Early Target Genes of IL-12 and STAT4 Signaling in Th Cells Riikka J. Lund, Zhi Chen, Joonas Scheinin and Riitta Lahesmaa This information is current as of September 28, 2021. J Immunol 2004; 172:6775-6782; ; doi: 10.4049/jimmunol.172.11.6775 http://www.jimmunol.org/content/172/11/6775 Downloaded from

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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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Early Target Genes of IL-12 and STAT4 Signaling in Th Cells1

Riikka J. Lund,2*† Zhi Chen,*‡ Joonas Scheinin,* and Riitta Lahesmaa*

IL-12 signaling through STAT4 is essential for induction of optimal levels of IFN-␥ production and commitment of Th1 cells. The molecular mechanism that controls how IL-12 and STAT4 signaling induces Th1 differentiation is poorly described. To identify the early target genes of IL-12 and STAT4 signaling, oligonucleotide arrays were used to compare the gene expression profiles of wild-type and STAT4-knockout murine Th cells during the early Th1 differentiation. According to the results, 20 genes were regulated in an IL-12- and STAT4-dependent manner. Importantly, Ifn␥ was clearly the first gene induced by IL-12 in a STAT4- dependent manner. Most of the other defects in gene expression in STAT4-knockout cells were seen after 48 h of Th1 polarization. In addition to IL-12 signaling mediated by STAT4, STAT4-independent induction of a number of genes was observed immediately ␥ in response to Th1 induction. This induction was at least in part driven by IFN- independently of STAT4. Importantly, addition Downloaded from of exogenous IFN-␥ into Th1 cell cultures of STAT4-knockout cells restored the defect in IFN-␥ production further demonstrating the critical role of IFN-␥ in early Th1 differentiation. The Journal of Immunology, 2004, 172: 6775–6782.

nterleukin-12 is a involved in regulation of cell- by IFN-␥ and STAT1 signaling during the activation of Th cells mediated immune responses and induction of Th1 differen- independently of STAT4 (20–22). Activation of T-bet during Th1 tiation. The effects of IL-12 are mediated through IL-12R differentiation leads to remodeling of the Ifn␥ locus, induction of I http://www.jimmunol.org/ consisting of two subunits IL-12R␤1 and IL-12R␤2 (1). IL- IFN-␥ production, and IL-12R␤2 expression essential for STAT4- 12R␤2, which transmits the signals inside the cell, is not expressed mediated IL-12 signaling (18, 22, 23). on naive Th cells, but is induced in response to Ag stimulation and Although both STAT1 and STAT4 signaling are contributing to is selectively down-regulated during Th2 differentiation (2, 3). the early Th1 polarization, only the requirement of STAT4 for Triggering of IL-12R leads to induction of tyrosine phosphoryla- long-term Th1 development has been clearly demonstrated (10, tion and DNA binding of 2, , and 11). However, the exact role of STAT4 in the early induction of STAT4 (4–6). Also STAT1, STAT3, and STAT5 are tyrosine Th1 cell differentiation is still unclear. Recent studies indicate that phosphorylated in response to IL-12 (6–10). However, only instead of being the primary factor inducing Th1 differentiation,

STAT4 has been shown to be necessary for the long-term com- STAT4 would rather be involved in enhancing initial IFN-␥ pro- by guest on September 28, 2021 mitment of Th1 cells, as mice deficient for STAT4 show impaired duction to optimal levels (21–23). Whether the effects of STAT4 Th1 and enhanced Th2 differentiation (10, 11). In addition to IL- are restricted to regulation of IFN-␥ or whether it also regulates 12, the only known to induce STAT4 phosphorylation other factors involved in inducing Th1 differentiation is unknown. are IFN-␣ and IL-23 (12–16). Previously described target genes of STAT4 include macrophage Although STAT4 has been shown to be required for the long- inflammatory protein-1␣ (Mip-1␣, Scya3), macrophage inflamma- term Th1 differentiation and IFN-␥ production, cells deficient for tory protein-1␤ (Mip-1␤, Ccl4, Scya4, Act-2), IL-1RA (Il1rn), IFN both STAT6 and STAT4 can differentiate to functional IFN-␥- regulatory factor 1 (IRF1), Ets-related (ERM), producing Th1 cells (17). Transcription factor T-box expressed in CCR5, and IL-18R (24–27). In a recent study, the target genes of T cells (T-bet)3 may be involved in this STAT4-independent Th1 IL-12 and STAT4 in already polarized Th1 cells were described differentiation. T-bet has been shown to be required for Th1 dif- (28). The aim of this study was to elucidate the mechanism of early ferentiation and its expression is sufficient to induce IFN-␥ pro- Th1 differentiation by identifying the immediate and early up- duction in Th cells (18, 19). Initial expression of T-bet is induced stream genes regulated in response to IL-12 and STAT4 signaling.

Materials and Methods *Turku Centre for Biotechnology, Turku University and Åbo Akademi, †Turku Grad- Mice uate School of Biomedical Sciences, Turku University, and ‡Drug Discovery Grad- uate School, Turku, Finland STAT4-knockout mice and wild-type controls with a BALB/cJ background Received for publication November 11, 2003. Accepted for publication March were purchased from The Jackson Laboratory (Bar Harbor, ME). The mice 23, 2004. used in the studies were sacrificed at the age of 6–8 wk. The costs of publication of this article were defrayed in part by the payment of page Cell cultures charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The murine mononuclear cells were isolated from spleen single cell sus- 1 This work was supported by the Academy of Finland, Turku Graduate School of pension using Lympholyte-M gradient (Cedarlane Laboratories, Hornby, Biomedical Sciences, Drug Discovery Graduate School, Ida Montin Foundation, the Canada). The CD4ϩ cells were further purified using magnetic MACS Finnish Society of Allergology and Immunology, National Technology Agency of beads (Miltenyi Biotec, Bergisch Gladbach, Germany). For induction of Finland. Th1 polarization pooled CD4ϩ cells were cultured in Iscove’s DMEM 2 Address correspondence and reprint requests to Dr. Riikka J. Lund, Turku Centre for containing 10% FCS, nonessential amino acids, and 2-ME (Life Technol- Biotechnology, University of Turku and Åbo Akademi, P.O. Box 123, FIN-20520, ogies, Paisley, Scotland) in the presence of plate-bound anti-mouse CD3 Turku, Finland. E-mail address: [email protected].fi (315 ng/well), soluble anti-mouse CD28 (500 ng/ml; BD PharMingen, San 3 Abbreviations used in this paper: T-bet, T-box expressed in T cells; IRF, IFN reg- Diego, CA), recombinant mouse IL-12 (10 ng/ml; R&D Systems, Minne- ulatory factor; Thp, Th precursor. apolis, MN) and rat anti-mouse IL-4 (10 ␮g/ml; BD PharMingen). Part of

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 6776 TARGET GENES OF STAT4 IN Th CELLS the cells were activated with anti-CD3 and -CD28 and cultured in “neutral” PMA (50 ng/ml; Calbiochem, La Jolla, CA) and ionomycin (500 ng/ml; conditions in the presence of rat anti-mouse IL-4 (10 ␮g/ml; BD PharM- Calbiochem). Part of the cells were maintained as unstimulated controls ingen), rat anti-mouse IL-12 (10 ␮g/ml; BD PharMingen), and hamster cultured otherwise identically to stimulated cells. After stimulation, the anti-mouse IFN-␥ (10 ␮g/ml; BD PharMingen). The cultures were con- cells were washed (0.5% BSA ϩ 0.01% azide in PBS) and incubated with ducted in parallel for both the cells deficient for STAT4 and the wild-type anti-CD4-PE (Caltag Laboratories, Burlingame, CA) for 15 min. The cells controls. Samples were collected at the time points of 0, 2, 6, and 48 h. For were washed and fixed with 4% paraformaldehyde for 15 min after which the real-time RT-PCR or cytokine secretion assay, the cells were cultured they were permeabilized (0.5% saponin ϩ 0.5% BSA ϩ 0.01% azide in in the presence of plate-bound anti-mouse CD3 (315 ng/well), soluble anti- PBS, pH 7) for 10 min and washed again. After the permeabilization, mouse CD28 (500 ng/ml), and indicated combinations of cytokines IL-4, intracellular cytokine staining was performed with anti-IFN-␥-FITC (BD IL-12, IFN-␥ (500 U; BD PharMingen) and neutralizing Abs for IL-12, Biosciences, San Jose, CA) for 10–20 min. The cytokine profiles of the IFN-␥, and IL-4. cells were studied with FACScan and CellQuest software (BD Oligonucleotide array experiments Biosciences). The total RNAs were isolated using the TRIzol method (Invitrogen, Carls- bad, CA) and were further purified with the Qiagen RNAeasy minikit (Qia- gen, Valencia, CA). For the Affymetrix sample preparation, 5 ␮g of total Results RNA was used as starting material. The sample preparation was performed Genes regulated immediately in response to Th1-polarizing according to the instructions and recommendations provided by the man- stimuli ufacturer (Affymetrix, Santa Clara, CA). The samples were hybridized to MG-U74Av2 arrays containing 12,488 probes for different genes. The data Affymetrix murine U74Av2 arrays, representing probes for was analyzed with Affymetrix Microarray Suite version 5 (MAS5) soft- ϳ10,000 different genes, were used to study the target genes of ware and filtered according to recommendations of the manufacturer. IL-12 and STAT4 signaling during the early polarization of Th1 lym- Downloaded from Briefly, at the expression level, the statistical algorithm used by MAS5 phocytes. To identify the genes regulated by IL-12, cells cultured in defined each gene-specific probe set to be present, absent, or marginal. At ϩ ϩ ϩ the comparison level, each probe set was classified as not changed, in- Th1 conditions (anti-CD3 anti-CD28 IL-12 anti-IL-4) were creased, marginally increased, decreased, or marginally decreased. The compared with the CD3 ϩ CD28-activated cells cultured in the “neu- genes that were absent in both treatments compared or the genes that were tral” conditions (anti-CD3 ϩ anti-CD28 ϩ anti-IFN-␥ ϩ anti-IL-12 not changed between the comparisons were excluded from the results. Fur- ϩ anti-IL-4). thermore, only the genes that presented a consistent change in two or three Altogether 16 genes were immediately up-regulated in the cells biological repeats and showed fold changes of Ն2orՅϪ2 (signal log ratio http://www.jimmunol.org/ Ն1orՅϪ1) in at least one of the time points studied were considered as polarized to differentiate to Th1 cells for 2 or 6 h compared with differentially expressed. the CD3/CD28-activated cells (Table II). These early response Real-time quantitative RT-PCR genes included Iigp-pending, Icsbp, Irf1, Gbp1, Gbp2, AA816121, Tgtp, Ifi1, Igtp, Gtpi-pending, Ifi47, Irf4, Ly6a, Cxcl9, Il12rb1, and Primers and probes were designed for the genes studied using Primer Ex- Ifn␥. The strongest induction by IL-12 was seen in the up-regula- press software (Applied Biosystems, Foster City, CA). Gene expression ␥ levels were measured for the selected panel of genes (Table I) at each time tion of Ifn . point using TaqMan real-time quantitative PCR (RT-PCR; ABI Prism To determine which of these genes regulated in response to Th1 7700; Applied Biosystems) as previously described (29). A TaqMan PCR induction were also targets of STAT4, expression profiles of wild- Core Reagent (Applied Biosystems) was used for the preparation of the type and STAT4-knockout cells cultured in Th1 conditions were by guest on September 28, 2021 RT-PCR mixtures. The primers and probes (MedProbe, Oslo, Norway) were used in the final concentrations of 300 and 200 nM, respectively. The compared. Of the 16 genes that were immediately induced in re- steps in the quantitative RT-PCR were 50°C for 2 min, 95°C for 10 min, sponse to IL-12 treatment, only Ifn␥ was clearly regulated through 95°C for 15 min, and 60°C for 1 min, and altogether 40 cycles were run. STAT4 signaling. In addition, induction of Cxcl9 and Ly6a by All measurements were performed in duplicate in two separate runs using IL-12 was defective in the STAT4-knockout cells compared with samples derived from at least two or three biological repeats. the wild-type cells. All the other immediate IL-12-regulated genes Intracellular cytokine staining and flow cytometry were induced by IL-12, also in STAT4-knockout cells, indicating For the cytokine secretion assay the cells cultured for 7 days were restim- that early induction of these other genes is not dependent of ulated at a density of 1 ϫ 106 in 0.5 ml in IMDM ϩ 10% FCS containing STAT4 (Table II).

Table I. Primers and probes used in real-time RT-PCR

1) 5Ј-6(FAM)-PROBE-(TAMRA)-3Ј 2) 5Ј-PRIMER 1- 3Ј GenBank Accession Symbol 3) 5Ј-PRIMER 2- 3Ј

K00083 Ifn␥ 1) 5Ј-AGGATGCATTCATGAGTATTGCCAAGTTTGA-3Ј 2) 5Ј-CAGCAACAGCAAGGCGAAA-3Ј 3) 5Ј-CTGGACCTGTGGGTTGTTGAC-3Ј M21065 Irf1 1) 5Ј-CTCCGAAGCCGCAACAGACGAGG-3Ј 2) 5Ј-GTGTCACCCATGCCTTCCA-3Ј 3) 5Ј-AGCTTCATAAGGTCTTCGGCTATC-3Ј U11692 Irf4 1) 5Ј-ACCCGGACGACAATGGACAGAGGA-3Ј 2) 5Ј-ACCTGGACCAGGTCCTGTTTC-3Ј 3) 5Ј-GGCTCAGCAACTTCTCAATGTTC-3Ј NM_008320 Icsbp (Irf8)1)5Ј-TCCTCAATCTCTGAGCGGCCACACT-3Ј 2) 5Ј-GCATGAGCGAAGTTCCTGAGAT-3Ј 3) 5Ј-ATGTACTCATCCACAGAAGGTTCCT-3Ј X59728 Gas5 1) 5Ј-CACATTGCGCTCGCTCTGTTATCCAG-3Ј 2) 5Ј-ATGAAGGCTTACGAGGACTCGT-3Ј 3) 5Ј-GCCTCAAACTCCACCATTTCAT-3Ј 3) 5Ј-TTATTTGTACCTTTCAAACTGTCTATCCA-3Ј NM_021792 Iigp-pending 1) 5Ј-ATCCTTCCAGCCAATCCTCTGCTTCAG-3Ј 2) 5Ј-GTCATTGAAAAGAAGCGGCAAT-3Ј 3) 5Ј-TCAGAGAAGGGATGATATTCACTAGGT-3Ј The Journal of Immunology 6777

Table II. Genes regulated immediately in response to Th1-polarizing stimuli (regulation in wild-type and STAT4-knockout cells is shown in parallel)

Th1ϩ/ϩ vs Th0ϩ/ϩ Th1Ϫ/Ϫ vs Th0Ϫ/Ϫ Th1Ϫ/Ϫ vs Th1ϩ/ϩ

Gene ID Functiona 2h 6h 48h 2h 6h 48h 2h 6h 48h

K00083 Ifn␥b Cytokine 3.9c 4.6 3.6 2.4 Ϫ4.3 Ϫ4.9 Ϫ3.4 AJ007971 Iigp-pendingb GTPase 5.2 2.1 3.0 4.1 3.6 8.2 Ϫ1.6 2.7 AA914345 Iigp-pendingb GTPase 3.6 2.5 3.7 3.0 6.5 Ϫ1.9 2.6 M21065 Irf1 Transcription factor 4.6 2.0 1.7 3.7 2.4 1.9 U53219 Igtp GTPase 4.2 1.7 1.7 3.9 2.3 2.7 1.5 M32489 Icsbp1 Transcription factor 3.8 2.7 2.2 3.1 2.4 2.4 M55544 Gbp1 GTPase 3.5 2.4 1.8 3.0 3.2 2.7 1.7 AJ007970 Gbp2 GTPase 3.1 2.4 1.1 2.9 3.1 3.0 1.6 AA816121 3.0 1.4 2.0 2.6 1.9 2.0 1.6 X04653 Ly6a Lymphocyte Ag 2.0 2.6 3.4 2.8 9.0 Ϫ1.5 L38444 Tgtpb GTPase 6.8 1.7 5.7 2.4 3.2 2.4 U19119 Ifi1 6.8 1.4 5.9 1.9 2.4 1.8 AJ007972 Gtpi-pending GTPase 3.9 1.7 3.9 2.1 1.8 1.8 M63630 Ifi47 A/GTP binding 3.6 1.4 Ϫ1.4 2.9 1.8 1.9 U20949 Irf4 Transcription factor 2.1 1.4 1.3 M34815 Cxcl9 Cytokine 4.9 Ϫ1.7 U23922 II12rb1 Cytokine 2.4 2.5 Downloaded from

a Putative or known function based on annotations (41). b Over 2-fold difference detected repeatedly in the gene expression between wild-type (ϩ/ϩ) and STAT4-knockout (Ϫ/Ϫ) cells cultured in Th1 conditions. c Average fold change value. Also changes Ͻ2-fold are shown, if the change was seen repeatedly.

Genes regulated in response to Th1 polarizing stimuli after 48 h in all samples studied was Ifn␥. In the wild-type cells, Ifn␥ was http://www.jimmunol.org/ In addition to the immediately regulated genes, there were 57 strongly induced by IL-12, but in STAT4-knockout cells the ex- genes regulated in response to IL-12 after 48 h of polarization. The pression levels in Th1 conditions were at the same level as in CD3 ϩ genes affected by Th1 induction after 48 h of polarization included CD28-activated wild-type or STAT4-knockout cells cultured in 29 up-regulated and 28 down-regulated genes belonging to diverse “neutral” conditions. functional categories (Table III). Ifn␥ and IFN-regulated genes Iigp-pending and Icsbp were the only genes for which the over A subset of genes becomes up-regulated in response to IL-12 in 2-fold induction by IL-12 was maintained throughout all time the absence of STAT4 points studied (2, 6, and 48 h). In addition to the genes that were induced by IL-12 and STAT4 ␥ by guest on September 28, 2021 In addition to Ifn , of the genes that became regulated by IL-12 signaling, direct comparison of wild-type and STAT4-knockout after 48 h of Th1 differentiation, only Acadl, Gas5, Furin, Rrad, cells cultured in Th1-polarizing conditions revealed another group and Gja1 were clearly regulated (over 2-fold change) by STAT4. of genes that seemed to become expressed at enhanced levels in These genes were induced by IL-12 in wild-type cells, but not in response to IL-12 in the absence of STAT4 (Table IV, Genes that STAT4-deficient cells. Expression of genes Acadl and Gas5 was become up-regulated in response to IL-12 in the absence of defective in the STAT4-knockout mice already at the Th precursor STAT4). The genes Iigp-pending and Tgtp that were immediately (Thp) level (data not shown). Moreover, the induction of genes induced in response to IL-12 were among these genes. Also, how- Ctla2b, Serpin5, and Plac8 in response to IL-12 in the STAT4- ever, new similar genes for which the induction by IL-12 was seen knockout mice was not as strong as in wild-type mice. Thus, in- later (after 48 h) were identified. These genes included Ifit1, duction of these 8 later targets genes of IL-12 was dependent of Trim30, Isg15, Ifit2, Ms4a4c, Mx1, AA959954, Isg20, and Ifi202a. STAT4 (summarized in Table IV, IL-12/STAT4-induced genes). Some of the other genes regulated by IL-12 after 2, 6, or 48 h were also slightly differentially expressed by wild-type and Validation of the results with quantitative real-time RT-PCR STAT4-knockout Th1 cells (Tables II and III). These genes in- To validate the results, expression of a subset of genes including cluded immediate IL-12-regulated genes Igtp, Gbp1, Gbp2, Ifn␥, Irf1, Irf4, Icsbp, Gas5, and Iigp-pending was studied with AA816121, Ly6a, Ifi1, Gtpi-pending, Ifi47, and Cxcl9. Similarly, a real-time RT-PCR (Fig. 1). Concordant with the Affymetrix re- subset of genes regulated by IL-12 after 48 h of Th1 polarization sults, Ifn␥ was expressed at a lower level in the absence of STAT4 displayed small differences between wild-type and STAT4-knock- compared with the wild type. In the wild-type cells, the induction out cells. Such genes included Ifi202b, Ccr5, Ctla2b, AI853444, compared with the STAT4-knockout cells was 3.6-times higher Timp1, Casp6, Nfil3, Mcm5, Igh-6, Ebna1bp2, Scd1, Ifi30, Ephb4, already after 2 h and 6-times higher after6hofTh1polarization. and Slfn2. These genes did not fulfill the filtering criteria to be In STAT4-knockout cells induced to differentiate to the Th1 di- classified as STAT4-regulated genes, as no reproducible differ- rection during the first 6 h, the expression level of Ifn␥ remained ences (over 2-fold) between wild-type and STAT4-deficient cells at the same level as in wild-type cells cultured in neutral or Th2 were detected. However, as these genes showed smaller differ- conditions. Also, the reduced expression of Gas5 in STAT4- ences between wild-type and STAT4-knockout Th1 cells, it cannot knockout cells, compared with the wild-type cells cultured in Th1 be excluded that they would be regulated by STAT4. It is also conditions, was confirmed with real-time RT-PCR. A fold differ- possible that expression of these genes becomes indirectly affected ence as high as 12.7 ( p Ͻ 0.05) in the expression of Gas5 was seen in STAT4-knockout cells as a result of abnormal expression of already in Thp cells. Based on Affymetrix results, gene Iigp-pend- earlier STAT4 target genes, such as Acadl, Gas5,orIfn␥. ing showed increased (2.7-fold, p Ͻ 0.05) expression in the ab- Importantly, the only gene that was clearly regulated by IL-12 in sence of STAT4 after 48 h of Th1 polarization compared with the a STAT4-dependent manner at all time points (2, 6, and 48 h) and wild type. In TaqMan analysis, this difference was seen at low 6778 TARGET GENES OF STAT4 IN Th CELLS

Table III. Genes regulated in response to Th1-polarizing stimuli after 48 h

Th1ϩ/ϩ vs Th0ϩ/ϩ Th1Ϫ/Ϫ vs Th0Ϫ/Ϫ Th1Ϫ/Ϫ vs Th1ϩ/ϩ

Gene ID Functiona 2h 6h 48h 2h 6h 48h 2h 6h 48h

X61385 Tcf7 Transcription factor Ϫ1.2b Ϫ1.8 Ϫ2.2 Ϫ2.1 AF052505 Ccl22 Chemokine Ϫ1.4 Ϫ2.8 Ϫ2.4 AF109906 Ϫ2.4 2.4 1.3 M31418 Ifi202b 1.4 6.7 1.9 19.2 2.5 U54705 Serpinb5c Serine protease inhibitor 15.5 6.5 Ϫ4.1 X94151 Ccr5 11.8 Ϫ2.5 AF022990 Ccr5 Chemokine receptor 4.5 6.3 Ϫ1.9 AV370035 Ccr5 Chemokine receptor 1.8 7.1 6.6 Ϫ2.0 AA790307 Plac8c 7.1 4.5 Ϫ2.4 X15591 Ctla2ac 4.7 2.7 Ϫ2.3 AF014941 Ctsw Enzyme 4.5 X15592 Ctla2b 4.2 2.9 Ϫ2.1 M63801 Gja1c Gap junction protein 3.7 Ϫ2.1 D86232 Ly6c 3.6 3.6 Ϫ1.6 AF065947 Ccl5 Chemokine 3.1 7.2 X83106 Mad Transcription factor 3.0 AI853240 D230016N13Rik 2.9 Downloaded from AF055638 Gadd45g MAPK signaling 2.8 1.9 2.4 Ϫ2.6 AV319920 LOC269796 2.7 1.7 1.7 X62502 Ccl4 Chemokine 2.7 3.6 1.6 AI853444 2610042L04Rik 2.6 3.6 1.6 AI853444 2610042L04Rik 4.0 3.5 1.8 M12302 Gzmb Enzyme 2.5 1.9 3.6 Ϫ1.4 Ϫ

V00755 Timp1 Enzyme inhibitor 2.4 2.2 http://www.jimmunol.org/ Y13087 Casp6 Enzyme 2.4 1.7 Ϫ2.1 AI842264 2610311I19Rik 2.2 AI837679 Ormdl3 2.2 1.6 AI837621 Tm4sf13 2.1 1.8 AA688938 2410004N09Rik 2.1 AI464596 4930553M18Rik 2.0 1.9 M35247 H2-T17 2.0 2.1 U83148 Nfil3 Transcription factor 2.0 1.6 Ϫ1.8 U21489 Acadlc 2.0 Ϫ3.4 Ϫ3.5 Ϫ3.1 AI849615 Gas5c 1.9 Ϫ4.3 Ϫ4.1

AV377350 Rnh1 1.9 1.7 by guest on September 28, 2021 X54056 Furinc 1.8 Ϫ1.5 Ϫ2.1 AF084466 Rradc 1.8 Ϫ2.5 AA614914 TgoIn1 1.8 AW121031 D11Ertd175e Stuctural component Ϫ1.9 AF053367 Pdlim1 Transcription cofactor Ϫ2.1 AI839988 Atp1a1 ATPase Ϫ2.2 Ϫ1.8 AW123952 Atp1a1 ATPase Ϫ3.1 Ϫ2.2 D26090 Mcm5 ATPase, transcription Ϫ2.3 1.5 AB031292 Plp2 Ϫ2.3 1.1 V00821 Igh-6 Ag binding Ϫ2.3 Ϫ1.6 1.4 V00817 Igh-6 Ag binding Ϫ2.3 Ϫ1.5 AI845934 Ebna1bp2 Ϫ2.4 2.0 M21285 Scd1 Fatty acid synthesis Ϫ2.4 Ϫ1.6 1.8 M21285 Scd1 Fatty acid synthesis Ϫ2.2 Ϫ1.8 1.6 D90374 Apex1 Enzyme Ϫ2.4 V00833 H2-Ea MHC class II receptor activity Ϫ2.6 Ϫ3.6 J03928 PfkI Enzyme Ϫ2.6 AI838951 2610017G09Rik Ϫ2.7 AI846553 1110020C13Rik Ϫ3.0 M80423 Ϫ3.0 1.6 AI844520 Ifi30 Ag processing Ϫ3.2 AI837006 Cotl1 Cytoskeletal component Ϫ3.3 D49691 Lsp1 Cytoskeletal regulator Ϫ3.4 Ϫ1.6 U55060 Lgals9 Cell adhesion Ϫ3.5 1.6 1.4 U49739 Myo6 Cytoskeletal regulator Ϫ3.5 L24495 Tnfrsf7 Cell surface receptor Ϫ3.8 D00472 Cfl1 Cytoskeletal regulator Ϫ4.0 AW045533 Fdps Enzyme Ϫ4.4 X00496 Ii Chaperone activity Ϫ4.5 Ϫ2.4 U06834 Ephb4 Intracellular signaling Ϫ4.5 1.7 X52643 H2-Aa MHC class II receptor activity Ϫ5.0 Ϫ4.1 AF099973 Slfn2 Inhibitor of cell proliferation Ϫ5.2 1.4 AF099973 Slfn2 Inhibitor of cell proliferation Ϫ5.7 1.3

a Putative or known function based on Gene Ontology annotations (41); MAPK, mitogen-activated protein kinase. b Average fold change value. Also changes Ͻ2-fold were included, if the change was seen repeatedly. c Over 2-fold difference detected repeatedly in the gene expression between wild-type and STAT4-knockout cells cultured in Th1 conditions. The Journal of Immunology 6779

Table IV. Genes regulated by both IL-12 and STAT4

Th1ϩ/ϩ vs Th0ϩ/ϩ Th1Ϫ/Ϫ vs Th0Ϫ/Ϫ Th1Ϫ/Ϫ vs Th1ϩ/ϩ

Gene ID Functiona 2h 6h 48h 2h 6h 48h 2h 6h 48h

IL-12/STAT4-induced genes K00083 Ifn␥ Cytokine 3.9b 4.6 3.6 2.4 Ϫ4.6 Ϫ4.9 Ϫ3.4 U21489 Acadl Oxidoreductase 2.0 Ϫ3.4 Ϫ3.5 Ϫ3.1 AI849615 Gas5 1.9 Ϫ4.3 Ϫ4.1 X54056 Furin Subtilase, tyrosine kinase 1.8 Ϫ1.5 Ϫ2.1 AF084466 Rrad Ras GTPase superfamily 1.8 Ϫ2.5 M63801 Gja1 Gap-junction 3.7 Ϫ2.1 X15591 Ctla2a 4.7 2.7 Ϫ2.3 U54705 Serpinb5 Serine protease inhibitor 15.5 6.5 Ϫ4.1 AA790307 Plac8 7.1 6.7 Ϫ2.4 Genes that become up-regulated in response to IL-12 in the absence of STAT4 AA914345 Iigp-pending GTPase 3.6 2.5 3.7 3.0 6.5 2.6 AJ007971 Iigp-pending 4.1 2.1 3.0 4.1 3.6 11.7 Ϫ1.6 2.7 L38444 Tgtp GTPase 6.8 1.7 5.7 2.4 3.2 2.4 U43084 Ifit1 Ϫ1.4 2.9 4.1

J03776 Trim30 DNA binding 1.5 2.9 1.7 Downloaded from X56602 Isg15 1.5 4.5 2.0 U43085 Ifit2 1.7 3.0 2.2 AI462516 Ms4a4c 2.5 2.1 M21038 Mx1 GTPase 6.3 4.1 AA959954 9130009C22Rik Helicase, ATP binding 1.5 1.9 2.0 AW122677 Isg20 Exonuclease 1.3 2.5 2.7 M31418 Ifi202a NA 1.4 6.7 1.9 19.2 2.5 http://www.jimmunol.org/ a Putative or known function based on Gene Ontology annotations (41). b Average fold change value. Also changes Ͻ2-fold were included, if the change was seen repeatedly.

levels in only two of three biological repeats (fold changes 1.8, 1.7, Addition of IFN-␥ to STAT4-knockout Th1 cultures restores the and Ϫ2.2). Furthermore, at the 2-h time point Iigp-pending was defect in IFN-␥ production and enhances IFN-␥ production in preferentially expressed in wild-type cells (fold change 1.8, p Ͻ the absence of IL-12

0.05). Thus, the increased expression of Iigp-pending in the ab- by guest on September 28, 2021 The most interesting observation of this study was that Ifn␥ was sence of STAT4 could not be confirmed. the only gene for which the regulation by IL-12 and STAT4 sig- Affymetrix arrays did not detect any significant differences be- naling was seen at all the time points, indicating that it was the tween wild-type and STAT4-knockout cells cultured in Th1 con- earliest target of IL-12 and STAT4. Therefore, our hypothesis also ditions in the expression of Irf1, Irf4, and Icsbp, although Irf1 has was that the long-term defect in Th1 commitment in STAT4- been previously reported to be directly induced by STAT4 (24, knockout mice was due to the lack of optimal IFN-␥ levels during 30). The STAT4-independent induction of Irf1 was confirmed with real-time RT-PCR. Irf4 and Icsbp were both expressed temporarily early polarization. To test this hypothesis, we cultured wild-type at higher levels in wild-type cells cultured in Th1 conditions after and STAT4-knockout cells in neutral or in Th1 conditions in the ␥ ␥ 6 h of polarization compared with the cells deficient for STAT4. presence and absence of exogenous IFN- to see whether IFN- Irf4 was expressed Ϫ1.9-fold less ( p Ͻ 0.05) in wild-type Thp was able to restore the defect in Th1 polarization in STAT4-knock- cells compared with the STAT4-knockout cells. After2hofTh1 out cells. induction, Irf4 was induced 1.27-fold ( p Ͻ 0.05) and, after 6 h, In the cells activated with anti-CD3 and anti-CD28 and cultured ϩ ϩ 2.13-fold ( p Ͻ 0.05) more in wild-type cells than in knockout for one week in neutral conditions (Th0: anti-CD3 anti-CD28 cells. Icsbp was expressed at 1.7- to 5.8-fold higher levels ( p ϭ anti-IL-12 ϩ anti-IFN-␥ ϩ anti-IL-4), restimulation with PMA 0.115) in wild-type cells cultured for6hinTh1conditions than in and ionomycin induced similar levels of IFN-␥ production both in the STAT4-knockout cells. However, in all other time points, the wild-type and STAT4-knockout cells (Fig. 3a). As expected, in the induction of these genes was independent of STAT4. cells polarized with IL-12 to the Th1 direction (anti-CD3 ϩ anti- CD28 ϩ IL-12 ϩ anti-IL-4), IFN-␥ production in response to PMA and ionomycin was highly increased in wild-type cells, IFN-␥ induces expression of Irf1, Irf4, and Icsbp during early whereas in STAT4-deficient cells the production remained at the Th1 polarization independently of IL-12 basal level (Fig. 3b). Importantly, addition of exogenous IFN-␥ to Irf1, Irf4, and Icsbp were induced in response to IL-12 during early the cells cultured in Th1 conditions (anti-CD3 ϩ anti-CD28 ϩ Th1 induction, but regulation by STAT4 was not clear. Therefore, IL-12 ϩ IFN-␥ ϩ anti-IL-4) was able to restore the defect in in- we decided to study further whether early induction could be duction of IFN-␥ production in STAT4-knockout cells, whereas in driven by IFN-␥ instead of IL-12. The expression of Irf1, Irf4, and wild-type cells addition of IFN-␥ had no effect (Fig. 3c). Further- Icsbp was studied with real-time RT-PCR in the presence and ab- more, in the absence of STAT4 and IL-12, IFN-␥ alone (anti-CD3 sence of IFN-␥ as indicated in Fig. 2. Interestingly, the RT-PCR ϩ anti-CD28 ϩ anti-IL-12 ϩ anti-IL-4 ϩ IFN-␥) was able to analysis revealed that the immediate induction of Irf1, Irf4, and enhance IFN-␥ production to the similar levels measured in wild- Icsbp was indeed driven by IFN-␥ and IL-12 alone was unable to type Th1 cells. Interestingly, in wild-type cells, addition of IFN-␥ induce expression of these genes. alone did not have any effect and the production of IFN-␥ was 6780 TARGET GENES OF STAT4 IN Th CELLS Downloaded from http://www.jimmunol.org/

FIGURE 1. Validation of the results with real-time RT-PCR. CD4ϩ cells were isolated from spleen of wild-type and STAT4-knockout by guest on September 28, 2021 BALB/cJ mice. The cells were activated with plate-bound anti-CD3 and soluble anti-CD28 and cultured for 0, 2, 6, 24, 48 h, or 7 days in the presence of different cytokine combinations or neutralizing Abs as indi- cated in the figure. The gene expression levels were measured for the selected genes including Ifn␥, Gas5, Iigp-pending, Irf1, Irf4, and Icsbp with real-time RT-PCR. The gene expression levels were compared with ,ء .the levels in Thp cells and are represented as fold changes in the figure Statistically significant differences in gene expression levels between wild- type and STAT4-knockout cells cultured in Th1 conditions (paired t test: p Ͻ 0.05).

FIGURE 2. Immediate induction of Irf1, Irf4, and Icsbp is driven by comparable to that measured in the cells cultured in neutral con- IFN-␥. CD4ϩ cells were isolated from spleen of wild-type BALB/cJ mice. ditions (Fig. 3d). The cells were activated with plate-bound anti-CD3 and soluble anti-CD28 and were cultured for 0, 2, or6hinthepresence of different cytokine Discussion combinations or neutralizing Abs as indicated in the figure. The gene ex- Previous studies have shown IL-12 to be the key cytokine directing pression levels were measured for the genes Irf1, Irf4, and Icsbp with Th1 polarization (31). The effects of IL-12 are mediated through real-time RT-PCR. The gene expression levels were compared with the ,ء .STAT1, STAT3, STAT5, and STAT4 signaling of which only levels in Thp cells and are represented as fold changes in the figure STAT4 is required for Th1 differentiation (4–11). However, the Statistically significant differences in the gene expression levels in cells cultured under Th1 conditions in the absence or presence of IFN-␥ (paired mechanism detailing how IL-12 induces the Th1 differentiation has t test: p Ͻ 0.05). not been clear. In the current study, altogether 73 genes were iden- tified to be regulated in response to IL-12 during the first two days of Th1 polarization. Sixteen of these 73 genes were induced by IL-12 in wild-type mice already after 2 or6hofpolarization (Ta- driving the induction of immediate genes during the initiation of ble II). This suggests that these genes are likely to be regulated in Th1 differentiation. Interestingly, all of the 16 immediate IL-12- response to the first upstream factors, such as STAT4, activated in regulated genes, with the exception of unknown AA816121, have Th1 conditions. However, of these immediate IL-12-regulated previously been described to be regulated by IFNs. In fact it is genes only IFN-␥ was clearly regulated through STAT4 signaling possible that these early induced STAT4-independent genes are during the initiation of Th1 polarization. This indicates that in up-regulated in response to IFN-␥ and STAT1, as IFN-␥ was neu- addition to STAT4, other upstream factors are also involved in tralized in cultures of the CD3 ϩ CD28-activated cells used as a The Journal of Immunology 6781

tion factors Irf1, Irf4, and Icsbp, which have essential roles in regulation of Th1 differentiation (21–23, 32–37). The expression profiles between the first hours and 2 days of Th1 polarization were different from each other and the only genes that were up-regulated over 2-fold in Th1 conditions at all time points (2, 6, and 48 h) were Ifn␥, Iigp-pending, and Icsbp. The number of IL-12-regulated genes was increased after 2 days of polarization probably due to activation of secondary response fac- tors or as a consequence of enhancement of IL-12 signaling in response to IL-12R induction. This is concordant with the previous studies that have shown that the subset of receptor IL-12R␤2, which transmits the signals inside the cell, is not expressed on naive Th cells, but is induced in response to Ag stimulation (2, 3). The defects in gene expression in the STAT4-knockout mice were mainly seen after 48 h. Of the genes that were regulated in response to IL-12 after 48 h (Table III), Acadl, Gas5, Furin, Rrad, Gja1, Ctla2b, Serpinb5, and Plac8 were regulated in a STAT4- dependent manner (summarized in Table IV, IL-12/STAT4-in-

duced genes). Acadl and Gas5 showed impaired expression al- Downloaded from ready at the Thp stage and thus could also have implications in the early Th1 differentiation and defective gene expression in the STAT4-knockout mice. The role of Acadl and Gas5 in immune response is unknown. Acadl is implicated in fatty acid metabolism (38, 39). Gas5 is preferentially expressed in growth-arrested cells,

but is not believed to encode for a protein (38, 39). Also, the role http://www.jimmunol.org/ of these other six genes induced by IL-12 and STAT4 in Th1 differentiation is unknown. In addition to IL-12- and STAT4-induced genes, expression of a subset of 11 genes was enhanced in response to IL-12 in the absence of STAT4 (Table IV, Genes that become up-regulated in response to IL-12 in the absence of STAT4). Nearly all the genes in this group were known IFN-regulated genes. Interestingly, ex- FIGURE 3. IFN-␥ is able to partly restore its own production in ϩ pression of most of these genes including Iigp-pending, Tgtp, STAT4-knockout mice. CD4 cells were isolated from spleen of wild-type Trim30, Ifit1, Isg15, Isg20, and Ifi202a is repressed by IL-4 during by guest on September 28, 2021 BALB/cJ mice or STAT4-knockout mice. The cells were activated with early Th2 polarization. Furthermore, genes Isg15, Isg20, Tgtp, plate-bound anti-CD3 and soluble anti-CD28 and cultured for 7 days in the presence of indicated cytokine combinations or neutralizing Abs (a) anti- Trim30, and Ifi202a are also repressed by STAT6 (40). Thus, the CD3 ϩ anti-CD28 ϩ anti-IL-4 ϩ anti-IL-12 ϩ anti-IFN-␥; b, anti-CD3 ϩ down-regulation of these genes must be important both for Th1 anti-CD28 ϩ IL-12 ϩ anti-IL-4; c, anti-CD3 ϩ anti-CD28 ϩ IL-12 ϩ and Th2 differentiation. anti-IL-4 ϩ IFN-␥; d, anti-CD3 ϩ anti-CD28 ϩ IFN-␥ ϩ anti-IL-12 ϩ As the major defect in STAT4-knockout cells was in reduced anti-IL-4). For the intracellular cytokine detection with anti-IFN-␥-FITC, expression levels of Ifn␥, we studied whether the defect in Th1 the cells were restimulated with PMA and ionomycin. Isotype controls polarization was a consequence of the reduced levels of IFN-␥ were used as controls to calculate the number of IFN-␥-producing cells. during the immediate response. The flow cytometric analysis dem- onstrated that addition of exogenous IFN-␥ to the Th1 cultures of STAT4-knockout cells restored the defect in IFN-␥ production, but control. However, IFN-␥ was not neutralized in the cells polarized in wild-type cells addition of IFN-␥ had no effect. This indicates to the Th1 direction. Thus, IFN-␥/STAT1 signaling could be reg- that in the absence of STAT4, IFN-␥ is able to compensate for the ulating induction of these genes. As a conclusion, initiation of Th1 IL-12- and STAT4-mediated induction of its own production, re- differentiation involves STAT4-dependent induction of Ifn␥ and placing the role that is normally conducted by IL-12. This was up-regulation of a subset of genes, which are primarily induced in further supported by the observation according to which IFN-␥ a STAT4-independent manner, presumably through STAT1 alone was able to induce normal levels of IFN-␥ production in the signaling. Th1 conditions in STAT4-knockout cells, but not in wild-type Among the genes that were immediately up-regulated in cells cells. Thus, it seems that the ability of IFN-␥ to induce its own cultured in Th1-inducing conditions, there were three transcription production in the absence of STAT4 is normally inhibited by factors, Irf1, Irf4, and Icsbp. These genes were shown to be in- STAT4. Interestingly, according to the Affymetrix results in duced in fact by IFN-␥ and not by IL-12. All these Irf1, Irf4, and STAT4 knockout cells, a subset of IFN-regulated genes were ab- Icsbp transcription factors play an essential role in Th1 differen- normally induced in response to Th1 induction. It is possible that tiation. Mice deficient for Irf1 have impaired Th1 differentiation in the absence of an inhibitory effect of STAT4, the induction of and show a defect in responsiveness to IL-12 (32, 33). Irf4 is these genes is driven by basal levels of IFN-␥ produced by the required for Th2 differentiation to induce Gata-3 expression in Th2 knockout cells. The enhancement of IFN-␥ production by itself in cells and inhibit Th1 development (34, 35). Icsbp is required for STAT4-knockout cells is also consistent with the previous obser- production of IL-12, generation of IFN-producing cells and an vation demonstrating that IFN-␥ was able to induce expression of optimal amount of CD8␣ϩ dendritic cells, which preferentially T-bet in STAT4-knockout cells (21, 22). promote Th1 differentiation (36, 37). Thus, early induction of Th1 Although the requirement of STAT4 in the development of ef- differentiation involves IFN-␥-mediated up-regulation of transcrip- fector Th1 cells has been demonstrated, the role of STAT4 during 6782 TARGET GENES OF STAT4 IN Th CELLS early Th1 polarization has been unclear (10, 11, 21, 22). In the 18. Szabo, S. J., S. T. Kim, G. L. Costa, X. Zhang, C. G. 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