IL-12, but Not IFN-?, Promotes STAT4 Activation and Th1
Development in Murine CD4?T Cells Expressing a Chimeric
Murine/Human Stat2 Gene1
Meredith E. Persky,* Kenneth M. Murphy,†‡and J. David Farrar2*
Humans and mice have evolved distinct pathways for Th1 cell development. Although IL-12 promotes CD4?Th1 development in
both murine and human T cells, IFN-?? drives Th1 development only in human cells. This IFN-??-dependent pathway is not
conserved in the mouse species due in part to a specific mutation within murine Stat2. Restoration of this pathway in murine T
cells would provide the opportunity to more closely model specific human disease states that rely on CD4?T cell responses to
IFN-??. To this end, the C terminus of murine Stat2, harboring the mutation, was replaced with the corresponding human Stat2
sequence by a knockin targeting strategy within murine embryonic stem cells. Chimeric m/h Stat2 knockin mice were healthy, bred
normally, and exhibited a normal lymphoid compartment. Furthermore, the murine/human STAT2 protein was expressed in
murine CD4?T cells and was activated by murine IFN-? signaling. However, the murine/human STAT2 protein was insufficient
to restore full IFN-?-driven Th1 development as defined by IFN-? expression. Furthermore, IL-12, but not IFN-?, promoted acute
IFN-? secretion in collaboration with IL-18 stimulation in both CD4?and CD8?T cells. The inability of T cells to commit to Th1
development correlated with the lack of STAT4 phosphorylation in response to IFN-?. This finding suggests that, although the C
terminus of human STAT2 is required for STAT4 recruitment and activation by the human type I IFNAR (IFN-??R), it is not
sufficient to restore this process through the murine IFNAR complex. The Journal of Immunology, 2005, 174: 294–301.
tokines (1). These developmental cues are directed by innate cy-
tokines secreted by professional APCs responding to pathogens (2,
3). For example, macrophages and dendritic cells responding to
Gram-negative bacteria secrete high levels of IL-12 and IL-18 (in-
nate cytokines) and present Ag to naive CD4?T cells (4). IL-12
directs T cell development to the Th1 phenotype (5) through the
activation of a key second messenger, STAT4 (6, 7). The Th1
phenotype is characterized by secretion of high concentrations of
IFN-? at sites of inflammation. Furthermore, IFN-? secreted by
Th1 cells mediates the elimination of both intracellular and extra-
cellular bacterial pathogens through the activation of granulocytes
and phagocytic cells.
The importance of Th1 cells in the immune response to patho-
gens is highlighted by the fact that this developmental pathway is
conserved in mammalian species. For CD4?T cells, IL-12 sig-
naling and STAT4 activation is a conserved pathway that regulates
the first steps of commitment to IFN-? expression (6–9). However,
in humans, in addition to IL-12, type I IFNs (IFN-??) also signal
defining hallmark of an adaptive immune response is
the orchestrated development of naive CD4?T cells into
effector populations that secrete distinct subsets of cy-
through STAT4 (10–12) and promote IFN-? secretion in CD4?T
cells (12–15). Furthermore, this pathway is not conserved and/or is
not efficient in murine CD4?T cells (11, 12). Although recent
studies have called this observation into question (16–18), clearly
the murine (m)3IFN-??R (IFNAR) is inefficient at promoting Th1
development and requires extremely high concentrations of IFN-?
to detect this effect in murine T cells. The lack of an experimen-
tally tractable mouse model of IFN-?-driven Th1 development is
a major barrier to understanding human immune responses to
pathogens that promote IFN-?? secretion, such as viruses.
Our previous studies have provided a molecular explanation for
the lack of IFN-??-dependent Th1 development in mouse CD4?
T cells (11, 19, 20). Unlike the IL-12R, our studies of the human
(h)IFNAR demonstrated that recruitment and activation of STAT4
by the hIFNAR is mediated by STAT2 (11). STAT2 is recruited to
the hIFNAR by an Src homology 2 (SH2)-dependent interaction
with phosphorylated Y466 within the hIFNAR1 subunit (21, 22).
In contrast, although STAT4 requires an intact SH2 domain for
efficient activation by the hIFNAR (19), it does not interact di-
rectly with any phosphorylated tyrosine residues within either the
hIFNAR1 or R2 subunit (11). Rather, STAT4 recruitment occurs
in a STAT2-dependent manner, possibly by an either direct or
indirect interaction with Y833 and Y841 within the C terminus of
human STAT2 (19). This interaction does not occur in mouse due
to a significant mutation and insertion of a repetitive minisatellite
sequence within the C terminus of murine STAT2. As such, murine
STAT2 fails to mediate STAT4 activation by the hIFNAR. Impor-
tantly, expression of a chimeric m/h STAT2 molecule, whereby the C
terminus of the murine sequence has been replaced with the human
counterpart, restores IFN-??-dependent STAT4 phosphorylation in
STAT2-deficient human fibroblasts (19).
*Center for Immunology and Department of Molecular Biology, University of Texas
Southwestern Medical Center, Dallas, TX 75390;†Department of Pathology and Cen-
ter for Immunology, and‡Howard Hughes Medical Institute, Washington University
School of Medicine, St. Louis, MO 63110
Received for publication April 16, 2004. Accepted for publication October 26, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by grants from Howard Hughes Medical Institute and the
National Institutes of Health awarded to K.M.M., and by a grant from the Leukemia
and Lymphoma Society and start-up funds from Howard Hughes Medical Institute
awarded to J.D.F.
2Address correspondence and reprint requests to Dr. J. David Farrar, Center for Immu-
nology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard,
Dallas, TX 75390-9093. E-mail address: David.Farrar@UTSouthwestern.edu
3Abbreviations used in this paper: m, murine; h, human; KI, knockin; IFNAR, IFN-
??R; SH2, Src homology 2; ES cell, embryonic stem cell; ISGF3, IFN-sensitive gene
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
Based on these results and the lack of IFN-??-dependent Th1
responses observed in murine CD4?T cells (12, 23), we proposed
that mice and humans might respond differently to pathogens such
as viruses that primarily evoke IFN-?? secretion, as opposed to
IL-12, from innate cells (20). A definitive test of this hypothesis
involves reconstructing the IFN-?? pathway to activate STAT4 in
murine T cells. As a first step, we report here the development of
a chimeric m/h Stat2 knockin (KI) mouse. The exon encoding the
murine STAT2 C terminus, exon 23, was replaced with the cor-
responding human sequence by a targeted mutation of murine em-
bryonic stem (ES) cells. Surprisingly, and in contrast to recent
reports, we found that murine CD4?T cells from m/h Stat2 KI
mice were not able to activate STAT4 or commit to IFN-? expres-
sion in response to mIFN-?, even at extremely high concentrations
of the cytokine. These results would suggest that an additional
species-specific component is required for the efficient recruitment
of STAT4 to the mIFNAR.
Materials and Methods
IL-12p40?/?(24) were purchased from The Jackson Laboratory and back-
crossed five generations onto the DO11.10 TCR transgenic (BALB/c back-
Generation of m/h Stat2 KI mice
A 129/SvJ bacterial artificial chromosome clone harboring the murine
Stat2 gene was obtained by screening a bacterial artificial chromosome
library with a probe generated from the 5? end of the murine Stat2 cDNA
(Genome Systems). A 20-kb fragment of the 3? region of murine Stat2 was
subcloned into pBluescript (Stratagene) and extensively characterized by
restriction mapping and sequencing. The 5? region of the targeting con-
struct was assembled with three segments that included the replacement of
exon 23 sequence with the corresponding human Stat2 C terminus sequence.
A 4-kb HindIII/AflII genomic fragment was cloned upstream of a 450-bp
AflII/BamHI-digested human Stat2 PCR fragment that was amplified with
primers 5?-GCTGCAGCAGCCTCTGGAGCTTAAGCAGGATTCAGA and
GGTCC. The fusion of these two fragments at the AflII site maintained the
correct reading frame of exon 23. A 150-bp BglII-digested PCR fragment
from the exon/intron junction of exon 23 was amplified with primers
5?-GCCTGAGATCTTGCAGCAGATTAGCGTGGAGG and 3?-GAT
GAAGGAGATCTTTGGGGCTCACGTTTTGGC. This exon/intron joint
fragment was cloned at the 3? BamHI site at the end of the human Stat2
sequence described above, and this final 4.7-kb fragment was cloned into
the XhoI site of the pLNTK targeting vector. Together, these three com-
ponents created the 5? region of homologous recombination and effectively
replaced the murine Stat2 C terminus with the human counterpart that
included a stop codon at the end of this sequence. The remaining segment
of exon 23 was preserved within this altered exon to ensure proper splicing
to exon 24, which encodes the natural polyadenylation sequence. The 3?
end of homologous recombination consisted of a 4-kb XhoI fragment that
included the remaining segment of intron 23 through to the end of the last
exon 24. This fragment was cloned into the SalI site within pLNTK.
Transfected RW-4 ES cells (26) were placed in selection with G418.
Correctly targeted clones were identified by Southern blotting with probes
derived from genomic sequence outside the regions of homologous recom-
bination. The PGK-neorcassette was removed by transient infection of ES
clones with adenovirus vector expressing Cre recombinase. ES cells were
injected into C57BL/6 blastocysts and implanted into pseudopregnant
Swiss Black females. Chimeric male offspring were mated to C57BL/6
females to determine germline transmission. Founders that were determined to
transmit the chimeric allele were then mated to DO11.10 (BALB/c back-
ground) for five generations. In addition, a second line was maintained on the
129 background by first crossing founders to 129/SvJ followed by intercross
breedings to maintain this colony. Experimental groups were generated from
crosses of heterozygous KI parents to generate both homozygous KI and
genomic PCR with primers 5?-GTGGACGAGCTGCAGCAG, 3?-ATAC
CATGCATAGTGTG, and 3?-CTAGTCCTCAGAAGGTATCAAGAGTC
T cell cultures
Spleen and mesenteric lymph node cells from wild-type and m/h Stat2
KI ? DO11.10 mice were cultured in IMDM supplemented with 10% FBS,
L-glutamine (200 ?M), nonessential amino acids (10 ?M each), sodium
pyruvate (100 ?M), 2-ME (50 ?M), and penicillin/streptomycin (100 U/ml
each) (HyClone). CD4?T cells were activated with OVA peptide (0.3
mM) and IL-2 (50 U/ml) under Th1 (anti-IL-4 (11B11; 10 ?g/ml) and
rmIL-12 (10 U/ml; R&D Systems)), Th2 (anti-IL-12 (Tosh; 10 ?g/ml) and
rmIL-4 (100 U/ml)), or under neutralizing conditions (anti-IL-4 plus anti-
IL-12) in the absence or presence of IFN-? (typically 1000 U/ml; R&D
Systems). Cells were activated for 3 days and split 1:8 into medium con-
taining additional IL-2 (50 U/ml) and cultured for an additional 4 days.
For CD8?T cell cultures, splenocytes and lymph node cells were iso-
lated from m/h Stat2 KI mice (129/SvJ background), and CD8?T cells
were purified by flow-cytometric sorting. Purified CD8 T cells were acti-
vated with Con A in the presence of irradiated BALB/c splenocytes and
IL-2 (100 U/ml) in complete IMDM for 3 days. Cells were diluted 1/10 on
day 3 in medium containing additional IL-2 (100 U/ml) and rested to day 7.
Intracellular cytokine staining and flow-cytometric analysis
Resting T cell cultures were restimulated in medium containing PMA (50
ng/ml) and ionomycin (1 ?M) for 4 h. In some cases, cells were restim-
ulated with recombinant cytokines rmIL-12 (10 U/ml), rmIL-18 (50 ng/
ml), and rmIFN-? (1000 U/ml). Brefeldin A (1 ?g/ml; Sigma-Aldrich) was
added during the last 2 h of stimulation. Activated cells were collected, fixed
in 4% formalin, permeabilized with 0.05% saponin, and stained with fluoro-
chrome-conjugated anti-IL-4 (11B11) and anti-IFN-? (R46A2) mAbs
(Caltag). Relative fluorescence was measured by flow-cytometric analysis us-
ing a FACSCalibur instrument (BD Biosciences) with emission compensation
Detection of IFN-? by ELISA has been described previously (27). Briefly,
Immulon 1B microtiter plates (ThermoLabsystems) were coated with pu-
rified mAb R46A2 (2 ?g/ml; Caltag), and IFN-? protein was detected with
a biotin-conjugated secondary mAb XMG1.2 (0.5 mg/ml; Caltag). Com-
plexes were detected by incubation with streptavidin-HRP followed by
detection with 3,3?,5,5?-tetramethylbenzidine substrate.
Nuclear extracts were prepared from T cells activated for 30 min with
either medium alone, IL-12 (10 U/ml), or rmIFN-? (A) (1000 U/ml) as
previously described (11). To detect STAT2-containing IFN-sensitive gene
factor-3 (ISGF3) complexes, 3 ?g of nuclear extracts were incubated in 20
?l of binding reaction (10 mM Tris-Cl (pH 7.5), 50 mM NaCl, 1 mM DTT,
1 mM EDTA, 5% (v/v) glycerol, and 3 ?g of poly(dI:dC) containing 1 ?
105cpm32P-labeled double-stranded IFN-stimulated regulatory element
oligonucleotide (GGGGGAAAGGGAAACCGAAACTGAACCCC). Re-
actions were incubated at room temperature for 30 min followed by the
addition of a supershifting Ab to some reactions. Complexes were resolved
on nondenaturing 4.5% acrylamide gels and visualized by autoradiography,
as previously described.
Immunoprecipitation and Western blotting
Resting T cell cultures (day 7, described above) were restimulated with
either medium alone, or with medium containing rmIL-12 (10 U/ml) or
rmIFN-? (A) (1000 U/ml) for 30 min at 37°C. Cells were harvested,
washed once in cold PBS, and lysed with 1 ml of lysis buffer (0.15 M NaCl,
1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and 50 mM Tris-Cl (pH
8.0)). Cell lysates were immunoprecipitated sequentially with anti-STAT4
(5 ?g/ml; SC-486; Santa Cruz) and anti-STAT1 (5 ?g/ml; SC-346) poly-
clonal Abs in the presence of protein A-Sepharose (Amersham Bio-
sciences). Immunoprecipitates were resolved by SDS-PAGE and immuno-
blotted with a peroxidase-conjugated anti-phosphotyrosine Ab (RC20;
Upstate Biotechnology). Immunoreactive complexes were detected by
chemiluminescence. To detect equivalent STAT precipitation, blots were
stripped and incubated again with polyclonal Abs anti-STAT4 (SC-486) or
anti-STAT1 (SC-346) and with a peroxidase-conjugated goat anti-rabbit Ig
295 The Journal of Immunology
Generation of m/h Stat2 KI mice
We previously demonstrated that a chimeric m/h STAT2 molecule
could restore IFN-??-dependent STAT4 activation when ex-
pressed in STAT2-deficient human fibroblasts (U6A cells) (19). To
reconstruct this pathway in murine CD4?T cells, we wished to
express the same molecule from the endogenous murine Stat2 lo-
cus as that expressed in our in vitro studies (Fig. 1A). The diver-
gence of sequence similarity between murine and human Stat2
begins within the first 50 nt of exon 23. The targeting construct
was designed such that the region of sequence divergence within
exon 23 was replaced with the cDNA sequence encoding the hu-
man STAT2 C terminus, including a stop codon (Fig. 1, A and B).
The PGK-neorcassette was flanked by loxP sites and placed within
the middle of intron 23. Following in vitro Cre-mediated deletion,
only a single 12-nt loxP site was left within intron 23 and did not
interfere with proper splicing of the message to exon 24, encoding
the natural polyadenylation signal. Homologous recombination
within ES cells was detected by Southern blotting with a genomic
DNA probe from the 3? end of the Stat2 gene outside the region of
recombination (Fig. 1C). Generation of chimeric m/h Stat2 KI
mice was performed by injection of targeted ES cells into
C57BL/6 blastocysts and implantation into pseudopregnant fe-
males. Germline transmission was confirmed for three highly chi-
meric founders. Offspring were routinely monitored for the pres-
ence of the KI allele by genomic PCR analysis (Fig. 1D).
Both heterozygous and homozygous m/h Stat2 KI mice were
fertile, produced normal-sized litters, and were of normal size and
activation in human cells (19) and is the basis for the KI targeting construct. B, The divergence between the murine and human sequences begins within
exon 23. The targeting construct replaces this sequence with the human counterpart that contains the natural stop codon. The intron/exon structure is
maintained following Cre-mediated deletion of the neorcassette that is flanked by loxP sites. C, Genomic DNAs from progeny (lanes 3–5) were probed
with a segment of exon 24 resulting in a 14-kb band for the wild-type (WT) allele and a 4.5-kb band for the KI allele. D, Genomic DNAs from progeny
(lanes 7–14) were amplified with one sense primer and two antisense primers resulting in a 850-bp band corresponding to the wild-type allele (lane 4) and
250- and 900-bp bands corresponding to the KI allele (lanes 5–6; see B).
Generation of m/h Stat2 KI mice. A, A chimeric m/h Stat2 molecule spliced just past the SH2 domain restores IFN-??-dependent STAT4
296EFFECT OF A CHIMERIC STAT2 MOLECULE ON Th1 DEVELOPMENT
weight. Lymphocyte analysis of thymus, mesenteric lymph nodes,
and spleen revealed a normal complement of CD4?and CD8?
cells, indicating that T cell development was unaltered by the Stat2
modification (data not shown). Additionally, we found that splenic
B cells (B220?) and monocytes (CD11b?) were also normal in
m/h Stat2 KI mice (data not shown).
The chimeric m/h STAT2 molecule is expressed and functional
in murine CD4?T cells
Our previous studies demonstrated that both the full-length murine
STAT2 and the m/h STAT2 chimeric molecules could be recruited
and activated by the hIFNAR (19). However, it is possible that
activation of murine STAT2 by the mIFNAR requires specific se-
quences within the murine STAT2 molecule that would be abol-
ished by the expression of the chimeric m/h STAT2. Thus, two
criteria must be met before IFN-??-dependent STAT4 activation
can be assessed: the m/h STAT2 molecule must be 1) expressed in
CD4?T cells and 2) activated by the mIFNAR. Northern blot
analysis of RNA isolated from purified CD4?T cells demon-
strated a gene-dose-dependent expression of murine Stat2 in the
wild-type and m/h Stat2 heterozygous backgrounds that hybridized
with a probe specific for the murine Stat2 C terminus (Fig. 2A,
upper panel). This probe contained a 20-nt overlap of sequence
within the C terminus that is not divergent between mouse and
human, and explains the low degree of hybridization seen in the
homozygous KI (Fig. 2A, upper panel, lane 3). Furthermore, a
probe from the C terminus of human Stat2 hybridized to a band
corresponding to the m/h Stat2 mRNA in both heterozygous and
homozygous KI T cells (Fig. 2A, middle panel).
The m/h Stat2 mRNA was translated into a protein of the correct
molecular mass and was recognized by an anti-STAT2 Ab specific
for the C terminus of human STAT2 (Fig. 2, B and C). In addition,
this Ab was also capable of specifically immunoprecipitating the
chimeric m/h STAT2 molecule from CD4?T cell lysates. A pre-
vious report of the STAT2 knockout demonstrated that ablation of
STAT2 dramatically decreased the expression of other STAT mol-
ecules, including STAT1. However, the m/h STAT2 molecule de-
scribed in this study did not alter expression of STAT1 in murine
T cells (Fig. 2B).
IFN-??-mediated STAT2 phosphorylation is accompanied by
concomitant phosphorylation of STAT1 and the assembly of the
ISGF3 complex containing a heterotrimer of STAT1, STAT2, and
IFN regulatory factor 9 (28). We detected the formation of this
complex in murine T cells by EMSA. ISGF3 was induced by mu-
rine IFN-? (A) in both wild-type and m/h Stat2 KI T cells (Fig.
2D). Furthermore, a supershift complex was identified in heterozy-
gous and homozygous KI T cells by incubation of nuclear extracts
with an anti-STAT2 Ab specific for the human STAT2 C terminus.
Collectively, these data demonstrate that the chimeric m/h Stat2
gene is expressed and translated into protein in murine T cells.
Furthermore, the chimeric m/h STAT2 molecule is functionally
activated by the mIFNAR and capable of binding DNA.
Expression of m/h STAT2 is not sufficient to restore
IFN-??-dependent Th1 development
Commitment of CD4?T cells to high levels of IFN-? secretion
occurs, in part, by a STAT4-dependent process (29). As a first step
in measuring the reconstitution of IFN-??-dependent signaling for
Th1 development, we crossed the m/h Stat2 KI to the DO11.10
TCR (TCR) transgenic on the BALB/c background (25). In these
mice, the majority of T cells are selected by I-Adand respond to
a single peptide derived from the chicken OVA protein. Thus, in
vitro T cell cultures from these mice can be activated by a single
Ag (OVA peptide) under well-defined cytokine conditions. To as-
sess developmental commitment to the Th1 phenotype, lymph
node and splenic T cells were activated with OVA peptide and
IL-2 in the absence or presence of specific cytokines and/or anti-
cytokine Abs for 3 days. Cells were split into new medium con-
taining IL-2 for an additional 4 days before restimulation and cy-
tokine measurements. As expected, primary activation of cells
under typical Th1-inducing conditions (?-IL-4 plus IL-12) led to
robust IFN-? secretion upon secondary activation of cells with
either PMA/ionomycin (Fig. 3A, condition no. 2), or with plate-
bound anti-CD3 (B). Activation with rmIFN-? (A) (Fig. 3, A and
B, condition no. 3) induced a 2- to 4-fold increase in the percentage
of cells capable of producing IFN-? when compared with cells
developing under neutralizing conditions (Fig. 3, A and B, condi-
tion no. 1). Although rmIFN-? (A) (specific activation of murine
ecule. A, Total RNA isolated from wild-type, m/h Stat2 heterozygous, and
homozygous splenocytes, and human Hut78 cells were probed with cDNA
corresponding to the murine STAT2 C terminus (top panel), the human
STAT2 C terminus (middle panel), and GAPDH (bottom panel). B, Whole-
cell lysates from wild-type, and m/h Stat2 KI homozygous splenocytes
were probed with an anti-STAT2 Ab specific for the C terminus of human
STAT2 and an anti-STAT1 polyclonal Ab. C, Whole-cell lysates from
wild-type and m/h Stat2 heterozygous splenocytes were immunoprecipi-
tated with an anti-STAT2 Ab specific for the C terminus of human STAT2.
The resulting blot was probed with the same anti-STAT2 Ab. D, Enriched
CD4?T cells from wild-type, heterozygous and homozygous m/h Stat2 KI
mice were activated for 30 min with rmIFN-? (A) (1000 U/ml). Nuclear
extracts from activated cells were incubated with a32P-labeled IFN-stim-
ulated regulatory element probe in the presence or absence of an anti-
STAT2 Ab specific for the human C terminus.
Expression and activation of the m/h STAT2 chimeric mol-
297The Journal of Immunology
cells) was more active than rhIFN-? (A/D) (active on both murine
and human cells) at promoting some cells to commit to IFN-?
secretion, the induction of Th1 development relative to the effects
of IL-12 was very low. However, similar levels of IFN-? secretion
were observed in wild-type and m/h Stat2 KI T cells regardless of
primary stimulation conditions.
Recent studies have suggested that murine T cells have the ca-
pacity to respond to IFN-?? for IFN-? production (16–18). How-
ever, in some cases, those experiments were performed with rel-
atively high concentrations of IFN-? (50,000–100,000 U/ml)
compared with the levels normally used to assess human T cell
differentiation (500–1,000 U/ml). Based on these observations,
wild-type and m/h Stat2 KI T cells were tested for their ability to
commit to Th1 development in response to increasing concentra-
tions of IFN-?. In this study, T cell cultures were activated in the
presence of anti-IL-12 (Tosh) (30) and increasing concentrations
of rmIFN-? (A) ranging from 100 to 100,000 U/ml (see Fig. 5, C
and D). In contrast to previous studies, we found no difference in
the levels of IFN-? secreted upon secondary activation when com-
paring cells activated under neutralizing conditions (anti-IL-12)
and cells activated with IFN-? at any concentration. In addition,
there were no significant differences in the percentage of cells ca-
pable of secreting IFN-? between wild-type and m/h Stat2 KI T
cells. The overall quantity of IFN-? secreted by cells polarized in
the presence of increasing concentrations of rmIFN-? (A) was
confirmed by ELISAs (Fig. 3D) and further demonstrated that
IFN-? failed to promote Th1 polarization in m/h Stat2 KI T cells.
In addition to the developmental effects exerted by STAT4 ac-
tivation, an additional STAT4-dependent pathway regulates acute
induction of IFN-? gene transcription within fully differentiated
Th1 cells. Combinatorial treatment of Th1 cells with IL-12 plus
IL-18 induces sustained levels of IFN-? secretion in the absence of
TCR activation (31, 32). This dual signaling pathway is dependent
upon STAT4 activation and is conserved between murine and hu-
man Th1 cells (14, 33). In human Th1 cells, IFN-? plus IL-18 also
induce acute IFN-? secretion, in part, due to the activation of
STAT4 (33). Although the m/h STAT2 molecule failed to mediate
IFN-??-dependent Th1 development, it was possible that the acute
pathway for IFN-? gene expression in fully differentiated Th1 cells
was restored by this genetic modification. This hypothesis was
tested by activating DO11.10?T cells from wild-type and m/h
Stat2 KI mice in the presence of IL-12 for 7 days to promote Th1
development. These cells were then restimulated in the presence of
either IL-12 plus IL-18 or with rmIFN-? (A) plus IL-18 for 24 h
(Fig. 4). Consistent with our prior results, we found that dual stim-
ulation with IFN-? plus IL-18 did not promote IFN-? secretion
from either wild-type or m/h Stat2 KI T cells.
The C terminus of human STAT2 is not sufficient to mediate
IFN-??-dependent STAT4 activation
In human cells, species-specific determinants within the human
STAT2 C terminus were necessary for recruiting STAT4 to the
hIFNAR (19). To test the sufficiency of this domain in recruiting
STAT4 to the mIFNAR, we assessed STAT4 phosphorylation in
wild-type and m/h Stat2 KI T cells. DO11.10?T cells were acti-
vated with OVA peptide and IL-12 for 7 days to generate cells that
could respond to subsequent activation with IL-12 as a positive
control. Resting Th1 cells were restimulated with either medium
alone, IL-12, or with rmIFN-? (A). STAT4 and STAT1 tyrosine
phosphorylation was determined by immunoblotting (Fig. 5).
IL-12 was active in both wild-type and KI Th1 cells to promote
STAT4 phosphorylation; however, rmIFN-? did not exhibit such
activity. The failure of IFN-? to activate STAT4 was not due to a
general lack of IFNAR activation, because STAT1 was robustly
CD4?T cells. DO11.10?splenocytes and lymph nodes from wild-type,
heterozygous and homozygous m/h Stat2 KI mice were stimulated with
OVA peptide and the indicated anti-cytokine Abs or cytokines. A and B,
Cells were restimulated with PMA/ionomycin (A) or plate-bound anti-CD3
(B) and analyzed for IFN-? expression by intracellular staining and flow
cytometry. Live cells were gated on CD4?T cells. C and D, DO11.10?
splenocytes and lymph nodes from wild-type, homozygous m/h Stat2 KI,
and IL-12p40?/?mice were stimulated with OVA peptide in the presence
of anti-IL-12 (Control), IL-12 (10 U/ml), or with anti-IL-12 and increasing
concentrations of rmIFN-? (A) as indicated in the figure. Cells were re-
stimulated for 4 h (C) or 24 h (D) on day 7, and IFN-? expression was
measured on by intracellular cytokine staining (C) and ELISA (D). Each
experimental point was performed in triplicate, and the data are represen-
tative of three independent experiments.
IFN-? does not induce Th1 development in m/h Stat2 KI
298EFFECT OF A CHIMERIC STAT2 MOLECULE ON Th1 DEVELOPMENT
phosphorylated in both wild-type and KI T cells (Fig. 5, lower
panel). Furthermore, previous studies with both human and murine
STAT2-deficient cells demonstrated that STAT1 activation is de-
pendent upon the presence of a functional STAT2 molecule (22,
34, 35). Thus, we conclude that the chimeric m/h STAT2 molecule
is functional to recruit and activate STAT1, but not STAT4, in
murine T cells. Taken together, this study demonstrates that, al-
though sequences within the C terminus of STAT2 are required in
human cells, this domain is not sufficient to promote STAT4 re-
cruitment and activation by the mIFNAR.
IFN-? fails to promote IFN-? secretion in murine CD8?T cells
A recent report has suggested that IFN-?? can promote STAT4
phosphorylation and IFN-? secretion from murine T cells in a
murine model of lymphocytic choriomeningitis virus infection
(16). However, their system relied primarily on the secretion of
IFN-? by CD8?T cells. Thus, it was possible that IFN-? signaling
for IFN-? secretion was more efficient in murine CD8?than in
CD4?T cells. Based on those results, we wished to determine
whether IFN-?-driven IFN-? secretion was more efficient in mu-
rine CD8?cells that expressed the m/h STAT2 chimeric molecule.
Unlike CD4?T cells, murine CD8?T cells do not require STAT4
signaling to become competent to secrete high levels of IFN-?
upon restimulation through the TCR (29). However, acute IL-12/
IL-18-mediated IFN-? secretion from CD8?T cells remains de-
pendent upon STAT4 activation. Thus, for these experiments,
CD8?T cells were purified from wild-type and m/h Stat2 KI mice
(on 129/SvJ background) by flow-cytometric sorting. Cells were
activated with irradiated allogeneic BALB/c splenocytes in the
presence of Con A for 3 days followed by dilution in medium
containing additional IL-2 and rested to day 7. CD8?T cells were
then washed extensively, restimulated with recombinant cytokines,
and analyzed for IFN-? secretion by both intracellular cytokine
staining (Fig. 6A) and ELISA (B).
Stimulation of CD8?cells with either IL-12 or IFN-? alone did
not lead to significant increases in either the percentage of cells
capable of expressing IFN-? (Fig. 6A, b, d, h, and j) or accumu-
lation of IFN-? in the culture supernatants (B), as expected. IL-18
stimulation led to a marked increase in the percentage of IFN-?-
secreting cells (Fig. 6A, c and i), and this effect was enhanced by
combined stimulation with IL-12 (A, e and k) but not with IFN-?
(f and l). However, only IL-12 plus IL-18 activation led to a sig-
nificant accumulation of IFN-? in the culture supernatants during
a 24-h stimulation (Fig. 6B), indicating that the percentage of cells
capable of expressing IFN-? in response to IL-18 or IL-18 plus
IFN-? was transient and not sustained. Furthermore, the levels of
IFN-? secreted in response to IL-18 were not significantly different
from the levels observed in response to combined stimulation with
IL-18 plus IFN-?. These data demonstrate that, unlike IL-12,
IFN-? stimulation has no direct effect on acute induction of IFN-?
gene expression in CD8?T cells. Furthermore, we found no sig-
nificant difference in either the percentage of cells capable of ex-
pressing IFN-? or the accumulation of IFN-? within the culture
supernatants of wild-type vs m/h Stat2 KI CD8?cells responding
to IFN-? stimulation.
A central role for STAT4 in promoting Th1 development and
type I responses in vivo has been demonstrated directly in Stat4-
deficient mice (6, 7) as well as in other genetic backgrounds that
influence STAT4 activation such as IL-12R knockouts (36, 37).
Early evidence of species-specific IFN-??-induced STAT4 acti-
vation (11, 12, 23, 38) correlated well with the known biological
responses when comparing mouse and human T cells. Collec-
tively, these correlations predict that any receptor, including the
IFNAR, that can activate STAT4 within CD4?T cells would have
the capacity to drive IFN-? gene expression in both mouse and
human T cells. STAT4 activation by the hIFNAR involves the
presence of activated STAT2 (11), and this interaction maps to a
nonconserved region within the STAT2 C terminus (19). In this
study, we demonstrated that, although this sequence within STAT2
is required to recruit and activate STAT4 within human cells, it is
cretion in m/h Stat2 KI CD4?T cells. DO11.10?splenocytes and lymph
node cells from wild-type, heterozygous and homozygous m/h Stat2 KI
mice were stimulated with OVA peptide under Th1-inducing conditions
(IL-12 plus IFN-? plus anti-IL-4) for 3 days and rested in medium con-
taining IL-2 until day 7. Cells were restimulated as indicated in the figure
and analyzed for IFN-? secretion by intracellular staining and flow cytom-
etry. Live cells were gated on CD4?T cells.
IFN-? does not synergize with IL-18 to induce IFN-? se-
?-dependent STAT4 tyrosine phosphorylation in murine CD4?T cells.
DO11.10?lymph node and spleen cells from wild-type (lanes 1–3) and
m/h Stat2 KI (lanes 4–6) mice were stimulated with OVA peptide in
Th1-inducing conditions (anti-IL-4, IFN-?, and IL-12) for 3 days and split
1:8 in medium containing IL-2. On day 7, cells were washed and restim-
ulated for 30 min at 37°C with either medium alone (lanes 1 and 4), or with
medium containing IL-12 (10 ng/ml; lanes 2 and 5) or with rmIFN-? (A)
(1000 U/ml; lanes 3 and 6). Cell lysates were prepared and immunopre-
cipitated with anti-STAT4 (SC-486) and anti-STAT1 (SC-346; Santa Cruz)
polyclonal Abs. Immunoprecipitates were immunoblotted for both phos-
photyrosine (P-Y; RC20) and for STAT4 and STAT1 as indicated in the
The C terminus of hSTAT2 is not sufficient to restore IFN-
299The Journal of Immunology
not sufficient to promote STAT4 phosphorylation or Th1 commit-
ment within murine CD4?T cells.
In our initial studies of the hIFNAR, we considered the possi-
bility that STAT4 was recruited directly to the IFNAR via inter-
actions with the cytoplasmic domain of the receptor subunits (11).
Indeed, neither the IFNAR1 nor -R2 subunits are well conserved
between mouse and human. However, we demonstrated that
STAT4 did not interact with any potential phosphotyrosine resi-
dues within either the hIFNAR1 or -R2 subunit by a phosphopep-
tide competition EMSA assay (11). Recently, several studies have
demonstrated a unique requirement for STAT N-terminal domains
that regulate receptor-proximal activation. For example, in human
cells, the STAT2 N-domain mediates a direct interaction with the
IFNAR2 cytoplasmic domain, and this interaction is formed before
cytokine activation (39). This preassociated complex facilitates cy-
tokine-mediated phosphorylation of STAT2.
An analogous mechanism might be operative for STAT4. First,
several reports have demonstrated that the STAT4 N-domain is
required for efficient phosphorylation in response to both IL-12
and IFN-? (40–42). Indeed, transgenic expression of STAT4 lack-
ing the N-domain fails to reconstitute IL-12-driven STAT4 phos-
phorylation or Th1 development when crossed to the STAT4-de-
ficient background (42). Crystallographic data have demonstrated
that the STAT4 N-domain exists as a latent dimer through homo-
typic interaction (43). Although this structure has been re-evalu-
ated (41, 44), the existence and purpose of this latent STAT4 dimer
in cytokine-driven phosphorylation has been recently examined. In
this study, instead of completely removing the N-domain from the
primary STAT4 sequence, specific residues that mediate N-domain
dimer formation were mutated and expressed within both human
fibroblasts and murine Stat4-deficient T cells (41). These experi-
ments revealed that mutation of these critical N-domain residues
abrogated STAT4 phosphorylation in response to both IFN-? (in
human fibroblasts) and IL-12 (in murine T cells), suggesting a
common mechanism for recruitment of STAT4 to both the IL-12R
and the hIFNAR. Whether STAT4, like STAT2, preassociates with
either the IL-12R or the hIFNAR has not been reported yet. How-
ever, due to the significant sequence divergence within both the
IFNAR1 and -R2 subunits between mouse and human, it is ex-
pected that the conserved STAT4 N-domain would interact in a
species-specific manner with the cytoplasmic domain of the hIF-
NAR. If this is the case, then the hIFNAR cytoplasmic domain
would represent a second species-specific component necessary
for the recruitment and activation of STAT4. Furthermore, this
possibility would also explain why the modification of STAT2
described here was not sufficient to activate STAT4 by the mIF-
NAR in murine CD4?T cells.
In contrast to our initial characterization of the species-specific
link between IFN-?? signaling and STAT4 activation, recent re-
ports have suggested that IFN-?? can activate STAT4 and pro-
mote IFN-? expression in murine T cells (16, 17). These studies
used relatively high concentrations of IFN-? to detect this effect.
Based on this observation, we considered the possibility that, al-
though the mIFNAR was inefficient at activating STAT4, this ef-
fect could be overcome by titrating IFN-? to very high concen-
trations. However, we found that wild-type, m/h Stat2 KI, and
IL-12p40-deficient CD4?T cells did not secrete significant levels
of IFN-? when activated in the presence of rmIFN-? (A) at any
concentration (up to 100,000 U/ml; Fig. 3, C and D). It has been
suggested that the use of different IFN-? subtypes could account
for this discrepancy. However, Berenson et al. (45) recently dem-
onstrated that, although mIFN-? (A) was more active than
rhIFN-? (A/D) at promoting weak STAT4 phosphorylation (as
observed by Nguyen et al. (16)), neither of these IFN-? subtypes
was able to induce Th1 commitment within CD4?T cells, and the
present study confirms this observation. In addition, Nguyen et al.
(16) reported elevated STAT4 phosphorylation in response to
IFN-?? in murine CD8?cells compared with enriched CD4?T
cretion in m/h Stat2 KI CD8?T cells. CD8?T cells were purified by
flow-cytometric sorting from lymph nodes of wild-type and m/h Stat2 KI
mice (on 129/SvJ background). Cells were stimulated with Con A (5 ?g/
ml) in the presence of irradiated BALB/c splenocytes for 3 days and rested
in medium containing IL-2 until day 7. Cells were restimulated with re-
combinant cytokines as indicated in the figure for 4 h (A) or 24 h (B), and
IFN-? expression was determined by intracellular cytokine staining (A) and
by ELISA (B).
IFN-? does not synergize with IL-18 to induce IFN-? se-
300 EFFECT OF A CHIMERIC STAT2 MOLECULE ON Th1 DEVELOPMENT
cells. Although a STAT4-dependent, IL-12-independent mecha-
nism for commitment to IFN-? secretion exists for CD8?T cells
(29), we found no evidence that CD8?T cells could secrete IFN-?
in response to acute stimulation with IFN-? in either the absence
or presence of IL-18. Thus, based on our present findings, we
conclude that IFN-? does not promote efficient STAT4 phosphor-
ylation or IFN-? expression in murine T cells even in the presence
of a humanized STAT2 molecule. A direct comparison of IFN-
??-dependent STAT4 phosphorylation (11, 12) and Th1 develop-
ment (12) between mouse and human CD4?T cells has been de-
scribed in detail, and forms the basis of the present study.
However, given the recent controversy using different in vivo and
in vitro model systems, this issue clearly warrants further study.
We thank Erik Geissal and Loderick Matthews for excellent technical as-
sistance, Alec Cheng and Barry Sleckman for help with ES cell targeting,
Michael White for blastocyst injections, and Angela Mobley for assistance
with flow cytometry. We thank Theresa Murphy, Douglas Tyler, Ann
Davis, and Nishant Sahni for helpful discussions, and Christoph Wu ¨lfing
and Lora Hooper for critically reading the manuscript.
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301The Journal of Immunology