By
From the * Department of Immunology, Department of Pathology, St. Jude Children's Research
Hospital, Memphis, Tennessee 38105; and the § Department of Pediatrics,
Department of Pathology,
University of Tennessee, Memphis, Tennessee 38163
The murine -herpesvirus 68 has many similarities to EBV, and induces a syndrome comparable to infectious mononucleosis (IM). The frequency of activated CD8+ T cells (CD62Llo) in
the peripheral blood increased greater than fourfold by 21 d after infection of C57BL/6J (H-2b)
mice, and remained high for at least a further month. The spectrum of T cell receptor usage
was greatly skewed, with as many as 75% of the CD8+ T cells in the blood expressing a V
4+
phenotype. Interestingly, the V
4 dominance was also seen, to varying extents, in H-2k, H-2d,
H-2u, and H-2q strains of mice. In addition, although CD4 depletion from day 11 had no effect
on the V
4 bias of the T cells, the V
4+CD8+ expansion was absent in H-2IAb-deficient congenic mice. However, the numbers of cycling cells in the CD4 antibody-depleted mice and
mice that are CD4 deficient as a consequence of the deletion of MHC class II, were generally lower. The findings suggest that the IM-like disease is driven both by cytokines provided by
CD4+ T cells and by a viral superantigen presented by MHC class II glycoproteins to
V
4+CD8+ T cells.
The murine Infectious mononucleosis (IM) is a debilitating condition
of adolescents resulting from primary infection with EBV.
The disease is characterized by lymph node enlargement
and the prolonged presence of greatly increased numbers of
activated CD8+ T cells in peripheral blood, after an initial
influenza-like phase reflecting the entry of EBV via the
oropharyngeal/respiratory mucosa. Apart from the viral etiology, the pathogenesis of this selective lymphocytosis is
not understood. Few of the circulating CD8+ T cells can
be shown to be EBV-specific, while the virus persists as a
latent infection in predominantly B, rather than T, lymphocytes (14). Analysis of the pathogenesis of MHV-68-
induced IM described in this report suggests a mechanism
involving both cytokines and a putative viral superantigen.
Mice.
Female C57BL/6J (B6, H-2b), B10.BR (H-2k), BALB/cJ
(H-2d), B10.PL (H-2u), and B10.Q (H-2q) mice were purchased
from Jackson Laboratory (Bar Harbor, ME). The CD2 mice that
are functionally negative for the H-2IAb gene (19) were bred at
St. Jude Children's Research Hospital (Memphis, TN), under license from GenPharm Intl. (Mountain View, CA). Mice were
infected with MHV-68 at 6-10 wk of age, and then maintained
under otherwise specific pathogen-free conditions in BL-3 containment. In some studies, B6 mice were thymectomized at 3 wk
of age.
Virus Stocks.
The original stock of MHV-68 (clone G2.4)
was obtained from Dr. A.A. Nash (Edinburgh, U.K.) as a cellfree lysate derived from infected baby hamster kidney cells. This
was then propagated in owl monkey kidney fibroblasts (ATCC
1566CRL; American Type Culture Collection, Rockville, MD).
Infection and Sampling.
Anesthetized (Avertin, 2,2,2,tribromoethanol) mice were infected intranasally with 400 PFU of MHV-68
at 6-10 wk of age, and sampled at various times after infection.
Blood was obtained from the axilla or retroorbital sinus of anesthetized mice.
Cell Cycle Analysis.
The cell cycle analysis of CD8+ T lymphocytes was performed as previously described (20). In brief,
cells were stained with FITC-conjugated antibodies to CD8 In Vivo T Cell Depletion and Flow Cytometry.
B6 mice were depleted of CD4+ T cells by in vivo treatment with GK1.5 mAb at
2-3-d intervals, as previously described (21, 22), beginning 11 d
after infection. Depletion was assessed by flow cytometry using
RM4-4, which is not blocked by GK1.5 (PharMingen). Activated
CD8+ lymphocytes were assessed by two-color staining with
CD62L (MEL-14-FITC), or CD44 (IM7-FITC) and CD8- Previous data have established that respiratory challenge of C57BL/6J (B6) mice with MHV-68
results in acute viral infection that is eliminated by CD8+
T cells by day 13. Persistent, latent infection is established in B cells, which is accompanied by a significant splenomegaly (9, 12, 13, 25). In the current studies, the profile of PBL
was analyzed at various stages of infection. Interestingly, the data showed a greater than fourfold increase in the frequency of activated/memory (26) CD8+CD44hiCD62Llo
PBL from day 21 after infection (Fig. 1 A), which is reminiscent of the IM that is frequently (~50% of cases) a consequence of EBV infection in humans (30). This IM-like
blood picture was also seen in mice that had been thymectomized as adults (Fig. 1 B), suggesting that the tendency
for human IM to be more common in adolescents is not
due to the provision of new thymic emigrants.
The T cell activation seen in the peripheral blood is likely
to be a consequence of events occurring in lymphoid tissue. Analysis of splenic lymphocytes showed that although
the spleens were enlarged, the relative prevalence of the
CD8+ subset remained fairly constant (Fig. 2 A), indicating
that all categories of lymphocytes are cycling at a high rate
(Fig. 2 B). However, the frequencies of T cells in S to G2 + M phase were generally greater for the CD8+ than the
CD4+ subset of T cells, with the difference being most apparent at the later time points (days 18 and 25 after infection, Fig. 2 B), corresponding to the expansion of CD8+
CD62Llo T cells in the PBL, first observed at day 21 after
infection (Fig. 1 A).
There are several possible mechanisms to explain virusinduced massive proliferation in lymphoid tissue that could
lead to an IM-like profile in the blood. First, it is well
known that viral infection results in clonal expansion of cytotoxic T lymphocyte precursors (CTLp) specific for viral
peptides + class I MHC glycoproteins (31, 32). However,
it has been shown that not all of the activated CD8+ T cells
in the peripheral blood of EBV-induced IM patients are virus specific (30, 33). Thus, additional mechanisms must contribute to the CD8+ T cell expansion. One possibility is
cytokine-induced bystander activation. For example, it has
been shown that cytokines contribute to T cell proliferation during viral infection (20, 34, 35). Another possibility
is superantigen-driven T cell proliferation. Superantigens
are potent stimulatory molecules secreted by microbial pathogens that cause V As a first step in determining the mechanism of T cell activation, the TCR-V
This marked TCR skewing could reflect a dominant
V Alternatively, the V Table 1.
V-herpesvirus 68 (MHV-68)1 is classified as
a type 2
-herpesvirus (
HV; references 1, 2), along
with Herpesvirus saimiri (3), and a novel
HV that has recently been implicated in Kaposi's sarcoma (4). However, the disease process induced in mice infected intranasally with MHV-68 is much more similar to the syndrome
associated with prototypic human type 1
HV, EBV in
people (7), than to that caused by the T lymphotrophic
H. saimiri in nonhuman primates (8). The key characteristic
is that MHV-68 replicates in the epithelial cells of the respiratory tract, with subsequent infection of B cells in lymphoid tissue (9). The productive growth phase in the
lung cells is terminated by the CD8+ T cell response within
10-13 d. Little, if any, infectious virus can be recovered directly from homogenized lymphoid tissue, although reactivation of latent MHV-68 in B cells occurs readily after cocultivation on susceptible fibroblast monolayers (9).
(536.72; PharMingen, San Diego, CA), permeabilized with ethanol,
and the DNA was stained with propidium iodide. The cells were
analyzed in two-color mode on the FACScan® (Becton Dickinson, San Jose, CA) using Modfit/Winlist software (Verity Software Inc., Topsham, ME). The program determines the status of
individual CD8+ lymphocytes as being in G0/G1, S, or G2 + M
by estimating the DNA content of intact nuclei.
(536.72-PE) (PharMingen). Lymphocytes were phenotyped using
mAb to B220, CD4 (GK1.5), and CD8-
(53-6.72) (PharMingen). TCR-V
usage was assessed using a panel of mAbs specific
for V
2-14, as previously described (23, 24).
Respiratory Infection with MHV-68 Induces an IM-like Syndrome in B6 Mice.
Fig. 1.
Phenotypic analysis of CD8+ T cells from peripheral blood
of MHV-68-infected B6 mice. CD8+ PBL (hatched bars) from intact (A)
and adult thymectomized (B) mice were assessed for the activation phenotype, CD62Llo (open bars), and CD44hi (stippled bars) at various time
points after infection, as indicated. Percentages are based on total PBL.
[View Larger Version of this Image (49K GIF file)]
Fig. 2.
Phenotype and activation profile of splenic lymphocytes
from MHV-68-infected B6 mice. The percentage of CD4+ (closed bars),
CD8+ (hatched bars), and B220+ (cross-hatched bars) splenic lymphocytes
(A) and the proportion of cycling cells within those subsets (B) were determined at various time points after infection.
[View Larger Version of this Image (45K GIF file)]
-specific T cell expansion (36).
The existence of an EBV-encoded superantigen has been
suggested by some groups (36), but remains controversial (39).
Profile of Activated T Cells.
profile was examined. The results showed a striking predominance of V
4+ T cells among the CD8+ T cells in the
peripheral blood, and a compensatory decrease in all other
V
s (Fig. 3 A). This V
4 expansion was also seen in splenic
CD8+ T cells (Fig. 3 B), but not in peripheral lymph nodes
(data not shown). The V
4 expansion among the CD8+
peripheral blood T cells was not evident in the first 2 wk of infection, but was consistently seen at day 21 after infection, although the magnitude of the increase varied in individual B6 mice (Fig. 4 A). A variable and much reduced
V
4 expansion was also transiently observed in CD4+ T cells
from peripheral blood (Fig. 4 B) and spleen (data not shown). Elevated levels of V
4+CD8+ T cells were still
observed at 90 d after infection (Fig. 4 A).
Fig. 3.
TCR-V usage. TCR-V
usage in CD8+ T cells isolated
from peripheral blood (A) and spleen (B) of MHV-68-infected (closed
bars) and uninfected (open bars) B6 mice analyzed 21 d after infection was
determined by two-color staining using a panel of V
-specific mAb and
anti-CD8. The data are expressed as the percentage of CD8+ T cells expressing a particular TCR-V
among total CD8+ T cells.
[View Larger Version of this Image (17K GIF file)]
Fig. 4.
Kinetics of V4 expression after MHV-68 infection. Percentage of V
4+ T cells among CD8+ T cells (A) and CD4+ T cells (B)
from peripheral blood of individual B6 mice at various time points after
MHV-68 infection. 3-9 mice were analyzed at each time point, but, in
many cases, the values are so close as to be indistinguishable on the graph.
The percent V
4+ T cells among total CD4+ and CD8+ T cells was
determined by two-color flow cytometry using mAb specific for V
4,
CD4, and CD8. Note that the scale for the y-axis differs in the two panels.
[View Larger Version of this Image (17K GIF file)]
4 usage of H-2Kb- or H-2Db-restricted MHV-68-specific CD8+ CTLp. Repertoire analysis of CTL specific for
a variety of viruses shows that diversity in the recognition
of a particular peptide + MHC class I glycoprotein can
range from very limited to very diverse (24, 42). Preferential usage of a single TCR-
/
pair has been described
for a long-term EBV-specific CTL response (47), and recently, oligoclonal expansion of T cells in IM patients was
reported (39). There is currently no virus-specific CTL assay developed for MHV-68. However, stimulation of T cells
with MHV-68, by a standard limiting dilution protocol,
showed that the prevalence of microcultures containing effectors capable of CD3-
-dependent CTL activity (48, 49)
ranged from 1:500-1:2000 (Tripp, R.A., and P.C. Doherty,
unpublished observations), comparable to the level described for other viruses (20). However, these CTLp frequencies may reflect a gross underestimate if highly activated CD8+ T cells are being driven to apoptosis after in
vitro stimulation, as has been described for EBV (50, 51).
We have observed that the activated V
4+CD8+ T cells
from MHV-68-infected mice are short-lived ex vivo (data not shown).
4 bias might be the consequence of
a superantigen-driven response, as superantigens stimulate
T cells in a V
-specific manner. Superantigens bind to
MHC class II (52), but, in contrast to T cell recognition
of conventional viral antigen, T cell responses to superantigens are generally not MHC restricted (58). Therefore,
as a first step in examining a role for a viral superantigen in
the T cell activation, we examined the MHC haplotype dependence of the elevated V
4 pattern seen in B6 mice.
TCR profiles were determined for MHV-68-infected H-2k,
H-2d, H-2u, and H-2q mice. The data show a clear V
4
expansion among CD8+ PBL (Table 1), but not CD4+
PBL (data not shown) in mouse strains representing each of
the haplotypes. Therefore, although we are currently unable to rule out the possibility of a conventional viral peptide that promiscuously binds to MHC class I (61), the data
suggest that the V
4 expansion seen during the IM phase
of MHV-68 infection is driven by a virally-encoded superantigen.
4 Expression among CD8+ T Cells in
MHV-68-infected Mice
Mouse
strain
MHC
haplotype
% V
4+CD8+
Days after
infection
Naive
MHV-68-infected
C57BL/6J
H-2b
3.9 ± 0.2
46.8 ± 12.6
24
BALB/c
H-2d
9.0 ± 1.2
29.2 ± 8.9
41
B10.BR
H-2k
3.0 ± 0.8
11.1 ± 3.3
24-28
B10.PL
H-2u
4.4 ± 1.6
22.0 ± 8.1
21
B10.Q
H-2q
3.9 ± 0.9
8.4 ± 2.4
28
*
PBL were obtained from control mice or MHV-68-infected mice at
the indicated time points after infection. The percentage of V 4+CD8+
T cells among total CD8+ T cells was determined by two-color flow
cytometry using biotinylated mAb specific for TCR-V
4+(KT4) and
FITC-conjugated mAb specific for CD8 (53-6.72), using standard protocols.
Previous studies established that the splenomegaly characteristic of MHV-68
infection is greatly diminished in mice that lack CD4+ T
cells (9) and MHC class II glycoproteins (13). Therefore, we analyzed CD4-deficient MHC class II /
mice for
evidence of IM. The results showed that both the extent of
MHV-68-induced CD8+ T cell proliferation in the spleen
(Fig. 5 A) and the relative prevalence of CD8+CD62Llo
T cells in the blood (Fig. 5, B and C) were much lower
than in the MHC class II +/+ controls. Furthermore, the
pattern of TCR-V
4+CD8+ dominance associated with
the increase in frequency for the CD8+CD62Llo set in the
PBL (Fig. 5 B) was not observed for MHC class II
/
mice (Fig. 5 C). These differences between the MHC class
II +/+ and
/
mice could reflect the absence of the
H-2IAb MHC class II glycoprotein, perhaps because of a
requirement for MHC class II presentation of a viral superantigen (62), and/or because of a role for cytokines produced by MHV-68-immune CD4+ T cells in the antiviral
CD8+ T cell response (63). To test this, +/+ mice were
treated in vivo with the GK1.5 mAb to CD4 from day 11 after virus challenge. The data show that this treatment decreased the extent of CD8+ T cell cycling in the spleen
from ~28% in intact mice to ~16 and 9% at days 17 and
23 after infection, respectively (Table 2). However, >80%
of the blood CD8+ T cells still showed the characteristic
IM-like CD62Llo profile, and there was little effect on the
prevalence of TCR-V
4+ T cells in the CD8+ PBL at 23 d
after infection (Table 2). These data raise the possibility that
the high frequency of V
4+CD8+ T cells in the peripheral
blood is a consequence of selective protection from apoptosis rather than a direct expansion. However, the data are
most consistent with selective expansion of V
4+CD8+ T
cells. First, although cycling is dramatically reduced, it is
not eliminated, and is still elevated compared with naive
animals, in which <5% of CD8+ T cells are cycling (Fig. 5
A and data not shown). In particular, ~16% of CD8+ T cell
in the spleen are cycling at day 17 in CD4-depleted animals, a time point just before the dramatic increase in percentage of V
4+CD8+ T cells (Fig. 4). Second, there is no
evidence for the massive reduction in numbers of CD8+ T
cells that would be necessary to account for the compensatory increase in V
4+CD8+ T cells (Table 2). Thus, the
data suggest that eliminating >90% of the CD4+ T cells
through the time that the IM-like phase of MHV-68 infection is developing did not prevent the emergence of the
prominent TCR-V
4+ CD8+CD62Llo population. Cytokines derived from the CD4+ population are not, therefore, primarily responsible for the selective expansion of the
V
4+ CD8+ T cells.
|
However, there does appear to be a role for CD4+ T
cells in the pathogenesis of IM. The numbers of cycling
CD8+ T cells in the spleen were much lower in both the
MHC class II /
(Fig. 5 A) and CD4-depleted mice
(Table 1). In addition, the frequency of the CD8+ PBL was
not significantly increased as a consequence of infection in
either group of CD4-deficient, MHV-68-infected mice
(Fig. 5 C and Table 2). It should be noted that the elevated
CD8 frequency in the MHC class II
/
mice is evident
before infection, and thus reflects a compensatory increase
because of the lack of CD4+ T cells rather than an increase
as a consequence of infection. These data suggest that although CD4+ T cells are not involved in the specific V
4
expansion, they play a part in the generalized proliferation
and activation of the CD8+ subset. Thus, the IM phase of
MHV-68 infection appears to be a consequence of at least
two separate activation events: a generalized, CD4-dependent, presumably cytokine-driven expansion that precedes, but does not control, the later, perhaps superantigen-driven, expansion of V
4+CD8+ T cells. The relationship between
cell cycling in the spleen and activated CD8+ T cells in the
peripheral blood is likely to be complex, but is an important issue for understanding the pathogenesis of IM, and
thus warrants further investigation.
Initial characterization of MHV-68 has revealed striking biological similarities to EBV. For example, major aspects of the pathogenesis of viral infection are similar in humans and mice, including the initial acute respiratory infection and the establishment of viral latency in B cells (9).
In the current studies, we describe a syndrome of T cell
activation that occurs late in infection, well after the clearance of infectious virus and the establishment of latent infection of B cells. The activated T cells in the peripheral
blood are predominantly CD8+ and express a CD62Llo-,
CD44hi-activated phenotype, with as many as 75% of the
CD8+ T cells expressing V4+ TCR. These activated cells
are a reflection of a more generalized activation in the enlarged spleen, in which CD4+ and B220+ cells are also activated. The activated phenotype is sustained in vivo for
>2 mo (Figs. 1 and 4). This pathology has two key features
in common with EBV-induced IM, namely, activated CD8+
T cells in the peripheral blood and splenomegaly. Taken
together with previously identified similarities between
MHV-68 and EBV infection (10), the similarities in the IM
profile strengthen the relevance of MHV-68 as an experimental mouse model for EBV.
The availability of a mouse model of a -herpesvirus-
induced IM promises to be a valuable tool in understanding
the pathogenesis of the disease. For example, these initial
studies show that the T cell-activation profile is unaltered
in adult thymectomized mice, suggesting that availability of
newly emerging thymocytes is not necessary for IM. In another example, we have been able to establish the relationship between initial infection and the onset of IM. Although this has been difficult to ascertain in EBV-induced
IM because, in most cases, IM is the first clinical evidence
of infection, the mouse model clearly indicates that the onset of IM is a late event in the viral infection, beginning as
early as 2-3 wk after infection, well after the clearance of infectious virus from the lung. Finally, it has been long
known that activated CD8+ T cells characteristic of EBVinduced IM are not all virus-specific, and the cause of the
lymphoproliferation has been extensively investigated. There
are conflicting reports supporting a role for viral antigens
(17, 39, 66), superantigens (37, 38), and nonspecific activation (17, 18, 67) in the generation of activated CD8+ T cells.
An intriguing possibility suggested by the late kinetics of
CD8+ T cell activation is that the IM is a consequence of
new viral antigen expression during the establishment of latency. The MHV-68 virus model will allow us to directly
address the mechanism of CD8+ T cell activation. Although
we don't yet know whether these T cells are specific for virus, our current studies suggest that both a TCR-mediated
event, characterized by V
4+CD8+ T cell expansion, and
a generalized T cell activation that is probably mediated by
CD4-dependent cytokines, are involved in the MHV-68-
induced IM.
The selective expansion of V4+CD8+ T cells described
in this report has two intriguing characteristics. First, the
effect is not MHC-restricted, in that V
4 expansion is seen
among activated CD8+ T cells in the peripheral blood and
spleen of MHV-68-infected mouse strains expressing five
different MHC haplotypes. Second, the V
4+CD8+ expansion is MHC class II-dependent because it is not observed in MHC class II
/
mice, but does occur in mice
that were depleted of CD4+ T cells by in vivo antibody administration. There are two possible explanations for the
MHC-unrestricted, V
4+ T cell expansion. The first possibility is an oligoclonal T cell response to a conventional
viral antigen. The MHC promiscuity could be explained
by the existence of a dominant epitope capable of binding
multiple class I haplotypes, as has been described (61). The
second possibility is superantigen-driven T cell activation. We believe the data are most consistent with a superantigen-driven response since V
-specific, class II-dependent,
MHC-unrestricted stimulation of T cells are hallmarks of
superantigen activation. As additional support of this, the
response appears to be relatively independent of the
chain of the TCR (Blackman, M.A., unpublished observations) and T cell responses to superantigens are characteristically independent of the non-V
components of the
TCR (68).
Despite the fact that the V4-specific T cell activation
fits several basic criteria for superantigen-driven responses,
it is unusual that CD8+ T cells are preferentially activated.
Superantigens characteristically stimulate both CD4+ and
CD8+ T cells (69, 70), or in the case of weak superantigens, CD4+ T cells are preferentially activated (71). It is
possible that the putative MHV-68 superantigen preferentially activates CD8+ T cells. In support of this possibility, a
superantigen-like activity in Toxoplasma gondii was shown to
selectively activate CD8+ T cells under some experimental
conditions (72, 73). It is also possible that MHV-68 activates both CD4+ and CD8+ T cells, but the CD4+ T cells
are preferentially driven to apoptosis, resulting in the selective retention of the CD8+ T cells. Selective apoptosis of
CD4+ T cells after staphylococcal enterotoxin B stimulation
has been described (74). This possibility is consistent with
the modest elevation of V
4+CD4+ T cells seen in some
experiments (Fig. 4 B). Thus, although we cannot eliminate the possibility that the V
4+CD8+ expansion represents a restricted response to a conventional viral peptide
with promiscuous MHC class I binding, the V
-specific, MHC class II-dependent, non-MHC-restricted, TCR-
chain-independent V
4+CD8+ activation during the IM
phase of MHV-68 infection is most readily explained by
the expression of a viral superantigen.
One trivial explanation for the V4 expansion is that the
effect is mediated by a retroviral superantigen. Perhaps the
MHV-68 infection is activating an endogenous superantigen
or the virus stock is contaminated with superantigen-expressing murine retroviruses. Although we cannot formally rule
out these possibilities, we think that they are unlikely. First,
no endogenous retrovirus specific for V
4+ T cells has
been identified. Also, if the mice were harboring an unknown V
4-specific endogenous retrovirus, it would be
expected that V
4+ T cells would be deleted in these
mouse strains, which was not observed (Table 1). Recently,
an exogenous mouse mammary tumor virus (MMTV) that
activates V
4+ T cells was described in the swiss IBM moro
mouse strain (75). However, this MMTV was shown to require the presence of MHC class II I-E molecules, which
are absent in B6 mice, for efficient superantigen presentation. Second, with regard to the possibility that the MHV-68
virus stock is contaminated with murine retroviruses, plaquepurified virus has been cultivated in owl monkey kidney fibroblast cells, which would not be permissive for MMTV
replication and would not be a source for introduction of
MMTV into the MHV-68 virus stock. In addition, the delayed and sustained expansion of V
4+ T cells seen after infection with MHV-68 are not consistent with the kinetics of superantigen expression during a typical MMTV infection (76).
Earlier studies have suggested the presence of a viral
superantigen expressed by EBV. Smith et al. described a
V-specific expansion in the peripheral blood of patients
with IM, which was absent in two cases examined after
resolution of the acute phase of the disease (37). Other
studies, however, have not shown a V
-specific expansion
either in vivo during IM nor after in vitro stimulation with
EBV-infected cells (40, 41). More recently, Sutkowski et al.
reported an MHC class II-dependent, but MHC-unrestricted proliferation of naive T cells in response to EBVtransformed B cells in which the virus was reactivated (38).
Analysis of early activation markers indicated a selective
V
13 activation. In contrast, in another recent study, Callan et al. showed clonal or oligoclonal populations of activated T cells in the peripheral blood of IM patients, in
which the V
-specific expansion varied among individuals, suggesting viral antigen-driven proliferation (39). It should be noted that variation in the V
profile from individual
patients does not in itself rule out a role for a viral superantigen. It is possible that there is sequence variation in different isolates, analogous to MMTV, resulting in expansion of
different V
populations of T cells. However, in support of
a conventional virus-specific response, Callan et al. showed
that, in some cases, the specificity of the
chain was identical to that identified in EBV-reactive T cell clones by an
independent group (39).
There are also reports of viral superantigens in other herpesviruses. For example, evidence for a CMV-encoded superantigen has been reported (77). In addition, an open
reading frame of the H. saimiri genome, ORF 14, which
has significant homology to a mouse mammary tumor virus-encoded superantigen (78), encodes a protein that
binds to MHC class II molecules and stimulates T cell proliferation, although the V profile of the proliferating T
cells has not been reported (79).
It is interesting to speculate on the role of a putative superantigen in MHV-68 infection. In the case of MMTV
superantigens, expression of the viral superantigen is essential for productive infection (62, 80). MHC class II
molecules on virally infected B cells present a superantigen
that activates a subset of T cells expressing the appropriate
V element. The activated T cells subsequently promote
the proliferation and differentiation of infected B cells, resulting in clonal expansion and the establishment of memory cells that serve as a stable reservoir for the virus (81). It is clear from our studies that expression of the putative superantigen is not essential for infection or the establishment
of latency of MHV-68 because MHC class II
/
mice
still became infected and established latency (13), although
they did not exhibit IM (Fig. 5). It is possible that superantigen-driven T cell activation is required for expansion of
the pool of latently infected B cells. A role for a putative
EBV superantigen in establishing an equilibrium between
latency and activation, resulting in the maintenance of a
constant EBV burden, has been postulated (38). In this
light, more detailed studies of latency and malignancy in
MHV-68-infected B6 and MHC class II
/
mice should
be revealing (13). An intriguing feature of the putative
MHV-68 viral superantigen is that the effect on the TCRV
4+CD8+ T cells is not apparent by day 14, when all
lytic virus has been cleared from epithelial sites, and only
becomes obvious from ~2 wk after evidence of latent
MHV-68 infection is first detected in B lymphocytes. It is
likely that the putative superantigen is a viral gene product
that binds to MHC class II glycoproteins on the surface of
persistently infected B cells but, if this is so, the kinetics of
IM development suggest either that the protein is not expressed during the acute stage of the disease, or that the
emergence of the TCR-V
4+CD8+ T cells is in some way
inhibited during the time that the effectors that deal with
the lytic phase of the infection are operating. One possible
scenario is that the superantigen is not expressed on infected epithelial cells during acute infection, but can be expressed on B cells during reactivation of latent virus.
In conclusion, we have described an IM phase of MHV-68
infection of mice characterized by at least two activation
events: a CD4-dependent expansion that is presumably
cytokine dependent, and a preferential expansion of V4+
CD8+ T cells, perhaps driven by a viral superantigen. This
MHV thus provides a valuable experimental model for understanding the pathogenesis of IM and the equilibrium between the lytic and latent stages of herpesvirus infection.
Address correspondence to Marcia A. Blackman, Department of Immunology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105.
Received for publication 20 November 1996 and in revised form 25 February 1997.
These experiments were supported by National Institutes of Health grants CA21765, AI38349, P30 CA21765, and by the American Lebanese Syrian Associated Charities.We thank Anthony McMickle for help with the flow cytometry.
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