of February 13, 2018.This information is current as

Expression Genebcl6, and tbx21, ifngRepression of

Blimp-1 Attenuates Th1 Differentiation by

L. CalameMagnusdottir, Gabriele Grunig, Rocio K. Perez and Kathryn Luisa Cimmino, Gislaine A. Martins, Jerry Liao, Erna

http://www.jimmunol.org/content/181/4/2338doi: 10.4049/jimmunol.181.4.2338

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Blimp-1 Attenuates Th1 Differentiation by Repression of ifng,tbx21, and bcl6 Gene Expression

Luisa Cimmino,* Gislaine A. Martins,† Jerry Liao,† Erna Magnusdottir,‡ Gabriele Grunig,†

Rocio K. Perez,‡ and Kathryn L. Calame1*§

T cell-specific deletion of Blimp-1 causes abnormal T cell homeostasis and function, leading to spontaneous, fatal colitis in mice.Herein we explore the role of Blimp-1 in Th1/Th2 differentiation. Blimp-1 mRNA and protein are more highly expressed in Th2cells compared with Th1 cells, and Blimp-1 attenuates IFN-� production in CD4 cells activated under nonpolarizing conditions.Although Blimp-1-deficient T cells differentiate normally to Th2 cytokines in vitro, Blimp-1 is required in vivo for normal Th2humoral responses to NP-KLH (4-hydroxy-3-nitrophenylacetyl/keyhole lymphocyte hemocyanin) immunization. Lack of Blimp-1in CD4 T cells causes increased IFN-�, T-bet, and Bcl-6 mRNA. By chromatin immunoprecipitation we show that Blimp-1 bindsdirectly to a distal regulatory region in the ifng gene and at multiple sites in tbx21 and bcl6 genes. Our data provide evidence thatBlimp-1 functions in Th2 cells to reinforce Th2 differentiation by repressing critical Th1 genes. The Journal of Immunology, 2008,181: 2338–2347.

D ifferentiation of the CD4 Th cell lineages is dictatedby the strength of antigenic stimulation, cytokine en-vironment, and the complement of transcription fac-

tors activated by these processes. T-bet and GATA-3 transcrip-tion factors are master regulators of Th1 and Th2 lineagedifferentiation, respectively; Foxp3 is critical for regulatory Tcell development and function, and, more recently, ROR�t (ret-inoid-related orphan receptor �t) has been shown to act as amaster regulator of Th17 development (1–3). Additionally,complicated regulatory pathways and feedback loops cooperatewith the master regulators to establish and maintain CD4 dif-ferentiation states.

Th polarization into Th1 and Th2 lineages can be divided intohierarchical steps of initiation, reinforcement, and maintenance(1). Initiation of lineage differentiation begins upon T cell recog-nition of Ag presented by APCs and response to the initial cyto-kine environment. Reinforcement of commitment is driven by theup-regulation of cytokine production, positive feedback loops, andrepression of genes necessary for the alternative fate. Maintenanceof the differentiated state is conferred by heritable epigeneticchanges. Transcription factors activated by TCR-signaling suchas AP-1, NF-�B, and NFAT can differentially induce bothIFN-� and IL-4 expression directly or via the induction of T-betand GATA-3, respectively (1, 4). In the case of Th1 differen-tiation, T-bet can be induced both by IFN-� or IL-12 throughthe transcription factors STAT-1 and STAT-4, respectively, todirectly reinforce expression of the IFN-� gene while simulta-neously repressing GATA-3 function (2, 5, 6). GATA-3 acti-vates expression at the Th2 cytokine locus of IL-4, IL-5, and

IL-13 genes (7) and can autoactivate its own expression bothindependently and as a result of IL-4-mediated STAT-6 signal-ing (8). How T-bet and GATA-3 compete to drive Th1 or Th2differentiation has been intensively studied and has laid thefoundation for understanding how other transcription factorsinfluence the way CD4 lineage decisions are made.

The transcriptional repressor B lymphocyte-induced maturationprotein-1 (Blimp-1)2 has recently been identified as a regulator ofT cell homeostasis and function (9, 10). Encoded by the prdm1gene, Blimp-1 is well established as a master regulator of plasmacell differentiation and maintenance (11, 12). Blimp-1 also plays arole in multiple developmental checkpoints in nonlymphoid lin-eages including germ cell formation during embryogenesis (13–15), myeloid differentiation (16), keratinocyte maturation (17), andsebocyte differentiation (18).

In the T cell lineage Blimp-1 is strongly induced upon TCRactivation (10), with IL-2 playing an important role in the induc-tion (19). In two independent studies, T cell-specific deletion ofBlimp-1 in mice caused spontaneous colitis (10) and a multiorganinflammatory disease (9). These autoimmune pathologies were at-tributed in part to altered homeostasis within the CD4 T cell lin-eage, including hyperproliferation in response to TCR stimulation,increased IL-2 and IFN-� production, and decreased IL-10 pro-duction, possibly contributing to diminished regulatory T cellfunction in vivo (9, 10).

Blimp-1 is a transcriptional repressor. Interestingly, most of theknown direct targets of Blimp-1 are also transcription regulators,explaining in part its ability to control extensive developmentalprograms (11). In B cells Blimp-1 directly represses myc, spiB,CIITA, id3, and pax5. In the epidermis it represses myc, fos, nfat5,and dusp16 (17). It has also been shown to repress p53 in sometumor cell lines (20).

*The Institute of Human Nutrition, †Department of Microbiology, ‡Department ofBiological Sciences, and §Department of Biochemistry and Molecular Biophysicis,College of Physicians and Surgeons, Columbia University, New York, NY 10032

Received for publication March 18, 2008. Accepted for publication June 9, 2008.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 Address correspondence and reprint requests to Dr. Kathryn Calame, Department ofBiochemistry and Molecular Biophysicis, College of Physicians and Surgeons, Co-lumbia University, 701 168th Street, Hammer Health Science Center 1202, Manhat-tan, NY 10032. E-mail address: KLC1@columbia.edu

2 Abbreviations used in this paper: Blimp-1, B lymphocyte-induced maturation pro-tein-1; Bcl-6, B cell lymphoma-6; ChIP, chromatin immunoprecipitation; CNS, con-served noncoding sequence; IP, immunoprecipitation; MFI, mean fluorescence inten-sity; NP-KLH, 4-hydroxy-3-nitrophenylacetyl/keyhole lymphocyte hemocyanin.

Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00

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Direct targets for Blimp-1 in T cells have not yet been identified.However, it is known that B cell lymphoma-6 (Bcl-6) mRNA iselevated in Blimp-1-deficient CD4 T cell effectors (10). Mutualrepression by Bcl-6 and Blimp-1 in B cells is important for main-taining mutually exclusive programs of germinal center B cells andplasma cells (21). In T cells, Bcl-6 repression is involved in Th1differentiation by repressing Th2 cytokine expression. It decreasesGATA-3 protein levels (22), represses IL-5 transcription (23), andcompetes for binding at STAT-6 recognition sequences in IL-4-responsive genes (24, 25).

The goal of the present study was to learn how Bcl-6 andBlimp-1 interact during Th1/Th2 development and to determinethe molecular mechanism(s) by which Blimp-1 alters expres-sion of cytokines important for Th1 vs Th2 differentiation andfunction. We show that Blimp-1 is most highly expressed inTh2 cells and that mice lacking Blimp-1 in CD4 T cells exhibitimpaired humoral Th2 responses, establishing a role forBlimp-1 in Th2 differentiation. We also show that ifng, tbx21,and bcl6 are direct targets of Blimp-1-dependent repression inCD4 T cells, providing a molecular basis for the idea thatBlimp-1 helps to oppose Th1 differentiation during Th2 lineagecommitment.

Materials and MethodsMice

Prdm1�/� or prdm1fl/fl mice (26) were crossed with mice expressing CD4-Cre� (27) or Lck-Cre� (28) to generate Cre�prdm1�/� or Cre�prdm1fl/fl

mice. Bcl-6�/� mice were described previously (29). Mice were housed inthe barrier facility of Columbia University (New York, NY). All experi-ments were approved by the Institutional Animal Care and Use Committeeof Columbia University.

CD4� T cell purification and CFSE labeling

Naive CD4� T cells were purified from the spleen and lymph nodes of4-wk-old prdm1�/� or prdm1fl/fl mice expressing CD4-Cre� or Lck-Cre� and Bcl-6�/� or Bcl-6�/� littermates by flow cell-sorting forCD4�CD62LhighCD44low cells. Staining with the division-sensitive dyeCFSE (Molecular Probes) was performed as previously described (10)before stimulation in vitro for 4 days with plate-bound anti-CD3 (5�g/ml), anti-CD28 (2.5 �g/ml), and recombinant human IL-2(25 U/ml).

FACS analysis and intracellular cytokine staining

FACS plots were gated on live, non-autofluorescent cells before anal-ysis. Cells were stained with fluorochrome-conjugated Abs specific forCD3, CD4, CD8, CD25, CD44, CD62L, Thy-1.2, IFN-� (eBioscience),IL-2, IL-4, or IL-10 (BD Pharmingen). For intracellular staining, naiveCD4 T cells were stimulated for either 4 – 6 days in the presence ofplate-bound anti-CD3 (5 �g/ml), anti-CD28 (2.5 �g/ml), and 25 U/mlof recombinant human IL-2 followed by restimulation for 6 h with PMA(20 ng/ml) and ionomycin (1 �g/ml) (Sigma-Aldrich) and monensin(GolgiStop; BD Pharmingen) to block cytokine secretion during thefinal 4 h of stimulation. Cells were fixed with 4% paraformaldehyde andwere made permeable with 0.5% saponin for intracellular staining withcytokine Abs.

4-Hydroxy-3-nitrophenylacetyl/keyhole lymphocyte hemocyanin(NP-KLH) immunizations

Mice aged 4–6 wk were injected i.p. with 100 �g of alum-precipitatedNP-KLH (Biosearch Technologies) in PBS. Imject Alum was purchasedfrom Pierce. Five to seven mice were included per genotype.

ELISA

NP-specific IgM, IgG1, and IgG2a in immunized mice for primary re-sponses were detected by ELISA as previously described using NP-BSA(Biosearch Technologies)-coated plates (68).

In vitro CD4 Th cell differentiation

For in vitro polarization, naive CD4 T cells were seeded at 1 � 106 cells/mlin 24-well plates coated with 5 �g/ml anti-CD3, 2.5 �g/ml anti-CD28 (BDPharmingen), and 25 U/ml of recombinant human IL-2 in RPMI 1640 withthe addition of murine IL-12 (10 ng/ml) and anti-IL4 (10 �g/ml) or murineIL-4 (1000 U/ml) and anti-IFN-� (10 �g/ml).

Western blot analysis

CD4 T cells were cultured for 2 wk under polarizing conditions de-scribed above, lysed in RIPA buffer (50 mM HEPES (pH 7.6), 1 mMEDTA, 0.7% sodium deoxycholate, 1% Nonidet P-40, and 0.5 MLiCl2), and 50 �g of protein from the whole-cell extract was separatedby 8% SDS-PAGE and Western blotted using mouse monoclonal anti-Blimp-1 (26), anti-GATA-3, and anti-�-tubulin Abs (Santa CruzBiotechnology).

Quantitative RT-PCR

Total RNA was isolated according to the manufacturer’s instructions byusing TRIzol reagent (Invitrogen) from in vitro-activated CD4 T cellsor sorted CD4 T cell subsets, and reverse transcription was performedusing SuperScript III (Invitrogen) following the manufacturer’s instruc-tions. For quantitative PCR analysis, the cDNA was diluted 5-fold inwater and 5 �l was amplified for 40 cycles using the ABI 7700 (AppliedBiosystems) sequence detection system with the following primers:IFN-�, forward 5�-GCTTTGCAGCTCTTCCTCAT and reverse 5�-GTCACCATCCTTTTGCCAGT; T-bet, forward 5�-GGTGTCTGGGAAGCTGAGAG and reverse 5�-CCACATCCACAAACATCCTG; Blimp-1,forward 5�-GACGGGGGTACTTCTGTTCA and reverse 5�-GGCATTCTTGGGAACTGTGT; Bcl-6, forward 5�-CTGCAGATGGAGCATGTTGT and reverse 5�-CACCCGGGAGTATTTCTCAG; and 18S, for-ward 5�-TCAAGAACGAAAGTCGGAGG and reverse 5�-GGACATCTAAGGGCATCACA.

Chromatin immunoprecipitation (ChIP) and quantitative PCR

CD4 T cells were grown for 6 days in culture and restimulated for 4 hwith 20 ng/ml PMA and 1 �g/ml ionomycin before harvesting. ChIPassays were performed as previously described (30). Briefly, 30 – 40 �106 cells were used per ChIP after crosslinking with 1% formaldehydeat room temperature for 10 min, quenching with 125 mM glycine, nu-clear lysis and chromatin fragmentation by sonication at 4°C. Preim-mune serum bled from rabbits before immunization with Blimp-1 an-tigenic peptide was used as a control for anti-Blimp-1 rabbit polyclonalserum generated in the laboratory (31). Abs were bound to protein ADynabeads (Dynal), immunoprecipitations were performed overnight at4°C, and washed in RIPA buffer. Protein-DNA crosslinks were reversedat 65°C overnight, and precipitated DNA was treated with proteinase Kand RNase before phenol chloroform extraction and ethanolprecipitation.

Analysis of sequence homology and putative Blimp-1 binding siteswas performed using the ECR browser (http://ecrbrowser.dcode.org/)and rVista 2.0. Quantitative PCR analysis was performed with FastStartSYBR Green Master mix (Roche) using the Stratagene MX3000 real-time PCR system. Relative fold-enrichment was determined by calcu-lating the immunoprecipitation (IP) efficiency (ratio of the amount ofimmunoprecipitated DNA to that of the input sample) of anti-Blimp-1normalized to control antiserum. The primer sequences used are shownin Table I.

ResultsBlimp-1 decreases the number of IFN-�-producing CD4� cellsand the amount of IFN-� made per cell

Since our previous work showed that murine CD4� T cells lackingBlimp-1 had elevated IFN-� production (10) and because IFN-� isa key component in Th1 differentiation, we wanted to explore thepotential role of Blimp-1 in the commitment and/or establishmentof Th1 vs Th2 cells. First, we studied the role of Blimp-1 in IFN-�production in more detail. Naive CD4 T cells (CD44�CD62L�)were purified from the spleen and lymph nodes of 4-wk-old BlimpCKO mice ( prdm1flox/flox with either Lck-Cre or CD4-Cre) andanalyzed for intracellular IFN-� following stimulation with

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anti-CD3, anti-CD28, and IL-2 for 6 days and subsequent restimu-lation. As shown in Fig. 1, A and B, the fraction of cells producingIFN-� was 2-fold higher in Blimp-1 CKO CD4 T cells comparedwith controls, and the IFN-�-producing cells from the CKOs alsoshowed a 2-fold higher mean fluorescence intensity (MFI), indi-cating elevated IFN-� production on a per cell basis. The fractionof cells expressing IFN-� increases in frequency with successivecell cycles (32); therefore, a previously observed proliferative ad-vantage in Blimp-1 CKO CD4 T cells (10) might explain increasedIFN-� production during activation in culture. To determine therelationship between IFN-� production and cell division inBlimp-1 CKO cells, naive CD4 T cells were stained with the di-vision-sensitive dye CFSE before culture for 4 days under varioustypes of stimulation, comparing IFN-� production as a functionof proliferation. Consistent with our previous findings, Blimp-1CKO CD4 T cells are more proliferative than wild-type controlcells under weak stimulatory conditions (10). Stimulation for 4days with anti-CD3 alone gave rise to 6-fold more IFN-�-pro-duction in the Blimp CKO cultures compared with a 2-foldincrease with anti-CD3 and anti-CD28 (Fig. 1C). However,upon stimulation with anti-CD3, anti-CD28, and IL-2, despiteequivalent rates of proliferation, the frequency of IFN-�-pro-ducing cells was 1.5-fold higher in the CKO in total culture(Fig. 1C) and, with one exception, at each round of division(Fig. 1D). These results show a cell cycle-independent effect ofBlimp-1 on the number of cells producing IFN-�. Additionally,the increased IFN-� MFI per cell in the CKO CD4 T cellsindicates that each CKO cell makes more IFN-�. Thus, we con-clude that Blimp-1 inhibits both the fraction of cells makingIFN-� and the amount of IFN-� made per cell.

Blimp-1 is more highly expressed in Th2 cells

Increased IFN-� production by Blimp-1-deficient CD4 T cellsstimulated under neutral conditions indicates that Blimp-1 maynormally play a role in Th2 differentiation. The expression patternof Blimp-1 under Th1 and Th2 polarizing conditions was thereforeanalyzed in wild-type CD4 T cells to determine whether the ex-pression pattern of Blimp-1 was consistent with a role in Th2differentiation.

Blimp-1 steady-state mRNA, measured by quantitative RT-PCR, was 5-fold higher in Th2 cells compared with Th1 cells

after 6 days in culture (Fig. 2A). Furthermore, Blimp-1 protein,analyzed by Western blotting of CD4 T cells cultured for 2 wkunder Th1 and Th2 polarizing conditions, showed moreBlimp-1 in Th2 cells compared with Th1 cells (Fig. 2B).

Blimp-1 mRNA was also analyzed in CD4 T cells followingstimulation with different cytokines important for Th cell differ-entiation. Naive CD4 T cells were cultured for 6 days either withanti-CD3 and anti-CD28 stimulation alone or with added IL-2,IL-4, IL-12, or IFN-�. Stimulation with anti-CD3, anti-CD28, andexogenous IL-2 increased Blimp-1 mRNA modestly comparedwith TCR stimulation alone. IL-4 induced Blimp-1 mRNA max-imally under these culture conditions (Fig. 2C), while IFN-� ac-tually inhibited induction of Blimp-1 mRNA (Fig. 2C). Thus,Blimp-1 mRNA is induced by IL-4 and repressed by IFN-� sig-naling, consistent with highest expression in Th2 cells. These dataare consistent with the idea that Blimp-1 is induced in Th2 cellsand plays a role in Th2 commitment or function.

Blimp-1 is required for a normal Th2 humoral response in vivo,but not for Th2 differentiation in vitro

To test if Blimp-1 deficiency alters differentiation of Th cells invitro, naive CD4 T cells were stimulated for 6 days with plate-bound anti-CD3, anti-CD28, and IL-2 with the addition of ei-ther IL-12 and anti-IL-4 or IL-4 and anti-IFN-� to promote Th1or Th2 polarization, respectively. There was no significant dif-ference in the fraction of IFN-�-producing cells between thewild-type and CKO Th1 cultures, and, similarly, under Th2culture conditions, equivalent fractions of IL-4-producing cellswere present (Fig. 3A). However, there was a 3–5-fold decreasein the number of IL-10-producing cells upon Th2 differentiationin Blimp CKO cultures (Fig. 3B), consistent with the decreasedIL-10 production observed in Blimp-1 CKO CD4 T cell effec-tors ex vivo and under neutral conditions of stimulation in vitro(10). Although the fraction of IFN-�- or IL-4-producing cellswas not significantly different between the wild-type and CKOTh cell cultures (Fig. 3C), the average MFI for IFN-� was 1.6-fold higher for Blimp-1 CKO cells under Th1 conditions, show-ing that these cells produce more IFN-� (supplementary Fig.

Table I. Sequences used

Site Location (bp) Forward Primer Reverse Primer

ifng1 5� �31,884 CCTGGCCCTCTCCTGTAAG GGGGCTATAGGAATGGCATA2 5� �27,497 GGCTAGACCAGCCAATAAGC TCTGGCCCTTCCTAGCTGTA3 5� �21,572 CACCTGGGGTGAAAAGAAAT GTGAATCCCCAGAGAAGCAG4 5� 5,592 GAACGGGTGGAGGAGGTAA GCCAAGGTGTGATTGCATTA5 3� 7,480 TGGGTTTTACCTATCATCAT GGGCACAGACTAATCTCAAC6 3� 14,895 GAAAGTTGGCGAGAAACAGG TCTGAATCCCTTGCTGACCT7 3� 16,011 TCCACACACCCCTCCATT CCAAGGACTTCCTCTCCTCA

tbx211 5� �8,673 GGTGGGTGAAGGAAGATCTA GACTGGGGACAAGAAGATGC2 5� �627 CTGAACACTGACAGCCAAGC AAGAAGATTGCACCCAAAGC3 Intron 1 1,637 CTCCACAGCCACCTACACTG GGGACTAGCGTAAGCCACTG4 Intron 1 10,872 TATGGAGGTGGTGGTTGTCA CCAGGAAAAGCAGAAGTTGG5 3� 3,846 TGCCAGCCATACCTGTTAAG GAGAGAGAGAGAGAGAGAGG6 3� 4,378 CCTCATCTCGGCTCCTTTC CTATGGGCGATGTCAAGCTA

bcl61 5� 4,778 CAGCCACCCTGAGTTTACAA CGTTCCAGCACTGTTTTGAA2 Intron 1 2,117 CTGGCTTCCTCTGCAGTTC CTGTCTCTGGATGGATCACG3 Intron 1 5,648 TGAGGCAGGACACGTAACAG GCGGGAATGACACAGAGATA4 3� UTR838 TTGTCTAAAATGCCTCCGTGT CATTTCTCCTTCTGCAGCTTT5 3� 4,993 ACAGCCAGGAGAGCTTTACCG TGTTTGCTTTCTGGCATTTG

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1A).3 Thus, we conclude that Blimp-1 is not required for Th1 orTh2 cell polarization in vitro, although in its absence, expres-sion of IL-10 is abnormal.

Polarization in vitro provides saturating amounts of polarizingcytokines and is unlikely to reflect the more subtle signals thatdetermine Th1/Th2 polarization in vivo. To determine whetherBlimp-1 is important for a Th2 response in vivo, we immunizedmice with NP-KLH in alum, which is known to cause a Th2-dependent response primarily of IgG1 (33). Sera from immunizedmice were measured over the course of 21 days for NP-specificIgM, IgG1, and IgG2a by ELISA. IgG1 was decreased �2-fold inCKO mice at each time point, indicating that the Th2 responseswere impaired (Fig. 4B). Interestingly, CKO mice also exhibited2–3-fold higher titers of NP-specific IgM compared with controlmice (Fig. 4A). The IgG2a response of the CKO mice was similarto controls late in the response, but 1.5–2-fold higher at days 12and 18 (Fig. 4C). It may be that the IgM and IgG2a responses are

elevated because the T cell cytokine milieu in the germinal centeris not normal, but this hypothesis requires further study. These datashow that Blimp-1 is required for a normal Th2 humoral responseto NP-KLH in alum.

Blimp-1 represses IFN-� and T-bet mRNA expression inactivated CD4 T cells

We wanted to explore the molecular mechanisms responsible forthe role of Blimp-1 in Th2 differentiation. The data showing ele-vated IFN-� production in CKO CD4� cells suggested thatBlimp-1 might repress IFN-� or other genes expressed in Th1cells. In CD4� cells, IFN-� transcription is activated by TCR sig-naling (34, 35), and subsequent IFN-� secretion initiates moreIFN-� production via a positive feedback loop involving the IFN-�R-STAT-1 pathway. Activated STAT-1 also induces the tran-scription of tbx21, encoding T-bet, and T-bet further induces ifngtranscription and interferes with GATA-3 function, thus reinforc-ing IFN-� production and Th1 polarization (5, 36, 37). To inves-tigate the possible role of Blimp-1 in expression of IFN-� and3 The online version of this article contains supplemental material.

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FIGURE 1. Blimp-deficient CD4 T cellsproduce higher amounts of IFN-� on a percell basis. A, Representative FACS plots ofnaive CD4 T cells cultured for 6 days withanti-CD3, anti-CD28, and IL-2 and restim-ulated for 6 h with PMA and ionomycin fol-lowed by intracellular cytokine staining forIFN-�. B, Average percentage and MFI ofIFN-�-producing cells in wild-type (openbar) and CKO (gray bar) CD4 Th cell cul-tures (n � 6). C, IFN-� production and pro-liferation measured by CFSE staining ofwild-type and CKO CD4 T cells cultured for4 days with combinations of anti-CD3, anti-CD28, and IL-2. Cells were restimulatedwith PMA and ionomycin for 6 h before har-vesting for intracellular cytokine staining ofIFN-�. Representative histograms for prolif-eration between wild-type (gray) and CKO(open) T cells are overlaid and representa-tive dot plots are shown with the total fre-quency and MFI of IFN-�-producing cellsper condition. D, Percentage of IFN-�-pro-ducing cells per cell division in WT (openbar) and CKO (gray bar) CD4 T cells stim-ulated with anti-CD3, anti-CD28, and IL-2.Data are representative of two experiments.

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T-bet mRNAs, we employed quantitative RT-PCR. During acti-vation in vitro under nonpolarizing conditions, total mRNA wascollected at various times and analyzed for IFN-� and T-bet

steady-state mRNA in wild-type and CKO CD4 T cells. IFN-�mRNA was increased 2–3-fold in Blimp-1-deficient CD4 T cellscompared with wild-type cells, in naive cells, and in activated cellsat each time during the 6 day stimulation and upon restimulationwith PMA and ionomycin at days 3 and 6 of culture (Fig. 5A).T-bet mRNA was also elevated in the CKO, most noticeably at day2, with 5–6-fold increased T-bet expression in CKO CD4 T cellscompared with wild-type cells and upon restimulation with PMAand ionomycin after 3 days in culture (Fig. 5B). Further evidencefor a requirement for Blimp-1 to repress IFN-� expression wasobtained by quantitative RT-PCR in which the levels of IFN-�mRNA from CKO Th1 and Th2 cells were observed to be 5-foldand 3-fold increased, respectively, as compared with wild-typecells (supplementary Fig. 1B). Collectively, these data showthat Blimp-1 normally represses expression of IFN-� and T-betmRNA. Under neutral cytokine conditions, acute stimulation ofnaive CD4 T cells for 24 h with PMA and ionomycin alsoinduced 8-fold higher levels of IFN-� and 15-fold higher levelsof T-bet mRNA in Blimp-1 CKO cells (Fig. 5C). Since stimu-lation by PMA and ionomycin bypasses early TCR signalingevents, this provides evidence that Blimp-1 regulation occursdownstream of PKC activation and Ca2� mobilization and sug-gests that Blimp-1 might directly repress IFN-� and T-betexpression.

Blimp-1 and Bcl-6 repress one another in CD4 T cells

Bcl-6 is known to play a role in repressing Th2 differentiation (22,29, 38). Furthermore, in B cells, Bcl-6 and Blimp-1 repress oneanother, resulting in mutually exclusive transcriptional programsthat maintain cells either in a germinal center state, where Bcl-6 isexpressed, or in a plasma cell state, where Blimp-1 is present (21).In CD4� T cells, Bcl-6 is induced by activated STAT-1 (39) andBcl-6 mRNA expression is elevated in Blimp-1 CKO CD4 T celleffectors purified ex vivo (10), suggesting that Blimp-1 also re-presses Bcl-6 in T cells, which, we hypothesized, might also beimportant for Th1 vs Th2 differentiation.

We measured Bcl-6 mRNA by quantitative RT-PCR in cellsstimulated in nonpolarizing conditions for 6 days. Lack of Blimp-1

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FIGURE 2. Blimp-1 is more highly expressed in Th2 cells andBlimp-1 mRNA expression is differentially regulated by polarizing cy-tokines. A, Blimp-1 steady-state mRNA levels in CD4 T cells stimu-lated for 6 days under Th1 and Th2 culture conditions (n � 5 withSEM). B, Western blot analysis of Blimp-1 and GATA-3 protein levelsnormalized to �-tubulin in CD4 T cells cultured for 2 wk under Th1 andTh2 conditions. C, Blimp-1 steady-state mRNA levels in CD4 T cellsstimulated for 6 days with plate-bound anti-CD3 and anti-CD28 in thepresence of IL-2, IL-4, IL-12, and IFN-� cytokines. Data representaverages and SEM of three experiments.

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had no effect on the early induction of Bcl-6 mRNA; however, byday 6, without restimulation, Bcl-6 mRNA was 2-fold higher inCKO cells and 2–3-fold increased in CKOs upon restimulation atday 3 and day 6 compared with wild-type controls (Fig. 6A). Thisprovides further evidence that Blimp-1 represses Bcl-6 expressionin CD4 T cells.

We also wanted to determine whether Bcl-6 represses Blimp-1mRNA in T cells as it does in B cells. Ex vivo naive, memory, andeffector CD4 T cell populations were sort-purified from 4-wk-oldbcl6�/� mice, and Blimp-1 mRNA was determined by quantitativeRT-PCR. As previously shown by our laboratory, Bcl-6 mRNA is3-fold higher in Blimp-1 CKO effector CD4 T cells (Fig. 6B).Additionally, we found that Blimp-1 mRNA was �2.5- fold higherin Bcl-6 KO CD4 effectors (Fig. 6C).

Under polarizing culture conditions, Blimp-1 levels are highestin Th2 cells (Fig. 2, A and B). Under the same conditions we findthat Bcl-6 levels are 3-fold higher in Th1 compared with Th2 cells(Fig. 6D). The levels of Bcl-6 mRNA were also analyzed in CD4T cells following stimulation with cytokines important for Th cell

differentiation. Naive CD4 T cells were cultured for 6 days eitherwith anti-CD3 and anti-CD28 alone or in addition to IL-2, IL-4,IL-12, or IFN-�. Stimulation with anti-CD3 and anti-CD28 in thepresence of IL-2, IL-4, or IL-12 caused no change in the level ofsteady-state Bcl-6 mRNA (Fig. 6E). However, IFN-� was able toincrease the level of Bcl-6 mRNA �7-fold higher than anti-CD3and anti-CD28 stimulation alone (Fig. 6E). These data show thatBlimp-1 and Bcl-6 mutually repress each other in CD4 T cells.Bcl-6 mRNA is more highly expressed in Th1 cells compared withTh2 cells and high levels are maintained upon stimulation in thepresence of IFN-�.

Blimp-1 binds directly to the ifng, tbx21, and bcl6 genes

To determine whether Blimp-1 directly represses ifng, tbx21, orbcl6 transcription, potential Blimp-1 binding sites in these geneswere identified using the Vista genomic analysis tool (40, 41) andtested for Blimp-1 binding in vivo by ChIP (Fig. 7). CandidateBlimp-1 binding sites were chosen based on their similarity topreviously identified sites in other Blimp-1 targets and to the ex-perimentally determined consensus binding site (42). For the ChIPexperiments, binding to an irrelevant site on the gene snail3 wasused as a negative control, as well as an irrelevant site within eachtarget gene. Known targets of Blimp-1 in B cells, myc and id3,

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FIGURE 5. IFN-� and T-bet mRNA expression levels are elevated inBlimp-1-deficient CD4 T cells. Quantitative PCR analysis of steady-statemRNA levels of (A) IFN-� and (B) T-bet from flow cell-sorted wild-type(open bar) and CKO (gray bar) naive CD4 T cells stimulated for 6 dayswith plate-bound anti-CD3(5 �g/ml), anti-CD28 (2.5 �g/ml), and IL-2 (20U/ml) and restimulated (RS) at days 3 and 6 with PMA (20 ng/ml) andionomycin (1 �g/ml) for 4 h. Data represent averages and SEM of fourexperiments and were analyzed using a Students paired t test with p values�0.05 (�) or �0.01 (��) as indicated. C, Steady-state mRNA levels ofIFN-� and T-bet in CD4 T cells activated under nonpolarizing conditionsfor 24 h with PMA and ionomycin (Iono). Data are representative of twoexperiments.

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were also tested but only the myc gene was bound by Blimp-1 inCD4 T cells (Fig. 7).

Seven candidate sites were tested in the ifng gene: sites 3, 4,6, and 7 are conserved between mice and humans, whereas sites1 and 2 are not conserved and site 5 contains an irrelevantsequence (Fig. 8A). None of the sites tested were found to lo-calize to previously described proximal regulatory regions ofthe ifng gene (43, 44). However, sites 3– 6, excluding the irrel-evant site 5, fall within or near distal conserved noncoding se-quence (CNS) regions 22 kb and 5 kb upstream of, and 17 kbdownstream of, the ifng gene, which have been shown to reg-ulate the expression of IFN-� in T cells (45). Blimp-1 bindingsite 3 is within the region of the IFN-� locus known as CNS-22that was recently shown to be essential for IFN-� expression inT cells (46) (Fig. 7A).

In tbx21, five potential Blimp-1 binding sites and one irrel-evant site were tested by ChIP. Of those tested, sites 2 and 3 areconserved between mice and humans, whereas sites 4 – 6 are not(Fig. 8B). Blimp-1 binding on the tbx21 gene was most highlyenriched for the conserved site 2, �600 bp upstream of thetranscriptional start site, and sites 3 and 5, �1.5 kb and 11 kbdownstream of exon 1 within the first intron of the tbx21 gene(Fig. 7A). These data, in conjunction with the expression dataabove, demonstrate that ifng and tbx21 are direct targets ofBlimp-1 repression in CD4 T cells.

Four candidate sites in bcl6 were analyzed, including an ir-relevant site 5 kb downstream of the last bcl6 exon (site 5), usedas a negative control (Fig. 8C), along with an irrelevant sitefrom snail3. Blimp-1 bound reproducibly to the bcl6 gene atsite 1, located 5 kb upstream of the first exon (Fig. 7B). Thus,Blimp-1 directly represses bcl6 in T cells.

It has been previously shown that Blimp-1 can repress Bcl-6when ectopically expressed in activated B cells (21), but directrepression of bcl6 by Blimp-1 has not been demonstrated in Bcells. To determine whether bcl6 was also a direct target ofBlimp-1 repression in B cells, ChIP was performed for theBlimp-1 binding sites identified in T cells using P3X plasma-cytoma cells. In these B cells, both myc and id3 were repro-ducibly bound by endogenous Blimp-1 at their known bindingsites, as was site 1 on the bcl6 gene (Fig. 7C). These data showthat bcl6 is a direct target of Blimp-1 repression in both CD4 Tcells and in B cells.

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FIGURE 6. Blimp-1 and Bcl-6 mRNA expression is inversely regulatedin CD4 T cells. Steady-state levels of Bcl-6 mRNA were measured byquantitative RT-PCR in (A) CD62LhighCD44low flow cell-sorted wild-type(open bar) and CKO (gray bar) naive CD4 T cells stimulated for 6 dayswith plate-bound anti-CD3, anti-CD28, and IL-2 and restimulated (RS) atdays 3 and 6 with PMA and ionomycin for 4 h. Data represent averages andSEM of four experiments. B, Steady-state levels of Bcl-6 mRNA from flowcell-sorted peripheral naive (N), memory (M), and effector (E) CD4 T cellsfrom Blimp-1 wild-type (open bar) and CKO (gray bar) mice and (C)Blimp-1 mRNA levels from Bcl-6�/� (open bar) and Bcl-6�/� (gray bar)mice. Data represent averages and SEM of four experiments and wereanalyzed using a Student’s paired t test with p values �0.05 (�) indicated.D, Bcl-6 steady-state mRNA levels in CD4 T cells stimulated for 6 daysunder Th1 and Th2 culture conditions (n � 5 with SEM) and with (E)plate-bound anti-CD3 and anti-CD28 in the presence of IL-2, IL-4, IL-12,and IFN-� cytokines. Data represent averages and SEM of threeexperiments.

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FIGURE 7. Blimp-1 binds directly to regions in the ifng, tbx21, andbcl6 genes in CD4 T cells. Naive CD4 T cells for ChIP were stimulated for6 days in vitro with anti-CD3, anti-CD28, and IL-2 followed by restimu-lation for 4 h with PMA/ionomycin before crosslinking. Quantitative PCRanalysis of immunoprecipitated DNA is displayed as relative fold-enrich-ment compared with control Abs for sites investigated within the (A) ifngand tbx21 genes and bcl6 genomic loci in (B) CD4 T cells and (C) P3Xplasmacytoma cells. Irrelevant sites in the snail3 gene and within eachtarget gene serve as negative controls and are indicated by the open bar.Previously identified Blimp-1 binding sites within myc, as well as id3genes, were used as a positive control. Data represent averages and SEMof four experiments.

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DiscussionWe have shown that the transcriptional repressor Blimp-1 is mosthighly expressed in Th2 cells and directly represses transcriptionof three genes that encode proteins with critical roles in Th1 dif-ferentiation: IFN-�, T-bet, and Bcl-6. Furthermore, the Th2 IgG1response to immunization with NP-KLH in alum is defective inmice lacking Blimp-1 in their T cells.

Blimp-1 repression of ifng

IFN-� is a critical cytokine for commitment to the Th1 lineage andfor establishment of feedback loops that reinforce Th1 lineagecommitment. Many activators but few repressors are known forifng, which is induced by both TCR stimulation and cytokinesIL-12 and IL-18. Th1 selective expression of IFN-� is regulated byT-bet binding in both the proximal promoter (43, 44) and in distalregulatory elements of the ifng gene (46–48). Many activators ofthe ifng gene have been identified in the proximal promoter region,including STATs, NFAT, NF-�B, AP-1, Oct-1, and T-bet (43, 44,49–51), but only Yin-Yang 1 (YY-1) has been shown to directlyrepress ifng gene expression (52). Later, however, this was foundto result in transactivation of IFN-� expression in partnership withnearby NFAT sites (49). Foxo3a and Foxj1 can indirectly repressIFN-� expression by directly repressing NF-�B (53, 54). Thus,Blimp-1 is one of the first direct repressors of ifng to be iden-tified. Interestingly, the site recognized by Blimp-1, �22 kbupstream of the ifng transcription start site, is in a conservedregion that was shown to have histone 3 K4 methylation in Th1cells but histone 3 K27 methylation in Th2 cells (55). Further-more, this region has been shown to be required for IFN-�expression in CD4 T cells, CD8 T cells, and NK cells (46),suggesting that Blimp-1 regulation of Th1 differentiation couldextend beyond the setting of Th cells.

Blimp-1 also increases IL-10 production, evidenced by lowerIL-10 production in CKO CD4 T cells (Fig. 3) (10). However, thisprobably involves regulatory as well as nonregulatory CD4 cellsand is predicted to proceed by an indirect mechanism, sinceBlimp-1 is a transcriptional repressor in all situations studied thusfar. Although we have not explored the mechanism of Blimp-1-dependent IL-10 regulation in this study, it is likely that in additionto direct repression by Blimp-1, increased IL-10 is another mech-anism by which Blimp-1 attenuates IFN-� production.

Blimp-1-dependent repression of tbx21, encoding T-bet

T-bet is the master transcription factor initiating and maintainingTh1 differentiation by driving high level of IFN-� expression. Inaddition to ifng, T-bet also induces additional genes in the Th1program, including those encoding IL-12R�2, HLX, and T-betitself. When ectopically expressed in Th2 cells, T-bet reprogramsthem to a Th1 phenotype (56) both by inducing a Th1 program andby interfering with the Th2 program. Interference with the Th2program occurs via a tyrosine phosphorylated form of T-bet thatinterferes with the ability of the master Th2 transcription factorGATA-3 to bind its cognate sites (57). Thus, by repressing tbx21and ifng, Blimp-1 attenuates expression of both the master Th1transcription factor and the critical Th1 cytokine.

Both STAT-1 and NF-�B can induce tbx21 expression; how-ever, studies in T cells have not yet confirmed their cis-activatingpotential (6, 54, 58). Notch1 signaling activated by DCs can con-vert a basal repressor of T-bet, the recombination signal bindingprotein Jk (RBJk), into an activator that will drive T-bet expression(59, 60). Additionally, the forkhead transcription factor Foxj1 canindirectly repress T-bet induction and Th1 differentiation by re-pressing NF-�B activation (54). While GATA-3 can reverse Th1differentiation and block T-bet activity, it also does so indirectly bydownregulating STAT-4 (61). We have shown that Blimp-1 bindsdirectly to a region containing a conserved consensus binding sitein the proximal promoter of the tbx21 gene and to regions con-taining conserved and nonconserved consensus sites in the firstintron. Binding of Blimp-1 to the tbx21 gene provides a mecha-nism by which this repressor can directly regulate Th1/Th2 polar-ization; however, future studies using retroviral transduction couldprovide further insight into the role of Blimp-1 in this process.

Mutual repression by Blimp-1 and Bcl-6

Data in this paper, in conjunction with previous reports (21, 31),provide strong evidence that Blimp-1 and Bcl-6 mutually repressone another by direct repression in both T and B cells. In B cells,this is important for mutually exclusive establishment of germinalcenter vs plasmacytic states of B cell differentiation (62, 63). Al-though it was known that Bcl-6 directly repressed prdm1 in B cells(31, 64), the data presented herein provide the first evidence thatBlimp-1 directly represses bcl6 in B cells.

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FIGURE 8. Schematic representa-tion of sites investigated within the (A)ifng, (B) tbx21, and (C) bcl6 murinegenomic loci. Blimp-1 recognition se-quences are shown for the site mostgreatly enriched by ChIP within eachgene. Conserved noncoding sequences(CNS) are indicated by an asterisk andlocations are shown in bases.

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In CD4 T cells, our data support the notion that Blimp-1-de-pendent repression of bcl6 is important for mutually exclusive Th1vs Th2 commitment. Bcl-6 is more highly expressed in Th1 cellsthan in Th2 cells (65, 66) and plays a role in Th cells of attenuatingthe expression or function of Th2 genes. Bcl-6-deficient mice haveeosinophilic inflammation caused by elevated Th2 cytokines (29),and Bcl-6 competes with STAT-6 for binding to Th2 genes (24,25). Bcl-6 also functions by other mechanisms in Th1 cells. Itbinds to a silencer region in il5 and directly represses its transcrip-tion (23). It induces degradation of GATA-3 by a poorly under-stood mechanism, leading to decreased expression of IL-10 (22).Finally, it directly represses prdm1, and we show in this work thatBlimp-1 represses Th1 genes ifng and tbx21. Thus, repression ofbcl6 by Blimp-1 would block the ability of Bcl-6 to repress Th2gene expression. Reciprocally, repression of prdm1 by Bcl-6blocks the ability of Blimp-1 to repress Th1 gene expression. Thisnegative feedback loop between Blimp-1 and Bcl-6 in CD4 T cellsis reminiscent in some ways of the feedback loop between T-betand GATA-3, which also reinforces Th1 or Th2 differentiationstates. Further studies using retroviral transduction of Blimp-1 orBcl-6 in CD4 T cells will help to determine the extent to which Thlineage choice depends on direct mutual repression by these an-tagonistic transcription factors.

Importance of Blimp-1 in Th2 differentiation

We and others have shown that IL-4 signaling increases Blimp-1mRNA expression (Fig. 2) (19). These data, along with the findingthat Blimp-1 mRNA is highest in Th2 cells (Fig. 2A), support aparticular role for Blimp-1 in Th2 cells.

Blimp-1-dependent repression of ifng, tbx21, and bcl6 provide amolecular mechanism for how Blimp-1 may function during Th2polarization to repress Th1 genes. Indeed, an important role forBlimp-1 in Th2 responses is supported by our demonstration thatthe response of CKO mice to immunization with NP-KLH in alum,an adjuvant causing a predominantly IgG1 Th2 response (33), isdefective, showing reduced IgG1 and increased IgM. It is alsoconsistent with the acute colitis the CKO mice develop, becausecolitis is usually an aberrant Th1 response. However, since CKOmice show abnormally activated T cells as early as 4–5 wk it ispossible that they were already predisposed to a Th1-like response,having an environment that disfavors Th2 responses.

However, Blimp-1 is not strictly required for Th2 differentiationbecause Blimp-1-deficient CD4 cells develop normally into IL-4-producing cells when stimulated with strongly polarizing condi-tions (IL-4 and anti-IFN-�) in vitro (Fig. 3A). Rather, we think thatthe role of Blimp-1 is to attenuate Th1 responses, thereby enhanc-ing the commitment and establishment of Th2 responses in vivo. Inthis way its role is similar to that of Bcl-6. Bcl-6 KO CD4 T cells(67) polarize normally in vitro in contrast to their spontaneouscytokine skewing toward Th2 cell differentiation in vivo. In vitropolarization bypasses the effect of imbalances in endogenous cy-tokine production; therefore, unlike the obligate requirement forSTAT-1 and STAT-6 or T-bet and GATA-3 to potentiate cytokinesignaling, Blimp-1 and Bcl-6 are repressors that attenuate Th dif-ferentiation during differentiation in vivo when signals for Th1 vsTh2 polarization are likely to be more subtle.

In summary, we have shown that Blimp-1 attenuates Th1 dif-ferentiation by directly repressing transcription of the signaturecytokine, IFN-�, and the master regulator of Th1 cell development,T-bet. Additionally, Blimp-1 also represses Bcl-6, thus expandingfrom B cells to T cells the settings in which these two transcrip-tional repressors can antagonize one another to promote alternativecell fates.

AcknowledgmentsWe greatly appreciate the work of Kristie Gordon and members of theColumbia FACS Facility for guidance and expertise in flow cytometry. Wethank members of the Calame laboratory for helpful advice.

DisclosuresThe authors have no financial conflicts of interest.

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