Activating 3 Is a Positive Regulator of Human IFNG Expression

This information is current as Sanna Filén, Emmi Ylikoski, Subhash Tripathi, Anne West, of October 1, 2021. Mari Björkman, Joel Nyström, Helena Ahlfors, Eleanor Coffey, Kanury V. S. Rao, Omid Rasool and Riitta Lahesmaa J Immunol 2010; 184:4990-4999; Prepublished online 19 March 2010; doi: 10.4049/jimmunol.0903106 Downloaded from http://www.jimmunol.org/content/184/9/4990

Supplementary http://www.jimmunol.org/content/suppl/2010/03/19/jimmunol.090310 http://www.jimmunol.org/ Material 6.DC1 References This article cites 63 articles, 33 of which you can access for free at: http://www.jimmunol.org/content/184/9/4990.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Activating Transcription Factor 3 Is a Positive Regulator of Human IFNG

Sanna File´n,*,† Emmi Ylikoski,*,‡ Subhash Tripathi,* Anne West,* Mari Bjo¨rkman,* Joel Nystro¨m,* Helena Ahlfors,*,x Eleanor Coffey,* Kanury V. S. Rao,{ Omid Rasool,* and Riitta Lahesmaa*,‖

IL-12 and IL-18 are essential for Th1 differentiation, whereas the role of IFN-a in Th1 development is less understood. In this microarray-based study, we searched for that are regulated by IFN-a, IL-12, or the combination of IL-12 plus IL-18 during the early differentiation of human umbilical cord blood CD4+ Th cells. Twenty-six genes were similarly regulated in response to treatment with IL-12, IFN-a, or the combination of IL-12 plus IL-18. These genes could therefore play a role in Th1 lineage decision. Transcription factor activating transcription factor (ATF) 3 was upregulated by these cytokines and selected for further +

study. Ectopic expression of ATF3 in CD4 T cells enhanced the production of IFN-g, the hallmark cytokine of Th1 cells, whereas Downloaded from small interfering RNA knockdown of ATF3 reduced IFN-g production. Furthermore, ATF3 formed an endogenous complex with JUN in CD4+ T cells induced to Th1. Chromatin immunoprecipitation and luciferase reporter assays showed that both ATF3 and JUN are recruited to and transactivate the IFNG promoter during early Th1 differentiation. Collectively, these data indicate that ATF3 promotes human Th1 differentiation. The Journal of Immunology, 2010, 184: 4990–4999.

aive CD4+ Th lymphocytes are capable of differentiating The precise role of type I IFNs in STAT4-dependent Th1 differ- http://www.jimmunol.org/ into functionally distinct subsets. These include IFN-g, entiation and IFN-g production has yet to be resolved. Initially, it N IL-2, and TNF-b–secreting Th1 cells, IL-4, IL-5, and IL- was shown by Brinkmann et al. (17) that human CD4+ T cells 13–secreting Th2 cells (1), IL-17, IL-17F, IL-21, IL-22, and TNF- produce increased levels of IFN-g in the presence of IFN-a, which secreting Th17 cells, (2–6), and naturally occurring CD4+CD25+ suggests that type I IFNs favor Th1 differentiation. Later, IFN-a/b Foxp3+ T regulatory cells (7–11). The differentiation process is in synergy with IL-18 was reported to induce IFN-g production by initiated by Ag encounter (i.e., activation via TCR/CD28) and T cell blasts (18). Furthermore, in the same report, IFN-a was shown controlled by the cytokine environment (reviewed in Ref. 12). IL- to increase IFNG mRNA synthesis in human T cells. There is also 12R–mediated STAT4 signaling is the key event in Th1 lineage clear evidence that type I IFNs are able to tyrosine phosphorylate by guest on October 1, 2021 commitment, as shown by the studies using knockout mice de- and activate STAT4 in primary human T cells (19, 20). However, the ficient for IL-12 (13), IL-12Rb2 (14), or Stat4 (15), which all ex- ability of IFN-a to optimally drive the invitro polarization of human hibit impaired Th1 responses and reduced IFN-g production. IL-18 Th1 cells has been called into question (21). Although IL-12 in- is a cytokine that is also intrinsically involved in Th1 differentia- duced sustained phosphorylation of STAT4, the response to IFN-a tion. It synergizes with IL-12 in the induction of IFN-g, although it was only transient and not sufficient for efficient Th1 polarization. is unable to drive Th1 polarization on its own (16). We hypothesized that genes regulated by several Th1-inducing cytokines are likely to be important for Th1 differentiation. To study this, we profiled the cytokine-induced (IFN-a, IL-12, or the com- *Turku Centre for Biotechnology, University of Turku and A˚ bo Akademi University; bination of IL-12 plus IL-18) gene expression during the early stages †Drug Discovery Graduate School and ‡Turku Graduate School for Biomedical Sci- of human umbilical cord blood CD4+ T cell differentiation. Oligo- ences, University of Turku; xNational School for Informational and Structural Bi- { ∼ ology, A˚ bo Akademi University, Turku, Finland; International Centre for Genetic microarrays with probes for 22,000 genes were used. Engineering and Biotechnology, New Delhi, India; and ‖Immune Disease Institute, This resulted in the identification of 26 genes with a similar ex- Harvard Medical School, Boston, MA 02115 pression pattern in response to the studied cytokines. Several of the Received for publication September 28, 2009. Accepted for publication February 17, genes identified have not been previously associated with Th1 dif- 2010. ferentiation, although the expected regulation of known marker This work was supported by the National Technology Agency of Finland, the Acad- emy of Finland, Turku University Hospital Fund, Turku University Foundation, Ida genes was also observed. Activating transcription factor (ATF) 3 Montin Foundation, Oskar O¨ flund Foundation, and Hengityssairauksien Tutkimus- was upregulated by all the cytokines studied. It is a member of the sa¨a¨tio¨. ATF/CREB family of transcription factors, and its functional role in The sequences presented in this article have been submitted to the National Center human Th cell differentiation has not previously been addressed. In for Biotechnology Information Gene Expression Omnibus (www.ncbi.nlm.nih.gov/ geo) under accession number GSE20198. this report, we show that in Th1-promoting conditions, ATF3 pos- itively regulated IFNG gene expression and Th1 differentiation. Address correspondence and reprint requests to Dr. Riitta Lahesmaa, Turku Centre for Biotechnology, University of Turku and A˚ bo Akademi University, P.O. Box 123, FI-20521 Turku, Finland. E-mail address: riitta.lahesmaa@btk.fi Materials and Methods The online version of this article contains supplemental material. Primary CD4+ T cell isolation and culture Abbreviations used in this paper: act, anti-CD3/anti-CD28 activation; ATF, activating transcription factor; ChIP, chromatin immunoprecipitation; N.D., not detected; o/n, Lymphocytes were isolated from heparinized human umbilical cord blood overnight; siRNA, small interfering RNA; SLR, signal log2 ratio. samples (Turku University Hospital, Turku, Finland) or from buffy coats from healthy blood donors (Red Cross Finland Blood Service, Helsinki, Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 Finland) by Ficoll-Paque gradient centrifugation (Amersham Pharmacia www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903106 The Journal of Immunology 4991

Biotech, Uppsala, Sweden). CD4+ T cell enrichment was performed using IFNG_fwd: 59-atgtgcccattaagcgtttg-39 and IFNG_rev: 59-agctgatcaggtc- anti-CD4–conjugated magnetic beads (Dynal, Oslo, Norway). Cells were caaagga-39 using human gDNA as template. Nested sets of primers were cultured in Yssel’s medium (22) supplemented with 1% human AB serum used to clone IFNG promoter fragments of differing length to KpnI-HindIII– (Red Cross Finland Blood Service). Cells were activated with either plate- digested pGL4.10[luc2] vector (Promega, Madison, WI). The sequences of bound anti-CD3 (2.5 mg/ml for coating) and 0.5 mg/ml soluble anti-CD28 the nested primers were: IFNG_1147_f: 59-atatggtaccgaccaaattgagatgcac- (Immunotech, Marseille, France) or with 100 ng/ml PHA (Murex Diag- 39; IFNG_940_f: 59-atatggtacctacaccaccatgcctg-39; IFNG_730_f: 59-atatg- nostics, Chatillon, France) and irradiated CD32/B7 transfected mouse L gtaccatgtcaaataccttattaac-39; IFNG_510_f: 59-atatggtaccttactaaccaactctgat- fibroblasts. A total of 2.5 ng/ml IL-12 (R&D Systems, Minneapolis, MN), 39; IFNG_292_f: 59-atatggtaccgggaggtacaaaaaaattt-39; IFNG_+38_r: 59- 25 ng/ml IL-18 (Medical & Biological Laboratories, Nagoya, Japan), and atataagcttcttctaatagctgatcttca-39; IFNG_1147_as: 59-atataagcttgaccaaattga- 100 U/ml highly purified human leukocyte IFN-a (Red Cross Finland gatgcac-39; and IFNG_+38_as: 59-atatggtacccttctaatagctgatctt-39. Deletion Blood Service) were used to induce Th1 polarization and 10 ng/ml IL-4 mutations of the potential ATF3/JUN binding sites were generated on (R&D Systems) to promote Th2 differentiation. For neutral Th0 con- pIFNG1147-GL4.10 using Phusion Site-Directed Mutagenesis Kit (Finn- ditions, no polarizing cytokines were added. Where indicated, 3 mg/ml zymes, Espoo, Finland). The primers used for the generation of the mutations neutralizing anti–IFN-g (clone 25718, R&D Systems) was added to the were: IFNG_del1_f: 59-cctccctagtagctgagattacaggca-39; IFNG_del1_r: 59- culture media. To promote proliferation, 17 ng/ml IL-2 (R&D Systems) agaatggcttgaacccagaaggtg-39; IFNG_del2_f: 59-attttaccagggcgaagtggggag- was added on the second day of culture to 7-d cultures. 39; IFNG_del2_r: 59-tcattcacaaagcacattgatcatttgc-39; IFNG_del3_f: 59-cgt- caaaggacccaaggagtctaaagg-39; IFNG_del3_r: 59-cattatgcccacctgtgccattct- Microarray studies 39; IFNG_del4_f: 59-cctcaggagacttcaattaggtataaatacc-39; IFNG_del4_r: 59-tcacaagttttttaagatgagatggtg-39 ; IFNG_del5_f: 59-agtgccttcaaagaatcccac- Cells were harvested at different time points and lyzed in TRIzol Reagent cag-39; and IFNG_del5_r: 59-caccattcaaggactggaaatttttttg-39. (Invitrogen, Carlsbad, CA). Total RNAwas further purified using an RNeasy Small interfering RNA (siRNA) oligonucleotides targeting ATF3 (ATF3- minikit (Qiagen, Valencia, CA). Sample preparation for GeneChip oligo- siRNA3: 59-cauuugauauacaugcucatt-39; ATF3-siRNA4: 59-gaaaccu- nucleotide array hybridizations was performed according to Affymetrix’s cuuuauccaacatt-39; and ATF3-siRNA-new2: 59-gaaggaacauugcagagcu-39) instructions using 5 mg total RNA as starting material. Samples were and control (scramble-siRNA1: 59-gcgcgcuuuguaggauucguu-39) (all from Downloaded from hybridized to HG-U133A arrays (Affymetrix, Santa Clara, CA). Micro- Sigma/Proligo, Evry Cedex, France) were used in knockdown studies. array Suite 5.1 software (Affymetrix) was used for data analysis. Data was normalized using the global normalization method and the expression Nucleofection, dead cell removal, and enrichment of analysis settings predefined by the manufacturer. Changes in gene ex- transfected cells pression levels between two samples were considered significant if 21 $ signal log2 ratio (SLR) $ 1, which equals a 2-fold change. The protocol for nucleofection, dead cell removal, and enrichment of transfected cells is described elsewhere (26). Briefly, 5 mg plasmids pATF3- Quantitative real-time RT-PCR (TaqMan) IRES2-H-2Kk and empty vector pIRES2-H-2Kk or 1.5 mg oligonucleotide http://www.jimmunol.org/ siRNAs targeting ATF3 or control (scramble) were used per 4 to 5 3 106 Total RNA was isolated from cells cultured in polarizing conditions with an m RNeasy Mini Kit (Qiagen), and cDNA synthesis was performed from 0.5 mg cells suspended in 100 l Opti-MEM I (Invitrogen). Postnucleofection, the DNase I treated total RNA using SuperScript II (Invitrogen). The primer cells were let to rest 20 h in RPMI 1640 supplemented with 10% FBS, and probe sequences used in TaqMan analysis were as follows: pIFNG- penicillin plus streptomycin, and 2 mM L-glutamine. When the cells were nucleofected with plasmid DNA, a Dead Cell removal kit (Miltenyi Biotec, fwd: 59-ctcgaaacagcatctgactcctt-39; pIFNG-rev: 59-tgtccaacgcaaagcaataca- k 39; probe-IFNG: 59-(FAM)-tgctggcgacagttcagccatcac-(TAMRA)-39; pEE- Bergisch Gladbach, Germany) and anti-H-2K –coated magnetic beads (Miltenyi Biotec) were used to remove dead cells and enrich the trans- F1A1-fwd: 59-ctgaaccatccaggccaaat-39; pEEF1A1-rev: 59-gccgtgtggcaatc- fected cells. Cells transfected with oligonucleotide siRNAs were not sor- caat-39; and probe-EEF1A1: 59-(FAM)-agcgccggctatgcccctg-(TAMRA)- 39. TaqMan analyses were performed with an ABI Prism 7700 Sequence ted. In vitro Th1 differentiation of the transfected cells was performed by Detector (Applied Biosystems, Foster City, CA) as described earlier (23). using anti-CD3/anti-CD28 activation and IL-12 as a polarizing cytokine as by guest on October 1, 2021 All analyses were performed in duplicate in two separate runs using described above. samples from at least three cultures. Housekeeping gene EEF1A1 was used Jurkat cell transfections as a reference for normalization (24). Jurkat E6-1 or Jtag cell lines were maintained in RPMI 1640 supplemented Western blotting with 10% FCS, penicillin/streptomycin, and 2 mM L-glutamine. A total of 3 6 m Cells were lyzed in SDS sample buffer (62 mM Tris-HCl [pH 6.8], 2% w/v 5 10 cells were transfected by electroporation with 5–15 g plasmid SDS, 10% v/v glycerol, 50 mM DTT, and 0.1% w/v bromphenol blue), and DNA, and the cells were rested overnight (o/n). The next day, the cells were activated with 50 ng/ml PMA (Calbiochem, San Diego, CA) and were separated by SDS-PAGE by a standard Laemmli method (25). 1 mg/ml PHA (Sigma-Aldrich). When the effect of ATF3/JUN over- The separated proteins were transferred to nitrocellulose membranes (Hy- expression on IFNG gene transcription was studied, the cells were har- bond ECL, Amersham Biosciences, Buckinghamshire, U.K.) and blocked k with 5% nonfat dried milk and 0.1% Tween 20/TBS. All Ab incubations were vested after 24 h of activation and H-2K –positive cells were sorted using magnetic beads as described elsewhere (26). Cells used in transactivation performed in blocking solution, and the following Abs/antiserum were used: k 1:1000 anti-ATF3 (rabbit IgG, C-19; Santa Cruz Biotechnology, Santa Cruz, assays were not H-2K selected. CA), 1:800 anti-JUN (mouse IgG2a, clone 3; BD Biosciences, Franklin Flow cytometric analysis of intracellular cytokines Lakes, NJ), 1:7500 goat anti-rabbit Ig-HRP (BD Biosciences). and 1:10,000 goat anti-mouse IgG-HRP (Santa Cruz Biotechnology). The ECL detection Sample preparation for flow cytometric analysis of intracellular cytokine system (Amersham Biosciences) was used for visualization of proteins. staining was performed as previously described (26). Briefly, cells harvested b-actin was blotted to control for equal loading with 1:10,000 anti–b-actin 7 d after culture were stimulated with 5 ng/ml PMA (Calbiochem) and 0.5 (AC-15; Sigma-Aldrich, St. Louis, MO). pg/ml ionomycin (Sigma-Aldrich) in Yssel’s medium. A portion of the cells was incubated in Yssel’s medium only and used as an unstimulated control. Plasmid constructs and small interfering RNA oligonucleotides After 2 h of stimulation, 10 mg/ml brefeldin A (Alexis Biochemicals, ATF3 was cloned into pcDNA3.2/V5/GW/D-TOPO vector (Invitrogen) Lausanne, Switzerland) was added to all samples, and incubation was according to the manufacturer’s instructions using PCR primers pATF3- continued for 3 h. Cells were washed and fixed with 4% (w/v) para- fwd: 59-caccatgatgcttcaacacc-39 and pATF3-rev: 59-ttagctctgcaatgttccttc-39 formaldehyde/PBS and permeabilized with 0.5% (w/v) saponin/PBS. Anti- human–IFN-g-FITC (B27; Caltag Laboratories, Burlingame, CA) was used and cDNA prepared from total RNA isolated from human T cells as for staining of the intracellular IFN-g, and mouse IgG1-FITC (Caltag template. The Gateway Vector Conversion System (Invitrogen) was used to modify pIRES2-H-2Kk vector (26) into Gateway destination vector Laboratories) was used as an isotypic control. Cells were analyzed with an pIRES2-GW-H-2Kk. ATF3 was subcloned from pcDNA3.2 vector into FACSCalibur Flow Cytometer (BD Biosciences). k pIRES2-GW-H-2K via pDONR/Zeo vector (Invitrogen) generating vector IFN-g cytokine assay pATF3-IRES2-H-2Kk. PCR primers pJUN-fwd-EcoRI: 59-atcgaattcatga- ctgcaaagatggaaacgac-39 and pJUN-rev-BamHI: 59-atcggatcctcaaaatgtttg- Human umbilical cord blood CD4+ T cells that were nucleofected with caactgctgcg-39 were used to directly clone JUN into EcoRI-BamHI– siRNA oligonucleotides were induced toward Th1 as described above. digested pIRES2-H-2Kk to create plasmid pJUN-IRES2-H-2Kk. Supernatants were collected after 24 h of culture for analysis of IFN-g Cloning of IFNG promoter constructs illustrated in Fig. 7Awas started by production. After 7 d of stimulation, the cells were treated with 5 ng/ml amplifying a 1.25-kb fragment from human IFNG promoter with primers PMA and 0.5 pg/ml ionomycin for 24 h and the supernatants collected. A 4992 ATF3 REGULATES IFNG EXPRESSION

Bio-Plex Cytokine Assay kit (Bio-Rad, Hercules, CA) was used for IFN-g Proteins in the DNA sample were removed by incubation with 120 ml detection according to the manufacturer’s instructions, and the assays were proteinase K solution (2% glycogen, 5% proteinase K stock solution, and analyzed with Luminex 100 IS 2.2 (Luminex, Austin, TX). TE) for 2 h at 37˚C. The DNA was then extracted twice with phenol and once with 24:1 chloroform/isoamyl alcohol. The sample was adjusted to Nuclear extracts and ATF3/JUN coimmunoprecipitations 200 mM NaCl. After ethanol precipitation, the DNA was dissolved in 30 ml TE containing 10 mg DNase-free RNase A and incubated for 2 h at 37˚C. Human umbilical cord blood CD4+ T cells were isolated, activated with The DNA was further purified with a Qiagen PCR kit (Qiagen). Primers anti-CD3 and anti-CD28, and cultured in the presence or absence of IL-12 F1: 59-cttgcagtgagccgagattg-39, R1: 59-ccagcctaggtgacagagtaagat-39, F2: or IL-4 or in the absence of cytokines as described above. Cells were 59-gaccaaattgagatgcaccgtagg-39, R2: 59-cacttcgccctggtaaaatgtt-39, F3: 59- harvested after 48 h, and nuclear extracts were prepared as described be- cattctttccttgctttctggtc-39, and R3: 59-tgcttcttttacatatgggtcctg-39 were used low. The cells were washed twice with PBS and lyzed in 250 ml lysis for PCR amplification. buffer (21.8 mM HEPES [pH 7.9], 0.2% Tween-20, 1 mM NaF, 1 mM Na3VO3, 1 mM DTT, 1 mM PMSF, and protease inhibitor Complete Mini Transactivation assays [Roche, Basel, Switzerland]). After 2 min incubation, cells were centri- fuged, and cytoplasmic fractions were collected. A total of 100 ml nuclear Jurkat Jtag cells (27) were used in transactivation assays, and they were transfected as described above. IFNG promoter constructs were co- lysis buffer (21.8 mM HEPES (pH 7.9), 430 mM NaCl, 1% glycerol, 1 mM k k NaF, 1 mM Na VO , 1 mM DTT, 1 mM PMSF, and protease inhibitor transfected with either pATF3-IRES2-H-2K or pJUN-IRES2-H-2K 3 3 m 3 Complete Mini [Roche]) was added, and the samples were incubated on plasmids alone or with both. A total amount of 15 g plasmid DNA/5 106 cells was used in transfections. The values of luciferase assays of the ice for 1 h. Thereafter, the samples were sonicated, centrifuged, and the mutation analysis were normalized by the transfection efficiency values soluble fractions collected. Nuclear extracts were precleared with 100 ml k G-Sepharose 4 Fast Flow beads (Amersham Biosciences) for 2 h at (i.e., the percentage of H-2K –positive cells). Britelite plus (PerkinElmer, 4˚C. The samples were spun, and supernatants were collected. A total of Waltham, MA) was used to measure luciferase activity, and the results 1.5 mg anti-ATF3 Ab and 10 ml JUN antiserum (polyclonal anti-JUN was were read with a Victor 1420 Multilabel Counter (PerkinElmer). Downloaded from raised in rabbits inoculated with rGST-human-JUN [5–89 aa]) were used Prediction of ATF3/JUN binding sites for immunoprecipitation; 1.5 mg anti-RANKL Ab (rabbit IgG, FL-317; Santa Cruz Biotechnology) was used as control. Ab incubations were done Putative ATF3/JUN binding sites in the human IFNG promoter (21147 to o/n at 4˚C. A total of 50 ml protein G-Sepharose beads were added for +38) were analyzed with both MatInspector (Genomatix Software, Mu- collecting the protein-Ab complexes, and the samples were incubated 1 h nich, Germany) and AliBaba 2.1 (Biobase, Wolfenbuettel, Germany). at 4˚C. The beads were washed three times in 1 ml PBS, and 20 ml SDS sample buffer was added for eluting the proteins in 95˚C for 5 min. Accession numbers

The Affymetrix gene expression data were deposited to the NCBI Gene http://www.jimmunol.org/ Chromatin immunoprecipitation assay Expression Omnibus (www.ncbi.nlm.nih.gov/geo) with accession number GSE20198. Freshly isolated human umbilical cord blood CD4+ T cells were induced toward Th1 and Th2 as described above. Poststimulation, the protein-DNA complexes were cross-linked with 1/10 volume of cross-linking solution Results (50 mM HEPES-KOH [pH 7.5], 100 mM NaCl, 1 mM EDTA, 0.5 mM IFN-a–induced IFNG gene expression peaks at 6 h of EGTA, and 11% formaldehyde) for 10 min and quenched with 1/20 vol- stimulation ume of 2.5 mM glycine solution for 10 min, after which the cells were washed twice with ice-cold PBS. The cells were pelleted, and nuclear Because the induction of IFNG gene expression by type I IFNs has fractions were prepared. The cell pellet was resuspended in 30 ml lysis been controversial, we were interested in studying how potent buffer 1 (50 mM HEPES KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, by guest on October 1, 2021 human leukocyte IFN-a is in inducing IFNG transcription in hu- 10% glycerol, 0.5% Nonidet P-40, and 0.25% Triton X-100 with protease + + inhibitor Complete Mini [Roche]) and mixed for 10 min at 4˚C. Post- man umbilical cord blood CD4 T cells. CD4 cells, purified using centrifugation at 2000 3 g for 10 min at 4˚C, the cell pellet was re- magnetic selection, were activated with immobilized anti-CD3 suspended in 24 ml lysis buffer 2 (200 mM NaCl, 1 mM EDTA [pH 8], and soluble anti-CD28 and cultured in the presence or absence of 0.5 mM EGTA, and 10 mM Tris [pH 8], with protease inhibitor) at room 100 U/ml IFN-a. In addition, 3 mg/ml neutralizing Ab against temperature for 10 min. Postcentrifugation at 2000 3 g for 10 min at 4˚C, the pellet was resuspended in 10 ml lysis buffer 3 (1 mM EDTA [pH 8], IFN-g was used to inhibit the induction of IFNG gene by autocrine 0.5 mM EGTA [pH 8], and 10 mM Tris [pH 8], with protease inhibitor). IFN-g production. Anti–IFN-g (9 mg/ml) has previously been The chromatin was sheared by sonication, and the fragmentation was ana- shown to effectively block IFNGR-induced T-bet expression and lyzed with agarose gel electrophoresis. The majority of the DNA fragments STAT1 phosphorylation in human CD4+ T cells (28). The 3 mg/ml were in the 400–1000 bp range. Cell debris was removed by centrifugation at 10,000 3 g for 10 min, and the supernatant was collected for chromatin anti–IFN-g used in this study was also effective in blocking T-bet immunoprecipitation (ChIP). A total of 20 ml chromatin supernatant was induction after 24 h of cell culture in Th1-inducing conditions retained as the DNA input control in the following PCR reaction. (Supplemental Fig. 1). Cells were harvested for quantitative real- Magnetic beads were used for ChIP, and 50 ml sheep anti-rabbit IgG- time RT-PCR analysis of IFNG mRNA at the time points of 0, 6, conjugated Dynabeads (Dynal) were conjugated with 10 mg anti-ATF3 (C- 12, 24, and 48 h. Although anti-CD3/anti-CD28 activation was 19; Santa Cruz Biotechnology), 10 ml polyclonal anti-JUN, or 5 mg control rabbit IgG (sc-2027; Santa Cruz Biotechnology) in PBS containing 5 mg/ml a potent inducer of IFNG transcription, IFN-a signaling BSA o/n at 4˚C. Beads were collected by centrifugation, washed three times greatly enhanced the gene induction, which peaked first at 6 h, was with PBS/BSA, and resuspended in 100 ml PBS/BSA for immunoprecipi- diminished at 12 h, and induced again at 48 h poststimulation (Fig. tation. Equal volumes of soluble chromatin were divided into sample and 1). The expression peak at 6 h was concordant with the finding control and adjusted to 0.1% Triton X-100, 0.1% sodium deoxycholate, and 1 mM PMSF, mixed with 100 ml magnetic beads prebound to the Ab and that IFN-a was able to induce only transient STAT4 phosphory- incubated at 4˚C o/n. The magnetic beads were collected, and the supernatant lation (21). was removed. To remove nonspecifically bound material, the beads were resuspended to 1 ml RIPA buffer (50 mM HEPES [pH 7.6], 1 mM EDTA, Microarray studies revealed a set of genes regulated in a similar 0.7% sodium deoxycholate, 1% Nonidet P-40, and 0.5 M LiCl, with protease manner by IL-12, IL-18, and IFN-a inhibitor mixture) at 4˚C. The beads were collected and washed with RIPA buffer a total of 10 times. After washing once with 1 ml TE buffer, the beads To identify which genes are regulated in a similar fashion by IFN-a, were collected by centrifugation at 2000 3 g for 3 min and resuspended in IL-12, and the combination of IL-12 and IL-18, microarray studies 50 ml elution buffer (50 mM Tris [pH 8], 10 mM EDTA, and 1% SDS). To were conducted using HG-U133A GeneChip arrays. Human um- elute precipitated chromatin from the beads, the tubes were incubated at 65˚ bilical cord blood CD4+ cells were cultured as described in Ma- 3 g for 30 min with constant agitation and centrifuged for 1 min at 2000 .A terials and Methods, and total RNA was isolated from cells total of 40 ml eluted supernatants were removed and mixed with 120 mlTE buffer. Similarly, 20 ml chromatin input control was mixed with 140 mlTE harvested at different time points. During the first hours of cell buffer. Cross-links were reversed by incubation at 65˚C o/n. culture, few changes in gene expression can be observed in the The Journal of Immunology 4993

FIGURE 1. IFN-a–induced IFNG expression peaks at 6 h. Human cord blood CD4+ T cells were activated with anti-CD3/anti-CD28 (act) in the presence/absence of human leukocyte IFN-a. Neutralizing anti–IFN-g was added to both cultures. IFNG gene expression was studied using quanti- tative real-time RT-PCR. Results were normalized against the reference gene EEF1A1 and shown as fold difference versus naive Thp cells. Data are averages from three independent cell cultures. Statistical significance was determined using Student t test. presence of IL-12 (29) due to the absence of IL12Rb2 on the cell Downloaded from surface of naive Thp cells. Accordingly, the time point of 48 h was chosen for studies on the effect of IL-12 and IL-12 plus IL-18 on gene expression. However, IFN-a signaling is fully functional already in naive Thp cells and, thus, time points of 2, 6, and 48 h FIGURE 2. Twenty-six genes were similarly regulated by IFN-a and the were chosen for IFN-a–treated samples. combination of IL-12 plus IL-18. Human CD4+ cells were activated with When the IL-12 plus IL-18–induced gene expression changes anti-CD3 and anti-CD28 Abs and induced to differentiate with IL-12 plus http://www.jimmunol.org/ were directly compared with those induced by IL-12 (anti-CD3/ IL-18 or IFN-a. Cells treated with IL-12 plus IL-18 were harvested after anti-CD28 activation plus IL-12 plus IL-18 versus anti-CD3/anti- 48 h and cells treated with IFN-a after 2, 6, or 48 h. The genes regulated in CD28 activation plus IL-12), no reproducible changes between response to IFN-a treatment at different time points were directly com- pared with those regulated by IL-12 plus IL-18 after 48 h. Genes showing two independent analyses were observed using an SLR cutoff 2 similar regulation in response to the different treatments were selected for level of 1 $ SLR $ 1 (i.e., a 2-fold change). However, IL-18 further examination. The Venn diagrams show how the genes with similar seemed to function as an enhancer of the effects caused by IL-12. expression patterns were regulated by IFN-a during the time-scale studied. In general, it was observed that most of the genes regulated by the combination of IL-12 and IL-18 behaved similarly. The response GIMAP4, SLC2A3, FOSL2, ZBED2, CTH, and TBX21 (28–30, 32– by guest on October 1, 2021 was only less pronounced than that resulting from IL-12 treatment 40, 41). Genes upregulated and novel in this context were alone. Activation of cells with a combination of IL-12 and IL-18 FLJ12377, GNA15, SERPINB1, DDIT4, and ENPP2. caused the expression of 50 genes to change significantly and reproducibly when compared with activation only. The data are ATF3 protein is differentially expressed in Th1 and Th2 cells provided as supplemental data (Supplemental Table I). ATF3 was chosen for further studies because its function in human IFN-a induced significant changes in the expression of 172 Th1 and Th2 differentiation has previously not been examined. 2 genes ( 1 $ SLR $ 1) in at least two of the three time points Western blot analysis showed that although ATF3 protein was vir- studied. The expression of 66 of these changed 4-fold or more, and tually absent in naive cord blood CD4+ Thp cells, its expression was among these, there were several genes known to be regulated by rapidly induced after 2 h of TCR activation with anti-CD3 and anti- IFN-a, as well as genes previously unknown to be targets of IFN- CD28 mAbs (data not shown). Inducing Th1 polarization with IFN- a receptor signaling. These data are provided as supplemental data a, IL-12, or with IL-12 plus IL-18 further upregulated ATF3 protein (Supplemental Table II). expression, whereas addition of IL-4 downregulated the ATF3 We were most interested in elucidating which genes are regu- protein expression. This differential regulation of ATF3 protein lated in a similar manner by IFN-a, IL-12, and the combination of became detectable after 12 h of culture and was prominent at 24 h IL-12 plus IL-18. We hypothesized that such genes could play an and 48 h (Fig. 3). The presence of neutralizing anti–IFN-g in the important role in the polarization of Thp cells along the Th1 lineage. Direct comparisons between the sets of genes that were found to be regulated in response to IFN-a with the genes regu- lated by the combination of IL-12 and IL-18 revealed that the expression patterns of 26 genes were similar. These genes are presented in Fig. 2 and Supplemental Table III. LAIR2, SOCS2, and AQP3 were downregulated in response to IL- 12, IL-18, and IFN-a receptor signaling and have previously been associated with Th1/Th2 differentiation (30, 31). CECR1 was also downregulated in response to IL-12, IL-12 plus IL-18, and IFN-a FIGURE 3. ATF3 protein is differentially expressed during the early and to our knowledge has not previously been associated with Th stages of Th1 or Th2 cell polarization. Human cord blood CD4+ T cells functions. Genes that were upregulated in response to IL-12, IL-12 were cultured under the conditions indicated for 0, 24, or 48 h. Proteins plus IL-18, and IFN-a treatment and previously known to be dif- were separated on a standard SDS-PAGE gel, transferred to nitrocellulose ferentially regulated in Th1/Th2 cells included NFIL3, LIF, NKG7, membrane, and blotted with rabbit anti-ATF3. b-actin expression was used IL18RAP, 1L12RB2, GZMB, IFNG, GZMH, ADAM19, BCL6, ATF3 as a loading control. Data are representative of four independent analyses. 4994 ATF3 REGULATES IFNG EXPRESSION culture media demonstrated that the observed regulation of ATF3 and IL-12) for 7 d. The ability of the cells to produce IFN-g after expression was not dependent on autocrine IFN-g production. restimulation with PMA plus ionomycin was studied using in- tracellular cytokine staining and flow cytometry (Fig. 4A). The ATF3 is a positive regulator of IFN-g production and T-bet and percentage of IFN-g–positive cells, especially cells that stained JUN expression highly positive for IFN-g, was higher in the cell population ex- To study the effects of ATF3 overexpression on Th1 differentiation, pressing ectopic ATF3 than in the mock-transfected cells. The two human peripheral blood CD4+ T cells were transiently transfected distinct IFN-g–positive cell populations are typically detected by either with pATF3-IRES2-H-2Kk vector or with an empty vector, intracellular staining and flow cytometry (21, 42, 43) and can be pIRES2-H-2Kk. Dead and apoptotic cells were removed, after considered as IFN-g+ (high) and IFN-g+ (low) cell populations. which the cells expressing the H-2Kk selection marker were sorted To confirm the role of ATF3 in IFN-g expression by an alter- with magnetic beads. Overexpression of ATF3 protein was detected native approach, specific siRNA oligonucleotides targeting ATF3 by Western blotting and was shown to be dependent on the amount mRNA were designed. Scrambled sequence was used as a control. of pATF3-IRES2-H-2Kk used for nucleofections (Supplemental Human cord blood CD4+ cells were transiently transfected with Fig. 2). For differentiation studies, the cells were cultured in Th1- the siRNA oligonucleotides and subsequently induced to differ- promoting conditions (i.e., in the presence of anti-CD3/anti-CD28 entiate toward Th1 by anti-CD3/anti-CD28 activation and IL-12.

FIGURE 4. ATF3 promotes Th1 differen- tiation by inducing IFN-g production. A, The Downloaded from ATF3 gene was introduced into peripheral blood CD4+ cells in pATF3-IRES2-H-2Kk vector by nucleofection. Cells nucleofected with an empty vector pIRES2-H-2Kk were used as controls. Dead cells were removed, and H-2Kk–positive transfected cells were http://www.jimmunol.org/ sorted by magnetic beads. Cells were cultured for 7 d in Th1-polarizing conditions, after which half of the cells were restimulated with PMA plus ionomycin, whereas the other half was left unstimulated. Cells were stained for intracellular IFN-g production and analyzed with a flow cytometer. The values represent the percentage of cells stained positive for IFN-g production and the geometrical mean of fluorescence intensity. Data are represen- by guest on October 1, 2021 tative of three independent analyses. B–D, Human cord blood CD4+ T cells were tran- siently transfected with siRNA oligonucleo- tides targeted against ATF3. Scrambled sequence was used as a control. Sub- sequently, the cells were activated with anti- CD3/anti-CD28 and induced to differentiate toward Th1 in the presence of IL-12 for 7 d. B, ATF3 knockdown as well as T-bet and JUN expression was assayed by Western blotting after 0, 24, and 48 h of stimulation. Data are representative of three to five in- dividual experiments and obtained using ATF3-siRNA-new2. C, The relative ATF3, T- bet, and JUN protein expression was quanti- fied from three to five individual cultures, and the figure shows the average values with error bars indicating the SD. D, Supernatants were collected 24 h poststimulation for IFN-g as- say, and reduction in the amount of secreted INF-g was observed in five out of seven in- dependent experiments. The averages of these five are shown in the figure. Cells cul- tured for 7 d were restimulated with PMA and ionomycin for 24 h, after which the su- pernatants were harvested for IFN-g assay. The figure shows average reduction of IFN-g secretion from six out of eight independent experiments in which the reduction was ob- served. Error bars indicate SD. N.D., not detected. The Journal of Immunology 4995

Because ATF3 expression was not detectable by Western blotting in undifferentiated cells, the efficacy of the knockdown was ex- amined from stimulated cells. The residual expression of ATF3 protein varied depending on the oligonucleotide used, but was on average 57% and 46% after 24 h and 48 h of differentiation, re- spectively (Fig. 4B,4C). The effect of ATF3 knockdown on IFN-g production was measured from the supernatant by cytokine bead assay after 24 h of stimulation as well as after restimulation of cells cultured for 7 d (Fig. 4D). These experiments verified the role of ATF3 as a positive regulator of IFN-g expression, because the selective knockdown of ATF3 resulted in reduced amounts of IFN-g pro- duction. Consistently, the knockdown of ATF3 also resulted in diminished amounts of T-bet expression (Fig. 4B,4C). Most likely, this was due to the decreased level of IFN-g.However,itis also possible that ATF3 regulated T-bet expression directly by binding to its promoter or via other signaling routes. JUN is a known positive regulator of ATF3 transcription (44–47), and there is also implication that ATF3 may control JUN ex- pression (48). Furthermore, a recent report described ATF2 as Downloaded from a positive regulator of JUN expression in human T cells (49). Thus, we examined the effect of ATF3 knockdown on JUN ex- pression also and discovered that the ATF3 knockdown resulted in decreased expression of JUN (Fig. 4B,4C). Importantly, JUN expression was not affected by ATF3 knockdown at the time of FIGURE 5. ATF3/JUN dimer is formed in CD4+ cells induced to differ- http://www.jimmunol.org/ activation (0 h sample), thus indicating that JUN was not an off- entiate along Th1 lineage and induces IFNG expression in Jurkat cells. A, target of ATF3 siRNA. Human cord blood CD4+ T cells were activated using anti-CD3 and anti- CD28 (act) and induced to polarize toward Th1 lineage by IL-12 or toward ATF3 and JUN dimerize upon IL-12 stimulation Th2 lineage by IL-4. After 48 h, the cells were harvested and nuclear frac- ATF3 homodimers are known to act as transcriptional repressors tions were prepared. Coimmunoprecipitations were performed using 1.5 mg (50), but heterodimers ATF3/JUN or ATF3/JUND possess tran- anti-ATF3 Ab or 10 ml anti-JUN antiserum. A total of 1.5 mg anti-RANKL scriptional activator function (51, 52). Our results clearly in- Ab was used as a control. One sample was treated with no Abs. Im- munoprecipitated protein complexes were separated by SDS-PAGE, trans- dicated that ATF3 was a positive regulator of IFNG transcription. ferred to nitrocellulose membrane, and blotted with anti-JUN mAb. As This allowed us to hypothesize that to form an activating complex, controls, T-bet and GATA3 were detected from total cell lysates (10% of by guest on October 1, 2021 ATF3 dimerizes with JUN, a transcription factor induced by Th1 input) to demonstrate that the cells have been induced to polarize properly. differentiation (Supplemental Table I). To test the hypothesis, we The results showed that JUN coimmunoprecipitated with ATF3 in cells in- conducted coimmunoprecipitation assays of nuclear extracts of duced to differentiate toward the Th1 lineage but not in cells induced to Th2. cord blood CD4+ cells induced to differentiate toward Th1 and The membrane was also blotted with anti-ATF3,but the resulting bands were Th2 lineages for 48 h. Both anti-ATF3 and anti-JUN Abs were masked by unspecific signals resulting from protein G (data not shown). No used for immunoprecipitation, and the protein/Ab complexes were mAbs for ATF3were available at the time of study. Data are representative of isolated using protein G-Sepharose beads. Proteins were eluted to three individual experiments. B, Jurkat cells were transfected with indicated SDS sample buffer and resolved by SDS-PAGE. Immunoblotting plasmids and rested for 24 h. The cells were activated with PMA/PHA, harvested after 24 h, and H-2Kk–positive cells were selected using magnetic assays showed (Fig. 5A) that endogenous ATF3 and JUN coim- beads. Quantitative real-time RT-PCR analysis showed that although ectopic munoprecipitated in cells induced to polarize to Th1 direction but expression of either ATF3 or JUN increased IFNG mRNA expression, co- not in cells polarized to Th2 direction. transfection of both transgenes further increased IFNG expression. Data are To test the hypothesis further, we cotransfected pJUN-IRES2-H- the average of five individual experiments. Statistical significance was k k 2K and pATF3-IRES2-H-2K vectors into Jurkat cells to study evaluated with Student t test. Error bars indicate SEM. the resulting induction of IFNG transcription by quantitative real- time RT-PCR. Posttransfection, the cells were rested o/n and ac- tivated with PMA/PHA. The cells were harvested after 24 h of human umbilical cord blood CD4+ T cells induced to differentiate activation. Real-time RT-PCR analysis showed (Fig. 5B) that al- toward Th0, Th1, and Th2 for 9 h as well as undifferentiated Thp though both JUN and ATF3 transfection enhanced IFNG tran- cells to study the recruitment of ATF3 and JUN to the IFNG pro- scription compared with the empty vector, the induction induced moter in the early stages of Th differentiation. Reproducible binding by cotransfection was greater than that induced by either of the of both ATF3 and JUN was observed with a primer pair that transgenes alone. amplified a 855-bp fragment of the distal promoter (from 21147 to 2293) (Fig. 6A). The band intensities were quantified with a den- ATF3 and JUN bind and transactivate human IFNG promoter sitometer, and the data from anti-ATF3– and anti-JUN–precipitated ChIP assays were performed to study the recruitment of ATF3 and samples were normalized to control IgG. The data demonstrated that JUN to the cis regulatory elements of the IFNG promoter. Abs the recruitment of both ATF3 and JUN to this region was specific for specific for ATF3 and a JUN-specific antiserum was used. Primers Th1-promoting conditions and statistically significant (p = 0.004 were designed to amplify both the proximal and distal elements of and p = 0.006, respectively) (Fig. 6B). Some binding of ATF3 and the human IFNG promoter. Three different regions were studied: JUN to the other regions (from 21763 to 21084 and from 2458 to from 21763 to 21084, from 21147 to 2293, and from 2458 to +216) was also detected, but this binding was neither reproducible +216. The ChIP assays were performed on nuclear fractions of nor specific for Th1-inducing conditions (data not shown). 4996 ATF3 REGULATES IFNG EXPRESSION

FIGURE 6. ATF3 and JUN are recruited to the IFNG promoter in Th1-promoting conditions. Umbilical cord blood CD4+ T cells were induced to differentiate to- ward Th0, Th1, or Th2 in the presence of anti–IFN-g for 9 h. ChIP was performed with both anti-ATF3 and JUN antiserum. Reproducible binding of ATF3 and JUN was detected in Th1- promoting conditions. PCR was performed using primers amplifying the IFNG promoter from 21147 to 2293. A, A representative result from at least three independent experiments is shown. B, The band intensities were quantified with a densitometer, and the data from anti-ATF3 and anti- JUN precipitated samples were normalized against control IgG. Error bars indicate SEM. ppp , 0.01. act, anti-CD3/anti-CD28 activation. Downloaded from http://www.jimmunol.org/ Although ATF3 and JUN were able to form a protein complex in 2311 (59-agagtcaaca-39, del2), a JUN site from 2200 to 2191 Th1-inducing conditions, and they both were recruited to the IFNG (59-gtctgtctcat-39, del3), an ATF site from 262 to 252 (59-aaa- promoter, the data do not enable us to make conclusions about the tacgtaat-39, del4), and a CREB site from 2256 to 2249 (59- possibility that the two factors may bind in a cooperative fashion. tgaagtaaa-39, del5) (Fig. 7A). Deletion mutations were made to To further study the potential of ATF3 and JUN to bind and the pIFNG1147-GL4.10 plasmid to study the functionality of transactivate the IFNG promoter, we designed and exploited lu- these sites. As indicated above, the putative binding sites are in ciferase reporter assays. As the ChIP analysis showed binding of italics, and the deleted regions are underlined. Each of the mutated both ATF3 and JUN on promoter areas 1.1 kb upstream of the constructs had one intact putative binding site and deletions in the by guest on October 1, 2021 IFNG gene transcription initiation site, the luciferase assays were four other sites. One construct had deletions in all five putative designed to cover the promoter from 21147 to +38 (Fig. 7). The sites. The mutation constructs were cotransfected to Jtag cells with promoter fragments were cloned in front of the luciferase gene ATF3 and JUN plasmid constructs as above. The results (Fig. 7C, (luc2) (Fig. 7A), and the IFNG promoter constructs were co- 7D) indicated that deletion of all five sites was needed to abolish transfected to Jurkat Jtag cells with either ATF3 or JUN over- binding of ATF3 to the promoter, and, thus, the binding was expression vectors or both of them. The resulting luminescence shown to be cooperative. However, JUN was still able to bind and signals were compared with the signal obtained by cotransfection transactivate transcription, suggesting that additional binding sites of the IFNG promoter constructs with an empty vector (Fig. 7B). exist for other JUN complexes. Also, when all five binding sites When ATF3 was cotransfected alone with any of the IFNG pro- were deleted, ATF3 inhibited JUN binding, possibly by seques- moter constructs, the luciferase activity was increased only by 1.2- tering JUN away from the promoter. to 1.3-fold, which may indicate that ATF3 is not able to bind the promoter alone. JUN overexpression caused a .10-fold increase Discussion in the luciferase signal. The increase in luciferase activity was To find potential new players involved in the initiation of human Th1 expected, because Jurkat cells express endogenously both ATF3 differentiation, we used transcriptomics and identified a panel of 26 and JUN (data not shown) as well as other AP-1 transcription genes that are regulated in a similar manner by IL-12, IFN-a, and the factors that are able to form heterodimers with ATF3 and JUN. combination of IL-12 plus IL-18 inhuman umbilicalcordblood CD4+ Most importantly, cotransfecting both ATF3 and JUN over- cells. The set of the genes regulated by Th1-inducing cytokines in- expression vectors with the promoter constructs resulted in all cluded genes known and new in this context. ATF3 was significantly cases in higher luciferase activity than transfecting JUN alone. upregulated by all the cytokines studied. Differential expression of When evaluated with the Student t test, these differences were also ATF3 in human Th1-inducing conditions has previously not been statistically significant (p , 0.02) in all cases but one (pIFNG940- reported. The expression of ATF3 was also increased at the protein GL4.10, p = 0.14). The results also showed that most likely there level in Th1-promoting conditions. Furthermore, its expression was is more than one functional binding site for ATF3 and JUN in the found to be downregulated by IL-4, a Th2-promoting cytokine. IFNG promoter because the average signal obtained with co- ATF3 is a member of the mammalian ATF/CREB protein family transfection of the longest promoter construct with ATF3 and JUN of transcription factors. ATF3 is a stress-inducible gene. However, was 1.4-fold when compared with the average signal obtained little is known about the signaling pathways involved in the in- with the shortest promoter construct (p = 0.025). duction of ATF3 as well as the role of ATF3 in stress responses. ATF3 and JUN binding sites on IFNG promoter were analyzed, Previous work from our group demonstrated that ATF3 is regu- and five putative sites were found: an AP-1 site from 2978 to lated via the IL-4R-induced STAT6 signaling pathway in murine 2968 (59-tcctgcctcagccct-39, del1), an AP-1 site from 2320 to CD4+ cells and that its transcript is more abundant in Th2 cells The Journal of Immunology 4997 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 7. ATF3 and JUN transactivate the IFNG promoter. A,Upto∼1.1 kb fragment of the human IFNG promoter was cloned in front of the luc2 gene in pGL4.10 vector. Sequence analysis revealed five putative ATF3/JUN binding sites, which are marked with arrows. B, The IFNG promoter constructs were cotransfected to Jurkat Jtag cells with either pATF3-IRES2-H-2Kk or pJUN-IRES2-H-2Kk or both. The cells were activated with PMA plus PHA the next day, and the luminescence signals were read 24 h postactivation. The figure shows average values from two to seven independent cultures. C, The mutated IFNG promoter constructs were cotransfected to Jurkat Jtag cells with pATF3-IRES2-H-2Kk or pJUN-IRES2-H-2Kk or both. Each promoter construct had only one intact binding site left. One construct had deletions in all putative binding sites. D, Deletion of all five putative ATF3/JUN binding sites abolished the ability of ATF3 to transactivate the IFNG promoter. Statistical significance was determined with Student t test. Error bars indicate SEM. pp , 0.05; ppp , 0.01. than in Th1 cells (32). ATF3 gene expression has also been re- (58). Gilchrist et al. (58) showed that ATF3 was induced in the ported to be induced by both IFN-g and IL-4 in murine macro- lungs of OVA-challenged mice and that ATF3-deficient mice phages (53), by pegylated IFN-a in PBMCs (54) and by IL-12 in showed more severe symptoms of asthma than wild-type mice. murine NK cells (55). A study using systems biology approaches Consistent with our previous studies in mouse cells (32), they identified ATF3 as a negative regulator of TLR-stimulated murine demonstrated that the expression of ATF3 in CD4+ mouse T cells macrophage responses (56). In that study, ATF3, as a part of was upregulated by Th2 cytokines but not by Th1 cytokines. They a transcriptional complex also containing members of the NF-kB further showed that ATF3 bound the Th2 cytokine in the transcription factors, was shown to act as a transcriptional re- chromatin of mouse Th2 cells and that ATF3 inhibited IL-4, IL-5, pressor of Il6 and Il12b upon macrophage activation. Furthermore, and IL-13 secretion. Whitmore et al. (57) showed that ATF3 gene expression was up- Importantly, there are no previous data on ATF3 expression or regulated by TLR signaling in mouse and human APCs and that function in human CD4+ T cells in response to Th1/Th2-inducing ATF3 acted as a negative regulator of IL-12 and IL-6 production cytokines. We demonstrated in this study that, in contrast to mouse by macrophages. ATF3 has also been shown to be a negative CD4+ T cells, in human CD4+ cells, ATF3 protein levels were regulator of IFNG gene expression in mouse NK cells (55). Re- clearly higher in cells induced to differentiate along a Th1 lineage cently, the role of ATF3 was studied in a mouse asthma model than along a Th2 lineage and that ATF3 positively regulated 4998 ATF3 REGULATES IFNG EXPRESSION human IFNG gene expression and Th1 differentiation. Thus, using spond to IL-12 and IL-18. This in turn efficiently drives Thp cells a different mechanism, ATF3 plays a similar role in human and toward the Th1 lineage. mouse CD4+ Th cell differentiation: to drive the Th1/Th2 balance toward the Th1 lineage. Acknowledgments AP-1 transcription factors consist of JUN and FOS family We thank Paula Suominen, Arja Reinikainen, Miina Miller, Outi Melin, proteins that can form homo- or heterodimers not only with each Sarita Heinonen, Matti Raitio, and the Finnish DNA Microarray Centre other, but also with proteins of the ATF/CREB or MAF protein at Turku Centre for Biotechnology. We also thank Robert Moulder for re- families (59). AP-1 transcription factors are induced upon acti- vising the language, the Turku University hospital staff for collecting the vation of naive Thp cells, and the different complexes have se- umbilical cord blood samples, and Dr. Anjana Rao for a careful review lective functions in the regulation of effector Th cell and comments on the manuscript. differentiation (60, 61). In general, the ATF3 homodimer is known to act as a repressor of transcription. However, the function of Disclosures ATF3 is dependent on its binding partner because selective het- The authors have no financial conflicts of interest. erodimers ATF3/JUN and ATF3/JUND have been shown to result in transcriptional activation (51, 52). JUN is a known positive References regulator of IFNG gene expression (62, 63). JUN is able to form 1. Mosmann, T. R., H. Cherwinski, M. W. Bond, M. A. Giedlin, and R. L. Coffman. heterodimers with ATF2 that have been shown to bind and 1986. Two types of murine helper T cell clone. I. Definition according to profiles transactivate the IFNG promoter (49, 62, 63). As JUN was also of lymphokine activities and secreted proteins. J. Immunol. 136: 2348–2357. upregulated by IL-12 and IL-12 plus IL-18 in our microarray 2. Liang, S. C., X. Y. Tan, D. P. Luxenberg, R. Karim, K. Dunussi-Joannopoulos,

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IFN-γ was effective in blocking T-bet induction at concentrations 3 and 9 µg/ml. act: anti-CD3/anti-CD28 activation.

Figure S2. ATF3 overexpression in CD4+ T cells is dependent on the amount of pATF3-IRES2-H-2Kk plasmid used for nucleofections. Human peripheral blood

CD4+ cells were isolated using gradient centrifugation and magnetic beads coated with anti-CD4 antibody. The cells were nucleofected with different amounts of pATF3-IRES2-H-2Kk plasmid. After overnight resting dead and apoptotic cells were removed and cells expressing H-2Kk molecule were enriched. The amount of ATF3 overexpressed was detected by Western blotting.

Table S1. IL-12 together with IL-18 regulated the differential expression of 50

genes in cord blood CD4+ cells. CD4+ cells were isolated from human umbilical

cord blood by density gradient centrifugation and magnetic bead selection. Cells were

activated using anti-CD3 and anti-CD28 antibodies to mimic TCR stimulation.

Cytokines IL-12, combination of IL-12 + IL-18 or IFN-α were added to induce Th1

differentiation. Cells were harvested at the indicated time points and GeneChip

analyses were performed using HG-U133A chips. Cytokine-induced gene expression

levels were compared to those of cells activated only through CD3 and CD28. The

results are shown as Signal Log2 Ratios (SLR). The data is filtered showing

significant changes induced by the combination of IL-12 + IL-18. Additionally, the

transcriptional regulation induced by IL-12 or IFN-α are also shown.

act + IFN-α vs. act act + IL-12 Representative Gene act + IL-12 + IL-18 vs. Public ID Gene Title Symbol vs. act, 48 h act, 48 h 2 h 6 h 48 h NM_017806 hypothetical protein FLJ20406 FLJ20406 -2.1 -1.2 -2.4 -2 U17074 cyclin-dependent kinase inhibitor 2C (p18, inhibits CDKN2C -2.2 -1.3 -0.9 CDK4) NM_017424 cat eye syndrome region, candidate 1 CECR1 -1.7 -1.2 -1.8 -1.1 -1 AI221950 leucine rich repeat neuronal 3 LRRN3 -1.3 -0.9 -1.7 -1.1 0.5 -0.6 AI922599 vimentin VIM -1.4 -1.5 -1.6 -1.8 -0.5 NM_002288 leukocyte-associated Ig-like receptor 2 LAIR2 -1.1 -1.3 -1.5 -1.5 -1 -1.6 NM_000960 prostaglandin I2 (prostacyclin) receptor (IP) PTGIR -0.9 -0.9 -1.3 -1.1 -0.4 -0.5 AB004903 suppressor of cytokine signaling 2 SOCS2 -1.2 -1.5 -1.2 -1.8 0.8 -1.1 NM_001311 cysteine-rich protein 1 (intestinal) CRIP1 -1.1 -1 -1.2 -1.1 -0.6 -0.7 -0.8 NM_002961 S100 binding protein A4 (calcium protein, S100A4 -1 -1.2 -1.1 -1.2 -0.6 calvasculin, metastasin, murine placental homolog) NM_003877 suppressor of cytokine signaling 2 SOCS2 -0.9 -1.1 -1.1 -1.2 0.7 -1 N74607 aquaporin 3 AQP3 -1.7 -0.6 -1.1 -1 -0.6 -0.8 -1 AF027205 serine protease inhibitor, Kunitz type, 2 SPINT2 -1 -0.6 -1 -1 -0.7 AU145019 GRP1-binding protein GRSP1 GRSP1 1.2 1.6 0.9 2 3.1 1.2 BE906572 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- GALNT10 0.7 0.6 1 1 0.8 acetylgalactosaminyltransferase 10 (GalNAc-T10) AV705244 hypothetical protein FLJ12377 FLJ12377 0.8 1.7 1 1.6 1.9 2.2 0.7 NM_001945 diphtheria toxin receptor (heparin-binding epidermal DTR 1 1.1 1.1 0.7 0.9 growth factor-like growth factor) NM_000376 vitamin D (1,25- dihydroxyvitamin D3) receptor VDR 0.6 0.7 1.1 1.4 0.8 NM_013351 T-box 21 TBX21 1.1 1.4 1.1 1.3 3.5 1.8 0.8 NM_002971 special AT-rich sequence binding protein 1 (binds to SATB1 0.8 0.8 1.2 1 -0.3 0.5 nuclear matrix/scaffold-associating DNA's) NM_005384 nuclear factor, interleukin 3 regulated NFIL3 1.1 1.3 1.2 1.5 1.3 1.3 0.9 NM_002309 leukemia inhibitory factor (cholinergic differentiation LIF 1 1.6 1.2 2 2 2.8 factor) NM_005601 natural killer cell group 7 sequence NKG7 0.9 2 1.2 1.9 1.3 Y13786 a disintegrin and metalloproteinase domain 19 (meltrin ADAM19 1 1.1 1.3 1.3 1.4 0.6 beta) U49396 P2X, ligand-gated , 5 P2RX5 1.1 1.8 1.3 2 0.4 NM_018326 immunity associated protein 4 GIMAP4 1.3 1.2 1.3 1.1 1.2 0.7 1.1 NM_003037 signaling lymphocytic activation molecule family SLAMF1 0.8 0.8 1.4 1.1 -0.6 0.7 member 1 AI554300 serine (or cysteine) proteinase inhibitor, clade B SERPINB1 1.8 1.2 1.4 1.5 2 0.9 (ovalbumin), member 1 NM_001674 activating transcription factor 3 ATF3 1.2 1.2 1.5 1.9 1 1.8 1 NM_002068 nucleotide binding protein (G protein), alpha GNA15 0.9 0.6 1.5 1 1.1 15 (Gq class) AB040875 solute carrier family 7, (cationic amino acid SLC7A11 0.9 1.5 1 -0.3 0.7 transporter, y+ system) member 11 AL110298 solute carrier family 2 (facilitated glucose transporter), SLC2A14 1 1 1.5 1.1 0.5 0.4 0.8 member 14 AA488687 solute carrier family 7, (cationic amino acid SLC7A11 1.1 1.5 1.2 0.6 transporter, y+ system) member 11 AF052094 endothelial PAS domain protein 1 EPAS1 1.6 1.6 2.7 -0.9 AI631159 solute carrier family 2 (facilitated glucose transporter), SLC2A3 1.5 1 1.6 1.2 0.5 1.1 member 3 AL080184 insulin induced gene 2 INSIG2 1.3 1 1.6 1.2 0.4 NM_030666 serine (or cysteine) proteinase inhibitor, clade B SERPINB1 1.3 1.1 1.6 1.4 1.6 0.9 0.9 (ovalbumin), member 1 BG491844 v-jun sarcoma virus 17 oncogene homolog (avian) JUN 1.2 1.4 1.7 1.8 -0.1 -0.5 NM_002569 furin (paired basic amino acid cleaving ) FURIN 1.3 1.1 1.7 1.1 0.6 0.4 NM_019058 DNA-damage-inducible transcript 4 DDIT4 1.5 1.3 1.7 1.5 0.5 1.3 1.3 NM_013332 hypoxia-inducible protein 2 HIG2 1.1 0.8 1.7 1.6 0.6 NM_002984 chemokine (C-C motif) ligand 4 CCL4 2 2.4 2.6 0.6 1.5 M63310 annexin A3 ANXA3 2 1.5 1.9 1.6 BE550486 solute carrier family 2 (facilitated glucose transporter), SLC2A3 1.7 1 2 1.4 0.3 1.5 member 3 NM_005204 mitogen-activated protein kinase kinase kinase 8 MAP3K8 1.3 2 1.6 L35594 ectonucleotide pyrophosphatase/ 2 ENPP2 0.9 2 1 1 () N36408 FOS-like antigen 2 FOSL2 1.5 2.1 1.6 1.1 1.6 NM_001706 B-cell CLL/lymphoma 6 ( finger protein 51) BCL6 1.9 0.7 2.4 1.1 -0.5 1 NM_003853 interleukin 18 receptor accessory protein IL18RAP 2.6 2 2.6 2.4 1.4 0.6 NM_001559 interleukin 12 receptor, beta 2 IL12RB2 2.5 2.7 2.7 3 2.9 2.2 2.2 NM_024508 , BED domain containing 2 ZBED2 2.3 1.2 2.7 1.5 -0.7 1.4 M36118 granzyme H (cathepsin G-like 2, protein h-CCPX) GZMH 3 1.7 2.8 2.6 1.6 NM_016081 palladin KIAA0992 1.5 3 1.3 J03189 granzyme B (granzyme 2, cytotoxic T-lymphocyte- GZMB 3.1 1.8 3.6 1.6 2.2 2 3.3 associated serine 1) AL354872 cystathionase (cystathionine gamma-lyase) CTH 4.2 1.1 5 1.3 3.2 M29383 interferon, gamma IFNG 5.5 4.2 6.2 5.6 3 1.8 NM_003856 interleukin 1 receptor-like 1 IL1RL1 2.1 1.7 1.3 -0.4

Table S2. The expression of 66 genes changed four-fold or more in response to

IFN-α during at least two of the three time points studied. Human umbilical cord blood CD4+ cells were enriched using density gradient centrifugation and magnetic bead selection. The cells were activated with anti-CD3/anti-CD28 antibodies and cultured in the presence of highly purified human leukocyte IFN-α for the indicated timepoints. GeneChip analyses were performed using HG-U133A microarrays. IFN-α induced gene expression levels were compared to those of cells that were activated via TCR only and the data is shown as Signal Log2 Ratio.

act + IFN-α vs. act Representative Public ID Gene Title Gene Symbol 2 h 6 h 48 h NM_001769 CD9 antigen (p24) CD9 -3.1 -2.2 N35112 ATPase, Class V, type 10A ATP10A 2 2.5 AK002064 DNA polymerase-transactivated protein 6 DNAPTP6 2 3.6 1.2 NM_002309 leukemia inhibitory factor (cholinergic differentiation LIF 2 2.8 factor) X83493 tumor necrosis factor receptor superfamily, member 6 TNFRSF6 2 2.2 0.8 NM_001775 CD38 antigen (p45) CD38 2.1 2.5 0.9 NM_018381 hypothetical protein FLJ11286 FLJ11286 2.1 2.2 0.2 NM_024989 hypothetical protein FLJ12377 FLJ12377 2.1 2.6 1.4 NM_005781 activated p21cdc42Hs kinase ACK1 2.2 2.1 J03189 granzyme B (granzyme 2, cytotoxic T-lymphocyte- GZMB 2.2 2 3.3 associated serine esterase 1) NM_014322 opsin 3 (encephalopsin, panopsin) OPN3 2.2 2.4 NM_002535 2'-5'-oligoadenylate synthetase 2, 69/71kDa OAS2 2.3 2.7 NM_005082 zinc finger protein 147 (estrogen-responsive finger ZNF147 2.4 2 protein) NM_009587 lectin, galactoside-binding, soluble, 9 (galectin 9) LGALS9 2.5 2.7 NM_002759 protein kinase, interferon-inducible double stranded PRKR 2.5 2.9 0.7 RNA dependent NM_022750 zinc finger CCCH type domain containing 1 ZC3HDC1 2.5 2.4 AI862559 angiopoietin-like 6 ANGPTL6 2.6 2 U03891 apolipoprotein B mRNA editing enzyme, catalytic APOBEC3A 2.6 2.1 polypeptide-like 3A NM_017631 hypothetical protein FLJ20035 FLJ20035 2.6 2.8 1.1 NM_014963 KIAA0963 protein KIAA0963 2.7 4.7 AI765383 KIAA1466 protein KIAA1466 2.7 2.4 NM_003113 nuclear antigen Sp100 SP100 2.7 2.1 0.4 NM_002922 regulator of G-protein signalling 1 RGS1 2.8 2.9 NM_002970 spermidine/spermine N1-acetyltransferase SAT 2.8 2.4 0.5 M55580 spermidine/spermine N1-acetyltransferase SAT 3 3 0.3 AA969194 SP110 nuclear body protein SP110 3.1 2.1 0.7 BC004395 apolipoprotein L, 2 APOL2 3.3 2 U88964 interferon stimulated gene 20kDa ISG20 3.4 4.6 1.1 AW129593 tudor repeat associator with PCTAIRE 2 PCTAIRE2BP 3.4 2.4 0.5 AF280094 SP110 nuclear body protein SP110 3.4 2.2 NM_004510 SP110 nuclear body protein SP110 3.4 2.1 0.4 NM_004509 SP110 nuclear body protein SP110 3.5 2.3 0.4 NM_001565 chemokine (C-X-C motif) ligand 10 CXCL10 3.6 5.6 1.4 NM_017654 FLJ20073 protein FLJ20073 3.6 2.3 AV755522 hypothetical protein FLJ38348 FLJ38348 3.6 2.8 0.7 NM_022873 interferon, alpha-inducible protein (clone IFI-6-16) G1P3 3.6 4 0.8 BC001356 interferon-induced protein 35 IFI35 3.6 4.8 0.4 NM_006187 2'-5'-oligoadenylate synthetase 3, 100kDa OAS3 3.7 3.7 0.7 BE971383 spermidine/spermine N1-acetyltransferase SAT 3.7 3.6 AF323540 apolipoprotein L, 1 APOL1 3.8 4.7 NM_002463 myxovirus (influenza virus) resistance 2 (mouse) MX2 3.8 4.3 0.8 AW474434 tumor necrosis factor (ligand) superfamily, member TNFSF10 3.8 2.6 1.1 10 NM_022168 melanoma differentiation associated protein-5 MDA5 4 2.5 0.7 U57059 tumor necrosis factor (ligand) superfamily, member TNFSF10 4 3.1 0.8 10 NM_018641 carbohydrate (chondroitin 4) sulfotransferase 12 CHST12 4.1 2.3 NM_004335 bone marrow stromal cell antigen 2 BST2 4.2 5 0.4 NM_004030 interferon regulatory factor 7 IRF7 4.2 3.7 0.7 BC000080 promyelocytic leukemia PML 4.2 4.2 0.4 BE888744 interferon-induced protein with tetratricopeptide IFIT2 4.4 4.4 repeats 2 N47725 interferon-induced protein with tetratricopeptide IFIT5 4.4 2.6 0.9 repeats 5 NM_002201 interferon stimulated gene 20kDa ISG20 4.4 6.4 1.4 NM_021105 phospholipid scramblase 1 PLSCR1 4.5 4 1 NM_014314 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide RIG-I 4.5 4.3 0.9 NM_016323 cyclin-E binding protein 1 CEB1 4.6 3.8 0.9 NM_017523 XIAP associated factor-1 HSXIAPAF1 4.6 3.9 0.7 NM_016817 2'-5'-oligoadenylate synthetase 2, 69/71kDa OAS2 4.6 3.7 0.9 AF063612 2'-5'-oligoadenylate synthetase-like /// 2'-5'- OASL 4.6 3.8 1 oligoadenylate synthetase-like AI825926 phospholipid scramblase 1 PLSCR1 4.6 3.7 0.9 NM_014398 lysosomal-associated 3 LAMP3 4.8 2.9 1.2 NM_024119 likely ortholog of mouse D11lgp2 LGP2 4.8 4.5 NM_002346 lymphocyte antigen 6 complex, locus E LY6E 4.8 3.1 0.6 NM_015376 RAS guanyl releasing protein 3 (calcium and DAG- RASGRP3 4.8 5.8 regulated) NM_002675 promyelocytic leukemia PML 4.9 4.4 NM_003810 tumor necrosis factor (ligand) superfamily, member TNFSF10 4.9 2.6 0.6 10 NM_016381 three prime repair 1 TREX1 4.9 2.2 NM_012420 interferon-induced protein with tetratricopeptide IFIT5 5 2.6 repeats 5 NM_017912 hypothetical protein FLJ20637 FLJ20637 5.2 4.5 1 NM_006417 interferon-induced protein 44 IFI44 5.3 4.3 0.9 NM_005101 interferon, alpha-inducible protein (clone IFI-15K) G1P2 5.4 3.7 0.7 NM_002462 myxovirus (influenza virus) resistance 1, interferon- MX1 5.5 4.4 1.1 inducible protein p78 (mouse) NM_003733 2'-5'-oligoadenylate synthetase-like OASL 5.5 3.8 0.8 NM_001549 interferon-induced protein with tetratricopeptide IFIT4 5.6 3.8 0.9 repeats 4 NM_002534 2',5'-oligoadenylate synthetase 1, 40/46kDa OAS1 6.1 5 0.8 NM_022147 28kD interferon responsive protein IFRG28 6.2 2.4 NM_016816 2',5'-oligoadenylate synthetase 1, 40/46kDa OAS1 6.4 4 0.6 BE049439 interferon-induced protein 44 IFI44 6.8 2.8 1.5 NM_006820 chromosome 1 open reading frame 29 C1orf29 6.9 4.3 1.5 AI337069 viperin cig5 6.9 4.3 1.7 NM_017414 ubiquitin specific protease 18 USP18 7.4 4.7 1.4 NM_001548 interferon-induced protein with tetratricopeptide IFIT1 10 5.6 2.1 repeats 1 NM_001559 interleukin 12 receptor, beta 2 IL12RB2 2.2 2.2

Table S3. 26 genes were regulated similarly in response to IL-12, IL-12 + IL-18

and IFN-α treatments. CD4+ cells were isolated from human umbilical cord blood

by density gradient centrifugation and magnetic bead selection. Cells were activated

using anti-CD3 and anti-CD28 antibodies and Th1 differentiation was induced by IL-

12, combination of IL-12 + IL-18 or IFN-α. GeneChip analyses were performed using

HG-U133A chips. Cytokine-induced gene expression levels were compared to those

of cells activated only and the results are shown as Signal Log2 Ratios (SLR).

act + IFN-α vs. act act + IL-12 + Representative Gene act + IL-12 IL-18 vs. act, Public ID Gene Title Symbol vs. act, 48 h 48 h 2 h 6 h 48 h NM_017424 cat eye syndrome chromosome region, CECR1 -1.7 -1.2 -1.8 -1.1 -1 candidate 1 NM_002288 leukocyte-associated Ig-like receptor 2 LAIR2 -1.1 -1.3 -1.5 -1.5 -1 -1.6 AB004903 suppressor of cytokine signaling 2 SOCS2 -1.2 -1.5 -1.2 -1.8 0.8 -1.1 NM_003877 suppressor of cytokine signaling 2 SOCS2 -0.9 -1.1 -1.1 -1.2 0.7 -1 N74607 aquaporin 3 AQP3 -1.7 -0.6 -1.1 -1 -0.6 -0.8 -1 AV705244 hypothetical protein FLJ12377 FLJ12377 0.8 1.7 1 1.6 1.9 2.2 0.7 NM_013351 T-box 21 TBX21 1.1 1.4 1.1 1.3 3.5 1.8 0.8 NM_005384 nuclear factor, interleukin 3 regulated NFIL3 1.1 1.3 1.2 1.5 1.3 1.3 0.9 NM_002309 leukemia inhibitory factor (cholinergic LIF 1 1.6 1.2 2 2 2.8 differentiation factor) NM_005601 natural killer cell group 7 sequence NKG7 0.9 2 1.2 1.9 1.3 Y13786 a disintegrin and metalloproteinase ADAM19 1 1.1 1.3 1.3 1.4 0.6 domain 19 (meltrin beta) NM_018326 immunity associated protein 4 GIMAP4 1.3 1.2 1.3 1.1 1.2 0.7 1.1 AI554300 serine (or cysteine) proteinase inhibitor, SERPINB1 1.8 1.2 1.4 1.5 2 0.9 clade B (ovalbumin), member 1 NM_001674 activating transcription factor 3 ATF3 1.2 1.2 1.5 1.9 1 1.8 1 NM_002068 guanine nucleotide binding protein (G GNA15 0.9 0.6 1.5 1 1.1 protein), alpha 15 (Gq class) AI631159 solute carrier family 2 (facilitated SLC2A3 1.5 1 1.6 1.2 0.5 1.1 glucose transporter), member 3 NM_030666 serine (or cysteine) proteinase inhibitor, SERPINB1 1.3 1.1 1.6 1.4 1.6 0.9 0.9 clade B (ovalbumin), member 1 NM_019058 DNA-damage-inducible transcript 4 DDIT4 1.5 1.3 1.7 1.5 0.5 1.3 1.3 BE550486 solute carrier family 2 (facilitated SLC2A3 1.7 1 2 1.4 0.3 1.5 glucose transporter), member 3 L35594 ectonucleotide ENPP2 0.9 2 1 1 pyrophosphatase/ (autotaxin) N36408 FOS-like antigen 2 FOSL2 1.5 2.1 1.6 1.1 1.6 NM_001706 B-cell CLL/lymphoma 6 (zinc finger BCL6 1.9 0.7 2.4 1.1 -0.5 1 protein 51) NM_003853 interleukin 18 receptor accessory protein IL18RAP 2.6 2 2.6 2.4 1.4 0.6 NM_001559 interleukin 12 receptor, beta 2 IL12RB2 2.5 2.7 2.7 3 2.9 2.2 2.2 NM_024508 zinc finger, BED domain containing 2 ZBED2 2.3 1.2 2.7 1.5 -0.7 1.4 M36118 granzyme H (cathepsin G-like 2, protein GZMH 3 1.7 2.8 2.6 1.6 h-CCPX) J03189 granzyme B (granzyme 2, cytotoxic T- GZMB 3.1 1.8 3.6 1.6 2.2 2 3.3 lymphocyte-associated serine esterase 1) AL354872 cystathionase (cystathionine gamma- CTH 4.2 1.1 5 1.3 3.2 lyase) M29383 interferon, gamma IFNG 5.5 4.2 6.2 5.6 3 1.8