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*
Department of Applied Biological Chemistry, University of Tokyo, and
Department of Microbiology and Immunology, Nippon Medical School, Tokyo, Japan;
Department of Immunology, Tokai University School of Medicine, Isehara, Kanagawa, Japan; and
Division of Immune Regulation, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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expression, which was also
detected at 50 nM Ag, increased with increasing doses. The expression
patterns of mRNA for the Th2-specific transcription factors GATA-3 and
c-Maf were parallel to that of IL-4. These expression profiles were not
altered by the addition of anti-IL-4 plus anti-IL-12 mAbs,
suggesting that cytokine receptor signaling is not essential. Naive
CD4+ T cells stimulated with 5 nM Ag elicited IgM secretion
from cocultured B cells, whereas those stimulated with 50 nM Ag or more
elicited apoptosis of B cells. This may be because at lower doses of Ag
(5 nM), naive CD4+ T cells express CD40 ligand and OX40,
whereas at higher doses (50 nM), they express Fas ligand. Clearly, the
expression of each type of molecule depends on the Ag dose, and
different molecules had different expression patterns. Thus, in the
primary response, naive CD4+ T cells can exhibit different
functions depending on the dose of Ag. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Numerous studies of T cell activation have revealed that T cells exhibit different amounts and types of functional responses in response to subtle differences in antigenic stimulation. One line of evidence supporting this concept is derived from experiments using antigenic peptides with subtle structural alterations, called altered peptide ligands (1, 2, 3, 4, 5), which can induce partial activation of T cells, e.g., cytokine production or cytolysis can be induced without proliferation (6, 7). T cell responses can also be altered by changing the doses of Ag used. Several studies have demonstrated that distinct effector functions of T cells, such as cytolysis, expression of activation markers, cytokine production, and proliferation, are elicited sequentially with increasing doses of conventional TCR agonistic ligands (8, 9, 10). The effects of different structures or amounts of Ag have been mostly examined by using long-term cultured T cells.
In naive CD4+ T cells from TCR-transgenic mice, differences in structure or amount of an antigenic ligand profoundly affect the polarized differentiation into the Th1/Th2 effector cells (11, 12, 13, 14). These results suggest that differences in the primary response of naive CD4+ T cells could affect their subsequent differentiation into the Th1/Th2 phenotypes. However, the primary response of naive CD4+ T cells to altered structure or amount of a specific Ag has not been well documented.
We have previously demonstrated a distinct activation profile, such as Th1- or Th2- cytokine secretion and helper activity, in the primary response of naive CD4+ T cells to altered peptide ligands (15). However, it is still unclear how the differences in the dose of a specific Ag affect this primary response of naive CD4+ T cells. In the present study we have investigated multiple readouts, i.e., the expression of early activation markers, cytokine responses, the ability to modulate B cell responses, and the expression of surface molecules. We show that naive CD4+ T cells can exhibit distinct activation patterns and functions depending on the dose of the specific Ag. Furthermore, our results clearly showed that cytokines and cell surface molecules are expressed with different dose-dependent patterns.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OVA323–339-specific TCR-transgenic mice (OVA23-3) were produced as described previously (16). Recombination-activating gene (RAG)2-2-/- mice were originally donated by Dr. Y. Shinkai (Institute for Virus Research, Kyoto University, Kyoto, Japan) (17). These mice were maintained by back-crossing to BALB/c mice in the animal facility at University of Tokyo (Tokyo, Japan). OVA23-3 mice were crossed with RAG-2-/- mice to generate TCR-transgenic RAG-2-/- mice. BALB/c mice (female, 6 wk old) were purchased from Japan CLEA (Tokyo, Japan).
Peptide and Ab
OVA323–339 (ISQAVHAAHAEINEAGR) was
prepared by solid phase peptide synthesis using an automated peptide
synthesizer (model 430A; PE Applied Biosystems, Foster City, CA) and
was purified by reverse phase HPLC. Anti-IL-4 (11B11), anti-IFN-
(R4-6A2), anti-IL-12 (C17.8), and hamster IgG (UC8-1B9) mAbs were
purified from ascites on a protein G column (Pharmacia Biotech,
Uppsala, Sweden). Biotin-anti-CD3 (145-2C11), biotin-anti-CD40
ligand (anti-CD40L; MR1), FITC-anti-CD4 (GK1.5),
biotin-anti-CD69 (H1.2F3), biotin-anti-CD25 (7D4), anti-Fas
ligand (anti-FasL; MFL3), and anti-OX40 ligand (anti-OX40L;
M134L) mAb, and control rat IgG1 (R3-34) and rat IgG2b (R35-38) were
purchased from BD PharMingen (San Diego, CA). PE-anti-OX40 (OX86)
mAb was purchased from Immunotech (Marseilles, France).
PE-anti-B220 (RA3-6A2) mAb was purchased from Life Technologies
(Gaithersburg, MD).
Preparation of naive CD4+ T cells, resting B cells, and APC
Naive CD4+ T cells (CD62LhighCD4+ T cells) were purified from OVA23-3 mice, or from RAG-2-/- OVA23-3 mice by positive selection using FITC-anti-CD4 mAb, a MACS anti-FITC multisort kit (Miltenyi Biotec, Bergish Gladbach, Germany), and CD62L microbeads (Miltenyi Biotec) as previously described (15). Splenic B cells were purified from BALB/c mice by positive selection using anti-B220 microbeads (Miltenyi Biotec). The B cells were further fractionated on discontinuous Percoll gradients (50/60/70%) to obtain small resting B cells (60/70% interface). T cell-depleted splenocytes as APC were prepared from spleen cells of BALB/c mice by negative selection using anti-Thy1.2 microbeads (Miltenyi Biotec) and were treated with 50 µg/ml mitomycin C (Sigma-Aldrich, St. Louis, MO), except in the B cell apoptosis assay. Isolated naive CD4+ T cells, resting B cells, and APC were routinely >96% CD4+CD62Lhigh, >98% B220+, and <5% Thy1.2+, respectively.
T cell proliferation assay
T cell proliferation assay was performed in 96-well flat-bottom plates. Naive CD4+ T cells (1 x 105/well) in a total volume of 200 µl were cultured with 0–5000 nM OVA323–339 in the presence of APC (3 x 105/well) for 96 h. Proliferation was assessed by measuring the incorporation of [3H]thymidine (37 kBq/well) during final 24 h of culture.
Cytokine secretion assay
Naive CD4+ T cells (3 x
105/well) in a total volume of 1 ml were cultured
with 0–5000 nM OVA323–339 in the presence of
APC (9 x 105/well) in 48-well plates. The
culture supernatants were recovered after 72 h for assaying IL-4
and IFN-. The cytokine concentration was determined by means of a
two-site ELISA as described previously (15). The pairs of
primary capture mAbs and biotinylated secondary detection mAbs used
were as follows: BVD4-1D11 (BD PharMingen) and BVD4-24G2 (BD
PharMingen) for IL-4, and R4-6A2 (BD PharMingen) and XMG1.2 for
IFN-. XMG1.2 was purified from ascites and biotinylated.
In vitro Ab production assay
Naive CD4+ T cells (1 x 105/well) and resting B cells (1 x 105/well) in a total volume of 200 µl were cocultured with 0–5000 nM OVA323–339 in the presence of APC (3 x 105/well). In functional blocking assays, neutralizing mAb (5 µg/ml) specific for cytokines or surface molecules, and the isotype control Ab (5 µg/ml), were added at the beginning of culture. The culture supernatants were collected 7 days later. The levels of total IgM were measured by two-site ELISA as described previously (15). Goat anti-mouse IgM Ab (Cappel-Organon Teknika, Durham, NC) and alkaline phosphatase-conjugated goat anti-mouse IgM Ab (Zymed Laboratories, South San Francisco, CA) were used as the primary and secondary Ab, respectively.
Flow cytometric analysis of expression of surface markers
Naive CD4+ T cells (1 x 106/well) in a total volume of 2 ml were cultured with 0–5000 nM OVA323–339 in the presence of APC (3 x 106/well) in 24-well plates for 12, 24, or 48 h. Cells were harvested and kept on ice until fluorescence staining. T cells were stained with FITC-conjugated anti-CD4 mAb together with PE-conjugated anti-OX40 mAb, biotinylated anti-CD40L, anti-CD69, or anti-CD25 mAb. PE-conjugated streptavidin (Life Technologies) was used for detection of bound biotinylated mAb. Dead cells were excluded by propidium iodide (PI) staining. Flow cytometric analysis was performed on FACSort with CellQuest software (BD Biosciences, Mountain View, CA). Data were gated for viable CD4+ T cells.
Measurement of TCR down-regulation
APC were cultured with 0–5000 nM OVA323–339 overnight and washed to remove excess Ag. Naive CD4+ T cells (1 x 105/well) in a total volume of 200 µl were cultured in 96-well round-bottom plates with these pulsed APC (3 x 105/well). Cells were harvested after 12 h. T cells were stained with FITC-conjugated anti-CD4 and biotinylated anti-CD3 mAb, followed by incubation with Red670-conjugated streptavidin (Life Technologies). The levels of surface CD3 expression were measured with FACSort as described above.
Detection of apoptotic B cells
Naive CD4+ T cells (3 x 105/well) and T cell-depleted splenocytes (9 x 105/well) in a total volume of 1 ml were cocultured with 0–5000 nM OVA323–339 in 48-well plates. In functional blocking assays, anti-FasL mAb (5 µg/ml) or control hamster IgG (5 µg/ml) was added at the beginning of the cultures. Cells were harvested at 96 h and stained with FITC-conjugated annexin V (Roche, Mannheim, Germany), PI, and PE-conjugated anti-B220 mAb. Their staining profile was analyzed using FACSort.
Quantitative RT-PCR
Naive CD4+ T cells (1 x 106/well) in a total volume of 2 ml were cultured with 0–5000 nM OVA323–339 in the presence of APC (3 x 106/well) in 24-well plates. In some experiments for measurement of expression levels of mRNA for cytokines and transcription factors, neutralizing mAb (10 µg/ml) specific for IL-4 or IL-12, or the isotype control Ab (10 µg/ml), were added at the beginning of the culture. Cells were harvested at various time points, and total RNA was prepared using an RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Reverse transcription was performed using RNase H-deficient reverse transcriptase (Life Technologies) and oligo(dT)12–18 primers (Promega, Madison, WI).
The Light Cycler PCR and real-time detection system (Roche) was
used for amplification and on-line quantification. The pairs of primers
used were as follows: GAPDH sense, 5'-TGAACGGGAAGCTCACTGG-3'; GAPDH
antisense, 5'-TCCACCACCCTGTTGGTGTA-3'; FasL sense,
5'-TTGTGATCAACGAAGCTGG-3'; FasL antisense,
5'-CCAGCTTCGTTGATCACAA-3'; CD40L sense,
5'-AGTCACCTTCTGCTCTAATCGG-3'; CD40L antisense,
5'-CCAACTCTGTGGATCACTTGG-3'; OX40 sense,
5'-GTGTACACAGTGCAACCATCG-3'; OX40 antisense,
5'-TTCTGTCCTCACAGACTGCG-3'; GATA-3 sense,
5'-GAAGGCATCCAGACCCGAAAC-3'; GATA-3 antisense,
5'-ACCCATGGCGGTGACCATGC-3'; c-Maf sense,
5'-AGCAGTTGGTGACCATGTCG-3'; c-Maf antisense,
5'-TGGAGATCTCCTGCTTGAGG-3'; IL-4 sense,
5'-CGAAGAACACCACAGAGAGTGAGCT-3'; IL-4 antisense,
5'-GACTCATTCATGGTGCAGCTTATCG-3'; IFN- sense,
5'-AGCGGCTGACTGAACTCAGATTGTAG-3'; and IFN- antisense,
5'-GTCGCTTCGTTGATCACAA-3'.
The hybridization probe format was used to quantify the amplified
fragment. Hybridization probes consisted of two different short
oligonucleotides that hybridize close to each other in an internal
sequence of the amplified fragment during the annealing phase of PCR
cycles. One probe was labeled at the 5' end with the Light Cycler
Red640 (LC Red640) fluorophore, and the other was labeled at the 3' end
with FITC. The pairs of hybridization probes used are as follows:
GAPDH, 5'-CTGAGGACCAGGTTGTTGTCTCCTGCGA-FITC-3' and 5'-LC
Red640-TTCAACAGCAACTCCCACTCTTCCACC-3'; FasL,
5'-TTGTGGTTTAGGGGCTGGTTG-FITC-3' and 5'-LC
Red640-TGCAAGACTGACCCCGGAAG-3'; CD40L,
5'-AGCTGGGAGGAACTGTGGGTAT-FITC-3' and 5'-LC
Red640-TGCCGCCTTGAGTAAGATTCT C-3'; OX40,
5'-TTTCTCCAGGCAACAACCAGG-FITC-3' and 5'-LC Red640-CTGCAAGCCCTGGACCAA
T-3'; GATA-3, 5'-AGCTGCTCTTGGGGAAGTCCT-FITC-3' and 5'-LC
Red640-CAGCGCGTCATGCACCTTT-3'; c-Maf,
5'-TTTTCAGGGTCCGCCTCTTCTGFITC-3' and 5'-LC
Red640-TTCAGTCGGATCACCTCCTCCTTG3'; IL-4,
5'-CTCTAGTGTTCTCATGGAGCTG-FITC-3' and 5'-LC
Red640-AGAGACTCTTTCGGGCTTTTCG-3'; and IFN-,
5'-CTGGCCCGGAGTGTAGACAT-FITC-3' and 5'-LC Red640-TCCTCCCATCA
GCAGCACTC-3'.
The level of GAPDH mRNA was used to normalize the amounts of assayable RNA in each sample. The data are shown as the relative expression index compared with the lowest amount of mRNA detected.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used TCR-transgenic OVA23-3 mice bearing a TCR specific for
OVA323–339 (16) to facilitate the
analysis of primary activation of naive CD4+ T
cells. Naive CD4+ T cells
(CD62LhighCD4+ T cells)
derived from OVA23-3 mice were stimulated with varying doses of
OVA323–339 in the presence of
H-2d splenic APC. As early activation events,
expression of CD69 or CD25 and TCR down-regulation were measured
12 h after stimulation (Fig. 1
).
Up-regulation of CD69 or CD25 expression on CD4+
T cells was significant upon stimulation with
OVA323–339 at 5 nM and above. However, induction
of TCR down-regulation required stimulation at 500 nM and above. Naive
CD4+ T cells derived from
RAG-2-/- OVA23-3 mice showed
fundamentally the same results (Fig. 1, •), demonstrating that a
minor contaminating population of T cells expressing endogenous TCR did
not affect the response of OVA23-3 T cells.
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We subsequently examined proliferation and cytokine secretion of
naive CD4+ T cells during their primary response
to stimulation with varying doses of OVA323–339
in the presence of T cell-depleted splenocytes as APC.
CD4+ T cells incorporated
[3H]thymidine upon stimulation with
OVA323–339 at 5 nM and above (Fig. 2A). However, secretion of
IL-4 and IFN- was only detectable with
OVA323–339 at 50 nM and above (Fig. 2, B and C). A maximum amount of IL-4 and a minimum
amount of IFN- were secreted by stimulation at 50 nM among all
tested doses. When cells were stimulated at doses higher than 50 nM,
decreasing IL-4 secretion and increasing IFN- secretion were
observed in a dose-dependent fashion. We also observed similar
dose-dependent changes in cytokine responses of naive
CD4+ T cells derived from
RAG-2-/- OVA23-3 mice (Fig. 2, •).
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by ELISA at 24 h of culture, we decided to measure the
levels of mRNA of IL-4 and IFN- 24 h after stimulation with
varying doses of OVA323–339 by means of a
quantitative RT-PCR (Fig. 3, A
and B). Expression of IL-4 mRNA was detected upon
stimulation with the Ag at 5 nM and above. Again, a bell-shaped
response was observed for IL-4 mRNA, with the maximum expression at 50
nM. Conversely, IFN- mRNA was induced by antigenic stimulation at 50
nM and above, although some IFN- mRNA was detectable even in the
cells cultured without Ag. The amount of IFN- mRNA was increased in
a dose-dependent manner. In terms of the pattern of dose-dependent
expression, the data obtained by ELISA and those obtained by RT-PCR
were essentially the same, although the expression of IL-4 mRNA was
detected at 5 and 5000 nM Ag only by an RT-PCR assay.
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We reasoned that a dose-dependent change in IL-4 expression should
be accompanied by changes in the expression of the Th2-specific
transcription factors, GATA-3 and c-Maf (18, 19). Fig. 3
C shows that the expression of mRNA for GATA-3 increased
upon stimulation with the Ag at 0.5–50 nM in a dose-dependent manner,
which, in turn, decreased at 500-5000 nM. Expression of c-Maf mRNA was
detected upon stimulation with 5 nM and above (Fig. 3D).
Both transcripts again showed a bell-shaped profile, with maximum
expression at 50 nM. These results indicate that the expression
patterns of these transcription factors correspond to that of
IL-4.
Effect of IL-4R or IL-12R signaling on the expression profile of cytokines and Th2-specific transcription factors
It has been well documented that signals via IL-4R or IL-12R
strongly affect functional differentiation of naive
CD4+ T cells (20, 21, 22, 23). Thus, it
might be possible that the Ag dose-dependent changes in the expression
profile of cytokines and Th2-specific transcription factors shown above
were affected by signaling via IL-4R and/or IL-12R. To test this
possibility, we investigated expression of mRNA for IL-4, IFN-,
GATA-3, and c-Maf in the presence of neutralizing anti-IL-4 mAb
and/or anti-IL-12 mAb. As shown in Fig. 3, E–H, we
observed fundamentally the same dose-dependent changes as previously
even in the presence of both anti-IL-4 mAb and anti-IL-12
mAb.
When either anti-IL-4 mAb or anti-IL-12 mAb was added to the
culture, the amounts of mRNA for IFN- and GATA-3 were somewhat
affected. By adding anti-IL-4 mAb, mRNA for IFN- was increased
(Fig. 3F). By adding anti-IL-12 mAb, mRNA for GATA-3 was
increased, and IFN- mRNA was decreased (Fig. 3, F and
G). Although we have not measured the amounts of IL-4 and
IL-12 secreted in those cultures, these results indicate that some
amounts of these cytokines may have been secreted from tested
CD4+ T cells or T cell-depleted splenocytes.
However, even when the cytokine balance was altered by adding either of
these mAb, this did not change the shape of the Ag dose-related
expression profile of mRNA for each molecule. Interestingly, the
expression of c-Maf was not significantly affected (Fig. 3H).
Modulation of B cell activation by naive CD4+ T cells stimulated with varying doses of the Ag
We next investigated the ability of naive
CD4+ T cells primed by stimulation with different
doses of the Ag to modulate B cell activation. Fig. 4 shows in vitro IgM secretion in a
coculture system of resting polyclonal B cells, naive
CD4+ T cells, T cell-depleted APC, and varying
doses of OVA323–339. A maximum amount of IgM
secretion was found in culture with 5 nM Ag (Fig. 4, 
and •).
However, the amount of IgM secreted was decreased at 50 nM and above in
a dose-dependent manner. We observed a similar pattern of IgM secretion
using naive CD4+ T cells derived from
RAG-2-/- OVA23-3 mice (Fig. 4, •).
Significant IgM secretion was not observed in the absence of
CD4+ T cells (Fig. 4, 
).
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, and •). Compared with the
medium-only control, the percentage of viable B cells was increased in
the presence of 5 nM Ag, but decreased in the presence of Ag at 50 nM
and above. We confirmed that naive CD4+ T cells
derived from RAG-2-/- OVA23-3 mice
exhibited the same results (Fig. 5, •). In the absence of naive
CD4+ T cells, the presence of Ag did not change
the percentage of viable B cells (Fig. 5, ).
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Numerous studies have revealed that the surface molecules on
activated T cells, such as CD40L, OX40, and FasL, can regulate T
cell-dependent activation of B cells (24, 25). We next
analyzed the expression of these molecules on naive
CD4+ T cells upon stimulation with varying doses
of OVA323–339 by flow cytometry and quantitative
RT-PCR. Flow cytometry (Fig. 6A) showed that surface CD40L
and OX40 were expressed upon stimulation at 5 nM Ag and above.
Quantitative RT-PCR (Fig. 6B) gave similar results. In
contrast, FasL expression could not be detected by FACS analysis on
CD4+ T cells at any time points and at any Ag
doses even in the presence of a metalloproteinase inhibitor of KB8301
(26) (data not shown), but mRNA for FasL was induced at 50
nM Ag and above (Fig. 6B).
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Finally, we used functional blocking assays to examine how the
cytokines and cell surface molecules described above could contribute
to the ability of naive CD4+ T cells to modulate
B cell activation. Fig. 7 shows the
effect of the addition of anti-IL-4 mAb, anti-CD40L mAb,
anti-OX40L mAb, or a mixture of these mAbs on in vitro Ab
production by B cells cocultured with naive CD4+
T cells, APC, and OVA323–339 at 5 nM. IgM
secretion was markedly inhibited by the addition of one or a mixture of
mAbs, suggesting that each of these molecules, IL-4, CD40L, and OX40,
was indispensable for helper activity of naive
CD4+ T cells.
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, Ag-induced expression of FasL mRNA was seen upon
stimulation with the peptide at 50 nM and above. We therefore tested
whether the lower frequency of viable B cells seen when the Ag was
higher than 50 nM was due to Fas-mediated apoptosis. As shown in Fig. 8A, the addition of
anti-FasL mAb that can inhibit Fas-mediated apoptosis
(27), increased the frequency of viable B cells.
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B).
The addition of anti-FasL mAb alone did not significantly affect
the levels of Ab secretion. We then tested Ab secretion in the presence
of anti-IFN- mAb, because significant IFN- secretion was
observed for a culture of CD4+ T cells stimulated
with 50 nM Ag and above. The addition of anti-IFN- mAb did not
affect Ab secretion. However, coaddition of anti-FasL and
anti-IFN- mAbs increased Ab secretion significantly when cells
were stimulated with the Ag at 50 nM and above. Collectively, these
data showed that in the presence of the Ag at 5 nM, naive
CD4+ T cells could be activated to help B cells
to secrete Ab through the expression of IL-4, CD40L, and OX40, while in
the presence of the Ag at 50 nM and above, these T cells suppressed the
secretion of IgM from B cells by inducing Fas-mediated apoptosis and
secreting IFN-. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, showing the profile
of early activation, cytokine responses, cocultured B cell responses,
and expression of relevant molecules. Our results clearly show that
different doses of Ag induced expression of different combinations of
cytokines and cell surface molecules. Each of the molecules was
expressed according to a dose-dependent pattern. Thus, naive
CD4+ T cells can exhibit different functions
depending on the dose of the specific Ag.
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and FasL; and
50–500 nM for induction of TCR down-regulation. Above these levels,
the magnitude of these responses increased with the dose. Therefore,
these activation events appear to be elicited according to a certain
hierarchy. However, the expression profile of IL-4, GATA-3, and c-Maf
was totally different from the profile of other molecules. The
bell-shaped expression profile of these molecules cannot be explained
simply by the hierarchical induction of the T cell responses with
increasing Ag dose. This type of expression profile has not been found
for any other molecule produced by T cell clones. Thus, this
bell-shaped pattern may be characteristic of naive T cells. Otherwise,
the bell-shaped expression profiles may be because Ag at >50 nM
stimulates the expression of molecules that can strongly suppress the
expression of IL-4, GATA-3, and c-Maf. For cloned T cells, it has been demonstrated that TCR down-regulation is an essential feature of T cell activation, and that there is a precise correlation between the number of TCR molecules triggered and the type of T cell responses (9, 28, 29, 30). However, using naive CD4+ T cells, we could not find such a correlation, because activation responses occurred at 50 nM Ag and below, but TCR down-regulation required Ag at 500 nM and more. Lezzi et al. (31) have demonstrated that in naive CD4+ T cells, the extent of TCR engagement does not always mirror the T cell responses elicited. Cai et al. (32) have compared the requirements for induction of TCR down-regulation vs T cell activation in naive CD8+ T cells and concluded that TCR down-regulation appears to be neither obligatory nor sufficient for activation of naive T cells. Taking these data and ours together, the extent of TCR down-regulation may not correlate well with functional responses in naive T cells. Furthermore, we observed a slight increase, rather than a decrease, in TCR expression when cells were stimulated with Ag at 0.5–50 nM. Cai et al. (32) have also observed an increase in TCR expression when T cells were stimulated with Ag at a limited range of doses. Both TCR internalization into T cells and the appearance of new TCR on their surfaces may occur. At 0.5–50 nM, the number of internalized TCR molecules should be smaller than that of newly expressed TCR molecules. It has been shown that T cell activation requires down-regulation of only a small proportion of total TCR molecules (9, 29). Thus, even if the total number of surface TCR molecules increased, T cell responses could still be triggered depending on the number of TCR molecules internalized.
Previous reports have clearly demonstrated the effect of Ag doses on functional differentiation of naive CD4+ T cells into Th1 or Th2 cells. Hosken et al. (11) showed that low or high dose Ag induced differentiation of naive CD4+ T cells into Th2-type cells, while intermediate doses induced Th1 development. Constant et al. (12) demonstrated in a different Ag system that low doses favored the development of Th2 cells, whereas high doses favored the development of Th1 cells. Both studies demonstrated that distinct patterns of cytokine secretion were induced after a secondary stimulation of T cells that had already been stimulated with varying doses of the Ag. Although they investigated the effect of cytokine secretion in the primary response on functional Th1/Th2 differentiation by means of functional blocking assays with anti-cytokine mAb, the cytokine secretion profile at the primary response was not shown. In this study we could clearly demonstrate that distinct Th1- or Th2-type cytokines can be elicited early in the primary response of naive CD4+ T cells depending on the dose of Ag, although the amount of cytokines secreted was lower than that from previously stimulated T cells.
Our data indicated that transcription of GATA-3 and a c-Maf gene
corresponds to the expression of IL-4 (Fig. 3), although the molecular
mechanism for the transcriptional regulation is unknown. The changes in
the expression levels of these transcription factors seem to be
responsible for the distinct cytokine responses. Cytokine milieu could
influence the amounts of transcription factor and cytokine mRNA,
because amounts of mRNA for GATA-3 and IFN- were changed by adding
either anti-IL-4 mAb or anti-IL-12 mAb (Fig. 3, E–H). However, the expression profiles of GATA-3 and c-Maf
as well as those of IL-4 and IFN- were not changed in the presence
of either or both of these mAbs (Fig. 3, E–H). Thus, in the
absence of signals via IL-4R and/or IL-12R, the strength of the signals
via TCR/CD3, which should correlate with Ag dose, directly determines
the amounts of these transcription factors. Expression of c-Maf mRNA
was not significantly affected by adding anti-IL-4 mAb or
anti-IL-12 mAb. This is consistent with the previous reports
showing that c-Maf can be induced solely by signals transmitted via the
TCR (18, 33, 34). Recent evidence showed STAT6-independent
activation of GATA-3 (35). Furthermore, it has been
reported that naive CD4+ T cells can transcribe
both IL-4 and IFN- genes within hours after stimulation in a
STAT4/STAT6-independent manner (36, 37, 38, 39). These data are in
line with our current results demonstrating that in the primary
responses of naive CD4+ T cells, IL-4R/IL-12R
signaling is not essential for the observed Ag dose-dependent change in
the expression profiles of cytokines.
In contrast, several recent studies have indicated a critical role of
cytokine receptor signaling in functional Th differentiation, rather
than in IL-4/IFN- expression by naive T cells during primary
responses. Kemeny et al. (40) have shown that IL-12 or
IL-4 is required for later maturation into Th1 or Th2 effectors, but
not for acquisition of the Th1 or Th2 phenotype at an earlier phase of
initial activation. Furthermore, Reiner et al. (41)
clearly demonstrated that a Th1-specific transcription factor, T-bet,
can induce Th1 development without STAT4 activation, and that
IL-12/STAT4 promotes selective survival and proliferation of committed
T cells. From their and our findings, we favor the idea that the extent
of TCR stimulation, correlating with the Ag dose, first dictates the
differentiation into Th1 or Th2 cells by modulating the expression of
transcription factors such as GATA-3, c-Maf, and T-bet, and that
extracellular cytokines then act as selective growth and survival
factors for TCR-stimulated T cells.
We found a distinct threshold for the induction of CD40L, OX40, and
FasL expression in naive CD4+ T cells (Fig. 6).
The expression pattern of CD40L and OX40 was parallel to that of CD69
and CD25, with the same minimal dose (0.5–5 nM) required for
activation and dose-dependent increases (Figs. 1 and 6). Previous
studies have shown that CD40L and OX40 are induced rapidly within
24 h of antigenic stimulation (42, 43, 44, 45) and that their
expression is regulated primarily by signals through TCR (42, 43). Therefore, CD40L and OX40 as well as CD69 and CD25 could be
early activation markers that are induced with the least concentration
of Ag. In contrast, we have little information about the regulation of
FasL expression on naive CD4+ T cells. Most
studies have been conducted with established T cell lines or T cell
hybridomas (46, 47, 48). Recently, Norian et al.
(49) investigated the transcriptional regulation of FasL
in freshly isolated T cells from transgenic mice, in which the murine
FasL promoter controls the expression of a luciferase reporter gene.
They demonstrated that maximal FasL promoter activation occurs only
after prolonged T cell stimulation and requires costimulation through
CD28. They further showed that an antigenic stimulation that generates
robust proliferation of T cells induces only modest FasL promoter
activity. These findings are consistent with our results, in that the
expression of FasL mRNA required stimulation at higher doses (>50 nM)
than those necessary for proliferation and expression of other surface
molecules (Fig. 6). The requirement of high doses of Ag for FasL
expression means that relatively low doses of Ag can stimulate naive
CD4+ T cells, causing clonal expansion and
differentiation, and can avoid activation-induced cell death via the
Fas-FasL system.
The functional blocking assays with anti-IL-4, CD40L, OX40L, FasL,
and IFN- mAbs revealed that different doses of Ag elicit distinct
activation profiles of naive T cells, which, in turn, determine the
fate of cocultured B cells (Figs. 7 and 8). One might argue that our
observation did not reflect physiological T-B interaction in vivo,
because our assay was conducted with Ag-nonspecific polyclonal B cells.
However, our results may suggest one of the mechanisms that regulate
the Ag-independent activation of B cells. Previous reports have
demonstrated that B cells stimulated via CD40 were extremely sensitive
to Fas-mediated apoptosis, whereas dual ligation of CD40 and surface Ag
receptors made them resistant (50, 51). In B cells
activated through LPS or CD40 stimulation, IFN- inhibits Ab
secretion in the absence of surface Ig stimulation, but induces Ab
secretion in the presence of surface Ig stimulation
(52, 53, 54, 55). These previous data are consistent with our
current findings, in that neutralization of IFN- activity as well as
blockade of B cell apoptosis are required to increase IgM production in
cultures with high dose Ag (Fig. 8). Importantly, these previous data
and our current study indicate that the fate of B cells interacting
with T cells is largely dependent on whether their B cell receptor is
engaged. Therefore, in our system FasL- and IFN--mediated inhibition
of B cell activation may be one of the mechanisms by which bystander
activation of B cells with nonrelevant Ag specificity is avoided. In
contrast, our data showed that naive CD4+ T
cells, cultured with low dose Ag activated polyclonal B cells rather
than killing them, suggesting that bystander activation of B cells is
not regulated. Further investigation using B cells of the same Ag
specificity as T cells will clarify the biological relevance of our
observations.
In summary, the present study demonstrates that different doses of Ag induced naive CD4+ T cells to express distinct cytokines and cell surface molecules, and that each of the molecules was expressed according to a dose-dependent pattern. Skewed cytokine expression could be elicited even in the absence of cytokine receptor signaling. Thus, naive CD4+ T cells can exhibit quite different functions in their primary response by recognizing differences in the amount of specific Ag. The present data will be helpful in providing a deeper understanding of the mechanism of Ag-driven activation and differentiation of naive CD4+ T cells.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: RAG, recombination-activating gene; CD40L, CD40 ligand; FasL, Fas ligand; OX40L, OX40 ligand; PI, propidium iodide.
Received for publication July 20, 2001. Accepted for publication January 22, 2002.
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