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T Cell Differentiation Leads to Default Production of IFN-: Failure to Down-Regulate IL-12 Receptor ß2-Chain Expression1
Sections of
*
Rheumatology and
Pulmonary and Critical Care Medicine, Department of Medicine,
Department of Pathology, and
§
Section of Immunobiology, Yale School of Medicine, New Haven, CT 06520
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells secrete Th1- and Th2-like cytokines that help
mediate innate and acquired immunity. We have addressed the mechanism
whereby murine T cells acquire the capacity to differentially
produce such cytokines. Splenic T cells could be polarized into
IFN-- or IL-4-secreting cells in vitro; however, in contrast to
CD4+ 
ß T cells, T cells predominantly produced
IFN-, even in the presence of IL-4, a finding independent of genetic
background. Like CD4+ Th1 cells, IFN--producing cells
expressed the IL-12 receptor ß2-chain after activation in
the presence of IL-12; however, unlike Th2 cells, IL-4-primed T
cells also expressed this receptor, even in the absence of IFN- and
despite the presence of the transcription factor GATA-3. IL-12 also
induced IL-4-primed T cells to proliferate and to translocate
Stat3/Stat4, indicating signaling through the IL-12 receptor. These
molecular events can account for the predominant production of IFN-
by T cells in the presence of IL-12, despite the availability of
IL-4. Early and predominant production of IFN- by T cells
likely is critical for the roles that these cells play in protection
against intracellular pathogens and in tumor
immunity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß T cells is
determined by the distinct patterns of cytokines that they secrete. Th1
cells secrete IFN-, TNF-, and IL-2 and promote inflammatory and
cellular immune responses against intracellular microbes, while Th2
cells secrete IL-4, IL-5, IL-10, and IL-13, induce
IgG1- and IgE-mediated humoral responses, and are
important for the elimination of large extracellular parasites such as
helminths and nematodes (1, 2). Many studies both in vitro
and in vivo have highlighted the predominant role of cytokines in
directing the functional differentiation of CD4+
T cell precursors during the initiation of antigenic stimulation. IL-12
and IL-4 drive differentiation of naive CD4+ T cells toward
Th1 or Th2 effector cells, respectively (3, 4, 5).
Th1 and Th2 CD4+ ß T cells are tightly
cross-regulated so that development of one subset is inhibited by
cytokines produced by the other. The Th2 cytokines IL-4 and IL-10
suppress Th1 development by inhibiting production of IFN- and IL-12,
whereas IFN- limits the outgrowth of Th2 cells (6, 7).
The molecular mechanisms mediating Th1 vs Th2 regulation rely on the
counterbalance between expression of the IL-12 receptor
ß2-chain (IL-12R
ß2)3
on Th1 cells and GATA-3 transcription in Th2 cells. The IL-12R
ß2-chain is a recently identified second
component of the IL-12 receptor (8), which mediates
IL-12-induced Stat3/Stat4 phosphorylation and subsequently IFN-
secretion (9). IL-12R ß2
expression can be up-regulated by IFN- and down-regulated by IL-4
(10). In contrast, GATA-3 is selectively expressed in Th2
cells (11, 12). GATA-3 transcription can promote Th2
cytokine secretion and inhibit IFN- production and IL-12R
ß2 expression, thus leading to the extinction
of the IL-12 signaling pathway during early Th2 development, providing
a mechanism that allows the stable commitment to the Th2 phenotype
(13, 14, 15).
T cells have unique features in comparison to ß T cells. It
has now become clear that T cells recognize nonpeptide and
nonprocessed bacterial and environmental Ags (16, 17), as
well as stress-associated Ags expressed on epithelial cells and on
certain tumor lines and primary carcinomas (18, 19).
Recognition of self-associated molecules induced by local infection or
cell transformation would enable these T cells to monitor multiple
insults to the host epithelium (20). In addition, T
cells elaborate chemokines to recruit inflammatory cells and secrete
cytokines that mediate both innate immunity and acquired immunity
(21, 22). For example, T cells produce IFN- and
IL-4 in vivo in response to Th1- or Th2-stimulating pathogens,
respectively (23), and both Th1 and Th2 T cell
clones have been obtained in vitro (24). Production of
IFN- by T cells may also be critical for tumor immunity
(19, 25), and such cells from mouse spleen selectively
express this cytokine, along with IL-2 and TNF-, after stimulation
through the TCR (26).
Although cytokine production by T cells appears to be important
for both host defenses and tumor immunity, the molecular mechanisms for
differentiation of T cells into IFN--producing or
IL-4-secreting cells are undefined, nor has it been established whether
the control of differentiation of these cells is mediated by the IL-12R
ß2-chain and/or by GATA-3. Thus, in the present
study, we have analyzed the differentiation of T cells in vitro
in comparison to CD4+ ß T cells. We
demonstrated that T cells from C57BL/6 (B6) and from BALB/c mice
can be polarized into Th1-like or Th2-like cytokine-secreting
cells in the presence of IL-12 or IL-4, respectively; however, striking
differences between T cells and CD4+
ß T cells were observed. IL-12 was dominant over IL-4 for T
cell differentiation in both B6 and BALB/c mice. Like ß Th1 cells,
IFN--producing T cells up-regulated the IL-12R
ß2 after activation in the presence of IL-12;
however, in contrast to ß Th2 cells, IL-4-producing T cells
also expressed this receptor, even in the absence of IFN- and
despite the presence of the transcription factor GATA-3. The latter
cells also proliferated IFN- after IL-12 stimulation, in the context
of Stat3/Stat4 phosphorylation. Taken together, these results indicate
that T cells fundamentally differ from ß T cells in
response to exogenous cytokines, including the molecular events that
lead to production of Th1- vs Th2-like cytokines. The predominant
production of IFN- by T cells likely is critical for the
roles that these cells play in protection against intracellular
pathogens and in tumor immunity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6), B6 TCR ß-deficient (TCR
ß-/-) (4–6 wk of age), B6 IFN--deficient
(IFN--/-), and BALB/c mice were purchased
from The Jackson Laboratory (Bar Harbor, ME). AND TCR transgenic mice
on the B10.BR background, expressing an TCR-ß recognizing pigeon
cytochrome C (27), were graciously provided by Steve
Hedrick (University of California at San Diego) via Kim Bottomly (Yale
University School of Medicine, New Haven, CT). All animals were
maintained under specific pathogen-free conditions at the Yale
University School of Medicine.
Cytokines and Abs
Recombinant murine IL-2, recombinant murine IL-4, and
recombinant murine IL-12 were purchased from R&D Systems (Minneapolis,
MN). Anti-IL-4 and anti-IFN- mAb were kindly provided by Dr.
Magarian Blander and Dr. Charles Janeway (Section of Immunobiology,
Yale University School of Medicine). The following anti-mouse mAb
used for phenotypic and cytokine analyses were purchased from
PharMingen (San Diego, CA): purified anti-CD3e (no azide/low
endotoxin, 145-2C11, hamster IgG), purified anti-CD28 (no azide/low
endotoxin, 37.51, hamster IgG), FITC-conjugated or biotinylated
anti- TCR (GL3, hamster IgG), FITC-anti-CD4 (GK1.5, rat
IgG2b), PE-anti-CD62L (L-selectin, MEL-14, rat IgG2a),
CyChrome-anti-CD44 (IM7, rat IgG2b), FITC-anti-IFN- (XMG1.2, rat
IgG1), PE-anti-IL-4 (11B11, rat IgG1), PE-anti-IL-5 (TRGK5, rat
IgG1), and PE-anti-IL-10 (JES5-16E3, rat IgG2b). Anti-Stat3,
-Stat4, and -Stat5 were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA).
Preparation of T cells and AND TCR transgenic
CD4+ T cells
T cells were purified from B6 TCR
ß-/- mice splenocytes using a magnetic
activated cell sorter (MACS) according to the instructions of the
manufacturer. Simply, cells were labeled with FITC-anti- TCR
Ab followed with anti-FITC magnetic beads (Miltenyi Biotec,
Germany). Labeled cells were eluted from the cell separation column.
For naive T cell enrichment, column-purified T cells were
further labeled with anti-CD44-CyChrome and anti-CD62L-PE.
CD44low and CD62Lhigh cells
were then gated and sorted using flow cytometry (Vantage; Becton
Dickinson, San Jose, CA). For AND TCR+
CD4+ T cell purification, splenocytes from AND
TCR transgenic B10.BR mice (4–6 wk of age) were labeled with
FITC-anti-CD4, followed with anti-FITC magnetic beads, as
described above. The purity of both and
CD4+ T cells were >95%. T cells from B6,
BALB/c, and B6 IFN--/- mice were first
enriched from splenocytes with positive selection via a MACS column,
then further sorted by flow cytometry as above.
Cytokine polarization for T cells and AND TCR
CD4+ T cells
Splenocytes (2 x 106/ml) from B6 TCR
ß-/- mice were cultured in complete Click’s
medium (10% heat-inactivated FBS) with PHA (3 µg/ml) in the presence
of either IL-4 (20 ng/ml) and anti-IFN- (10 µg/ml) to promote
T cells that produced IL-4 (Th2 differentiation) or IL-12 (5
ng/ml) and anti-IL-4 (10 µg/ml) for promotion of T cells
producing IFN- (Th1 development). IL-2 was added to the culture
medium at day 3. At day 6, cells were washed and restimulated with
plate-bound anti-CD3 (10 µg/ml) and soluble anti-CD28 (1
µg/ml) for intracellular cytokine staining as described below or
further cultured as described in the figure
legends. For polarization,
sorted naive or purified T cells were stimulated with
plate-bound anti-CD3 and soluble anti-CD28 in the presence of
cytokines and/or anti-cytokine Abs, with further restimulation as
described above. For AND TCR+ transgenic
CD4+ T cells, purified transgenic cells (1
x 106/ml) were primed with coated anti-CD3
and soluble anti-CD28 under the same cytokine conditions as
described above. CD4+ T cells were also
restimulated for cytokine staining and further cultured as described
for T cells.
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Cultured T cells and CD4+ T cells
under Th1- or Th2-priming conditions were washed and restimulated with
coated anti-CD3 and soluble anti-CD28 in the presence of IL-2.
After 4 h of culture, brefeldin A (1 µM; PharMingen, San Diego,
CA) was added to cultures to enhance intracellular cytokine
accumulation. Two hours later (total culture of 6 h), cells were
washed with PBS, fixed with 2% formaldehyde in PBS, permeabilized with
0.5% (w/v) saponin, stained with cytokine-specific Abs, and detected
by flow cytometry. For three-color staining (for cell lines in bulk
cultures in the first week only), cells were washed after 6 h
culture and labeled with biotinylated-anti-mouse TCR
followed with streptavidin-CyChrome. After washing twice, labeled cells
were fixed and permeabilized as above. For cytokine staining, cells
were incubated with FITC-anti-mouse IFN- and PE-anti-mouse
IL-4, IL-5, or IL-10 in saponin buffer at room temperature for 1
h. Cells were washed twice with saponin buffer, and further washed with
PBS without saponin, resuspended with PBS, and analyzed using a
FACScaliber flow cytometer (Becton Dickinson). Data were displayed as
dot plots of FITC (x-axis) and PE (y-axis)
fluorescence (log scales). Quadrant markers were positioned to include
>99% of control Ig-stained cells in the lower left quadrant (data not
shown).
RT-PCR
Cultured T cells or AND TCR+
transgenic CD4+ T cells were used for
determination of mRNA encoding the IL-12R ß2 or
ß-tubulin by RT-PCR. Total RNA was extracted using RNeasy kit
(Qiagen, Santa Clara, CA) following the instructions of the supplier.
RNA was reverse transcribed using a kit from Stratagene (La Jolla, CA)
according to the instructions of the manufacturer. PCR amplification
using different primers was performed in 50-µl volumes with 1 U of
Taq polymerase (Perkin-Elmer, Norwalk, CT) using a 96-well
thermocycler (MJ Research, Cambridge, MA). The sequences of the primers
were: IL-12R ß2,
5'-AAAGCCAACTGGAAAGCATTCG-3' and 5'-AGTTTTGAGTCAGGGTCTCTGC-3'; and
ß-tubulin, 5'-GGCGCCCTCTGTGTAGTGGCCTTTGGCCCA-3' and
5'-CAGGCTGGTCAATGTGGCAACCAGATCGGT-3'. PCR products were electrophoresed
through a 1.5% agarose gel and visualized by ethidium bromide
staining.
RNA isolation and Northern analysis
Total cellular RNA was prepared by using Trizol (Life
Technologies, Paisley, U.K.) according to the instructions of the
manufacturer. Six micrograms of RNA from each sample were fractionated
on a formaldehyde agarose gel and transferred to a nylon membrane
(Micron Separations, Westboro, MA). A 360-bp DNA fragment
(BglI-ClaI) derived from the murine GATA-3 cDNA
not containing the zinc finger domain (kindly provided by Dr. James D.
Ergel) was labeled with [-32P]dCTP using a
random primer DNA labeling kit (Boehringer Mannheim, Mannheim,
Germany). Hybridization was performed by QuikHyb (Stratagene) according
to the instructions of the manufacturer.
T cell proliferation
Both CD4+ T cells and T cells were
activated in the presence of IL-4 and anti-IFN- for 6 days.
Cells were then washed two times, cultured with different cytokines for
48 h without TCR triggering, and pulsed with
[3H]thymidine (20 µCi/ml) 18 h before
harvesting. Uptake of radiolabeled thymidine (cpm) was measured in a
liquid scintillation counter.
Gel shift assay
The double-stranded 32P-labeled oligonucleotide 5'-GTGCATTTCCCGTAAATCTTGTCTACAATTC-3' (m67) and annealed complementary oligonucleotide were used as described (28). Binding reactions with whole-cell extracts and EMSA on 4% polyacrylamide gels were also performed as described previously (28).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells differentiate into IFN--producing cells and
IL-4-producing cells
To assess T cells differentiation in vitro, splenocytes
from B6 TCR ß-/- mice were stimulated with
the mitogen PHA in the presence of IL-12 and anti-IL-4, or IL-4 and
anti-IFN-, for 6 days. Cells were then restimulated with
plate-coated anti-CD3 and anti-CD28, followed by intracellular
cytokine staining. Under Th1-priming conditions, 
70% of T
cells produced IFN-, whereas <1% were IL-4+
or IL-10+ (Fig. 1
A). In comparison, after Th2
priming nearly 26% of T cells stained positive for IL-4;
however, almost 15% were IFN-+. T
cells sorted from T cell-intact B6 mice had an identical pattern of
cytokine production under the same polarization conditions (data not
shown), indicating that T cells from ß T cell-intact and
ß T cell-deficient B6 mice have the same functional properties in
terms of cytokine production in vitro.
Only 30–40% of splenic T cells isolated from young (4–6 wk of
age) B6 TCR ß-/- mice or T cell-intact mice
were CD62Lhigh CD44low, a
naive phenotype with lower cell turnover in comparison to
CD62Llow CD44high cells
(29, 30). To rule out the possibility that the phenotype
of polarized T cells was a consequence of outgrowth of cells
previously activated in vivo, we next asked if naive T cells,
sorted according to the CD44 and CD62L surface markers, could be
polarized to IFN-+ or
IL-4+ cells. Comparison was made to naive
CD4+ ß T cells isolated from AND TCR
transgenic mice (27). Under Th1 priming conditions
identical with those described above, nearly 26% of
CD4+ cells became IFN-+,
while <1% of the cells stained for IL-4 (Fig. 1B) or IL-5
(data not shown). Under Th2-priming conditions, 8% of naive
CD4+ cells became IL-4+ and
<1% cells were IFN-+.
Like CD4+ ß T cells, naive T cells
predominantly became IFN-+ (67%) with little
or no IL-4+ cells (0.34%) under Th1-priming
conditions. In contrast to CD4+ Th2 cells,
however, about 5% of naive T cells sorted from B6 spleens
became IL-4+ and 28% became
IFN-+ after IL-4 and anti-IFN-
treatment (Fig. 1B), consistent with the bulk culture
results (Fig. 1A). In total, these data imply that naive
T cells predominantly produce Th1 cytokines upon activation in
the presence of polarizing cytokines, even in the presence of IL-4 and
anti-IFN-, a striking difference from ß T cells.
To determine whether these findings were a consequence of the B6
genetic background, total splenic T cells were sorted from
BALB/c mice, which typically produce strong Th2 responses (31, 32). Cells were cultured under the same polarizing conditions,
followed by restimulation and intracellular cytokine staining as
described above. As for B6 mice, T cells from TCR-intact BALB/c
mice became primarily IFN-+ cells under
Th1-priming conditions; however, there were substantial numbers of
IFN-+ cells, about 2-fold as many as
IL-4+ cells, after Th2 priming (Fig. 1C). These data further support the notion that T
cells appear to largely default to a Th1-like phenotype, a result that
is not dependent upon genetic background.
IL-12 is dominant over IL-4 in T cell differentiation
Stimulation of CD4+ ß T cells by
anti-CD3, or by specific peptide for TCR transgenic cells, in the
presence of IL-4 and IL-12 leads to a Th2 phenotype; e.g., IL-4 is
dominant over IL-12 in ß T cell differentiation (33, 34). To determine the effects of the combination of IL-4 and
IL-12 on T cells, CD62Lhigh
CD44low naive T cells, purified by sorting
from spleens of B6 TCR ß-/- mice, and total
T cells purified from spleens of TCR-intact BALB/c mice were
cultured with coated anti-CD3 and soluble anti-CD28 in the
presence of both cytokines. After 6 days of culture, cells were
restimulated followed by intracellular cytokine staining. Parallel
cultures of purified CD4+ AND T cells were also
studied. As expected, CD4+ ß T cells exposed
to both IL-4 and IL-12 more commonly produced IL-4 and IL-5 than
IFN- (Fig. 2A): the ratio
of IL-4+ to IFN-+ cells
was >3-fold and that of IL-5+ to
IFN-+ was >9-fold (Fig. 2B),
indicative of a Th2 phenotype and consistent with published results
(33, 34). In contrast, under identical conditions,
T cells from both B6 and BALB/c mice had a reversed IL-4 (IL-5)/IFN-
ratio, with predominant production of IFN- and much less IL-4 or
IL-5 (Fig. 2A). IFN-/IL-4 and IFN-/IL-5 ratios were
>13- and 15-fold, respectively, for B6 mice, and >8- and 13-fold,
respectively, for BALB/c mice (Fig. 2B). This striking
phenotypic difference between T cells and
CD4+ ß T cells strongly suggested that
T cells default to a Th1-like pathway under the influence of
IL-12, even in the presence of IL-4. In these experiments,
IL-4-producing and IFN--producing ß CD4+
and T cells had similar means fluorescence intensity, an
indicator of the amount of cytokines produced per cell
(35).
Both Th1- and Th2-primed T cells express the IL-12R
ß2 subunit
The IL-12R ß2 subunit is necessary for
IL-12 signaling through the Janus kinases/Stat pathway (8, 9). IL-4 can selectively down-regulate expression of this
receptor subunit in polarized Th2 CD4+ cells
(10, 36). Because IL-12 appears to be dominant over IL-4
for T cell differentiation (Fig. 2), in contrast to what is
found for ß T cells, we next asked if T cells, especially T
cells activated in the presence of IL-4 and anti-IFN-, expressed
the IL-12R ß2 subunit. Total RNA was extracted
from both IL-12-primed and IL-4-primed T cells 6 days after
priming in vitro, and the presence of the IL-12R
ß2 subunit mRNA was sought using RT-PCR, with
ß-tubulin transcription as a control. As an additional control, total
RNA from both Th1 and Th2 CD4+ T cells was also
prepared. Both IL-12-primed and IL-4-primed T cells expressed
the IL-12 ß2-chain, as did cells exposed to
both IL-4 and IL-12 (Fig. 3A,
lanes 4–6). This result was in contrast to
CD4+ T cells, in which only Th1 cells, but not
Th2 cells nor cells primed with IL-12 plus IL-4, expressed this
receptor subunit (Fig. 3A, lanes 1–3).
It was conceivable that the failure to down-regulate IL-12R
ß2 expression was secondary to incomplete
skewing to the IL-4-producing phenotype. Therefore,
IFN--/- mice were employed to induce a
population of T cells producing IL-4 but not IFN-. Purified
T cells from B6 IFN--/- mice were
activated with anti-CD3 and anti-CD28 in the presence of IL-4.
CD4+ T cells isolated from
IFN--/- mice were also primed as a control.
Cells were then restimulated for cytokine staining, with RNA extracted
for RT-PCR to assess IL-12R ß2 expression.
Under Th2-priming conditions, only IL-4+ and
IL-5+ cells were observed in cultures, with no
IFN-+ cells, confirming the genotype of the
mice (data not shown). Nevertheless, the IL-12R
ß2 was expressed by IL-4-primed T cells
from both IFN--/- and wild-type mice,
respectively (Fig. 3B, lanes 3 and 4),
indicating that IL-12R ß2 expression on
IL-4-primed T cells was independent of IFN-. In contrast,
this receptor was only expressed in CD4+ cells
primed with IL-12 plus anti-IL-4 (Th1 cells) and not Th2 cells in
the control cultures (Fig. 3B, lanes 1 and
2). This is consistent with previous evidence that
IL-12-primed CD4+ T cells may express IL-12R
ß2 independently of IFN-
(10).
To examine the kinetics of IL-12R ß2
expression, naive T cells as determined by surface phenotype
were next sorted from B6 TCR ß-/- mice,
followed by culture under different cytokine conditions. Total RNA was
extracted from the cultured cells at serial time points, as well as
directly from sorted naive and memory T cells, reverse
transcribed, and amplified by PCR. Notably, IL-12R
ß2 was expressed on T cells with both
naive and activated phenotypes (Fig. 3C, lanes 1
and 2), with further up-regulation on days 3 and 5, even in
the presence of IL-4.
GATA-3 does not counterbalance IL-12-induced IFN- secretion by
T cells
When naive CD4+ T cells are stimulated to
differentiate along the Th1 or Th2 pathway, GATA-3 expression is
up-regulated in Th2 cells but is down-regulated in Th1 cells (11, 12). Because IL-12 extinguishes GATA-3 production in
CD4+ T cells in a Stat4-dependent manner, and
because IL-12 is dominant over IL-4 in T cell differentiation,
we next determined GATA-3 expression in T cells under polarizing
conditions described above, with naive CD4+
ß T cells as a control. GATA-3 expression was barely detectable in
or ß T cells directly isolated from spleens (Fig. 4A, lanes 1 and
2). In comparison, GATA-3 expression was clearly
up-regulated in IL-4-primed T cells but not in IFN--primed
T cells, a pattern similar to that found in
CD4+ ß T cells, as reported previously (Fig. 4A, lanes 10 and 6) (11).
Notably, T cells cultured with IL-4 plus IL-12, which
predominantly produce IFN-, displayed high levels of GATA-3,
comparable to its expression in Th2-primed T cells (Fig. 4A, compare lanes 14 and 10). A
similar profile of GATA-3 expression was observed when sorted naive
T cells, as determined by surface staining, were cultured with
IL-4, or a combination of IL-12 plus IL-4 (Fig. 4B).
Collectively, these results indicate that IL-4 induces GATA-3
expression in T cells as it does in CD4+
cells; however, IL-4-mediated up-regulation of GATA-3 expression does
not inhibit IL-12R ß2 expression in T
cells, in contrast to findings in ß T cells, where this
transcription factor inhibits IL-12 driven IFN- secretion (14, 15). Also, despite IL-12 dominance in regard to overall cytokine
production from T cells after exposure to both IL-12 and IL-4,
IL-4 is able to induce GATA-3 expression in these cells.
IL-12-primed cells do not produce Th2 cytokines upon IL-4
stimulation
Previous studies have shown that CD4+ ß
Th1 cells can be converted to a Th2 phenotype in the presence of IL-4
at the level of cell populations (37, 38). To determine
whether such a shift also occurs in T cells, we added IL-4 to T
cells previously polarized to IFN- production. Upon re-exposure of
Th1 CD4+ ß T cells to IL-4, the Th1
phenotype reverts to a Th2 pattern, as evidenced by an increased
percentage of IL-10+ (Fig. 5) and IL-4+ cells
(data not shown). In contrast, IFN--producing T cells were
unable to respond to restimulation with IL-4 without significant
amounts of IL-4+ and IL-10+
cells (<0.5%) induced in comparison to the primary culture. These
data further support a default Th1 pathway for T cell
differentiation.
IL-4-primed T cells proliferate in response to IL-12
restimulation and signal through Stat3/Stat4
To further demonstrate that the IL-12R
ß2-chain expressed on IL-4-primed T
cells is functional, we next asked if IL-12 could induce IL-4-driven
T cells to proliferate. Here, T cells were primed in the
presence of IL-4 and anti-IFN- for 6 days. After washing, cells
were cultured with different cytokines as indicated, without further
TCR triggering (Fig. 6A).
CD4+ ß Th2 cells were used as a control.
Notably, T cells primed in the presence of IL-4 and
anti-IFN- proliferated in response to IL-12 plus IL-2, in that
cells cultured with both the latter cytokines had substantially
increased proliferation compared with cells cultured with IL-2 alone,
and equivalent to that induced by addition of IL-2 plus IL-4
(39). In contrast, CD4+ Th2 cells
did not respond to IL-2 plus IL-12, with proliferation in the presence
of the latter cytokine similar to that induced by IL-2 alone and much
less than IL-2 plus IL-4.
Next, we asked if IL-12 signaled through Stat4 to induce IFN-
production in IL-4-primed T cells, assessing translocation of
this transcription factor in gel shift assays (Fig. 6B).
T cells isolated from B6 wild-type mice and B6
IFN--/- mice were primed with IL-4, with or
without anti-IFN- for 6 days. After washing, cells then treated
for 20 min with IL-12 and analyzed for Stat activation by gel shift
assay using the high-affinity binding site of Stats, m67, as a probe
(27). IL-12 treatment of IL-4-primed T cells led to
the activation of Stat proteins (Fig. 6B, lane
2). To exclude IFN-+ cells in the
IL-4-primed T cell cultures, Stat activation was also tested in
IL-4-primed cells from IFN--/- mice.
A similar pattern of activated Stat factors was observed in these cells
(Fig. 6B, lane 3). Stat3 and Stat4 as components
of the IL-12-induced DNA complexes were shown by anti-Stat Ab
supershift reactions (Fig. 6B, lanes 9 and
10). The pattern of Stat activation was indistinguishable
whether cells were activated under IL-12 or IL-4 conditions and
subsequently stimulated with IL-12 (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells can be directed
to produce either IFN- or IL-4 in vitro after exposure to IL-12 or
IL-4, respectively, analogous to ß T cells. Previous studies have
shown that T cells are capable of producing Th1 (IFN-) and
Th2 (IL-4) cytokines in vivo (23, 24, 26, 40, 41). The in
vitro approach allowed us to identify fundamental differences in
cytokine production between these two T cell lineages. First, in
contrast to ß CD4+ T cells (33, 34), IL-12 is dominant over IL-4 in T cell
differentiation, a finding independent of genetic background with
similar responses in the B6 and BALB/c strains and one that is
apparently a consequence of constitutive expression of the IL-12R
ß2-chain. Second, IL-4-primed T cells
maintained the expression of the IL-12R
ß2-chain, independent of IFN-, with intact
IL-12 signaling despite the presence of the transcription factor
GATA-3.
The most striking difference between ß and T cells
demonstrated here was their response to IL-12. To understand the
mechanism of IL-12 dominance over IL-4 in T cell
differentiation, we investigated the expression of IL-12R
ß2 and GATA-3 transcription in T cells under
Th1 and Th2 polarization conditions. GATA-3 can suppress the expression
of the IL-12R ß2 subunit, extinguishing
IL-12-mediated signaling (3, 13, 14), thus inhibiting
IFN- production by Th1 cells (14, 15). Conversely,
IL-12 can repress GATA-3 transcription and inhibit IL-4 production by
Th2 cells (5, 14). Notably, we found that T cells
under both Th1- and Th2-priming conditions expressed the IL-12R
ß2-chain (Fig. 3A). To exclude the
possibility that the failure to down-regulate IL-12R
ß2 expression on IL-4-primed T cells was
secondary to incomplete skewing to an IL-4-producing phenotype with
contamination by IFN--producing cells, we used IL-4-primed T
cells from IFN--deficient mice. Such cells also had expression of
this chain (Fig. 3B). This is in contrast to the finding
that in CD4+ ß T cells primed in the
presence of IL-4, the IL-12R ß2-chain is
maintained only in the presence of IFN- (10). This
contrast raises the possibility that, because the majority of splenic
T cells are TCR coreceptor negative, CD4- and/or CD8-mediated
signaling may down-regulate IL-12R ß2,
especially in the setting of IL-4 signaling.
GATA-3 mRNA synthesis in T cells followed a similar pattern to
that in ß T cells in that it was up-regulated in IL-4-primed
T cells but not in IFN--producing cells (Fig. 4). However,
T cells in cultures exposed to IL-4 plus IL-12 had both IL-12R
ß2 expression (Fig. 3) and GATA-3 transcription
(Fig. 4), despite the presence of a large number of IFN--secreting
cells (Fig. 2). These results indicate that in T cells,
IL-12-mediated IFN- secretion is dissociated from IL-4-induced
GATA-3 expression. The dissociation between GATA-3 and IL-12R
ß2 expression in T cells implies that
the regulation of ß and T cells fundamentally differ in
terms of responsiveness to exogenous cytokines. Considerable progress
has been made in identifying transcription factors that control helper
cell development, especially for Th2 cells (42). The c-Maf
proto-oncogene (43), NF-AT, and a novel nuclear Ag,
NF-AT-interacting protein 45 kDa (NIP45) (44, 45), all
have been demonstrated to promote Th2 development and IL-4 secretion.
Further studies on the effects of these novel transcription factors on
T cell differentiation, especially how they counterbalance
IL-12-induced IFN- secretion, will give us additional information
about the distinction between ß and T cells.
IL-12 dominance over IL-4 on T cells was also shown in the
secondary response. We showed that IL-12-primed T cells, 70% of
which were IFN--secreting cells, failed to produce Th2 cytokines
after IL-4 restimulation. This result is also dramatically different
from that found in CD4+ ß T cells. Polarized
murine ß Th1 populations after a week of IL-12 stimulation can be
converted to Th0/Th2-like populations by IL-4 in vitro (37, 38). Indeed, IFN--producing ß T cells have been isolated
and converted to IL-4 and, especially, IL-10-producing cells after
restimulation with IL-4 (46). In our system,
CD4+ Th1 cells were also induced to become
IL-10+ and IFN-+
IL-10+ double positive, consistent with previous
reports. Surprisingly, however, IFN--producing T cells showed
no response to IL-4 restimulation, retaining their phenotype (Fig. 5).
In addition, IL-4-primed T cells maintained response to IL-12
restimulation, through active IL-12R ß2 subunit
expression. This receptor was fully capable of transmitting an IL-12
signal, even after IL-4 priming, in that IL-12 together with IL-2
induced IL-4-cultured T cells, but not
CD4+ ß T cells, to proliferate to a similar
extent as IL-4 plus IL-2 (Fig. 6A). Moreover, IL-12 induced
IL-4-primed T cells to translocate Stat4, even in the absence of
IFN- (Fig. 6B), further supporting the notion that
IL-12-mediated signaling was intact.
Our results indicate that although T cells default toward
IFN- production, they can be induced to secrete IL-4 in vitro (Fig. 1), as they do in vivo. Indeed, recent work in an allergic airway
inflammation model that has pointed to the critical role of T
cells in CD4+ Th2 development, implying that
these cells may serve as a source of IL-4, depending on the site and
model of immunization (41). We emphasize that our studies
have focused upon splenic T cells and that other subsets of
these cells, for example those found at various epithelial surfaces,
may have different phenotypes. Even in the spleen, other factors may
affect T cell differentiation. For example, CD4 has been
implicated in the requirement for IL-4 secretion (47, 48);
however, the majority of splenic T cells are
CD4-CD8-, a fact perhaps
contributing to the findings herein. The strength of TCR signaling may
also affect ß T cell differentiation (49), although
we do not know if such findings are applicable to T cells in
vivo. We do note that in our in vitro work, the same concentration of
anti-CD3 Abs were used for activation of both and ß T
cells. In addition, in the presence of APC (bulk cultures of
splenocytes), T cells secreted more IL-4 than purified naive
T cells incubated with anti-CD3 and anti-CD28 in the
absence of APC (Fig. 1, A and B). Thus, APC in
splenocyte culture appear to be capable of providing additional stimuli
such as cytokines or costimulatory molecules that aid development
of IL-4-producing T cells. This is consistent with the general
notion that the generation of a Th2 response is more dependent on
costimulation than is generation of a Th1 response (50, 51).
What are the biologic consequences of the predominant production of
IFN- by T cells? This cytokine is critical for host defenses,
especially protection from intracellular pathogens and for tumor
immunity (1, 25), and its secretion by T cells is
apparently critical for its function. For example, T cells play
an important role in early protection from experimental
Mycobacterium tuberculosis infection through IFN-
secretion (52, 53). These T cells also contribute to the
regulation of NK cell-mediated innate resistance against another
intracellular pathogen, Listeria monocytogenes
(54). Recently, T cells were shown to recognize MHC
class I-related molecules MICA and MICB, which are induced by stress
resulting from infection or injury, or in cellular transformation
(18, 19). These molecules have more recently been shown to
be stimulatory ligands for the NK cell receptor NKG2D, which is also
found on T cells (55, 56). Recognition of MICA and
MICB on transformed cells by human T cells leads to IFN-
production, presumably aiding in tumor immunity (19, 25).
Our data demonstrate that T cells, in the presence of IL-12 and
despite the availability of IL-4, are intrinsically programmed to
predominantly produce IFN-, presumably after contact with
stress-related molecules expressed on infected or transformed cells.
This program would lead to early and appropriate defense mechanisms in
the host.
Early production of IFN- by T cells also apparently
contributes to the regulation of ß T cell-mediated specific immune
responses. NK1.1+ T (NK T) T cells have a
constitutively activated surface phenotype (57), similar
to T cells. The former cells, while capable of IFN-
production, produce an early burst of IL-4 after anti-CD3 injection
(58). This leads us to speculate that in the early phase
of immune response, the balance between T cell activation, with
early IFN- production needed for clearance of intracellular
pathogens and for tumor immunity, and NK T cells, with early IL-4
production needed for humoral responses and clearance of extracellular
organisms, may be critical for regulating specific ß
CD4+ T cell responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Joe Craft, Box 208031, 610 LCI, 333 Cedar Street, New Haven, CT 06520-8031. E-mail address:
3 Abbreviations used in this paper: IL-12R ß2, IL-12 receptor ß2-chain; B6, C57BL/6; IFN--/-, IFN- gene deficient; MACS, magnetic-activated cell sorter; TCR ß-/-, TCR ß-chain deficient.
Received for publication October 25, 1999. Accepted for publication January 13, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production and diminishes interleukin 4 inhibition of such priming. Proc. Natl. Acad. Sci. USA 90:10188. inhibits the proliferation of Th2 but not Th1 murine helper T lymphocyte clones. J. Immunol. 140:4245.[Abstract]
production from developing Th1 cells in addition to inducing IL-4 and IL-5 levels. Clin. Immunol. 91:134.[Medline]
T cells recognize alkylamines derived from microbes, edible plants, and tea: implications for innate immunty. Immunity 11:57.[Medline]
T cells. Science 279:1737. T cells of MICA and MICB. Proc. Natl. Acad. Sci. USA 96:6879. T cells. Science 266:1253. T cells: implications for the recruitment of inflammatory cells to damaged epithelia. J. Immunol. 157:985.[Abstract]
T-cell bridge: linking innate and acquired immunity. Nat. Med. 4:764.[Medline]
and interleukin-4 in response to Th1- and Th2-stimulating pathogens by T cells in vivo. Nature 373:255.[Medline]
cell clones can be defined phenotypically and functionally as Th1/Th2 cells and illustrate the association of CD4 with Th2 differentiation. J. Immunol. 160:1965.-dependent tumor surveillance system in immunocompetent mice. Proc. Natl. Acad. Sci. USA 95:7556.-expressing peripheral T cells. J. Immunol. 142:3754.[Abstract]
T cells. J. Exp. Med. 187:357.8 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice. Immunity 6:541.[Medline]
and is inhibited by transforming growth factor-ß. Eur. J. Immunol. 24:793.[Medline]
+ T cells. J. Immunol. 156:232.[Abstract]
T cells in allergic airway inflammation. Science 280:1265. versus IL-4 and IL-10: selective induction of IL-10 by sequential stimulation of naive Th cells with IL-12 and IL-4. J. Immunol. 161:2825. T cells and ß T cells in tuberculosis. Eur. J. Immunol. 25:2877.[Medline]
ß and T lymphocytes to immunity against Mycobacterium bovis bacillus Calmette Guerin: studies with T cell receptor-deficient mutant mice. Eur. J. Immunol. 25:838.[Medline]
T lymphocytes. Infect. Immun. 64:1744.[Abstract]
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