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Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA 02215;
New England Regional Primate Research Center, Southboro, MA 01772
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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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Bacterial superantigens including staphylococcal enterotoxin B (SEB)3 have been implicated in the pathogenesis of selected human diseases. In humans, SEB is associated with food poisoning and nonmenstrual toxic shock syndrome (14, 15). Patients with nonmenstrual toxic shock syndrome have been found to have SEB-producing staphylococcal infections in injured or mucosal tissues (14, 15, 16). In contrast, mice do not appear to be susceptible to the bacterial superantigen-mediated diseases seen in humans. Mice injected with an extremely large dose of SEB or other bacterial superantigens usually do not develop emesis or a lethal toxic shock syndrome (3, 17). These observations have raised the possibility that superantigen-mediated in vivo effects in humans may be different from those in mice.
In vivo studies of superantigen-driven alterations in TCR Vß repertoires in patients with toxic shock syndromes have yielded conflicting results. In one study an expansion but not deletion of Vß2-expressing lymphocyte subpopulations was detected in PBMC of the patients with staphylococcal toxic shock syndrome (18). In another study, however, decreases in the reactive Vß-expressing cell subpopulations without a prior expansion of these cells was demonstrated in the blood of the patients with severe group A streptococcal infections and streptococcal toxic shock syndrome (19). This discrepancy between these studies may be explained, at least in part, by the difficulties in recruiting the patients exposed to superantigens and optimally obtaining blood samples from those patients. Further studies are needed to characterize the superantigen-driven T lymphocyte responses in humans and their role in disease pathogenesis.
Nonhuman primates have proven powerful animal models for exploring immunologic and pathogenic events in bacterial and viral infections in humans. In fact, we have recently employed a macaque model to study TCR Vß-expressing lymphocyte subpopulation responses in various pathogenic infections (20, 21, 22, 23, 24, 25). In the present study we utilized the rhesus monkey model to study SEB superantigen-induced T lymphocyte responses in the blood, lymph nodes, and spleen. Moreover, we performed the molecular analyses of TCR Vß repertoires to characterize superantigen-mediated changes in clonal representation in PBMC of the monkeys.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rhesus monkeys (Macaca mulatta), three to five years old, were used in these studies. These animals were maintained in accordance with the guidelines of the Committee on Animals for Harvard Medical School and the Guide for the Care and Use of Laboratory Animals (National Academy Press, 1996).
SEB injections
rSEB was purchased from Toxin Technology (Sarasota, FL). SEB was aliquoted in a 0.1 mg/µl PBS solution, stored at -25°C, and thawed immediately before injection. Three different doses (0.1 µg, 0.8 µg, and 2.5 µg per kilogram body weight) were tested in rhesus monkeys. Injection of a monkey with 0.1 µg/kg of SEB induced a subtle expansion of reactive Vß+ cells, whereas administration of 2.5 µg/kg of SEB resulted in a lethal toxic shock syndrome overnight after the challenge. An amount equal to 0.8 µg/kg of SEB stimulated a striking in vivo expansion of reactive Vß+ cells in PBMC of monkeys. Therefore, two unanesthetized monkeys (073 and 099) were injected i.m. and five animals i.v. under the light Ketamine-induced anesthesia, with 0.8 µg SEB per kilogram body weight. After SEB injection, the monkeys were followed for the signs of clinical illness. The unanesthetized monkeys developed an emetic syndrome 15 to 20 min after SEB injection. As controls, two normal rhesus monkeys were inoculated i.v. with the culture supernatant derived from a virus-free CEMX174 cell line. PBL were obtained from the animals on days 0, 3, 8, and 15 following inoculation and then assessed for TCR Vß gene family expression. No perturbation in TCR Vß repertoire was seen in PBL derived from the normal monkeys after the cell supernatant inoculation when compared with the Vß expression by their PBL obtained before inoculation (23).
Isolation and fractionation of lymphocyte populations in blood, lymph nodes, and spleen
PBMC were isolated from heparinized blood of the monkeys using Ficoll/diatrizoate gradient centrifugation. Peripheral lymph nodes were obtained by standard biopsy procedures before and after SEB injection and were carefully teased to generate single cell suspensions. Biopsies of spleen were done through a small wedge biopsy procedure. CD4+ or CD8+ lymphocytes were purified using anti-CD4 or anti-CD8 Ab-conjugated Dynabeads (Dynal, Great Neck, NY), as described previously (21). PBMC or lymph node cells, as well as spleen cells, were incubated with these immunomagnetic beads for 30 min at room temperature and then selected in two cycles with a magnetic particle concentrator.
mAbs and mAb staining
The following anti-human Vß mAbs that cross-reacted with
corresponding macaque Vß+ cells were used:
FITC-conjugated anti-TCR Vß3.1, FITC-conjugated TCR Vß5.2, 5.3,
and FITC-conjugated anti-TCR Vß12 (T Cell Diagnostics, Cambridge,
MA), as well as FITC-conjugated anti-TCR Vß19 (currently
designated anti-TCR Vß 17, Immunotech, Westbrook, MA). Other mAbs
used included phycoerythrin (PE)-conjugated anti-monkey CD3 (FN18,
Biosource, Camarillo, CA), PE-conjugated anti-human CD4 (Ortho
Diagnostic Systems, Raritan, NJ), PE-conjugated anti-human CD8
(Dako Corporation, Carpinteria, CA) and FITC-conjugated anti-human
CD20 (Coulter, Hialeah, FL). Each of the FITC-conjugated anti-Vß
mAbs was paired individually in staining with PE-conjugated
anti-CD4 and PE-conjugated anti-CD8 mAbs, respectively. To
define the TCR-
ß + CD8+ cells the FITC-conjugated
anti-CD3 mAb was paired in staining with the PE-conjugated
anti-CD8+ mAb. Whole blood staining was employed
following the instructions of the immunolysing kit, ImmunoPrep
(Coulter). Single cell suspensions from lymph nodes and spleen were
stained using standard methods.
Flow cytometric analysis and complete blood cell counts (CBC)
Two-color flow cytometric analyses were performed on an XL flow cytometer (Coulter). Lymphocytes were gated by means of forward and side scatters, and up to 20,000 gated cells were analyzed. The frequency of TCR Vß+ cells was determined as the percentage of Vß+ CD4+ cells in total CD4+ lymphocytes and Vß+ CD8+ cells in total CD8+ lymphocytes. CBCs were performed on a hematologic analyzer, the Coulter T 540.
mRNA extraction and cDNA synthesis
mRNA was extracted from these unfractionated or fractionated lymphocytes using guanidinium thiocynate and oligo(dT)-spun columns (mRNA extraction kit; Pharmacia, Piscataway, NJ). The first strand cDNA was synthesized in a 20-µl final volume at 42°C for 1 h using approximately 0.2 to 1 µg of mRNA, 1 µg of random hexanucleotides, and 5 U of reverse transcriptase (Promega, Madison, WI). The samples were heated for 5 min at 95°C to terminate the reaction.
PCR-based analysis of TCR Vß gene expression
A semiquantitative PCR-based method was employed as previously
described to determine the relative expression of the 24 Vß gene
families in monkey lymphocytes (21, 22, 23, 24). The cDNA isolated from each
lymphocyte sample was aliquoted into 25 tubes, each containing a sense
Vß family-specific and an antisense Cß primer. As an internal
control, each reaction tube also contained a pair of primers that
amplified a 105-bp fragment of the constant region of macaque TCR
-chain. A 25-cycle PCR reaction was performed in a 30-µl volume
containing 300 nM of each Vß and Cß primer, 3 nM of the 5' and 3'
C primers, 1 U of Taq polymerase (Perkin-Elmer, Norwalk,
CT), and 32P-end-labeled C and Cß primers (3 x
105 cpm for each reaction). The cycle conditions were 1
min at 95°C, 55°C, and 72°C, respectively. The radiolabeled PCR
products were electrophoresed through a 5% polyacrylamide gel, dried,
and exposed to x-ray film. The separated Vß, Cß, and C bands
were measured for their radioactivity using an Ambis 100 (Ambis, San
Diego, CA). The cpm of individual Vß bands were normalized by
dividing the cpm for each Vß band by the cpm of its associated
internal control C band. The relative intensity of individual Vß
gene families was expressed as the number of cpm present in any one of
the Vß families divided by the total cpm present in the repertoire
surveyed.
TCR-ß CDR3 length display
CDR3 length display was conducted as described previously (22). Briefly, 24 Vß family-bearing cDNAs were amplified by PCR using the individual Vß-specific primers and a Cß-specific primer as described previously (22). The second round of PCR was performed using nested Vß primers and a Cß primer, designed as described (22). The sequences for Vß3 and Vß14 nested primers are described previously (22). Those of Vß15, Vß18, and Vß19 are as follows: Vß15 NS, 5'-ACA TCT ATG TAC CTC TGT G-3'; Vß16 NS, 5'-TTC TGG AGT TTA TTT CTG TG-3'; Vß17 NS, 5'-TCA GCA GCT TAT TTC TGT G-3'; Vß18 NS, 5'-TCA GGC TTC TAT CTC TGT G-3'; Vß19 NS, 5'-ACA GCT TTC TAT CTC TGT G-3'. The Cß primer was labeled at its 5' end with 32P. The first round PCR products were amplified for 15 cycles in the following condition: 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The amplified TCR ß-chains bearing various CDR3 lengths were visualized as series of radiolabeled bands, 3 bases apart, on a 6% polyacrylamide sequencing gel.
Molecular cloning and sequencing of TCR-ß CDR3 length-bearing cDNA
This was done using a PCR-based cloning technique (20, 21, 22, 23, 24). The predominant CDR3 length-bearing cDNA, as observed by CDR3 length display, was cut from the sequencing gel using the autoradiogram for guidance. The gel piece containing the cDNA was melted for 3 min in the TE buffer at 100°C. The cDNA was recovered by conventional ethanol precipitation. The recovered cDNA was then amplified by PCR using individual nested Vß primers (as described above) containing an EcoRI restriction site and the Cß primer containing an XbaI restriction site. As a control, the same CDR3 length-bearing cDNA recovered from the CD4+ or CD8+ PBL sampled at different time points from each of the three monkeys was amplified by PCR to isolate the corresponding TCR-ß CDR3-bearing cDNA. PCR was performed as previously described (22) for 30 cycles. To minimize PCR-generated misincorporation, pfu DNA polymerase was used in the PCR reactions. The PCR products were digested with EcoRI and XbaI and ligated into the plasmid pSP65 (Promega) for cloning and sequencing.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vitro studies were initiated to characterize the SEB-driven
stimulation and expansion of macaque T lymphocyte subpopulations. PBMC
obtained from two rhesus monkeys were stimulated with SEB or a control
mitogen, PHA, and then assessed for the expression of individual Vß
gene families by PCR-based TCR Vß repertoire analysis (21, 22, 23, 24).
PHA-stimulated cells exhibited no significant change in TCR Vß
repertoire when compared with the individual Vß gene family
expression in the unstimulated PBMC. In contrast, SEB stimulated a
striking expansion of both CD4+ and CD8+
lymphocyte subpopulations expressing TCR Vß3, -14, -15, -18, and -19
gene families. Up to 65% of SEB-stimulated PBMC were the T lymphocyte
subpopulations expressing the SEB-reactive Vß gene families (Fig. 1
). In fact, the pattern of SEB-reactive
TCR Vß families employed by macaque T lymphocytes resembled that of
their human counterparts (1) (The human TCR Vß18 and -19 gene
families have recently been redesignated as Vß20 and 17,
respectively.). Interestingly, the macaque TCR Vß12-expressing
lymphocyte subpopulation failed to proliferate and expand after SEB
stimulation, whereas human Vß12-expressing cells expanded in the
SEB-stimulated PBMC. In fact, flow cytometry analysis using
anti-TCR Vß3, -5, -12, and -19 Abs confirmed the results obtained
by PCR-based quantitation (data not shown). These observations indicate
that SEB stimulated an expansion of macaque CD4+ and
CD8+ T lymphocyte subpopulations expressing selected TCR
Vß gene families.
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We then sought to characterize the in vivo responses of T
lymphocyte subpopulations to SEB in the macaques. Upon SEB challenge,
monkeys showed a striking down-regulation of reactive
Vß+ cell subpopulations in PBL. In the blood
compartment, the SEB-reactive Vß3+ and
Vß19+ lymphocytes became undetectable as early as 2
h through 2 days after SEB injection (Tables
I and II;
Fig. 2,
B and C). In the first 24 h, absence
of these reactive cell subpopulations in the circulation coincided with
a decrease in total lymphocyte counts, CD3+,
CD4+, and CD8+ T lymphocytes, and a relative
increase in CD20+ B lymphocytes. The reduction of
circulating T lymphocytes was likely to account for the observed
absence of Vß3+ and Vß19+ cells. On day 2
after SEB challenge, however, the lack of detectable reactive cell
subpopulations in the circulation was not consistent with the changes
in CD4+, CD8+, and CD20+ cell
populations. A down-regulation of Vß3 and Vß19 was still evident
despite up to 70% recovery of the number of total lymphocytes as well
as CD4+, CD8+, and CD20+
lymphocytes (Tables I and II). CD3 expression was also recovered
significantly at this time point. Furthermore, SEB-driven
down-regulation of circulating T lymphocytes appeared to mainly affect
the reactive Vß+ cell subpopulations, since
SEB-nonreactive Vß5+ and Vß12+ cells were
still detected by the flow cytometry in PBMC (Table II). Similarly, TCR
Vß repertoire analysis demonstrated the consistent reduction in the
SEB-reactive TCR Vß gene family-expressing lymphocyte subpopulations,
while lymphocyte subpopulations expressing those TCR Vß families
other than Vß3, -14, -15, -18, and -19 displayed no significant
decrease in PBMC of the SEB-injected monkeys (Fig. 2D). Thus, the SEB-induced hyperacute response was
characterized by a disturbed homeostasis of migration and recirculation
of the reactive Vß-expressing PBL subpopulations.
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B and C). Moreover, the down-regulation of
reactive Vß+ lymphocytes was associated with decreased
expression of CD3, CD4, and CD8, as well as an increased expression of
CD20 in three of the four animals, although their expressions decreased
or increased to a lesser extent in the lymphoid tissues than in the
circulation. Therefore, while reactive Vß+ PBL
subpopulations disappeared in the circulation in the hyperacute
response to SEB, evidence that these cell subpopulations migrated to
the lymphoid tissues could not readily be demonstrated.
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Following the hyperacute down-regulation of the selected
Vß-expressing cell subpopulations, a transient expansion of these
lymphocyte subpopulations in the blood and lymphoid tissues of the
SEB-injected monkeys was observed. In the blood, the expansion of the
selected Vß-expressing cell subpopulations was evident on day 3
through day 7 following SEB injection. Surprisingly, the SEB
superantigen drove a more striking expansion of the reactive
Vß+ CD8+ than CD4+ PBL
subpopulations. As much as a sevenfold increase in the percentage and
ninefold increase in the absolute number of the reactive
Vß3+ and Vß19+ CD8+ cell
subpopulations could be detected in PBMC obtained from these animals on
day 4 or 5 after SEB injection (Fig. 2
, A, B, and
C). In contrast, less than a 3.7-fold, and in most
cases a 2-fold increase in these SEB-reactive Vß+
CD4+ cell subpopulations was seen in PBMC of the monkeys
following SEB injection (Fig. 2, B and C).
Similarly, PCR-based Vß repertoire analysis consistently showed a
more profound expansion of the selected Vß-expressing
CD8+ than CD4+ lymphocyte subpopulations (Fig. 2D). These results suggest that the SEB superantigen
favored proliferation and expansion of circulating CD8+ PBL
subpopulations expressing the reactive Vß gene families in rhesus
monkeys.
SEB drove a major expansion of the reactive Vß family-expressing CD4+ lymphocyte subpopulations in peripheral lymphoid tissues.
Superantigen-driven proliferation and expansion of T
lymphocyte subpopulations may be more profound in the peripheral
lymphoid tissues than in PBMC since the activated cells may migrate to
lymphoid tissues to proliferate. In fact, SEB superantigen stimulated a
more profound proliferation and activation of the selected
Vß-expressing lymphocytes in lymph nodes and spleens than in PBMC of
the monkeys. The monkeys exhibited a marked lymphadenopathy and
splenomegaly following SEB injection. Up to a fivefold increase in the
number of total lymphocytes was noted in a single enlarged lymph node
when compared with the cell number in a lymph node obtained from the
monkeys before SEB injection. Interestingly, while SEB favored the
proliferation of the reactive Vß-expressing CD8+
cell subpopulations in the blood, a preferential expansion of
CD4+ cell subpopulations expressing the reactive Vß
families was seen in the lymph nodes and spleens of the monkeys. In
monkey 213 up to a 6-fold increase in percentage and approximately
19-fold increase in absolute number of the reactive Vß3+
or Vß19+ CD4+ cell subpopulations were
demonstrated in the peripheral lymph nodes, whereas only a 2-fold
increase in percentage and a 3-fold increase in absolute number of
these Vß+ CD4+ cell subpopulations were
observed in the blood (Fig. 3, A and B).
The expanded Vß3+ and Vß19+
CD4+ lymphocyte subpopulations were 3.5 to 5 times greater
than the corresponding Vß+ CD8+ cell
subpopulations in lymph nodes and spleens of the injected monkey.
Before SEB injection, Vß3+ and Vß19+
CD4+ cells were present in a about twofold greater number
than the corresponding CD8+ cell subpopulations (Figs. 2A and 3B). Such major expansion of the
reactive Vß+ CD4+ lymphocyte subpopulations
was also seen in the enlarged lymph nodes obtained from the other three
monkeys after SEB injection (Fig. 3, A, B, and
C). In fact, the expansion of the selected Vß
family-expressing cell subpopulations was most evident in the spleens
of the studied monkeys. Up to 32% of CD4+ spleen cells
expressing Vß3.1 and Vß19 were detected by immune flow cytometry,
and 72% of the cell subpopulations expressing Vß3, -14, -15, -18,
and -19 families were demonstrated by PCR-based repertoire analysis
(Fig. 3, A, B, and C). Finally,
the SEB-driven expansion of the reactive Vß-expressing lymphocyte
subpopulations was more prolonged in lymph nodes than in the blood of
the monkeys. The expanded Vß+ lymphocyte subpopulations
were detected in the lymph nodes as long as 10 days after SEB injection
(Fig. 3, A and B). These results,
therefore, demonstrated that the SEB superantigen stimulated a more
profound activation and expansion of lymphocytes in lymph nodes and
spleens than in the blood of the rhesus monkeys. In these lymphoid
tissues, SEB induced a preferential expansion of the reactive
Vß-expressing CD4+ lymphocyte subpopulations.
SEB-mediated activation events resulted in a prolonged decrease in expression of the reactive Vß-expressing CD4+, but not CD8+, lymphocyte subpopulations.
The third phase of SEB-induced changes in TCR Vß repertoires was
characterized by a rapid loss of the expanded lymphocyte subpopulations
in the blood of the monkeys. Those expanded Vß-expressing cell
subpopulations declined to baseline levels or even further
approximately 8 days after SEB injection. About a 50% decrease in
Vß3+ and Vß19+ CD4+ cell
subpopulations was observed in the blood of the injected monkeys when
compared with the relative and absolute number of these cells before
SEB injection (Fig. 2, B and C). PCR-based
Vß repertoire analysis also showed this decrease in the expression of
reactive Vß family-expressing cells (Fig. 2D). The
decrease in the reactive Vß-expressing CD4+ PBL
subpopulations was still detected in the circulation 60 days after SEB
injection. Interestingly, the decrease in the selected Vß
family-expressing cell subpopulations appeared to be more striking and
prolonged in the lymph nodes than in the blood (Fig. 3, A
and B). The decreased representation of the
SEB-reactive Vß-expressing cell subpopulations could be detected in
lymph nodes of a monkey up to 6 mo after SEB injection (Fig. 3A). In contrast to the depletion of reactive
Vß+ CD4+ cells, however, no decrease in the
reactive Vß-expressing CD8+ lymphocyte subpopulations was
seen in the circulation or lymph nodes of the monkeys.
Some reactive Vß+ cell clones appeared to be more resistant than others to superantigen-mediated depletion following superantigen-induced activation and expansion.
TCR-ß CDR3 length display and ß cDNA sequencing were
employed to characterize SEB-induced T cell responses at a molecular
level. Interestingly, SEB-reactive Vß-expressing lymphocyte
subpopulations underwent profound changes in clonal distribution in
early phases of depletion, whereas the nonreactive Vß-expressing cell
subpopulations displayed no clonal changes after SEB injection. The
multiple symmetric CDR3 lengths employed by SEB-reactive
Vß-expressing lymphocyte subpopulation remained unchanged during the
striking expansion of those Vß-expressing lymphocyte subpopulations,
indicating an SEB-driven polyclonal expansion (Fig. 4, A and B).
However, these same reactive Vß-expressing lymphocyte subpopulations
exhibited an asymmetric distribution or a single length dominance in
their CDR3 lengths on days 8 through 10 after SEB injection. This
corresponded with the time these Vß family-expressing cell
subpopulations rapidly declined to about 50% of their baseline levels
in the blood (Fig. 4A). Such changes in CDR3 lengths
were observed in both CD4+ and CD8+ cell
subsets expressing reactive Vß gene families, although these changes
were more striking in Vß-expressing CD8+ cells. The
changes in CDR3 lengths employed by the reactive Vß-expressing cell
subpopulations were no longer detected in PBMC obtained on day 15 after
SEB injection.
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C).
These reactive Vß+ cell clones could still be detected in
the corresponding CDR3 length-bearing cell subpopulations 2 and 3 weeks
after SEB injection. In contrast, polyclonal sequences were identified
in the corresponding Vß-CDR3 length-bearing cells in the blood
obtained before and during the striking expansion of the
Vß+ cell subpopulations (Fig. 4B).
These results provide molecular evidence demonstrating that, in the
initial phase of SEB-mediated deletion of the Vß-expressing cell
subpopulations, some residual clone(s) among the Vß family-expressing
cell subpopulations displayed a clonal dominance or prolonged expansion
in those cell subpopulations, suggesting that they were more resistant
to the deletion event. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The initial disappearance of SEB-reactive cells in monkeys contrasts with observations found in the mice injected with SEB or staphylococcal enterotoxin A (SEA). In monkeys, SEB-reactive cell subpopulations become undetectable in the blood and decrease in expression of reactive TCR in lymph nodes and spleens of the monkeys from 2 h to 2 days after SEB challenge. In contrast, murine-reactive Vß+ cells rapidly proliferate and expand in lymphoid tissues within one or two days of SEB and SEA injection (13, 14, 26, 27, 28). Several factors may contribute to the prolonged absence of reactive Vß+ cell subpopulations in the blood and lymphoid tissues of monkeys. SEB may alter expression of adhesion molecules on the surface of the reactive Vß-expressing cell subpopulations, resulting in adherence of these cell subpopulations to microvascular endothelia (29, 30). In addition, cytokines produced by SEB-stimulated cells may drive the migration of these reactive Vß-expressing cell subpopulations to the endothelial system (31). This may explain why some nonreactive Vß-expressing cell subpopulations are also reduced in number in the blood during this period of time. Furthermore, the absence of the detectable reactive Vß+ cells may also be due to the SEB-mediated internalization of the reactive TCR on the surface of cells (32). This proved to be a true case on day 2 after SEB injection in that, at this time point, the expression of Vß3 and Vß19 remained low in the recirculating CD4+ and CD8+ cells in the blood. Finally, we cannot exclude the possibility that SEB induced an early death of the reactive Vß+ cell subpopulations (33). By whatever mechanism, the findings in the present study suggest that SEB superantigen interferes with the homeostasis in migration and recirculation of lymphocyte subpopulations in macaques.
The extent and duration of the Vß-expressing cell subpopulation expansion in response to SEB stimulation were more marked in monkeys than in mice. It has been shown in mice that Vß expansion is evident on day 2 through day 4 after SEB challenge, with a maximum of 5- to 10-fold increase in the single Vß8+ cell subpopulation in the lymphoid tissues. The SEB doses used in those studies were 500-fold greater than that employed in this study. We have shown that injection of mice with 0.8 µg per kilogram body weight of the SEB stock used in the present studies, the same dose used in monkeys, failed to induce a detectable expansion of reactive Vß8+ cell subpopulation. A 500-fold increase in SEB dose resulted in only a 6-fold increase in absolute number of Vß8+ cells in lymphoid tissues of mice (data not shown). In contrast, a 12- to 19-fold increase in the absolute number of individual Vß+ CD4+ cell subpopulations was readily demonstrated in lymph nodes of monkeys injected with 0.8 µg/kg of SEB. Total reactive Vß-expressing cell subpopulations account for 78% of the entire T lymphocytes in the lymphoid tissues of monkeys at the time of peak proliferation. Furthermore, expansion of reactive Vß+ cell subpopulations can be detected in lymph nodes up to 10 days after SEB challenge. This great sensitivity of T cell response to SEB superantigen is consistent with the susceptibility of the SEB-exposed monkeys and humans to the clinical diseases. In fact, we found that a monkey developed a lethal shock syndrome after receiving a 2.5 µg/kg dose of SEB (see Materials and Methods). Other groups have also reported that monkeys develop a shock syndrome following SEB administration (34, 35, 36, 37). The affinity of trimolecular interaction between SEB, MHC, and TCR components may determine the differences between the murine and primate species in immune responses and clinical manifestations after SEB exposure.
This study indicates that lymph nodes and spleen are major sites for SEB-induced lymphocyte activation and exhaustion. A preferential expansion and exhaustion of the reactive Vß+ CD4+ cell subpopulations was demonstrated in the lymphoid tissues. In contrast, preferential proliferation of the corresponding Vß+ CD8+ cell subpopulations was found in the blood compartment. The mechanism underlying the dichotomy of Vß+ CD4+ and CD8+ cell responses in different anatomical compartments is not apparent. The reactive Vß+ CD4+ cell subpopulations in the blood may be more predisposed to activation-induced exhaustion following SEB challenge than the corresponding Vß+ CD8+ cells. On the other hand, APC in different compartments may contribute to the differential expansions of reactive Vß+ CD4+ and CD8+ cell subpopulations in response to SEB superantigen. Dendritic cells in lymph nodes and spleens may be more efficient in the presentation of SEB and activation of the reactive CD4+ lymphocyte subpopulations (36, 37). Furthermore, adhesion molecules expressed by the activated CD4+ cells may facilitate a migration of these cells to lymphoid tissues. Finally, the microenvironment in lymph nodes and spleen may favor the proliferation and prolonged expansion of reactive Vß+ CD4+ lymphocyte subpopulations. Further studies are needed to elucidate the regulatory factors dictating the differential responses in the different lymphoid compartments.
The molecular analysis of TCR-ß CDR3 in PBMC of the monkeys
demonstrated that lymphocyte clones in reactive Vß-expressing cell
subpopulations evolved differently in their responses to SEB. The
majority of Vß-expressing cell subpopulations underwent a polyclonal
expansion, followed by a rapid exhaustion in response to SEB
superantigen. However, a minority of the reactive Vß+
cells in the blood of the SEB-injected monkeys sustained clonal
expansions at a time when a rapid depletion of the majority of expanded
Vß-expressing cell subpopulations took place. These monoclonal or
oligoclonal expansions detected during the transitional phase of
depletion are probably not driven by MHC class I- or II-restricted T
cell responses to the SEB peptides, since these expansions were seen
only in the SEB-reactive Vß+ cell subpopulations. Several
possibilities may be considered as explanations for this prolonged
clonal expansion and resistance to the activation-induced depletion.
The TCR -chain components employed by these Vß+ clones
may provide a proliferative advantage in the interaction with MHC class
II and SEB. This possibility is supported by recent studies
demonstrating the contribution of the TCR -chain to the superantigen
recognition (38, 39, 40). In addition, these selected Vß+
clones may acquire the resistance to the superantigen-induced
exhaustion if they have been primed by other conventional antigens
before SEB challenge (41). Finally, distinct profiles of cytokine
production in these Vß+ cell clones may favor their
prolonged proliferation. Additional studies are needed to clarify the
biologic significance of these long-proliferating clones in response to
SEB superantigen.
The present study indicates that SEB superantigen-mediated in vivo effects in macaques involve disordered homeostasis of migration and recirculation of lymphocytes in the blood as well as massive activation and exhaustion of these cells in the lymphoid tissues. These findings may mirror similar T lymphocyte responses in humans, suggesting that these changes contribute to bacterial superantigen-induced diseases in higher primates.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Zheng W. Chen, Harvard Medical School, Beth Israel Deaconess Medical Center, 330 Brookline Avenue RE113, East Campus, Boston, MA 02215. E-mail address:
3 Abbreviations used in this paper: SEB, staphylococcal enterotoxin B; CDR3, complementary determining region 3; PE, phycoerythrin; CBC, complete blood cell.
Received for publication October 9, 1997. Accepted for publication January 12, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression on Mlsa superantigen-specific T cell responses. J. Exp. Med. 175:1405. chain influences reactivity to Mls-1 in Vß8.1 transgenic mice. J. Immunol. 149:887.[Abstract]
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 Y. Shen, L. Shen, P. Sehgal, D. Huang, L. Qiu, G. Du, N. L. Letvin, and Z. W. Chen Clinical Latency and Reactivation of AIDS-Related Mycobacterial Infections J. Virol., December 15, 2004; 78(24): 14023 - 14032. [Abstract] [Full Text] [PDF]
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 W. Stohl, D. Xu, S. Zang, K. S. Kim, L. Li, J. A. Hanson, S. A. Stohlman, C. S. David, and C. O. Jacob In vivo staphylococcal superantigen-driven polyclonal Ig responses in mice: dependence upon CD4+ cells and human MHC class II Int. Immunol., October 1, 2001; 13(10): 1291 - 1300. [Abstract] [Full Text] [PDF]
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Z. W. Chen, Y. Shen, D. Zhou, M. Simon, Z. Kou, D. Lee-Parritz, L. Shen, P. Sehgal, and N. L. Letvin In Vivo T-Lymphocyte Activation and Transient Reduction of Viral Replication in Macaques Infected with Simian Immunodeficiency Virus J. Virol., May 15, 2001; 75(10): 4713 - 4720. [Abstract] [Full Text]
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 Z. W. Chen, Y. Li, X. Zeng, M. J. Kuroda, J. E. Schmitz, Y. Shen, X. Lai, L. Shen, and N. L. Letvin The TCR Repertoire of an Immunodominant CD8+ T Lymphocyte Population J. Immunol., April 1, 2001; 166(7): 4525 - 4533. [Abstract] [Full Text] [PDF]
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 Z. C. Kou, J. S. Puhr, M. Rojas, W. T. McCormack, M. M. Goodenow, and J. W. Sleasman T-Cell Receptor Vbeta Repertoire CDR3 Length Diversity Differs within CD45RA and CD45RO T-Cell Subsets in Healthy and Human Immunodeficiency Virus-Infected Children Clin. Vaccine Immunol., November 1, 2000; 7(6): 953 - 959. [Abstract] [Full Text] [PDF]
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Z. W. Chen, Y. Shen, Z. Kou, C. Ibegbu, D. Zhou, L. Shen, P. Morrison, C. Bogle, H. M. McClure, A. J. Nahmias, et al. Prolonged Dominance of Clonally Restricted CD4+ T Cells in Macaques Infected with Simian Immunodeficiency Viruses J. Virol., August 15, 2000; 74(16): 7442 - 7450. [Abstract] [Full Text]
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