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Departments of
* Immunobiology and
Laboratory Medicine, Yale University 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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain, a lack of
phosphorylation of ZAP70, linker for activation of T cells, and
phospholipase C-
1 and a weak, transient pattern of calcium
mobilization, while agonist peptides induce a full and sustained
pattern of signaling (13, 14, 15, 16). Although weak, transient TCR signals correlate with the development of Th2 cells, recent studies suggest that weak signaling per se is not sufficient for Th2 differentiation, but rather a specific signaling component is required. For example, mice deficient in CD4, lck, or Itk fail to develop normal Th2 responses in vitro and in vivo (17, 18, 19, 20, 21, 22). The failure to observe Th2 differentiation in these cases correlates with decreased calcium flux. In terms of CD4 signaling, this is especially evident during low potency signaling where CD4 coreceptor function is more important for TCR-mediated signal transduction (19). Together, these data suggest that the level of calcium mobilization provides an important signal to initiate the development of Th2 cells.
Although these studies have added significantly to our understanding of Th cell differentiation, it remains to be determined how these early signaling events (within minutes of TCR signaling) translate into discrete, cytokine-secreting effector populations (6–7 days after priming). One approach is to study the lineage-specific cytokine genes and their associated transcription factors and determine how they might be regulated by specific TCR signals. Multiple factors have been identified that appear to regulate IL-4 production and are preferentially associated with Th2 differentiation, including GATA-3, STAT6, and c-maf (23, 24). Overexpression of either STAT6, GATA-3, or c-maf leads to the induction of Th2 cytokines in developing Th1 cells, suggesting a key role for each in Th2 cytokine regulation (25, 26, 27). Engagement of the IL-4R by IL-4 leads to the activation of STAT6 and the subsequent up-regulation of GATA-3 (28). Increased GATA-3 expression correlates with commitment to the Th2 pathway and is observed by 48 h after priming CD4+ T cells (26, 29). In contrast, c-maf appears to be a relatively late activator of IL-4 transcription because its expression is not up-regulated until 5–7 days after priming (30). While these transcription factors are clearly important for generating Th2 cells, their activation and up-regulation appear to be primarily cytokine driven and not TCR driven.
Transcription factors that might be regulated by the strength of TCR
signaling and are implicated in the expression of several cytokine
genes are the NFATs. NFAT target sequences have been identified in the
promoters of multiple cytokine genes, including IL-2, IL-4, IL-3,
GM-CSF, and TNF-
(31). Several studies have
demonstrated a crucial role for NFATc (NFATc1 or NFAT2), NFATp (NFATc2
or NFAT1), and NFAT4 (NFATc3) in the regulation of Th1 and Th2 cytokine
gene transcription. NFATc-deficient mice exhibit a selective decrease
in IL-4 production and impaired IgG1 and IgE production, suggesting a
positive role for NFATc in the regulation of IL-4 responses (32, 33). In contrast, mice deficient in NFATp (34, 35, 36)
display a modest increase in Th2 development, which is greatly
accentuated in mice deficient in NFAT4 as well (37). The
increase in IL-4 production in these mice has been attributed to
unopposed NFATc activity and suggests that NFATp and/or NFAT4 may
function to down modulate or inhibit IL-4 transcription. Although these
findings are important to our understanding of the factors involved
during Th2 differentiation, they all involve the use of KO mice and
thus do not address the issue of how these signals emerge during the
priming of a naive CD4+ T cell.
Because the NFATs are exquisitely regulated by the level of calcium mobilization within a cell (38, 39), we postulated that this may provide a mechanism by which the strength of TCR signaling may regulate the development of Th1 and Th2 effector cells. Ag stimulation through the TCR triggers the release of intracellular calcium and the subsequent activation of calcineurin (40, 41). Calcineurin dephosphorylates cytoplasmic NFAT family members, allowing their nuclear translocation where they cooperatively bind their target sequences together with AP-1 proteins and initiate transcription (42). The export of NFATs from the nucleus is equally sensitive to the changes in calcium concentration such that a drop in calcium levels causes the proteins to become rephosphorylated and shuttled back to the cytoplasm (43, 44). Consequently, the degree of calcium mobilization initiated by TCR signaling may affect NFAT translocation and, in turn, NFAT-dependent gene transcription.
In this paper we demonstrate that the strength of TCR signaling in naive CD4+ T cells differentially regulates the nuclear translocation of NFAT family members. By modulating the potency of TCR signaling with an APL, we can alter the relative ratio of NFATp and NFATc localization in the nucleus and regulate the transcription of IL-4 message within 48 h of naive T cell priming. The induction of IL-4 transcription in this system is independent of STAT6 signaling and occurs before an increase in GATA-3 expression, which is STAT6 dependent. In contrast, stimulation with a strong agonist peptide increases the NFATp binding activity and down-regulates GATA-3 expression, thus inhibiting early IL-4 transcription. These data provide a model for how the potency of TCR signaling can influence the generation of Th1 and Th2 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The TCR-transgenic mice in which CD4+ T cells express a TCR specific for moth cytochrome c peptide (MCC) in the context of I-Ek or I-Eb have been previously described (45). These mice were bred in our facilities and maintained as heterozygotes on a B10.A (5R), B10.BR, or C57BL/6 (B6) background. STAT6-/- mice on a B6/129 background (46) were generously provided by J. Ihle (St. Jude’s Research Center, Memphis, TN) and backcrossed onto a B6 TCR+/- background to generate homozygous STAT6-/- and heterozygous TCR+/- mice. B10.A (5R) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred in our facility. All TCR mice used in these studies were 6–8 wk old.
Peptides
MCC (81–103) and its APL, K99R, were synthesized by the W. M. Keck Foundation Biotechnology Resource Laboratory (New Haven, CT). The sequence of the MCC peptide is VFAGLKKANERADLIAYLKQATK and the sequence of the K99R peptide is VFAGLKKANERADLIAYLRQATK.
Preparation of APCs and CD4+ T cells
T cell-depleted APC were prepared by Ab-mediated complement
lysis of B10.A (5R) splenocytes as previously described
(47). The APC were treated with 50 µg/ml mitomycin C
(Sigma-Aldrich, St. Louis, MO) before use. CD4+ T
cells from lymph nodes and spleens of TCR-transgenic mice were isolated
by immunomagnetic negative selection (47) using Abs
against CD8, CD32/CD16, B220, MHC class II, and NK cells, followed by
incubation with anti-mouse and anti-rat Ig-coated magnetic
beads (Polysciences, Warrington, PA). Purity of the recovered
V11+CD4+ T cells was
85–95% as determined by staining with anti-CD4 and
anti-V11 mAbs. Naive CD4+ T cells were
obtained from this population by sorting for
Pgp-1low and Mel14high
cells using a FACSVantage SE cell sorter (BD Bioscences, Mountain View,
CA). The resultant population was 99% pure for both
Mel14high and Pgp-1low.
Equivalent results were obtained using either total
CD4+ T cells or FACS sorted, naive
CD4+ T cells.
In vitro stimulation of TCR-transgenic CD4+ T cells
Induction of naive T cell differentiation was performed as
previously described (10), with slight modifications.
Briefly, mitomycin C-treated APC (1 x
106/ml) were incubated with
CD4+ T cells (5 x
105/ml) from TCR-transgenic mice, peptide (MCC at
5 µg/ml or K99R at 10–15 µg/ml), rIL-2 (25 U/ml), and
anti-IFN- Ab. When stimulating TCR-transgenic
CD4+ T cells from the B10.BR background,
peptide-primed APC were used by incubating APC with 50 µM APL or
agonist peptide for 3 h at 37°C, followed by three washes. rIL-2
and anti-IFN- Ab were also included in the primary culture.
After 4 days of priming, T cells were harvested and dead cells were
removed by gradient centrifugation. Viable T cells were then incubated
for a rest period of 2 days. For secondary culture, rested T cells
(5 x 105/ml) were restimulated with fresh
APC (1 x 106/ml) and 5 µg/ml MCC peptide
for an additional 2-day period, after which supernatants were collected
for cytokine analysis. The presence of IL-4 and IFN- in the
supernatants was determined using ELISA kits from Endogen (Cambridge,
MA). Calculated values are expressed as means ± SEM. In some
experiments, cyclosporin A (CsA) or ionomycin (Calbiochem, San Diego,
CA) was added during the primary culture. For CsA, T cells were
preincubated with the drug for 30 min at 37°C at the indicated
concentrations (12.5–25 ng/ml) before the addition of APCs and
peptide. Ionomycin (10–100 nM) was added to the T cells at the same
time as the APCs and peptide.
RNA analysis of primary transcripts
After 2 days in the primary culture, cells were harvested and
dead cells were removed by gradient centrifugation. Total cellular RNA
was isolated using TRIzol reagent (Life Technologies, Frederick, MD).
Cytokine RNA levels were analyzed by RNase protection assay (RPA) using
the RiboQuant multiprobe kit with the mCK-1 template (BD PharMingen,
San Diego, CA). Five micrograms of total RNA was used in each reaction.
To ensure equal loading, transcript levels for two housekeeping genes
(L32 and GAPDH) were quantified for each sample with a GS-525 Molecular
Imager System and Molecular Analyst software (Bio-Rad, Hercules, CA).
For Northern blot analysis, 10 µg of total RNA from each sample was
fractionated on 1.2% agarose/formaldehyde gels, transferred to
-Probe GT membranes (Bio-Rad), and hybridized with the indicated
cDNA probes in QuikHyb buffer (Stratagene, La Jolla, CA). The GATA-3
cDNA probe was a 460-bp HindIII-SacI fragment
derived from pBS-GATA3 construct kindly provided by R. Flavell
(Yale University, New Haven, CT). The mouse GAPDH and 18S rRNA cDNA
probes were purchased from Ambion (Austin, TX) and were used to monitor
total RNA loading of each sample. UV shadowing of the membrane to
visualize 28S and 18S ribosomal RNA (rRNA) was also performed to ensure
equivalent sample loading.
Calcium mobilization
Calcium signaling following Ag-specific stimulation was monitored as described previously (48). Briefly, CD4+ T cells loaded with 5 µM fluo-3/AM ester (Molecular Probes, Eugene, OR) were plated by centrifugation in 96-well plates at a concentration of 5 x 105 cells/50 µl. The cells were then scanned using the ACAS 570 video laser cytometer (Meridian Instruments, Okemos, IL). After initiation of scanning, 4 x 106 T-depleted splenocytes, pulsed with 20 µM peptide, were added to the CD4+ T cells. The initial average fluorescence of each cell was digitized and normalized to 1, and the results are expressed as changes in normalized fluorescence intensity of individual cells over time. The percentage of responding cells was determined by dividing the number of cells demonstrating an increase in intracellular calcium of >50% by the total number of scanned cells.
Preparation of nuclear extracts
CD4+ T cells were stimulated with peptide and APC for 48 h, as described above. Viable cells were collected by gradient centrifugation. After washing in cold PBS, the cell pellet was resuspended in 250 µl buffer A (10 mM HEPES (pH 7.9), 3 mM MgCl2, 10 mM NaCl, 0.1 mM EDTA, 300 mM sucrose, 0.5 mM DTT, and a mixture of protease inhibitors) and incubated on ice for 10 min. Next, 25 µl of 1% Nonidet P-40 solution was added and mixed carefully. Cells were immediately centrifuged at 800 x g for 1 min. The nuclear pellet was washed in 200 µl of buffer A and centrifuged again at 800 x g for 1 min. Pelleted nuclei were resuspended in 50 µl of buffer B (20 mM HEPES (pH 7.9), 3 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT, and a mixture of protease inhibitors) and incubated on ice for 15 min. The suspension was vortexed briefly and centrifuged at 16,000 x g for 5 min. Supernatants were stored at -70°C and protein concentrations were determined using the BCA Protein Assay kit from Pierce (Rockford, IL).
EMSA
EMSAs for NFATs were performed with 3 µg of nuclear extract
incubated with 1 µg of poly(dI:dC) in 20 µl of 1x binding buffer
(10 mM HEPES (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 10%
glycerol, 0.1% Nonidet P-40, 0.5 mg/ml BSA, 1 mM benzamidine, and 1 mM
DTT) with 7.5 x 105 cpm of a
32P-labeled probe for 20 min at room temperature.
Complexes were resolved on nondenaturing 6% polyacrylamide gels
(37.5:1 cross-linking ratio) in 0.5x TBE for 2.5 h at 140 volts.
For supershift reactions, nuclear extracts were preincubated for 15 min
on ice with the appropriate Abs before the addition of the labeled
probe. For detection of NFAT binding, a double-stranded oligonucleotide
from the murine IL-4 promoter (-88 to -60) was used:
5'-CTGGTGTAATAAAATTTTCCAATGTAAAC-3'. The supershifting Abs for
NFATp (clone 4G6-G5) and NFATc (clone 7A6) were from Santa Cruz
Biotechnology (Santa Cruz, CA). NF-
B EMSAs were performed with 5
µg of nuclear extract incubated with 2 µg of poly(dI:dC) in 20 µl
of 1x binding buffer (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA,
5% glycerol, 1 mg/ml BSA, 3 mM GTP, 1 mM benzamidine, and 1 mM DTT)
with 5 x 105 cpm of a
32P-labeled probe for 30 min at room temperature.
Complexes were resolved on nondenaturing 6% polyacrylamide gels (29:1
cross-linking ratio) in 0.5x TBE for 2.5 h at 140 volts. The
sequence for the NF-B probe (derived from the murine intronic
enhancer) is 5'-GATCAGAGGGGACTTTCCGAGG-3'.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using an in vitro system for T cell differentiation with
TCR-transgenic CD4+ T cells, we have previously
shown that altering the affinity of the peptide/MHC ligand can
selectively induce Th1 or Th2 differentiation. Priming with the agonist
peptide (MCC), i.e., a strong TCR signal, generates Th1 cells
(10). In contrast, an APL (K99R), which lowers
the TCR affinity for the antigenic complex by 
300-fold but does not
change the affinity of binding to MHC class II (49),
induces predominantly Th2 differentiation with a low level of Th1
differentiation (10). This observation is best illustrated
by cytokine production upon restimulation of the primed
CD4+ T cells with the agonist peptide. As shown
in Fig. 1
A,
CD4+ T cells primed initially with MCC
differentiated into IFN--producing or Th1 effector cells. In
contrast, priming with K99R generated predominantly IL-4-secreting or
Th2 effector cells.
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B). In striking contrast, priming
with K99R primarily induces transcription of the Th2 cytokines, IL-4,
IL-13, and IL-9. Phosphor imaging analysis from five independent
experiments clearly demonstrates IL-4 induction is specific to
stimulation with K99R (Fig. 1C, upper panel) and
represents a 3- to 6-fold increase over MCC (Fig. 1C,
lower panel). The reduced IL-2 transcription in the APL
cultures, compared with agonist cultures, agrees with previous findings
in T cell clones that APLs are poor initiators of IL-2 production
(51, 52, 53). As shown in Fig. 1D, GATA-3 mRNA
levels correlate with the level of IL-4 transcription such that GATA-3
message is only up-regulated in the presence of a low-affinity TCR
signal.
To determine whether the early IL-4 transcription observed with the APL
was actually due to TCR signaling differences, STAT6-deficient
CD4+ T cells were stimulated with K99R to exclude
IL-4 signaling from exogenous sources. Importantly,
CD4+ T cells primed with K99R transcribe IL-4 in
the absence of STAT6 (Fig. 2A,
lane 2). While the induction of IL-4 is reduced compared
with that seen with wild-type (WT) CD4+ T cells,
it is clearly specific for the APL and represents a nearly 3-fold
increase over background (Fig. 2B). Similarly, WT
CD4+ T cells primed with K99R, in the presence of
anti-IL-4 Ab to also prevent signaling through the IL-4R,
transcribed IL-4 message at a level comparable to the STAT6-deficient
cells (Fig. 2, A and B). Of note, IL-4
transcription under these circumstances occurs without an increase in
the basal level of GATA-3 expression (Fig. 2C), indicating
that increased levels of GATA-3 are dependent upon STAT6 signaling but
are not absolutely required to induce low levels of IL-4. As cells
become fully committed to the Th1 pathway, GATA-3 expression becomes
down-regulated, as seen when STAT6-deficient cells are primed with a
high-affinity TCR signal (Fig. 2C, lane 3, and
data not shown). These data suggest that the small amount of GATA-3
present in naive CD4+ T cells is sufficient to
promote low levels of IL-4 transcription. Moreover, they strongly argue
that the TCR signal itself, and not responsiveness to exogenous IL-4,
induces the initial IL-4 expression.
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We next asked what about the TCR signaling pattern is different
between agonist and APL that might preferentially induce IL-4
transcription when a weak signal is given to a naive
CD4+ T cell. One of the fundamental differences
previously observed in early TCR signaling events is the magnitude and
pattern of calcium mobilization (15, 16, 54). Analysis of
intracellular calcium flux in individual T cells following TCR
engagement with the two different peptides is shown in Fig. 3. The agonist peptide induces a strong,
sustained calcium flux from the majority of responding cells. In
striking contrast, K99R induces a weak and often transient calcium
flux. As seen in Fig. 3C, the number of responding cells is
greatly diminished during low-affinity peptide stimulation compared
with stimulation with a high-affinity ligand (25% for K99R vs
50% for MCC, Fig. 3C). In addition, most of the
MCC-responding cells (90%) showed a sustained calcium flux lasting
>5 min, whereas the cells responding to K99R exhibited a mixed pattern
of calcium mobilization (Fig. 3D). Within the population of
APL-stimulated cells that generated a sustained calcium flux, the
pattern of calcium mobilization was often shorter in duration than seen
within the MCC-stimulated cells (compare the lower single cell tracings
in Fig. 3B).
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A) and subsequently inhibits
Th2 differentiation (Fig. 4C). These data suggest that the
transient calcium flux observed following APL stimulation is an
important component of biochemical signals leading to the development
of Th2 cells.
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, B and D, demonstrate that the presence of
ionomycin during APL stimulation inhibits IL-4 transcription as well as
Th2 differentiation. Furthermore, the decrease in IL-4 transcription
leads to an increase in Th1 development as observed by the dramatic
increase in IFN--producing cells (Fig. 4D), analogous to
stimulation with an agonist peptide. These findings provide support for
our earlier observation that the generation of Th2 cells by APL
stimulation is sensitive to both CsA and ionomycin (16)
and extend those analyses to demonstrate a direct effect on early
transcriptional events during priming of naive
CD4+ T cells. Together, these and published data
suggest that a weak or transient pattern of calcium signaling is
important for the induction of early IL-4 transcription and subsequent
Th2 differentiation. Low-potency TCR signals alter the NFATc/p ratio
Calcium signaling is an extremely important regulator of NFAT
family members such as NFATc and NFATp, which are two factors predicted
to have opposing roles in the regulation of IL-4 expression. As such,
we asked whether the distinct patterns in calcium mobilization observed
with the two peptides corresponded to a difference in specific NFAT DNA
binding activity. As demonstrated in Fig. 5A, there is a notable
difference in NFATp vs NFATc DNA binding activity, depending on the
potency of the TCR signal. A strong TCR signal (MCC) correlates with
abundant NFATp and NFATc binding. In contrast, a weak TCR signal (K99R)
results in poor NFATp but significant NFATc levels, indicating a
differential sensitivity of the two proteins to calcium/calcineurin
activation. Of note, the differences in NFAT binding activity
correlated with the nuclear localization of NFATc and NFATp, as
determined by Western blot analysis of nuclear fractions (data not
shown). NF-B, which is regulated, in part, by the
calcium/calcineurin pathway but also by protein kinase C, demonstrated
fairly similar levels of DNA binding activity, indicating the strength
of the TCR signal did not have a significant effect on its activity
(Fig. 5B). Densitometric analyses of the relative amounts of
nuclear NFAT indicate that priming with an agonist peptide yields at
least a 2-fold increase in the amount of NFATp relative to APL-primed
cells, while NFATc activity is more similar between the two priming
conditions (Fig. 5C, left panel). Furthermore, a
comparison of the ratio of NFATc/NFATp binding activity from three
independent experiments revealed an extremely reproducible ratio
(p = 0.02) for the two priming conditions (Fig. 5C, right panel). MCC-primed T cells typically
have a 1.8:1 ratio of NFATc:NFATp, whereas K99R induces a ratio of
3.8:1. The significance of these findings is underscored by reports of
NFAT knockout mice that predict a positive role for NFATc and a
negative role for NFATp in the regulation of IL-4 transcription. As
such, our data predict that the strength of the TCR signal can
influence the balance of the two NFAT proteins and thus affect Th
differentiation.
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A demonstrates that NFAT DNA
binding activity during APL priming is extremely sensitive to CsA,
likely due to the predicted low level of calcineurin activity
associated with a transient calcium flux. At the lowest dose of CsA,
NFATp and nearly all NFATc DNA binding activity is inhibited. Of note,
NF-B nuclear translocation is unaffected by these low doses of CsA.
In contrast, treatment with ionomycin during priming with APL leads to
an increase in the amount of both NFATp and NFATc activity at the
higher dose of ionomycin, which mimics the pattern of agonist peptide
signaling (Fig. 6B). In fact, densitometric analysis of the
levels of NFATc vs NFATp clearly demonstrates a dose-dependent switch
in the ratio of NFATc/NFATp (Fig. 6C; 3.5:1 without
ionomycin and 1.7:1 with 100 nM ionomycin). Similar treatment with
ionomycin during MCC priming clearly has no effect on the NFATc/p ratio
even though ionomycin promotes additional NFAT translocation (Fig. 6D).
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. Together, these data
indicate that pharmacologically mediated changes in calcineurin/NFAT
signaling can alter the nuclear localization of NFATs toward a ratio of
NFATc/p that will inhibit early IL-4 transcription. Moreover, they
provide a mechanistic basis for how the strength of the TCR signal can
regulate the balance of NFAT family members to a ratio that is
permissive for IL-4 transcription and Th2 differentiation. As such, our
data provide evidence for the hypothesis that the balance between NFAT
family members is crucial to the differentiation of naive
CD4+ T cells. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because the vast majority of studies looking at the regulation of Th cell commitment use cytokine skewing to generate Th1 and Th2 cells, our finding that an APL selectively induces IL-4 transcription within 48 h of priming a naive CD4+ T cell provides a critical tool for further studying the regulation of Th cell differentiation. Importantly, the induction of early IL-4 transcription is dependent on the strength of the TCR signal but independent of signaling through the IL-4R/STAT6 complex, which highlights several important points.
First, an exogenous source of IL-4 is not required to initiate IL-4 transcription in a naive CD4+ T cell and thus is not dependent upon STAT6 signaling. Several groups have found that STAT6-deficient mice are unable to generate Th2 inflammatory responses and are profoundly impaired in generating IL-4-secreting effector cells in vivo and in vitro (46, 55, 56). However, these studies were analyzing late events in the generation of Th2 responses such as cytokine production upon restimulation and IgE production. Our analysis within 48 h of priming a CD4+ T cell with an APL indicates STAT6 is not an early mediator of IL-4 transcription because we can block IL-4R signaling by using either STAT6-deficient CD4+ T cells or anti-IL-4 Abs and still detect IL-4 message. This finding agrees with other recent reports showing that IL-4 can be made in the absence of STAT6 signaling (57, 58, 59, 60). Importantly, though, STAT6 is clearly necessary for the up-regulation of GATA-3 and the generation and expansion of IL-4-secreting Th2 effector populations in our system (data not shown).
Second, the potency of the TCR signal provides specific signals that are necessary to initiate early IL-4 transcription. We and others have previously found that weak TCR signals preferentially induce Th2 development while strong TCR signals promote Th1 differentiation (61). Moreover, it has become evident that weak signals alone are not enough to generate Th2 cells but that a specific signaling event is required and is linked to calcium signaling in that only a weak, transient pattern of calcium mobilization correlates with Th2 development. This idea initially arose from the finding that CsA treatment of naive CD4+ T cells during priming with an APL inhibited Th2 but not Th1 differentiation (16). Further support for calcium providing an important signal for Th2 differentiation came from mice deficient in CD4, lck, or Itk. In all cases, Th2 development was severely impaired and correlated with a decreased calcium flux (17, 18, 19, 20, 21, 22). However, increasing the level of calcium mobilization induced by an APL with ionomycin, so as to mimic an agonist peptide or sustained calcium signaling, led to inhibition of early IL-4 transcription, suggesting that the potency of the calcium signal can differentially regulate IL-4 transcription.
Our ability to alter early IL-4 transcription by manipulating the calcium/calcineurin/NFAT pathway with pharmacological reagents highlights the importance of this pathway during Th cell differentiation. Inhibition of NFAT activation by CsA during APL priming results in an inhibition of IL-4 transcription, supporting the well-accepted notion that IL-4 transcription is NFAT dependent. Together, these data strongly imply that a certain threshold of calcium signaling is necessary for the initiation of IL-4 transcription but that strong calcium signals such as those induced by an agonist peptide can inhibit IL-4 transcription and Th2 differentiation.
This kind of model can be explained by the differential regulation of NFATc/p translocation based on the potency of the TCR signal. While a weak TCR signal imparts selective NFATc nuclear translocation and IL-4 transcription, a strong TCR signal promotes both NFATc and NFATp nuclear translocation but fails to induce IL-4 transcription. Similarly, ionomycin treatment during APL stimulation inhibited IL-4 transcription and correlated with an increase in nuclear NFATp. The significance of these observations is underscored by the fact that high levels of nuclear NFATc are not simply sufficient to induce IL-4 but the absence or low level of NFATp is also required. For example, stimulation with an agonist peptide induces both NFATc and NFATp translocation but does not induce IL-4 transcription. These data are consistent with the NFATp-deficient mouse models, which suggested a negative role for NFATp in the regulation of IL-4 (35, 36). In these studies, priming CD4+ T cells in the absence of NFATp led to an accumulation of IL-4 message by 48 h.
In contrast to the inhibitory role for NFATp, NFATc is thought to be a
positive regulator of IL-4 transcription given that NFATc-deficient T
cells are impaired in their ability to make IL-4 or differentiate into
Th2 cells (32, 33). In support of this idea, doses of CsA
that prevented nuclear translocation of NFATc during priming with APL
correlated with a lack of IL-4 transcription. Because calcineurin can
also regulate the activity of NF-B and c-Jun N-terminal kinase (JNK)
(62, 63, 64), the CsA-mediated effects on early IL-4 message
may be due to indirect effects on these two pathways, though the
likelihood of such seems rather limited. Whereas NFAT activation
absolutely requires a calcium stimulus, NF-B and JNK are only
partially dependent upon a calcium signal and can be activated via
other pathways (62, 65, 66). Analysis of NF-B
activation indicated that the strength of TCR signal did not have a
differential effect on its nuclear localization in that agonist- and
APL-primed cells expressed similar levels of p65 and p50 in the nucleus
at 48 h. In addition, the low doses of CsA used in this study did
not inhibit NF-B translocation in our system. Consistent with these
findings, Dolmetsch et al. (39) demonstrated that NFATc/p
nuclear translocation is more sensitive to calcium signals than NF-B
and JNK activation.
There are a number of circumstances during naive T cell priming in which there is no preexisting cytokine microenvironment (2). Under these conditions, we propose that the strength of the TCR signal becomes extremely important in regulating the balance between the development of Th1 and Th2 cells. In this study, we find that the potency of the TCR signal differentially regulates the nuclear translocation of NFATc and NFATp and, in turn, influences cytokine gene transcription. These findings suggest a model in which differences in TCR-mediated signals affect the relative balance of different NFAT family members to either promote or inhibit a low level of IL-4 gene transcription. After high-potency stimulation, early IL-4 transcription and Th2 differentiation are inhibited due to high levels of NFATp and low levels of GATA-3 expression. In contrast, stimulation with low-potency ligands induces less NFATp but sufficient NFATc levels in the nucleus to promote IL-4 transcription. This small amount of IL-4 feeds back through the IL-4R to activate STAT6 and up-regulate GATA-3 expression, which enable the full development of Th2 effector cells.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Immunology, George Washington University, Washington, DC 20037.
3 Address correspondence and reprint requests to Dr. Kim Bottomly, Department of Immunobiology, Yale University School of Medicine, 310 Cedar Street LH408, P.O. Box 208011, New Haven, CT 06520-8011. E-mail address: Kim.Bottomly{at}yale.edu
4 Abbreviations used in this paper: APL, altered peptide ligand; WT, wild type; MCC, moth cytochrome c peptide; CsA, cyclosporin A; RPA, RNase protection assay; JNK, c-Jun N-terminal kinase.
Received for publication December 20, 2001. Accepted for publication February 13, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-transgenic model. J. Exp. Med. 182:1579. by T cells. Proc. Natl. Acad. Sci. USA 92:9510. and lack of Zap 70 recruitment in APL-induced T cell anergy. Cell 79:913.[Medline]
Phosphorylation without Zap-70 activation induced by TCR antagonists or partial agonists. Science 267:515.-producing CD4+ T cells following activation of naive CD4+ T cells. J. Immunol. 158:1085.[Abstract]
B/MAD3, an inhibitor of NF-B. EMBO J. 13:861.[Medline]
to activate JNK and IL-2 promoter in T lymphocytes. EMBO J. 17:3101.[Medline]
B kinase and NF-B in T lymphocytes. J. Biol. Chem. 274:22923.This article has been cited by other articles:
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