IL-12 signaling through STAT4 is essential for induction of optimal levels of IFN-γ 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, Ifnγ 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-γ independently of STAT4. Importantly, addition of exogenous IFN-γ into Th1 cell cultures of STAT4-knockout cells restored the defect in IFN-γ production further demonstrating the critical role of IFN-γ in early Th1 differentiation.

Interleukin-12 is a cytokine involved in regulation of cell-mediated immune responses and induction of Th1 differentiation. The effects of IL-12 are mediated through IL-12R consisting of two subunits IL-12Rβ1 and IL-12Rβ2 (1). IL-12Rβ2, which transmits the signals inside the cell, is not expressed on naive Th cells, but is induced in response to Ag stimulation and is selectively down-regulated during Th2 differentiation (2, 3). Triggering of IL-12R leads to induction of tyrosine phosphorylation and DNA binding of Janus kinase 2, tyrosine kinase 2, and STAT4 (4, 5, 6). Also STAT1, STAT3, and STAT5 are tyrosine phosphorylated in response to IL-12 (6, 7, 8, 9, 10). However, only STAT4 has been shown to be necessary for the long-term commitment of Th1 cells, as mice deficient for STAT4 show impaired Th1 and enhanced Th2 differentiation (10, 11). In addition to IL-12, the only cytokines known to induce STAT4 phosphorylation are IFN-α and IL-23 (12, 13, 14, 15, 16).

Although STAT4 has been shown to be required for the long-term Th1 differentiation and IFN-γ production, cells deficient for both STAT6 and STAT4 can differentiate to functional IFN-γ-producing Th1 cells (17). Transcription factor T-box expressed in T cells (T-bet)3 may be involved in this STAT4-independent Th1 differentiation. T-bet has been shown to be required for Th1 differentiation and its expression is sufficient to induce IFN-γ production in Th cells (18, 19). Initial expression of T-bet is induced by IFN-γ and STAT1 signaling during the activation of Th cells independently of STAT4 (20, 21, 22). Activation of T-bet during Th1 differentiation leads to remodeling of the Ifnγ locus, induction of IFN-γ production, and IL-12Rβ2 expression essential for STAT4-mediated IL-12 signaling (18, 22, 23).

Although both STAT1 and STAT4 signaling are contributing to the early Th1 polarization, only the requirement of STAT4 for long-term Th1 development has been clearly demonstrated (10, 11). However, the exact role of STAT4 in the early induction of Th1 cell differentiation is still unclear. Recent studies indicate that instead of being the primary factor inducing Th1 differentiation, STAT4 would rather be involved in enhancing initial IFN-γ production to optimal levels (21, 22, 23). Whether the effects of STAT4 are restricted to regulation of IFN-γ or whether it also regulates other factors involved in inducing Th1 differentiation is unknown. Previously described target genes of STAT4 include macrophage inflammatory protein-1α (Mip-1α, Scya3), macrophage inflammatory protein-1β (Mip-1β, Ccl4, Scya4, Act-2), IL-1RA (Il1rn), IFN regulatory factor 1 (IRF1), Ets-related transcription factor (ERM), CCR5, and IL-18R (24, 25, 26, 27). In a recent study, the target genes of IL-12 and STAT4 in already polarized Th1 cells were described (28). The aim of this study was to elucidate the mechanism of early Th1 differentiation by identifying the immediate and early upstream genes regulated in response to IL-12 and STAT4 signaling.

STAT4-knockout mice and wild-type controls with a BALB/cJ background were purchased from The Jackson Laboratory (Bar Harbor, ME). The mice used in the studies were sacrificed at the age of 6–8 wk.

The murine mononuclear cells were isolated from spleen single cell suspension using Lympholyte-M gradient (Cedarlane Laboratories, Hornby, Canada). The CD4+ cells were further purified using magnetic MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany). For induction of Th1 polarization pooled CD4+ cells were cultured in Iscove’s DMEM containing 10% FCS, nonessential amino acids, and 2-ME (Life Technologies, Paisley, Scotland) in the presence of plate-bound anti-mouse CD3 (315 ng/well), soluble anti-mouse CD28 (500 ng/ml; BD PharMingen, San Diego, CA), recombinant mouse IL-12 (10 ng/ml; R&D Systems, Minneapolis, MN) and rat anti-mouse IL-4 (10 μg/ml; BD PharMingen). Part of the cells were activated with anti-CD3 and -CD28 and cultured in “neutral” conditions in the presence of rat anti-mouse IL-4 (10 μg/ml; BD PharMingen), rat anti-mouse IL-12 (10 μg/ml; BD PharMingen), and hamster anti-mouse IFN-γ (10 μg/ml; BD PharMingen). The cultures were conducted in parallel for both the cells deficient for STAT4 and the wild-type controls. Samples were collected at the time points of 0, 2, 6, and 48 h. For the real-time RT-PCR or cytokine secretion assay, the cells were cultured in the presence of plate-bound anti-mouse CD3 (315 ng/well), soluble anti-mouse CD28 (500 ng/ml), and indicated combinations of cytokines IL-4, IL-12, IFN-γ (500 U; BD PharMingen) and neutralizing Abs for IL-12, IFN-γ, and IL-4.

The total RNAs were isolated using the TRIzol method (Invitrogen, Carlsbad, CA) and were further purified with the Qiagen RNAeasy minikit (Qiagen, Valencia, CA). For the Affymetrix sample preparation, 5 μg of total RNA was used as starting material. The sample preparation was performed according to the instructions and recommendations provided by the manufacturer (Affymetrix, Santa Clara, CA). The samples were hybridized to MG-U74Av2 arrays containing 12,488 probes for different genes. The data was analyzed with Affymetrix Microarray Suite version 5 (MAS5) software and filtered according to recommendations of the manufacturer. Briefly, at the expression level, the statistical algorithm used by MAS5 defined each gene-specific probe set to be present, absent, or marginal. At the comparison level, each probe set was classified as not changed, increased, marginally increased, decreased, or marginally decreased. The genes that were absent in both treatments compared or the genes that were not changed between the comparisons were excluded from the results. Furthermore, only the genes that presented a consistent change in two or three biological repeats and showed fold changes of ≥2 or ≤−2 (signal log ratio ≥1 or ≤−1) in at least one of the time points studied were considered as differentially expressed.

Primers and probes were designed for the genes studied using Primer Express software (Applied Biosystems, Foster City, CA). Gene expression levels were measured for the selected panel of genes (Table I) at each time point using TaqMan real-time quantitative PCR (RT-PCR; ABI Prism 7700; Applied Biosystems) as previously described (29). A TaqMan PCR Core Reagent kit (Applied Biosystems) was used for the preparation of the RT-PCR mixtures. The primers and probes (MedProbe, Oslo, Norway) were used in the final concentrations of 300 and 200 nM, respectively. The steps in the quantitative RT-PCR were 50°C for 2 min, 95°C for 10 min, 95°C for 15 min, and 60°C for 1 min, and altogether 40 cycles were run. All measurements were performed in duplicate in two separate runs using samples derived from at least two or three biological repeats.

Table I.

Primers and probes used in real-time RT-PCR

GenBank AccessionSymbol1) 5′-6(FAM)-PROBE-(TAMRA)-3′
2) 5′-PRIMER 1-3′
3) 5′-PRIMER 2-3′
K00083 Ifnγ 1) 5′-AGGATGCATTCATGAGTATTGCCAAGTTTGA-3′ 
  2) 5′-CAGCAACAGCAAGGCGAAA-3′ 
  3) 5′-CTGGACCTGTGGGTTGTTGAC-3′ 
M21065 Irf1 1) 5′-CTCCGAAGCCGCAACAGACGAGG-3′ 
  2) 5′-GTGTCACCCATGCCTTCCA-3′ 
  3) 5′-AGCTTCATAAGGTCTTCGGCTATC-3′ 
U11692 Irf4 1) 5′-ACCCGGACGACAATGGACAGAGGA-3′ 
  2) 5′-ACCTGGACCAGGTCCTGTTTC-3′ 
  3) 5′-GGCTCAGCAACTTCTCAATGTTC-3′ 
NM_008320 Icsbp (Irf81) 5′-TCCTCAATCTCTGAGCGGCCACACT-3′ 
  2) 5′-GCATGAGCGAAGTTCCTGAGAT-3′ 
  3) 5′-ATGTACTCATCCACAGAAGGTTCCT-3′ 
X59728 Gas5 1) 5′-CACATTGCGCTCGCTCTGTTATCCAG-3′ 
  2) 5′-ATGAAGGCTTACGAGGACTCGT-3′ 
  3) 5′-GCCTCAAACTCCACCATTTCAT-3′ 
  3) 5′-TTATTTGTACCTTTCAAACTGTCTATCCA-3′ 
NM_021792 Iigp-pending 1) 5′-ATCCTTCCAGCCAATCCTCTGCTTCAG-3′ 
  2) 5′-GTCATTGAAAAGAAGCGGCAAT-3′ 
  3) 5′-TCAGAGAAGGGATGATATTCACTAGGT-3′ 
GenBank AccessionSymbol1) 5′-6(FAM)-PROBE-(TAMRA)-3′
2) 5′-PRIMER 1-3′
3) 5′-PRIMER 2-3′
K00083 Ifnγ 1) 5′-AGGATGCATTCATGAGTATTGCCAAGTTTGA-3′ 
  2) 5′-CAGCAACAGCAAGGCGAAA-3′ 
  3) 5′-CTGGACCTGTGGGTTGTTGAC-3′ 
M21065 Irf1 1) 5′-CTCCGAAGCCGCAACAGACGAGG-3′ 
  2) 5′-GTGTCACCCATGCCTTCCA-3′ 
  3) 5′-AGCTTCATAAGGTCTTCGGCTATC-3′ 
U11692 Irf4 1) 5′-ACCCGGACGACAATGGACAGAGGA-3′ 
  2) 5′-ACCTGGACCAGGTCCTGTTTC-3′ 
  3) 5′-GGCTCAGCAACTTCTCAATGTTC-3′ 
NM_008320 Icsbp (Irf81) 5′-TCCTCAATCTCTGAGCGGCCACACT-3′ 
  2) 5′-GCATGAGCGAAGTTCCTGAGAT-3′ 
  3) 5′-ATGTACTCATCCACAGAAGGTTCCT-3′ 
X59728 Gas5 1) 5′-CACATTGCGCTCGCTCTGTTATCCAG-3′ 
  2) 5′-ATGAAGGCTTACGAGGACTCGT-3′ 
  3) 5′-GCCTCAAACTCCACCATTTCAT-3′ 
  3) 5′-TTATTTGTACCTTTCAAACTGTCTATCCA-3′ 
NM_021792 Iigp-pending 1) 5′-ATCCTTCCAGCCAATCCTCTGCTTCAG-3′ 
  2) 5′-GTCATTGAAAAGAAGCGGCAAT-3′ 
  3) 5′-TCAGAGAAGGGATGATATTCACTAGGT-3′ 

For the cytokine secretion assay the cells cultured for 7 days were restimulated at a density of 1 × 106 in 0.5 ml in IMDM + 10% FCS containing PMA (50 ng/ml; Calbiochem, La Jolla, CA) and ionomycin (500 ng/ml; Calbiochem). Part of the cells were maintained as unstimulated controls cultured otherwise identically to stimulated cells. After stimulation, the cells were washed (0.5% BSA + 0.01% azide in PBS) and incubated with anti-CD4-PE (Caltag Laboratories, Burlingame, CA) for 15 min. The cells were washed and fixed with 4% paraformaldehyde for 15 min after which they were permeabilized (0.5% saponin + 0.5% BSA + 0.01% azide in PBS, pH 7) for 10 min and washed again. After the permeabilization, intracellular cytokine staining was performed with anti-IFN-γ-FITC (BD Biosciences, San Jose, CA) for 10–20 min. The cytokine profiles of the cells were studied with FACScan and CellQuest software (BD Biosciences).

Affymetrix murine U74Av2 arrays, representing probes for ∼10,000 different genes, were used to study the target genes of IL-12 and STAT4 signaling during the early polarization of Th1 lymphocytes. To identify the genes regulated by IL-12, cells cultured in Th1 conditions (anti-CD3 + anti-CD28 + IL-12 + anti-IL-4) were compared with the CD3 + CD28-activated cells cultured in the “neutral” conditions (anti-CD3 + anti-CD28 + anti-IFN-γ + anti-IL-12 + anti-IL-4).

Altogether 16 genes were immediately up-regulated in the cells polarized to differentiate to Th1 cells for 2 or 6 h compared with the CD3/CD28-activated cells (Table II). These early response genes included Iigp-pending, Icsbp, Irf1, Gbp1, Gbp2, AA816121, Tgtp, Ifi1, Igtp, Gtpi-pending, Ifi47, Irf4, Ly6a, Cxcl9, Il12rb1, and Ifnγ. The strongest induction by IL-12 was seen in the up-regulation of Ifnγ.

Table II.

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

Gene IDFunctionaTh1+/+ vs Th0+/+Th1−/− vs Th0−/−Th1−/− vs Th1+/+
2 h6 h48 h2 h6 h48 h2 h6 h48 h
K00083 Ifnγb Cytokine 3.9c 4.6 3.6   2.4 −4.3 −4.9 −3.4 
AJ007971 Iigp-pendingb GTPase 5.2 2.1 3.0 4.1 3.6 8.2 −1.6  2.7 
AA914345 Iigp-pendingb GTPase 3.6 2.5  3.7 3.0 6.5 −1.9  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  −1.5  
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 −1.4 2.9 1.8    1.9 
U20949 Irf4 Transcription factor 2.1   1.4     1.3 
M34815 Cxcl9 Cytokine  4.9      −1.7  
U23922 II12rb1 Cytokine receptor  2.4   2.5     
Gene IDFunctionaTh1+/+ vs Th0+/+Th1−/− vs Th0−/−Th1−/− vs Th1+/+
2 h6 h48 h2 h6 h48 h2 h6 h48 h
K00083 Ifnγb Cytokine 3.9c 4.6 3.6   2.4 −4.3 −4.9 −3.4 
AJ007971 Iigp-pendingb GTPase 5.2 2.1 3.0 4.1 3.6 8.2 −1.6  2.7 
AA914345 Iigp-pendingb GTPase 3.6 2.5  3.7 3.0 6.5 −1.9  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  −1.5  
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 −1.4 2.9 1.8    1.9 
U20949 Irf4 Transcription factor 2.1   1.4     1.3 
M34815 Cxcl9 Cytokine  4.9      −1.7  
U23922 II12rb1 Cytokine receptor  2.4   2.5     
a

Putative or known function based on Gene Ontology annotations (41 ).

b

Over 2-fold difference detected repeatedly in the gene expression between wild-type (+/+) and STAT4-knockout (−/−) cells cultured in Th1 conditions.

c

Average fold change value. Also changes <2-fold are shown, if the change was seen repeatedly.

To determine which of these genes regulated in response to Th1 induction were also targets of STAT4, expression profiles of wild-type and STAT4-knockout cells cultured in Th1 conditions were compared. Of the 16 genes that were immediately induced in response to IL-12 treatment, only Ifnγ was clearly regulated through STAT4 signaling. In addition, induction of Cxcl9 and Ly6a by IL-12 was defective in the STAT4-knockout cells compared with the wild-type cells. All the other immediate IL-12-regulated genes were induced by IL-12, also in STAT4-knockout cells, indicating that early induction of these other genes is not dependent of STAT4 (Table II).

In addition to the immediately regulated genes, there were 57 genes regulated in response to IL-12 after 48 h of polarization. The genes affected by Th1 induction after 48 h of polarization included 29 up-regulated and 28 down-regulated genes belonging to diverse functional categories (Table III). Ifnγ and IFN-regulated genes Iigp-pending and Icsbp were the only genes for which the over 2-fold induction by IL-12 was maintained throughout all time points studied (2, 6, and 48 h).

Table III.

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

Gene IDFunctionaTh1+/+ vs Th0+/+Th1−/− vs Th0−/−Th1−/− vs Th1+/+
2 h6 h48 h2 h6 h48 h2 h6 h48 h
X61385 Tcf7 Transcription factor  −1.2b −1.8   −2.2   −2.1 
AF052505 Ccl22 Chemokine  −1.4 −2.8   −2.4    
AF109906    −2.4 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   −4.1 
X94151 Ccr5 Chemokine receptor   11.8      −2.5 
AF022990 Ccr5 Chemokine receptor   4.5   6.3   −1.9 
AV370035 Ccr5 Chemokine receptor  1.8 7.1   6.6   −2.0 
AA790307 Plac8c    7.1   4.5   −2.4 
X15591 Ctla2ac    4.7   2.7   −2.3 
AF014941 Ctsw Enzyme   4.5       
X15592 Ctla2b    4.2   2.9   −2.1 
M63801 Gja1c Gap junction protein   3.7      −2.1 
D86232 Ly6c    3.6   3.6   −1.6 
AF065947 Ccl5 Chemokine   3.1   7.2    
X83106 Mad Transcription factor   3.0       
AI853240 D230016N13Rik    2.9       
AF055638 Gadd45g MAPK signaling   2.8 1.9  2.4   −2.6 
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 −1.4   
V00755 Timp1 Enzyme inhibitor   2.4      −2.2 
Y13087 Casp6 Enzyme   2.4   1.7   −2.1 
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   −1.8 
U21489 Acadlc    2.0    −3.4 −3.5 −3.1 
AI849615 Gas5c    1.9    −4.3  −4.1 
AV377350 Rnh1    1.9   1.7    
X54056 Furinc    1.8     −1.5 −2.1 
AF084466 Rradc    1.8      −2.5 
AA614914 TgoIn1    1.8       
AW121031 D11Ertd175e Stuctural component   −1.9       
AF053367 Pdlim1 Transcription cofactor   −2.1       
AI839988 Atp1a1 ATPase   −2.2   −1.8    
AW123952 Atp1a1 ATPase   −3.1   −2.2    
D26090 Mcm5 ATPase, transcription   −2.3      1.5 
AB031292 Plp2    −2.3      1.1 
V00821 Igh-6 Ag binding   −2.3   −1.6   1.4 
V00817 Igh-6 Ag binding   −2.3   −1.5    
AI845934 Ebna1bp2    −2.4      2.0 
M21285 Scd1 Fatty acid synthesis   −2.4   −1.6   1.8 
M21285 Scd1 Fatty acid synthesis   −2.2   −1.8   1.6 
D90374 Apex1 Enzyme   −2.4       
V00833 H2-Ea MHC class II receptor activity   −2.6   −3.6    
J03928 PfkI Enzyme   −2.6       
AI838951 2610017G09Rik    −2.7       
AI846553 1110020C13Rik    −3.0       
M80423     −3.0      1.6 
AI844520 Ifi30 Ag processing   −3.2       
AI837006 Cotl1 Cytoskeletal component   −3.3       
D49691 Lsp1 Cytoskeletal regulator   −3.4   −1.6    
U55060 Lgals9 Cell adhesion   −3.5   1.6   1.4 
U49739 Myo6 Cytoskeletal regulator   −3.5       
L24495 Tnfrsf7 Cell surface receptor   −3.8       
D00472 Cfl1 Cytoskeletal regulator   −4.0       
AW045533 Fdps Enzyme   −4.4       
X00496 Ii Chaperone activity   −4.5   −2.4    
U06834 Ephb4 Intracellular signaling   −4.5      1.7 
X52643 H2-Aa MHC class II receptor activity   −5.0   −4.1    
AF099973 Slfn2 Inhibitor of cell proliferation   −5.2      1.4 
AF099973 Slfn2 Inhibitor of cell proliferation   −5.7      1.3 
Gene IDFunctionaTh1+/+ vs Th0+/+Th1−/− vs Th0−/−Th1−/− vs Th1+/+
2 h6 h48 h2 h6 h48 h2 h6 h48 h
X61385 Tcf7 Transcription factor  −1.2b −1.8   −2.2   −2.1 
AF052505 Ccl22 Chemokine  −1.4 −2.8   −2.4    
AF109906    −2.4 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   −4.1 
X94151 Ccr5 Chemokine receptor   11.8      −2.5 
AF022990 Ccr5 Chemokine receptor   4.5   6.3   −1.9 
AV370035 Ccr5 Chemokine receptor  1.8 7.1   6.6   −2.0 
AA790307 Plac8c    7.1   4.5   −2.4 
X15591 Ctla2ac    4.7   2.7   −2.3 
AF014941 Ctsw Enzyme   4.5       
X15592 Ctla2b    4.2   2.9   −2.1 
M63801 Gja1c Gap junction protein   3.7      −2.1 
D86232 Ly6c    3.6   3.6   −1.6 
AF065947 Ccl5 Chemokine   3.1   7.2    
X83106 Mad Transcription factor   3.0       
AI853240 D230016N13Rik    2.9       
AF055638 Gadd45g MAPK signaling   2.8 1.9  2.4   −2.6 
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 −1.4   
V00755 Timp1 Enzyme inhibitor   2.4      −2.2 
Y13087 Casp6 Enzyme   2.4   1.7   −2.1 
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   −1.8 
U21489 Acadlc    2.0    −3.4 −3.5 −3.1 
AI849615 Gas5c    1.9    −4.3  −4.1 
AV377350 Rnh1    1.9   1.7    
X54056 Furinc    1.8     −1.5 −2.1 
AF084466 Rradc    1.8      −2.5 
AA614914 TgoIn1    1.8       
AW121031 D11Ertd175e Stuctural component   −1.9       
AF053367 Pdlim1 Transcription cofactor   −2.1       
AI839988 Atp1a1 ATPase   −2.2   −1.8    
AW123952 Atp1a1 ATPase   −3.1   −2.2    
D26090 Mcm5 ATPase, transcription   −2.3      1.5 
AB031292 Plp2    −2.3      1.1 
V00821 Igh-6 Ag binding   −2.3   −1.6   1.4 
V00817 Igh-6 Ag binding   −2.3   −1.5    
AI845934 Ebna1bp2    −2.4      2.0 
M21285 Scd1 Fatty acid synthesis   −2.4   −1.6   1.8 
M21285 Scd1 Fatty acid synthesis   −2.2   −1.8   1.6 
D90374 Apex1 Enzyme   −2.4       
V00833 H2-Ea MHC class II receptor activity   −2.6   −3.6    
J03928 PfkI Enzyme   −2.6       
AI838951 2610017G09Rik    −2.7       
AI846553 1110020C13Rik    −3.0       
M80423     −3.0      1.6 
AI844520 Ifi30 Ag processing   −3.2       
AI837006 Cotl1 Cytoskeletal component   −3.3       
D49691 Lsp1 Cytoskeletal regulator   −3.4   −1.6    
U55060 Lgals9 Cell adhesion   −3.5   1.6   1.4 
U49739 Myo6 Cytoskeletal regulator   −3.5       
L24495 Tnfrsf7 Cell surface receptor   −3.8       
D00472 Cfl1 Cytoskeletal regulator   −4.0       
AW045533 Fdps Enzyme   −4.4       
X00496 Ii Chaperone activity   −4.5   −2.4    
U06834 Ephb4 Intracellular signaling   −4.5      1.7 
X52643 H2-Aa MHC class II receptor activity   −5.0   −4.1    
AF099973 Slfn2 Inhibitor of cell proliferation   −5.2      1.4 
AF099973 Slfn2 Inhibitor of cell proliferation   −5.7      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 <2-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.

In addition to Ifnγ, of the genes that became regulated by IL-12 after 48 h of Th1 differentiation, only Acadl, Gas5, Furin, Rrad, and Gja1 were clearly regulated (over 2-fold change) by STAT4. These genes were induced by IL-12 in wild-type cells, but not in STAT4-deficient cells. Expression of genes Acadl and Gas5 was defective in the STAT4-knockout mice already at the Th precursor (Thp) level (data not shown). Moreover, the induction of genes Ctla2b, Serpin5, and Plac8 in response to IL-12 in the STAT4-knockout mice was not as strong as in wild-type mice. Thus, induction of these 8 later targets genes of IL-12 was dependent of STAT4 (summarized in Table IV, IL-12/STAT4-induced genes).

Table IV.

Genes regulated by both IL-12 and STAT4

Gene IDFunctionaTh1+/+ vs Th0+/+Th1−/− vs Th0−/−Th1−/− vs Th1+/+
2 h6 h48 h2 h6 h48 h2 h6 h48 h
IL-12/STAT4-induced genes            
 K00083 Ifnγ Cytokine 3.9b 4.6 3.6   2.4 −4.6 −4.9 −3.4 
 U21489 Acadl Oxidoreductase   2.0    −3.4 −3.5 −3.1 
 AI849615 Gas5    1.9    −4.3  −4.1 
 X54056 Furin Subtilase, tyrosine kinase   1.8     −1.5 −2.1 
 AF084466 Rrad Ras GTPase superfamily   1.8      −2.5 
 M63801 Gja1 Gap-junction   3.7      −2.1 
 X15591 Ctla2a    4.7   2.7   −2.3 
 U54705 Serpinb5 Serine protease inhibitor   15.5   6.5   −4.1 
 AA790307 Plac8    7.1   6.7   −2.4 
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 −1.6  2.7 
 L38444 Tgtp GTPase 6.8 1.7  5.7 2.4 3.2   2.4 
 U43084 Ifit1   −1.4    2.9   4.1 
 J03776 Trim30 DNA binding   1.5   2.9   1.7 
 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 
Gene IDFunctionaTh1+/+ vs Th0+/+Th1−/− vs Th0−/−Th1−/− vs Th1+/+
2 h6 h48 h2 h6 h48 h2 h6 h48 h
IL-12/STAT4-induced genes            
 K00083 Ifnγ Cytokine 3.9b 4.6 3.6   2.4 −4.6 −4.9 −3.4 
 U21489 Acadl Oxidoreductase   2.0    −3.4 −3.5 −3.1 
 AI849615 Gas5    1.9    −4.3  −4.1 
 X54056 Furin Subtilase, tyrosine kinase   1.8     −1.5 −2.1 
 AF084466 Rrad Ras GTPase superfamily   1.8      −2.5 
 M63801 Gja1 Gap-junction   3.7      −2.1 
 X15591 Ctla2a    4.7   2.7   −2.3 
 U54705 Serpinb5 Serine protease inhibitor   15.5   6.5   −4.1 
 AA790307 Plac8    7.1   6.7   −2.4 
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 −1.6  2.7 
 L38444 Tgtp GTPase 6.8 1.7  5.7 2.4 3.2   2.4 
 U43084 Ifit1   −1.4    2.9   4.1 
 J03776 Trim30 DNA binding   1.5   2.9   1.7 
 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 
a

Putative or known function based on Gene Ontology annotations (41 ).

b

Average fold change value. Also changes <2-fold were included, if the change was seen repeatedly.

Some of the other genes regulated by IL-12 after 2, 6, or 48 h were also slightly differentially expressed by wild-type and STAT4-knockout Th1 cells (Tables II and III). These genes included immediate IL-12-regulated genes Igtp, Gbp1, Gbp2, AA816121, Ly6a, Ifi1, Gtpi-pending, Ifi47, and Cxcl9. Similarly, a subset of genes regulated by IL-12 after 48 h of Th1 polarization displayed small differences between wild-type and STAT4-knockout cells. Such genes included Ifi202b, Ccr5, Ctla2b, AI853444, Timp1, Casp6, Nfil3, Mcm5, Igh-6, Ebna1bp2, Scd1, Ifi30, Ephb4, and Slfn2. These genes did not fulfill the filtering criteria to be classified as STAT4-regulated genes, as no reproducible differences (over 2-fold) between wild-type and STAT4-deficient cells were detected. However, as these genes showed smaller differences between wild-type and STAT4-knockout Th1 cells, it cannot be excluded that they would be regulated by STAT4. It is also possible that expression of these genes becomes indirectly affected in STAT4-knockout cells as a result of abnormal expression of earlier STAT4 target genes, such as Acadl, Gas5, or Ifnγ.

Importantly, the only gene that was clearly regulated by IL-12 in a STAT4-dependent manner at all time points (2, 6, and 48 h) and in all samples studied was Ifnγ. In the wild-type cells, Ifnγ was strongly induced by IL-12, but in STAT4-knockout cells the expression levels in Th1 conditions were at the same level as in CD3 + CD28-activated wild-type or STAT4-knockout cells cultured in “neutral” conditions.

In addition to the genes that were induced by IL-12 and STAT4 signaling, direct comparison of wild-type and STAT4-knockout cells cultured in Th1-polarizing conditions revealed another group of genes that seemed to become expressed at enhanced levels 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). The genes Iigp-pending and Tgtp that were immediately induced in response to IL-12 were among these genes. Also, however, new similar genes for which the induction by IL-12 was seen later (after 48 h) were identified. These genes included Ifit1, Trim30, Isg15, Ifit2, Ms4a4c, Mx1, AA959954, Isg20, and Ifi202a.

To validate the results, expression of a subset of genes including Ifnγ, Irf1, Irf4, Icsbp, Gas5, and Iigp-pending was studied with real-time RT-PCR (Fig. 1). Concordant with the Affymetrix results, Ifnγ was expressed at a lower level in the absence of STAT4 compared with the wild type. In the wild-type cells, the induction compared with the STAT4-knockout cells was 3.6-times higher already after 2 h and 6-times higher after 6 h of Th1 polarization. In STAT4-knockout cells induced to differentiate to the Th1 direction during the first 6 h, the expression level of Ifnγ remained at the same level as in wild-type cells cultured in neutral or Th2 conditions. Also, the reduced expression of Gas5 in STAT4-knockout cells, compared with the wild-type cells cultured in Th1 conditions, was confirmed with real-time RT-PCR. A fold difference as high as 12.7 (p < 0.05) in the expression of Gas5 was seen already in Thp cells. Based on Affymetrix results, gene Iigp-pending showed increased (2.7-fold, p < 0.05) expression in the absence of STAT4 after 48 h of Th1 polarization compared with the wild type. In TaqMan analysis, this difference was seen at low levels in only two of three biological repeats (fold changes 1.8, 1.7, and −2.2). Furthermore, at the 2-h time point Iigp-pending was preferentially expressed in wild-type cells (fold change 1.8, p < 0.05). Thus, the increased expression of Iigp-pending in the absence of STAT4 could not be confirmed.

FIGURE 1.

Validation of the results with real-time RT-PCR. CD4+ cells were isolated from spleen of wild-type and STAT4-knockout 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 indicated in the figure. The gene expression levels were measured for the selected genes including Ifnγ, Gas5, Iigp-pending, Irf1, Irf4, and Icsbp with real-time RT-PCR. The gene expression levels were compared with 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 < 0.05).

FIGURE 1.

Validation of the results with real-time RT-PCR. CD4+ cells were isolated from spleen of wild-type and STAT4-knockout 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 indicated in the figure. The gene expression levels were measured for the selected genes including Ifnγ, Gas5, Iigp-pending, Irf1, Irf4, and Icsbp with real-time RT-PCR. The gene expression levels were compared with 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 < 0.05).

Close modal

Affymetrix arrays did not detect any significant differences between wild-type and STAT4-knockout cells cultured in Th1 conditions in the expression of Irf1, Irf4, and Icsbp, although Irf1 has been previously reported to be directly induced by STAT4 (24, 30). The STAT4-independent induction of Irf1 was confirmed with real-time RT-PCR. Irf4 and Icsbp were both expressed temporarily at higher levels in wild-type cells cultured in Th1 conditions after 6 h of polarization compared with the cells deficient for STAT4. Irf4 was expressed −1.9-fold less (p < 0.05) in wild-type Thp cells compared with the STAT4-knockout cells. After 2 h of Th1 induction, Irf4 was induced 1.27-fold (p < 0.05) and, after 6 h, 2.13-fold (p < 0.05) more in wild-type cells than in knockout cells. Icsbp was expressed at 1.7- to 5.8-fold higher levels (p = 0.115) in wild-type cells cultured for 6 h in Th1 conditions than in the STAT4-knockout cells. However, in all other time points, the induction of these genes was independent of STAT4.

Irf1, Irf4, and Icsbp were induced in response to IL-12 during early Th1 induction, but regulation by STAT4 was not clear. Therefore, we decided to study further whether early induction could be driven by IFN-γ instead of IL-12. The expression of Irf1, Irf4, and Icsbp was studied with real-time RT-PCR in the presence and absence of IFN-γ as indicated in Fig. 2. Interestingly, the RT-PCR analysis revealed that the immediate induction of Irf1, Irf4, and Icsbp was indeed driven by IFN-γ and IL-12 alone was unable to induce expression of these genes.

FIGURE 2.

Immediate induction of Irf1, Irf4, and Icsbp is driven by IFN-γ. CD4+ cells were isolated from spleen of wild-type BALB/cJ mice. The cells were activated with plate-bound anti-CD3 and soluble anti-CD28 and were cultured for 0, 2, or 6 h in the presence of different cytokine combinations or neutralizing Abs as indicated in the figure. The gene expression levels were measured for the genes Irf1, Irf4, and Icsbp with real-time RT-PCR. The gene expression levels were compared with the levels in Thp cells and are represented as fold changes in the figure. ∗, Statistically significant differences in the gene expression levels in cells cultured under Th1 conditions in the absence or presence of IFN-γ (paired t test: p < 0.05).

FIGURE 2.

Immediate induction of Irf1, Irf4, and Icsbp is driven by IFN-γ. CD4+ cells were isolated from spleen of wild-type BALB/cJ mice. The cells were activated with plate-bound anti-CD3 and soluble anti-CD28 and were cultured for 0, 2, or 6 h in the presence of different cytokine combinations or neutralizing Abs as indicated in the figure. The gene expression levels were measured for the genes Irf1, Irf4, and Icsbp with real-time RT-PCR. The gene expression levels were compared with the levels in Thp cells and are represented as fold changes in the figure. ∗, Statistically significant differences in the gene expression levels in cells cultured under Th1 conditions in the absence or presence of IFN-γ (paired t test: p < 0.05).

Close modal

The most interesting observation of this study was that Ifnγ was the only gene for which the regulation by IL-12 and STAT4 signaling was seen at all the time points, indicating that it was the earliest target of IL-12 and STAT4. Therefore, our hypothesis also was that the long-term defect in Th1 commitment in STAT4-knockout mice was due to the lack of optimal IFN-γ levels during early polarization. To test this hypothesis, we cultured wild-type and STAT4-knockout cells in neutral or in Th1 conditions in the presence and absence of exogenous IFN-γ to see whether IFN-γ was able to restore the defect in Th1 polarization in STAT4-knockout cells.

In the cells activated with anti-CD3 and anti-CD28 and cultured for one week in neutral conditions (Th0: anti-CD3 + anti-CD28 + anti-IL-12 + anti-IFN-γ + anti-IL-4), restimulation with PMA and ionomycin induced similar levels of IFN-γ production both in wild-type and STAT4-knockout cells (Fig. 3,a). As expected, in the cells polarized with IL-12 to the Th1 direction (anti-CD3 + anti-CD28 + IL-12 + anti-IL-4), IFN-γ production in response to PMA and ionomycin was highly increased in wild-type cells, whereas in STAT4-deficient cells the production remained at the basal level (Fig. 3,b). Importantly, addition of exogenous IFN-γ to the cells cultured in Th1 conditions (anti-CD3 + anti-CD28 + IL-12 + IFN-γ + anti-IL-4) was able to restore the defect in induction of IFN-γ production in STAT4-knockout cells, whereas in wild-type cells addition of IFN-γ had no effect (Fig. 3,c). Furthermore, in the absence of STAT4 and IL-12, IFN-γ alone (anti-CD3 + anti-CD28 + anti-IL-12 + anti-IL-4 + IFN-γ) was able to enhance IFN-γ production to the similar levels measured in wild-type Th1 cells. Interestingly, in wild-type cells, addition of IFN-γ alone did not have any effect and the production of IFN-γ was comparable to that measured in the cells cultured in neutral conditions (Fig. 3 d).

FIGURE 3.

IFN-γ is able to partly restore its own production in STAT4-knockout mice. CD4+ cells were isolated from spleen of wild-type BALB/cJ mice or STAT4-knockout mice. The cells were activated with 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-CD3 + anti-CD28 + anti-IL-4 + anti-IL-12 + anti-IFN-γ; b, anti-CD3 + anti-CD28 + IL-12 + anti-IL-4; c, anti-CD3 + anti-CD28 + IL-12 + anti-IL-4 + IFN-γ; d, anti-CD3 + anti-CD28 + IFN-γ + anti-IL-12 + anti-IL-4). For the intracellular cytokine detection with anti-IFN-γ-FITC, the cells were restimulated with PMA and ionomycin. Isotype controls were used as controls to calculate the number of IFN-γ-producing cells.

FIGURE 3.

IFN-γ is able to partly restore its own production in STAT4-knockout mice. CD4+ cells were isolated from spleen of wild-type BALB/cJ mice or STAT4-knockout mice. The cells were activated with 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-CD3 + anti-CD28 + anti-IL-4 + anti-IL-12 + anti-IFN-γ; b, anti-CD3 + anti-CD28 + IL-12 + anti-IL-4; c, anti-CD3 + anti-CD28 + IL-12 + anti-IL-4 + IFN-γ; d, anti-CD3 + anti-CD28 + IFN-γ + anti-IL-12 + anti-IL-4). For the intracellular cytokine detection with anti-IFN-γ-FITC, the cells were restimulated with PMA and ionomycin. Isotype controls were used as controls to calculate the number of IFN-γ-producing cells.

Close modal

Previous studies have shown IL-12 to be the key cytokine directing Th1 polarization (31). The effects of IL-12 are mediated through STAT1, STAT3, STAT5, and STAT4 signaling of which only STAT4 is required for Th1 differentiation (4, 5, 6, 7, 8, 9, 10, 11). However, the mechanism detailing how IL-12 induces the Th1 differentiation has not been clear. In the current study, altogether 73 genes were identified 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 or 6 h of polarization (Table II). This suggests that these genes are likely to be regulated in response to the first upstream factors, such as STAT4, activated in Th1 conditions. However, of these immediate IL-12-regulated genes only IFN-γ was clearly regulated through STAT4 signaling during the initiation of Th1 polarization. This indicates that in addition to STAT4, other upstream factors are also involved in driving the induction of immediate genes during the initiation of Th1 differentiation. Interestingly, all of the 16 immediate IL-12-regulated genes, with the exception of unknown AA816121, have previously been described to be regulated by IFNs. In fact it is possible that these early induced STAT4-independent genes are up-regulated in response to IFN-γ and STAT1, as IFN-γ was neutralized in cultures of the CD3 + CD28-activated cells used as a control. However, IFN-γ was not neutralized in the cells polarized to the Th1 direction. Thus, IFN-γ/STAT1 signaling could be regulating induction of these genes. As a conclusion, initiation of Th1 differentiation involves STAT4-dependent induction of Ifnγ and up-regulation of a subset of genes, which are primarily induced in a STAT4-independent manner, presumably through STAT1 signaling.

Among the genes that were immediately up-regulated in cells cultured in Th1-inducing conditions, there were three transcription factors, Irf1, Irf4, and Icsbp. These genes were shown to be induced in fact by IFN-γ and not by IL-12. All these Irf1, Irf4, and Icsbp transcription factors play an essential role in Th1 differentiation. Mice deficient for Irf1 have impaired Th1 differentiation and show a defect in responsiveness to IL-12 (32, 33). Irf4 is required for Th2 differentiation to induce Gata-3 expression in Th2 cells and inhibit Th1 development (34, 35). Icsbp is required for production of IL-12, generation of IFN-producing cells and an optimal amount of CD8α+ dendritic cells, which preferentially promote Th1 differentiation (36, 37). Thus, early induction of Th1 differentiation involves IFN-γ-mediated up-regulation of transcription factors Irf1, Irf4, and Icsbp, which have essential roles in regulation of Th1 differentiation (21, 22, 23, 32, 33, 34, 35, 36, 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 Ifnγ, 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 factors 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-12Rβ2, 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-induced genes). Acadl and Gas5 showed impaired expression already 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 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, expression of most of these genes including Iigp-pending, Tgtp, Trim30, Ifit1, Isg15, Isg20, and Ifi202a is repressed by IL-4 during early Th2 polarization. Furthermore, genes Isg15, Isg20, Tgtp, Trim30, and Ifi202a are also repressed by STAT6 (40). Thus, the down-regulation of these genes must be important both for Th1 and Th2 differentiation.

As the major defect in STAT4-knockout cells was in reduced expression levels of Ifnγ, we studied whether the defect in Th1 polarization was a consequence of the reduced levels of IFN-γ during the immediate response. The flow cytometric analysis demonstrated that addition of exogenous IFN-γ to the Th1 cultures of STAT4-knockout cells restored the defect in IFN-γ production, but in wild-type cells addition of IFN-γ had no effect. This indicates that in the absence of STAT4, IFN-γ is able to compensate for the IL-12- and STAT4-mediated induction of its own production, replacing the role that is normally conducted by IL-12. This was further supported by the observation according to which IFN-γ alone was able to induce normal levels of IFN-γ production in the Th1 conditions in STAT4-knockout cells, but not in wild-type cells. Thus, it seems that the ability of IFN-γ to induce its own production in the absence of STAT4 is normally inhibited by STAT4. Interestingly, according to the Affymetrix results in STAT4 knockout cells, a subset of IFN-regulated genes were abnormally induced in response to Th1 induction. It is possible that in the absence of an inhibitory effect of STAT4, the induction of these genes is driven by basal levels of IFN-γ produced by the knockout cells. The enhancement of IFN-γ production by itself in STAT4-knockout cells is also consistent with the previous observation demonstrating that IFN-γ was able to induce expression of T-bet in STAT4-knockout cells (21, 22).

Although the requirement of STAT4 in the development of effector Th1 cells has been demonstrated, the role of STAT4 during early Th1 polarization has been unclear (10, 11, 21, 22). In the current study, we have for the first time examined the role of STAT4 in regulation of gene expression during the first steps of Th1 differentiation in the global scale. The results demonstrate that IFN-γ indeed is the first gene induced by IL-12 in a STAT4-dependent fashion and thus is likely to be the primary force inducing Th1 polarization in response to IL-12. Importantly, we demonstrate that the defect in Th1 polarization in STAT4-knockout mice can be restored by adding exogenous IFN-γ to the developing Th1 cells highlighting the importance of the cytokine in Th1 polarization.

We thank Marju Niskala, Outi Melin, and Miina Miller for technical assistance and Elizabeth Carpelan for language revision.

1

This work was supported by the Academy of Finland, Turku Graduate School of Biomedical Sciences, Drug Discovery Graduate School, Ida Montin Foundation, the Finnish Society of Allergology and Immunology, National Technology Agency of Finland.

3

Abbreviations used in this paper: T-bet, T-box expressed in T cells; IRF, IFN regulatory factor; Thp, Th precursor.

1
Presky, D. H., H. Yang, L. J. Minetti, A. O. Chua, N. Nabavi, C. Y. Wu, M. K. Gately, U. Gubler.
1996
. A functional interleukin 12 receptor complex is composed of two β-type cytokine receptor subunits.
Proc. Natl. Acad. Sci. USA
93
:
14002
.
2
Szabo, S. J., A. S. Dighe, U. Gubler, K. M. Murphy.
1997
. Regulation of the interleukin (IL)-12R β2 subunit expression in developing T helper 1 (Th1) and Th2 cells.
J. Exp. Med.
185
:
817
.
3
Rogge, L., L. Barberis-Maino, M. Biffi, N. Passini, D. H. Presky, U. Gubler, F. Sinigaglia.
1997
. Selective expression of an interleukin-12 receptor component by human T helper 1 cells.
J. Exp. Med.
185
:
825
.
4
Bacon, C. M., D. W. McVicar, J. R. Ortaldo, R. C. Rees, J. J. O’Shea, J. A. Johnston.
1995
. Interleukin 12 (IL-12) induces tyrosine phosphorylation of JAK2 and TYK2: differential use of Janus family tyrosine kinases by IL-2 and IL-12.
J. Exp. Med.
181
:
399
.
5
Bacon, C. M., E. F. Petricoin, 3rd, J. R. Ortaldo, R. C. Rees, A. C. Larner, J. A. Johnston, J. J. O’Shea.
1995
. Interleukin 12 induces tyrosine phosphorylation and activation of STAT4 in human lymphocytes.
Proc. Natl. Acad. Sci. USA
92
:
7307
.
6
Jacobson, N. G., S. J. Szabo, R. M. Weber-Nordt, Z. Zhong, R. D. Schreiber, J. E. Darnell, Jr, K. M. Murphy.
1995
. Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4.
J. Exp. Med.
181
:
1755
.
7
Szabo, S. J., N. G. Jacobson, A. S. Dighe, U. Gubler, K. M. Murphy.
1995
. Developmental commitment to the Th2 lineage by extinction of IL-12 signaling.
Immunity
2
:
665
.
8
Yu, C. R., J. X. Lin, D. W. Fink, S. Akira, E. T. Bloom, A. Yamauchi.
1996
. Differential utilization of Janus kinase-signal transducer activator of transcription signaling pathways in the stimulation of human natural killer cells by IL-2, IL-12, and IFN-α.
J. Immunol.
157
:
126
.
9
Gollob, J. A., E. A. Murphy, S. Mahajan, C. P. Schnipper, J. Ritz, D. A. Frank.
1998
. Altered interleukin-12 responsiveness in Th1 and Th2 cells is associated with the differential activation of STAT5 and STAT1.
Blood
91
:
1341
.
10
Kaplan, M. H., Y. L. Sun, T. Hoey, M. J. Grusby.
1996
. Impaired IL-12 responses and enhanced development of Th2 cells in Stat4-deficient mice.
Nature
382
:
174
.
11
Thierfelder, W. E., J. M. van Deursen, K. Yamamoto, R. A. Tripp, S. R. Sarawar, R. T. Carson, M. Y. Sangster, D. A. Vignali, P. C. Doherty, G. C. Grosveld, J. N. Ihle.
1996
. Requirement for Stat4 in interleukin-12-mediated responses of natural killer and T cells.
Nature
382
:
171
.
12
Cho, S. S., C. M. Bacon, C. Sudarshan, R. C. Rees, D. Finbloom, R. Pine, J. J. O’Shea.
1996
. Activation of STAT4 by IL-12 and IFN-α: evidence for the involvement of ligand-induced tyrosine and serine phosphorylation.
J. Immunol.
157
:
4781
.
13
Nguyen, K. B., W. T. Watford, R. Salomon, S. R. Hofmann, G. C. Pien, A. Morinobu, M. Gadina, J. J. O’Shea, C. A. Biron.
2002
. Critical role for STAT4 activation by type 1 interferons in the interferon-γ response to viral infection.
Science
297
:
2063
.
14
Freudenberg, M. A., T. Merlin, C. Kalis, Y. Chvatchko, H. Stubig, C. Galanos.
2002
. Cutting edge: a murine, IL-12-independent pathway of IFN-γ induction by Gram-negative bacteria based on STAT4 activation by type I IFN and IL-18 signaling.
J. Immunol.
169
:
1665
.
15
Parham, C., M. Chirica, J. Timans, E. Vaisberg, M. Travis, J. Cheung, S. Pflanz, R. Zhang, K. P. Singh, F. Vega, et al
2002
. A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rβ1 and a novel cytokine receptor subunit, IL-23R.
J. Immunol.
168
:
5699
.
16
Oppmann, B., R. Lesley, B. Blom, J. C. Timans, Y. Xu, B. Hunte, F. Vega, N. Yu, J. Wang, K. Singh, et al
2000
. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12.
Immunity
13
:
715
.
17
Kaplan, M. H., A. L. Wurster, M. J. Grusby.
1998
. A signal transducer and activator of transcription (Stat)4-independent pathway for the development of T helper type 1 cells.
J. Exp. Med.
188
:
1191
.
18
Szabo, S. J., S. T. Kim, G. L. Costa, X. Zhang, C. G. Fathman, L. H. Glimcher.
2000
. A novel transcription factor, T-bet, directs Th1 lineage commitment.
Cell
100
:
655
.
19
Szabo, S. J., B. M. Sullivan, C. Stemmann, A. R. Satoskar, B. P. Sleckman, L. H. Glimcher.
2002
. Distinct effects of T-bet in TH1 lineage commitment and IFN-γ production in CD4 and CD8 T cells.
Science
295
:
338
.
20
Grogan, J. L., M. Mohrs, B. Harmon, D. A. Lacy, J. W. Sedat, R. M. Locksley.
2001
. Early transcription and silencing of cytokine genes underlie polarization of T helper cell subsets.
Immunity
14
:
205
.
21
Lighvani, A. A., D. M. Frucht, D. Jankovic, H. Yamane, J. Aliberti, B. D. Hissong, B. V. Nguyen, M. Gadina, A. Sher, W. E. Paul, J. J. O’Shea.
2001
. T-bet is rapidly induced by interferon-γ in lymphoid and myeloid cells.
Proc. Natl. Acad. Sci. USA
98
:
15137
.
22
Afkarian, M., J. R. Sedy, J. Yang, N. G. Jacobson, N. Cereb, S. Y. Yang, T. L. Murphy, K. M. Murphy.
2002
. T-bet is a STAT1-induced regulator of IL-12R expression in naive CD4+ T cells.
Nat. Immunol.
3
:
549
.
23
Mullen, A. C., F. A. High, A. S. Hutchins, H. W. Lee, A. V. Villarino, D. M. Livingston, A. L. Kung, N. Cereb, T. P. Yao, S. Y. Yang, S. L. Reiner.
2001
. Role of T-bet in commitment of TH1 cells before IL-12-dependent selection.
Science
292
:
1907
.
24
Galon, J., C. Sudarshan, S. Ito, D. Finbloom, J. J. O’Shea.
1999
. IL-12 induces IFN regulating factor-1 (IRF-1) gene expression in human NK and T cells.
J. Immunol.
162
:
7256
.
25
Ouyang, W., N. G. Jacobson, D. Bhattacharya, J. D. Gorham, D. Fenoglio, W. C. Sha, T. L. Murphy, K. M. Murphy.
1999
. The Ets transcription factor ERM is Th1-specific and induced by IL-12 through a Stat4-dependent pathway.
Proc. Natl. Acad. Sci. USA
96
:
3888
.
26
Iwasaki, M., T. Mukai, C. Nakajima, Y. F. Yang, P. Gao, N. Yamaguchi, M. Tomura, H. Fujiwara, T. Hamaoka.
2001
. A mandatory role for STAT4 in IL-12 induction of mouse T cell CCR5.
J. Immunol.
167
:
6877
.
27
Lawless, V. A., S. Zhang, O. N. Ozes, H. A. Bruns, I. Oldham, T. Hoey, M. J. Grusby, M. H. Kaplan.
2000
. Stat4 regulates multiple components of IFN-γ-inducing signaling pathways.
J. Immunol.
165
:
6803
.
28
Hoey, T., S. Zhang, N. Schmidt, Q. Yu, S. Ramchandani, X. Xu, L. K. Naeger, Y. L. Sun, M. H. Kaplan.
2003
. Distinct requirements for the naturally occurring splice forms Stat4α and Stat4β in IL-12 responses.
EMBO J.
22
:
4237
.
29
Lund, R. J., E. K. Ylikoski, T. Aittokallio, O. Nevalainen, R. Lahesmaa.
2003
. Kinetics and STAT4- or STAT6-mediated regulation of genes involved in lymphocyte polarization to Th1 and Th2 cells.
Eur. J. Immunol.
33
:
1105
.
30
Coccia, E. M., N. Passini, A. Battistini, C. Pini, F. Sinigaglia, L. Rogge.
1999
. Interleukin-12 induces expression of interferon regulatory factor-1 via signal transducer and activator of transcription-4 in human T helper type 1 cells.
J. Biol. Chem.
274
:
6698
.
31
Manetti, R., F. Gerosa, M. G. Giudizi, R. Biagiotti, P. Parronchi, M. P. Piccinni, S. Sampognaro, E. Maggi, S. Romagnani, G. Trinchieri, et al
1994
. Interleukin 12 induces stable priming for interferon γ (IFN-γ) production during differentiation of human T helper (Th) cells and transient IFN-γ production in established Th2 cell clones.
J. Exp. Med.
179
:
1273
.
32
Lohoff, M., D. Ferrick, H. W. Mittrucker, G. S. Duncan, S. Bischof, M. Rollinghoff, T. W. Mak.
1997
. Interferon regulatory factor-1 is required for a T helper 1 immune response in vivo.
Immunity
6
:
681
.
33
Taki, S., T. Sato, K. Ogasawara, T. Fukuda, M. Sato, S. Hida, G. Suzuki, M. Mitsuyama, E. H. Shin, S. Kojima, et al
1997
. Multistage regulation of Th1-type immune responses by the transcription factor IRF-1.
Immunity
6
:
673
.
34
Tominaga, N., K. Ohkusu-Tsukada, H. Udono, R. Abe, T. Matsuyama, K. Yui.
2003
. Development of Th1 and not Th2 immune responses in mice lacking IFN-regulatory factor-4.
Int. Immunol.
15
:
1
.
35
Lohoff, M., H. W. Mittrucker, S. Prechtl, S. Bischof, F. Sommer, S. Kock, D. A. Ferrick, G. S. Duncan, A. Gessner, T. W. Mak.
2002
. Dysregulated T helper cell differentiation in the absence of interferon regulatory factor 4.
Proc. Natl. Acad. Sci. USA
99
:
11808
.
36
Giese, N. A., L. Gabriele, T. Doherty, D. Klinman, L. Tadesse-Heath, C. Contursi, S. L. Epstein, H. C. r. Morse.
1997
. Interferon (IFN) consensus sequence-binding protein, a transcription factor of the IFN regulatory factor family, regulates immune responses in vivo through control of interleukin 12 expression.
J. Exp. Med.
186
:
1535
.
37
Schiavoni, G., F. Mattei, P. Sestili, P. Borghi, M. Venditti, H. C. Morse, 3rd, F. Belardelli, L. Gabriele.
2002
. ICSBP is essential for the development of mouse type I interferon-producing cells and for the generation and activation of CD8α+ dendritic cells.
J. Exp. Med.
196
:
1415
.
38
Kurtz, D. M., R. J. Tolwani, P. A. Wood.
1998
. Structural characterization of the mouse long-chain acyl-CoA dehydrogenase gene and 5′ regulatory region.
Mamm. Genome
9
:
361
.
39
Raho, G., V. Barone, D. Rossi, L. Philipson, V. Sorrentino.
2000
. The gas 5 gene shows four alternative splicing patterns without coding for a protein.
Gene
256
:
13
.
40
Chen, Z., R. Lund, T. Aittokallio, M. Kosonen, O. Nevalainen, R. Lahesmaa.
2003
. Identification of novel IL-4/Stat6-regulated genes in T lymphocytes.
J. Immunol.
171
:
3627
.
41
Ashburner, M., C. A. Ball, J. A. Blake, D. Botstein, H. Butler, J. M. Cherry, A. P. Davis, K. Dolinski, S. S. Dwight, J. T. Eppig, et al
2000
. Gene ontology: tool for the unification of biology: the Gene Ontology Consortium.
Nat. Genet.
25
:
25
.