The Journal of Immunology, 2001, 167: 5429-5438.
Copyright © 2001 by The American Association of Immunologists
Highly Activated CD8+ T Cells in the Brain Correlate with Early Central Nervous System Dysfunction in Simian Immunodeficiency Virus Infection1
Maria Cecilia Garibaldi Marcondes,
E. M. E. Burudi,
Salvador Huitron-Resendiz,
Manuel Sanchez-Alavez,
Debbie Watry,
Michelle Zandonatti,
Steven J. Henriksen and
Howard S. Fox2
Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037
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Abstract
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One of the consequences of HIV infection is damage to the CNS. To
characterize the virologic, immunologic, and functional factors
involved in HIV-induced CNS disease, we analyzed the viral loads and T
cell infiltrates in the brains of SIV-infected rhesus monkeys whose CNS
function (sensory evoked potential) was impaired. Following infection,
CNS evoked potentials were abnormal, indicating early CNS disease. Upon
autopsy at 11 wk post-SIV inoculation, the brains of infected animals
contained over 5-fold more CD8+ T cells than did uninfected
controls. In both infected and uninfected groups, these
CD8+ T cells presented distinct levels of activation
markers (CD11a and CD95) at different sites: brain > CSF >
spleen = blood > lymph nodes. The CD8+ cells
obtained from the brains of infected monkeys expressed mRNA for
cytolytic and proinflammatory molecules, such as granzymes A and B,
perforin, and IFN-
. Therefore, the neurological dysfunctions
correlated with increased numbers of CD8+ T cells of an
activated phenotype in the brain, suggesting that virus-host
interactions contributed to the related CNS functional
defects.
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Introduction
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The
best experimental model to mimic human AIDS is SIV infection in
macaques. SIV infection shares several qualities with HIV-1 infection,
such as cell type tropism, immunosuppression, and disease of the CNS
(1, 2). Both SIV and HIV gain access to the CNS early in
the infection (3, 4, 5). Varying degrees of CNS pathology may
result, the most severe form, SIV or HIV encephalitis, being
characterized by lesions bearing multinucleated giant cells, microglial
nodules, infiltrating macrophages, and gliosis (2, 6, 7).
The infected cells in the CNS are of the monocytic lineage (6, 8, 9). Virus can be detected in the brain within 2 wk after
infection, when acute viremia peaks. In the early stages of
experimental SIV infection, viral expression in the brain is localized
mainly in perivascular mononuclear cells and within the leptomeninges
(4, 10). Later, viral RNA can be detected in the
leptomeninges and in brain infiltrating/perivascular macrophages and
multinucleated giant cells (3, 9, 10, 11). Of several
mechanisms proposed to account for this CNS infection, the "Trojan
horse" model, in which infected monocytes and/or T cells migrate into
the brain, has received much support (12, 13).
Approximately one-third of HIV-1-infected individuals develop the
associated cognitive/motor disorder, also known as the AIDS dementia or
neuroAIDS (13, 14, 15). Furthermore, HIV alters sleep
patterns, psychomotor activity, and sensory evoked potentials
(EPs)3
(16, 17, 18). In humans, progressive motor and cognitive
abnormalities are thought to be initiated by the entry of HIV into the
CNS (19). Similarly, in SIV-infected rhesus monkeys,
functional CNS deficiencies in sensory EPs, behavioral/cognitive
ability, and motor skills have been found by us and others
(20, 21, 22, 23). In the rhesus/SIV model, the reduction of virus
load in the periphery by antiviral treatment restored
neurophysiological normalcy, perhaps, in part, because this treatment
lowered the number of infected macrophages and T cells entering the CNS
(20). However, abnormalities in movement and temperature
were unchanged by antiviral treatment. Because cells of the
macrophage/microglia lineage (but not neurons) are infected in the CNS,
the neurological consequences of the CNS infection are related to
indirect mechanisms, such as viral products or molecules produced by
the host in response to the virus. However, the triggering events
remain unknown.
Tissue damage is frequently associated with host cell-mediated (T cell)
responses (24). Virus-specific CD8+
CTLs play a central role in the immune response against viruses, in
general, by eliminating virus-infected cells. Macaques additionally
have CD8+ cells that lack the CD3 complex and
exhibit NK cell activity (25). The importance of
CD8+ cells in controlling SIV infection in
monkeys was shown when the viral load increased and progression of
disease was accelerated in animals whose CD8+
cells were depleted by Ab treatment (26).
Most of the specific effector CTL generated during viral infections
undergo apoptosis, but a fraction of them remains in the circulation,
constituting a pool of memory cells. These memory cells promptly and
efficiently respond to a second specific stimulation. The expression of
CD45RA can be used in rhesus macaques to define whether previously
primed cells are mediating a secondary response. Resting naive
CD8+ cells, not primed by Ag, express high levels
of CD45RA, but memory cells express far less (27).
However, this low expression of CD45RA does not discriminate memory
cells in the resting state from those that are activated. Instead, the
expression of receptors for homing to endothelium of inflamed sites,
such as CD11a (LFA-1), give some clue about the activation status of
these cells (28). The expression of CD95 (Fas) also
increases on activated cells, and its binding to its ligand (FasL) may
induce apoptosis or proliferation of CD8+ cells
(29). CD28 can also be used to define the pattern of
activation. The T cell Ag CD28 is an important provider of
costimulatory signals to TCR-mediated activation, thereby regulating
proliferation, preventing the induction of anergy or apoptosis, and
driving the pattern of cytokine production (30). The
expansion of CD8+CD28-
cells in HIV infection suggests that such cells arise in response to
the virus (31). In fact, the
CD8+CD28- cells can
present characteristics of end-stage effector CTL, such as expression
of perforin, granzymes A and B, and IFN-
, and they exhibit cytolytic
activity (32).
We previously identified SIV-specific CD8+ CTL in
the cerebrospinal fluid (CSF) of SIV-infected macaques very early after
the infection and in the brain at autopsy (33). Yet the
contribution of the CD8+ cells and other T cell
subpopulations in the progression of CNS functional deficits remains
unknown.
Therefore, the present work was designed to evaluate the brain
infiltrate and the phenotype of T cells in the CNS compared with those
in the periphery, at a relatively early stage of SIV-induced disease,
for a possible correlation with neurological dysfunction. The results
showed altered EPs and a drastic increase in the number of activated
CD8+ T cells expressing predominantly a memory
and a cytolytic phenotype in the brains of animals with early SIV
infection. These data suggest that SIV infection induces a chronic
immune response in the CNS that mediates injury to the brain.
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Materials and Methods
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Animals
Rhesus monkeys, free from SIV, type D simian retrovirus, and
herpes B virus were obtained from Covance (Alice, TX) and Charles River
Breed Laboratories (Key Lois, FL). All animal experiments were
performed with approval from the Institutional Animal Care and Use
Committee. Blood samples were drawn from the femoral vein, and plasma
was separated by centrifugation from cells in EDTA-anticoagulated
blood. CSF was obtained from the cisterna magna. CSF samples containing
>0.1% blood contamination (determined by RBC counts) were discarded.
Necropsy was performed after terminal anesthesia. During necropsy,
animals were perfused intracardially with sterile PBS containing 1 U/ml
heparin before tissue samples were taken for cell isolation, virus
quantification, and formalin fixation for histology. Animals 350, 352,
353, and 354 were sacrificed at 73, 75, 77, and 80 days postinoculation
(p.i.), respectively, and comprised the 11-wk p.i. group. Animals 335,
346, and 347 were sacrificed at 13 days p.i. and comprised the 2-wk
p.i. group.
Viral infection
A cell-free stock of SIV (SIVmac182) obtained after serial
passage of SIV-infected microglia was the inoculum used
(34). Animals received 0.25 ml of SIVmac182 stock, diluted
into RPMI 1640 for injection, containing 5 ng/ml p27 (gag) Ag.
Viral quantitation
To measure virus load, SIV RNA in plasma, CSF, and brain tissue
was determined by using the quantitative branched DNA (bDNA) signal
amplification assay. Bayer Reference Testing Laboratory (Emeryville,
CA) performed this assay. Each sample was measured in duplicate and is
reported as an average value. Virus was also recovered from the CSF in
a coculture assay. Cells were separated from the CSF by centrifugation,
and the cells and cell-free supernatant were separately added to
cultures of CEMx174 cells; all cultures were maintained for at least 3
wk for examination for syncytia formation.
Electrophysiology
Eletrophysiological analysis of brainstem auditory EP (BSAEP)
and auditory EP (AEP) was performed on ketamine (20 mg/kg)-anesthetized
animals as described previously (35). Positive waves in
BSAEP were denoted as P1, P2a, P2b, P3, P4, and P5. In AEP, two
positive (P1 and P2) and two negative (N1 and N2) waves were
identified. Averaged peak latencies were calculated and compared
individually between animals and as group means using ANOVA.
Peripheral mononuclear cells
Buffy coats obtained from centrifugation of EDTA-anticoagulated
blood, and cell suspensions from spleen, deep cervical, and inguinal
lymph nodes, passed through a nylon mesh, were submitted to a
Ficoll-Isopaque (Pharmacia Biotech, Uppsala, Sweden) gradient
centrifugation for isolation of the mononuclear fraction. After
washing, cells were enumerated in a Coulter Z2 (Coulter, Miami, FL) and
resuspended in complete RPMI 1640, containing 10% FCS at a
concentration of 107 cells/ml, for labeling or
freezing.
Brain cells
The meninges were carefully removed, and the brain was minced
and transferred to a sterile cell sieve with a 40-mesh wire screen. A
sterile plunger was used to force this material through the sieve, and
the disassociated material was collected by centrifugation at 470
x g for 15 min at 4°C. The supernatants were aspirated,
and each pellet brought to a final volume of 30 ml with HBSS, after
which 28 U/ml of DNase I and 500 U/ml of collagenase II were added.
Digestion was performed at 37°C in a shaking water bath for 1 h.
Afterward, the cells were pelleted at 470 x g for 10
min and then washed in HBSS containing 1% FCS. The cell pellet was
resuspended in 6.4 ml of a 1.122 g/ml Percoll (Pharmacia Biotech)
solution and brought to a final volume of 30 ml with HBSS, resulting in
1.033 g/ml Percoll. Eight milliliters of 1.088 g/ml Percoll was
underlayed, and this gradient was centrifuged at 1200 x
g for 20 min at 20°C. The cells at the 1.033/1.088
interface were collected, washed, and quantified in a Coulter
Z2.
Flow cytometry
Cells isolated as described (2 x 105
to 10 x 105) were stained with 50 µl
mixtures of Abs diluted according to a previous titration in staining
buffer (HBSS with 2% FCS and 0.01% NaN3). The
Abs used for the staining were anti-monkey CD3-biotin (clone FN-18;
Biosource International, Camarillo, CA) followed by streptavidin-PerCP
or streptavidin-APC (BD PharMingen, San Diego, CA), anti-human
CD8-PE (clone DK25; DAKO, Carpinteria, CA), anti-CD11a-FITC
(clone 25.3.1; Immunotech, Westbrook, ME), anti-CD28-FITC (clone
CD28.2; Immunotech), anti-CD95-FITC (cloneDX2; Caltag, Burlingame,
CA), anti-Mac-1-PE (clone M1/70(9) Roche, Indianapolis, IN),
and anti-CD4-PE (clone OKT4; hybridoma obtained from the American
Type Culture Collection (Manassas, VA), with the secreted Ab purified
and PE-conjugated in the laboratory). Isotype controls (BD PharMingen)
were also used. The cells were then processed through a FACScan flow
cytometer before analysis of data with CellQuest software (BD
Immunocytometry Systems, San Jose, CA).
Magnetic columns
Cells isolated from the blood or lymphoid tissues were
resuspended to the concentration of 2 x 107
in 200 µl of PBS, and 50 µl of anti-CD8 MACS Microbeads
(Miltenyi Biotec, Auburn, CA) were added. The cells were incubated at
4°C for 20 min, washed once, and passed twice through magnetic
columns according to the manufacturers instructions for the obtention
of a CD8-enriched population. The cells were pelleted and frozen for
RNA extraction.
RNA extraction and RT-PCR
The RNA was extracted from the cell pellets on a Bead-Beater
(Biospec Products, Bartlesville, OK) with an Ambion Totally RNA kit
(Ambion, Austin, TX), as per the manufacturers instructions. A volume
equivalent to 1 µg was subjected to reverse transcription using 125
ng of random primers, 5x first-strand buffer, 0.1 M of DTT, 10 mM of
dNTPs, and 200 U of Superscript II (Life Technologies, Grand Island,
NY), in a final volume of 20 µl. The samples were then incubated at
42°C for 50 min followed by inactivation at 90°C for 5 min. To
design primers for rhesus CD8
-chain, granzymes A and B, and
perforin, fragments of cDNA derived from activated rhesus monkey
lymphocytes were PCR-amplified using primers derived from human
sequences available in GenBank. These products were molecularly cloned,
and three to seven clones of each product were sequenced using an
Applied Biosystems automated sequencer at the TSRI Protein and Nucleic
Acids Core Facility. Primers were then designed from the monkey cDNA
sequences obtained, as well as the rhesus IFN-
sequence available in
GenBank, with the aid of the MacVector program (Accelrys, San Diego,
CA). Next, amounts of cDNA to be used in the PCR were normalized
after determining the relative amounts of 18 S cDNA in the sample by
real-time quantitative PCR (primers and probe from Perkin-Elmer, Foster
City, CA) using a Smart Cycler (Cepheid, Sunnyvale, CA). Finally, the
normalized cDNA was amplified with the following primers: CD8
,
5'-CATGG TGAAG AGGTG GCACA GGAGA and 3'-TGGAC TTCTT GGTGG GCTGG GCA
(72°C, 205-bp product); perforin, 5'-ACTTT GCAGC CCAGA AGACC CAC and
3'-CCGTA GTTGG AGATG AGCCT GAGGT (71°C, 193-bp product); granzyme A,
5'-GGGTG GGGGA AGATT CACAA TAGTG C and 3'-CCCTC GGAAA ACACC GTTAC ACAAC
A (60°C, 219-bp product); granzyme B, 5'-GGAAG ATCAA ACGTG CAAAT CCCG
and 3'-GGAGG CTTGC CATTT CTTTG TCCAT (60°C, 177-bp product); and
IFN-
, 5'-ACGGG ATGAC TTTGA AAAGC TGACC and 3'-CACTG GGATG CTCTT
CGACC TGC (72°C, 165-bp product).
Histopathology and immunohistochemistry
Tissues were fixed in 10% formalin, embedded in paraffin, and
cut into 5-µm sections. After H&E staining, sections from each tissue
block were examined microscopically. Representative sections of brain
(plus lymph node and spleen controls) were immunohistochemically
stained for leukocyte common Ag (LCA) as described
(20).
Statistical analysis
After establishing the normality and homogeneity of variances,
the samples were submitted to one- or two-way ANOVA, depending on the
number of criteria to be tested, followed by a multiple comparisons
t test (Tukey) when appropriate. Only the samples that
exhibited a confidence interval of 95% or more were accepted as
significant. The tests were performed by SigmaStat version 2.0 for
Windows (SPSS Science, Chicago, IL).
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Results
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Rhesus monkeys infected with SIVmac182 were monitored for CNS
function (EP activity), blood cellularity, and viral load. When
evaluated by bDNA, viremia peaked in the plasma at the 14th day p.i.,
then stabilized at a lower level (Fig. 1
A). In the CSF, the temporal
pattern of viral load was similar to that in plasma for two animals
(monkeys 350 and 353), another animal (354) never had virus detectable
by bDNA tests of the CSF at any time (Fig. 1
B). Early time
point CSF samples from monkey 352 were too small for quantitation by
bDNA analysis, but CSF from all four animals was positive in coculture
assays on day 14 p.i., suggesting that virus was present in the
CSF simultaneously with the peak load in the plasma of all four
animals.

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FIGURE 1. Virus load in the plasma (A) and CSF (B)
during the course of SIV infection in rhesus monkeys followed for 11 wk
following viral inoculation. Samples of blood and CSF periodically
collected from SIV-infected monkeys were analyzed for the presence and
quantification of virus copies by the bDNA assay, given as the
log10 transformation of the number of RNA equivalents per
milliliter. Values shown at the axis break are below the limits of
detection (<2.7).
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To detect neuronal dysfunction, the monkeys were also analyzed for
alterations by the measurement of eletrophysiologic EPs. For this,
auditory sensory evoked responses were obtained for analysis of
latencies of the waves. As early as the first assessment (27 days p.i.)
all four animals presented delays in at least one of the sensory evoked
potential measures, and both the brainstem (BSAEP) and cortical (AEP)
measures were abnormal in all animals at sacrifice (11 wk p.i.). Fig. 2
A shows that the latency of
the BSAEP P5 increased during the infection in comparison to each
animals individual preinoculation baseline, and Fig. 2
B
similarly shows the increase in the latency of the AEP N2 auditory time
following viral inoculation. Representative waveforms before and after
infection for the BSAEP are shown in Fig. 2
C. These data are
consistent with previous observations of early abnormal sensory EPs
after infection.

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FIGURE 2. SIV-induced EP abnormalities. A, Latency for BSAEP P5 in
each SIV-inoculated monkey. Preinoculation baseline is the average of
four measurements taken before infection. B, Latency for
the AEP N2 wave, baseline as in A. C,
Representative pattern of BSAEP waveforms from monkey 352 before and
after SIV infection (day 0 and day 69). The position of the P5 latency
is indicated by arrows.
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During the course of SIV infection, circulating
CD4+ and CD8+ T cells were
monitored by lymphocyte counts and FACS analysis, and the CD4:CD8 ratio
was calculated. The CD4:CD8 ratio in the blood declined acutely,
reaching a nadir on day 14 p.i. (0.69), followed by a gradual
rise, but never reaching the preinfection level. The main cause was an
increase in the absolute number of CD8+ T cells,
peaking at 2 wk p.i., whereas the number of CD4+
T cells remained stable throughout the infection. Peripheral blood
CD8+ lymphocytes were also analyzed for CD11a
expression, which increased to an average of 65% by the 69th day
postinfection compared with the preinfection average of 35%.
Therefore, SIV infection apparently favored the relative enlargement of
an activated CD8+ population (Fig. 3
). Because the number of
CD4+ T cells with CD11a phenotype did not change
(Fig. 3
), no generalized activation of T cells occurred.

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FIGURE 3. Kinetics of CD11a expression on the surface of peripheral
CD4+ and CD8+ T lymphocytes after SIV infection
in rhesus macaques. Blood samples were obtained periodically from
SIV-infected animals and analyzed for the expression of CD11a by flow
cytometry. Cells were gated on CD4 or CD8+CD3+
cells. Values represent the average ± SD of the four infected
animals in each time point.
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To investigate whether T cell infiltration into the brain correlated
with the observed functional abnormalities, the animals were sacrificed
at 11 wk p.i., when the EP assessments were abnormal and the viral load
measurements indicated that infection had stabilized. Blood cells were
cleared from the CNS by perfusion, and brains were collected and
processed for histology as well as measurement of viral load and
isolation of lymphocytes from the CNS.
According to histopathological analysis, SIV encephalitis was not
present, and the CNS had no focal lesions, although infiltrating
lymphoid cells were noted and subsequently verified by reactivity with
LCA. The LCA-positive cells were easily detected on brain sections from
infected animals but were rare in uninfected controls. Positively
stained mononuclear cells formed foci in the perivascular area or were
scattered diffusely within the parenchyma (Fig. 4
). No differences were observed in the
intensity or distribution of infiltrating cells among individual
infected animals.

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FIGURE 4. Histopathology of the CNS of an 11-wk-SIV-infected monkey
immunohistochemically stained for LCA. The presence of leukocytes can
be observed in the brain sections (from animal 350) either in the
perivascular area (A), forming foci (B),
or as scattered infiltrates within the parenchyma (C and
D). AC are from the brainstem, observed
at x20 magnification. D is from the cortex, observed at
x40 magnification.
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bDNA analysis of RNA isolated from five separate regions of the CNS
revealed that three of the four SIV-infected animals had virus in at
least one segment of the brain (none was detected in samples from
monkey 350), but one animal (353) had virus in all five regions (Table I
). Calculation of the number of
CD3+CD8+ cells isolated
from the CNS revealed that an increased number of
CD8+ T cells was found in the brains relative to
controls, but the intensity of the CD8+
infiltrate was not necessarily proportional to the amount of virus
detected in the brain tissue (Table I
). Thus, no link was evident
between the amount of virus and the number of
CD8+ T cells at this stage of SIV infection.
Because SIV has been shown to enter the brain within the first 2 wk
following viral inoculation, a separate group of three (335, 346, 347)
animals was sacrificed at 2 wk p.i., corresponding to the peak of
viremia. High levels of plasma and CSF virus (7.79 ± 0.68 and
5.72 ± 0.42 log viral load/ml, respectively), as well as
activated blood CD3+CD8+
cells (58.2 ± 7.4% CD11ahigh, compared
with 28.2 ± 19.5% preinoculation) were present at this stage.
Relative to the animals sacrificed at 11 wk p.i., at least 10-fold more
virus was present in the brain, and virus could be found in all areas
of the brain tested, yet fewer
CD3+CD8+ cells were present
in the brain than in the 11 wk p.i. animals (Table I
).
The phenotypes of lymphocytes infiltrating the CNS was
determined by flow cytometry and identification of surface markers that
define the subpopulations and their activation status. Fig. 5
A depicts the relative
percentage of T cell types in the brains of individual monkeys. The
most dramatic expansion in both absolute and relative counts was found
in the CD3+CD8+ cell
population in the 11 wk p.i. animals; such infected animals had a
significant 5.4-fold increase (F = 38.797;
p < 0.001) relative to the number in controls. Amounts
of other lymphocyte subpopulations also increased somewhat, but not to
a level of statistical significance. That is,
CD3+CD4+ T cells increased
2.3-fold (F = 1.811; p = 0.218) and
CD3-CD8+ cells 2.5-fold
(F = 1.108; p = 0.371) in the brains of
11-wk-infected animals compared with control brains. Although the
number of CD3+CD8+ T cells
in the brain of the 2 wk p.i. group was approximately twice that found
in uninfected control animals, such a change was not statistically
significant (F = 38.797; p =
0.403).

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FIGURE 5. Distribution of T cell subpopulations in the brain parenchyma of
SIV-infected and uninfected control monkeys. A,
Percentage of indicated lymphocyte subpopulations in the brain.
B, Absolute numbers of CD8+ T cells in the
brain and the expression of CD45RA. Single-cell suspensions originating
from the brain were analyzed by flow cytometry, gating on
lymphocytes.
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A particularly notable change was the increased number of
CD3+CD8+ cells expressing a
low level of CD45RA, which is considered a memory phenotype. In the
brain, 11 wk of infection induced an average 6.7-fold increase of
CD3+CD8+CD45RAlow
(memory phenotype) cells and a 4.0-fold increase in the absolute
numbers of CD3+CD8+ cells
expressing CD45RAhigh (naive phenotype) (Fig. 5
B). The increase in both memory and naive
CD8+ cells in 11 wk was statistically significant
in comparison to controls (F = 16.669 and
F = 10.334, respectively; p < 0.001).
In contrast, the smaller increase in
CD3+CD8+ cells found at 2
wk p.i. was evenly divided between memory and naive cells. In the CNS
of SIV-infected monkeys, a preferential enrichment of the
CD8+ T cells observed at 11 wk presumably favored
those expressing a memory phenotype.
Furthermore, the foregoing increase of CD8+ T
cells resulting from SIV infection was also observed in peripheral
sites. Fig. 6
presents the CD4:CD8 ratios
in the blood, spleen, and lymph nodes, all of which exhibited lower
values following 2 or 11 wk of infection relative to controls.
Comparison of CD4:CD8 T cell ratios in the brain vs peripheral regions
showed that infected as well as uninfected monkeys had a predominance
of CD8+ T cells in the brain (Fig. 6
).

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FIGURE 6. CD4:CD8 ratio in several organs of controls and SIV-infected monkeys.
Single-cell suspensions from the indicated sites were analyzed by flow
cytometry, cells were gated on the CD3+ T cell
subpopulation. The ratio between the subpopulations was calculated.
Values represent the average ± SD of the five uninfected
controls, three animals at the 2 wk and four at 11 wk after SIV
infection.
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The naive/memory phenotype of the CD8+ T cells
was also analyzed in uninfected as well as in 11 wk p.i. animals at
sites other than the CNS. At all sites except the brain, the proportion
of cells expressing memory phenotype (low levels of CD45RA) was higher
in infected monkeys than in controls (Fig. 7
), suggesting that the infection led to
a systemic enrichment of memory cells. The comparison of
CD8+ T cells from different sites within the same
group of animals revealed that in control monkeys, the percent of
memory CD8+ T cells (expressing
CD45RAlow) in the brain was significantly higher
(F = 10.334; p < 0.01) than in
the periphery (Fig. 7
). However, in the SIV-infected monkeys,
the percent of memory CD8+ T cells was comparable
at all sites analyzed. In the CNS, 11 wk-infected and control animals
had the same level of predominance of memory CD8+
T cells, but their absolute number differed greatly.

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FIGURE 7. Percentage of CD8+ T cells expressing memory phenotype
(CD45RAlow) in several locations from uninfected controls
and 11 wk SIV-infected monkeys. Single-cell suspensions originating
from the indicated sites were analyzed by flow cytometry, cells were
gated on the CD8+CD3+ T cell subpopulation.
Values represent the average ± SD of the five uninfected controls
and four 11-wk-SIV-infected animals.
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To better characterize these CD8+ T cells, we
compared the phenotype of activation markers expressed on the
CD3+CD8+ cells obtained
from different sites in uninfected control animals to those at 11 wk
p.i. Generally, the percent of cells expressing CD11a, which defines
activation status, tended to be higher in infected animals than in
controls (Fig. 8
A), especially
in the spleen and lymph nodes, where the differences were statistically
significant (F = 8.733; p < 0.001).
Additionally, both infected and control animals had a significantly
higher (F = 26.521; p < 0.001)
percentage of CD8+ T cells expressing
CD11ahigh in the CNS than in the periphery. Close
to 100% of the cells in the CNS expressed high levels of CD11a, but in
peripheral sites of control as well as infected monkeys, the proportion
of cells was always much below that level. The level of CD95 expression
did not differ statistically between controls and infected monkeys for
each individual site analyzed, but CD8+ T cells
from the CNS tended to have a higher level of CD95 expression than
those in the periphery (Fig. 8
B). The expression of CD28 was
also evaluated on CD8+ cells from the CNS and
other sites in SIV-infected monkeys and controls (Fig. 8
C)
but the differences were not significant. CD8+ T
cells in the CNS seem to differ phenotypically, then, from those in the
periphery, and the SIV-induced changes in the CNS do not involve
differences on the expression of the surface markers that we analyzed.
Similar to the animals infected for 11 wk, the
CD8+ T cells in the brain of 2-wk-infected
animals did not differ from uninfected controls for the level of
expression of the activation markers analyzed (Fig. 8
D).

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FIGURE 8. Phenotypic characterization of the CD8+ T cells in
different sites of SIV-infected and control monkeys. A,
Percent of CD8+ T cells expressing high levels of CD11a.
B, Percent of CD8+ cells expressing high
levels of CD95. C, Percent of CD8+ T cells
negative for expression of CD28. D, Percent of
CD8+ T cells in the brain expressing high levels of
CD11ahigh levels of CD95, negative for expression of CD28,
and low levels of CD45RA. Single-cell suspensions originating from the
indicated sites were analyzed by flow cytometry, cells were gated on
the CD8+CD3+ T cell subpopulation. Values
represent the average ± SD of the five uninfected controls, three
animals at 2 wk (D only), and four animals at 11 wk
after SIV infection.
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Fig. 9
represents examples of
fluorescence intensity histograms of the surface markers we
investigated. The differences between controls (Fig. 9
A) and
SIV-infected monkeys (Fig. 9
B) were strongest for CD11a and
CD95 expression. Clearly, the uninfected controls expressed lower
levels of these markers and fewer cells with an abundance of these
markers than SIV-infected animals. This was true at all sites analyzed,
except the brain. Although control animals varied considerably in CD28
expression, CD8+ T cells from infected animals
had less CD28 than CD8+ cells from three of five
control animals, regardless of site.

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|
FIGURE 9. Representative FACS histograms exemplifying the expression of
activation markers. The values shown are for one representative animal
from each group (control uninfected and SIV-infected). Single-cell
suspensions originating from the indicated sites were analyzed by flow
cytometry, cells were gated on the CD8+CD3+ T
cell subpopulation. The numbers between keys (defining M2) represent
the percentage of cells situated in that range of expression. The
numbers above each histogram represent the geometric mean of the
fluorescence intensity for the whole subpopulation, indicating the
average level of expression.
|
|
Because CD8+ T cells from the brain presented a
more highly activated phenotype than those in the periphery, we
determined whether these brain cells produced cytolytic and potentially
neurotoxic molecules. The cells isolated from the brain were enriched
for CD8+ cells by a magnetic positive selection.
RNA extracted from these CD8+ cells was then
analyzed for detection of gene expression by RT-PCR. As a result, mRNA
for granzymes A and B, perforin, and IFN-
, all agents of tissue
destruction, was identified in the CD8+
cells from the brains of SIV-infected animals at both 2 and 11 wk p.i.
(Fig. 10
). In contrast, the mRNA of
CD8+ cells obtained from the brains of control
animals contained heterogeneous and variable expression patterns of the
analyzed molecules.
 |
Discussion
|
|---|
Here we used a microglia isolate of SIV to infect rhesus macaques
for the purpose of correlating relevant immune cells within and outside
the brain with CNS infection and dysfunction. The brain of each
infected animal was examined for the infiltration of lymphoid cells.
Immunohistochemical analysis of several brain segments from infected
animals showed the presence of mononuclear leukocytes in the parenchyma
and in the perivascular area. These infiltrating cells were
predominantly CD3+CD8+ T
cells, a subset that was dramatically increased in the brain tissue of
the infected animals at 11 wk p.i. compared with uninfected
controls.
Periodic evaluation of T cell subpopulations in the circulation of the
infected animals over an 11-wk-period showed an enlargement of the CD8
compartment, peaking at 2 wk p.i., although the
CD4+ cells remained unaltered. Indeed, a burst of
proliferation is found in the CD8+ T cell
compartment within the first few weeks of SIV infection (27, 36). Our study as well as those of others show that these
CD8+ T cells express high levels of CD11a on the
surface (37, 38), indicating that these cells become
activated and able toadhere to endothelial cells, possibly
followed by transmigration to tissue parenchyma. In parallel with the
changes in blood CD8+ T cells at 2 wk p.i., a
corresponding mild increase in the number of cells is found in the
brain at 2 wk p.i. However, at 11 wk p.i., while the number of
CD8+ T cells in the circulation has declined from
the 2 wk p.i. peak, the CD8+ T cell presence in
the brain has increased to over 5-fold in number compared with
controls.
In peripheral sites such as the blood, spleen, and lymph nodes, the
CD4:CD8 ratio of the infected animals decreased in comparison to
control animals. This finding suggests that the virus infection induced
an expansion of the CD8 compartment relative to CD4 that was not
restricted to the brain, but was a systemic phenomenon, confirming
previous observations in the SIV model and also in HIV
(39). However, the CD4:CD8 ratio in the brains of
SIV-infected monkeys and their uninfected counterparts was not
statistically different and, in both, was lower than in the periphery.
This evidence points to the predominance of CD8+
T cells in the CNS even under nonpathological conditions, as has been
found in the CSF of monkeys (40) and brains of mice
(41).
Naive CD8 cells in monkeys express high levels of CD45RA, but memory
cells lose these molecules from the surface (27). However,
an effector CD8+ T cell does not necessarily
present low levels of CD45RA, because cells with naive phenotype can
have effector activity, with expression of high levels of CD11a.
Effector CD8+ T cells initiate transcription of
cytokines such as IFN-
early in the activation process. Other
effector molecules such as granzymes A and B are expressed upon
acquisition of cytotoxic function, and their expression is a useful
marker of CTL activation in vivo (32, 42). Here, we found
that brain-infiltrating CD8+ cells expressed mRNA
for the cytotoxic granule proteins perforin, granzyme A and granzyme B,
as well as for IFN-
, indicating an effector phenotype at 2 and 11 wk
after infection. CD8+ T cells can regulate
IFN-
expression in an Ag-specific manner, expressing it when in
contact with their cognate Ag (42). The expression pattern
we found here is consistent with our previous immunohistochemical and
in situ hybridization studies identifying granzyme B and IFN-
expression in the brains of SIV-infected monkeys (20, 43),
and our finding of infiltrating SIV-specific cells with CTL activity in
the brains of infected monkeys (33).
Despite having a similar pattern of surface marker expression,
CD8+ cells in the brains of uninfected animals
have an inconsistent or mixed pattern of mRNA expression for the
proinflammatory and cytolytic molecules. This suggests that the few CD8
cells in the brain of uninfected animals are circulating cells not
carrying out an effector function, but may represent cells in what is
termed a surveillance mode (44). Highly activated cells,
silent in terms of effector molecule production, can circulate in the
blood and tissues, switching to an effector mode upon Ag stimulation.
At the second week after infection, although only a limited increase in
the number of CD8+ T cells in the brain was
observed, the cells that got into the brain were indeed qualitatively
different, because the expression of these molecules was consistent and
homogeneous. At 11 wk, a highly significant increase in the
CD8+ T cell population occurred with maintenance
of a consistent and homogeneous level of production of effector
molecules. These findings suggest that the CD8 cells that are present
in the brain of SIV-infected animals are qualitatively as well as
quantitatively distinct from controls.
Certainly, the increased number of
CD3+CD8+ T cells at 11 wk,
a relatively early stage of infection, was a most remarkable finding in
terms of brain cell populations. However, the fact that we identified
1) far greater expression of activation markers on brain-derived
CD8+ T cells than on those in the periphery and
2) expression of RNA to cytolytic and proinflammatory molecules adds a
functional dimension to the quantitative changes. Immunohistochemical
and FACS studies by others have indeed shown that
CD8+ T cells are present in the brains of
SIV-infected macaques both at the acute, and later stages of the
disease (3, 45, 46). The CD8+ T
cells that migrate into the brain can have a protective antiviral role
(45), as suggested by the lowering of brain viral load in
the 11 wk vs 2 wk p.i. animals and as described for other CNS viral
infections such as influenza encephalitis (47). However, a
pathogenic role of CD8+ T cells in virus-induced
CNS disease has been suggested not only by us for SIV-infected monkeys
(33), but for HTLV-I in humans (48), as well
as mouse hepatitis virus, Semliki, Borna, and Theilers virus
infections in rodents (49, 50, 51, 52).
It is notable that there was a temporal coincidence between peripheral
and CNS events related to the peak of virus load, but not to the
increase of CD8+ T cells. There is a time lag
between the expansion of CD11ahigh
CD8+ T cells in the periphery and their
accumulation into the brain. This suggests that the increase of
CD8+ T cells in the brain is not exclusively due
to a systemic circumstance of activation and expansion of the CD8
subset, although such a change is potentially reflected in the mild
increase in brain CD8+ T cells found at 2 wk p.i.
It is recognized that activated T cells can cross the unaltered
blood-brain barrier into the brain, independent of Ag specificity, as
part of general immune surveillance of the CNS (53). Naive
T cells can also enter an inflamed CNS (54). However, the
retention and accumulation of T cells in the CNS requires Ag
recognition (55, 56), as does effector molecule production
(54). Thus, following infection and the consequent Ag
stimulation, the CD8+ T cell population observed
in the brain has changed from one mainly composed of cells in the
surveillance mode to one of effector activity, capable of producing
molecules that can target infected cells, but also with the capacity of
inducing tissue damage. The CNS is a specialized tissue with a very low
degree of functional redundancy and is thus quite sensitive to cellular
injury that can compromise the ability of the brain to carry out its
critical role.
Overall, our data support the concept that activated cells are present
in the CNS. With respect to the expression of CD45RA in our
experiments, the percentage of naive and memory
CD8+ T cells in the brain was not statistically
different between SIV-infected and control animals. This was due to the
fact that uninfected controls presented a higher percentage of
CD8+ T cells with memory phenotype in the brain
in comparison to the periphery. Interestingly, recent data indicate
that specific CD8+ memory and effector T cells
migrate to nonlymphoid tissues (57). In other experimental
models, particularly in rodents, specificity and even MHC restriction
were not crucial factors for T cells to infiltrate the CNS, suggesting
that SIV-specific cells are not the only ones to enter the brain
(53, 54). However, the CD8+
lymphocytes we found in the brain of either infected or control monkeys
were close to 100% CD11ahigh and
CD95high, indicating an activation status
independent of the expression of memory markers (28).
Although studies have indicated that low levels of CD28 expression
correlate with effector function and infection (31), we
saw no such differences, at least not to the level of significance, for
the expression of this marker in the infected monkeys. In part, this
may have resulted from the uneven expression of CD28 in the uninfected
animals. However, not all effector cells exhibit low levels of CD28, as
SIV-specific CTL, identified by tetramer binding studies, are divided
for the expression of this marker (38). Others have shown
a population of CD8+CD28+
cells that expanded during viral infection, produced IFN-
and
perforin, and had effector function. This cell subset, as opposed to
the CD28- population, retained the ability to
proliferate (58). The nature of these cells in the
periphery as well as the brain has yet to be examined in SIV-infected
subjects. Our results emphasize that alterations in CNS T cell
subpopulations soon after infection result in an abnormally large
number of activated CD8+ T cells in the brain;
this may increase the concentrations of cytolytic and proinflammatory
cytokines to a pathological level.
The purified brain CD8+ cells obtained from
SIV-infected animals expressed the mRNA for perforin, granzymes A and
B, important molecules involved in the apoptotic process and cell
lysis, as well as for IFN-
. Proinflammatory cytokines, among them
IFN-
, are known to induce an increase in the expression of adhesion
molecules like ICAM-1 on endothelial cells (59). ICAM-1 is
the ligand for CD11a (LFA-1) expressed on the leukocyte cell surface.
The up-regulation of this molecule may play a role in the adhesion of
leukocytes to the endothelial cell, a step that precedes the
transmigration to the tissue, especially if LFA-1 expression is also
increased on the cell surface, as we observed here on
CD8+ T lymphocytes. Thus, IFN-
can contribute
to the migration of CD11ahigh
CD8+ T cells to the parenchyma. Additionally,
IFN-
can regulate cell traffic into the brain by inducing chemokine
synthesis (60) and can function as a potent
macrophage/microglia activator that induces the production of
potentially neurotoxic molecules.
Our results suggest that the SIV infection promotes a numerical
increase of CD8+ T lymphocytes with the capacity
to enter the CNS. The accumulation of such cells in the brain, despite
having a protective role, can potentially induce cellular damage in the
brains of SIV-infected monkeys and incite the functional CNS
abnormalities characteristic of this and similar infections.
 |
Acknowledgments
|
|---|
We thank Curtis Phillipson and Cathie York-DeFalco for technical
assistance, Joe Trotter, Allan Saluk, and Mary Cleary for helpful
suggestions on FACS techniques, Dr. Lisa Madden for help with
statistical analysis and for a critical review of the manuscript, Dr.
Michael Buchmeier for stimulating discussion, and Phyllis Minick and
Michael Gilliatt for editorial assistance.
 |
Footnotes
|
|---|
1 This work was supported by National Institutes of Health Grants MH59468, MH61224, and MH62261 (to H.S.F.) and DA12444 (to S.J.H.). This is manuscript number 14001-NP from The Scripps Research Institute. 
2 Address correspondence and reprint requests to Dr. Howard S. Fox, Department of Neuropharmacology, The Scripps Research Institute, 10550 North Torrey Pines Road, CVN 8, La Jolla, CA 92037. E-mail address: hsfox{at}scripps.edu 
3 Abbreviations used in this paper: EP, evoked potential; CSF, cerebrospinal fluid; p.i., postinoculation; bDNA, branched DNA; BSAEP, brainstem auditory EP; AEP, auditory EP; LCA, leukocyte common Ag. 
Received for publication May 10, 2001.
Accepted for publication August 31, 2001.
 |
References
|
|---|
-
Desrosiers, R. C.. 1990. The simian immunodeficiency viruses. Annu. Rev. Immunol. 8:557.[Medline]
-
Burudi, E. M. E., H. S. Fox. 2001. Simian immunodeficiency virus model of HIV-induced central
nervous system dysfuntion. M. J. Buchmeier, and I. Campbell, eds. Advances in Virus Research, Vol. 56 431. Academic Press, .
-
Chakrabarti, L., M. Hurtrel, M. A. Maire, R. Vazeux, D. Dormont, L. Montagnier, B. Hurtrel. 1991. Early viral replication in the brain of SIV-infected rhesus monkeys. Am. J. Pathol. 139:1273.[Abstract]
-
Lackner, A. A., P. Vogel, R. A. Ramos, J. D. Kluge, M. Marthas. 1994. Early events in tissues during infection with pathogenic (SIVmac239) and nonpathogenic (SIVmac1A11) molecular clones of simian immunodeficiency virus. Am. J. Pathol. 145:428.[Abstract]
-
Wiley, C. A., E. Masliah, M. Morey, C. Lemere, R. DeTeresa, M. Grafe, L. Hansen, R. Terry. 1991. Neocortical damage during HIV infection. Ann. Neurol. 29:651.[Medline]
-
Kure, K., K. M. Weidenheim, W. D. Lyman, D. W. Dickson. 1990. Morphology and distribution of HIV-1 gp41-positive microglia in subacute AIDS encephalitis: pattern of involvement resembling a multisystem degeneration. Acta Neuropathol. 80:393.[Medline]
-
Navia, B. A., E. S. Cho, C. K. Petito, R. W. Price. 1986. The AIDS dementia complex. II. Neuropathology. Ann. Neurol. 19:525.[Medline]
-
Wiley, C. A., R. D. Schrier, J. A. Nelson, P. W. Lampert, M. B. Oldstone. 1986. Cellular localization of human immunodeficiency virus infection within the brains of acquired immune deficiency syndrome patients. Proc. Natl. Acad. Sci. USA 83:7089.[Abstract/Free Full Text]
-
Williams, K. C., S. Corey, S. V. Westmoreland, D. Pauley, H. Knight, C. deBakker, X. Alvarez, A. A. Lackner. 2001. Perivascular macrophages are the primary cell type productively infected by simian immunodeficiency virus in the brains of macaques: implications for the neuropathogenesis of AIDS. J. Exp. Med. 193:905.[Abstract/Free Full Text]
-
Sharer, L. R., J. Michaels, M. Murphey-Corb, F. S. Hu, D. J. Kuebler, L. N. Martin, G. B. Baskin. 1991. Serial pathogenesis study of SIV brain infection. J. Med. Primatol. 20:211.[Medline]
-
Lackner, A. A., M. O. Smith, R. J. Munn, D. J. Martfeld, M. B. Gardner, P. A. Marx, S. Dandekar. 1991. Localization of simian immunodeficiency virus in the central nervous system of rhesus monkeys. Am. J. Pathol. 139:609.[Abstract]
-
Haase, A. T.. 1986. Pathogenesis of lentivirus infections. Nature 322:130.[Medline]
-
Price, R. W., B. J. Brew. 1988. The AIDS dementia complex. J. Infect. Dis. 158:1079.[Medline]
-
Heaton, R. K., R. A. Velin, J. A. McCutchan, S. J. Gulevich, J. H. Atkinson, M. R. Wallace, H. P. Godfrey, D. A. Kirson, I. Grant. 1994. Neuropsychological impairment in human immunodeficiency virus infection: implications for employment. HNRC Group. HIV Neurobehavioral Research Center. Psychosom. Med. 56:8.[Abstract/Free Full Text]
-
Bacellar, H., A. Munoz, E. N. Miller, B. A. Cohen, D. Besley, O. A. Selnes, J. T. Becker, J. C. McArthur. 1994. Temporal trends in the incidence of HIV-1-related neurologic diseases: Multicenter AIDS Cohort Study, 19851992. Neurology 44:1892.[Abstract/Free Full Text]
-
Sacktor, N. C., R. H. Lyles, R. L. Skolasky, D. E. Anderson, J. C. McArthur, G. McFarlane, O. A. Selnes, J. T. Becker, B. Cohen, J. Wesch, E. N. Miller. 1999. Combination antiretroviral therapy improves psychomotor speed performance in HIV-seropositive homosexual men: Multicenter AIDS Cohort Study (MACS). Neurology 52:1640.[Abstract/Free Full Text]
-
Darko, D. F., J. C. Miller, C. Gallen, J. White, J. Koziol, S. J. Brown, R. Hayduk, J. H. Atkinson, J. Assmus, D. T. Munnell, et al 1995. Sleep electroencephalogram delta-frequency amplitude, night plasma levels of tumor necrosis factor
, and human immunodeficiency virus infection. Proc. Natl. Acad. Sci. USA 92:12080.[Abstract/Free Full Text]
-
Pagano, M. A., P. E. Cahn, M. L. Garau, C. A. Mangone, H. A. Figini, A. A. Yorio, M. C. Dellepiane, M. G. Amores, H. M. Perez, A. D. Casiro. 1992. Brain-stem auditory evoked potentials in human immunodeficiency virus- seropositive patients with and without acquired immunodeficiency syndrome. Arch. Neurol. 49:166.[Abstract]
-
Kolson, D. L., E. Lavi, F. Gonzalez-Scarano. 1998. The effects of human immunodeficiency virus in the central nervous system. Adv. Virus Res. 50:1.[Medline]
-
Fox, H. S., M. R. Weed, S. Huitron-Resendiz, J. Baig, T. F. Horn, P. J. Dailey, N. Bischofberger, S. J. Henriksen. 2000. Antiviral treatment normalizes neurophysiological but not movement abnormalities in simian immunodeficiency virus-infected monkeys. J. Clin. Invest. 106:37.[Medline]
-
Horn, T. F. W., S. Huitron-Resendiz, M. R. Weed, S. J. Henriksen, H. S. Fox. 1998. Early physiological abnormalities after simian immunodeficiency virus infection. Proc. Natl. Acad. Sci. USA 95:15072.[Abstract/Free Full Text]
-
Murray, E. A., D. M. Rausch, J. Lendvay, L. R. Sharer, L. E. Eiden. 1992. Cognitive and motor impairments associated with SIV infection in rhesus monkeys. Science 255:1246.[Abstract/Free Full Text]
-
Raymond, L. A., D. Wallace, J. K. Marcario, R. Raghavan, O. Narayan, L. L. Foresman, N. E. Berman, P. D. Cheney. 1999. Motor evoked potentials in a rhesus macaque model of neuro-AIDS. J. Neurovirol. 5:217.[Medline]
-
Silverstein, A. M., N. R. Rose. 2000. There is only one immune system! The view from immunopathology. Semin. Immunol. 12:173.[Medline]
-
Sopper, S., C. Stahl-Hennig, M. Demuth, I. C. Johnston, R. Dorries, V. ter Meulen. 1997. Lymphocyte subsets and expression of differentiation markers in blood and lymphoid organs of rhesus monkeys. Cytometry 29:351.[Medline]
-
Schmitz, J. E., M. J. Kuroda, S. Santra, V. G. Sasseville, M. A. Simon, M. A. Lifton, P. Racz, K. Tenner-Racz, M. Dalesandro, B. J. Scallon, et al 1999. Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes. Science 283:857.[Abstract/Free Full Text]
-
Kaur, A., C. L. Hale, S. Ramanujan, R. K. Jain, R. P. Johnson. 2000. Differential dynamics of CD4+ and CD8+ T-lymphocyte proliferation and activation in acute simian immunodeficiency virus infection. J. Virol. 74:8413.[Abstract/Free Full Text]
-
Hoflich, C., W. D. Docke, A. Busch, F. Kern, H. D. Volk. 1998. CD45RAbright/CD11abright CD8+ T cells: effector T cells. Int. Immunol. 10:1837.[Abstract/Free Full Text]
-
Suzuki, I., S. Martin, T. E. Boursalian, C. Beers, P. J. Fink. 2000. Fas ligand costimulates the in vivo proliferation of CD8+ T cells. J. Immunol. 165:5537.[Abstract/Free Full Text]
-
Powell, J. D., J. A. Ragheb, S. Kitagawa-Sakakida, R. H. Schwartz. 1998. Molecular regulation of interleukin-2 expression by CD28 co-stimulation and anergy. Immunol. Rev. 165:287.[Medline]
-
Fiorentino, S., M. Dalod, D. Olive, J. G. Guillet, E. Gomard. 1996. Predominant involvement of CD8+CD28- lymphocytes in human immunodeficiency virus-specific cytotoxic activity. J. Virol. 70:2022.[Abstract]
-
Hamann, D., P. A. Baars, M. H. Rep, B. Hooibrink, S. R. Kerkhof-Garde, M. R. Klein, R. A. van Lier. 1997. Phenotypic and functional separation of memory and effector human CD8+ T cells. J. Exp. Med. 186:1407.[Abstract/Free Full Text]
-
von Herrath, M., M. B. Oldstone, H. S. Fox. 1995. Simian immunodeficiency virus (SIV)-specific CTL in cerebrospinal fluid and brains of SIV-infected rhesus macaques. J. Immunol. 154:5582.[Abstract]
-
Watry, D., T. E. Lane, M. Streb, H. S. Fox. 1995. Transfer of neuropathogenic simian immunodeficiency virus with naturally infected microglia. Am. J. Pathol. 146:914.[Abstract]
-
Prospero-Garcia, O., L. H. Gold, H. S. Fox, I. Polis, G. F. Koob, F. E. Bloom, S. J. Henriksen. 1996. Microglia-passaged simian immunodeficiency virus induces neurophysiological abnormalities in monkeys. Proc. Natl. Acad. Sci. USA 93:14158.[Abstract/Free Full Text]
-
Sopper, S., U. Sauer, J. G. Muller, C. Stahl-Hennig, V. ter Meulen. 2000. Early activation and proliferation of T cells in simian immunodeficiency virus-infected rhesus monkeys. AIDS Res. Hum. Retroviruses 16:689.[Medline]
-
Rosenberg, Y. J., A. Cafaro, T. Brennan, J. G. Greenhouse, K. McKinnon, S. Bellah, J. Yalley-Ogunro, S. Gartner, M. G. Lewis. 1997. Characteristics of the CD8+ lymphocytosis during primary simian immunodeficiency virus infections. AIDS 11:959.[Medline]
-
Kuroda, M. J., J. E. Schmitz, W. A. Charini, C. E. Nickerson, M. A. Lifton, C. I. Lord, M. A. Forman, N. L. Letvin. 1999. Emergence of CTL coincides with clearance of virus during primary simian immunodeficiency virus infection in rhesus monkeys. J. Immunol. 162:5127.[Abstract/Free Full Text]
-
Rosenberg, Y. J., A. O. Anderson, R. Pabst. 1998. HIV-induced decline in blood CD4/CD8 ratios: viral killing or altered lymphocyte trafficking?. Immunol. Today 19:10.[Medline]
-
Sopper, S., S. Hemm, J. Meixensberger, C. Coulibaly, C. Stahl-Hennig, G. Hunsmann, B. Fleckenstein, V. ter Meulen, R. Dorries. 1993. Dynamics of the immune system response in cerebrospinal fluid and blood of SIVmac-infected rhesus monkeys. J. Med. Primatol. 22:138.[Medline]
-
Brabb, T., P. von Dassow, N. Ordonez, B. Schnabel, B. Duke, J. Goverman. 2000. In situ tolerance within the central nervous system as a mechanism for preventing autoimmunity. J. Exp. Med. 192:871.[Abstract/Free Full Text]
-
Slifka, M. K., F. Rodriguez, J. L. Whitton. 1999. Rapid on/off cycling of cytokine production by virus-specific CD8+ T cells. Nature 401:76.[Medline]
-
Lane, T. E., M. J. Buchmeier, D. D. Watry, H. S. Fox. 1996. Expression of inflammatory cytokines and inducible nitric oxide synthase in brains of SIV-infected rhesus monkeys: applications to HIV-induced central nervous system disease. Mol. Med. 2:27.[Medline]
-
Slifka, M. K., J. L. Whitton. 2000. Antigen-specific regulation of T cell-mediated cytokine production. Immunity 12:451.[Medline]
-
Sopper, S., U. Sauer, S. Hemm, M. Demuth, J. Muller, C. Stahl-Hennig, G. Hunsmann, V. ter Meulen, R. Dorries. 1998. Protective role of the virus-specific immune response for development of severe neurologic signs in simian immunodeficiency virus-infected macaques. J. Virol. 72:9940.[Abstract/Free Full Text]
-
Boche, D., E. Khatissian, F. Gray, P. Falanga, L. Montagnier, B. Hurtrel. 1999. Viral load and neuropathology in the SIV model. J. Neurovirol. 5:232.[Medline]
-
Stevenson, P. G., S. Hawke, C. R. Bangham. 1996. Protection against lethal influenza virus encephalitis by intranasally primed CD8+ memory T cells. J. Immunol. 157:3065.[Abstract]
-
Biddison, W. E., R. Kubota, T. Kawanishi, D. D. Taub, W. W. Cruikshank, D. M. Center, E. W. Connor, U. Utz, S. Jacobson. 1997. Human T cell leukemia virus type I (HTLV-I)-specific CD8+ CTL clones from patients with HTLV-I-associated neurologic disease secrete proinflammatory cytokines, chemokines, and matrix metalloproteinase. J. Immunol. 159:2018.[Abstract]
-
Morris, M. M., H. Dyson, D. Baker, L. S. Harbige, J. K. Fazakerley, S. Amor. 1997. Characterization of the cellular and cytokine response in the central nervous system following Semliki Forest virus infection. J. Neuroimmunol. 74:185.[Medline]
-
Sobbe, M., T. Bilzer, S. Gommel, K. Noske, O. Planz, L. Stitz. 1997. Induction of degenerative brain lesions after adoptive transfer of brain lymphocytes from Borna disease virus-infected rats: presence of CD8+ T cells and perforin mRNA. J. Virol. 71:2400.[Abstract]
-
Murray, P. D., K. D. Pavelko, J. Leibowitz, X. Lin, M. Rodriguez. 1998. CD4+ and CD8+ T cells make discrete contributions to demyelination and neurologic disease in a viral model of multiple sclerosis. J. Virol. 72:7320.[Abstract/Free Full Text]
-
Wu, G. F., A. A. Dandekar, L. Pewe, S. Perlman. 2000. CD4 and CD8 T cells have redundant but not identical roles in virus-induced demyelination. J. Immunol. 165:2278.[Abstract/Free Full Text]
-
Hickey, W. F., B. L. Hsu, H. Kimura. 1991. T-lymphocyte entry into the central nervous system. J. Neurosci. Res. 28:254.[Medline]
-
Krakowski, M. L., T. Owens. 2000. Naive T lymphocytes traffic to inflamed central nervous system, but require antigen recognition for activation. Eur. J. Immunol. 30:1002.[Medline]
-
Hirschberg, D. L., G. Moalem, J. He, F. Mor, I. R. Cohen, M. Schwartz. 1998. Accumulation of passively transferred primed T cells independently of their antigen specificity following central nervous system trauma. J. Neuroimmunol. 89:88.[Medline]
-
Zeine, R., T. Owens. 1992. Direct demonstration of the infiltration of murine central nervous system by Pgp-1/CD44high CD45RBlow CD4+ T cells that induce experimental allergic encephalomyelitis. J. Neuroimmunol. 40:57.[Medline]
-
Masopust, D., V. Vezys, A. l. Marzo, L. Lefrancois. 2001. Preferential localization of effector memory cells in nonlymphoid tissue. Science 291:2413.[Abstract/Free Full Text]
-
Fiorentini, S., S. Licenziati, G. Alessandri, F. Castelli, S. Caligaris, M. Bonafede, M. Grassi, E. Garrafa, A. Balsari, A. Turano, A. Caruso. 2001. CD11b expression identifies CD8+CD28+ T lymphocytes with phenotype and function of both naive/memory and effector cells. J. Immunol. 166:900.[Abstract/Free Full Text]
-
Shen, J., T. T. SS, L. Schrieber, N. J. King. 1997. Early E-selectin, VCAM-1, ICAM-1, and late major histocompatibility complex antigen induction on human endothelial cells by flavivirus and comodulation of adhesion molecule expression by immune cytokines. J. Virol. 71:9323.[Abstract]
-
Tran, E. H., E. N. Prince, T. Owens. 2000. IFN-
shapes immune invasion of the central nervous system via regulation of chemokines. J. Immunol. 164:2759.[Abstract/Free Full Text]
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