By
From * Roche Milano Ricerche, I-20132 Milan, Italy; and Department of Inflammation/
Autoimmune Diseases, Hoffmann-La Roche Inc., Nutley, New Jersey 07110
Interleukin-12 (IL-12), a heterodimeric cytokine produced by activated monocytes and dendritic cells, plays a crucial role in regulating interferon (IFN)- production and in the generation of IFN-
-producing T helper 1 (Th1) cells. Here we show that the IL-12 receptor (IL12R)
2 subunit, a recently cloned binding and signal transducing component of the IL-12R,
is expressed on human Th1 but not Th2 clones and is induced during differentiation of human
naive cells along the Th1 but not the Th2 pathway. IL-12 and type I but not type II interferons
induce expression of the IL-12R
2 chain during in vitro T cell differentiation after antigen receptor triggering. The selective expression and regulation of the IL-12R
2 subunit may help
to understand the basis of Th1/Th2 differentiation and may provide therapeutic options for altering the Th1/Th2 balance in several immuno-pathological conditions such as autoimmune
diseases and allergies.
CD4+ T helper cells can be divided into three major
subsets termed Th1, Th2, and Th0, based on the pattern of lymphokines they produce. Th1 cells produce IFN- In the present paper, we have studied IL-12 signaling
and IL-12 receptor expression and regulation during differentiation of human naive T cells into the Th1 and the Th2
subsets. As previously reported in the murine system, IL-12
induces tyrosine phosphorylation of the transcription factor
Stat4 in human Th1, but not Th2 cells. IL-12-dependent
signaling in human Th1 cells correlates with the selective expression of the transcripts encoding the signaling component
of the IL-12 receptor (IL-12R) Generation of Th1 and Th2 Lines from Cord Blood Leukocytes.
Human neonatal leukocytes were isolated from freshly collected,
heparinized, neonatal blood by Ficoll-Paque (Pharmacia Biotech AB, Uppsala, Sweden) density gradient centrifugation (18). Neonatal T cell preparations were >70% CD45RA+ and 50-60%
CD4+. Th1 and Th2 cell lines were generated by stimulating
cord blood leukocytes with 2 µg/ml PHA (Wellcome, Beckenham, UK) in the presence of 2 ng/ml IL-12 (Hoffmann-La
Roche Inc., Nutley, NJ) and 200 ng/ml neutralizing anti-IL-4
antibodies (No. 18500D; PharMingen, San Diego, CA) for Th1
cultures, or 200 U/ml IL-4 (PharMingen) and 2 µg/ml neutralizing anti-IL-12 antibodies 17F7 and 20C2 (provided by M. Gately, Hoffmann-La Roche Inc.) for Th2 cultures, respectively. The cord blood-derived T cell lines were 60-90% CD4+. The
remaining cells were CD8+ T cells. Cells were washed on day 3 and expanded in RPMI 1640 medium supplemented with 5%
FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin-streptomycin, and containing 100 U/ml IL-2 (HoffmannLa Roche Inc.). Cells were washed again on day 10 and 105 cells
were restimulated in 96-well round-bottom plates for 24 h with
plate-bound anti-CD3 antibodies (clone TR66; reference 19) to
measure IFN- Purification and Stimulation of CD45RA+ T Cells.
PBMCs were
isolated from human blood buffy coats from healthy donors by
Ficoll-Paque (Pharmacia Biotech AB) density gradient centrifugation. Monocytes were depleted by two rounds of plastic adherence and B cells were depleted by adherence to nylon wool as
described (18). CD45RA+ T cells were isolated by two rounds of
immunomagnetic negative selection with a mixture of the following monoclonal antibodies: anti-CD16 (B73.1; reference 21),
anti-CD45RO (UCHL-1; reference 22) and anti-HLA-DR
(1-1C4; reference 23). The suspension was incubated with goat
anti-mouse IgG-coated Dynabeads (Dynal, Inc., Geat Neck, NY)
and exposed to a magnetic field using a magnetic particle concentrator (Dynal, Inc.) according to the manufacturer's instructions. The
purity of the CD3/CD45RA+ T cells using this procedure was typically >98% as determined by flow cytometry. Purified CD45RA+
T cells were stimulated with plate-bound anti-CD3 mAbs (TR66; reference 19) without the addition of cytokines or in the presence of IL-12 (2 ng/ml) or IL-4 (200 U/ml).
T Cell Clones.
Th1 and Th2 cells generated from cord blood
lymphocytes were cloned by limiting dilution as described (24).
In brief, 0.3 cells/well were plated in 96-well plates in the presence of irradiated (5,000 rads) allogeneic PBMCs, PHA (2 µg/ml),
and IL-2 (100 U/ml). Positive wells were restimulated and expanded and cytokine production of individual clones was measured as above. Three T cell clones that produced IFN- Cell Extracts, Immunoprecipitations, and Western Blot Analysis.
Th1 and Th2 lines generated from cord blood lymphocytes were
harvested on day 10 after priming. Th1 (CB13) and Th2 (CB21) clones were harvested on day 13 after restimulation. 5 × 106 cells
were washed and incubated for 15 min at 37°C in 1 ml RPMI with 5% FCS with or without 2 ng/ml IL-12. Cells were washed
once in PBS before lysing the cell pellet in 250 µl IP buffer (10 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, pH 8.0, 1 mM
EGTA, pH 8.0, 1% NP-40, 0.25% sodium deoxycholate, 10 µg/ml
aprotinin, leupeptin, and NaF, 1 mM AEBSF and sodium orthovanadate). The lysate was incubated 30 min on a shaker at 4°C
and insoluble debris was removed by centrifugation (13,000 rpm,
4°C, 30 min). Stat4 was immunoprecipitated with rabbit polyclonal
anti-Stat4 antisera (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
and resolved by SDS-PAGE. After transfer to nitrocellulose, blots
were probed with anti-phosphotyrosine antibody 4G10 (Upstate
Biotechnology Inc., Lake Placid, NY), and immunoreactive bands
were visualized using the enhanced chemiluminescence Western
blotting system (Amersham, Milan, Italy), according to the company's protocols. To control for equal protein loading, blots were
stripped and reprobed with anti-Stat4 antisera.
Ribonuclease Protection Assays.
A 327-bp DNA fragment encompassing the cytoplasmic region of the human IL-12R Scatchard Analysis of [125I]IL-12 Binding to Human Th1 and Th2
Cell Lines.
Th1 and Th2 lines were generated as described
above. [125I]IL-12 binding to human Th1 and Th2 cell lines was
determined as described previously (28). Specific binding was analyzed using the LIGAND program (29) and plotted by the Scatchard method.
To characterize the role of IL-12 and other
cytokines in the differentiation of T helper cell subsets, we
have generated Th1 and Th2 lines by stimulating human
cord blood leukocytes with mitogen in the presence of
IL-12, and neutralizing anti-IL-4 mAb or IL-4, and neutralizing anti-IL-12 mAb, respectively. This protocol allows the establishment of human T cell lines with strongly
polarized cytokine production. Neonatal T cells primed
under the Th1 conditions produced up to 2 ng/ml of IFN-
We next examined whether the defect in IL-12 signaling
observed in Th2 cells is due to a differential expression of
IL-12 receptors in the two cell types. To date, two IL-12R
subunits, termed
We next analyzed IL-12R expression by radioligand
binding assays. Scatchard analysis of specific [125I]IL-12 binding to human cord blood derived Th1 and Th2 cell lines is
shown in Fig. 5. In Th1 cell lines, both high (KD of 27 pM, 140 sites/cell) and low affinity (KD of 5 nM, 450 sites/cell) [125I]IL-12 binding sites were measured. In contrast, Th2
cells lines exhibited only low affinity [125I]IL-12 binding
sites (KD of 2 nM 220 sites/cell). The absence of measurable high affinity [125I]IL-12 binding to the human Th2 cell
lines is in agreement with the observed lack of IL-12R
To determine whether IL-12 directly influences expression of the IL-12R In the mouse, IL-4 has been shown to block the development of naive CD4+ T cells into Th1 effectors. The
block is complete if IL-12 is not present in the priming culture, but incomplete if IL-12 is present (9). Consistent with
this observation, we found that cord blood lymphocytes
primed in the presence of IL-4 and IL-12 together developed into cells secreting both IL-4 (1.1 ng/ml) and IFN-
In addition to IL-12,
IFN- Our data indicate the presence of two major signaling
pathways influencing IL-12R The IL-12R
and predominantly promote cell-mediated immune responses, whereas Th2 cells, which produce IL-4, IL-5, and
IL-13, provide help for some B cell responses. IL-4 in particular is the major inducer of B cell switching to IgE production (1), and therefore plays a crucial role in allergic reactions involving IgE and mast cells. Th cells producing
cytokines typical of both Th1 and Th2 clones have also
been described in both the murine and human system, and
they have been named Th0 (2). The differential development of these subsets is a major determinant of the outcome of physiological, as well as pathological, immune responses including autoimmune, allergic, and infectious
diseases (3, 4). Thus, understanding the mechanisms underlying Th cell differentiation is essential for therapeutic manipulation of the cytokine phenotype in disease conditions.
Differentiation towards the Th1 or Th2 phenotype has been
shown to be directed by exogenous cytokines present at
the time of antigen exposure. IL-4 promotes Th2 development (5, 6), whereas IL-12 produced by APCs is a potent
inducer of Th1 cells (7). Consistent with the necessity of
IL-12 for induction of Th1 cell differentiation is the finding that IL-12 p40
/
mice are defective in IFN-
production and almost completely lack the ability to generate a
Th1 response (10). Conversely, knockout of the IL-4 gene
resulted in deficient Th2 responses (11, 12). Furthermore,
the crucial role of IL-12 and IL-4 signaling in the differentiation of Th subsets has been analyzed more recently in
mice deficient in the signal transducers and activators of
transcription 6 and 4 (Stat6 and Stat4)1. These proteins are
thought to mediate functional responses to IL-4 and IL-12,
respectively. Stat6-deficient T lymphocytes fail to differentiate into Th2 cells in response to IL-4 (13), and the
analysis of Stat4
/
T cells revealed an impaired production
of IFN-
upon antigen triggering, indicative of a defect in
Th1 differentiation (16, 17).
2 and with the presence of high affinity IL-12 binding sites selectively on Th1 cells. IL-12R
2 transcripts, which were absent in naive T cells,
were found as early as 24 h after mitogen stimulation. IL-12
and type I but not type II IFNs induce expression of IL-12R
2 mRNAs, whereas IL-12R
2 transcripts could not be
detected in cells stimulated in the presence of IL-4. The selective expression and regulation of the IL-12 receptor
2
subunit may help understand the molecular events controlling Th cell development.
and IL-4 in culture supernatants by ELISA assays
(provided by H. Gallati, F. Hoffmann-La Roche AG, Basel, Switzerland; reference 20). To test the effects of type I and type II
interferons on T helper cell differentiation, T cell lines were generated by stimulating cord blood leukocytes with PHA in the
presence of the indicated cytokines or anti-cytokine antibodies as
above. IFN-
or IFN-
(1,000 U/ml; F. Hoffmann-La Roche
AG) were added at the time of priming to the cultures. Cytokine
production was determined on day 10 as above.
but not
IL-4 were categorized as Th1, and five T cell clones able to produce IL-4 but not IFN-
were categorized as Th2 clones (data
not shown).
2 subunit (25) and a 553-bp EcoRI/BamHI fragment derived from the
IL-12R
1 cDNA (26) were subcloned into pGEM 3Z. The
constructs were linearized with EcoRI and radiolabeled antisense
transcripts were synthesized with SP6 polymerase and a commercial kit according to the manufacturer's protocol (Promega Corp.,
Madison, WI). RNA was extracted from Th1 and Th2 lines or
clones using Ultraspec total RNA extraction reagent (Biotecx Laboratories Inc., Houston, TX) as described (27). The antisense RNA probes were hybridized to 10 µg total RNA and ribonuclease protection assays were performed with a commercial kit
(Ambion Inc., Austin, TX) according to the company's protocol.
Products were resolved on 6% denaturing polyacrylamide gels
and the protected fragments were visualized by autoradiography.
The radioactivity present in the protected fragments was also quantitated using a MolecularImager (Bio-Rad Labs., Richmond, CA).
An 18S RNA probe was used as a control for equal RNA loading.
IL-12 Induces Stat4 Phosphorylation in Human Th1 but Not
Th2 Cells.
and <0.2 ng/ml IL-4 upon restimulation with plate-bound
anti-CD3 mAb, whereas neonatal T cells primed in the
presence of IL-4 and anti-IL-12 mAb produced 1.2 ng/ml of IL-4 and <0.02 ng/ml IFN-
(Fig. 1 A). Subsequent
cloning of the Th1 and the Th2 lines resulted in a series of
T cell clones with either a Th1 or a Th2 polarized cytokine
profile, respectively (Fig. 1 B). Using a murine TCR transgenic system, Szabo et al. observed that IL-12 selectively
activated the Janus kinase Jak2, and the signal transducers
and activators of transcription Stat3 and Stat4 in Th1, but
not in differentiated Th2 cells (30). To ascertain whether
human Th1 and Th2 subsets also differ in IL-12 signaling,
we examined Stat4 expression and tyrosine phosphorylation in response to IL-12 in Th1 and Th2 lines and clones.
As shown in Fig. 2, IL-12 treatment induced tyrosine
phosphorylation of Stat4 selectively in Th1 but not in Th2
cells, although both cell types expressed comparable amounts
of the Stat4 protein (Fig. 2, bottom) required for IL-12 signaling (31, 32). Similarly, after IL-12 stimulation, Jak2 (33),
which was also expressed at comparable levels in Th1 and
Th2 cells, was phosphorylated only in Th1 cells (data not
shown).
Fig. 1.
Cytokine profiles of cord blood derived Th1 and Th2 lines
and of established human T cell clones. (A) IFN- and IL-4 production by Th1 and Th2 lines generated from cord blood lymphocytes. Cells were washed on day 10 after priming, 105 cells were restimulated in 96well round-bottom plates for 24 h with plate-bound anti-CD3 antibodies
(clone TR66; reference 19), to determine IFN-
and IL-4 in culture supernatants by ELISA assays. The results shown are representative of five
independent experiments. (B) IFN-
and IL-4 production by established
Th1 (CB13) and Th2 (CB21) clones.
[View Larger Version of this Image (31K GIF file)]
Fig. 2.
IL-12-dependent signaling in human Th1 and Th2 cell lines
and clones. IL-12 induces Stat4 phosphorylation in human Th1 but not
Th2 cells. Th1 and Th2 lines generated from cord blood lymphocytes as
in Fig. 1 A were harvested on day 10 after priming. Th1 (CB13) and Th2
(CB21) clones were harvested on day 13 after restimulation. 5 × 106 cells
were washed and incubated 15 min at 37°C in medium with or without
2 ng/ml IL-12 followed by the preparation of whole cell extracts and immunoprecipitation with anti-Stat4 antiserum. Precipitated proteins were
separated by SDS-PAGE (8%), transferred to nitrocellulose, and probed
with anti-phosphotyrosine antibody 4G10 (anti-P-Y, top). As a control for
Stat4 expression, blots were stripped and reprobed with anti-Stat4 antibodies (bottom).
[View Larger Version of this Image (23K GIF file)]
2 Chain in Th1 Cells.
1 and
2, have been identified. IL-12R
1 or
2, independently expressed in COS cells, bind human
IL-12 with low affinity (KD = 3-5 nM), whereas coexpression of IL-12R
1 and
2 in COS cells gives rise to high
affinity binding sites (KD = 50 pm) (25, 26). These studies
have also shown that the IL-12R
2 is the signaling component of the IL-12R, in agreement with the fact that the
IL-12R
2, in contrast to the IL-12R
1, contains tyrosine residues in its cytoplasmic domain (25). We analyzed the
mRNA expression levels for the IL-12R
1 and
2 subunits by ribonuclease protection assays in both Th1 and
Th2 lines at different time points after priming and in Th1
and Th2 clones at day 13 after restimulation. Whereas IL-12R
1 transcripts were expressed in similar amounts in both
Th1 and Th2 cells (Fig. 3, A and B), the transcripts coding
for the IL-12R
2 subunit were selectively expressed in established Th1, but not Th2 lines (Fig. 3 A) and clones (Fig.
3 B). Maximum expression in Th1 cells was seen between day 3 and day 8, and declined thereafter. Th2 cells, in contrast, expressed little IL-12R
2 transcripts on day 3, and
none were detected after day 8 (Fig. 3 A). A key question is
whether cytokine signaling is required for IL-12R induction or whether stimulation via the TCR/CD3 complex is
sufficient to induce IL-12R in naive T cells. IL-12R transcripts were therefore analyzed in purified CD45RA+ T cells
before and after stimulation with plate-bound anti-CD3 mAb.
IL-12R transcripts were not found in naive T cells (Fig. 4,
lane 2), but could be detected as early as 24 h after T cell activation (Fig. 4, lane 9). Priming of naive T cells in the
presence of IL-12 resulted in enhanced expression of IL-12R
2 transcripts in this population (Fig. 4, lane 13); priming
of cells in the presence of IL-4, in contrast, resulted in very
low levels of IL-12R
2 transcripts (Fig. 4, lane 14). Thus,
antigen receptor triggering is sufficient to make naive T cells
susceptible to IL-12 signaling.
Fig. 3.
Transcripts encoding IL-12 receptor subunits in human Th1
and Th2 cells. (A) IL-12R 2 transcripts are selectively expressed in human Th1 cells. Th1 and Th2 lines generated from cord blood lymphocytes were harvested on the indicated time after priming. In lanes 9 and
10, cells were washed on day 19 and incubated for 48 h in medium containing 2 ng/ml IL-12 before RNA extraction on day 21. Transcripts encoding the IL-12R
1 subunit (top), the IL-12R
2 subunit (middle) and
an 18S RNA gene fragment as loading control (bottom) were quantitated
with ribonuclease protection assays as described in Materials and Methods.
(B) Expression of IL-12R
1 and
2 mRNAs in human Th1 (CB13) and
Th2 (CB21) T cell clones. Th1 and Th2 cells were incubated on day 11 after restimulation for 48 h with or without 2 ng/ml IL-12, followed by
RNA extraction on day 13. Quantitation of the IL-12R
1 and
2 transcripts in IL-12 treated and untreated Th1 and Th2 cells was performed
by RNase protection assays.
[View Larger Versions of these Images (37 + 30K GIF file)]
Fig. 4.
Antigen receptor triggering induces expression of IL-12R 1
and
2 mRNAs in purified CD45RA+ T cells. CD45RA+ T cells were
purified by negative selection from buffy coats as described in Materials and
Methods. Purified CD45RA+ T cells (5 × 106) were stimulated with platebound anti-CD3 mAb with or without the addition of IL-12 (2 ng/ml)
or IL-4 (200 U/ml). RNA was extracted from unstimulated CD45RA+
T cells (lane 2) or at the indicated time after CD3 stimulation. Transcripts
encoding IL-12R subunits were quantitated in RNase protection assays.
[View Larger Version of this Image (39K GIF file)]
2
expression in these cells. On the contrary, similar levels of
high and low affinity IL-12 binding were measured in
mouse Th1 and Th2 cell lines (30 and data not shown).
This difference in IL-12 binding behavior in the mouse and
human systems may reflect the dominant role mouse IL-12R
1 plays in binding IL-12 (34), whereas in humans, both
IL-12R
1 and
2 appear to contribute equally to IL-12
binding (25). These results demonstrate that the IL-12R
2
subunit is selectively expressed in Th1 cells where it is induced upon T cell activation. Moreover, our data suggest
that the lack of IL-12R
2 chain expression could be the
reason underlying the inability of IL-12 to activate the
Jak2-Stat4 pathway in Th2 cells.
Fig. 5.
Scatchard analysis of
[125I]IL-12 binding to human
Th1 and Th2 cell lines. Th1 and
Th2 lines were generated as described above. Scatchard analysis
was performed using [125I]labeled
human IL-12 binding on human
Th1 and Th2 cell lines on day 9 after priming as described in Materials and Methods. Comparable
[125I]IL- 12 binding data were
obtained in three independently
derived Th1 and Th2 cell lines.
[View Larger Version of this Image (11K GIF file)]
2 mRNA Expression during T Helper Cell Differentiation.
2 transcripts,
we added IL-12 to both Th1 and Th2 lines at day 19 after
priming. Culturing the cells in the presence of IL-12 for 48 h
resulted in a sevenfold increase in IL-12R
2 transcripts in
Th1 lines and a twofold increase in Th2 lines (Fig. 3 A,
lanes 9 and 10). These results were confirmed and extended using established Th1 and Th2 clones. IL-12 induced IL-12R
2 mRNA upregulation (threefold) in both
cell types (Fig. 3 B). Time course experiments revealed that
the IL-12 induced upregulation of the IL-12R
2 mRNA
in primed T cells was transient; maximal induction was
seen 24-48 h after the addition of IL-12, but returned to
the level of the control cultures after 96 h (data not shown). The IL-12-induced transient upregulation of the IL-12R
2 transcripts in Th2 clones may account for the transient
period of IFN-
production in cells with an established
Th2 phenotype (35, 36).
(0.9 ng/ml) (Fig. 6 A). These cells phosphorylate Stat4 in
response to IL-12 and accordingly express the IL-12R
2
mRNA, although to a lower level, as compared to Th1
cells (Fig. 6, B and C). In contrast, neonatal cells primed in
the presence of IL-4 and neutralizing anti-IL-12 mAb
completely lost IL-12R
2 expression (Figs. 3 A and 6 B)
and consequently, IL-12-mediated signaling (Figs. 2 and 6 C).
Although the mechanism by which IL-4 induces the generation of IL-12 nonresponsive Th2 cells remains to be
clarified, our results are consistent with a model in which IL-12 prevents the gradual extinction of the IL-12R
2
subunit that appears to follow exposure to IL-4. Alternatively, the presence of IL-12 or IL-4 could result in the
preferential expansion of IL-12 responsive Th1 cells or IL-12
nonresponsive Th2 cells, respectively.
Fig. 6.
Function of type I and II IFNs in the development of T
helper cell subsets and on the expression of the IL-12R 2 subunit. (A)
IFN-
induces Th1 development. T cell lines were generated by stimulating cord blood lymphocytes with PHA in the presence of the indicated
cytokines or anti-cytokine antibodies. IFN-
or IFN-
(1,000 U/ml)
were added at the time of priming to the cultures as indicated. Cytokine
production was determined on day 10. IFN-
, but not IFN-
, induces a
Th1 phenotype even in the presence of IL-4 and neutralizing anti-IL-12
antibodies. (B) IFN-
induced Th1 cells express IL-12R
2 transcripts.
RNA was extracted from T cell lines 10 d after priming in the presence of
the indicated cytokines or neutralizing anti-cytokine antibodies. RNase
protection assays were performed as described above. (C) IFN-
induced
Th1 cells are IL-12 responsive. T cell lines (Fig. 6 A) were harvested on
day 10 after priming. 8 × 106 cells were washed and incubated 15 min at
37°C in medium with or without 2 ng/ml IL-12 in 1 ml RPMI-FCS.
IL-12 induced Stat4 phosphorylation was determined as described above.
[View Larger Versions of these Images (25 + 29 + 27K GIF file)]
Induces IL-12R
2 mRNA Expression and IL-12
Responsiveness in Human T Cells.
and IFN-
have also been shown to influence Th1
cell development (37, 38). We therefore examined the effect of these cytokines on the regulation of the IL-12R
2
expression. The addition of IFN-
(Fig. 6 A) as well as
IFN-
(data not shown) to human cord blood leukocytes
stimulated under Th2-inducing conditions (IL-4 and anti-
IL-12 mAb) resulted in cells producing IFN-
(>1 ng/ml)
upon restimulation with anti-CD3 mAb (Fig. 6 A). IL-12R
2 mRNA was expressed in the IFN-
-treated T cell lines
(Fig. 6 B) and IL-12 induced Stat4 phosphorylation in
these cells (Fig. 6 C). In contrast, addition of IFN-
to cultures containing IL-4 and anti-IL-12 (Th2-inducing conditions) or addition of neutralizing anti-IFN-
mAb at the
time of Th1 priming had no significant effect on the pattern of Th cell development (Fig. 6 A and data not shown)
or IL-12R
2 expression (Fig. 6 B). IFN-
or IFN-
had
no significant effect on the expression of the IL-12R
1
transcripts (data not shown). Our findings differ from previous reports showing that in the mouse IFN-
but not
IFN-
influences Th1 cell development (38). Our data, together with the evidence that IFN-
in vitro favors the development of human allergen-specific T cells into a Th1
phenotype (37), suggest that type I and type II IFNs could
have different functional effects on human as compared to
mouse T cells. Indeed, preliminary results indicate that in human Th2 cells, both IFN-
and IFN-
are able to induce a much stronger (10-fold higher) phosphorylation of
Stat1 as compared to IFN-
(data not shown), suggesting a
quantitative rather than a qualitatitive difference in IFNinduced signaling in human and mouse cells.
2 expression. Triggering of
the antigen receptor on naive T cells is sufficient for the
initial expression of functional IL-12 receptors on activated
T cells. Depending on the cytokines present during the differentiation, T cells will then develop into IL-12-responsive Th1 cells or IL-12-nonresponsive Th2 cells. Analysis
of the IL-12R
2 promoter should provide insights into
the molecular mechanisms by which TCR triggering and
signals mediated by IL-12, IFN-
, IL-4, and other cytokines are integrated to regulate IL-12R
2 chain expression.
2 chain appears to be the first Th1 subset-
specific noncytokine gene to be identified to date. Here we
provide evidence that IFN-
, but not IFN-
, can induce
Th1 development in neonatal T cells even in the presence
of the Th2-inducing cytokine IL-4. We also demonstrate
that IL-12 and IFN-
, by inducing the upregulation of the
2 component of the IL-12R after antigen receptor triggering, play an important role in both the functional activity and development of Th1 cells. The understanding of
the molecular events controlling T cell development may
allow the selective manipulation of Th subsets in vivo. The
possibility of forcing expression in vivo of the IL-12R
2
by IFN-
or IL-12 treatment, for example, could have important therapeutic implications in Th2-mediated pathological conditions, such as allergies.
Address correspondence to Lars Rogge or Francesco Sinigaglia, Roche Milano Ricerche, Via Olgettina 58, I-20132 Milano, Italy. L. Barberis-Maino's present address is Institute for Molecular Biology, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.
Received for publication 2 December 1996.
1Abbreviations used in this paper: IL-12R, IL-12 receptor; Stat, signal transducers and activators of transcription.We would like to thank M. Gately for the IL-12 and anti-IL-12 antibodies, H. Gallati and R. Lang for the
IL-4 and IFN- ELISA assays, P. Vigano for the cord blood samples, R. Pardi for the mAbs, R. Germain
and E. Bianchi for critically reading the manuscript, and K. Murphy for communicating results before publication.
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