Downloaded from 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 6 SD. *p , 0.05, **p , 0.01. The Journal of Immunology 5

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. by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd.

decreased induction of Th1 cells by Ezh2 inhibition might be these results suggest that Ezh2 promotes Th1 cell differentiation associated with impaired activation of T cells in cultures, we through a mechanism independent of IL-4 and GATA3. examined the effects of Ezh2 deficiency on the activation and division of T-KO CD4+ T cells cultured under Th1-skewing Ezh2 associates with Th1 gene loci conditions. We found that in the absence of Ezh2, CD4+ Tn were Development of Th1 cells involves a complex mechanism (15). normally activated as evidenced by the upregulation of T cell ac- Th1 cell differentiation is initiated by IFN-g upregulation of T-bet, tivation markers CD25, CD44, and CD69 (Fig. 4C). Furthermore, which specifically activates Ifng transcription (15). IL-12 activa- http://classic.jimmunol.org like WT CD4+ T cells, T-KO CD4+ T cells underwent extensive cell tion of STAT4 is also important to promote Th1 cell differentiation division as evidenced by their dilution of fluorescence dye CFSE in (13–15). To understand the mechanism by which Ezh2 promoted cultures (Fig. 4D). These data suggest that in the absence of Ezh2, the development of Th1 cells, we first used ChIP assay to examine CD4+ T cells can be normally activated to undergo cell division and the presence of Ezh2 and histone methylation markers on Th1 expansion upon stimulation with Th1-skewing conditions. gene loci (e.g., Ifng, Tbx21, and Stat4). CD4+ Tn were isolated Previous studies suggest that IL-4 can reduce Th1 cell differ- from normal WT B6 mice and cultured under Th1-skewing con- Downloaded from entiation (35, 42). We found that in vitro activated T-KO T cells ditions for 7 d. Chromatin was prepared from both CD4+ Tn and produced more IL-4–producing T cells in frequency than their WT Th1 cells. As expected, the amount of H3K27me3 was markedly counterparts (Fig. 4A). It is possible that increased production of reduced at the promoter regions of Ifng, Tbx21, and Stat4 gene IL-4 in T-KO T cells might account for their impaired Th1 cell loci (Fig. 5A). In contrast, H3K4me3, which is a permissive his- differentiation. To test this possibility, we added neutralizing anti– tone methylation marker associated with gene activation (27, 43), IL-4 Ab into the cultures. Indeed, blockade of IL-4 efficiently was increased at the promoter region of these genes (Fig. 5A). inhibited the production of IL-4 by both WT and T-KO CD4+ These data are in agreement with previous observations that T cells (Fig. 4E). Seven days after cultures, there was no marked H3K27me3 strongly marked the promoter, intergenic, and 39UTR difference in frequency of IL-4–producing T cells between activated regions of TBX21 in CD4+ Tn, whereas differentiated Th1 cells WT and T-KO CD4+ T cells (Fig. 4E). In contrast, neutralizing IL-4 had reduced H3K27me3 but increased H3K4me3 at the TBX21 in cultures did not improve the ability of T-KO CD4+ Tcellsto regulatory regions (22, 27). produce IFN-g (Fig. 4E). Furthermore, we confirmed that both WT Interestingly, there was no significant reduction in the amount of and T-KO CD4+ T cells derived from Th1-skewing cultures ex- Ezh2 at both regulatory and promoter regions of these Th1 genes in pressed similar levels of GATA3 protein and mRNA (Fig. 4F). All differentiated Th1 cells compared with CD4+ Tn, with moderately 6 EPIGENETIC REGULATION OF Th1 CELLS MEDIATING APLASTIC ANEMIA

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,orStat4.(B) Schematic representation of the mouse Ifng, Tbx21,orStat4 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,orStat4 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.

increased amount of Ezh2 at the promoter region of Stat4 (Fig. promoter regions of these gene loci in T-KO Th1 cells compared by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. 5B). Real-time RT-PCR showed that Ezh2 was positively correlated with WT Th1 cells (Fig. 6A–C). Thus, despite the reduction of with expression of these Th1 genes (Fig. 5C). All CD4+ T cells pro- H3K27me3 at the promoter regions of these Th1 genes, its cata- ducing IFN-g alsoexpressedhighlevelsofEzh2(Fig.5D). lyzing enzyme Ezh2 remains associated with these regions. No- To further confirm that Ezh2 associated with Th1 gene loci, we tably, T-KO Th1 cells showed a higher amount of H3K4me3 at the prepared chromatin from WT and T-KO CD4+ T cells activated promoter regions of these Th1 gene loci than WT Th1 cells did under Th1-skewing conditions for 7 d. ChIP analysis revealed a (Fig. 6D). This suggests that loss of Ezh2 leads to the switch to a significantly reduced amount of Ezh2 and H3K27me3 at the permissive histone methylation signature for Th1 genes, which is http://classic.jimmunol.org

FIGURE 6. Ezh2 specifically binds to promoter regions of Downloaded from 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,orStat4 locus. The Journal of Immunology 7

favorable for their activation. However, our results showed that of T-bet protein in T-KO Th1 cells appeared not to be completely loss of Ezh2 impaired Th1 cell development (Fig. 2C). supported by moderate reduction of T-bet mRNA in these cells. We reasoned that loss of Ezh2 might lead to increased degradation Ezh2 regulates T-bet at both the transcriptional level and of T-bet protein in Th1 cells. To test this reasoning, we treated WT posttranslational level and T-KO Th1 cells with the proteasome inhibitor MG115 (44). To determine how Ezh2 regulated the expression of T-bet and The addition of the proteasome inhibitor MG-115 restored the ex- STAT4 in Th1 cells, we examined the expression of T-bet and pression of T-bet protein, but not STAT4 protein, in Ezh2-deficient STAT4 mRNA in WT and T-KO CD4+ T cell 7 d after culture Th1 cells (Fig. 7E), suggesting that T-bet protein is more susceptible under Th1-skewing conditions. CD4+ Tn were assessed as con- than STAT4 to proteasome-mediated degradation in Th1 cells trols. Seven days after culture under Th1-skewing conditions, lacking Ezh2. Altogether, Ezh2 promotes T-bet expression at T-KO CD4+ T cells expressed ∼1.8-fold less T-bet mRNA than transcriptional and posttranslational levels, largely with the latter. their WT counterparts did (Fig. 6A). STAT4 mRNA was slightly These results identify a novel and important role for Ezh2 to decreased in activated T-KO CD4+ T cells (Fig. 7A). In addition, regulate Th1 cells.

Ezh2 deficiency had no effect on the expression of Th1-related + Ifngr1 and Il12rb2 genes (Fig. 7B), two critical signaling mole- Introduction of T-bet into T-KO CD4 T cells fully rescues their cules upstream of T-bet and STAT4, respectively (15). These data differentiation into Th1 cells together with our observations that Ezh2 was positively associated To determine whether the downregulation of T-bet caused the with Tbx21 gene (Figs. 5, 6) indicate that Ezh2 promotes the impairment of Th1 cell development, we used MigR1 virus bicis- expression of T-bet at the transcriptional level. tronically encoding T-bet and GFP (named MigR1/T-bet) to infect To assess whether Ezh2 directly activated Tbx21 transcription, T-KO CD4+ T cells cultured under Th1-skewing conditions. T-KO we cotransfected 3T3 cells with pGL3-Tbx21 reporter and MigR1 CD4+ T cells infected with MigR1 encoding STAT4 and GFP viral plasmid encoding Ezh2 or empty MigR1 plasmid. These 3T3 (named MigR1/STAT4) or GFP alone (named MigR1/GFP) were cells were harvested 48 h after transfection and analyzed with the assessed in parallel. Expression of GFP allowed us to track cells Dual Luciferase system. Overexpression of Ezh2 resulted in mod- expressing T-bet or STAT4. WT CD4+ T cells were also infected erate induction of Tbx21 reporter activity (Fig. 7C). This finding with each of these viruses as controls. indicates that Ezh2 can directly activate Tbx21 transcription. We found that T-KO GFP-positive (GFP+)CD4+ T cells that We further verified whether loss of Ezh2 led to reduction of were derived from cultures infected by MigR1/T-bet, which STAT4 and T-bet protein in Th1 cells. In a comparison with ac- overexpressed T-bet, produced a similar percentage of IFN-g+ tivated WT CD4+ T cells, there was only minimal reduction of T cells and WT GFP+ CD4+ T cells derived from cultures infected STAT4 protein in these activated T-KO CD4+ T cells (Fig. 7D). by either MigR1/T-bet or MigR1/GFP (Fig. 8A, 8B). Interestingly, Most notably, T-KO CD4+ T cells showed ∼4-fold and 10-fold compared with WT GFP+CD4+ T cells infected with MigR1/GFP less T-bet protein at day 3 and day 7 after culture, respectively, or MigR1/STAT4, T-KO GFP+CD4+ T cells expressing STAT4 than their WT counterparts did (Fig. 7D). This dramatic reduction had significantly lower frequency of IFN-g+ T cells (Fig. 8A, 8B). by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from

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. 8 EPIGENETIC REGULATION OF Th1 CELLS MEDIATING APLASTIC ANEMIA

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 mean 6 SD. *p , 0.05, **p , 0.01, ***p , 0.001.

Furthermore, T-KO CD4+ T cells expressing STAT4 contained aftertransfer(Fig.9A,9B).Furthermore,GFP+ MigR1/T-bet- ∼40% less IFN-g+ T cells than T-KO CD4+ T cells expressing infected KO T cells (which expressed T-bet) had ∼4-fold more in T-bet (Fig. 8A, 8B). These data suggest that viral expression frequency of IFN-g–producing T cells than control GFP+ T-KO of T-bet fully rescues the ability of Ezh2-deficient CD4+ T cells T cells infected by MigR1/GFP (Fig. 9D). In contrast, transfer to differentiate into Th1 cells, whereas overexpression of STAT4 of MigR1/STAT4-infected T-KO T cells resulted in moderate only partially improves Th1 cell differentiation of activated T-KO decrease of BM cells, but not blood pancytopenia (Fig. 9A, 9B). T cells. Notably, GFP+ MigR1/STAT4-infected T-KO T cells (which To validate these observations, we highly purified GFP-positive expressed STAT4) failed to produce high levels of IFN-g (Fig. 9C, T-KO T cells from these cultures (Fig. 8C) and confirmed the 9D). Thus, Ezh2 regulation of T-bet is important for production overexpression of T-bet and STAT4 in these T-KO CD4+ T cells, of Th1 cells mediating AA. by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. respectively, using real-time RT-PCR (Fig. 8D). Furthermore, overexpression of T-bet in T-KO T cells induced significantly Discussion more Ifng transcripts than did overexpression of STAT4 (Fig. 8D). Our findings elucidate a new and critical role of Ezh2 in controlling Phosphorylation of STAT4 is critical for IFN-g production and pathogenic Th1 cell responses during AA process, which has not Th1 cell differentiation (13, 45). Western blot analysis showed been previously identified. Ezh2 is absolutely required for CD4+ that MigR1/STAT4-infected T-KO T cells expressed 7.5-fold more Th1 cells to mediate fatal AA. Ezh2 inhibition led to dramatic STAT4 protein and 1.2-fold more phosphorylated STAT4 than reduction of BM-destructive Th1 cells in vivo, decreasing BM- their WT counterparts did (data not shown). Thus, the reduction infiltrating Th1 cells, and protecting mice from BMF. Decreased of T-bet in activated T-KO CD4+ T cells is a major contributor capability of Ezh2-deficient T cells to differentiate into Th1 cells http://classic.jimmunol.org to their impaired development of Th1 cells. resulted from reduced expression of T-bet mRNA and protein, as We finally examined whether retroviral expression of T-bet could retroviral expression of T-bet in Ezh2-deficient CD4+ T cells fully rescue the ability of Ezh2-deficient T cells to induce AA. Both rescued their differentiation into Th1 cells. In contrast, ectopic CD4+ and CD8+ T cells were highly purified from T-KO B6 mice expression of STAT4 only partially restored the ability of Ezh2- and activated in vitro for 24 h, followed by infection with MigR1/ deficient CD4+ T cells to produce IFN-g. Interestingly, although T-bet, MigR1/STAT4, and MigR1/GFP, respectively. Thirty-six Ezh2 is known to act primarily as a gene silencer, it promoted the Downloaded from hours later, these infected T cells were harvested, assessed using expression of T-bet gene in Th1 cells via a mechanism of directly flow cytometry for the expression of GFP, and transferred into activating Tbx21 gene promoter. Furthermore, Ezh2 was required sublethally irradiated BDF1 mice. Each mouse received unfrac- to prevent proteasome-mediated degradation of T-bet protein in tionated T cells that contained similar numbers of GFP+ cells Th1 cells. This constellation of Ezh2 actions induced the optimal (0.8 3 106 CD4+ T cells plus 0.6 3 106 CD8+ T cells). BDF1 mice production of Th1 cells destructing BM cells, highlighting the receiving T-KO T cells transduced by either T-bet or STAT4 therapeutic potential of Ezh2 inhibition in controlling AA. survived .24 d after transfer, whereas all BDF1 mice receiving Because Ezh2 specifically catalyzes the repressive maker WT T cells infected by control MigR1/GFP died of AA within H3K27me3, a corollary belief is that Ezh2 may be required for 14 d (Fig. 9A and data not shown). This suggests that transfer of repressing cytokine gene expression (22, 46, 47). CD4+ Tn showed this amount of T-KO T cells expressing T-bet or STAT4 did moderate levels of H3K27me3 in the promoter regions of Ifng not cause lethal AA. However, as compared with control T-KO and Tbx21 gene loci (46, 48, 49, 27). Upon Th1 differentiation, T cells infected by MigR1/GFP, MigR1/T-bet–infected T-KO H3K27me3 was reduced in the promoter regions of Th1 genes T cells caused marked reduction of BM cells and peripheral (e.g., Ifng, Tbx21, and Stat4), whereas H3K4me3 was upregulated blood cells (i.e., platelets, RBCs, WBCs, and neutrophils) 24 d in these gene loci (27). We confirmed and further extended these The Journal of Immunology 9

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 (0.8 3 106 CD4+ T cells + 0.6 3 106 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.

observations that loss of Ezh2 caused the reduction of H3K27me3 for controlling appropriate Th1 cell differentiation through re- by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. at the Ifng, Tbx21, and Stat4 gene loci in Th1 cells, while in- versing and establishing new epigenetic states in T cells (52, 53). creasing the amount of H3K4me3 in these gene loci. However, T-bet recruited the histone methyltransferase SET7/9 to catalyze this skewed balance of histone methylation signature toward the permissive H3K4me2 at the Ifng promoter in Th1 cells (52, 53). transcription permission state at these Th1 gene loci did not lead Notably, T-bet recruited Jumonji domain–containing protein his- to increased expression of these genes in Ezh2-deficient CD4+ tone demethylase 3 (Jmjd3) to remove H3K27me3 across the Ifng T cells. In contrast, Ezh2 deficiency caused downregulation of locus (54). These data suggest that epigenetic regulation of Th1 Tbx21 mRNA. ChIP assay and reporter analysis verified that Ezh2 cell differentiation involves a complex and functional cooperation possessed the ability to directly activate Tbx21 transcription. This of histone methyltransferase, demethylase, and transcription fac- result is in sharp contrast to conventional thoughts that Ezh2 might tors (13, 52). In the current study, we show that Ezh2 plays a http://classic.jimmunol.org repress the expression of genes associated with effector differen- critical role in promoting optimal Th1 cell responses via a mech- tiation of Ag-driven T cells during immune response (22, 27, 46). anism of posttranslational regulation of T-bet. This effect of Ezh2 Past studies have shown that Ezh2 and other polycomb proteins appeared to be independent of its activity of methylating histone were unconventionally associated with effector cytokine genes proteins. Data from several studies indicate that Ezh2 can directly (e.g., Ifng and Il4) (25, 50). However, these studies did not report methylate signaling proteins to regulate gene transcription (40, whether Ezh2 influenced the expression of Tbx21 and the devel- 55). For example, Ezh2 methylates STAT3 in cancer cells, leading Downloaded from opment of Th1 cells in vivo during inflammatory responses (25, to enhanced STAT3 activity (55). Given the presence of high 50). Our data demonstrate that Ezh2 acted as an activator for in- amounts of Ezh2 at the promoter regions of Ifng gene locus in Th1 ducing optimal expression of Tbx21 during Th1 cell differentia- cells, we speculated that Ezh2 could play critical roles in stabi- tion. These findings are in agreement with observations by others lizing T-bet protein at this gene locus, thereby facilitating the that Ezh2 can activate some critical transcription factors in cancer T-bet regulation of Th1 cell differentiation. Formal experiments cells in a context-dependent manner (19, 51). are needed to address this point and to determine the mechanisms Intriguingly, we identified a previously unknown role for whereby Ezh2 protects T-bet from proteasome-mediated degra- Ezh2 in protecting proteasome-mediated degradation of T-bet dation in Th1 cells. protein in Th1 cells. Ezh2 deficiency led to increased degra- Accumulating evidence indicates that T-bet can be an effective dation of T-bet protein in Th1 cells. Addition of the proteasome target for the prevention and treatment of immune-mediated AA. inhibitor MG-115 restored the expression of T-bet protein in Abnormal expression of T-BET has been shown in human patients Ezh2-deficient Th1 cells. This effect of Ezh2 on protecting T-bet with AA (17). T cells from patients with AA who were refractory from proteasome-mediated degradation differs from the effect to immunosuppressive treatment expressed high levels of T-BET. of Ezh2 on repressing gene transcription (19). T-bet is crucial In contrast, patients with AA who responded to the therapy showed 10 EPIGENETIC REGULATION OF Th1 CELLS MEDIATING APLASTIC ANEMIA

low levels of T-BET in their circulating blood cells (17). Experi- 7. Young, N. S. 2000. Hematopoietic cell destruction by immune mechanisms in acquired aplastic anemia. Semin. Hematol. 37: 3–14. mental studies further indicate that genetic inhibition of T-bet leads 8. Young, N. S., and J. Maciejewski. 1997. The pathophysiology of acquired to decreased induction of AA in mice (6); however, so far, there is aplastic anemia. N. Engl. J. Med. 336: 1365–1372. no pharmacologic approach available to modulate T-bet. Further- 9. Young, N. S., A. Bacigalupo, and J. C. Marsh. 2010. Aplastic anemia: patho- physiology and treatment. Biol. Blood Marrow Transplant. 16(1, Suppl)S119– more, transcription factors are difficult drug targets (18). Given that S125. Ezh2 plays critical roles in the regulation of T-bet mRNA and 10. Blazar, B. R., W. J. Murphy, and M. Abedi. 2012. Advances in graft-versus-host protein in Th1 cells, we proposed that targeting Ezh2 may prove disease biology and therapy. Nat. Rev. Immunol. 12: 443–458. 11. Ferrara, J. L., J. E. Levine, P. Reddy, and E. Holler. 2009. Graft-versus-host advantageous to modulate T-bet and its controlled pathogenic T cell disease. Lancet 373: 1550–1561. inflammation. 12. Szabo, S. J., S. T. Kim, G. L. Costa, X. Zhang, C. G. Fathman, and Aberrant expression of Ezh2 has been associated with many L. H. Glimcher. 2000. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100: 655–669. types of malignant diseases such as prostate cancer, breast cancer, 13. Wilson, C. B., E. Rowell, and M. Sekimata. 2009. Epigenetic control of and lymphomas (19, 56, 57). Data from our previous studies in- T-helper-cell differentiation. Nat. Rev. Immunol. 9: 91–105. 14. Zhou, L., M. M. Chong, and D. R. Littman. 2009. Plasticity of CD4+ T cell dicate that Ezh2 is markedly increased in activated T cells (58, lineage differentiation. Immunity 30: 646–655. 59). Inhibition of Ezh2 leads to the reduction of graft-versus-host 15. Zhu, J., H. Yamane, and W. E. Paul. 2010. Differentiation of effector CD4 T cell disease in mice of allogeneic BMT (28). In these mouse models of populations (*). Annu. Rev. Immunol. 28: 445–489. 16. Miller, S. A., and A. S. Weinmann. 2010. Molecular mechanisms by which T-bet allogeneic BMT, infusion of donor BM cells fully rescues he- regulates T-helper cell commitment. Immunol. Rev. 238: 233–246. matopoiesis and thymopoiesis in lethally irradiated recipients (10, 17. Solomou, E. E., K. Keyvanfar, and N. S. Young. 2006. T-bet, a Th1 transcription 11), which prevents us from precisely assessing the impact of factor, is up-regulated in T cells from patients with aplastic anemia. Blood 107: 3983–3991. T cells in mediating BMF. Our experiments using the AA mouse 18. Dunker, A. K., and V. N. Uversky. 2010. Drugs for ‘protein clouds’: targeting model and in vitro cultures provide compelling evidence for intrinsically disordered transcription factors. Curr. Opin. Pharmacol. 10: 782– a T cell–intrinsic contribution of Ezh2 to the expression of Th1- 788. 19. Margueron, R., and D. Reinberg. 2011. The Polycomb complex PRC2 and its associated transcription factors and cytokines. Inhibition of Ezh2 mark in life. Nature 469: 343–349. in T cells through conditional deletion suppressed Th1 cell dif- 20. Bantignies, F., and G. Cavalli. 2006. Cellular memory and dynamic regulation of polycomb group proteins. Curr. Opin. Cell Biol. 18: 275–283. ferentiation and development of BMF. As several Ezh2-specific 21. Schuettengruber, B., D. Chourrout, M. Vervoort, B. Leblanc, and G. Cavalli. inhibitors have recently been discovered for experimental treat- 2007. Genome regulation by polycomb and trithorax proteins. Cell 128: 735–745. ment of cancer (60–63), it will be important to test whether these 22. He, S., Q. Tong, D. K. Bishop, and Y. Zhang. 2013. Histone methyltransferase and histone methylation in inflammatory T-cell responses. Immunotherapy 5: Ezh2-specific inhibitors may control AA in experimental models. 989–1004. In summary, we have identified the critical role of Ezh2 in 23. Hod-Dvorai, R., E. Jacob, Y. Boyko, and O. Avni. 2011. The binding activity of regulating Th1 responses during AA and the mechanism by which Mel-18 at the Il17a promoter is regulated by the integrated signals of the TCR and polarizing cytokines. Eur. J. Immunol. 41: 2424–2435. Ezh2 promotes Th1 cell responses mediating BMF in mice. Our 24. Jacob, E., R. Hod-Dvorai, O. L. Ben-Mordechai, Y. Boyko, and O. Avni. 2011. findings open new perspectives to study the importance of Ezh2 Dual function of polycomb group proteins in differentiated murine T helper in regulating the development of other lineages of CD4+ T cells (CD4+) cells. J. Mol. Signal. 6: 5. 25. Koyanagi, M., A. Baguet, J. Martens, R. Margueron, T. Jenuwein, and M. Bix. (e.g., Th2, Th17, Th9, and regulatory T cells). Indeed, a recent study 2005. EZH2 and histone 3 trimethyl lysine 27 associated with Il4 and Il13 gene has demonstrated that Ezh2 is involved in repressing the develop- silencing in Th1 cells. J. Biol. Chem. 280: 31470–31477. 26. Araki, Y., Z. Wang, C. Zang, W. H. Wood, III, D. Schones, K. Cui, T. Y. Roh, ment of Th9 cells (64). Furthermore, it will be important to investigate B. Lhotsky, R. P. Wersto, W. Peng, et al. 2009. Genome-wide analysis of histone by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. whether deregulated Ezh2 expression and activity are associated methylation reveals chromatin state-based regulation of gene transcription and with inflammatory disorders in humans, such as autoimmune function of memory CD8+ T cells. Immunity 30: 912–925. 27. Wei, G., L. Wei, J. Zhu, C. Zang, J. Hu-Li, Z. Yao, K. Cui, Y. Kanno, T. Y. Roh, diseases and chronic infections. W. T. Watford, et al. 2009. Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD4+ Acknowledgments T cells. Immunity 30: 155–167. 28. He, S., F. Xie, Y. Liu, Q. Tong, K. Mochizuki, P. E. Lapinski, R. S. Mani, We thank Jinfang Zhu (Molecular and Cellular Immunoregulation Unit P. Reddy, I. Mochizuki, A. M. Chinnaiyan, et al. 2013. The histone methyl- Laboratory of Immunology, National Institute of Allergy and Infectious transferase Ezh2 is a crucial epigenetic regulator of allogeneic T-cell responses Diseases, National Institutes of Health) and Cheong-hee Chang (Depart- mediating graft-versus-host disease. Blood 122: 4119–4128. ment of Microbiology and Immunology, University of Michigan) for 29. Chen, J., K. Lipovsky, F. M. Ellison, R. T. Calado, and N. S. Young. 2004. Bystander destruction of hematopoietic progenitor and stem cells in a mouse http://classic.jimmunol.org thoughtful discussion and critical reading of the manuscript. model of infusion-induced bone marrow failure. Blood 104: 1671–1678. 30. Chen, J., J. S. Brandt, F. M. Ellison, R. T. Calado, and N. S. Young. 2005. Defective stromal cell function in a mouse model of infusion-induced bone Disclosures marrow failure. Exp. Hematol. 33: 901–908. The authors have no financial conflicts of interest. 31. Chen, J., F. M. Ellison, M. A. Eckhaus, A. L. Smith, K. Keyvanfar, R. T. Calado, and N. S. Young. 2007. Minor antigen h60-mediated aplastic anemia is ame- liorated by immunosuppression and the infusion of regulatory T cells. J. References Immunol. 178: 4159–4168. Downloaded from 1. Scheinberg, P., and N. S. Young. 2012. How I treat acquired aplastic anemia. 32. Su, I. H., A. Basavaraj, A. N. Krutchinsky, O. Hobert, A. Ullrich, B. T. Chait, and Blood 120: 1185–1196. A. Tarakhovsky. 2003. Ezh2 controls B cell development through histone H3 2. Young, N. S., P. Scheinberg, and R. T. Calado. 2008. Aplastic anemia. Curr. methylation and Igh rearrangement. Nat. Immunol. 4: 124–131. Opin. Hematol. 15: 162–168. 33. Zhang, Y., J. P. Louboutin, J. Zhu, A. J. Rivera, and S. G. Emerson. 2002. 3. Zeng, W., J. P. Maciejewski, G. Chen, and N. S. Young. 2001. Limited hetero- Preterminal host dendritic cells in irradiated mice prime CD8+ T cell-mediated geneity of T cell receptor BV usage in aplastic anemia. J. Clin. Invest. 108: 765– acute graft-versus-host disease. J. Clin. Invest. 109: 1335–1344. 773. 34. Fang, T. C., Y. Yashiro-Ohtani, C. Del Bianco, D. M. Knoblock, S. C. Blacklow, 4. Roderick, J. E., G. Gonzalez-Perez, C. A. Kuksin, A. Dongre, E. R. Roberts, and W. S. Pear. 2007. Notch directly regulates Gata3 expression during T helper J. Srinivasan, C. Andrzejewski, Jr., A. H. Fauq, T. E. Golde, L. Miele, and 2 cell differentiation. Immunity 27: 100–110. L. M. Minter. 2013. Therapeutic targeting of NOTCH signaling ameliorates 35. Usui, T., R. Nishikomori, A. Kitani, and W. Strober. 2003. GATA-3 suppresses immune-mediated bone marrow failure of aplastic anemia. J. Exp. Med. 210: Th1 development by downregulation of Stat4 and not through effects on 1311–1329. IL-12Rbeta2 chain or T-bet. Immunity 18: 415–428. 5. Sloand, E., S. Kim, J. P. Maciejewski, J. Tisdale, D. Follmann, and N. S. Young. 36. Zhu, J., B. Min, J. Hu-Li, C. J. Watson, A. Grinberg, Q. Wang, N. Killeen, 2002. Intracellular interferon-gamma in circulating and marrow T cells detected J. F. Urban, Jr., L. Guo, and W. E. Paul. 2004. Conditional deletion of Gata3 shows by flow cytometry and the response to immunosuppressive therapy in patients its essential function in T(H)1-T(H)2 responses. Nat. Immunol. 5: 1157–1165. with aplastic anemia. Blood 100: 1185–1191. 37. Pearce, E. L., A. C. Mullen, G. A. Martins, C. M. Krawczyk, A. S. Hutchins, 6. Tang, Y., M. J. Desierto, J. Chen, and N. S. Young. 2010. The role of the Th1 V. P. Zediak, M. Banica, C. B. DiCioccio, D. A. Gross, C. A. Mao, et al. 2003. transcription factor T-bet in a mouse model of immune-mediated bone-marrow Control of effector CD8+ T cell function by the transcription factor Eomeso- failure. Blood 115: 541–548. dermin. Science 302: 1041–1043. The Journal of Immunology 11

38. Pear, W. S., J. P. Miller, L. Xu, J. C. Pui, B. Soffer, R. C. Quackenbush, a separate family member-specific transactivation domain. Mol. Cell. Biol. 27: A. M. Pendergast, R. Bronson, J. C. Aster, M. L. Scott, and D. Baltimore. 1998. 8510–8521. Efficient and rapid induction of a chronic myelogenous leukemia-like myelo- 53. Miller, S. A., A. C. Huang, M. M. Miazgowicz, M. M. Brassil, and proliferative disease in mice receiving P210 bcr/abl-transduced bone marrow. A. S. Weinmann. 2008. Coordinated but physically separable interaction with Blood 92: 3780–3792. H3K27-demethylase and H3K4-methyltransferase activities are required for 39. Zhu, J., D. M. Giannola, Y. Zhang, A. J. Rivera, and S. G. Emerson. 2003. NF-Y T-box protein-mediated activation of developmental gene expression. Genes Dev. cooperates with USF1/2 to induce the hematopoietic expression of HOXB4. 22: 2980–2993. Blood 102: 2420–2427. 54. Miller, S. A., S. E. Mohn, and A. S. Weinmann. 2010. Jmjd3 and UTX play 40. Su, I. H., M. W. Dobenecker, E. Dickinson, M. Oser, A. Basavaraj, a demethylase-independent role in chromatin remodeling to regulate T-box R. Marqueron, A. Viale, D. Reinberg, C. Wulfing,€ and A. Tarakhovsky. 2005. family member-dependent gene expression. Mol. Cell 40: 594–605. Polycomb group protein ezh2 controls actin polymerization and cell signaling. 55. Kim, E., M. Kim, D. H. Woo, Y. Shin, J. Shin, N. Chang, Y. T. Oh, H. Kim, Cell 121: 425–436. J. Rheey, I. Nakano, et al. 2013. Phosphorylation of EZH2 activates STAT3 41. Burbach, B. J., R. B. Medeiros, K. L. Mueller, and Y. Shimizu. 2007. T-cell signaling via STAT3 methylation and promotes tumorigenicity of glioblastoma receptor signaling to integrins. Immunol. Rev. 218: 65–81. stem-like cells. Cancer Cell 23: 839–852. 42. Ouyang, W., S. H. Ranganath, K. Weindel, D. Bhattacharya, T. L. Murphy, 56. Morin, R. D., N. A. Johnson, T. M. Severson, A. J. Mungall, J. An, R. Goya, W. C. Sha, and K. M. Murphy. 1998. Inhibition of Th1 development mediated by J. E. Paul, M. Boyle, B. W. Woolcock, F. Kuchenbauer, et al. 2010. Somatic GATA-3 through an IL-4-independent mechanism. Immunity 9: 745–755. mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lym- 43. Berger, S. L. 2007. The complex language of chromatin regulation during phomas of germinal-center origin. Nat. Genet. 42: 181–185. transcription. Nature 447: 407–412. 57. Varambally, S., S. M. Dhanasekaran, M. Zhou, T. R. Barrette, C. Kumar-Sinha, 44. Imamura, T., T. Haruta, Y. Takata, I. Usui, M. Iwata, H. Ishihara, M. Ishiki, M. G. Sanda, D. Ghosh, K. J. Pienta, R. G. Sewalt, A. P. Otte, et al. 2002. The O. Ishibashi, E. Ueno, T. Sasaoka, and M. Kobayashi. 1998. Involvement of heat polycomb group protein EZH2 is involved in progression of prostate cancer. shock protein 90 in the degradation of mutant insulin receptors by the protea- Nature 419: 624–629. some. J. Biol. Chem. 273: 11183–11188. 58. He, S., J. Wang, K. Kato, F. Xie, S. Varambally, S. Mineishi, R. Kuick, 45. Morinobu, A., M. Gadina, W. Strober, R. Visconti, A. Fornace, C. Montagna, K. Mochizuki, Y. Liu, E. Nieves, et al. 2012. Inhibition of histone methylation G. M. Feldman, R. Nishikomori, and J. J. O’Shea. 2002. STAT4 serine phos- arrests ongoing graft-versus-host disease in mice by selectively inducing apo- phorylation is critical for IL-12-induced IFN-gamma production but not for cell ptosis of alloreactive effector T cells. Blood 119: 1274–1282. proliferation. Proc. Natl. Acad. Sci. USA 99: 12281–12286. 59. Kato, K., S. Cui, R. Kuick, S. Mineishi, E. Hexner, J. L. Ferrara, S. G. Emerson, 46. Chang, S., and T. M. Aune. 2007. Dynamic changes in histone-methylation and Y. Zhang. 2010. Identification of stem cell transcriptional programs normally ‘marks’ across the locus encoding interferon-gamma during the differentiation expressed in embryonic and neural stem cells in alloreactive CD8+ T cells of T helper type 2 cells. Nat. Immunol. 8: 723–731. mediating graft-versus-host disease. Biol. Blood Marrow Transplant. 16: 751– 47. Wei, J., O. Duramad, O. A. Perng, S. L. Reiner, Y. J. Liu, and F. X. Qin. 2007. 771. Antagonistic nature of T helper 1/2 developmental programs in opposing pe- 60. Knutson, S. K., T. J. Wigle, N. M. Warholic, C. J. Sneeringer, C. J. Allain, ripheral induction of Foxp3+ regulatory T cells. Proc. Natl. Acad. Sci. USA 104: C. R. Klaus, J. D. Sacks, A. Raimondi, C. R. Majer, J. Song, et al. 2012. A 18169–18174. selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lym- 48. Boyle, A. P., S. Davis, H. P. Shulha, P. Meltzer, E. H. Margulies, Z. Weng, phoma cells. Nat. Chem. Biol. 8: 890–896. T. S. Furey, and G. E. Crawford. 2008. High-resolution mapping and charac- 61. Konze, K. D., A. Ma, F. Li, D. Barsyte-Lovejoy, T. Parton, C. J. Macnevin, terization of open chromatin across the genome. Cell 132: 311–322. F. Liu, C. Gao, X. P. Huang, E. Kuznetsova, et al. 2013. An orally bioavailable 49. Roh, T. Y., S. Cuddapah, K. Cui, and K. Zhao. 2006. The genomic landscape of chemical probe of the Lysine Methyltransferases EZH2 and EZH1. ACS Chem. histone modifications in human T cells. Proc. Natl. Acad. Sci. USA 103: 15782– Biol. 8: 1324–1334. 15787. 62. McCabe, M. T., H. M. Ott, G. Ganji, S. Korenchuk, C. Thompson, G. S. Van 50. Jacob, E., R. Hod-Dvorai, S. Schif-Zuck, and O. Avni. 2008. Unconventional Aller, Y. Liu, A. P. Graves, A. Della Pietra, III, E. Diaz, et al. 2012. EZH2 in- association of the polycomb group proteins with cytokine genes in differentiated hibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. T helper cells. J. Biol. Chem. 283: 13471–13481. Nature 492: 108–112. 51. Xu, K., Z. J. Wu, A. C. Groner, H. H. He, C. Cai, R. T. Lis, X. Wu, E. C. Stack, 63. Qi, W., H. Chan, L. Teng, L. Li, S. Chuai, R. Zhang, J. Zeng, M. Li, H. Fan, M. Loda, T. Liu, et al. 2012. EZH2 oncogenic activity in castration-resistant Y. Lin, et al. 2012. Selective inhibition of Ezh2 by a small molecule inhibitor prostate cancer cells is Polycomb-independent. Science 338: 1465–1469. blocks tumor cells proliferation. Proc. Natl. Acad. Sci. USA 109: 21360–21365. 52. Lewis, M. D., S. A. Miller, M. M. Miazgowicz, K. M. Beima, and 64. Wang, A., D. Pan, Y. H. Lee, G. J. Martinez, X. H. Feng, and C. Dong. 2013. A. S. Weinmann. 2007. T-bet’s ability to regulate individual target genes requires Cutting edge: Smad2 and Smad4 regulate TGF-b-mediated Il9 gene expression

by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. the conserved T-box domain to recruit histone methyltransferase activity and via EZH2 displacement. J. Immunol. 191: 4908–4912. http://classic.jimmunol.org Downloaded from