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Departments of
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Allergology and
Bacterial Infection, Institute of Medical Science, University of Tokyo, Minatoku, 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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expression of Th1 cells stimulated with B cell
APC than of the cells stimulated with anti-CD3 alone (2),
suggesting that some molecules on B cell APC cooperate with IL-12 for
IL-2R expression of Th1 cells.
B7-1 and B7-2 molecules are expressed on various APC, such as B cells
(5), macrophages (6), and dendritic cells (7), and work as
costimulators for T cell activation by binding to CD28 or CTLA-4 with
similar avidity (8). Although exogenous IL-12 was shown not to affect
the proliferation of naive T cells costimulated with B7-1, the
proliferation of Th1 clones was shown to be enhanced by the cooperation
of IL-12 and B7-1 (9). In our previous experiment, naive but not memory
CD4+ T cells were shown to require costimulation with B7-2
for the enhancement of IL-2R-chain expression by IL-12 (10). These
results indicate that the efficiency of cooperation of IL-12 and
B7-1/B7-2 is dependent upon the activation stage of Th cells. The
enhancement of Th1 clone proliferation mediated by IL-12/B7-1
cooperation was shown to be caused by the up-regulation of
IL-2R-chain expression on Th1 cells in an IL-2-independent mechanism
(11). The results suggest that B7-1 cooperates with IL-12 in IL-2R
expression by affecting the activation signal evoked by the
IL-12/IL-12R interaction or by inducing IL-12R expression itself.
IL-12R were identified on activated but not on resting NK and T cells
by the detection of cell-bound IL-12 using anti-IL-12 p40 (12). In
addition to the cDNA of one component of IL-12R, IL-12ß1 chain (13, 14), another ß-type receptor subunit cDNA was also cloned (14); this
subunit was designated IL-12Rß2 (15).
In the present experiments, we studied the requirement of B7-2 costimulation for IL-12R expression of naive and memory CD4+ T cells and Th1 clones stimulated with anti-CD3. The results indicated that both anti-CD3 and B7-2 stimulation are required for the expression of IL-12R, especially for IL-12Rß2 mRNA of CD44low naive T cells, and that both Th1 clones and CD45RBlow memory T cells expressed a low density of IL-12R without stimulation. The expression was augmented by anti-CD3 stimulation alone, and costimulation with B7-2 enhanced the expression further by augmenting both IL-12Rß1 and -ß2 chain mRNA accumulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C3H/He and C57BL/6 mice, purchased from Japan SLC Inc. (Shizuoka, Japan) and Charles River Inc. (Atsugi, Japan), respectively, were used at 7 to 8 wk of age.
Culture media
RPMI 1640 (Hazleton Biologics, Lenexa, KS) supplemented
with 10% FCS (CSL Ltd., Victoria, Australia), 5 x
10-5 M 2-ME, and 100 µg/ml kanamycin was used in
all experiments. Chinese hamster ovary cells (CHO) were maintained in
-MEM (Life Technologies, Gaithersburg, MD).
Abs and reagents
Anti-CD3
(145-2C11, hamster IgG) (16) and anti-B7-2
(GL-1, rat IgG2a) (17) were purified from ascites on protein A and
protein G columns (Pharmacia Fine Chemicals, Piscataway, NJ),
respectively. Anti-Thy 1.2 (HO-13.4, mouse IgM) (18) was used in the
form of ascites. FITC- or PE anti-CD4 (RM4-5, rat IgG2a),
anti-B7-1 (1G10, rat IgG2a), and unlabeled and biotinylated rat
IgG2a with an irrelevant specificity, used as controls for
anti-B7-1 and anti-B7-2, were all purchased from PharMingen
(San Diego, CA). PE-streptavidin and FITC-goat F(ab')2
anti-rat IgG were purchased from Life Technologies. Biotinylated
goat anti-streptavidin was purchased from Vector Laboratories, Inc.
(Burlingame, CA). mAbs specific for CD8 (53.6.72, rat IgG2a) (19), Ia
(M5/114, rat IgG2b) (20), heat-stable Ag (M1/69, rat IgG2a) (21),
Fc
RII/III (2.4G2, rat IgG2b) (22), and CD45RB (23G2, rat IgG2a)
(23), used for a Dynabead operation, and CD44 (KM201, rat IgG2a) (24),
used for FACS sorting, were in the form of culture supernatants.
Anti-murine IL-12 p40 (C17.8.20, rat IgG2a) (25), kindly provided by
Dr. G. Trinchieri (The Wistar Institute of Anatomy and Biology,
Philadelphia, PA), was biotinylated with NHS-biotin (Vector
Laboratories) and was used in flow cytometry. A hybrid molecule between
murine CTLA-4 and human IgG1 Fc portion (CTLA-4-Ig) (26) was kindly
provided by Drs. M. Murakami and T. Uede, Hokkaido University, Sapporo,
Japan. Human IgG1 (hIgG1) was purchased from Sigma-Aldrich Japan,
Tokyo, Japan. Murine rIL-12 was a generous gift from The Genetics
Institute (Cambridge, MA).
Cell lines
An OVA/I-Ab-specific Th1 clone, 35-9D, was established from C57BL/6 mouse lymph node cells and maintained by repeated Ag stimulation as previously described (27). CHO cells transfected with murine B7-2 cDNA cloned from a B-lymphoblastoid cell line library (B7-2-CHO) (10) were generously provided by Drs. M. Azuma and K. Okumura (Juntendo University School of Medicine, Tokyo, Japan) and used for experiments after fixation with 0.5% paraformaldehyde as previously described (11).
Preparations of naive and memory CD4+ T cells
CD4+ T cells were prepared by negative
selection using Dynabeads (Dynal AS, Oslo, Norway). Briefly, nylon
wool-passed spleen T cells from C3H/He mice were incubated with a
mixture of mAbs against CD8, Ia, heat-stable Ag, and FcRII/III and
then with Dynabeads coated with sheep anti-rat IgG for 20 min at
4°C. The T cells to which these mAbs did not react were collected by
two 10-min cycles of exposure to a magnetic field and were used as
CD4+ T cells. CD44low T cells were prepared
from the CD4+ T cells by sequential staining with
anti-CD44 and FITC-goat anti-rat IgG, followed by sorting in a
FACStar (Becton Dickinson, Mountain View, CA). A CD45 RBlow
population was obtained from the CD4+ T cells by treatment
with anti-CD45RB, followed by negative selection using Dynabeads.
The purities of both CD4+CD44low and
CD4+CD45RBlow populations were >95%, and
neither CD8+ nor Ia+ cells were detected in
these populations in flow cytometry. In the present experiments,
CD4+CD44low and
CD4+CD45RBlow cells were used as naive and
memory CD4+ T cells, respectively.
Preparation of splenic adherent cells (SAC)
SAC were enriched by adherence to a plastic dish and depletion of T cells. Briefly, spleen cells from C3H/He or C57BL/6 mice were incubated for 2 h at 37°C in a 100-mm culture dish containing RPMI 1640 with 10% FCS. After washing the dish to deplete nonadherent cells, adherent cells were detached by vigorous pipetting with PBS containing 0.6 mM EDTA, washed and depleted of residual T cells by treatment with anti-Thy1.2 plus baby rabbit complement, and used after 35 Gy irradiation. The resulting cells were mostly Mac1+ and B220+, and 80 to 90% of the cells were confirmed as strongly positive in B7-2. Expression of marginal density of B7-1 was also observed on the surface of these cells.
Stimulation of T cells
Naive and memory T cells, 3 x 105 cells/culture, were stimulated with plate-coated anti-CD3 in the presence of B7-2-CHO, CHO, or SAC from C3H/He mice in a flat-bottom 48-well plate. Th1 clone 35-9D cells, 1 x 105 cells/culture, were stimulated with 100 µg/ml OVA or plate-coated anti-CD3 in the presence of SAC from C57BL/6 mice, CHO, or B7-2-CHO.
Assay for IL-12R expression on T cells in flow cytometry
T cells were incubated with 100 ng/ml of rIL-12 in PBS containing 2% FCS and 0.1% NaN3 at 4°C for 40 min. Cells were washed three times and incubated sequentially with biotinylated anti-IL-12, PE-streptavidin, and biotinylated goat anti-streptavidin and then with PE-streptavidin. For a negative control of the fluorescence profile of IL-12R expression, T cells were incubated without rIL-12 and then treated as above by stimulating them with anti-CD3 in the presence of B7-2-CHO or CHO. However, when SAC were included in culture, biotinylated rat IgG2a with an irrelevant specificity was used as a control for biotinylated anti-IL-12, because SAC may produce IL-12 via CD40-CD40L interaction as described (4). All Abs and PE-streptavidin were used at 1 µg/ml. For flow cytometric analysis, 104 cells were analyzed on a FACScan (Becton Dickinson) after dead cells were excluded on the basis of propidium iodide staining. When SAC were used as accessory cells, FACS analysis was conducted after gating the CD4+ cells.
RNA preparation and cDNA synthesis
Total cellular RNA was prepared using the acid guanidinium-phenol-chloroform method (28). Two-tenths microgram of total RNA was reverse transcribed into cDNA for PCR amplification using oligo(dT) primer (Promega, Madison, WI) and Moloney murine leukemia virus reverse transcriptase (Life Technologies).
Competitive RT-PCR
Competitive RT-PCR was performed by adding a fixed amount of
competitor (1 µl) in PCR amplification of the target cDNA (1 µl) in
a 20-µl reaction mixture containing 1x PCR buffer, 200 nM dNTP, 2 mM
MgCl2, 200 nM primers, 50 µCi/ml
[-32P]dCTP, and 0.5U Taq polymerase
(AmpliTaq Gold, Perkin-Elmer/Cetus, Norwalk, CT) for 30 to
40 cycles, 1 min at 94°C, 1 min at 55°C, and 2 min at 72°C.
Competitive DNA fragments containing IL-12Rß1 and -ß2 and ß-actin
primer sequences were constructed using a PCR MIMIC construction kit
(Clontech, Palo Alto, CA) according to the manufacturer’s instruction.
These competitive fragments were designed to contain the same primer
templates as the target cDNA. Target cDNAs and the competitive
fragments were confirmed to be amplified with equal efficiency to each
other, as the ratios of target to competitor PCR products were proven
constant for 20 to 40 cycles in our preliminary experiments (data not
shown). The optimal competitor concentrations for semiquantitative
analysis were determined by amplifying target cDNA in the presence of
twofold serial dilutions of competitor. The following primers were used
for DNA amplification: IL-12Rß1 chain sense, 5'-CCA GCA CAG GAA CCA
CAC A-3'; -ß1 antisense, 5'-CAG AGA CGC GAA AAT GAT G-3'; IL-12Rß2
chain sense, 5'-AAT TCA GTA CCG ACG CTC TCA-3'; -ß2 antisense, 5'ATC
AGG GGC TCA GGC TCT TCA-3'; ß-actin sense, 5'-ATG GAT GAC GAT ATC GCT
G-3'; ß-actin antisense, 5'-CAT GAG GTA GTC TGT CAG GT-3'. The
primers for IL-12Rß1 and -ß2 chains were designed according to the
cDNA sequences of these chains, including the transmembrane portion, in
GenBank (accession No. U23922 for IL-12Rß1 chain, U64199 for
IL-12Rß2 chain). To estimate the amount of specific mRNA, 7 µl of
the RT-PCR mixture (30% of the portion) was electrophoresed through a
10 to 20% polyacrylamide gradient gel (Daiichi Kagaku Yakuhin, Tokyo,
Japan) and dried, and the radioactivity of the specific bands was
measured. The results of semiquantitation of target molecules are
presented as the ratio of target to competitor PCR products normalized
with that of ß-actin. Quantitation of specific mRNA for target
molecules was performed as described previously (29). Briefly, 1 µl
of target cDNA was amplified in the presence of serial dilutions of the
respective competitor, and the amount of competitors required to obtain
the same radioactivity of electrophoretic band of the target cDNA to
that of competitor was determined from a calibration line. The results
are presented as the number of mRNA molecules per microgram of total
cellular RNA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An OVA-specific Th1 clone, 35-9D, was assayed for IL-12R
expression using a rIL-12 binding assay system before and after
stimulation with OVA on SAC. The binding of a marginal amount of IL-12
was detected on the clone that has not received OVA stimulation;
however, the fluorescence profile of the clone was apparently shifted
to the right by the stimulation for 2 days with OVA on SAC but not with
SAC alone (Fig. 1
A).
When the clone was stimulated with various numbers of SAC in the
presence of OVA, the binding of rIL-12 to the clone was increased
depending upon the dose of SAC (Fig. 1B). These
results, confirmed reproducible in four repeated experiments, indicate
that stimulation with Ag on APC induces IL-12R expression on the Th1
clone.
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A, rIL-12 binding was
suppressed by the inclusion of anti-B7-2 to a concentration of 5
µg/ml. The binding was further suppressed when both anti-B7-1 and
anti-B7-2 were added in culture, although control IgG scarcely
suppressed the binding. We repeated this experiment twice under the
same conditions, and the results were essentially the same.
Concentrations of anti-B7-1 and anti-B7-2 used in these
experiments were determined to be optimal in preliminary experiments.
The inhibitory effect of anti-B7-1 was also examined in separate
experiments, and the results were variable from experiment to
experiment, although the addition weakly suppressed the binding
consistently. These results indicate that the B7-2 molecule on SAC
plays a major role in the induction of IL-12R expression of Th1 clones
stimulated with TCR ligation, although B7-1 may also contribute to the
expression.
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B.
To confirm the function of B7-2 in the enhancement of rIL-12 binding to
35-9D clone, the clone was stimulated with various concentrations of
anti-CD3 for 2 days in the presence of 1 x 105
cells/culture B7-2-CHO or CHO and assayed for rIL-12 binding to the
clone. The clone bound a small amount of rIL-12 without stimulation.
The binding was enhanced by anti-CD3 stimulation in the absence of
B7-2-CHO, peaked at 30 ng/well of anti-CD3, and declined with
additional amounts of anti-CD3. When 3 x 105
cells/culture B7-2-CHO were included in the cultures, the clone bound
more rIL-12 than the clone stimulated with anti-CD3 in the presence
of 3 x 105 cells/culture CHO (Fig. 3A). The incubation of
the clone with B7-2-CHO alone did not affect rIL-12 binding. In
separate experiments, the addition of CHO was confirmed not to affect
the rIL-12 binding to the clone stimulated with any concentration of
anti-CD3 tested (data not shown). When the clone was stimulated
with anti-CD3 in the presence of various numbers of B7-2-CHO,
binding of rIL-12 was increased in a B7-2-CHO dose-dependent manner
(data not shown).
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C, small amounts of IL-12Rß1 and -ß2
mRNAs were expressed in the clone without stimulation, and anti-CD3
stimulation in the absence of B7-2-CHO induced a weak accumulation of
mRNAs for both chains at 100 ng/well and for ß2 chain at 30 ng/well
also. The inclusion of CHO in cultures was confirmed not to affect the
accumulation of mRNAs for IL-12Rß1 and -ß2. The
accumulation of mRNAs for both chains was increased in the clone
stimulated with anti-CD3 plus B7-2-CHO, although incubation with
B7-2-CHO alone did not increase the mRNAs accumulation. These results
indicate that costimulation with anti-CD3 and B7-2-CHO efficiently
increases the accumulation of mRNAs for both IL-12Rß1 and -ß2
chains in the Th1 clone, although anti-CD3 stimulation alone
slightly increases the accumulation of these mRNAs depending upon the
dose of anti-CD3. Taken together, these results indicate that Th1 clone 35-9D requires costimulation with TCR ligation and B7-2 for the adequate accumulation of both IL-12Rß1 and -ß2 chain mRNAs and for the strong expression of IL-12R.
IL-12R expression of naive and memory CD4+ T cells stimulated with anti-CD3 in the presence or absence of SAC
IL-12R could not be detected on freshly isolated naive
CD4+ T cells. However, at least some small populations
of memory CD4+ T cells were found to express the receptor
in repeated experiments, although the amount of rIL-12 bound to the
receptor was very small. When naive T cells were stimulated with 100
ng/well anti-CD3 without APC for 3.5 days, they did not bind rIL-12
at all. However, they apparently bound a large amount of rIL-12 when
they were stimulated with anti-CD3 in the presence of 1 x
106 cells/culture SAC. On the other hand, the amount of
rIL-12 bound to the surface of memory T cells, at least of some
populations, was enhanced by stimulation for 3.5 days with 100 ng/well
anti-CD3 alone, and most of the memory T cells were enhanced in
rIL-12 binding by the addition of 3 x 105
cells/culture SAC (Fig. 4). The
enhancement of rIL-12 binding on both naive and memory T cells was
shown to be dependent upon the dose of SAC (data not shown).
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Effective costimulatory molecule on SAC for IL-12R expression of naive and memory CD4+ T cells stimulated with anti-CD3 plus SAC
To examine the effective costimulatory molecule on SAC for the
expression of IL-12R on splenic CD4+ T cells, naive
and memory T cells from spleen were stimulated with 100 ng/well
anti-CD3 for 3.5 days in the presence of SAC, and the effects of
the inclusion of anti-B7-1 and/or anti-B7-2 on rIL-12 binding
to these T cells were examined by flow cytometry. Concentrations of
both anti-B7-1 and anti-B7-2 used in this experiment were
confirmed in preliminary experiments to be optimal for suppression.
rIL-12 binding to both naive and memory T cells was inhibited by the
inclusion of anti-B7-2, but not anti-B7-1 (Fig. 5, A and B).
When both anti-B7-1 and anti-B7-2 were included in culture,
rIL-12 binding was suppressed a little more than it was by
anti-B7-2 alone. Similar results were obtained in three repeated
experiments. The difference in the level of suppression between
cultures containing both anti-B7-1 and anti-B7-2 and those with
anti-B7-2 alone was consistently marginal. These results indicate
that B7-2 on SAC plays a major role as a costimulatory molecule for the
induction of IL-12R expression of both naive and memory
CD4+ T cells.
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, C and D, rIL-12 binding to both naive and memory
T cells was inhibited in a dose-dependent manner by the inclusion of
CTLA-4-Ig, reaching a maximum level of inhibition at 3 µg/ml, both in
naive and memory T cells. The degree of inhibition by CTLA-4-Ig at the
maximum level was similar to that observed with anti-B7-1 plus
anti-B7-2 in both naive and memory T cells. Taken together, these results strongly indicate that costimulation with B7-2 plays an important role in the expression of IL-12R on both naive and memory CD4+ T cells.
Effective costimulation with B7-2-CHO for IL-12R expression of naive and memory CD4+ T cells stimulated with anti-CD3
To confirm that B7-2 costimulation plays an important role in
IL-12R expression of naive and memory T cells, both naive and memory T
cells were stimulated for 4 and 3 days, respectively, with 100 or 1000
ng/well anti-CD3 in the presence of B7-2-CHO or CHO, and assayed
for the binding of rIL-12. As shown in Figure 6, naive T cells bound a small amount of
rIL-12 when they were stimulated with 1000 ng/well anti-CD3, but
not with 100 ng/well anti-CD3. When B7-2-CHO were included in
culture, rIL-12 was found to bind to naive T cells stimulated with 100
ng/well anti-CD3, and the amount of rIL-12 bound to the cells was
increased with the number of B7-2-CHO. The amount of rIL-12 bound to
the naive T cells stimulated with 1000 ng/well was also increased
depending upon the number of B7-2-CHO. The addition of CHO did not
affect the rIL-12 binding of the T cells stimulated with anti-CD3,
and naive T cells incubated with B7-2-CHO alone did not bind rIL-12
either (data not shown). On the other hand, freshly prepared memory T
cells bound a small amount of rIL-12 without stimulation, as shown in
Figure 4 (mean fluorescence ratio, 1.27–1.35), and the amount of
rIL-12 bound to memory T cells was enhanced by anti-CD3 stimulation
even in the absence of B7-2-CHO. When memory T cells were stimulated
with anti-CD3 in the presence of B7-2-CHO, the amount of rIL-12
bound to the cells was increased depending upon the number of B7-2-CHO
(Fig. 6). Incubation with B7-2-CHO alone did not enhance the binding of
rIL-12 to memory T cells (data not shwon). These results indicate that
B7-2 costimulation plays a critical role in the efficient expression of
IL-12R on both naive and memory T cells.
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. When naive T cells
were stimulated with 100 ng/well or a lesser concentration of
anti-CD3, they did not bind rIL-12; however, they bound a small
amount of rIL-12 upon stimulation with 300 or 1000 ng/well of
anti-CD3. The rIL-12 binding was remarkably increased by the
inclusion of B7-2-CHO in the cultures (Fig. 7A).
Consistent with rIL-12 binding, IL-12Rß1 mRNA expression was observed
in naive T cells stimulated with 300 or 1000 ng/well of anti-CD3 in
the absence of B7-2-CHO; however, IL-12Rß2 mRNA was not expressed in
the cells stimulated with anti-CD3 in the absence of B7-2CHO. When
B7-2-CHO was included in the culture, IL-12Rß1 mRNA accumulation was
induced or markedly enhanced, and IL-12Rß2 mRNA expression was also
induced in naive T cells stimulated with 100 ng/well or more
concentrations of anti-CD3 (Fig. 7E). The amount
of rIL-12 binding to memory T cells was increased with the dose of
anti-CD3 in the absence of B7-2-CHO, and the binding was augmented
by the addition of B7-2-CHO at any concentration of anti-CD3 tested
(Fig. 7B). Small amounts of IL-12Rß1 and -ß2
chain mRNAs were expressed in freshly isolated memory T cells, and the
expression of IL-12Rß1 and-ß2 mRNAs was increased by stimulation
with 300 ng/well or more and 100 ng/well or more of anti-CD3,
respectively. The accumulation of both IL-12Rß1 and -ß2 chain mRNAs
was enhanced by the inclusion of B7-2-CHO in cultures of memory T cells
stimulated with any concentration of anti-CD3 (Fig. 7F).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression
(11), proliferation, and IFN- production (9). B7-2 stimulation was
also shown to cooperate with IL-12 in IL-2R expression of naive and
memory CD4+ T cells (10). These effects of B7-1/B7-2
stimulation are mainly caused by an IL-2-independent mechanism,
although IL-2 may participate in the enhancement somewhat, because CD28
stimulation augments the IL-2 production of T cells (29). B7-1 and B7-2
on APC are well known to exert similar costimulatory functions in T
cell activation (30, 31). However, the expression of B7-1 and B7-2 on
APC has been shown to be regulated independently. The expression of
B7-2 was up-regulated more efficiently than that of B7-1, both in time
kinetics and density, by various stimulations (31, 32), indicating that
B7-2 plays a more important role than B7-1 in T cell activation in an
early phase of the interplay between Th cells and APC. As described in
Materials and Methods, SAC used in our experiments expressed
a greater density of B7-2 than B7-1, and their expression of B7-1
was only marginal. In addition, the inhibitory effects of
anti-B7-1 on IL-12R expression were variable in our preliminary
experiments. Therefore, we directed our attention to the B7-2 molecule
on APC. In our present experiments, both Th1 clone and memory
CD4+ T cells were shown to faintly express IL-12R without
stimulation. Although the expression was enhanced by stimulation with
various concentrations of anti-CD3 alone, costimulation with
anti-CD3 and B7-2-CHO was required for the maximum expression of
IL-12R. In contrast to these T cell populations, naive T cells bound
only a marginal amount of IL-12 upon stimulation with high doses of
anti-CD3, and they expressed a comparable density of IL-12R to
memory T cells only when B7-2-CHO were included in cultures. IL-12R has been shown to consist of at least two chains, ß1 (13) and ß2 (15). COS-7 cells cotransfected with genes for these molecules express both low and high affinity binding sites for IL-12 (15). In naive T cells, neither IL-12Rß1 nor -ß2 chain mRNA expression could be detected without stimulation. The accumulation of IL-12Rß1 but not -ß2 mRNA was observed when they were stimulated with high doses of anti-CD3, suggesting that IL-12R expressed on naive T cells by stimulation with high doses of anti-CD3 is of low affinity, because high affinity IL-12R has to be composed of both ß1 and ß2 chains (15). Consistent with this notion, the proliferation of CD4+CD44low naive T cells stimulated with anti-CD3 was not enhanced by IL-12 in our previous experiments (10). The accumulation of mRNAs for both the ß1 and ß2 chains and the binding of considerable amount of IL-12 to cell surface were observed when naive T cells were stimulated with anti-CD3 plus B7-2-CHO. These results indicate that B7-2, and possibly B7-1 also, stimulation plays a crucial role in the expression of a high affinity and functional IL-12R in naive T cells.
Freshly prepared memory T cells bore a low density of IL-12R in
flow cytometric analysis. Consistent with this finding, the expression
of mRNAs for IL-12Rß1 and -ß2 chains could be detected in memory T
cells without stimulation. When memory T cells were stimulated with
anti-CD3 alone, the binding of IL-12 was increased with the
accumulation of mRNAs for IL-12Rß1 and -ß2 chain. Costimulated with
anti-CD3 and B7-2-CHO, memory T cells bound a greater amount of
IL-12 than those stimulated with anti-CD3 alone, and the
accumulation of mRNAs for IL-12Rß1 and-ß2 chains was also enhanced
further. Similar results were obtained in a Th1 clone 35-9D.
CD45RBlow T cells used as memory T cells have been shown to
contain recently activated T cells (33). This finding might explain the
two peak formations in the fluorescence profile of IL-12 binding to
CD45RBlow T cells stimulated with anti-CD3 alone (Fig. 4). The cells that bound greater amounts of IL-12 when stimulated with
anti-CD3 could be those that were recently activated. Memory T
cells and recently activated T cells might differ in their requirement
for B7-2 stimulation for IL-12Rß1 and, especially, -ß2 mRNA
accumulation. When the accumulation of mRNAs for IL-12Rß1
and -ß2 chains was compared in Th1 clone and in naive and memory T
cells (Figs. 3 and 7). The difference in ß2 mRNA accumulation seemed
to be more apparent than that in ß1 mRNA accumulation. Therefore,
IL-12Rß1 and -ß2 accumulation in these cells, stimulated with the
optimum conditions in terms of anti-CD3 and B7-2-CHO, was estimated
by quantitative RT-PCR. As shown in Table I, no difference was observed in the
accumulation level of ß1 mRNA; however, the ß2 mRNA accumulation
was variable depending upon the activation stages of these cell
populations. Naive T cells accumulated minimally, and Th1 clone
maximally. These results may suggest that the costimulation required
for their IL-12-dependent activation is regulated by the efficiency of
ß2 mRNA accumulation.
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The enhancement of IL-12R expression of T cells by B7-2 costimulation
may explain the previous finding that B7-1 costimulation synergized
with IL-12 in proliferation (9) and IL-2R expression of murine Th1
clones (11) and also of splenic CD4+ T cells (10). In human
PHA blasts, anti-CD28 stimulation was also shown to synergize with
IL-12 in inducing their proliferation (34). It has been claimed that
B7–CD28 interaction delivers a positive signal, whereas B7–CTLA-4
interaction delivers a negative signal for T cell activation. However,
CTLA-4 has recently been shown to be critically involved in
B7-1-mediated costimulation for both naive and memory murine
CD4+ T cells (35). The results suggest the possibility that
CTLA-4 may mediate a positive signal for IL-12R expression. Roles of
CD28 and CTLA-4 in IL-12R expression remains to be studied. The
IL-12-induced proliferation of human T cells stimulated with lectin was
largely IL-2 independent, and Ab against IL-2 or IL-2R marginally
inhibited their proliferation (36), although the maximum proliferation
of preactivated T cells induced by IL-12 was much less than that
induced by IL-2 (36, 37). In some murine T cell clones, IL-12 was shown
to induce IL-2-independent proliferation (38), although the expression
of functional IL-2R, and therefore IL-2-dependent proliferation, was
enhanced by IL-12 in all of our own Th1 clones (2). These results may
suggest that IL-12R uses different chain combinations for different
functions or different affinities.
Expression of B7-2 on various APC has been shown to be regulated by
different stimulations. B cells express B7-2 through the engagement of
surface Ig with Ag (31, 32), stimulation with cytokines such as IL-2,
IL-4, or IFN- (39, 40), or CD40 cross-linking (41, 42). IFN- also
increases the expression of B7-2 on macrophages and peripheral blood
monocytes (31, 43). The production of these cytokines and CD40L
expression are well known to occur in T cells stimulated by interaction
with APC. Our present finding that B7-2 costimulation up-regulates
IL-12R expression of CD4+ T cells indicates that the
interplay between Th cells and APC plays an important role in the
ability of IL-12 to support T cell activation. Thus, T cells activated
with TCR ligation express CD40L, and a CD40-CD40L interaction leads to
IL-12 production (4) by regulating IL-12p40 transcription and
up-regulating B7-2 expression on APCs, and then, B7-2 stimulates IL-12R
expression of T cells. Cytokines produced by T cells stimulated with
the TCR ligation also enhance B7-2 expression of APC to stimulate
IL-12R expression on T cells. IL-12 is well documented as directing the
differentiation of Th1 cells. Therefore, our present results suggest
that costimulation of CD4+ T cells with B7 molecules on APC
plays an important role in inducing the differentiation of Th1 cells
through the induction and enhancement of IL-12R.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hideo Nariuchi, Department of Allergology, Institute of Medical Science, University of Tokyo, 4-6-1 Shiroganedai, Minatoku, Tokyo 108, Japan.
3 Abbreviations used in this paper: CD40L, CD40 ligand; hIgG1, human immunoglobulin G1; CTLA-4-Ig, hybrid molecule of CTLA-4 with hIgG1 Fc portion; CHO, Chinese hamster ovary cells; SAC, splenic adherent cells; PE, phycoerythrin.
Received for publication January 23, 1997. Accepted for publication October 27, 1997.
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References |
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