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Department of Allergology, Institute of Medical Science, University of Tokyo, Tokyo, Japan
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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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, and lymphotoxin and promote
cellular immune responses, whereas Th2 cells secrete IL-4, -5, -6, and
-13, and promote humoral immune responses, especially of IgE
(1). Th cell subset differentiation has been well
documented to be determined by cytokines, such as IL-12 and IL-4, in
milieu at an early activation stage of naive CD4+
T cells (2). IL-12 secreted by APC such as macrophages and
dendritic cells plays a pivotal role in the differentiation of Th1
cells, whereas IL-4 produced by naive CD4+ T
cells themselves and/or NK-T cells promotes the differentiation into
Th2 cells. TGF-ß has been shown to promote the differentiation of Th1
subset (3) or Th1-like cells (4) independent
of IL-12 (5), although it was also reported to inhibit the
differentiation of naive CD4+ T cells toward Th1
cells induced by coordination of IL-12 and IFN-
(6).
In addition to the cytokine environment, several other factors such as
valency of TCR ligation, CD28-mediated costimulation, type of APC, and
genetic background (1, 7) have been shown to affect Th
subset differentiation. It is well established that
CD4+ T cells require costimulations for their
efficient activation (8). B7 family molecules, CD80 and
CD86, expressed on APC are well known costimulators for
CD4+ T cells (9). The interaction of
B7 molecules with CD28 on naive CD4+ T cells has
been shown to promote Th2 subset differentiation in vivo
(10) as well as in vitro (11, 12). CTLA-4 is
a molecule structurally related to CD28 and transiently expressed on
activated T cells to bind B7 family molecules (13). The
cross-linking of CTLA-4 with immobilized anti-CTLA-4 was shown to
suppress IL-4 and IFN- production of T cell clones
(14). Engagement of CTLA-4 with anti-CTLA-4 or CD80
was shown to inhibit IL-2 production of primary
CD4+ T cells (15, 16), suggesting a
negative regulatory role for CTLA-4 in T cell activation. CTLA-4 was
also suggested to regulate Th cell subset differentiation.
CD4+ T cells in CTLA-4-deficient mice were
polarized toward the Th2 subset (17). In vitro IL-4
production was shown to be augmented in CD4+ T
cells from mice injected with anti-CTLA-4 (18). IL-4
production of CTLA-4-deficient mouse spleen cells was shown to be
markedly enhanced, although IFN- production was also rather enhanced
(19). However, blockade of CTLA-4/B7 interaction in vivo
by anti-CTLA-4 was reported to result in the exacerbation of
experimental allergic encephalomyelitis
(EAE),3 a classical
Th1-mediated autoimmune disease (20, 21, 22). Thus, the role
of CTLA-4 in the Th cell subset polarization is not yet clear. Th1/Th2
polarization or balance could be determined by a variety of events,
especially in vivo. Therefore, in the present experiment, we examined
the role of CTLA-4/B7 interaction in Th cell subset polarization in in
vitro experiments using tumor cells expressing CD80 instead of
professional APC.
Our results indicate that CTLA-4 costimulation contributes to polarize naive CD4+ T cells toward the Th1 subset through different mechanisms including augmentation of TGF-ß1 and suppression of IL-4 production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female DBA/2 mice were obtained from Japan SLC (Shizuoka, Japan) and used at 7 to 8 wk of age.
Culture media
RPMI 1640 (JRH Biosciences, Lenexa, KS) supplemented with 10% FCS (Sanko Junyaku, Tokyo, Japan), 5 x 10-5 M 2-ME, and 100 µg/ml kanamycin was used for cultures throughout the present experiments. In some procedures, MEM (JRH Biosciences) with or without FCS was also used.
Abs and reagents
Culture supernatants containing
anti-I-Ad (M5/114, rat IgG2b), anti-heat
stable Ag (M1/69, rat IgG2b), anti-CD8 (53.6.72, rat IgG2a), and
anti-FcRII/III (2.4G2, rat IgG2b), and ascites containing
anti-Thy1.2 (HO-13.4, mouse IgM) were prepared in our laboratory
using respective hybridomas obtained from American Type Culture
Collection (Manassas, VA). Purified anti-CD28 (35.71, hamster IgG),
anti-CTLA-4 (UC10-4F10, hamster IgG), anti-CD80 (16-10A1,
hamster IgG), FITC-anti-CD44 (IM7, rat IgG2b), PE-anti-CD4
(GK1.5, rat IgG2b), -anti-IL-2Rß (TM-ß1, rat IgG2b),
-anti-B220 (RA3-6B2, rat IgG2a), -anti-CD45RB (16A, rat IgG2a),
-anti-CD62L (MEL-14, rat IgG2a), -anti-CTLA-4 (UC10-4F10), and
purified and PE-normal hamster IgG were all purchased from
PharMingen (San Diego, CA). FITC- and PE-goat IgG
F(ab')2 anti-rat IgG were purchased from
Biosource (Camerillo, CA) and Life Technologies (Gaithersburg, MD),
respectively. Anti-CD3 (145-2C11, hamster IgG) was purified from
ascites on a protein A column. Part of anti-CTLA-4, anti-CD80
and normal hamster IgG were digested into Fab with papain (Sigma, St.
Louis, MO), followed by the passage through a protein A column to
remove possibly remaining intact IgG and Fc fragment. These Fab
preparations were confirmed to form a single band with 50 kDa on
SDS-PAGE. The anti-CTLA-4 Fab was confirmed by flow cytometry to
block the binding of PE-anti-CTLA-4 to L cells transfected with
CTLA-4 cDNA. Fusion proteins composed of human IgG1 heavy chain
constant region and extracellular region of murine CD80 (CD80-Ig) or
CD86 (CD86-Ig) were generous gifts from Dr. T. Uede (Hokkaido
University, Hokkaido, Japan). Anti-IL-4 (11B11, rat IgG1) with
neutralizing activity was purified from ascites on a protein G column,
and an isotype-matched control mAb (anti-IgE, R35-72, rat IgG1) was
obtained from PharMingen. Anti-TGF-ß1, 2, 3 (code: 80-1835-03, mouse
IgG1) with neutralizing activity and an isotype-matched control mAb
(mouse IgG1) were obtained from Genzyme (Cambridge, MA) and PharMingen,
respectively. Murine rIL-2 was purified on an anti-IL-2 affinity
column from culture supernatant of Ag8.653 transfected with IL-2 cDNA
(23). Porcine TGF-ß1 was purchased from R&D Systems
(Minneapolis, MN).
Cells
Naive CD4+ T cells were purified as
described (24). Briefly, nylon wool column-passed spleen
cells were depleted of CD8+ cells,
I-Ad+ cells, heat stable
Ag+ cells, and
FcRII/III+ cells on a MACS column (Miltenyi
Biotech, Germany) to obtain CD4+ T cells. The
CD4+ T cells were stained with
FITC-anti-CD44, and CD44low cells were sorted
in a FACS Vantage (Becton Dickinson, Mountain View, CA). The sorted
cells were confirmed to be >99% CD4+
CD44low CD45RBhigh
Mel-14high, and CD8+ cells,
IL-2Rß+ NK-T cells, I-Ad+
macrophages/dendritic cells, or B220+ B cells
were not detected flow cytometrically in the preparations. These sorted
CD4+ T cells were used as naive
CD4+ T cells. Spleen cells depleted of T cells
(T-d spleen cells) by the treatment with anti-Thy1.2 and baby
rabbit complement as described (24) were irradiated 35 Gy
and used as accessory cells for secondary stimulation of
CD4+ T cells with anti-CD3. The irradiated
T-d spleen cells were confirmed not to produce detectable level of
IL-2, IL-4, IL-5, or IFN- after the stimulation with anti-CD3.
Splenic adherent cells (SAC) were prepared from the T-d spleen cells by
adherence to a plastic dish for 2 h as described (25)
and used as accessory cells after 35 Gy irradiation for priming of
naive CD4+ T cells. FcR+
murine mastocytoma P815 cells transfected with murine CD80 cDNA
(CD80-P815) or vector alone (mock-P815) (26) and L cells
transfected with CTLA-4 cDNA (CTLA-4-L cells) were generously provided
by Dr. H. Yagita (Juntendo University, Tokyo, Japan). Both CD80-P815
and mock-P815 were used as accessory cells for priming of naive
CD4+ T cells after fixation with 0.5%
paraformaldehyde in PBS for 10 min at room temperature.
Stimulation of CD4+ T cells
Priming of naive CD4+ T cells (2 x
105 cells/1 ml/culture) was conducted using 1
µg/ml anti-CD3 with irradiated SAC (2 x
105 cells/1 ml/culture) or paraformaldehyde-fixed
CD80-P815 (1 x 106 cells/1 ml/culture) in
the presence or absence of 1 µg/1 ml/culture anti-CD28 in a well
of 48-well plate (Iwaki Glass, Tokyo, Japan). To block the engagement
of CTLA-4 molecules of these T cells, 10 µg/ml anti-CTLA-4 Fab
was included in cultures. The CD4+ T cells primed
for 4 days in a well were distributed into four wells with fresh medium
containing 50 U/ml IL-2 and the corresponding Ab Fab to those in the
first 4 days culture. In some experiments, naive
CD4+ T cells were primed in a well of 48-well
plate coated with 300 ng/well anti-CD3, 2 µg/well anti-CD28,
and 2 µg/well anti-CTLA-4, and the cells primed for 4 days in a
well were expanded into four wells with fresh medium containing 50 U/ml
IL-2. Eight days after the priming as above, CD4+
T cells were recovered by a centrifugation over Ficoll. These primed T
cells (5 x 104 cells/250 µl/culture) were
restimulated with 1 µg/ml anti-CD3 in the presence of irradiated
T-d spleen cells (5 x 105 cells/250
µl/culture) in a 96-well plate (Becton Dickinson). Supernatants of
cultures for the priming were harvested for 48 h and assayed for
IL-2. Supernatants of cultures restimulated as described above for
24 h were assayed for IL-2, and those restimulated for 48 h
were assayed for IL-4, IL-5, and IFN-. Results are presented as
mean ± SD of triplicate cultures.
Cytokine assays
IL-2, IL-4, IL-5, and IFN- in culture supernatants were
assayed in ELISA using paired mAbs specific for the corresponding
cytokine according to the manufacturer’s instruction (PharMingen).
Standard curves were obtained using murine rIL-2 (Immugenex, Los
Angels, CA), rIL-4 (Life Technologies), rIL-5 (Dr. T. Koro, Department
of Immunology, University of Tokyo), and rIFN- (Toray Industry,
Tokyo, Japan). Lower detection limits of these assays were as follows:
IL-2, 4 pg/ml; IL-4, 10 pg/ml; IL-5, 5 pg/ml; IFN-, 150 pg/ml.
TGF-ß1 in culture supernatants was assayed by the TGF-ß1 Emax
ImmunoAssay System (Promega, Madison, WI), which is specific for the
active form of TGF-ß1. The detection limit of this assay was 32
pg/ml.
Competitive RT-PCR
Total cellular RNA was prepared using the acid guanidinium
thiocyanate-phenol-chloroform extraction method (27).
Four-tenths microgram of the total RNA was reverse-transcribed into
cDNA in a 50 µl reverse transcriptase reaction mixture as described
(28). Semiquantitative analyses of IFN-, IL-4, and
TGF-ß1 mRNA accumulation were performed as described
(29) with minor modifications. Briefly, PCR amplification
of target cDNA (1 µl) was performed in the presence of competitor (1
µl) in a 10-µl reaction mixture containing 1x PCR buffer, 200 nM
dNTP, 2 mM MgCl2, 400 nM primers, 50 µCi/ml
[
-32P]dCTP, and 0.5 U Taq
polymerase (AmpliTaq Gold, Perkin-Elmer Cetus, Norwalk, CT).
The optimal competitor concentrations were determined by amplifying
target cDNA in the presence of 2-fold serial dilutions of competitor.
The multiple competitor PQRS used in our assay was generously given by
Dr. R. M. Locksley (University of California, Los Angeles, CA)
(30). The PCR product was electrophoresed through a
10–20% polyacrylamide gradient gel (Daiichi Pure Chemicals, Tokyo,
Japan), and the radioactivity of the specific band was measured. The
results of semiquantitation of the target molecule are presented as the
ratio of target to competitor PCR products normalized with that of
hypoxanthine-guanine phosphoribosyltransferase (HPRT). The PCR cycling
conditions were 94°C for 1 min, 60°C for 1 min, and 72°C for 1
min for 35 cycles for IL-4 and TGF-ß1 cDNA and 28 cycles for HPRT
cDNA amplification. The primers used for cDNA amplification were as
described (30).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether an interaction of CTLA-4 with B7 family
molecules affects Th cell subset polarization, the interaction was
blocked by the inclusion of anti-CTLA-4 Fab in cultures when naive
CD4+ T cells were primed with anti-CD3 on
SAC. Eight days after the priming, they were stimulated with
anti-CD3 in the presence of T-d spleen cells, and their
supernatants were assayed for cytokines. Although the priming in the
presence of control IgG Fab polarized the Th cells toward the Th1
subset, the cells primed in the presence of anti-CTLA-4 Fab were
polarized toward the Th2 subset (Fig. 1
B). Consistent with the
results published previously (15), the inclusion of
anti-CTLA-4 Fab augmented IL-2 production of naive
CD4+ T cells upon priming (Fig. 1A),
suggesting that anti-CTLA-4 Fab included in cultures for priming
effectively blocked CTLA-4/B7 interaction. The addition of IgG Fab, a
control for anti-CTLA-4 Fab, was confirmed not to affect the
cytokine production profile of the naive CD4+ T
cells primed with anti-CD3 on SAC in either primary or secondary
response (data not shown). The concentration of anti-CTLA-4 Fab
used in this experiment was confirmed by flow cytometry to completely
inhibit binding of CD80-Ig or CD86-Ig to L cells expressing CTLA-4
(data not shown).
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Therefore, in the next experiment, naive CD4+ T
cells were primed with anti-CD3 together with
paraformaldehyde-fixed CD80-P815 cells in the presence of
anti-CTLA-4 Fab or control IgG Fab. IL-2 produced by naive
CD4+ T cells upon priming was significantly
enhanced in amount by the presence of anti-CTLA-4 Fab (Fig. 2A), and they were apparently
polarized toward the Th2 subset, although naive
CD4+ T cells were polarized toward the Th1 subset
without CTLA-4 blocking (Fig. 2B). Naive
CD4+ T cells were confirmed to express CTLA-4
within 24 h following the stimulation with anti-CD3 on
CD80-P815 cells (data not shown).
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Polarization of naive CD4+ T cells into the Th1 subset by CTLA-4 engagement
CD28-mediated costimulation has been shown to promote Th2 subset
differentiation of naive CD4+ T cells
(10, 11, 12). Therefore, in the above experiments, it is
possible that anti-CTLA-4 Fab promoted Th2 subset differentiation
by blocking the binding of CTLA-4 to B7 to increase the number of B7 to
stimulate CD28 on naive CD4+ T cells. To directly
examine the effect of CTLA-4 engagement on Th1 subset polarization,
naive CD4+ T cells were primed with anti-CD3
on CD80-P815 or mock-P815 cells in the presence of anti-CD28. The
CD4+ T cells primed in the presence of CD80-P815
cells were polarized toward the Th1 subset, although the cells primed
using mock-P815 cells were polarized toward the Th2 subset (Fig. 3B). The effect of CD80-P815
cells on the induction of Th1 subset polarization was abrogated by the
inclusion of anti-CTLA-4 Fab or anti-CD80 Fab in cultures
during priming (Fig. 3B). The dose of anti-CD28 was
confirmed in preliminary experiments to be enough to inhibit the
binding of CD80-Ig or CD80-P815 cells treated with
anti-FcRII/III to naive CD4+ T cells (data
not shown). IL-2 production of naive CD4+ T cells
during priming was significantly suppressed by the presence of
CD80-P815 cells (Fig. 3A), indicating that CD80 actually
engaged CTLA-4. When anti-CTLA-4 Fab or anti-CD80 Fab was
included in cultures for priming, the suppressive effect of CD80-P815
cells on IL-2 production was abrogated (Fig. 3A), indicating
that the CD80 molecules on P815 cells predominantly costimulate naive
CD4+ T cells via CTLA-4 in our system.
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A). Cross-linking of CTLA-4
during priming promoted the polarization of naive
CD4+ T cells toward the Th1 subset and suppressed
Th2 subset polarization (Fig. 4B).
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Augmentation and suppression of mRNAs for TGF-ß1 and IL-4 in naive CD4+ T cells by CTLA-4 engagement
Engagement of CTLA-4 on CD4+ T cells has
been reported to induce TGF-ß1 (34). Therefore, culture
supernatants of naive CD4+ T cells costimulated
with anti-CD3 and anti-CD28 in the presence of CD80-P815 or
mock-P815 cells were assayed for TGF-ß1 and also for IL-4, which is
well known to play critical role in the differentiation of the Th2
subset. The cells in these cultures were also assayed for the
accumulation of mRNAs for these cytokines. The active form of TGF-ß1
was not detected in ELISA (lower detection limit; 32 pg/ml) in any
culture supernatant of naive CD4+ T cells
stimulated for 4 days. However, TGF-ß1 mRNA accumulation was detected
in naive CD4+ T cells stimulated in the presence
of mock-P815 cells, and it was enhanced by the presence of CD80-P815
cells in cultures (Fig. 5). In contrast,
163 pg/ml IL-4 in average was detected in the culture supernatants of
naive CD4+ T cells primed for 4 days in the
presence of mock-P815. However, IL-4 was not detected in culture
supernatants of these T cells primed for 4 days in the presence of
CD80-P815 cells. In accordance with these results in ELISA, IL-4 mRNA
was accumulated in CD4+ T cells primed in the
presence of mock-P815 cells, and the accumulation was suppressed by the
presence of CD80-P815 cells (Fig. 5). The effects of CD80-P815 cells on
the accumulation of mRNAs for both cytokines in the
CD4+ T cells were abrogated by the inclusion of
anti-CTLA-4 Fab (data not shown). Furthermore, we obtained similar
results to those described above in IL-4 and TGF-ß1 mRNA accumulation
in naive CD4+ T cells stimulated with immobilized
anti-CD3, anti-CD28, and anti-CTLA-4 or control mAb (data
not shown).
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Roles of endogenous TGF-ß and IL-4 in the Th1 subset polarization induced by CTLA-4 engagement
We next examined the effects of neutralization of TGF-ß1 and
IL-4 during priming on the polarization of naive
CD4+ T cells toward the Th1 subset. The addition
of anti-TGF-ß during priming of naive CD4+
T cells with anti-CD3 and anti-CD28 in the presence of
CD80-P815 cells suppressed the polarization toward the Th1 subset,
although the suppression was not complete even with 40 µg/ml
anti-TGF-ß (Fig. 6). The addition
of anti-TGF-ß to naive CD4+ T cells
stimulated in the presence of mock-P815 cells did not affect the Th
cell subset polarization even at 20 µg/ml (Fig. 6).
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). These results indicate that the
promotion of Th1 subset polarization by CTLA-4 engagement with CD80 is
not simply due to the suppression of IL-4 production, although the
reduction of IL-4 production may impair the generation of the Th2
subset.
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Polarization toward the Th1 subset of naive CD4+ T cells primed in the presence of low concentrations of TGF-ß1
To examine whether such a low dose of TGF-ß1 as produced by
naive CD4+ T cells in response to CTLA-4
engagement promotes Th1 subset polarization, various concentrations of
exogenous TGF-ß1 were included in cultures of naive
CD4+ T cells during priming with anti-CD3 and
anti-CD28 in the presence of mock-P815 cells. These T cells were
restimulated with anti-CD3 on T-d spleen cells, and culture
supernatants were assayed for cytokines. The production of Th1
cytokines was augmented by the addition of 0.4 or 2 pg/ml TGF-ß1 but
it was decreased with more TGF-ß1. The addition of exogenous TGF-ß1
did not completely substitute for the effect of CTLA-4 engagement with
CD80 on P815 cells in terms of enhancement of Th1 cytokine production
in secondary response (Fig. 8A). In addition, Th2 cytokine
production was not affected by the addition of 0.08–2 pg/ml TGF-ß1.
When >10 pg/ml TGF-ß1 was added, both Th1- and Th2-type cytokine
production was suppressed in secondary response. Because CTLA-4
engagement suppressed IL-4 production of naive
CD4+ T cells, it is possible that IL-4 produced
by naive CD4+ T cells during priming in the
absence of CTLA-4 engagement interferes with the effect of exogenous
TGF-ß1 on the Th1 subset polarization. Therefore, anti-IL-4 was
included in cultures when naive CD4+ T cells were
primed as above in the presence of various concentrations of TGF-ß1,
and culture supernatants of these cells restimulated as above were
assayed for cytokines. Although the inclusion of anti-IL-4 reduced
the production of Th2 cytokines, the production of Th1 cytokines was
not augmented any more in the presence of exogenous TGF-ß1 (Fig. 8B). The dose of TGF-ß1 required for the optimal
augmentation of Th1 cytokine production was reduced to 0.4 pg/ml. The
doses of TGF-ß1 that augmented Th1-type cytokine production in
secondary culture did not significantly affect IL-2 production during
priming. Cytokine production profiles in secondary response of T cells
primed in the presence of control mAb were confirmed to be essentially
the same to those of the T cells primed in the absence of mAb (data not
shown).
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, indicate that
a small amount of TGF-ß1 produced by naive CD4+
T cells in response to CTLA-4 engagement promotes the differentiation
of the Th1 subset. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Consistent with our present results, CD4+ T cells in anti-CTLA-4-treated (18) or in CTLA-4-deficient mice (17) were shown to be polarized toward the Th2 subset. In these mice, it is possible that blocking or a deficiency of the CTLA-4 molecule made the reaction of CD80/CD86 to CD28 more efficient to promote Th2 subset differentiation, because CD28-mediated costimulation was well demonstrated to polarize CD4+ T cells toward the Th2 subset (10, 11, 12). Quite recently, CTLA-4 was also shown to be a potent inhibitor of Th2 subset differentiation induced by the CD28 costimulation using CTLA-4-/-CD80/CD86-/- mice (35), indicating a reciprocal regulation of CD4+ T cell differentiation by CD28 and CTLA-4 as well as of IL-2 production (15, 36).
However, the severity of EAE, a classical Th1-mediated autoimmune
disease model, was shown to be exacerbated through CTLA-4 blockade by
anti-CTLA-4 administration (20, 21, 22) in association
with the enhancement of IFN- and IL-2 production (20, 21). In addition, T cells from mice immunized with
GM-CSF-producing tumor cell vaccine in combination with anti-CTLA-4
treatment were shown to be enhanced in IFN- production
(37). These results suggest the enhancement of Th1
response by CTLA-4 blockade. CTLA-4 seems to regulate the effector
phase of EAE, because the disease was markedly exacerbated by the
anti-CTLA-4 administration after the onset of clinical symptoms
(20) or in mice transferred with T cells primed with
epitope peptide of myelin protein (21). IL-12 was well
documented to play an important role in the pathogenesis of EAE
(38). To induce EAE, mice are primed with myelin basic
protein or related peptide emulsified with CFA. A
Mycobacterium cell wall glycopeptide in CFA was shown to
stimulate IL-12 production (39). IL-12 is known to drive
naive CD4+ T cells toward the Th1 subset. GM-CSF,
which plays a critical role in tumor cell vaccination as mentioned
above, was also shown to promote IL-12 production of macrophages
(40). CD28 costimulation was shown to play an important
role in maintaining T cell response by inducing expression of
anti-apoptotic genes such as Bcl-xL
(41). In contrast, CTLA-4 costimulation inhibited clonal
expansion of CD4+ T cells in vivo
(42). Therefore, it is possible that CTLA-4 blockade in
mice with EAE and the tumor cell vaccine mentioned above promoted the
expansion of Th1 cells already polarized in the presence of IL-12,
although it remains a possibility that CTLA-4 blockade in vivo
polarized a Th1/Th2 balance into a Th1 dominance by an unknown
mechanism in complex events in autoimmunity or tumor immunity.
CD28 costimulation was shown to be required for optimal CTLA-4 expression of naive CD4+ T cells (43). CD28-/- CD4+ T cells expressed a low density of CTLA-4 (44). In our present experiments, naive CD4+ T cells stimulated with anti-CD3 alone did not express detectable level of CTLA-4; however, they apparently expressed CTLA-4 by the stimulation with anti-CD3 on CD80-P815 (data not shown), confirming that CD28 costimulation plays an important role in sufficient CTLA-4 expression for its function. In our experiments, costimulatory conditions during priming that induced a lower amount of IL-2 production of naive CD4+ T cells induced polarized toward the Th1 subset, and those that induced higher amounts of IL-2 production induced the Th2 subset differentiation. These results could be explained as following: CD4+ T cells effectively costimulated against CD3 and CD28 expressed sufficient density of CTLA-4 to drive them to the Th1 subset, but the engagement of a high density of CTLA-4 also inhibits IL-2 production independent of the Th1 differentiation, while the cells produced higher amount of IL-2 were polarized toward the Th2 subset due to the efficient CD28 costimulation and insufficient CTLA-4 engagement. IL-2 was also reported to be required for Th2 subset differentiation, because the Th2 subset could not be differentiated even in the presence of IL-4 when IL-2 in cultures was neutralized with mAb (45). However, in our experiments the addition of 50–200 U/ml exogenous IL-2 did not affect the Th1/Th2 balance polarized by the ligation of CTLA-4 with CD80 (data not shown). In addition, exogenous IL-2 did not recover the Th2 subset development of CD28-/- CD4+ T cells (12). These results suggest that IL-2 does not play a critical role, if any, in Th2 subset differentiation.
Recently, the interaction of ICAM-1 or ICAM-2 with LFA-1, either of which is on APC, has been reported to promote the development of Th1 subset through suppression of Th2 subset differentiation (46). ICAM-1 expressed on accessory cells was also shown to suppress Th2 cytokine production of naive CD4+ T cells (47). Because costimulation of T cells with ICAM-1 was shown to augment CTLA-4 expression (48), the effect of ICAM-1/-2 on the suppression of Th2 differentiation or of Th2 cytokine production mentioned above could be mediated by the augmentation of CTLA-4 expression on CD4+ T cells.
In our experiments, CTLA-4 costimulation augmented the accumulation of
TGF-ß1 mRNA and suppressed IL-4 production of naive
CD4+ T cells during priming. However, the active
form of TGF-ß1 was not detected in ELISA in culture supernatants of
the CD4+ T cells costimulated against CTLA-4.
Although the active form of TGF-ß1 was reported to be detected in
culture of CD4+ T cells (34), their
T cell preparations contained memory CD4+ T
cells. Memory CD4+ T cells produced TGF-ß1 more
than did naive CD4+ T cells. Naive
CD4+ T cells were shown not to produce levels of
the active form of TGF-ß1 detectable in ELISA (49). The
lower detection limit of our ELISA system was 32 pg/ml, and
concentrations of exogenous TGF-ß1 in cultures effectively polarizing
the Th subset toward Th1 were as low as 0.4–2 pg/ml. Taken together
with the result that the inclusion of anti-TGF-ß1 in cultures for
priming of naive CD4+ T cells partially reversed
the effect of CTLA-4 costimulation on Th1 subset differentiation, these
results indicate that TGF-ß1 produced by naive
CD4+ T cells contributes in part to the Th1
polarization induced by CTLA-4 costimulation. TGF-ß1 was shown to
play a role in the development of the Th1 subset or Th1-like cells from
freshly isolated CD4+ T cells stimulated with
staphylococcal enterotoxin B (3) or Con A
(4), respectively. Our results also suggest that a low
dose of TGF-ß1 promotes the Th1 differentiation without affecting Th2
subset development (Fig. 8), although CTLA-4 costimulation was reported
to inhibit Th2 subset differentiation (35). TGF-ß was
also reported to suppress Th1 subset development of naive
CD4+ T cells even in the presence of IL-12 and
IFN- (6). However, the doses of TGF-ß1 they added in
their culture were very high, and in our present experiments the high
dose of TGF-ß1 (50 pg/ml or more) suppressed the development of both
Th1 and Th2 subsets (Fig. 8).
FCS used in our experiments contained a biologically inactive form of TGF-ß1 (data not shown). However, the inclusion of Ab specific for the active form of TGF-ß1 in cultures for priming of naive CD4+ T cells without CTLA-4 costimulation did not affect their Th subset polarization, indicating that TGF-ß1 in FCS remains inactive during cultivation.
IL-4 is well known to support Th2 subset development. IL-4 production
of naive CD4+ T cells costimulated with
anti-CD3 and anti-CD28 was suppressed by the engagement of
CTLA-4 in terms of both ELISA and RT-PCR (Fig. 5), suggesting that the
CTLA-4 costimulation also contribute to the suppression of Th2 subset
development by reducing IL-4 production of CD4+ T
cells. CTLA-4 may inhibit CD28-mediated signals to preferentially
activate Th2 cytokine genes. Cross-linkage of CTLA-4 was indicated to
activate SHP-2, resulting in dephosphorylation of signaling proteins of
which phosphorylation was enhanced by CD28-cross-linkage
(50, 51, 52, 53). Therefore, it is possible that the CTLA-4
engagement merely inhibits the CD28-mediated signal, causing a
reversion to a default Th1 phenotype.
Taken all together, our present results indicate that the engagement of CTLA-4 on naive CD4+ T cells costimulated by ligations of CD3 and CD28 contributes to their polarization toward the Th1 subset through different mechanisms. Some of these mechanisms might contribute to the polarization of the Th subset in vivo dependent upon the microenvironment around individual CD4+ T cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Takuma Kato, Department of Allergology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail address:
3 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; CD80-P815 cells; P815 cells transfected with CD80 cDNA; CTLA-4-L cells, L cells transfected with CTLA-4 cDNA; HPRT, hypoxanthine-guanine phosphoribosyltransferase; SAC, splenic adherent cells; T-d spleen cells, T cell-depleted spleen cells.
Received for publication September 15, 1999. Accepted for publication January 26, 2000.
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) or
mock-P815 cells (
). They were harvested at indicated times after the
stimulation and assayed for accumulation of mRNAs by semiquantitative
RT-PCR. Radioactivities of the specific band in the gel were measured,
and the ratios of target to competitor PCR products normalized with
those HPRT products were shown. The data shown are representative of
three experiments with similar results.
