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Clinical and Diagnostic Laboratory Immunology, July 2000, p. 710-713, Vol. 7, No. 4
1071-412X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
CD4+ Lymphocyte-Mediated Suppression of
Cytomegalovirus Expression in Human Astrocytes
Maxim C.-J.
Cheeran,1,2
Genya
Gekker,1,3
Shuxian
Hu,1,3
Stephanie L.
Yager,1
Phillip K.
Peterson,1,2,3 and
James R.
Lokensgard1,3,*
Institute for Brain and Immune Disorders,
Minneapolis Medical Research Foundation,1 and
University of Minnesota Medical School,3
Minneapolis, Minnesota 55404, and the College of Veterinary
Medicine, University of Minnesota, St. Paul, Minnesota
551082
Received 17 December 1999/Returned for modification 25 February
2000/Accepted 8 May 2000
 |
ABSTRACT |
Cytomegalovirus-stimulated CD4+ lymphocytes from
seropositive but not seronegative donors suppressed viral gene
expression in primary human astrocytes. This suppressive activity was
mediated through soluble factors. These findings suggest that
CD4+ lymphocytes play a role in defense of the brain
against cytomegalovirus.
| |
TEXT |
Between 60 and 90% of the world
population is infected with human cytomegalovirus (CMV). Yet, despite
this high prevalence of infection, CMV brain disease is restricted to
those with severely impaired or underdeveloped immune systems (e.g.,
AIDS patients and the developing fetus). In human immunodeficiency
virus-infected patients, the clinical outcome of CMV retinal disease is
related to both CD4+ (29) and CD8+
(27) lymphocyte counts. CMV encephalitis, however, is
observed only in advanced stages of AIDS, when CD4+ T-cell
counts fall below 50 per mm3 (5).
Various model systems have been used to demonstrate that T lymphocytes,
both CD4+ and CD8+, are important immune
effectors responsible for protection against CMV (2, 6, 22, 26,
30). Control of CMV is not exclusive to any one lymphocyte
subset, and it appears that there is a hierarchical control by distinct
compartments of the immune system in different organs (24).
CMV is known to productively infect astrocytes (11, 13, 17,
23). However, little is known about the role of lymphocytes in
host defense against CMV in the central nervous system (CNS).
Cytotoxic T-cell (CTL) responses are important in controlling the
spread of CMV (2, 22, 26, 30), but when operating within the
CNS, they may be destructive rather than protective. CD4+ T
lymphocytes have been shown to play a crucial role in tissue sites like
the salivary gland, where CTLs have limited or no antiviral effect
(15). Hence in this study, we explored the hypothesis that
CD4+ T lymphocytes possess the ability to inhibit CMV gene
expression in human astrocytes.
Peripheral blood mononuclear cells (PBMC) from CMV-seropositive and
CMV-seronegative healthy donors were stimulated for 72 h in vitro
with CMV strain AD 169 (21). To determine if T lymphocytes possess antiviral properties, CD4+ and CD8+ T
cells were isolated from stimulated PBMC cultures using anti-CD4 and
anti-CD8 antibody-coated immunomagnetic beads (Dynabeads; Dynal, Inc.,
Lake Success, N.Y.) yielding lymphocyte populations with >95% purity
as determined by flow cytometry. Purified CD4+ or
CD8+ lymphocytes were added to human fetal astrocytes,
which were prepared as described previously (3). Seventy-two
hours after the lymphocyte-astrocyte cocultures were constituted, the
cultures were infected at a multiplicity of infection of 2.5 50%
tissue culture infective doses per cell with a recombinant CMV strain, RC256 (25), expressing 
-galactosidase from a viral
-promoter. Infected cells were harvested 72 h postinfection,
resuspended in phosphate-buffered saline (100 µl), and subjected to
three freeze-thaw cycles. The cell lysates were analyzed for
-galactosidase activity using CPRG (1 mg/ml;
5-bromo-4-chloro-3-indolyl--D-galactoside; Boehringer Mannheim, Indianapolis, Ind.) as a substrate
(13). Optical density values at 595 nm (OD595)
were used to determine differences in viral gene expression in cultures
with and without added lymphocytes.
Lymphocytes from seropositive donors suppress CMV gene expression
in human astrocytes.
CMV gene expression was markedly reduced in
cocultures containing astrocytes and lymphocytes isolated from
CMV-seropositive donors compared to cultures with untreated astrocytes
(Fig. 1). CD4+ lymphocytes
obtained from four seropositive donors dose-dependently suppressed
viral gene expression as measured by -galactosidase activity (Fig.
1A). Lymphocyte-to-astrocyte ratios of 0.25:1, 0.5:1, and 1:1
suppressed viral expression by (34.6 ± 24.3)%, (55.8 ± 15.9)%, and (84.2 ± 2.4)%, respectively (n = 5 experiments). In comparison, CD8+ lymphocytes from
seropositive donors suppressed CMV gene expression by (72.7 ± 4.94)% at a ratio of 1:1 (Fig. 1B). However, neither CD4+
nor CD8+ lymphocytes from five seronegative donors,
cocultured with astrocytes at the same ratios, suppressed CMV gene
expression in astrocytes (Fig. 1).
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FIG. 1.
Effect of lymphocytes on CMV gene expression in
astrocytes. CD4+ and CD8+ T cells were isolated
from PBMC obtained from CMV-seropositive as well as seronegative donors
and stimulated in vitro with CMV antigen. CD4+ (A) and
CD8+ (B) lymphocytes were cocultured with astrocytes at the
indicated ratios for 72 h prior to infection with the recombinant
CMV strain RC256. Cultures were collected 72 h postinfection and
assayed for -galactosidase activity. Data, expressed as the
percentages of viral expression compared to those in untreated infected
controls (means ± standard errors), were obtained from five
independent experiments with lymphocytes from seropositive and
seronegative donors using astrocytes from different brain specimens.
|
|
To rule out the possibility that CD4
+ lymphocyte-mediated
viral suppression was associated with cytotoxicity in cocultures,
MTT
[3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide]
uptake
assays (
9) were performed at all lymphocyte/astrocyte
ratios
for the duration of the experiment (6 days). MTT (1 mg/ml)
was added to
the cultures for 4 h at 37°C. The cultures were then
treated
with lysis buffer (20% sodium dodecyl sulfate and 50%
dimethyl
formamide, pH 4.7) overnight at 37°C. OD
570 readings,
representing the conversion of MTT to formazan in living cells
by
dehydrogenases, were similar at all coculture ratios, with
no
appreciable difference between control and lymphocyte-treated
astrocyte
cultures. OD
570 readings for cocultures containing
astrocytes
(10
5 cells) with CD4
+ T cells
(ratio, 1:1) or CD8
+ T cells (ratio, 1:1) were 2.8 ± 0.10 and 2.4 ± 0.16, respectively.
OD
570 readings for
astrocytes (10
5 cells) and lymphocytes (10
5
cells) cultured separately, representing baseline assay values,
were
2.6 ± 0.13 and 0.4 ± 0.01, respectively. These results
support
the hypothesis that CD4
+ lymphocytes possess the
ability to confer upon astrocytes noncytotoxic
protection against CMV.
This antiviral property is dependent on
prior exposure of the
lymphocyte donor to CMV antigen in vivo.
The ability of seropositive
donor lymphocytes to suppress CMV
gene expression may reflect an
effective CD4
+ T-cell memory response (
7)
capable of generating a unique
set of cytokines on subsequent
stimulation with CMV antigen (
12,
16).
Soluble factors mediate antiviral effect of CD4+
lymphocytes.
To determine if the noncytotoxic antiviral effect of
CD4+ lymphocytes was mediated by soluble factors, transwell
tissue culture inserts (Becton Dickinson, Franklin Lakes, N.J.) were
used to physically separate the CMV-stimulated lymphocytes from
astrocyte monolayers. Following viral infection, CMV expression was
suppressed by (58.05 ± 3.67)% (n = 3) when
CMV-stimulated CD4+ T cells from seropositive donors were
added to one side of the transwell culture system (Fig.
2). CD4+ T cells from
seronegative donors, however, had no antiviral effect in this system
(data not shown). On the other hand, CD8+ lymphocytes from
seropositive donors did not suppress CMV gene expression ([12.73 ± 1.02]%; n = 3) when separated from astrocytes by a
porous membrane (Fig. 2), suggesting that effective viral suppression
with these cells requires cellular contact.
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FIG. 2.
Soluble factors mediate the induction of an antiviral
state in astrocytes. CD4+ and CD8+ T cells were
maintained in culture with primary human astrocytes across a transwell
membrane for 72 h. The cultures were infected with CMV (RC256) and
assayed for -galactosidase activity 72 h postinfection. The
data, expressed as percents suppression (means ± standard errors)
compared with untreated infected controls, were obtained from three
independent experiments using astrocytes and T cells from different
donors.
|
|
Since the antiviral effects of CD4
+ T cells could be
mediated across a porous membrane, we tested the ability of cell-free
coculture supernatants to impart an antiviral state to astrocyte
cultures. Serial dilutions of cell-free supernatants from seropositive
lymphocyte-astrocyte cocultures, when added to fresh astrocytes,
suppressed CMV gene expression in a concentration-dependent manner
(Fig.
3). The decrease in CMV gene
expression from four seropositive
donor CD4
+
lymphocyte-astrocyte cocultures, when compared to untreated controls,
ranged from (8.33 ± 12.49)% (20% concentration) to (82.49 ± 5.17)%
(100% cell-free supernatants). However, cell-free
supernatants
from uninfected lymphocyte-astrocyte cocultures, using
CD4
+ lymphocytes from seronegative donors and
CD8
+ lymphocytes from both seropositive and seronegative
donors, did
not suppress viral gene expression (data not shown).
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FIG. 3.
Cell-free coculture supernatants confer an antiviral
state upon primary human astrocytes. Dilutions of supernatants from
CD4+ lymphocyte-astrocyte cocultures were applied to fresh
astrocyte cultures for 72 h and then infected with CMV (RC256).
Freeze-thawed lysates of infected cells were assayed for
-galactosidase activity 72 h postinfection. The data, expressed
as percents suppression compared to untreated infected astrocyte
cultures (means ± standard errors), were obtained from
independent experiments using CD4+ T cells from four
different seropositive donors.
|
|
We have attempted to inhibit the antiviral effects of coculture
supernatants using neutralizing antibodies to tumor necrosis
factor
alpha (TNF-
) and gamma interferon (IFN-
), two known antiviral
cytokines. Cell-free supernatants from CD4
+
lymphocyte-astrocyte cocultures were incubated with specific
antibodies
to TNF-
and/or IFN-
(10 µg/ml) at room temperature
for 30 min
prior to applying them to astrocyte cultures. This
treatment with
cytokine-specific antibodies did not significantly
abrogate the
antiviral activity of the coculture supernatants.
These data
demonstrate that the soluble factors mediating this
antiviral activity
are complex and not restricted to TNF-
and
IFN-
.
Host defense mechanisms against viral infections of the CNS are shaped
by the brain's limited capacity for antigen presentation
and
functional modulation of immune responses, designed to prevent
extensive damage in this vital nonregenerating tissue (for reviews,
see
references
8 and
20). Activated
lymphocytes routinely
enter the CNS, in an antigen-independent manner,
during immune
stimulation (
10) without associated
neuropathology (
18). Published
studies provide evidence that
lymphocytes also mediate clearance
of viral infections from the CNS
without conventional major histocompatibility
complex expression on
target cells and without massive cell death
(
1). We show
here that CD4
+ and CD8
+ T lymphocytes from
seropositive donors suppressed CMV gene expression
in astrocytes. The
suppression was not mediated by cytotoxic damage
of infected cells but
by soluble factors induced in the CD4
+ lymphocyte-astrocyte
cocultures. Similar experiments have indicated
that the soluble factors
derived from CMV-stimulated PBMC, which
mediate anti-CMV effects on
human fibroblasts, are IFNs and TNF
(
28). CD4
+
T-cell clones specific to CMV immediate-early proteins produce
TNF-
and IFN-
, which can inhibit viral replication in U373MG,
an
astrocyte cell line (
6). We have also shown in our
laboratory
that these proinflammatory cytokines inhibit CMV replication
in
primary human astrocytes (
4). In addition, experiments
performed
in vivo suggest that TNF-
and IFN-
may be responsible
for control
of CMV (
14,
19), particularly in areas where
CTLs have limited
function (
15).
This report indicates that lymphocytes mediate suppression of CMV in
primary human brain cells. The use of primary astrocytes,
in this
study, enabled us to evaluate the antiviral effects of
lymphocytes in a
relevant tissue type. Although the precise mechanisms
of viral
suppression are unknown, it is likely that one or more
cytokines are
involved in mediating this noncytotoxic antiviral
effect.
In conclusion, the results of this study suggest that CD4
+
lymphocytes may play a role in host defense of the brain against
CMV
infection. The lack of host defense in advanced AIDS, normally
provided
by CD4
+ lymphocytes, may allow viral replication in
astrocytes, resulting
ultimately in the necrotizing lesions seen during
CMV ventriculoencephalitis.
These findings also have implications in
developing immune-based
therapies for CMV brain infection. However,
successful development
of adoptive immunotherapies for CNS infections
will require a
much greater understanding of both viral pathogenesis
and neuroimmune
responses to the
virus.
| |
ACKNOWLEDGMENTS |
This study was supported in part by the United States Public Health
Service grants MH-57617, T-32-DA-07239, and NS-38836.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Minneapolis
Medical Research Foundation, 914 South 8th St., Bldg. D-3, Minneapolis, MN 55404. Phone: (612) 347-6849. Fax: (612) 337-7372. E-mail: loken006{at}tc.umn.edu.
| |
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Clinical and Diagnostic Laboratory Immunology, July 2000, p. 710-713, Vol. 7, No. 4
1071-412X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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