Promoter DNA Methylation Ifng Ontogeny and Influence Changes in Expression Across Th2 Γ Plasticity of IFN- STAT4 and T-Bet

The Journal of Immunology

STAT4 and T-bet Are Required for the Plasticity of IFN-g Expression across Th2 Ontogeny and Inﬂuence Changes in Ifng Promoter DNA Methylation

Christopher L. Williams,*,† Marcia M. Schilling,*,† Sung Hoon Cho,*,† Keunwook Lee,*,† Mei Wei,*,† Aditi,*,† and Mark Boothby*,†,‡

CD4+ T cells developing toward a Th2 fate express IL-4, IL-5, and IL-13 while inhibiting production of cytokines associated with other Th types, such as the Th1 cytokine IFN- g. IL-4–producing Th2 effector cells give rise to a long-lived memory population committed to reactivation of the Th2 cytokine gene expression program. However, reactivation of these effector- derived cells under Th1-skewing conditions leads to production of IFN-g along with IL-4 in the same cell. We now show that this ﬂexibility (“plasticity”) of cytokine expression is preceded by a loss of the repressive DNA methylation of the Ifng promoter acquired during Th2 polarization yet requires STAT4 along with T-box expressed in T cells. Surprisingly, loss of either STAT4 or T-box expressed in T cells increased Ifng promoter CpG methylation in both effector and memory Th2 cells. Taken together, our data suggest a model in which the expression of IFN-g by Th2-derived memory cells involves attenuation of epigenetic repression in memory Th2 cells, combined with Th1-polarizing signals after their recall activation. The Journal of Immu- nology, 2013, 191: 678–687.

D4+ T cells play central roles in the ability of the im- transcription factors lead to the subset-specific expression of cyto- mune system to adapt the nature of its responses to kines that characterize the CD4 effector cells. C different types of pathogens. Upon exposure to their Two of the best studied effector programs, both in terms of cognate Ag, signals from the TCR and the local cytokine milieu physiological impact and the mechanisms leading to their devel- drive naive CD4+ T cells to develop into one of several effector opment, are Th1 and Th2 (8, 9). Th1 effectors produce IFN-g, programs. Each effector type, commonlyreferredtoasalineage which activates macrophages to promote clearance of intracellular or subset, produces a set of hallmark cytokines while inhibiting pathogens (10, 11) and is involved in the pathogenesis of auto- the expression of cytokine genes characteristic of other effector immune diseases such as type 1 diabetes (12). The importance of types (1). Development along an effector lineage is, in large part, IFN-g in human health is illustrated by the increased susceptibility directed by cytokines signaling through their membrane-bound to mycobacterial infections in patients lacking a functional IFN-gR receptors at or around the time of Ag encounter (2, 3). The cell (13). Th1 polarization requires IL-12R signaling via STAT4 and an surface receptors for cytokines that can polarize populations of induction of T-box expressed in T cells (T-bet) expression (4, 6, 7). Th cells activate immediate-early transcription factors in the These transcription factors direct histone posttranslational mod- family of STAT proteins (4, 5). These, in concert with other ifications (14) along with chromatin remodeling of the Ifng gene transcription factors and signaling pathways, induce subset- (15) to allow for efficient gene transcription. In contrast, the Th2 or lineage-specific transcription factors sometimes considered effector program is characterized by repression of Ifng gene ex- master regulators (6, 7). The combined signals from the TCR, pression along with production of IL-4, IL-5, and IL-13 (8, 16). cytokine receptors, and the activity of the master regulator These Th2 cytokines direct responses against helminthic pathogens (17) and contribute to atopic diseases such as allergic asthma (18). Th2 polarization involves IL-4R signaling though STAT6 (19, 20), followed by induction of the transcription factor GATA3 (21, 22), *Department of Microbiology and Immunology, Vanderbilt University, Nashville TN to enable transcription of the Th2 cytokine genes. 37232; †Department of Pathology, Microbiology, and Immunology, Vanderbilt Uni- Beyond the activation of cytokine gene expression, each po- versity, Nashville TN 37232; and ‡Division of Rheumatology, Department of Med- icine, Vanderbilt University, Nashville TN 37232 larized Th program also involves the silencing of genes from Received for publication December 10, 2012. Accepted for publication May 7, 2013. opposing transcriptional programs (23). For instance, GATA3 mediates the silencing of the genes encoding IFN-g, IL-12Rb, and This work was supported by bridge funding from Vanderbilt University and by National Institutes of Health Grants R01 AI077528 (to M.B.); T32 DK07563 (to STAT4 via repressive histone modifications during the course of C.L.W.); and T32 AR59039 and T32 HL94296 (to M.M.S.). Th2 polarization (24). Conversely, cells developing toward Th1 Address correspondence and reprint requests to Dr. Mark Boothby, Department of effector function inhibit the production of IL-4 through inhibition Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, of GATA3 transcription (23, 25). The silencing of cytokines as- 1161 21st Avenue South, AA-4214 B MCN, Nashville, TN 37232. E-mail address: [email protected] sociated with other Th subsets also involves repressive DNA The online version of this article contains supplemental material. methylation. Thus, the proximal promoter of the Ifng gene is CpG Abbreviations used in this article: CFDA-SE, 5 (and 6)-carboxyfluorescein diacetate- methylated in Th2 clones (26) and primary effector cells (27–29) succinimidyl ester; ChIP, chromatin immunoprecipitation; DNMT, DNA methyl- when compared with Th1 counterparts. Moreover, methylation of transferase; IRES, internal ribosomal entry sequence; KO, knockout; me-, methyl-; the evolutionarily conserved CpG at 253 in this proximal Ifng T-bet, T-box expressed in T cells; WT, wild-type. promoter sufficed to abrogate its activity (29). Together, these Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 studies indicated that DNA methylation, especially at the 253 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203360 The Journal of Immunology 679

CpG of that Ifng promoter is a key aspect of repressing expression Cell culture and purification of GFP+ IL-4–producing effectors of this Th1 cytokine in Th2 effector cells. and adoptive transfers Immunological memory is a key feature of the adaptive immune DO11.10 cells were activated with OVA323–339 peptide, and all cells system and provides protection against reinfection after a first were cultured as described (35), with the following modifications. For exposure to a pathogen (30). After an Ag has been cleared, a Th1 culture conditions, cells were plated at 7 3 106 cellsper milliliter and fraction of responding cells survives as a long-lived population received 1 mg/ml OVA peptide, 5 ng/ml IL-12, and 3 mg/ml anti–IL-4 Ab 3 6 and appears to acquire a memory program differing from the 11B11. For Th2 cell cultures, cells were plated at 3.5 10 cellsper milliliter and received 0.5 mg/ml OVA peptide, 7.5 ng/ml IL-4, 3 mg/ml effector state (31). Because cytokine production by memory cells anti–IFN-g Ab, and 2 mg/ml anti–IL-12 Ab. Both Th1 and Th2 cell upon Ag exposure can instruct a new generation of immune ef- cultures were supplemented with IL-2 (50 U/ml 24 and 72 h after Ag fectors, the profile of cytokines produced by recall responses of stimulation). GFP+ Th2 effector cells were purified for transfer as de- a memory CD4 population can dictate its protective value in re- scribed (35). After 4 d of culture in Th2 conditions, 4get, DO11.10 cells [Tbx212/2, Stat42/2, or transcriptionally wild-type (WT)] were stained peat exposures to a given pathogen. For example, rapid produc- + with APC-conjugated anti-CD4 and PE-conjugated KJ1-26 (anti-DO11.10 tion of IL-4 by memory-phenotype CD4 T cells sufficed to guide TCR) Abs and flow sorted on a FACS Aria (BD, Franklin Lakes, NJ) to CD4+ T cells to adopt a Th2 effector program following exposure purify (.98.5%) viable DO11.10 KJ1-26+ CD4+,GFP+ cells. Prior to transfer to Leishmania major, leading to a failure to resolve the infection or DNA isolation for methyl-CpG analyses, these cells were cultured in (32). In contrast, IFN-g promotes resistance to such pathogens. Th2 conditions for 9–10 d after restimulation with APCs (4:1 with T cells) and 0.25 mg/ml OVA323–339 (13–14 d total). Th2-derived memory cells arise from IL-4–producing Th2 effectors; after acquisition of a relatively quiescent state, Ag Quantitation of cytokine production restimulation of these Th2-derived memory cells rapidly leads to Th cell cultures plated with APCs at a 1:4 ratio, and single-cell suspensions IL-4 production (33, 34). After stimulation and growth in Th1- of recipient spleens, were stimulated with 1 mg/ml OVA323–339. Cytokine biased conditions in vitro or in vivo, these reactivated memory concentrations in supernatant collected after 36 h were quantified using cells continue to produce IL-4, illustrating that Th2 memory cells a flow cytometric kit (Th1/Th2/Th17 cytokine bead array; BD Pharmin- retain a commitment to produce IL-4 (35). gen). Intracellular cytokines were analyzed by stimulating cells 18 h with OVA323–339 (1 mg/ml) in the presence of APCs, followed by GolgiStop Although production of IL-4 remains part of the programming (BD) (2–3 h), and staining in the presence of anti-CD16/32 (Fc Block; BD for Th2-derived memory cells, restimulation under Th1 conditions Pharmingen), as previously described (35). Viable cells were selected also drove these cells to produce substantial amounts of IFN-g (35– based on forward and side scatter characteristics, and fluorescence signals 37). These findings revealed that the nature of gene silencing as representing intracellular cytokines were determined in cells positive for the DO11.10 TCR and CD4. part of the Th program could be changed in memory cells, so that IFN-g and the Th2 cytokine genes can be coexpressed within an Bisulfite analysis of DNA methylation individual CD4 lymphocyte. However, almost nothing is known DNA isolated from flow-purified naive, effector (Th1, Th2, 13 d), and about the molecular mechanisms for this plasticity of program- memory CD4 T cells, or from tissues, was digested with BamH1 (New ming. Recent work indicated that, in addition to the Th1 master England BioLabs, Ipswich MA), bisulfite modified using the Imprint bi- regulator T-bet, IL-12 was required for the induction of Ifng gene sulfite modifying kit (Sigma-Aldrich, St. Louis, MO), then used as template expression after reactivation of memory Th2 populations (38). The in quadruplicate PCR performed using primer pairs specific for each modified strand sequence in the Ifng promoter (Supplemental Table I) (29). signal or signals downstream from IL-12 and essential for plas- The noncoding strand of the Ifng promoter was amplified with a single ticity of Ifng regulation are not established. Moreover, a key reaction, whereas the coding strand was amplified using a nested PCR. unanswered question regarding the mechanisms permitting IFN-g After pooling four tubes of separate amplification for each sample, specific production by Th2-derived memory cells is whether repressive PCR products were identified on agarose gels, extracted using the QIA- epigenetic modifications of the Ifng promoter that occur during quick Gel Extraction Kit (Qiagen, Valencia, CA), ligated with T Easy Vector (Promega, Madison, WI), and then transformed into JM109 cells Th2 polarization are maintained in the memory phases. In this (Agilent, Santa Clara, CA). For each of three independent biological article, we have tested whether STAT4 affects the ability of replicate cell samples and for each strand, 8–10 clones derived from each memory Th2 cells to express T-bet or IFN-g in Th1 recall con- reaction pool were sequenced and scored for the frequency of unmodified ditions. Further, we analyzed Ifng promoter DNA methylation in C residues in the CpG dinucleotides; the modification frequency for C residues outside of CpG dyads was verified as . 99% for all sample sets. naive, Th1-, Th2-, and Th2-derived memory CD4+ T cells, and explored the relationship between promoter methylation and the EMSA Th1-determining transcription factors STAT4 and T-bet. EMSAs were performed as reported (29, 39), except that extracts of Th1 cells developing from primary mouse CD4+ T cells were used. Methylated Materials and Methods upper-strand oligonucleotides were synthesized by Invitrogen (Grand Mice Island, NY). The upper-strand oligonucleotides were designated as unmethylated, methylated (me)C(253) hemimethylated, and trihemime- BALB/c Il4-IRES-Gfp [“4get”; internal ribosomal entry sequence (IRES)], thylated (meC at 253, 245, and 234) (Supplemental Table I). Each was 2 2 DO11.10 mice were bred with BALB/c Tbx21 (T-bet) / [knockout (KO)] annealed to an unmethylated lower-strand oligonucleotide after radio- or BALB/c Stat4 KO mice (The Jackson Laboratory). BALB/c-ByJ (The labeling with g-[32P]-ATP (Perkin-Elmer, Waltham MA) and T4 polynu- Jackson Laboratory) and athymic BALB/c nude (Foxn1/Foxn1) mice were cleotide kinase (New England BioLabs). For competition assays, unlabeled used as recipients for transfer experiments. Recipients were 4–6 wk old at competitor was added simultaneously with the labeled probe at molar the time of transfer. Mice were maintained in microisolator cages at ratios (competitor:probe) of 100:1, 10:1, and 1:1. Abs used for the a Vanderbilt University facility in accordance with Institutional Animal supershift assays were CREB1 (sc-186), ATF2 (sc-187), and c-Jun (sc-45) Care and Use Committee guidelines and an approved protocol. (Santa Cruz Biotechnology, Santa Cruz, CA). Reagents Transient transfection and reporter assays Fluorophore-conjugated and purified mAbs were obtained from BD Phar- Nucleofection was carried out via the Amaxa T cell kit (Lonza, Basel, mingen (San Jose, CA) and purified recombinant cytokines from Leinco (St. Switzerland) using a minimal Ifng promoter reporter P1P2-Luc (40) along Louis, MO) unless otherwise indicated. Purified 11B11 anti–IL-4 and with pCMV-Sport6-CREB1 or pCMV-Sport6. All results were normalized recombinant huIL-2 were obtained from the Biological Response Modifiers to GFP expression from the pMAX-GFP plasmid (Lonza) measured via Program (National Cancer Institute, Frederick MD). Anti–T-bet–eFluor 660 flow cytometry. Luciferase activity was measured using the Dual-Glo was obtained from eBioscience (San Diego, CA). Oligonucleotides were Luciferase Assay System (Promega) according to the manufacturer’s pro- synthesized by Invitrogen (Grand Island, NY) unless otherwise stated. tocols. 680 PLASTICITY OF Ifng PROMOTER DNA METHYLATION IN Th2 ONTOGENY

Chromatin immunoprecipitation assays assays, BrdU (1 mM) was added to Th2 cultures (days 2 and 13 after Ag activation), followed 4 h later by processing, direct immunofluorescent Chromatin immunoprecipitation (ChIP) assays were performed essentially anti-BrdU staining, and flow cytometry of KJ1-26+ CD4+ cells. as described previously (15). After cross-linking with formaldehyde, primary Th1 and Th2 cells were sonicated in a Bioruptor (Diagenode, Denville, Retroviral transduction NJ) to produce an average sheered DNA length of 400 bp. Immunoprecipi- tation was carried out using anti-AcH3(K9) (acetyl-lysine 9 of histone Retroviral transduction was carried out using as previously described (15). H3; #DAM1813175; Millipore), or CREB1 (sc-186X; Santa Cruz,), and In brief, retrovirus-containing supernatants from FNX packaging cells transfected with MSCV-IRES-Thy1.1 (MiT) or MSCV-T-bet-IRES-Thy1.1 the precipitates were analyzed by PCR using primers shown in Sup- + plemental Table I. (MIT-T-bet) were used to transduce established GFP DO11.10 Th2 cells 2 d after restimulation. Cells were then cultured under Th1 or Th2 conditions, followed by measurements of Ag-stimulated cytokine production Proliferation in vitro and in vivo or intracellular cytokines as above. Proliferation studies using CFSE partitioning and BrdU incorporation were carried out as described (39). For CFSE partitioning in vivo, Th2 cells were Results grown for 5 d, labeled with 5 (and 6)-carboxyfluorescein diacetate- Strand-biased acquisition of Ifng promoter methylation in Th2 succinimidyl ester (CFDA-SE; Invitrogen) (2.5 mM, 15 min) following effector cells manufacturer instructions, and then transferred into BALB/c recipients. Fluorescence was measured on donor-derived cells recovered 12 d after The finding that memory Th2 cells, unlike their effector-phase transfer, with gating as described above. For CFSE partitioning assays precursors, can respond to an inflammatory milieu by efficient in- in vitro, DO11.10 splenocytes were either labeled, Ag-stimulated, and duction of IFN-g production (35, 36) raises questions about the fate cultured 2 d, or activated with Ag, cultured in Th2 conditions (11 d, with one interim Ag stimulation, as for cells used in adoptive transfers), labeled of repressive DNA methylation observed in Th2 effectors once with CFDA-SE as above, and then cultured 2 d in IL-2–supplemented these cells transition to memory status. Accordingly, we used bi- medium before analysis by flow cytometry. For assays of BrdU incorpo- sulfite modification of DNA followed by strand-specific PCR to ration into donor cells in vivo, recipient mice were injected twice (72 h, analyze CpG methylation in Ifng promoter DNA of naive CD4+ 24 h before harvest; 3 mg i.p. per injection) with BrdU (Sigma-Aldrich) in + sterile saline. Cells harvested 12 d posttransfer were then processed as lymphocytes, Th1, and Th2 T cells. Naive CD4 and Th1 effector described (39) to detect Alexa-647 anti-BrdU (Invitrogen) in donor- (KJ1- cells showed little methylation of either strand of DNA upstream 26+) and recipient-derived CD4+ T cells by flow cytometry. For in vitro from the transcription start site (Fig. 1), whereas high methyla-

FIGURE 1. Asymmetrical increase in CpG methylation of the Ifng promoter in Th2 cells. Methylation of CpG residues was determined by sequencing individual subcloned products from strand-specific PCR of bisulfite-modified DNA from naive CD4+ T cells (A) and Th1 and Th2 cells (B). Shown are diagrams illustrating representative individual sequences, from biological replicates for each type of sample, generated from naive (A) or Th1 and Th2 cells (B). Filled circles (d) represent methylated cytosine, and open circles (s) the unmethylated residue. Analyses included at least eight independent clones from each of three independent biological replicates per sample. (C) Data for the coding (left) and noncoding (right) strands are shown, with each bar representing the mean (6 SEM) number of methylated CpGs per clone in all surveyed upstream CpGs (six total), the CpGs at 2205, 2190, -170, or the CpG triad at 253, 245, and 234. (D) Comparison of coding and noncoding strand methylation in Th2 effectors. (E) Methylation frequencies at the 253 CpG dinucleotide. (F) CFSE partitioning was measured 48 h after labeling cells that were either acutely stimulated (2 d total), or from 11-d Th2 cultures (13 d total). (G) Cells cultured under Th2 conditions for 2 d or 13 d were pulsed with BrdU for 4 h. The incorporation of BrdU into cellular DNA was measured by intracellular staining. Cells grown continuously in BrdU or unexposed to BrdU were used as staining controls. Results (F and G) are representative of two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 681 tion densities were found at two dinucleotides in exon 1, inde- Asymmetrical methylation affects transcription factor binding pendent of T cell differentiation, as expected (41, 42). In effector to the Ifng promoter Th2 cells (Fig. 1B, 1C), we found increased methylation of the On the basis of evidence that the Ifng promoter in many Th2 cells coding strand of the Ifng promoter, with a majority of samples can be in a state of asymmetrical methylation, we investigated 2 exhibiting modification of the key 53 CpG whose modifica- whether hemimethylation might affect transcription factor recruit- tion appeared to abrogate promoter activity (29). Surprisingly, ment to the Ifng promoter. EMSA using nuclear extracts of primary however, the noncoding strand was reproducibly and signifi- Th1 cells were carried out using unmethylated or hemimethylated cantly less methylated in Th2 effector cells relative to the probes (Fig. 2A). Both hemimethylated probes impaired the for- 2 coding strand (Fig. 1D), particularly at the 53 CpG (Fig. 1E). mation of the slower migrating complex (indicated by filled arrow, In light of this unexpected result, we tested samples including Fig. 2B). Competition assays using unlabeled competitor DNA DNA from a mouse brain and 3T3 cells, both of which would be confirmed that the mobility shift bands represented sequence- expected to have symmetrical hypermethylation of the Ifng specific binding; moreover, 10-fold more cold competitor was promoter, along with thymocytes and naive CD4 lymphocytes. needed to attenuate the slower migrating complex to the WT Brain and 3T3 cell DNA demonstrated a high density of sym- compared with the hemimethylated probe (Fig. 2C). To characterize metrical methylation across the surveyed region and in partic- this complex, we performed Ab blocking/supershift assays with the 2 ular at the crucial 53 CpG, whereas thymocytes, like naive unmethylated probe and Abs against CREB/ATF family members. CD4 T cells, exhibited little meC (Table I). The frequency of The upper band was impacted by anti-CREB1 (Fig. 2D), whereas non-coding strand DNA methylation in Th2 cells was too low Abs against ATF2 and c-Jun had no discernible effect, leading us simply to represent a lack of modification on one chromosome, to conclude that the slower migrating complex is predominantly e.g., from monoallelism (43). Separate analyses (Table I and formed by CREB1. Consistent with the hemimethylation observed later results) exclude a strand bias in the detection method as at the Ifng promoter having an impact on CREB1 recruitment the basis for the observation. Accordingly, we infer from these in vivo, ChIPs performed using anti-CREB1 Ab showed greater data that Ifng promoters were hemimethylated in these Th2 promoter occupancy in Th1 cells than did their Th2 counterparts effector cells. The asymmetry of CpG methylation might in (Fig. 2E). The decreased binding of CREB1 in effector-stage Th2 principle be observed because a high proportion of the Th2 cells, in which the Ifng gene is not active, would be consistent with population was actively in cell cycle and rapidly undergoing CREB1 function as a trans activator. To test if CREB1 can increase DNA synthesis. However, virtually no divisions were observed activity of the Ifng promoter in primary Th1 cells, we performed in CFSE analyses of the effector Th2 cells in the 2 d prior to the nucleofections of developing Th1 cells, using a minimal Ifng pro- time at which DNA samples were prepared (Fig. 1F). Moreover, moter reporter construct and either a CREB1 expression vector or only a small fraction of the population was in cell cycle, as an empty vector control (Fig. 2F). We found that CREB1 increased + indicated by a low frequency of BrdU cells at this time activity of the Ifng reporter construct. In sum, these findings show 13 d after the initial Ag activation (Fig. 1G). From these low that upper-strand hemimethylation of the CpG at -53 can impair frequencies of cycling cells, we conclude that at most a very binding of CREB1, a trans activator of the Ifng promoter. small minority of asymmetrical methylation observed in Th2 effectors could be due to DNA replication, whereas the re- Loss of Ifng methylation in Th2-derived memory cells mainder is an epigenetic feature of the Ifng locus at this stage in Th2-derived memory cells can produce IFN-g when exposed Th2 effectors. to Th1-skewing conditions during recall responses (35, 36). To

Table I. CpG methylation in naive CD4+ T cells, thymus, brain, and NIH 3T3 cells

CpG positiona

Sample Strand 2205 2190 2170 253 245 234 16 Naive CD4 Coding 11.4 (3.6)b 11.4 (3.6) 14.4 (3.6) 17.4 (3.9) 14.4 (3.6) 14.4 (3.6) 59.8 (6.1) Noncoding 17 (3.6) 10.8 (1.6) 6.25 (2.3) 17 (3.4) 15.3 (2) 15.3 (2) 90.9 (3.6) Thymus Coding 0 0 0 0 0 0 72.2 (3.5) Noncoding 0 0 0 0 0 0 71.3 (3.5) Brain Coding 100 86.6 (2) 86.6 (2) 100 100 100 100 Noncoding 95 (2.7) 76.7 (3) 100 95 (2.7) 100 91.7 (3.3) 83.3 (4.9) NIH 3T3 Coding 78.9 (3.2) 63.1 (3.8) 52.6 (1.8) 63.1 (0.9) 63.1 (0.9) 52.6 (3.5) 68.4 (2.8) Noncoding 62.5 (4.2) 36.3 (6.1) 43.8 (3.0) 56.3 (3.0) 50 (0.0) 50 (0.0) 87.5 (4.2)

Sample Strand Totalc 2205–170 253–34 Naive CD4 Coding 0.9 (0.25)d 0.4 (0.18) 0.52 (0.2) Noncoding 0.7 (0.34) 0.25 (0.14) 0.45 (0.22) Thymus Coding 0.11 (0.07) 0 (0) 0.11 (0.07) Noncoding 0 (0) 0 (0) 0 (0) Brain Coding 5.7 (0.12) 2.7 (0.12) 3 (0) Noncoding 5.6 (0.16) 2.6 (0.15) 2.9 (0.06) 3T3 Coding 3.8 (0.45) 2.1 (0.27) 1.8 (0.31) Noncoding 2.9 (0.53) 1.4 (0.26) 1.5 (0.35)

aPosition is presented as the distance in bases from the transcription start site. bData are presented as the mean (6SEM) percentage of samples methylated at a given position. cCluster of CpG dinucleotides being assayed. dMean (6SEM) number of methyl-CpGs per clone in a given cluster. 682 PLASTICITY OF Ifng PROMOTER DNA METHYLATION IN Th2 ONTOGENY

FIGURE 2. Asymmetrical methylation suffices to inhibit transcription factor binding to the Ifng promoter. (A) Schematic of the hemimethylated probes used. (B) Shown is an au- toradiograph representative of EMSA results after using probes that are unmethylated (lane 1), methylated at the coding strand C(253)pG (lane 2) or methylated at coding strand C(253)pG, C(245)pG, and C(234)pG (lane 3). (C)EMSA using unmethylated probe in competition with oligonucleotides that were unmethylated (lanes 2–4), hemimethylated at C(253) (lanes 5–7), or hemimethylated at all three upper-strand residues (lanes 8–10), at 1, 10, and 100-fold excess of competitor over probe. Shown is one result representative of three replicate assays. (D)Ab blocking/supershift assay against ATF2, c-Jun, CREB, or control (nonspecific IgG). Shown is one result representative of three replicate assays. (E) ChIPs using Abs specific for CREB1 or AcH3(K9). Eluted precipitates were analyzed for Ifng promoter sequences by PCR (left panel). Signals (mean 6 SEM) from three independent biological replicates were quantified (right panel) as product relative to input, as described in Materials and Methods.**p # 0.01; BLD, below limit of detection. (F) After nucleofection of primary Th1 cells with a minimal Ifng promoter–luciferase construct and pSport6 or pSport6-CREB1, luciferase activities were normalized to GFP expression. Shown are mean (6 SEM) data from three independent experiments. * p # 0.05.

investigate the relationship between this capacity and the repres- IL-12R elicits multiple intracellular signals (45, 46), and which sive methylation observed in primary Th2 cells, we prepared DNA of these is essential for the plasticity of gene expression is not from purified effector cells and their memory Th2 descendants known. Accordingly, we compared the amounts of IFN-g pro- (Fig. 3A). As expected, cells in the donor-derived memory pool in duced after recall stimulation and cultures of memory Th2 cells each type of recipient underwent homeostatic divisions after from Tbx212/2 and Stat42/2 T cells to that derived from parallel transfer (Fig. 3B), and these memory cells produced IFN-g after controls with normal transcriptional function (Fig. 5A, Supple- reactivation by Ag and growth in Th1 conditions (Fig. 3C). Weeks mental Fig. 1A). When cytokine production was elicited 1 wk after transfers into normal or lymphopenic BALB/c mice, donor- after recall restimulation with peptide Ag and culture under Th1 derived cells were purified from the recipient lymphoid organs. and Th2 conditions, samples of each transcription factor–deficient Strand-specific PCR analyses of bisulfite-modified donor-derived memory Th2 population produced substantially less IFN-g than cellular DNA showed that methylation of multiple sites decreased did matched WT controls (Fig. 5A). IFN-g production elicited (Fig. 4B) and the 253 CpG of the Ifng promoter coding strand after Th1-skewed recall was higher than background with each was almost completely unmethylated (Fig. 4A, 4C). These results type of knockout cell type. To assess the extent to which double- were independent of whether the recipients had normal endoge- producing (IL-4+ IFN-g+) cells could be generated from memory nous T cells or were lymphopenic (data not shown). These find- Th2 cells, we used intracellular staining for these cytokines (Fig. ings provide evidence of dynamic change in Ifng promoter 5B, 5C). Although subject to the likelihood that the limits of methylation, as the population of Th2 effectors yields a memory detection are more sensitive for secreted cytokine than intracel- Th2 subset. lular staining, these analyses consistently revealed almost no IFN- g+ donor-derived (KJ1-26+ CD4+) cells in the absence of either STAT4 is required for flexible IFN-g production STAT4 or T-bet (Fig. 5C). In sharp contrast, ample IL-4+ IFN-g+ The development of Th1 effector cells from naive CD4+ T cell CD4+ T cells were abundant (31% of donor T cells) when controls precursors is highly dependent on IL-12–induced STAT4 and, in with normal transcription factor genes were used (Fig. 5C). These most settings, on T-bet (4, 7, 44). IL-12 is required for the fac- data indicate that STAT4 is required in support of the capacity for ultative induction of IFN-g production by memory Th2 cells after memory Th2 cells to turn on IFN-g production to an extent similar recall stimulation in vitro and in vivo (35, 36, 38). However, the to that of T-bet. The Journal of Immunology 683

FIGURE 3. Elicitation of IFN-g production from memory Th2 cells (A) Schematic overview of the experimental design and generation of Th2- derived memory cells. (B) CFSE partitioning assay for DO11.10 cells transferred into WT (shaded) or nude recipients (outline) and recovered after 2 wk, gated on CD4+ DO11.10 TCR+ cells. (C) IFN-g secretion data are shown for memory Th2 cells at the time of harvest and after recall activation and growth (6 d) under Th1- or Th2-polarizing conditions, as indicated. Values in each individual sample were normalized to the frequencies of DO11.10 TCR+ CD4+ cell number to account for differences in cell prevalence. Shown are data from a single experiment representative of four independent replicates and consistent with earlier work (35, 36).

T-bet induction in developing Th1 cells is driven by STAT1 and NF-kB (47, 48), but STAT4 regulates a later, IL-12–dependent FIGURE 4. Altered DNA methylation of the Ifng promoter in effector- phase of T-bet expression in the development of primary Th1 derived memory Th2 cells. (A) Representative bisulfite sequencing results B responses (49, 50). Therefore, we also tested whether STAT4 is from freshly isolated memory cells are diagrammed as in Fig. 1A. ( )Mean required for Ifng plasticity in memory Th2 cells because it is es- methylated CpG dinucleotides per clone are shown as in Fig. 1C. The Th2 effector results are taken from Fig. 1 and are shown for comparison. (C) sential for T-bet induction. Consistent with prior work (49, 50), Methylation of the Ifng promoter 253 CpG in effector versus memory Th2 intracellular stains detected T-bet immunofluorescence in STAT4- cells is compared as in Fig. 1E. *** p , 0.001. deficient Th1 effectors at levels equivalent to those observed for Th2 effectors (Fig. 6A, top panel). After recall activation and culture under Th1 conditions, however, STAT4-deficient memory An alternative model is that the absence of T-bet or STAT4 led Th2 cells displayed more T-bet protein expression, with at least to a higher or more symmetric density of CpG methylation at the half of the cells exhibiting induction to Th1 levels (Fig. 6A, middle Ifng promoter during the development of Th2 effectors. Signifi- panel). This finding indicates that the regulation of T-bet expres- cant changes in upper-strand meCpG densities were not observed sion by STAT4 in this memory setting was not sufficient to explain (Supplemental Fig. 1B), but the methylation frequency of the non- the defect in Ifng induction. Together, the data show that the gene coding strand was strikingly higher for T-bet–null Th2 cells. In expression plasticity of memory Th2 cells—that is, elicitation of addition, STAT4-deficient cells had essentially symmetrical DNA IFN-g—requires independent input from STAT4 as well as T-bet. methylation (Fig. 6D) and increased noncoding strand methylation, especially at the crucial 253 CpG (Supplemental Fig. 1C). T-bet and STAT4 alter Ifng promoter methylation pattern but Moreover, meCpG densities in the Ifng promoter DNA from not homeostatic divisions transcription factor–deficient memory Th2 cells, recovered after Homeostatic divisions of cells in which asymmetrical methyl-CpG several weeks in vivo, were higher than those from WT memory marks were present could lead to descendants in which this re- controls (Fig. 6E; Supplemental Fig. 1D). Thus, T-bet and STAT4 pressive mark was absent from the Ifng promoter on both chro- each influenced Ifng promoter methylation in memory as well as mosomes. Accordingly, we tested whether the rates of division effector Th2 lymphocytes. 2 2 2 2 were slower for Tbx21 / or Stat4 / Th2 cells in recipient mice. Prior studies have supported several potential relationships CFSE partitioning assays showed no defect in rates of division for between T-bet and the capacity to produce IFN-g after Th2 dif- DO11.10 Th2 cells that were T-bet- or STAT4-deficient cells, ferentiation. In one study, most human CD4 T cells could switch compared with controls that were WT with respect to the tran- from polarized Th2 cytokine gene expression to turn on their scription factors (Fig. 6B). To compare proliferation of the tran- IFNG gene; a subset of the helper cells unable to exhibit such scriptionally deficient memory Th2 cells to WT controls long after flexibility was attributed to lack of T-bet expression (51). Parallel the transfer, recipient mice received BrdU and its incorporation work indicated that differentiation progressively reduced the ca- into DNA was measured. This analysis provided evidence that the pacity of such Th2 cells to turn on IFN-g expression in the transferred cells almost completely exited the cell cycle and that presence of forced T-bet expression (52). In light of the failure of low rates of S-phase entry were similar for all genotypes (Fig. 6C). T-bet–deficient Th2 cells to exhibit flexibility (Fig. 5A) and their 684 PLASTICITY OF Ifng PROMOTER DNA METHYLATION IN Th2 ONTOGENY

controls. We conclude that T-bet at a sufﬁcient level can overcome the block to IFN-g production by established Tbx212/2 Th2 cells. On the basis of collective ﬁndings, we propose (see Discussion) that at least two barriers to Ifng gene expression are present in effector Th2 cells but are reversible once cells have experienced a transition to memory state.

Discussion The capacity of memory cells derived from Th2 effectors to produce IFN-g in recall responses represents a naturally occurring form of cellular reprogramming. Apart from a requirement for IL- 12, type I IFNs, and the transcription factor T-bet (35, 38), nothing is known about the molecular mechanisms by which this plasticity of gene expression is effected. We have found that the Ifng promoter exhibits asymmetrical methylation in committed Th2 effectors. The coding strand DNA preferentially acquires signifi- cantly increased methylation relative to the low frequency of meCpG in naive CD4+ T cells and on the noncoding strand. A hemimethylated state created by such asymmetry suffices to impair CREB1 binding to an Ifng promoter sequence that is highly conserved and strongly required for promoter activity. Consistent with these data, nucleofection assays provide evidence that CREB1 is a trans activator of the Ifng promoter, and that this ubiquitously expressed transcription factor preferentially binds to the promoter in Th1 cells as compared with Th2 counterparts. Strikingly, CpG methylation of the Ifng promoter in memory Th2 cells was observed at a frequency little different from that of the naive progenitor. Inasmuch as promoter methylation is a strongly repressive mark, these findings suggest that loss of meCpG marks contributes to the plasticity of Ifng gene expression upon recall activation. In investigating the transcription factor requirements for this facultative production of IFN-g, we found that the IL-12– induced factor STAT4 is required along with T-bet. Surprisingly, increased densities of CpG methylation were observed in T-bet– deficient Th2 cells relative to WT controls, as well as in memory Th2 cells deficient in either of these essential transcription factors. We suggest that changes in the frequency of this repressive mark at promoters forms one—but not the only—part of the molecular FIGURE 5. Impact of STAT4 and T-bet on potential for IFN-g pro- basis for reprogramming of gene expression in memory Th2 cells 2 2 duction by memory Th2 cells. CD4+ Th2 cells of DO11.10 4get+ Tbx21 / after recall activation. + 2/2 or DO11.10 4get Stat4 mice were generated in parallel with DO11.10 Consistent with this component of our overall model, methyl- + 4get controls, flow purified, and transferred into recipients, as in Fig. 3A. ation of the coding stand of the Ifng promoter inhibited CREB1 Secreted IFN-g and IL-4 were measured at the time of recovery and after binding, as well as CREB1 trans-activated Ifng promoter activity recall activation and culture (6 d) under Th1 and Th2 conditions (A). 2 2 in primary Th1 cells. Previous work showed that Ifng promoter Results are displayed as mean (6SEM) IFN-g production by Tbx21 / or Stat42/2 cells relative to WT controls after Ag restimulation with DNA methylation inhibited mobility shift complexes of the OVA323–339 peptide. *p , 0.05, **p , 0.01, ***p , 0.001. Intracellular CREB/ATF family, and a more recent study used ChIP of the Th1 cytokine staining was performed after Ag stimulation with OVA323–339 clone AE7 to implicate ATF2 as a major factor in this scenario peptide at the time of cell recovery (B) and 1 wk after recall stimulation (29, 53). One likely factor in a difference of results is the use of and Th1 or Th2 culture, as indicated (C). Cytokine staining profiles are primary Th1 cells as opposed to a clone. The functional impact of shown for events within the donor-derived (KJ1-26+ CD4+) gate and CREB1 and other ATF transcription factors has been unclear, represent consistent observations in three biological replicates. perhaps in part because of a paucity of analyses in primary cells. CREB1 appeared to inhibit (53, 54) or increase (55) Ifng tran- Ifng promoter methylation pattern, we explored the impact of scription in T cells. However, CREB1 occupancy of the IFNG forcing expression of this transcription factor after Th2 differen- promoter enhanced transcription in human CD4+ T cells (56). tiation in its absence. Tbx212/2 Th2 cells were transduced with Furthermore, exposure to M. tuberculosis induced CREB1 binding a bicistronic retrovector (“MiT”) directing T-bet expression linked to the IFNG promoter in human CD4+ T cells, and RNAi-driven to Thy1.1, and compared with parallel transductions of the ret- depletion of CREB1 in these cells decreased in IFN-g production rovector without T-bet cDNA. After culture in Th1 conditions and (57). Thus, the balance of evidence indicates that CREB1 pro- restimulation with APCs and Ag, intracellular staining for IL-4 motes Ifng gene expression, so that asymmetrical methylation at and IFN-g (Fig. 6F) revealed that high-level T-bet expression the highly conserved CREB/ATF binding site likely contributes to forced IFN-g expression in Tbx212/2 Th2 cells. Consistent with the inhibition of IFN-g production in the developing Th2 cell. this finding, restimulation elicited substantial IFN-g production by Loss of such hemimethylation—for example—at the 253 site, a T-bet–transduced Tbx212/2 Th2 population as compared with would facilitate trans activator interactions. The Journal of Immunology 685

FIGURE 6. Impact of T-bet and STAT4 on DNA methylation at the Ifng promoter. (A) Intracellular stain for T-bet in primary Th1 cells (top panel) and recall-activated Th2-derived memory cells cultured (5 d) in Th1 (middle panel) or Th2 (lower panel) conditions. Results gated on CD4+ DO11.10 TCR+ cells are shown. (B)WT,Tbx212/2, and Stat42/2 Th2 cells were labeled with CFDA-SE and transferred into BALB/c nu/nu recipients for 2 wk. Flow results show the indicated parameter in the gate for CD4+ DO11.10 TCR+ cells. (C) In vivo BrdU incorporation into WT, Tbx212/2, and Stat42/2 Th2 cells 5 wk after their transfer into BALB/c recipients. Shown are representative ﬂow cytometry results of anti-BrdU signal in the CD4+ DO11.10 TCR+ lymphocyte gate. (D) Mean (6SEM) meCpG dinucleotides per clone are shown for Tbx212/2 and Stat42/2 Th2 effector cells, as in previous ﬁgures. WT Th2 effector results from Fig. 1 are shown for comparison. (E) Mean (6SEM) meCpG dinucleotides per sequenced clone for Tbx212/2 and Stat42/2 Th2 memory cells are shown, with WT results from Fig. 4B reproduced for comparison. (F and G) Tbx212/2 DO11.10 4get+ CD4+ cells cultured in Th2 conditions for 9 d were transduced with either MSCV-IRES-Thy1.1 (MIT) or MSCV-T-bet-IRES-Thy1.1 (MIT–T-bet), grown under Th1 conditions (1 wk), and analyzed as in Fig. 5 except that intracellular cytokines were analyzed in the Thy1.1+, CD4+, and Thy1.1neg, CD4+ cell gates (F). (G) Secreted IFN-g, measured as in Fig. 5A, was normalized to the number of Thy1.1+, CD4+, and DO11.10 TCR+ cells in the culture. Analyses of total culture production of IFN-g [MIT-T-bet, 164 (635) ng/ml versus MIT control, 21 (626) ng/ml; p = 0.02] yielded the same conclusion. Results are representative of two (A–C)or three (D–G) independent biological replicates. *p , 0.05, **p , 0.01, ***p , 0.001.

As in previous reports (26, 28, 29), our analyses indicate that differentiating conditions, absence of T-bet led to increased thymocytes and naive CD4 T cells have quite low levels of CpG methylation of the coding strand and substantially greater sym- methylation at the Ifng promoter. Thus, molecular processes that metry (i.e., increased noncoding strand meCpG). These results are part of Th2 differentiation include direction of de novo DNA suggest that, surprisingly, the low level of T-bet present early methylation to this site. In general, DNA methyltransferases after activation of naive CD4+ lymphocytes under Th2 con- (DNMTs) of the DNMT3 family (DNMT3a, b) appear to execute ditions (6, 7, 44) directly or indirectly impedes access of DNMTs the process of adding new marks. T cell activation via TCR to the Ifng promoter. Beyond this unexpected function, we infer engagement increased DNMT3a expression, and a conditional there is an additional aspect of T-bet in the molecular events loss-of-function study indicated that DNMT3a was important for underlying plasticity of Ifng gene expression. Although the repression of inappropriate cytokine genes in Th differentiation promoter methylation and its symmetry increased in T-bet–de- (58). Intriguingly, although memory cell generation and main- ficient Th2 cells, a second impediment to IFN-g production lies tenance were not analyzed, in vitro analyses of DNMT3a-de- in a block to expression of T-bet when Th2 effectors are switched ficient cells detected plasticity of cytokine production somewhat to Th1-promoting conditions without a period as memory cells. akin to what memory Th2 cells are able to execute naturally (35, This idea would be consistent with the correlative data from 36). Further analysis of this Dnmt3a0 model indicated that, switching experiments and measurements after single-cell cloning similar to the low density of meCpG marking of the Ifng pro- of human memory-phenotype CD4 T cells (51) as well as results of moter in memory Th2 cells in our analyses, the de-repressed T-bet transduction [Fig. 6F (52)]. in vitro effectors lacking DNMT3a had levels of Ifng promoter A complementary finding of our work identifies a functional methylation similar to those of naive CD4 T cells (59). DNA importance of STAT4 equal to that of T-bet in allowing memory replication naturally creates hemimethylation of CpG dyads, and Th2 cells to produce IFN-g along with Th2 cytokines after recall establishment or enforcement of symmetry is predominantly activation. The initial discovery of this flexibility indicated that executed by DNMT1 (60). Deletion of this DNMT early in T cell IL-12 was a crucial factor for the process (35, 38). 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