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Departments of
* Microbiology and Immunology and
Oral and Maxillofacial Surgery, School of Medicine, Tokyo Women’s Medical University, and
Medical Devices and Diagnostics Research Laboratories, Toray Industries, Ohtsu, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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3+CD4+ T cells exhibited a
high-level protracted expansion for 30 days, and SEA-reactive
V11+CD4+ T cells
exhibited a low-level protracted expansion. SEA-reactive
CD8+ counterparts exhibited only a transient
expansion. A similar difference in T cell expansion was also observed
in YPM-reactive T cell fractions in mice implanted with the YPM pump.
V3+CD4+ and
V11+CD4+ T cells
from mice implanted with the SEA pump exhibited cell divisions upon in
vitro restimulation with SEA and expressed surface phenotypes as memory
T cells. CD4+ T cells from mice implanted with
the SEA pump exhibited high IL-4 production upon in vitro restimulation
with SEA, which was due to the enhanced capacity of the SEA-reactive
CD4+ T cells to produce IL-4. The findings in
the present study indicate that, in mice implanted with a specific
SAGT, the level of expansion of the SAGT-reactive
CD4+ T cell fractions varies widely depending
on the TCR V elements expressed and that the reactive
CD4+ T cells acquire a capacity to raise a memory response.
CD8+ T cells are low responders to
SAGTs. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-selective manner
(1, 2, 3, 4, 5, 6), have been known as the primary pathogenic factors
of infectious diseases such as toxic shock syndrome (TSS)
(7, 8, 9, 10), neonatal TSS-like exanthematous disease (NTED)
(11, 12), and systemic Yersinia
pseudotuberculosis infection (13, 14, 15). In adult
patients with TSS, a high-level protracted expansion was observed in
TSS toxin-1 (TSST-1)-reactive V2+ T cells for
4–5 wk (Ref. 10 and data not shown). In the late
acute phase patients with Y. pseudotuberculosis infection, T
cell expansion was observed in one of three Y.
pseudotuberculosis-derived mitogen (YPM)-reactive
(V3+, V9+, and
V13+) T cells (15). These
findings suggested that SAGTs induced a protracted expansion in the
SAGT-reactive T cells and that the protracted expansion was
preferentially induced in limited fractions out of the entire
SAGT-reactive T cell population. However, this hypothesis has never
been supported in any experiments with mice.
Experiments using mice injected with SAGTs have shown that SAGTs
uniformly induce a transient expansion and anergy in the entire
SAGT-reactive T cell population shortly after the injection
(16, 17, 18, 19, 20, 21, 22). For example, only a transient expansion was
observed in virtually all staphylococcal enterotoxin A (SEA)-reactive
V3+ and V11+ T cell
fractions, irrespective of the CD4 or CD8 subsets in mice injected with
SEA (21, 22). The SEA-reactive T cells present in the
SEA-injected mice were anergic to restimulation with SEA
(21). The discrepancies in the T cell responses observed
between SAGT-injected mice and patients with SAGT-induced diseases
could be explained as follows. Patients with the SAGT-induced
infectious diseases would have been exposed to the pathogenic SAGTs
continuously for particularly long periods, whereas SAGT-injected mice
would have been exposed to them for only a short period. Implantation
of an osmotic pump filled with TSST-1, which delivers TSST-1
continuously for 
7 days, into rabbits reproduced pathologic changes
similar to those caused by TSS in humans, whereas injection of the same
dose of TSST-1 induced only small changes (23, 24). These
findings seem to support the relevance of the above explanation.
Continuous exposure of mice to SAGTs for specific long periods may
reproduce the T cell response seen in patients with SAGT-induced
infectious diseases.
In the present study, we analyzed the responses of several SEA-
or YPM-reactive T cell fractions in mice implanted with mini-osmotic
pumps filled with SEA (SEA pump) or YPM (YPM pump). The findings
indicate that in mice implanted with a specific SAGT, the SAGT-reactive
T cell fractions exhibited various response patterns ranging from
protracted to transient expansions depending on the TCR V elements
expressed and the CD4/CD8 subsets. The reactive
CD4+ T cells seem to have acquired a capacity to
raise a memory response. We discuss the mechanisms of SAGT-induced T
cell response in humans and mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male and female C57BL/6 mice and BALB/c mice were purchased from Japan SLC (Hamamatsu, Japan). Athymic mice were prepared by excising the thymus of 5-wk-old C57BL/6 mice, as described previously (21), and were used in experiments 3 wk later. These mice were used as osmotic pump recipients or as sources of spleen cells. The animal experiments done in the present study were approved by the ethical review committee of animal experiments of Tokyo Women’s Medical University.
Reagents and culture medium
SEA was purchased from Toxin Technology (Sarasota, FL). YPM was purified as a recombinant product from culture supernatants of Escherichia coli XL 1-Blue (Stratagene, La Jolla, CA) harboring pQE30-6 xhisypm by using Ni-NTA Agarose (Qiagen, Chatsworth, CA) followed by Sepharose Fast Flow (Pharmacia LKB Biotechnology, Tokyo, Japan) coupled with Ni2+, as reported previously (25, 26). CFSE was purchased from Molecular Probes (Leiden, The Netherlands). Monensin (GolgiStop) was purchased from BD PharMingen (San Diego, CA). A23187 and PMA were purchased from Sigma-Aldrich (St. Louis, MO). The RPMI 1640 culture medium used in the tissue cultures contained 100 U/ml penicillin and 100 µg/ml streptomycin, 10% FCS, and 5 x 10-5 M 2-ME.
Monoclonal Abs
mAbs 28-16-8S (anti-I-Ab/d, IgM), LR-1
(anti-B, IgM), HO13 (anti-thy-1.2, IgM), RL.172.4
(anti-CD4, IgM), 83.12.5 (anti-CD8, IgM), KJ25 (anti-V3,
IgG), RR3-15 (anti-V11, IgG), RR4-7 (anti-V6, IgG), KJ16
(anti-V8.1 and V8.2, IgG), F23.1 (anti-V8.1, -V8.2,
and -V8.3; IgG), and F23.2 (anti-V8.2, IgG) were used in the
present study, as described previously (21, 26).
PE-conjugated streptavidin was purchased from BD Biosciences
(Mountain View, CA). Anti-CD44; FITC-conjugated anti-CD4,
anti-CD8, anti-CD3, and anti-CD69 mAbs; FITC-conjugated
IFN-
; PE-conjugated anti-CD4, anti-CD8, and anti-IL-4
mAbs; and CyChrome-conjugated streptavidin were purchased from BD
PharMingen. FITC-conjugated goat anti-rat IgG and anti-mouse
IgG were purchased from Zymed Laboratories (South San Francisco, CA)
and Tago Scientific (Burlingame, CA), respectively.
Preparation of spleen cell fractions
Details of preparing splenic lymphoid cells have been described previously (21, 26). Briefly, CD4+ T cells and CD8+ T cells were obtained by treating C57BL/6 spleen cells with a combination of 83.12.5, 28-16-8S, LR-1, and guinea pig serum and a combination of RL.172.4, 28-16-8S, LR-1, and guinea pig serum, respectively. T cell-depleted spleen cells as accessory cells (ACs) were obtained by treating C57BL/6 spleen cells with a combination of HO13 and guinea pig serum. In the final step, viable cells were recovered by Percoll (Pharmacia Biotech, Uppsala, Sweden) gradient centrifugation (density, 1.055) of mAb-treated cells.
Osmotic pump implantation into mice
The osmotic pumps (ALZA, Palo Alto, CA) were filled with SAGTs and implanted into mice according to the manufacturer’s instructions, as reported previously (21). Briefly, model 2001 mini-osmotic pumps were filled with a 0.2-ml volume of SEA or YPM (SEA or YPM pump). Mice were anesthetized with an injection of 0.2 ml of 10% sodium pentobarbital, and a small s.c. incision pocket was created between the scapulae. The SAGT osmotic pump was inserted into the s.c. pocket, the skin incision was closed with sutures, and penicillin G (20,000 U) was s.c. injected shortly thereafter. In some experiments, the SEA pump was removed from SEA pump-implanted mice 10 days later.
Flow cytometric analysis
Cell preparations were examined for various immunologic
phenotypes by flow cytometry analysis, as described previously
(21, 26). To analyze the expression of TCR V elements
in CD4+ or CD8+ T cells
or the expression of CD44 and CD69 in SEA-reactive T cells, cells were
stained with combinations of biotin-conjugated anti-V3, -V6,
-V7, -V8, and -V11 mAbs and a mixture of PE-conjugated
streptavidin and FITC-conjugated anti-CD4, anti-CD8, or
anti-CD44 and CD69. Samples were analyzed with an Epics XL flow
cytometer (Beckman Coulter, Miami, FL).
DNA sequencing of V3+ TCR -chains
Total mRNA was extracted from several preparations of
CD4+ T cells by oligo(dT)-latex (Nippon Roche,
Tokyo, Japan) and reverse transcribed into cDNA at 42°C for 2 h
with RAV-2 reverse transcriptase and random hexamer primers
(Takara Biochemicals, Osaka, Japan). The cDNA was amplified on a
thermocycler (Program Tempcontrol System PC-700; Astec, Tokyo, Japan)
by using Taq DNA polymerase (Takara Biochemicals) and
oligonucleotide pairs specific for V3 (5'-CTCTGCTGAGTCCTTCAA-3') and
C (5'-GGTAGCCTTTTGTTTGTTTGC-3'), as reported previously (26, 27). The PCR products were ligated into a PCR2.1 vector
(Invitrogen, Carlsbad, CA) and transformed into XL 1-Blue
supercompetent cells (Stratagene). After random selection of
transformants, cloned plasmic DNAs were purified with a QIA-prep spin
mini prep kit (Qiagen) and were analyzed with an ABI PRISM 310 Genetic
Analyzer (PE Applied Biosystems, Foster City, CA).
Measurement of SEA in serum
The serum SEA concentration was measured by ELISA, as described previously (28). Briefly, 100-µl volumes of mouse samples after dilution were added to each well of polyclonal anti-SEA Ab-immobilized ELISA plates. After a 1-h incubation at 25°C, unbound reagents in the wells were removed, and HRP-labeled mouse mAbs to SEA in 100-µl volumes were added to all test wells. The plates were incubated for 30 min at 25°C. After removing unbound reagents, substrate solution containing 0.09% hydrogen peroxide and 0.2 mg/ml 3,3',5,5'-tetramethylbenzidine in 0.1 M citric acid, pH 3.5, was then added to the well in 100-µl volumes and incubated for 30 min at 25°C. The enzyme reaction was terminated by adding 100-µl volumes of 0.5 M sulfuric acid. The plates were read spectrophotometrically at 450 nm with a microplate reader.
Labeling of spleen cells with CFSE and analysis of T cell division
Spleen cells were labeled with CFSE as described by others
(29). Briefly, splenic CD4+ T cells
were suspended in PBS at a concentration of
107/ml, and CFSE was added to the cells to a
final concentration of 10 mM. After a 10-min incubation at 37°C, the
cells were washed with RPMI 1640 culture medium and incubated on ice
for 5 min to terminate the reaction. Cells stained with CFSE were
stimulated with 10 ng/ml SEA in the presence of irradiated ACs.
Three-color cytometric analysis of T cell division was performed by
gating on V3+CD4+ and
V11+CD4+ T cells.
Assay of amounts of cytokines in culture supernatants
For the cytokine production assays, various numbers of
CD4+ T cells, together with irradiated ACs, were
stimulated in 0.5-ml volumes with 10 ng of SEA per ml for various
periods in 48-well plates. Amounts of IFN- and IL-4 in the culture
supernatants were measured by the sandwich ELISA (BD PharMingen)
according to the manufacturer’s instructions. Data were presented as
nanograms of IFN- and IL-4 per milliliter.
Induction of cytokine production and intracellular cytokine analysis
Purified CD4+ T cells (2 x
106/ml) were stimulated with SEA (10 ng/ml) in
the presence of ACs (2 x 106/ml) for 2 days
and further stimulated with A23187 (0.4 µM) and PMA (10 ng/ml) for
4 h in the presence of monensin (GolgiStop). For intracellular
analysis of cytokine production, cells were first stained with
combinations of biotin-conjugated anti-V mAbs and
CyChrome-conjugated streptavidin. They were then fixed, permealized
with Cytofix/Cytoperm solution (BD PharMingen) according to the
manufacturer’s instructions, and stained with PE-conjugated
anti-IL-4 and FITC-conjugated IFN-. Samples were analyzed using
an Epics XL flow cytometer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SEA concentration in serum was measured in individual C57BL/6 mice
injected once with 10 µg of SEA and in mice implanted with the
10-µg SEA pump (Fig. 1
). According to
the instructions of the manufacturer, the mini-osmotic pump delivers
SEA continuously for 7 days. In mice implanted with the SEA pump, SEA
concentration peaked at 24 h after the implantation at a
substantial level and remained at measurable levels for another 5 days.
In mice injected with SEA, SEA concentration peaked at a high level in
as little as 3 h after the injection and thereafter declined
rapidly. Thus,it seems likely that the SEA pump implanted into
mice worked well to deliver SEA continuously for 7 days.
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As the first step of experiments to evaluate the effect of
implantation of the SEA pump on the SEA-induced T cell response,
C57BL/6 mice were injected with 10 µg of SEA once or were implanted
with an osmotic pump that contained several SEA doses. At various days
after the implantation, splenic T cells were prepared from individual
mice and were examined for expansion of SEA-reactive
V3+ and V11+ T
cells in the CD4/CD8 subsets. Data are presented as the percentages of
the SEA-reactive T cell fractions in whole splenic T cells.
All four SEA-reactive T cell fractions in mice injected with 10 µg of
SEA uniformly exhibited a transient expansion at low levels 2 days
after the injection (Fig. 2A),
as reported previously (21). By comparison, in mice
implanted with the 10-µg SEA pump,
V3+CD4+ T cells
expanded to 10 times the control by 6 days after pump implantation.
Their expanded state was maintained at similarly high levels for
another 14 days and reduced slightly thereafter (Fig. 2B).
V11+CD4+ T cells
expanded to 3.5 times the control by 6 days after the implantation, and
their expanded state was reduced gradually to a slightly higher level
than the control by 30 days after the implantation (Fig. 2B). A similar level of expansion was also seen in
V3+CD4+ T cells in mice
that carried the SEA pump for the first 10 days (data not shown).
V3+CD8+ and
V11+CD8+ T cells
exhibited only a transient expansion in low levels at 2 days after the
implantation. As for the effect on the T cell response of specific SEA
doses delivered by the osmotic pump, a high-level protracted expansion
was observed only in
V3+CD4+ T cells at 10
µg or more of SEA (Fig. 2C).
V11+CD4+ T cells did
not exhibit the high-level expansion as seen in
V3+CD4+ T cells even
at a 50-µg SEA dose. Both
V3+CD8+ and
V11+CD8+ T cells
exhibited only a transient expansion at a 50-µg SEA dose.
V6+CD4+ T cells and
V6+CD8+ T cells,
unreactive to SEA, remained at similar levels before and after the
implantation (data not shown). In addition, the expansion patterns of
V3+CD4+ and
V11+CD4+ T cells in
mice implanted with the SEA pump were not changed by thymectomy (Fig. 2B).
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3+CD4+ T cells are
high responders to SEA and that
V11+CD4+ T cells are
moderate responders, whereas two CD8+
counterparts are low responders.
Polyclonal nature of the expanded
V3+CD4+ T cells
The massive and prolonged expansion of
V3+CD4+ T cells in the
mice implanted with the SEA pump can be due to the polyclonal expansion
of heterogeneous
V3+CD4+ T cells or a
selective expansion of oligoclonal
V3+CD4+ T cells. To
determine which of the two mechanisms was responsible for the
expansion, we analyzed the sequences of 20 randomly selected cloned
PCR-amplified cDNAs of V3+ TCR -chain
genes, prepared from splenic CD4+ T cells from
mice implanted with the 10-µg SEA pump 6 or 30 days previously (Table I). The junctional (N-D-N) region was
highly heterogeneous in all of the V3+
-chain clones in the two mouse groups. The J region was also
composed of evenly heterogeneous J gene segments. The results
indicate that the expansion of
V3+CD4+ T cells in
mice implanted with the SEA pump was due to the polyclonal T cell
activation of the heterogeneous
V3+CD4+ T cells.
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We addressed the question of whether the T cell responses seen in
mice implanted with the SEA pump were also seen in mice exposed to
SAGTs other than SEA. BALB/c mice were implanted with a pump containing
300 µg of YPM, which activates V7+ and
V8+ mouse T cells (26, 30), and
were examined for expansion of them in individual mice. We used a large
amount of YPM in the osmotic pump because the stimulatory activity of
YPM on mouse T cells is 100- to 1000-fold lower than that of SEA
(13, 26, 30). Data were presented as the percentage of
respective YPM-reactive T cell fractions in splenic T cells (Fig. 3A) and as a ratio of the
values of the respective experimental groups to that in the control
mice (Fig. 3B).
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7+CD4+ T cells
expanded from 2.7 to 12% (4.5 times the control) by 10 days after the
implantation, and their expanded state was reduced gradually throughout
the examination period. In contrast, other YPM-reactive T cell
fractions such as
V7+CD8+,
V8+CD4+, and
V8+CD8+ T cells
exhibited a transient expansion or no expansion. We think that a lesser
level of expansion of
V7+CD4+ T cells in mice
implanted with the YPM pump compared with
V3+CD4+ T cells in
mice implanted with the SEA pump (Fig. 2
) was due to the much lower
activity of YPM to stimulate mouse T cells. The results indicated that
V7+CD4+ T cells are
high responders to YPM and that other YPM-reactive T cell fractions are
low responders.
Based on the findings obtained so far, it seems possible to state
generally that the level of expansion of CD4+ T
cell fractions reactive with a given SAGT varies in a wide range from a
protracted high-level expansion to a transient low-level expansion,
depending on the TCR V elements expressed.
CD8+ T cells are low responders to SAGTs,
irrespective of TCR V elements expressed. It seems possible to
speculate that V3+CD4+
T cells in a long-term expanded state in mice implanted with the SEA
pump were not in an anergic state, but acquired the ability to raise a
memory-type response upon restimulation with SEA. In the following
experiments, we analyzed surface phenotypes as memory cells and in
vitro SEA-induced proliferation and cytokine production in the
SEA-reactive CD4+ T cells in mice implanted with
the SEA pump.
Expression of CD44 and CD69 on SEA-reactive CD4+ T cells of mice implanted with the SEA osmotic pump
It has been proposed that memory T cells that experienced the antigenic stimulation express CD44 at a high level and CD69 at a low level (31). Information on the immunologic state of the SEA-reactive CD4+ T cells in mice implanted with the SEA pump would be key knowledge for the comprehensive understanding of the SAGT-induced T cell activation.
C57BL/6 mice were implanted with the 10-µg SEA pump and
CD4+ splenic T cells of individual mice were
examined for expression of CD44 and CD69 in
V3+CD4+ and
V11+CD4+ T cells for
26 days after the implantation. Data in one of several mice are
presented in Fig. 4. Expression of CD44
was low 6 days after the implantation in both
V3+CD4+ and
V11+CD4+ T cells and
increased to higher levels as time passed after the implantation (Fig. 4). Conversely, expression of CD69 was high at 1 day after the
implantation and decreased rapidly to the control level in both
V3+CD4+ and
V11+CD4+ T cells. We
obtained similar results in repeated experiments. The results suggest
strongly that memory T cells were generated in both
V3+CD4+ and
V11+CD4+ T cells in
mice implanted with the SEA pump.
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To directly analyze the proliferative capacity of
V3+CD4+ and
V11+CD4+ T cells of
mice implanted with the SEA pump, pooled splenic
CD4+ T cells from several C57BL/6 mice implanted
with the 10-µg SEA pump were stained with CFSE and stimulated in
vitro with SEA for 3 days in the presence of ACs. The samples after
harvest were analyzed for the intensity of CFSE fluorescence in these
two T cell fractions. When a T cell divides, the intensity of the CFSE
fluorescence decreases by about one-half and therefore provides an
accurate count of the cycles of cell division (29). Data
are presented as histograms of the cell division (Fig. 5A) and as percentages of
proliferating cells against cycles of cell division in
V3+CD4+ or
V11+CD4+ T cells (Fig. 5B).
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3+CD4+ T cells of the
controls and mice implanted with SEA pump, irrespective of the period
of the implantation, exhibited cell division (Fig. 5A).
However, some differences were found in cycles of cell division in
these mouse groups (Fig. 5B). The percentages of
V3+CD4+ T cells that
exhibited more than four cell divisions were much higher in the
controls than in mice implanted with the SEA pump at 15 or 25 days
after the implantation. Conversely, the percentages of
V3+CD4+ T cells that
exhibited less than two cell divisions were much lower in the controls
than in these mice (Fig. 5B).
V11+CD4+ T cells also
exhibited a similar response pattern. We obtained similar results in
repeated experiments. The results indicated that SEA-reactive CD4+ T cells of mice implanted with the SEA pump retained the capacity to divide after in vitro restimulation with SEA. However, the capacity of the two SEA-reactive T cell fractions to divide may have decreased as time passed after the implantation of the SEA pump.
Enhanced capacity to produce cytokines of the SEA-reactive CD4+ T cells in mice implanted with the SEA osmotic pump
It has been well established that CD4+ T
cells, when stimulated with Ags, are differentiated into memory T cells
composed of two major types of Th cells: Th1 cells, which
preferentially produce IFN-, and Th2 cells, which preferentially
produce IL-4, IL-5, and IL-10 (32, 33). Various numbers of
pooled splenic CD4+ T cells from C57BL/6 mice
implanted with the 10-µg SEA pump or from mice injected with 10 µg
of SEA were stimulated in vitro with SEA in three ways and examined for
production of IL-4 and IFN-.
First, CD4+ T cells from the experimental and
control mice were stimulated with SEA for 3 days, and culture
supernatants were examined for amounts of cytokines (Table II). Notably, the amount of IL-4 was
markedly increased in mice implanted with the SEA pump as time passed
after the pump implantation. It was quite high, particularly at 25–30
days after the implantation. Amount of IL-4 was only marginal in the
control mice and SEA-injected mice. Amount of IFN- was 2- to 5-fold
lower than the controls in mice implanted with the SEA pump at 6 days
and 15 days after the implantation, and conversely 3- to 5-fold higher
than the controls at 25–30 days after the implantation.
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) could be attributable either to the high
numbers of the SEA-reactive T cells contained in the cultures or to the
enhanced capacity of these T cells to produce cytokines. To address
this question, amounts of cytokines produced in cultures containing
various numbers of splenic T cells from the mice used in experiment 2
in Table II are plotted as the function of the total numbers of
V3+CD4+ and
V11+CD4+ T cells
contained (Fig. 6). We included
V11+CD4+ T cells as
well as V3+CD4+ T
cells in the calculation because
V11+CD4+ T cells in
mice implanted with the SEA pump expressed CD44 and exhibited the
capacity to proliferate after stimulation with SEA at similar levels as
V3+CD4+ T cells,
irrespective of the time of the pump implantation (Fig. 5). The level
of response curve for IL-4 production markedly increased as time passed
after the SEA pump implantation. Levels were 13 times the control at
6 days and 15 days after the implantation and 100 times the control
at 25 days after the implantation. As for IFN- production, the
level of the response curve was decreased to approximately one-fifth of
the control at 6 days and 15 days after the implantation and then
reached similar or slightly higher levels as the control at 25 days
after the implantation.
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). T cells harvested were examined for
cytokine production after being stained with several combinations of
appropriate mAbs to V elements, IL-4, and IFN-. The percentages
of V3+ T cells, which produced IL-4 but not
IFN- in whole T cells, were three times higher in the SEA
pump-implanted mice than in control mice. The percentages of
V3+ T cells that produced IFN- but not IL-4
and of those that produced both IL-4 and IFN-, however, were
approximately two to three times lower in the former mice than in the
latter. A similar response pattern was observed in
V11+ T cells. Changes of responses were not
observed in SEA-unreactive V8+ T cells between
the two groups.
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3+ T cell fraction, acquired the capability
to raise the memory-type response upon restimulation with SEA as time
passed after a protracted in vivo exposure with SEA. Th2-type memory T
cells may have become dominant over Th1-type T cells in these
mice. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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elements expressed. CD8+ T cells are a low
responders to SAGTs, irrespective of TCR V elements expressed. In
addition, CD4+ T cells in these mice acquired a
capacity to raise a memory response. These findings have never been
observed in mice given SAGTs by injection (16, 17, 18, 19, 20, 21, 22) but are
compatible with the observations in clinical studies of the
SAGT-induced infectious diseases (Refs. 10 and
15 and data not shown).
Previous studies have shown that the binding affinity of a complex of
SAGT and MHC class II molecules to TCRs play a critical role for SAGTs
to preferentially trigger T cells expressing particular V elements
(5). As for the capacity of T cells to expand after
stimulation with SAGTs in vivo,
V3+CD4+ and
V11+CD4+ T cells are
high and low in the reactivity to SEA, respectively, and
V7+CD4+ and
V8+CD4+ T cells
are high and low in the reactivity to YPM, respectively (Figs. 2 and 3). The results strongly suggest that the binding affinities of SEA or
YPM are somehow different between V3+ and
V11+ TCRs or between
V7+ and V8+ TCRs,
although these affinities are enough to trigger all of the reactive T
cell fractions. So far we do not know what factors determine the high
or low responsiveness of SEA- or YPM-reactive T cell fractions. This
question may be answered by the measurement of the binding affinities,
including the dissociation velocity, as proposed by others (34, 35), of a complex of SEA or YPM/MHC class II molecules to the
TCRs of the respective SEA- or YPM-reactive T cell fractions.
It is well established that CD4 and CD8 molecules contribute to the
stable interaction of a complex of Ag/MHC class II molecules with the
TCR molecule (36). As SAGTs activate T cells in direct
association with the MHC class II molecule on ACs (3, 5, 8, 9), it seems likely that the triggering force of SAGTs
to T cells is stronger for CD4+ T cells than for
CD8+ T cells. The findings in the present study
showing that CD4+ T cells were high responders
to SEA and YPM, whereas CD8+ T cells were low
responders (Fig. 2), were compatible with the above-mentioned traits of
CD4 and CD8 molecules. However, in patients with TSS,
V2+CD8+ T cells
exhibited a protracted expansion at similar levels as
V2+CD4+ T cells (L.
Chen, M. Koyanagi, K. Fukada, K. Imanishi, J. Yagi, H. Kato, T.
Miyoshi-Akiyama, R. Zhang, K. Miwa, and T. Uchiyama, manuscript in
preparation), indicating that CD8+ T
cells in humans are much more sensitive to the triggering force of
SAGTs than those in mice.
It seems unlikely that memory T cells were generated in SAGT-reactive T
cells in mice injected with SAGTs, because the reactive T cells
exhibited a transient expression of CD44 (37) and an
anergic state upon restimulation with SAGTs (16, 17, 18, 19, 20, 21). The
SEA-reactive CD4+ T cells in mice implanted with
the SEA pump exhibited a protracted expression of CD44 and a transient
expression of CD69 (Fig. 4) and exhibited the enhanced capacity to
produce IL-4 and IFN- upon in vitro restimulation with SEA (Table II
and Fig. 6). Preferential polarization to Th2-type cells was induced in
the SEA-reactive CD4+ T cells as time passed
after SEA pump implantation (Fig. 7). Several years ago, it was
reported that a Th2-type response was predominantly induced in mice
implanted with an osmotic pump filled with a low dose of a protein Ag
(38). So far we do not know whether a similar mechanism is
working in the responses induced by SAGT and a conventional Ag. It is
noteworthy that a capacity to divide the SEA-reactive
CD4+ T cells decreased as time passed after the
implantation of the SEA pump (Fig. 5). CD4+ T
cells from these mice cultured in vitro without stimulation by SEA for
24 or 48 h responded in a similar way, although an intensity of
response was markedly weak (data not shown). SEA-reactive
CD4+ T cells may have been losing the capacity to
divide in a time-dependent way as the intrinsic mechanism but not as
the result of protracted antigenic stimulation for 20 days or more,
although these T cells have been increasing the capacity to produce
cytokines. The study to address this subject in more detail is under
way. Taken together, the profiles observed in these T cells matched the
criteria of the memory T cells.
We think that the results in the present study give clues to
understanding the T cell response in patients with SAGT-induced
diseases. Because human V3 and V13 TCR elements have high
homologies to murine V7 and V8 TCR elements, respectively
(39), the findings in the present study support our
presumption that human V3+ T cells exhibit a
protracted expansion, and the other two fractions exhibited a transient
expansion in late acute phase patients with Y.
pseudotuberculosis infection. In contrast to
V2+ T cells in adult patients with TSS,
V2+ T cells in acute phase neonatal patients
with NTED exhibited only a transient expansion similar to SAGT-reactive
T cells in mice injected with SAGTs (16, 17, 18, 19, 20, 21, 22). We think
that immaturity of T cells in the neonatal periods is responsible for
the transient expansion of V2+ T cells in the
neonatal patients with NTED. Previously, we found that human cord blood
T cells were susceptible to anergy induction with TSST-1, whereas T
cells in adult donors were resistant (40, 41, 42). We
speculate that mouse
V3+CD4+ and
V7+CD4+ T cells would
not exhibit a protracted expansion in neonatal mice continuously
exposed to SEA or YPM over a long period. Lastly, examination to
address whether SAGT-reactive T cells in a long-term expanded state in
adult patients with TSS exhibit an enhanced production of cytokines
(especially Th2-type cytokines) to in vitro stimulation with TSS is
under way. Also, SAGTs have been implicated in the pathogenesis of
autoimmune diseases (43). The present experimental system
may provide a way to examine this possibility.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Takehiko Uchiyama, Department of Microbiology and Immunology, School of Medicine, Tokyo Women’s Medical University, 8-1 Kawada-Cho, Shinjuku-Ku, Tokyo 162-8666 Japan. E-mail address: tuchi{at}research.twmu.ac.jp
3 Abbreviations used in this paper: SAGT, superantigenic toxin; TSS, toxic shock syndrome; NTED, neonatal TSS-like exanthematous disease; TSST-1, TSS toxin-1; YPM, Yersinia pseudotuberculosis-derived mitogen; SEA, staphylococcal enterotoxin A; AC, accessory cell.
Received for publication June 18, 2001. Accepted for publication February 12, 2002.
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) or were injected i.p.
with 10 µg of SEA (•), and were monitored individually for the
concentration of SEA in serum.
), and
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in mice without thymectomy;
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in mice with thymectomy. C,
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