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,§
*
Division of Immunobiology, Research Institute for Biological Sciences, Science University of Tokyo, Noda City, Chiba, Japan;
Department of Molecular Immunology, Graduate School of Medicine, Chiba University, Chuo-ku, Chiba, Japan;
Department of Immunology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan;
§
CREST, Japan Science and Technology Corporation, Tokyo, Japan; and
¶
Cadus Pharmaceutical Corp., Tarrytown, NY 10591
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain,
and STAT6 following IL-4 stimulation, CD28 costimulation increased
IL-4R sensitivity without affecting its expression and binding
property. This evidence of the enhancement of IL-4R sensitivity
increases our understanding of how CD28 costimulation accelerates Th2
development. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and TNF-. Th1 cells are responsible for defense
against infectious intracellular microorganisms. The Th2 phenotype
secretes cytokines that help the proliferation and differentiation of B
cells, such as IL-4, IL-5, and IL-6, and are responsible for defense
against extracellular pathogens and the development of allergic immune
reactions (1, 2). The
CD4++ß+
T cells leaving the thymus have a naive phenotype and produce IL-2 and
a small amount of IL-3 and GM-CSF. The naive T cells differentiate into
either Th1 or Th2 phenotypes following the appropriate activation
signals. There is much evidence that Th cell differentiation may reflect the nature of the antigenic stimulation and the cytokine environment to which the cells have been exposed (2, 3). Most work addressing the role of cytokines in T cell differentiation has used in vitro priming systems. IL-12 is thought to be a major factor for promoting Th1 differentiation because the presence of IL-12 during the priming stage directly augments Th1 differentiation (4, 5, 6). In contrast, the presence of IL-4 in the priming culture promotes Th2 differentiation (7, 8, 9, 10). A number of recent studies using mice with germline disruptions strongly supports the evidence accumulated with in vitro priming systems. For instance, disruption of either the IL-4 gene or the STAT6 gene resulted in mice that failed to generate Th2 cells (11, 12, 13). STAT6 is known to be a transcriptional factor involved in the IL-4-mediated Jak/STAT signaling pathway (14, 15). Recently, we have described that the activation status of STAT6 differs between Th1 and Th2 cells, and that Th2 cell-specific IL-4 expression is determined by the inhibition of a silencer gene caused by activated STAT6 (16). Therefore, the IL-4-mediated Jak/STAT signaling pathway plays an important role in determining the development of Th1 and Th2 cells. In contrast, little is known about the nature of TCR signaling events during the priming stage for Th cell differentiation. Targeted disruption of the CD4 gene selectively impairs Th2 differentiation (17). Similarly, we have reported a requirement of Lck activity for Th2 differentiation using mice that overexpress a dominant negative form of Lck (18). However, despite these findings, there remain questions such as the nature of the association between Ag stimulation through TCR and the IL-4R signaling pathway.
In this study, we focus on role of costimulatory signals on Th cell differentiation and on how both Ag stimulation and IL-4 mutually regulate Th2 generation. Th1/Th2 polarization has been analyzed previously in transgenic (Tg)3 TCR systems (7, 8, 9, 19). However, the combination of Tg TCR and Ag/MHC complexes in certain genetic backgrounds seems to automatically determine the polarization pattern. For example, naive CD4+ T cells from OVA-specific TCR Tg, DO11.10 mice with BALB background preferentially differentiate into Th2 cells by antigenic stimulation without the addition of IL-4 (19, 20, 21). To exclude those biases, we used nontransgenic congenic mice, and Th cell differentiation was initiated by polyclonal activators such as Con A or anti-TCR. Stimulation with anti-TCR was not sufficient for Th2 differentiation, while activation with Con A generated a significant number of Th2 cells. Costimulatory molecules such as CD28 influence this difference. In general, antigenic stimulation involves both TCR and costimulatory signals, and the coordination of these two signals strongly affects IL-4R function during the Th cell differentiation process. Most importantly, we have obtained evidence that an increase in the sensitivity of IL-4R to IL-4 is caused by coincidental TCR and CD28 signaling and is required for Th2 differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c and C57BL/6 mice were purchased from Clea (Tokyo, Japan).
OVA-specific TCRß Tg mice (DO 11.10 Tg) were kindly distributed by
Dr. Dennis Loh (Roche, Nutley, NJ). C57BL/6 STAT6 knockout mice were
provided by Dr. Shizuo Akira (Hyogo College of Medicine, Hyogo, Japan)
(13).
Cytokines and Abs
The reagents (for IL-2, JES6-1A12 and JES6-5H4 biotin; for
IFN-, R4-1A12 and XMG1.2 biotin; and for IL-4, BVD4-1D11 and
BVD6-24G2 biotin) were purchased from PharMingen (La Jolla, CA) and
used for ELISA. The reagents for cytostaining analysis,
anti-IFN- (XMG1.2) FITC and anti-IL-4 (11B11) PE, were
obtained from PharMingen. Anti-IL-4R mAb (M-1) was purchased from
Genzyme (Cambridge, MA). Anti-IL-4 mAb (11B11) was generously gifted by
Dr. Waul (National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, MD). Anti-CD44 mAb (KM201) was
kindly provided by Dr. K. Miyake (Saga Medical College, Saga, Japan).
Anti-CD28 mAb (PV-1) was previously described (22). Mouse
rIL-4 was purchased from PeproTech (London, U.K.). The antigenic OVA
synthetic peptide (residues 323–339; ISQAVHAAHAEINEAGR) was
synthesized by BEX (Tokyo, Japan).
Preparation of CD4+ naive T cells and induction of Th cells
To isolate CD4+ naive T cells, spleen cells were incubated with anti-CD8 mAb (53-6.72) at 4°C and the cells were incubated on the plate-coated anti-mouse Ig to eliminate B and CD8+ T cells. The CD4+-enriched T cells were incubated with anti-CD44 mAb (KM201), followed by the cytotoxic killing treatment with Low-Tox-M rabbit complement (Cederlane Laboratories, Hornby, Ontario, Canada). These CD4+ naive T cell preparations contain more than 80% CD4+, CD44- T cells. APCs were prepared from spleen cells by the cytotoxic killing treatment with anti Thy-1.2 mAb (30H12) and complement. The CD4+ naive T cells were stimulated with either antigenic peptide, Con A, anti-TCR (H57-597), or anti-TCR plus anti-CD28 mAb (PV-1). Ag and Con A stimulation was performed in the presence of the splenic APC, while the anti-TCR stimulation was performed with plate-bound Ab. After 5 days, the primed CD4+ T cells were repurified by panning with anti-CD8 mAb and anti-mouse Ig and with anti-TCR mAb to induce cytokine production.
Intracellular cytokine staining
To prevent release of cytokines, the naive
CD4+ T cells were restimulated with anti-TCR
mAb for 6 h in the presence of 4 µM monensin (Sigma, St. Louis,
MO). Then, cells were fixed with 4% paraformaldehyde and permeabilized
with 0.5% Triton X. After blocking with PBS containing 3% BSA, cells
were stained with anti-IFN- (XMG1.2) FITC and anti-IL-4
(11B11) PE, as described previously (18). Flow-cytometric
analysis was performed on FACSort and CellQuest software (Becton
Dickinson, San Jose, CA).
Flow cytometry analysis
Cells were suspended in PBS supplemented with FCS and 0.1% sodium azide. In general, 106 cells were blocked with anti-FcR (2.4 G2) and stained using a standard method, as described previously (16). Flow cytometry analysis was performed with FACSort (Becton Dickinson) using CELL Quest software (Becton Dickinson).
Measurement of cytokine concentrations by ELISA
CD4+ naive T cells were stimulated with different stimuli, and after 24 h, the culture supernatants were harvested. Cytokine concentration in the supernatant was measured as described previously (18). Briefly, the supernatants were applied on the plastic plate coated with specific Ab against certain cytokines. After washing, the plate was probed with HRP-conjugated streptavidin (Zymed, San Francisco, CA) and developed with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Kirkegard & Perry Laboratories, Gaithersburg, MD). The 405 nm absorbance was measured by spectrophotometer (Bio-Rad Laboratories, Hercules, CA).
SDS-PAGE and Western blot analysis
Total cellular lysates of 2 x 106
cells were prepared in RIPA solution (50 mM Tris-HCl, 1% Nonidet P-40,
0.25% sodium-deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF,
aprotinin (1 µg/ml), leupeptin (1 µg/ml), pepstatin (1 µg/ml), 1
mM NaVO4, and 1 mM NaF) and separated by electrophoresis on 7.5%
SDS-PAGE. Jak3, the IL-4R-chain, and STAT6 were immunoprecipitated
from the cell lysates of 8 x 106 cells
using anti-Jak3 antisera (Upstate Biotechnology, Lake Placid,
NY), anti-IL-4R-chain mAb (M1) (Genzyme), and anti-STAT6 mAb
(R&D Systems, Minneapolis, MN). The samples were loaded and run on
7.5% SDS-PAGE gel. After electrotransfer to polyvinylidene difluoride
membrane, the blots were probed with HRP-conjugated
anti-phosphotyrosine Ab (HRP-RC20) (Transaction Laboratory,
Lexington, KY). To estimate protein concentration in lysates, the
phospho-blots of total cellular lysates were stripped with phosphate
buffer containing 2% SDS and 0.1 M 2-ME. The membranes were reprobed
with either anti-STAT6 (Transaction Laboratory) or anti-Jak3 Ab
(Santa Cruz Biotechnology, Santa Cruz, CA) and developed with
HRP-conjugated anti-mouse Igs and anti-rabbit Igs (Dako,
Glostrup, Denmark).
Measurement of IL-4R affinity
T cells were resuspended in 4°C HBSS at 1 x 107 cells/ml. IL-4 was labeled by enzyme reaction (glucoseoxidase and lactoperoxidase) with 125I, and labeled IL-4 was purified with PD-10 column. Cells were incubated with 125I-labeled mouse rIL-4 (600 Ci/mmol) for 2 h at 4°C and added separating solution (8:1 mixture of di-n-butyl phthalate and olive oil). After centrifuge for 2 min at 4000 rpm, the reaction solution and half of separating solution were discarded, and the rest of the solution was counted by Auto well gamma system (Aloka, Tokyo, Japan).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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First, it was important to characterize the Th development
properties in the systems. Naive CD4+ T cells
were obtained from OVA-specific TCR Tg (DO11.10) mice that
were BALB/c background and stimulated with antigenic peptide, Con A, or
immobilized TCR mAb (H57-597). Antigenic peptide and Con A stimulation
was performed in the presence of splenic APC, while anti-TCR
stimulation was performed with plate-bound Ab. After 5 days, the primed
CD4+ T cells were restimulated with anti-TCR
mAb for 6 h, and the Th1 and Th2 differentiation profile was
assessed by intracellular cytokine staining. In this study, we defined
Th1 cells as those producing IFN- but not IL-4, and the Th2 cells as
those producing IL-4 but not IFN-.
DO11.10 T cells showed significant Th2 development in response to
either antigenic peptide or Con A in the presence of BALB/c splenic APC
(Fig. 1
A). About 25% of the
population were Th2 cells after stimulation by the antigenic peptide
and Con A stimulation yielded a similar profile. Very little Th2
differentiation occurred following anti-TCR stimulation. To study
the possibility that the distinct polarization was due to the absence
of APC, the Th cells were induced by soluble anti-TCR in the
presence of APC. However, these treatments were again insufficient for
the generation of Th2 cells (Fig. 1, A and
B).
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B), while
initial activation by Abs against TCR or CD3 was insufficient for Th2
development (Fig. 1B). Similar frequencies of Th1 cells were
detected after stimulation with a range of immobilized anti-TCR Ab
from 1–300 µg/ml (data not shown), indicating that TCR cross-linking
with mAb was sufficient for Th1 differentiation, but not for Th2
differentiation.
The addition of exogenous IL-4 (10 ng/ml) into the anti-TCR
induction culture of BALB/c T cells resulted in the development of a
significant number of Th2 cells (Fig. 2A). IL-4 itself was
insufficient for Th2 generation, although all naive
CD4+ T cells expressed significant amounts of
IL-4R (Fig. 6A). We examined whether IL-4 was required
before or after TCR cross-linking to promote Th2 differentiation. To
test this, IL-4 was added at different time points into the
anti-TCR induction culture of CD4+ T cells
obtained from BALB/c and B10.D2 mouse. Significant numbers of Th2 cells
were generated when IL-4 was added either simultaneously with or after
the anti-TCR (Fig. 2B). The addition of IL-4 before TCR
stimulation failed to promote Th2 differentiation. These results
indicated that the sequence of a TCR activation signal followed by an
IL-4 signal should be critical for Th2 differentiation and suggested
that the TCR-mediated signal might modify the properties of the
IL-4R.
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CD28 costimulation is involved in Th2 differentiation and allows
naive T cells to respond to lower levels of TCR occupancy
(23, 24, 25, 26). CD28 costimulation was provided by the addition
of anti-CD28 mAb (PV-1) into the anti-TCR induction culture.
Soluble forms of this Ab activate the CD28-mediated signaling pathway
(22). Purified PV-1 mAb (0.1–10 µg/ml) was added into
the induction culture with plate-bound anti-TCR. As shown in Fig. 3A, CD28 costimulation with
anti-TCR resulted in a significant number of Th2 cells (11–18%).
In contrast, the Th1 frequency was decreased to almost half of that
generated by anti-TCR mAb alone. Similarly, CD28 costimulation
promoted significant enhancement of Th2 generation even in C57BL/6 (B6)
strain, which favors Th1 response. The proportion of Th2 cells in B6
mice was constantly between 3 and 10%; thus, the enhancement was more
pronounced in BALB/c mice (Fig. 3B, middle).
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B, upper). Next, the effect of CD28
costimulation was examined in C57BL/6 (B6) STAT6 knockout mice. In
STAT6 knockout mice, CD28 costimulation did not promote Th2
differentiation even after the addition of exogenous IL-4 (Fig. 3B, lower). Because Th1 generation occurred at
the same level (14%) as that observed in B6 mice, abolishment of Th2
differentiation was not due to a defect in the initial activation
signal. Taken together, these results indicate that the enhancement of
Th2 generation initiated by CD28 costimulation was dependent on the
IL-4R-mediated signaling pathway. CD28 costimulation promotes Th2 differentiation by alteration of the sensitivity threshold for IL-4
Next, we asked whether the inability of TCR cross-linking with mAb
to generate sufficient Th2 cells was due to the modest IL-4 production
or the low sensitivity of the IL-4R. To examine quantitative
differences in cytokine production promoted by different stimuli, naive
CD4+ T cells were stimulated by various doses of
Con A and anti-TCR mAb. To examine the effect of CD28
costimulation, anti-CD28 mAb was added into the anti-TCR
stimulation. The amounts of IL-2, IL-4, and IFN- in the culture
supernatants were measured at 24 h after stimulation. As shown in
Fig. 4A, T cells stimulated by
Con A produced over 10 times more IL-2 than the T cells stimulated by
100 µg/ml of anti-TCR. The T cells were capable of producing
detectable amounts of IL-4 following stimulation by Con A as well as
anti-TCR. Stimulation with 10 µg/ml of Con A induced 120 pg/ml of
IL-4, while the anti-TCR stimulation induced nearly 40 pg/ml.
Stimulation with either Con A or anti-TCR induced very low amounts
of IFN-. There were no quantitative differences in IFN-
production between Con A and the anti-TCR. The addition of a CD28
costimulation dramatically augmented IL-2 production by 10- to 20-fold,
while the same treatment modestly affected the IL-4 and IFN-
production. Because the enhancement of IL-4 production by the CD28
costimulation was only 2- to 3-fold, indicating that role of CD28
costimulation on Th2 development was not only an augmentation of IL-4
production.
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B). However, the addition of 1 ng/ml of IL-4 into the
induction culture with anti-TCR mAb fails to generate a significant
number of Th2 cells (Fig. 4C). The addition of a 20- to
100-fold excess amount of IL-4 was required to bring the Th2 frequency
to the level promoted by CD28 costimulation (Fig. 4C).
We directly compared the sensitivity of T cells to IL-4 after either
anti-TCR or anti-TCR plus CD28 costimulation. Th2 cells were
induced from BALB/c T cells by four different concentrations of
exogenous IL-4 (0.01–10 ng/ml) in the presence of anti-TCR with or
without CD28 costimulation. As shown in Fig. 4D,
left, when the induction culture was performed with TCR
cross-linking, 10 ng/ml of IL-4 was required for a significant number
of Th2 cells (13.5%), while CD28 costimulation promoted Th2
development even without the addition of IL-4. To examine Th2
differentiation initiated by exogenous IL-4, the concentration of
anti-CD28 mAb for the induction culture was reduced to 10 ng/ml.
Stimulation with 10 ng/ml of anti-CD28 mAb generated 6.7% Th2
cells without exogenous IL-4. The addition of 1 ng/ml IL-4 increased
the proportion of Th2 up to 13.2%, and 10 ng/ml IL-4 was sufficient
for the maximum level of Th2 development (26.2%). Stimulation with
anti-TCR required 100 times more IL-4 to generate the same number
of Th2 cells as anti-TCR with 10 ng/ml of anti-CD28
mAb.
To mimic normal antigenic stimulation, Th2 cells were induced from
DO11.10 Tg T cells by antigenic peptide in the presence of three
different concentrations of IL-4. The effect of IL-4 in these cultures
was very similar to that with the anti-TCR and CD28 costimulation
(Fig. 4D, left and right). These
results suggested that alteration of the sensitivity to IL-4 might be
one function of CD28 costimulation in initial T cell activation, and
this enhancement of the sensitivity to IL-4 might also occur in the
antigenic activation process.
TCR cross-linking enhances the expression and binding property of the IL-4R
We next studied the role of CD28 costimulation on modification of
IL-4R sensitivity. CD28 may simply enhance the cell surface expression
of the IL-4R-chain, which in turn increases the cell’s capacity.
Therefore, BALB/c T cells were prestimulated with anti-TCR and
anti-TCR plus anti-CD28 mAb for 36 h, and the expression
of IL-4R was investigated. The naive CD4+ T cells
expressed substantial levels of IL-4R, and the expression was
increased after TCR cross-linking by almost 2-fold (Fig. 5A, left and
middle). The addition of CD28 costimulation did not further
enhance the expression level (Fig. 5A,
right).
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B). Taken together, activation by TCR cross-linking
was sufficient to enhance the expression, and the binding property of
the IL-4R and CD28 costimulation did not affect these properties
further.
The expression of CD25 and CD69 as activation markers was markedly
up-regulated by Con A, anti-TCR, and anti-TCR plus
anti-CD28 mAb. The expression of CD69 reached its maximum level
with TCR cross-linking, and the effect of CD28 costimulation was very
slight (Fig. 5C). The maximum level of CD25 expression was
induced by Con A and TCR plus CD28 stimulation, and up-regulation by
TCR cross-linking was lower than that by the other two stimuli (Fig. 5C). The difference between TCR cross-linking and TCR plus
CD28 stimulation in CD25 expression may be explained by the amount of
IL-2 that was induced by these two stimuli. IL-2 regulates CD25
expression via STAT5 activation. The additional up-regulation by CD28
may be due to the striking enhancement of IL-2 production by CD28
stimulation. The protein expression of Jak3 and STAT6 had the same
profile as CD69 and IL-4R. The expression of Jak3 and STAT6 was
increased by TCR cross-linking between 1.2- to 2-fold and 8.3- to
24-fold in their densities, respectively (Fig. 6A, bottom panels).
Again, their expression was not enhanced any further by CD28
costimulation (Fig. 6A, bottom panels; Fig. 7A, bottom panels).
Thus, activation by TCR cross-linking was sufficient to result in the
maximum protein expression of IL-4R, CD69, Jak3, and STAT6.
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-chain, Jak3, and STAT6 initiated by IL-4We further tested the tyrosine phosphorylation profiles initiated by IL-4 after the initial activation. Naive CD4+ T cells from BALB/c mice were prestimulated with Con A, anti-TCR, and anti-TCR plus anti-CD28 mAb. All cultures were performed in the presence of anti-IL-4R mAb to avoid the effect of IL-4 secreted from naive CD4+ T cells after the primary stimulation. After 36 h, the cells were harvested and then cultured another 12 h in the absence of exogenous cytokines. The naive and the primed CD4+ T cells were activated with 10 ng/ml of IL-4 for 5–10 min, and the tyrosine phosphorylation patterns were studied.
The naive CD4+ T cells did not reveal detectable
tyrosine phosphorylation, while the stimulated T cells showed
significant phosphorylated bands when followed by IL-4 stimulation
(Fig. 6A). Strong phosphorylation bands were observed at
about 130–140 kDa and 100 kDa (Fig. 6A, arrows II and IV)
after prestimulation with either Con A or anti-TCR plus
anti-CD28. However, cells prestimulated with anti-TCR
showed weaker phosphorylation, indicating that TCR cross-linking
increased the phosphorylation status of IL-4R on naive T
cells to a detectable level. Addition of CD28 costimulation
further augmented the phosphorylation (Fig. 6A).
Tyrosine phosphorylations of the IL-4R-chain, Jak3, and STAT6 among
the same preparations in Fig. 6A were further examined by
immunoprecipitation (Fig. 6B). Either Con A or anti-TCR with
CD28 costimulation phosphorylated Jak3, while anti-TCR stimulation
initiated very little phosphorylation. The tyrosine phosphorylation of
Jak3 was increased 12.6-fold by the addition of a CD28 costimulation as
compared with that in TCR cross-linking alone. IL-4R and STAT6 were
phosphorylated by all three stimuli, but phosphorylation by
anti-TCR with CD28 costimulation was 1.5- and 6.3-fold more than
that with TCR cross-linking alone (Fig. 6B). The induction
culture with Con A that provided a significant Th2 generation
resulted in 7.6-, 1.2-, and 2.7-fold higher phosphorylation of Jak3,
the IL-4R-chain, and STAT6 compared with TCR cross-linking
alone.
To examine the correlation between IL-4-mediated tyrosine
phosphorylation and the Th2 differentiation caused by CD28
costimulation, naive T cells were prestimulated with anti-TCR and
three different amounts of anti-CD28 mAb (1–10% of the culture
supernatant containing 5 µg/ml of PV-1 mAb). After 36 h, the
cells were harvested and then cultured for another 12 h in the
absence of exogenous cytokines. The cells were divided into two groups,
and half of the cells were stimulated with IL-4 to study the tyrosine
phosphorylation patterns. The rest of cells were cultured for another 3
days to study Th2 development. As shown in Fig. 7, A and
B, tyrosine phosphorylation followed by IL-4 stimulation
correlated with the amount of anti-CD28 mAb, and the presence of
CD28 costimulation resulted in significant Th2 generation in a
dose-dependent manner.
Augmentation of IL-4-dependent tyrosine phosphrylation was also
observed in DO11.10 Tg CD4+ T cells initially
activated with 1 µM antigenic peptide (Fig. 7C). These
results indicated that initial activation by Ag was also able to
augment the receptor sensitivity against IL-4, and that the
coordination of TCR and CD28 signals might be involved in those
processes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our observations demonstrated that anti-TCR stimulation generated a detectable number of Th2 cells only if high concentrations of IL-4 were added. This prompted us to speculate that there could be quantitative differences in the magnitude of T cell activation by anti-TCR as compared with the initial activation in which costimulatory molecules might be involved. The Th1/Th2 polarization profile initiated by TCR cross-linking with mAb was consistent with previous reports indicating that the initial activation with anti-CD3 mAb was able to initiate the mRNA expression of Th1 cytokines, but not that of Th2 cytokines (27, 28). The initiation of Th1 dif-ferentiation appears to be less dependent on a CD28 costimulatory signal because Th1 responses could be developed in CD28-deficient mice or in mice in which CD28 costimulation was blocked by CTLA4Ig (29, 30).
In contrast, Th2 differentiation appears to be dependent on CD28
costimulation in an in vitro TCR cross-linking differentiation system.
The addition of CD28 costimulation promoted the generation of a
significant number of Th2 cells (Fig. 3). Our result was consistent
with previous reports demonstrating that CD28 costimulation was
required for the Th2 responses (29, 31, 32, 33, 34). Abbas
(35) and Thompson (36) have proposed a
possible explanation for how CD28 costimulation could affect the
initial commitment of naive CD4+ T cells.
Th1/Th2 polarization was determined by the strength of the initial
activation signal. Low Ag concentrations and low-dose infections tend
to induce Th1 differentiation preferentially, while high doses induce
Th2 differentiation (35, 36). CD28 costimulation could
increase the strength of the initial activation signal. This is also
supported by recent observations that CD28 costimulation is required
for the full activation of naive T cells in a TCR Tg system
(37). Our data were consistent with the concept that CD28
costimulation is required for Th2 differentiation and that it enhances
the strength of the initial activation signal.
Previous reports have shown that polarization of the Th1 and Th2 cells
may reflect both the cytokine environment to which the cells are
exposed and the nature of the antigenic stimulation (35).
The requirement for IL-4- and IL-4R-dependent signals to drive Th2
differentiation has been demonstrated by targeted disruption of the
IL-4 and STAT6 genes and by using recombinant cytokines
(7, 8, 9, 10). We have provided evidence that STAT6 activation
is essential to up-regulate IL-4 transcription in Th2 cells
(16). In con-trast, the signaling mechanisms initiated by
TCR stimulation that could influence IL-4R function are not well
understood. All naive T cells express significant numbers of IL-4R
without prior activation signals (Fig. 6A) (38, 39). The IL-4R expressed on naive T cells is functionally active
because IL-4 significantly up-regulated IL-4R expression
(38) and naive T cells can proliferate with IL-4 in the
presence of PMA (40). However, our results showed that
neither IL-4 stimulation alone nor IL-4 before antigenic stimulation
could initiate Th2 differentiation (Fig. 2B). Stimulation
with the TCR cross-linking increased expression levels of IL-4R (Fig. 6A). Only the addition of IL-4 simultaneously with or within
several days after TCR stimulation was able to support Th2
development (Fig. 2B). Thus, our data indicated that
the number and the nature of the IL-4R expressed on naive
CD4+ T cells are different from those on the
primed T cells. We propose that initial T cell activation events may be
responsible for the alteration of the sensitivity of the IL-4R to IL-4
and that this may be at the level of the Jak/STAT signaling pathway,
which is activated by IL-4. This hypothesis was supported by
observations that the total phosphotyrosine protein content and the
phosphotyrosine content of the IL-4R-chain, Jak3, and STAT6
generated by IL-4 were substantially increased by initial T cell
activation (Fig. 6, B and C).
TCR cross-linking was unable to promote significant Th2
differentiation. Anti-TCR stimulation resulted in the production of a
detectable amount of IL-4 but, in comparison with the CD28
costimulation cultures, the addition of a 20- to 100-fold excess amount
of IL-4 was required to generate Th2 cells. TCR cross-linking enhanced
the protein expression of Jak3 and STAT6, but only a minor alteration
occurred in IL-4R-mediated tyrosine phosphorylation. TCR cross-linking
with CD28 costimulation apparently enhanced the magnitude of the
initial activation event, as evidenced by increased IL-2 and IL-4
production (Fig. 4A). CD28 costimulation enhanced the IL-4
production induced by anti-TCR 2- to 5-fold. Moreover, even when
the shortage in the IL-4 amount was supplemented, the number of Th2
cells in the anti-TCR cultures did not reach the level that
generated in TCR plus CD28 stimulation (Fig. 4C). Therefore,
it is unlikely that the difference in Th2 development with or without
CD28 costimulation was due only to the difference in IL-4 production.
TCR cross-linking markedly up-regulates the protein synthesis of the
IL-4R, CD69, Jak3, and STAT6, but CD28 does not further enhance the
expression of those molecules. Moreover, there is no difference between
anti-TCR and anti-TCR plus CD28 in the cell surface expression
of the IL-4R and the binding affinity for IL-4. Therefore, major role
of CD28 costimulation may be an alteration of the IL-4R-mediated
tyrosine phosphorylation property. Strong phosphorylations were
consistently observed in the IL-4R-chain, Jak3, and STAT6 by the
addition of CD28 costimulation (Fig. 6, B and C,
and Fig. 7A), indicating that these stimuli together
influenced the IL-4R-mediated Jak/STAT cascade. Our data were
consistent with a previous report that CD28 costimulation was necessary
for the IL-4 responsiveness of Th2 (41) and provided
direct evidence at the biochemical level to explain the effect of CD28
costimulation on the IL-4R-mediated Jak/STAT cascade. However, we could
not directly address whether the modification of the IL-4-dependent
phosphorylation properties was simply consequence of the high magnitude
of activation signal or this was a specific outcome for CD28
costimulation. These questions remain to be answered for further
investigation.
Recent reports demonstrated that overexpression of Lck in a T cell
lymphoma led to constitutive activation of Jak1, Jak2, STAT3, and STAT5
(42), and that TCR activation resulted in the
phosphorylation of Jak3 in these cells (T. Saito, personal
communication). These findings also address the possibility that the
initial T cell activation process might affect the activation status of
the Jak/STAT cascade. Moreover, we and others have recently described
that the activation status of STAT6 in Th2 cells is distinct from that
in Th1 cells (16, 43). The Jak/STAT signaling cascade is
impaired in murine Th1 cells, while it is functional in Th2 cells. This
was consistently observed in cloned lines and in induced Th1 and Th2
cells. These results are also consistent with the hypothesis that a
qualitative difference in the IL-4R signaling pathway can influence the
differentiation pathway of Th cells. Indeed, the generation of Th2
cells by CD28 costimulation was totally dependent on the IL-4-mediated
pathway because Th2 cells were not generated in the situation that IL-4
and IL-4-mediated signaling were blocked (Fig. 3B). Thus,
CD28 costimulation may be required for promoting Th2 generation at the
IL-4 concentration that naive T cells can produce.
In our hypothesis, the presence of IL-4 at the initial activation
process is essential for Th2 differentiation. This raises the question
of which cells are the initial source of IL-4. One such source of
primary IL-4 is a small population of NK1.1+
cells, which may recognize Ags that are associated with CD1
(44). However, the importance of these cells in in vivo
Th2 responses to specific Ags is still unclear (3, 45).
Because many types of cells express IL-4R in the resting state, it is
possible that a small amount of secreted IL-4 was immediately utilized
by the cells expressing IL-4R. Thus, we assayed IL-4 production from
naive T cells in the presence of neutralizing Ab against IL-4R. Under
these conditions, detectable amounts of IL-4 (50–100 pg/ml) were
secreted by naive T cells (Fig. 4A), although we could not
exclude the possibility that our preparation contained
CD4+NK1.1+ cells. Moreover,
some previous reports have indicated that naive T cells express IL-4
mRNA and can produce a small amount of IL-4 upon primary activation
(46, 47, 48, 49). Further evidence that a Th2 phenotype is not
essential for IL-4 production by naive T cells is that T cells from
STAT6 knockout mice that are deficient in Th2 responses can secrete
IL-4 upon primary stimulation (unpublished data). Therefore, it appears
likely that naive CD4+ T cells are one possible
source of primary IL-4 and that an IL-4-dependent autocrine pathway is
necessary for Th2 differentiation.
In summary, our observations suggest that the following novel mechanisms may be operative during Th2 differentiation. Costimulatory molecules, in addition to the TCR signal, may play an important role in generating Th2 cells. The CD28 plus TCR cross-linking signals provide an optimal initiation signal to the naive T cells and influence the IL-4R-mediated Jak/STAT signaling cascade. We hypothesize that as a result of the alterations in IL-4R signaling molecules, the sensitivity of the IL-4R is enhanced so that it can respond to the small amount of IL-4 produced by naive CD4+ T cells. Only under all of those circumstances the IL-4R may generate sufficient signals in the primed naive T cells to drive Th2 differentiation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. M. Kubo, Division of Immunobiology, Research Institute for Biological Sciences, Science University of Tokyo, 2669 Yamazaki, Noda City, Chiba 278-0022, Japan. E-mail address:
3 Abbreviations used in this paper: Tg, transgenic; Jak, Janus kinase.
Received for publication October 14, 1998. Accepted for publication June 10, 1999.
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production and diminishes interleukin 4 inhibition of such priming. Proc. Natl. Acad. Sci. USA 90:10188.ß T-cell-receptor transgenic system. Proc. Natl. Acad. Sci. USA 89:6065.ß-TCR transgenic T cells is macrophage dependent. Int. Immunol. 5:371. and TNF- are required for IL-12-induced development of Th1 cells producing high levels of IFN- in BALB/c but not C57BL/6 mice. J. Immunol. 160:1708. production. J. Exp. Med. 179:299.-producing CD4+ T cells following activation of naive CD4+ T cells. J. Immunol. 158:1085.[Abstract]
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170 kDa corresponding to
the size of IRS-1 (165 kDa). The arrow II indicated 130
