Basic leucine zipper transcription factor, ATF-like (BATF)regulates epigenetically and energetically effectorCD8 T-cell differentiation via Sirt1 expressionShoko Kurodaa, Maya Yamazakib, Manabu Abeb, Kenji Sakimurab, Hiroshi Takayanagic,d, and Yoshiko Iwaia,e,1,2

aDepartment of Cellular Physiology and Immunology, Medical Top Track Program, Medical Research Institute, Tokyo Medical and Dental University, Tokyo113-8510, Japan; bDepartment of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata 951-8585, Japan; Departments of cCell Signalingand eMolecular Immunology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo 113-8549, Japan; and dJapanScience and Technology Agency (JST), ERATO, Takayanagi Osteonetwork Project, Tokyo 113-8549, Japan

Edited by Arthur Weiss, University of California, San Francisco, CA, and approved August 3, 2011 (received for review April 1, 2011)

CD8 T cells play a critical role in protection against viral infections.During effector differentiation, CD8 T cells dramatically changechromatin structure and cellular metabolism, but how energyproduction increases in response to these epigenetic changes isunknown. We found that loss of basic leucine zipper transcriptionfactor, ATF-like (BATF) inhibited effector CD8 T-cell differentiation.At the late effector stage, BATF was induced by IL-12 and requiredfor IL-12–mediated histone acetylation and survival of effector Tcells. BATF, together with c-Jun, transcriptionally inhibited expres-sion of the nicotinamide adenine dinucleotide (NAD+)-dependentdeacetylase Sirt1, resulting in increased histone acetylation of theT-bet locus and increased cellular NAD+, which increased ATP pro-duction. In turn, high levels of T-bet expression and ATP produc-tion promoted effector differentiation and cell survival. Theseresults suggest that BATF promotes effector CD8 T-cell differenti-ation by regulating both epigenetic remodeling and energy me-tabolism through Sirt1 expression.

AP-1 | chromatin remodeling | glycolysis | oxidative phosphorylation

CD8 T cells are one of the most important components ofprotective immunity against viral infections, and under-

standing their development is necessary for generating effectivevaccines (1–3).During effector differentiation, CD8 T cells dramatically

change gene expression and cellular metabolism (4). In naïve Tcells, most energy is generated in mitochondria through oxidativephosphorylation. Upon stimulation, T cells massively increaseglucose uptake and produce ATP by glycolysis to support rapidproliferation. At the late effector stage, inflammatory cytokinespromote effector CD8 T-cell differentiation through chromatinremodeling (5, 6), which increases ATP consumption to sustaingene expression and subsequent protein synthesis. However, howeffector T cells increase energy production in response to theseepigenetic changes is unknown.Basic leucine zipper transcription factor, ATF-like (BATF),

a member of the AP-1 family, forms heterodimers with Jun andblocks AP-1 transactivation (7–9). Unlike other AP-1 familymembers, BATF expression is restricted to lymphoid organs, andis induced in T cells and natural killer T cells upon stimulation.Although BATF-deficient mice show impaired Th17 and follic-ular Th differentiation and antibody (Ab) production (10, 11),the function of BATF in CD8 T-cell differentiation is unknown.In this study, we examined the role of BATF in effector CD8

T-cell development by generating knock-in mice. We show thatBATF is induced by IL-12 and required for IL-12–mediated histoneacetylation and survival of effector CD8 T cells. We demonstratethat BATF with c-Jun transcriptionally inhibits expression of theNAD+-dependent deacetylase Sirt1. Furthermore, BATFpromoteseffector CD8 T-cell differentiation by regulating both epigeneticremodeling and energy metabolism through Sirt1 expression.

ResultsBATF Deficiency Inhibits Effector CD8 T-Cell Differentiation. To ex-amine the role of BATF in CD8 T-cell responses in vivo, wegenerated a reporter mouse strain by inserting GFP cDNA intothe Batf locus (Fig. S1 A and B). These Batf gfp/gfp mice lackedBATF protein expression (Fig. S1C). Consistent with previousreports (10, 11), Batf gfp/gfp mice demonstrated a slight increase insplenic B-cell numbers (Fig. S1 D and E).To induce antigen-specific effector CD8 T cells in vivo, we

targeted a model antigen, ovalbumin (OVA), to DEC-205+

dendritic cells (DCs) with CD40 ligation (12–14). By in-corporating antigen within a monoclonal Ab to DEC-205, anendocytic receptor expressed on DCs, targeted antigen is takenup by DCs and presented to T cells (12). CD40 ligation inducesDC maturation, and mature DCs provide “signal 2” (cos-timulatory signal) and “signal 3” (inflammatory cytokine signal)to T cells by expressing high levels of B7 molecules and IL-12 (5,15–17). IL-12 promotes effector CD8 T-cell differentiation,probably through chromatin remodeling by histone acetylation(5). At day 3 after priming, similar numbers of OVA-specifictetramer+ CD8 T cells were detected in the spleens of Batf+/+

and Batf gfp/gfp mice (Fig. S2A). At day 5, however, OVA-specifictetramer+ CD8 T cells increased in Batf+/+ mice but not inBatf gfp/gfp mice (Fig. S2A). At day 7, the number of OVA-specifictetramer+ CD8 T cells was much lower in Batf gfp/gfp mice than inBatf+/+ mice (Fig. 1A and Fig. S2A). Similarly, the number ofOVA-specific IFN-γ+ CD8 T cells was much lower in Batf gfp/gfp

mice than in Batf+/+ mice (Fig. 1A). Moreover, the frequency ofCD62Llow and CD127 (IL-7R)low subsets in Batf gfp/gfp tetramer+

CD8 T cells was lower than in Batf+/+ tetramer+ CD8 T cells(Fig. S2B). These results suggest that BATF deficiency inhibitseffector CD8 T-cell differentiation at the late effector phase.To demonstrate the CD8 T-cell–intrinsic function of BATF

in vivo, we transferred Batf+/+ and Batf gfp/gfp CD8 T cells intoRag1−/− mice and immunized them with anti-DEC:OVA+anti-CD40 (Fig. S2C). After 7 d of priming, the number of OVA-specific tetramer+ CD8 T cells was much lower in the micetransferred with Batf gfp/gfp CD8 T cells than in the mice trans-ferred with Batf+/+ CD8 T cells. These results suggest that

Author contributions: Y.I. designed research; S.K. performed research; M.Y., M.A., K.S.,and H.T. contributed new reagents/analytic tools; S.K. analyzed data; and Y.I. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1Present address: Department of Molecular Immunology, Tokyo Medical and Dental Uni-versity, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.

2To whom correspondence should be addressed. E-mail: iwai.mim@tmd.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1105133108/-/DCSupplemental.

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Batf gfp/gfp CD8 T cells have an intrinsic defect in their ability todifferentiate into effector CD8 T cells.

IL-12 with Costimulation Induces BATF at the Late Effector Stage.Upon T-cell stimulation, BATF was slowly up-regulated andpeaked at 72 h, whereas c-Fos and c-Jun were down-regulated(Fig. S3A). To examine the effect of signals 2 and 3 on BATFexpression, CD8 T cells were stimulated with anti-CD3 in com-bination with anti-CD28 and IL-12. CD3/CD28 coligation inducedBATF, but CD3 ligation alone did not (Fig.1B). IL-12 treatmentalone also slightly increased BATF (Fig. S3B). However, IL-12treatment together with CD3/CD28 ligation induced much higherBATF expression than CD3/CD28 ligation alone at 72 h (Fig. 1B),whereas induction was not observed at 48 h (Fig. S3C). Theseresults suggest that IL-12 with costimulation induces BATF at thelate effector stage. Unlike BATF, IL-12 treatment with CD3/CD28 ligation slightly increased c-Jun but not c-Fos (Fig. 1B).

BATF Is Required for IL-12-Induced Cell Survival and HistoneAcetylation. IL-12 is known to promote survival of effector CD8T cells (5). To examine the effect of BATF on IL-12–induced cellsurvival, Batf gfp/gfp and Batf+/+ CD8 T cells were stimulated withanti-CD3/CD28 with or without IL-12. IL-12 had no effect oncell survival and proliferation at 48 h (Fig. 2A and Fig. S4 A andB). At 72 h, however, IL-12 treatment with CD3/CD28 ligationincreased the viability of Batf+/+ CD8 T cells, but not Batf gfp/gfp

CD8 T cells compared with CD3/CD28 ligation alone (Fig. 2Aand Fig. S4B). These results suggest that BATF is required forIL-12–induced effector CD8 T-cell survival.To examine the effect of BATF on IL-12–mediated histone

acetylation, Batf gfp/gfp and Batf+/+ CD8 T cells were stimulatedwith anti-CD3/CD28 with or without IL-12, and the per-cell levelof diacetylated histone H3, a marker of open chromatin, wasexamined by intracellular staining (18) (Fig. 2B and Fig. S4C).IL-12 had no effect at 48 h, but at 72 h, IL-12 treatment withCD3/CD28 ligation increased the percentage of acetylatedH3high population in Batf+/+ CD8 T cells, but not in Batf gfp/gfp

CD8 T cells relative to CD3/CD28 ligation alone. These resultssuggest that BATF is required for IL-12–mediated histoneacetylation.

BATF Promotes Histone Acetylation of the T-Bet Locus. There is ac-cumulating evidence that IL-12 promotes effector CD8 T-celldifferentiation through Tbx21 (T-bet) expression (19, 20). Wetherefore examined the histone acetylation level of the Tbx21locus using chromatin immunoprecipitation (ChIP) analysis (Fig.3A). Batf+/+ CD8 T cells stimulated with anti-CD3/CD28+IL-12showed much higher histone H3 acetylation levels of the Tbx21locus than Batf gfp/gfp CD8 T cells. When stimulated with anti-

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Fig. 1. BATF deficiency inhibits effector CD8 T-cell differentiation. (A)Batfgfp/gfp and Batf+/+ mice were primed with anti-DEC:OVA+anti-CD40 and7 d later, OVA-specific CD8+ T cells were examined by Kb/OVA257–264 tet-ramer or peptide-stimulated intracellular IFN-γ staining. The percentage(Left) and total number (Right) of OVA-specific CD8 T cells per spleen areindicated. (B) mRNA levels of the indicated genes in CD8 T cells stimulatedwith anti-CD3 in combination with anti-CD28 and IL-12 for 72 h. Data arepresented relative to expression levels in unstimulated cells. Data are rep-resentative of three independent experiments.

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Fig. 2. BATF is required for IL-12-mediated cell survival and histone acety-lation. (A) The percentage of PIlow population in Batf gfp/gfp and Batf+/+ CD8 Tcells stimulated with anti-CD3/CD28 with and without IL-12. (B) The per-centage of acetylated H3high population in Batf gfp/gfp and Batf+/+ CD8 T cellsstimulated with anti-CD3/CD28 with and without IL-12. Data are represen-tative of three independent experiments.

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Fig. 3. BATF promotes histone acetylation of the T-bet locus. (A) Batf gfp/gfp

and Batf+/+ CD8 T cells were stimulated with anti-CD3/CD28+IL-12 for 60 hand immunoprecipitated with antiacetyl H3 or control IgG. Purified ChIP andinput DNA were analyzed by semiquantitative PCR (Left) and real-time PCR(Right). ChIP DNA level was normalized to the level of input DNA. (B) mRNAlevels of the indicated genes in Batf gfp/gfp and Batf+/+ CD8 T cells stimulatedwith anti-CD3/CD28 alone or with IL-12 for 72 h. Data are presented relativeto expression levels in unstimulated Batf+/+ CD8 T cells. Data are represen-tative of two independent experiments.

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CD3/CD28+IL-12, Batf+/+ CD8 T cells showed higher mRNAexpression of T-bet and its target genes, such as Perforin, IFN-γ,and IL-12Rβ2, than Batf gfp/gfp CD8 T cells (Fig. 3B). These resultssuggest that BATF promotes histone acetylation of the Tbx21locus, resulting in high expression of T-bet and its target genes.Consistent with the FACS data showing that BATF-deficiencyreduces cell viability (Fig. 2A), Batf gfp/gfp CD8 T cells showedlower Bcl2 expression than Batf+/+ CD8 T cells (Fig. 3B).

BATF Overexpression Increases ATP Production. To examine themechanism by which BATF promotes histone acetylation, a sta-ble BATF-expressing murine T-cell line was established usingEL4 cells (Fig. S5A), which show AP-1 family gene expressionprofiles similar to those observed in activated T cells (Fig. S5B).EL4/BATF cells showed higher levels of acetylated H3 than EL4cells, suggesting that BATF promotes histone acetylation (Fig.4A). Among the histone deacetylase family genes, Sirt1 wassignificantly down-regulated in EL4/BATF cells relative to EL4cells (Fig. 4B). Sirt1 is a NAD+-dependent histone deacetylase(21, 22). NAD+ is the cosubstrate of Sirt1 and acts as a co-enzyme in redox reactions where it continuously cycles betweenNAD+ and NADH forms to produce ATP without being con-sumed (23, 24). EL4/BATF cells showed increased levels ofcellular NAD+, but not NADH, relative to EL4 cells (Fig. 4C).EL4/BATF cells showed higher ATP levels, higher glycer-aldehydes-3-phosphate dehydrogenase (GAPDH) activity, andhigher mitochondrial membrane potential (ΔΨm) than EL4 cells(Fig. 4 D–F). In addition, EL4/BATF cells showed higher glu-cose consumption and lactate production than EL4 cells (Fig.4G). These results suggest that BATF increases cellular NAD+,which in turn increases ATP production by promoting glycolysisand mitochondrial activity. The mRNA levels of other NAD-consuming enzymes were also examined (Fig. S5C). Parp1 wasslightly lower in EL4/BATF cells than in EL4 cells.

BATF Promotes Cell Survival by Increasing NAD+ Levels. We thenexamined the effect of BATF on ΔΨm of primary T cells (Fig.5A). After 72 h of stimulation, IL-12 treatment with CD3/CD28ligation increased the percentage of TMREhigh population inBatf+/+ CD8 T cells, but not in Batf gfp/gfp CD8 T cells relative toCD3/CD28 ligation alone. These results suggest that BATF isrequired for IL-12–mediated maintenance of ΔΨm. In addition,the loss of ΔΨm and cell death were correlated (Figs. 5A and2A), suggesting that only cells that maintain ΔΨm can survive.The surviving Batf gfp/gfp and Batf+/+ CD8 T cells showed similar

ATP levels (Fig. S6A), but a significant difference in their NAD+

levels was observed (Fig. 5B). In synergy with CD3/CD28 stim-ulation, IL-12 treatment increased NAD+ levels in Batf+/+ CD8T cells but not in Batf gfp/gfp CD8 T cells, whereas no difference inNADH levels was observed. These results suggest that BATFincreases cellular NAD+ levels. Moreover, treatment withNAD+ increased the viability of Batf gfp/gfp CD8 T cells stimu-lated with anti-CD3/CD28+IL-12 but did not affect Batf+/+

CD8 T cells (Fig. 5C), suggesting that NAD+ rescues BATF-deficient CD8 T cells from cell death.NAD+-consuming enzymes such as Sirt1 and Parp1 use

NAD+ as a substrate, and their inhibitors increase NAD+ levelsin activated T cells (25). Treatment with the Sirt1 inhibitorsnicotinamide (Nam) and Sirtinol, but not with the Parp1 in-hibitor 6-(5H)-phenanthridinone (PHE), increased the viabilityof Batf gfp/gfp CD8 T cells to Batf+/+ CD8 T-cell levels (Fig. S6B),suggesting that Sirt1 inhibition promotes the survival of activatedT cells. These results are consistent with the finding that loss ofSirt1 increases T-cell activation (26). Treatment with the Sirt1inhibitors, but not with NAD+, increased the viability of Batf+/+

CD8 T cells stimulated with anti-CD3/CD28+IL-12 (Fig. S6Band Fig. 5C), suggesting that Sirt1 inhibition may also promoteeffector T-cell survival through factors other than NAD+.To examine the role of BATF in metabolic flux, Batf gfp/gfp and

Batf+/+ CD8 T cells were stimulated with anti-CD3/CD28 withor without IL-12, and the levels of glucose and lactate in culturesupernatant were examined (Fig. 5D). IL-12 had no effect onglucose or lactate levels at 48 h. At 72 h, IL-12 treatment withCD3/CD28 ligation increased glucose consumption but not lac-tate production in Batf+/+ CD8 T cells relative to CD3/CD28ligation alone, suggesting that IL-12 promotes oxidative phos-phorylation. By contrast, IL-12 treatment with CD3/CD28 liga-tion did not increase either glucose uptake or lactate productionin Batf gfp/gfp CD8 T cells relative to CD3/CD28 ligation alone.These results suggest that BATF plays an important role in IL-12–induced metabolic shift to oxidative phosphorylation.

BATF with c-Jun Transcriptionally Inhibits Sirt1 Expression. Batf gfp/gfp

and Batf+/+ CD8 T cells showed similar Sirt1 expression levels,when stimulatedwithanti-CD3/CD28+IL-12 (Fig. S6C).BecauseTcells increase ATP consumption upon antigen stimulation (4), wehypothesized that T cells expressing high levels of Sirt1 might diefrom energy collapse, whereas T cells expressing low levels of Sirt1might selectively survive. To examine the role of BATF in Sirt1expression without T-cell activation (i.e., under baseline conditions

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Fig. 4. BATF overexpressionincreases ATP production. (A)Acetylated H3 levels in EL4(filled curve) and EL4/BATF(open curve) cells. (B) Compar-ison of histone deacetylasefamily gene expression be-tween EL4 and EL4/BATF cells.(C) Cellular NAD+ and NADHlevels, (D) cellular ATP levels,and (E) GAPDH activity in EL4and EL4/BATF cells. (F) ΔΨm ofEL4 (filled curve) and EL4/BATF(open curve) cells. (G) EL4 andEL4/BATF cells were plated in24-well plates at 1 × 105 cells/mL. After 18 h of culture, glu-cose (Left) and lactate (Right)levels were measured in culturesupernatant. Data are repre-sentative of three independentexperiments. *P < 0.05; **P <0.01; NS, not significant.

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with low levels of ATP consumption),Batf gfp/gfp andBatf+/+CD8Tcells were treated with IL-12 alone (Fig. 6A). Batf gfp/gfpCD8 T cellsshowed higher Sirt1 expression than Batf+/+ CD8 T cells. MemoryT cells have demonstrated resistance to apoptosis (27). Whenstimulated with anti-CD3/CD28+IL-12, Batf gfp/gfp CD44high mem-ory CD8 T cells showed significantly higher Sirt1 expression thanBatf+/+ CD44high memory CD8 T cells (Fig. S6D). These resultssuggest that BATF inhibits Sirt1 expression.We then investigated the molecular mechanism by which BATF

regulates Sirt1 expression. The Sirt1 locus has no conserved non-coding sequences in the 59 upstream region except the promoterregion (Fig. 6B). ChIP analysis using Batf+/+ CD8 T cells stimu-lated with anti-CD3/CD28+IL-12 revealed that BATF specificallybound to the Sirt1 promoter region (Fig. 6C). To examine whetherBATF regulates Sirt1 transcription, 293T cells were transfectedwith the Sirt1 reporter construct and BATF expression vector (Fig.6D). Expression of c-Jun decreased Sirt1 transcription. Coex-pression of BATF and c-Jun, but not BATF expression alone,decreased Sirt1 transcription, suggesting that BATF inhibits Sirt1transcription in concert with c-Jun. To identify the regulatoryelements important for negative regulation by these molecules,293T cells were transfected with BATF and c-Jun expressionvectors and reporter constructs containing mutations (Fig. 6E).

Mutation of the AP-1 motif (position −186) decreased basalpromoter activity and abolished the suppressive effect of c-Jun andBATF. These results suggest that the AP-1 motif (position −186)in the Sirt1 promoter region is important for basal Sirt1 tran-scription and negative regulation by c-Jun and BATF.

DiscussionOur study has revealed a unique mechanism of BATF-mediatedeffector CD8 T-cell differentiation (Fig. S7). We found thatBATF deficiency inhibits effector CD8 T-cell differentiation. IL-12 with costimulation induces BATF in activated T cells at thelate effector phase. IL-12 is one of the inflammatory cytokinesthat can provide a third signal required for effector CD8 T-celldifferentiation. We show that BATF is required for IL-12–mediated histone acetylation and survival of effector CD8 Tcells. BATF, together with c-Jun, transcriptionally inhibits Sirt1expression, resulting in increased histone acetylation of the T-betlocus and elevated cellular NAD+ levels, which increase T-betexpression and ATP production, respectively. In turn, high levelsof T-bet expression and ATP production promote effector dif-ferentiation and cell survival. Thus, BATF promotes effectorCD8 T-cell differentiation by regulating both epigeneticremodeling and energy metabolism through Sirt1 expression.We demonstrate the molecular mechanism by which BATF

promotes histoneacetylation.We identifiedSirt1 as oneof the targetgenesofBATF.Sirt1 isa class III histonedeacetylase that isuniquelydependent on NAD+ for catalysis, and plays an important role inepigenetic gene silencing (21, 22). BATF, together with c-Jun,negatively regulated Sirt1 transcription. We found that BATF wasrecruited to the Sirt1 promoter region, and that the AP-1 motif(position −186) in the Sirt1 promoter region was important forbasalSirt1 transcription andnegative regulationbyBATFandc-Jun.We show that BATF promotes histone acetylation of the T-bet

locus, resulting in high expression of T-bet and its target genes.

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Fig. 5. BATF promotes cell survival by increasing NAD+ levels. (A) The per-centage of TMREhigh population in Batf gfp/gfp and Batf+/+ CD8 T cells stim-ulated with anti-CD3/CD28 with and without IL-12. (B) Cellular NAD+ andNADH levels in Batf gfp/gfp and Batf+/+ CD8 T cells stimulated with anti-CD3/CD28+IL-12 for 60 h. (C) Batf gfp/gfp and Batf+/+ CD8 T cells were stimulatedwith anti-CD3/CD28+IL-12 for 24 h, and NAD+ was added for 48 h. Cell via-bility was examined by PI staining. (D) Batf gfp/gfp and Batf+/+ CD8 T cells wereplated in 96-well plates at 1 × 106 cells/mL and stimulated with anti-CD3/CD28 with and without IL-12 for the indicated times. Glucose (Left) andlactate (Right) levels were measured in culture supernatant. Data are rep-resentative of two independent experiments. *P < 0.05; NS, not significant.

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Fig. 6. BATF with c-Jun negatively regulates Sirt1 transcription. (A) Sirt1mRNA expression in Batf gfp/gfp and Batf+/+ CD8 T cells stimulated with IL-12 for48 h. Data are presented relative to expression levels in unstimulated Batf+/+

CD8 T cells. (B) The 59 regulatory region of Sirt1. A VISTA plot showing thecomparison of the Sirt1 loci between mouse and either human, rat, chicken, ordog. (C) ChIP analysis of BATF binding to the Sirt1 locus. Batf gfp/gfp and Batf+/+

CD8 T cells were stimulated with anti-CD3/CD28+IL-12 for 60 h and subjectedto ChIP analysis using control and anti-BATF Abs. (D and E) Luciferase activityof 293T cells transfected with the indicated expression vectors and the Sirt1reporter construct (D) or mutation-containing reporter constructs (E). Data arerepresentative of two independent experiments. *P < 0.05.

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T-bet is the master regulator of type I effector differentiationboth in CD4 and CD8 T cells (19, 28). By increasing T-bet ex-pression, BATF promotes effector T-cell differentiation. It hasbeen reported that T-bet expression is considerably enhancedand sustained in the presence of IL-12 (19, 29). We show that IL-12 induces BATF, and that BATF in turn induces high levels ofT-bet expression through chromatin remodeling. These resultssuggest that BATF plays an important role in IL-12–induced T-bet expression.Our study also revealed the mechanism by which BATF pro-

motes effector T-cell survival. During effector differentiation,the balance between ATP production and ATP consumptiondetermines T-cell fate: death or survival. BATF inhibited Sirt1expression, resulting in increased levels of cellular NAD+. Highlevels of NAD+ drive both glycolysis and the TCA cycle, fol-lowed by oxidative phosphorylation, resulting in rapid and effi-cient ATP production. Thus, BATF promotes effector T-cellsurvival by preventing energy collapse. Importantly, BATF isinduced by IL-12 at the late effector stage. In response to IL-12–induced epigenetic changes, BATF increases ATP production tosustain gene expression and subsequent protein synthesis.Intriguingly, BATF deficiency induced high levels of Sirt1

expression in memory CD8 T cells upon stimulation, but not innaïve CD8 T cells. Unlike naïve T cells, memory T cells areresistant to apoptosis (27) and do not require a third signal todevelop effector functions (6). We speculate that naïve andmemory T cells may have different energy requirements for ef-fector differentiation and survival. In primary CD8 T-cell re-sponse, we demonstrated that BATF acted downstream of IL-12and promoted effector CD8 T-cell differentiation through T-betup-regulation. In chronic infection, Quigley et al. (30), reportedthat the immunoinhibitory receptor PD-1 caused CD8 T-cellexhaustion by up-regulating BATF, whereas Kao et al. (31)reported that PD-1 expression was repressed by T-bet. Furtherinvestigation is required to determine the role of BATF inmemory responses and the signaling pathways that induce BATF.

Although Sirt1 has been reported to modulate T-cell activa-tion (26), the administration of Sirt1 inhibitors or activators isassociated with many side effects because Sirt1 is expressed inmany cell types. Because BATF expression is restricted to acti-vated lymphocytes, BATF may be a promising therapeutic targetto control effector T-cell generation for vaccine developmentand immunotherapies.

Materials and MethodsMice. Mice were used and maintained under specific pathogen-free con-ditions according to the guidelines of the institutional animal care committeeat Tokyo Medical and Dental University. C57BL/6 (B6) mice were obtainedfrom Japan SLC. Rag1−/− mice (B6 background) were obtained from TheJackson Laboratory. Generation of the Batf gfp allele is described in SIMaterials and Methods.

Cell Preparation, Stimulation, Flow Cytometry, Real-Time PCR, ChIP Assay, andLuciferase Assay. Detailed methods and primer sequences are provided in SIMaterials and Methods and Tables S1 and S2.

Metabolic Assays. Intracellular ATP content was measured using the luciferin-luciferase method (ATP Bioluminescence Assay Kit HS II; Roche) as recom-mended by the manufacturer. NAD+ and NADH levels were measured fromwhole-cell extracts using the NAD+/NADH Quantification kit (Biovision),according to manufacturer instructions. GAPDH activity was measured usingthe Kdalert GAPDH Assay kit (Ambion). Glucose and lactate levels in cellculture supernatants were measured using Glucose Assay kit II (Biovision)and Lactate Assay kit II (Biovision), respectively.

Statistics. All data are expressed as mean ± SD (n = 3 or more). Statisticalanalysis was performed using the unpaired two-tailed Student’s t test (*P <0.05; **P < 0.01; NS, not significant).

ACKNOWLEDGMENTS. We thank Drs. R. Shinkura, K. Okamoto, and M. Oh-hora for critical comments and Ms. E. Kushiya and R. Natsume for technicalassistance. This work was supported by the Program for Improvement ofResearch Environment for Young Researchers from Special Coordination Fundsfor Promoting Science and Technology (SCF) commissioned by the Ministry ofEducation, Culture, Sports, Science and Technology (MEXT) of Japan.

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