The Journal of Immunology, 1998, 160: 2896-2904.
Copyright © 1998 by The American Association of Immunologists
CTL Effector Function Within the Central Nervous System Requires CD4+ T Cells1
Stephen A. Stohlman2,*,
,
Cornelia C. Bergmann*,
,
Mark T. Lin
,
Daniel J. Cua
and
David R. Hinton*,
Departments of
*
Neurology and Molecular Microbiology,
Immunology, and
Pathology, University of Southern California, Los Angeles, CA 90033
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Abstract
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CTL responses induced during most viral infections are independent
of help derived from the CD4+ T cell population.
However, clearance of virus from the central nervous system (CNS)
during infection with the neurotropic JHM strain of mouse hepatitis
virus is inhibited in the absence of CD4+ T cells. Adoptive
transfer of activated CD8+ T cells with virus-specific
cytolytic activity into CD4+ T cell-depleted hosts
demonstrated that CD4+ T cells were one component of the
host response required for expression of CTL effector function(s)
within the CNS. Analysis of mice infected with the JHM strain of mouse
hepatitis virus demonstrated that, in contrast to CD8+ T
cells, few CD4+ T cells entered the brain parenchyma.
Although fewer CD8+ T cells entered the brain parenchyma in
mice depleted of CD4+ T cells, access of CTL was not
inhibited in the absence of CD4+ T cells. The number of
apoptotic lymphocytes in the CNS increased in the absence of
CD4+ T cells, suggesting that CTL enter the CNS during
viral infection in a CD4-independent manner. However, these cells
rapidly undergo apoptosis, indicating that expression of CTL effector
function with the parenchyma of the CNS is CD4 dependent. These data
raise the possibility that programmed cell death of CD8+ T
cells within the CNS is due to the increased Ag present in the CNS of
infected CD4 depleted mice or that autocrine cytokines, which maintain
CTL activity within peripheral tissues, are inhibited in the
microenvironment of the CNS.
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Introduction
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Vigorous
host immune responses are associated with recovery from acute
infectious diseases and subsequent resistance to reinfection. The
CD8+ CTL response predominates as the major effector
arm of immunity in both clearance and protection from many viral
infections (1, 2, 3). Although cytokines released from CD8+ T
cells contribute to viral clearance (1, 4, 5, 6, 7), in vitro and in vivo
evidence suggests that the major effector mechanism for limiting viral
infection is the ability of CTL to lyse infected target cells (8, 9, 10, 11, 12, 13).
It has been suggested that clearance of infections produced by
noncytopathic viruses such as lymphocytic choriomeningitis virus
(LCMV)3 is predominantly
mediated by CTL. By contrast, clearance of cytopathic viruses is more
dependent on the humoral response (2, 3). However, the roles of these
effectors in the immune response to infections that localize to
specific tissue sites are not well understood. Access of immune
effectors is restricted from various tissues (14, 15), especially in
relatively immune privileged sites such as the central nervous system
(CNS). The inability of CTL to clear virus from specific tissues is
readily apparent following the adoptive transfer of LCMV-specific CTL
into chronic carrier mice (16, 17). CTL were relatively inefficient at
reducing LCMV in brain, kidney, salivary gland, and testis, indicating
that virus clearance from tissues with tight endothelium is
substantially delayed. Although CTL induction and virus clearance
following LCMV infection are independent of CD4+ T cells
and Ab responses, clearance of persistent LCMV infection requires
CD4+ T cells (18, 19, 21, 22). The mechanism(s) for this
requirement is not clear; however, the role of CD4+ T cells
may be to prevent exhaustion of CD8+ T cells in the
presence of large amounts of Ag (3, 23), possibly via the secretion of
IL-2 (4, 19, 23), which is required for both clonal expansion and
prevention of apoptosis (24).
In this report we examined the requirement for CD4+ T cells
in virus clearance from the parenchyma of the CNS following infection
by the neurotropic JHM strain of mouse hepatitis virus (JHMV). Analysis
of immune infiltrates within the CNS of rodents infected with JHMV
support the importance of CTL in the antiviral response by
demonstrating that CD8+ T cell accumulation coincides with
declining virus titers (25, 26). The protective role of CTL during JHMV
infection of the CNS was recently confirmed by the effects of
adoptively transferred CTL specific for the JHMV nucleocapsid (N)
protein into lethally infected recipients (27). N-specific CTL mediated
protection via partial elimination of CNS virus replication. Whereas
CTL suppressed virus replication in astrocytes and microglia (27), they
appeared to have had little or no direct effect on virus replication in
oligodendroglia.
Induction of CTL and subsequent clearance of many viruses are
independent of CD4+ T cells (1, 2, 18, 28, 29). For
example, vaccinia virus, influenza virus, and LCMV are cleared via a
vigorous CTL response in CD4-depleted mice or in mice in which the
CD4+ T cell response has been eliminated by gene disruption
(29). By contrast, Rauscher leukemia virus infection and Japanese
encephalitis virus infection of the CNS require both CTL and
CD4+ T cells for protection (30, 31). CD8+
CTL-mediated clearance of JHMV from the CNS also appears to be
dependent on CD4+ T cell-mediated help. This idea is based
on the demonstration that adoptive transfer of a virus-specific
CD4+ T cell population mediated protection and viral
clearance from the CNS in an MHC class I-dependent manner (32). It has
been further supported by the absence of JHMV clearance from mice
depleted of CD4+ T cells (33) or mice in which class II has
been eliminated by gene deletion (34) and the demonstrations that both
virus-specific CD4+ and CD8+ T cell clones
protect from lethal JHMV infections (35, 36, 37). These data suggest that
the hosts immune response to CNS infection by JHMV includes a
CD4+ T cell component necessary for CTL function. Whether
this is an intrinsic property of a virus that exhibits cytotoxicity in
vitro, but little or no direct cytopathology in vivo, or whether this
is due to the relatively immune-privileged target organ of infection is
not clear. In support of the idea that the site may play a role in the
regulation of CTL function, excellent CTL activity can be detected in
mononuclear cells isolated directly from the CNS during acute JHMV
infection (38, 39, 40, 41). However, there appears to be little or no
JHMV-specific CTL activity in peripheral organs during JHMV
infection (38, 40, 41).
This report confirms that the ability of CTL to reduce JHMV replication
in the CNS is dependent on CD4+ T cells. Mice depleted of
CD4+ T cells show decreased CD8+ T cell
cytolytic activity following infection, suggesting that
CD4+ T cells contribute to but are not required for the
development of CTL effector function. Adoptive transfers of
BrdU-labeled CTL activated in vitro into infected CD4+ T
cell-deficient recipients showed that entry into the CNS parenchyma was
not inhibited, although the number of cells within the CNS that
expressed CD8 was reduced. These data were reconciled by demonstrating
the presence of increased numbers of apoptotic lymphocytes within the
CNS of CD4-depleted mice. These findings indicate that CD4+
T cells are required for the maintenance of CD8+ T cell
effector function within the CNS, but are not an absolute requirement
for either CTL induction or trafficking of CD8+ T cells
into the parenchyma during acute CNS viral infection.
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Materials and Methods
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Mice
BALB/c By (H-2d), mice were obtained from The
Jackson Laboratory (Bar Harbor, ME) and housed in isolator cages.
Donors were immunized as previously described (27) using 1 x
106 plaque-forming units JHMV injected i.p. Recipient and
control mice were infected intracranially with 100 plaque-forming units
of JHMV in 30 µl of PBS. This inoculum is uniformly fatal within 7 to
9 days postinfection. Recipients were depleted of CD4+ T
cells by i.p. injection of 200 µg of anti-CD4 mAb GK1.5 at -2,
0, and +2 days relative to infection as previously described (33). Mice
depleted of CD8+ T cells were injected on the same schedule
with 200 µg of anti-CD8 mAb 2.43. Both regimens resulted in
98% depletion of their respective phenotypes, as analyzed by flow
cytometry (see below), 4 days after the final Ab injection.
Viruses and cell lines
The DM strain, a plaque-purified isolate derived from a suckling
mouse brain pool that has plaque morphology and pathogenesis consistent
with those of parental JHMV (42) was propagated and plaque assayed
using the murine DBT astrocytoma cell line as previously described (27, 39). Virus titers were determined by homogenization of one-half the
brain in 2.0 ml of Dulbeccos PBS, pH 7.4, using Tenbroeck tissue
homogenizers. The remainder was used for histopathologic analysis (see
below). Following centrifugation at 1500 x g for 10
min at 4°C, supernatants were assayed immediately or were frozen at
-70°C. Virus titers were determined by plaque assay using monolayers
of DBT cells as previously described (27). The data presented are the
average titers of groups of at least three mice per point.
Induction and transfer of CTL effectors
Spleen cell suspensions were prepared from mice immunized 3 to 8
wk earlier. Spleen cells (1 x 108) were cultured
for 6 days at 37°C in 40 ml of RPMI 1640 medium supplemented with
10% FBS (Gemini Bioproducts, Calabasas, CA), 2 mM glutamine, 25
µg/ml gentamicin, 1 mM sodium pyruvate, 5 x 10-5 M
ß2-ME, and nonessential amino acids (RPMI complete) plus 10% rat Con
A supernatant (RCS) containing 25 mM
-methylmannopyranoside and 1
µM pN318335 peptide as previously described (27, 43).
The 18-mer peptide containing the CTL epitope was used in most
experiments due to enhanced solubility compared with that of the
optimal 9-mer peptide. Viable cells were purified by centrifugation
onto Lympholyte M (Accurate Chemicals, Westbury, NY) cushions and
washed twice before transfer into naive recipients. Total cells (2
x 107) or CD8+ T cells (1 x
107) purified by positive selection using magnetic beads
(see below) were transferred i.v. either 4 to 6 h before infection
or at 2 days postinfection for BrdU-labeled cells (day 3 of infection).
Recipients were challenged with JHMV within 6 h of adoptive
transfer. To label CD8+ T cells for analysis of trafficking
into the CNS, 50 nM BrdU (Sigma Chemical Co., St. Louis, MO) was added
during in vitro expansion of memory cells for the final 48-h incubation
(15). BrdU-labeled CD8+ T cells were purified by positive
selection, as described below, before i.v. transfer. Inclusion of 50 nM
BrdU in the culture medium for 48 h did not alter cytolytic
activity compared with that in untreated cultures.
Flow cytometry and T cell purification
Cell surface expression following in vitro activation was
examined using rat anti-CD4 (L3T4), rat anti-CD8 (53-5.8), rat
anti-CD11a (M17/4), and rat anti-CD62L (MEL-14; PharMingen, San
Diego, CA) followed by goat anti-rat FITC-F(ab')2
Ab (Caltag, South San Francisco, CA). Cells were labeled, washed,
resuspended in PBS containing 0.1% formaldehyde, and analyzed on a
FACStar (Becton Dickinson, Mountain View, CA). Ab plus C-mediated
depletions were conducted as previously described (27), using rat
anti-CD4 (mAb RL172.4) and anti-CD8 (mAb 31M).
CD8+ T cells were purified using MACS Microbeads conjugated
to anti-CD8 mAb (clone 53-6.7; Miltenyi Biotec, Auburn, CA).
Peptide-stimulated cells (1.5 x 108) were incubated
for 40 min at 4°C with 100 µl of a colloidal microbead solution.
The cell suspension was passed over a saturated steel wool column
equilibrated with ice cold-PBS containing 0.5% BSA (PBS/BSA) attached
to a MACS magnet (Miltenyi Biotec). The unabsorbed CD8-
fraction was collected by eluting the column with 5 vol of chilled
PBS/BSA at a slow flow rate regulated by a 23-gauge needle. The
absorbed CD8+ fraction was collected by removing the magnet
and eluting the cells with PBS/BSA. Typically, >20% of the cells
applied to the column were recovered in the CD8+ fraction.
Purity was
98% as determined by flow cytometry.
CD4+ T cells from cultures stimulated in vitro with peptide
pN318335 were purified using MACS microbeads conjugated
with anti-CD4 mAb (GK1.5; Miltenyi Biotec) as described above. For
proliferation, CD4+ cells (1 x 105) were
cultured in complete RPMI 1640 medium supplemented with 7 x
105 irradiated syngeneic spleen cells and 1% syngeneic
mouse serum in 96-well plates. Cells were stimulated with lysates of
UV-inactivated, JHMV-infected, DBT cells (36) or with 0.2 to 20 nM
peptides (pN318335 or pN318326) for 72
h at 37°C and pulsed with 2 µCi of [3H]dThd (ICN
Radiochemicals, Irvine, CA)/well for the last 16 h of culture.
Incorporation was measured by liquid scintillation spectroscopy.
Cytotoxicity assay
Spleen cell suspensions (1 x 108)
prepared from infected normal and CD4-depleted mice were cultured for 6
days at 37°C with 0.1 µM pN318335 in 40 ml of
complete RPMI 1640 medium supplemented with 10% FBS (Gemini
Bioproducts). Cytolytic activity was measured in a 4-h 51Cr
release assay as previously described (27, 43). J774.1
(H-2d) target cells were washed and labeled with 100 µCi
of Na51CrO4 (New England Nuclear, Boston, MA).
Washed target cells were preincubated for 15 min at 37°C with 0.5 to
1.0 µM peptide and added at a concentration of 1 x
104 in a 100-µl volume. Data are expressed as the percent
specific release, defined as (experimental release - spontaneous
release)/total (detergent release - spontaneous release). Maximum
spontaneous release values were
20% of the total release values.
Histology and cell trafficking
For histopathologic analysis mice were killed by
CO2 asphyxiation. Brains were removed and bisected in
the midcoronal plane. Brains and spinal cords were fixed for 3 h
in Clarks solution (75% ethanol and 25% glacial acetic acid) and
embedded in paraffin. Sections were stained with either hematoxylin and
eosin or luxol fast blue for routine examination. The distribution of
JHMV Ag was examined using immunoperoxidase staining (Vectastain-ABC
kit, Vector Laboratories, Burlingame, CA) and anti-JHMV mAb J.3.3
specific for the carboxyl terminus of the N protein (44). The
distribution of BrdU+ cells was determined on
paraffin-embedded sections by the immunoperoxidase method (Vectastains
ABC Kit) using mouse mAb BU-33 (Sigma). To identify CD4+
and CD8+ T cells, immunoperoxidase staining was performed
on acetone-fixed frozen sections using rat anti-CD4+
(L3T4, PharMingen) and rat anti-CD8+ (Ly-2,
PharMingen). Anti-CD4+ and CD8+ Ab were
detected with biotinylated rabbit anti-rat Ab preabsorbed with
mouse serum (Vector Laboratories). Visualization was achieved using the
ABC kit with 3-amino-9-ethylcarbazole as chromogen. Terminal
deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling was
used to examine the distribution of apoptotic cells according to the
suppliers instructions (Oncor, Gaithersburg, MD).
Quantitation of lymphoid cells identified as being CD4+,
CD8+, or BrdU+ was performed on
immunoperoxidase-stained tissue sections counterstained with
hematoxylin. Brain sections were cut in the midsagittal plane of one
hemisphere and included olfactory cortex, hippocampus, and cerebellum.
All positively labeled cells in the section were counted using a x40
objective, with separate computations for subarachnoid, perivascular,
and parenchymal regions. Apoptotic cells were counted in a similar
manner, except that the location of the cells was not differentiated
since the vast majority of apoptotic cells were intraparenchymal in
location.
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Results
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CD8+ T cells and viral clearance
Adoptive transfer of virus-specific CTL derived from
JHMV-immunized donors expanded in vitro with either N-derived peptide
pN318335 or pN318326 mediates MHC
class I-dependent protection from an otherwise lethal infection via the
reduction of JHMV replication within the CNS (27). Analysis of the
transferred population by flow cytometry showed the presence of
approximately equal numbers of activated (MEL-14low)
CD4+ and CD11ahigh CD8+ T cells. To
determine whether activated CD4+ T cells were specific for
the pN318335 peptide, CD4+ cells were
purified after 6 days in vitro stimulation with peptide and tested for
proliferation to the peptides containing the JHMV N-protein CTL epitope
(43). No stimulation was detected using either the 18-mer
pN318335 peptide or the 9-mer pN318326
peptide (data not shown). Minimal JHMV-specific proliferation
(stimulation index =
3) was detected, suggesting
non-Ag-specific IL-2-driven in vitro expansion of the CD4+
T cells. Although an N-specific proliferative response has previously
been demonstrated in BALB/c mice (45), these data indicate that neither
the optimal 9-mer nor the larger 18-mer peptide contains a
JHMV-specific, H-2d-restricted, CD4+ T cell
epitope. However, the presence of activated CD4+ T cells
suggested the possibility that this population might participate in
clearance of JHMV from the CNS (35, 36, 37). To test whether the
CD4+ T cells derived following in vitro activation played a
role in reduction, CD8+ T cells were either depleted by mAb
plus C or positively selected by magnetic bead purification and
transferred to JHMV-infected recipients. Depletion of CD8+
T cells eliminated the majority, but not all, of the antiviral activity
present in the CNS (Table I
). Cells
treated with C only retained the ability to reduce CNS virus
replication. These data are consistent with previous reports suggesting
that CD4+ T cells participate in the clearance of JHMV
(35, 36, 37). By contrast, the transfer of 1 x 107
affinity-purified CD8+ T cells was as effective as transfer
of 2 x 107 unfractionated cells containing
approximately 50% activated CD8+ T cells (Table I
). These
data confirm that CD8+ T cells are a major effector
population mediating the partial reduction of virus replication within
MHC class I-positive CNS cells of JHMV-infected mice (27). Furthermore,
these data indicate that the CD4+ T cells activated in
vitro are not as effective as CTL at reducing virus replication within
the CNS; however, CD4+ T cells activated in situ may
provide protection (35, 36, 37) by an as yet unknown mechanism(s), possibly
via secretion of IFN-
or Fas/Fas ligand-mediated cytolysis.
CD4+ T cells are required for CTL-mediated viral
clearance
The protection afforded by JHMV-specific CD4+ T
cell clones requires recipients with intact immune responses (35). In
vitro activated JHMV-specific CTL were therefore transferred into
untreated and immunosuppressed recipients to determine whether
CTL-mediated virus clearance requires participation of the hosts
immune response. A single dose of irradiation was administered to
recipients on the same day as the i.v. transfer of 2.5 x
107 splenocytes derived by in vitro stimulation of memory
cells with pN peptide. In contrast to untreated recipients,
immunosuppression prevented CTL-mediated reduction of JHMV within the
CNS regardless of whether the CTL were transferred i.v. or
intracranially (Table II
). In addition,
immunosuppressed recipients were not protected from fatal disease (data
not shown), similar to the prevention of CD4+ T
cell-mediated protection by immunosuppression (36). The inability of
CD8+ T cells to mediate complete viral clearance from the
CNS (27) or protect immunosuppressed recipients (Table II
) suggested
the participation of additional components of the hosts immune
response that were activated during infection.
CD4+ T cells were suggested to provide help during
JHMV-specific CTL induction (32, 33); however, transfer of in vitro
activated CTL was expected to obviate this requirement. To further
define the role of the hosts immune response in CTL-mediated
clearance, purified pN-specific CTL derived by in vitro expansion were
transferred into untreated and CD4-depleted recipients. The transfer of
CTL into untreated recipients reduced viral replication within the CNS
and protected recipients (Table III
). By
contrast, no viral clearance from the CNS was detected in CTL
recipients depleted of CD4+ T cells, nor were they
protected from lethal disease (Table III
). These data suggest that
CD4+ T cells provide a critical function during JHMV
infection other than providing help during CTL induction. Depletion of
either CD4+ or CD8+ T cells inhibits JHMV
clearance from the CNS (33), which suggested that CD4+ T
cells are required during induction of JHMV-specific CTL (32, 33). To
determine whether the induction of JHMV-specific CD8+ T
cells is dependent on CD4+ T cells, untreated and
CD4-depleted mice were infected with JHMV, and the cytolytic activity
of splenocytes was determined 6 days postinfection following
pN318335 peptide restimulation in vitro. Restimulation is
required as no cytolytic activity ex vivo is detectable in the spleen
or cervical lymph nodes during acute JHMV infection (38, 41). CTL
specific for the pN epitope were primed in the absence of
CD4+ T cells (Fig. 1
).
However, 16-fold more cells were required to achieve half-maximal
killing (E:T cell ratio =
100:1) compared with cells from
untreated mice (E:T cell ratio =
6:1). The poor cytolytic
activity of CTL derived from infected CD4-depleted donors was overcome
by the addition of exogenous IL-2 in the form of RCS during in vitro
incubation (Fig. 1
). These data suggest that CD4+ T cells,
although not required for CTL induction following JHMV infection, play
a role in the development of CD8+ T cell effector function,
presumably by providing help in the form of IL-2 secretion.

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FIGURE 1. Virus-specific CTL induction in CD4-depleted, JHMV-infected, BALB/c
mice. Untreated and CD4-depleted mice were infected i.c. with 100
plaque-forming units of JHMV, and 6 days postinfection, splenocytes
were restimulated with 1 µM pN peptide in vitro for 6 days. The
cytolytic activity of cells cultured in the absence (left
panel) or the presence (right
panel) of 5% RCS was tested on pN-coated J774.1 target
cells at the indicated E:T cell ratios. Cytolysis is shown as the
percent specific lysis of peptide-coated minus untreated J774.1
targets.
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Entry of CD8+ T cells and maintenance of effector
function
To examine the effects of CD4 and CD8 T cell depletion on the
immunopathology of JHMV infection, mice were depleted of either
CD4+ or CD8+ T cells as previously
described (33). Both treatments were >98% effective as determined by
flow cytometric analysis of splenic populations and inhibited the
clearance of JHMV from the CNS (data not shown). Mice depleted of
either CD4+ or CD8+ T cells showed reduced, but
not absent, mononuclear cell infiltrations and increased numbers of
viral Ag-positive cells compared with untreated controls (Fig. 2
), consistent with analysis of mice
rendered CD4 and CD8 deficient by gene ablation (34). These data
suggested the possibility that inhibition of CTL-mediated virus
clearance from the CNS in CD4-depleted recipients was not due to
inhibition of CTL trafficking into the CNS. To examine this
possibility, BrdU was incorporated into pN-specific CTL by incubation
with 50 nM BrdU for the final 48 h of a 6-day in vitro culture
(15). Positively selected CD8+ T cells were transferred to
both untreated and CD4-depleted recipients at 3 days postinfection
(15), and the recipients were killed 48 h later. This time point
was chosen because preliminary experiments showed a significant
reduction of labeled cells within the CNS >2 days after the transfer,
consistent with previous results examining the trafficking of
BrdU-labeled CTL into the livers of transgenic recipients expressing Ag
(15). No differences in BrdU+ cells were detected in the
lungs of recipients, suggesting that the efficacy of the i.v. transfers
was similar in all recipients (data not shown). No difference in either
the number or the distribution of BrdU+ in vitro activated
CTL was detected in the CNS of JHMV-infected recipients when comparing
the untreated and CD4-depleted groups (Fig. 3
).

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FIGURE 2. Encephalitis and viral Ag in CD4- and CD8-depleted mice. Control mice
(a and b) show perivascular
lymphoid infiltrates (a, arrowheads) and scattered
cells immunoreactive for viral Ag (a, arrows). At
higher magnification (b), a mixed population
of glial cells is positive for viral Ag. Mice depleted of either
CD4+ (c and d) or
CD8+ (e and f) T cells
showed reduced, but not absent, mononuclear cell infiltration
(c, arrow head) and increased numbers of viral
Ag-positive cells (arrows) compared with untreated controls
(a and b). At higher magnification
(d and f), similar populations of
glial cells were positive for viral Ag. Immunoperoxidase staining was
performed using the avidin-biotin-peroxidase complex method for viral
Ag (mAb J.3.3) with hematoxylin counterstain. Magnification, x110
(a, c, and e) and x440
(b, d, and f).
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FIGURE 3. Trafficking of BrdU-labeled CD8+ T cells in
CD4-depleted recipients. BrdU was incorporated into pN-specific CTL
cultures, and positively selected CD8+ T cells were
transferred to both untreated (a andb) and CD4-depleted (c andd) recipients at 3 days postinfection with JHMV.
Recipients were killed 48 h later. Both the number and the
distribution of BrdU-labeled cells were similar in the CNS parenchyma
of CD4-depleted (c and d) and
control animals (a and b). The
lymphoid morphology of the labeled cells is clearly seen at higher
magnification (b and d).
Immunoperoxidase staining was performed using the
avidin-biotin-peroxidase complex method and anti-BrdU Ab with
hematoxylin counterstain. Magnification, x110 (a andc) and x440 (b andd).
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These data demonstrated that highly activated CD8+ CTL gain
access to the CNS during JHMV infection independent of the presence of
CD4+ T cells. Furthermore, they suggested that CTL gain
access to the CNS of CD4-depleted mice, yet express little or no
effector function(s). One possible mechanism to account for the
apparent discrepancy is a requirement for CD4+ T cells in
maintaining CTL viability. To determine whether CD4+ T
cells play a role in CTL survival, the frequency and distribution of
CD4+ T cells, CD8+ T cells, and apoptotic cells
within the CNS of untreated and CD4-depleted CTL recipients were
examined. The majority of CD4+ T cells accumulate in the
perivascular and subarachnoid spaces during JHMV infection of untreated
mice, with few gaining access to the CNS parenchyma (Figs. 4
and 5a). By contrast,
approximately 50% of CD8+ T cells are found within the
brain parenchyma (Figs. 4
and 5
b). Consistent with
previous results (13), few T cells are undergoing apoptosis within the
CNS of JHMV-infected mice, and the majority of these are localized
within the parenchyma (Fig. 5
c). In CD4-depleted
mice, no CD4+ T cells could be detected in the CNS by
immunohistochemistry, consistent with the
98% depletion of splenic
CD4+ T cells following GK1.5 mAb treatment. The number of
CD8+ T cells infiltrating the CNS was reduced to
approximately 50% the number recruited into the CNS of mice with a
normal complement of CD4+ T cells. Although the overall
number of cells that stain for CD8 was reduced, the percentage of
CD8+ cells within the parenchyma was approximately the same
in the CD4-depleted and untreated groups (Fig. 4
). However, the number
of apoptotic cells in CD4-depleted mice increased approximately
threefold (Fig. 5
). Although a few apoptotic neurons were detected in
the CD4-depleted mice, the vast majority of the cells undergoing
apoptosis had the morphologic appearance of lymphocytes, consistent
with the near absence of apoptotic cells in the CNS of infected,
immunosuppressed mice (13). Although the identity of the apoptotic
cells could not be established with certainty, morphologic
characteristics, the absence of CD4+ T cells in these mice,
and the predominant perivascular distribution suggest that these cells
largely represent the CD8+ population. These results
suggest that activated CTL traffic normally into the CNS of
CD4-depleted recipients. By contrast, during infection of CD4-deficient
mice, fewer CD8+ T cells were identified in the CNS. The
reduction in CD8+ T cells appears to be due to increased
apoptosis of this population in the absence of CD4+ T
cells.

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FIGURE 4. Distribution of CD4+ and CD8+ T cells and
apoptotic cells with the CNS of JHMV-infected mice. Cells within the
perivascular and subarachnoid areas are compared with cells within the
parenchyma in untreated (left panel) and
CD4-depleted (right panel) mice.
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FIGURE 5. CD4+ and CD8+ T cells and apoptosis in the
CNS of CD4-depleted mice. During JHMV infection of untreated mice, the
majority of CD4+ T cells accumulate in the perivascular and
subarachnoid spaces (a; v, vessel lumen), with few
gaining access to the parenchyma. CD8+ cells are more
frequently found in an intraparenchymal location (b,
arrows). In control animals, few intraparenchymal T cells are
undergoing apoptosis (c, arrow). The number of
apoptotic cells in the CD4-depleted group (d, arrows)
increased approximately threefold compared with that in the untreated
mice (c). Most of the apoptotic cells are
consistent in appearance with lymphoctyes (c,
inset). Immunoperoxidase staining was performed using the
avidin-biotin-peroxidase complex method and anti-CD4 and
anti-CD8 Ab with hematoxylin counterstain. Apoptosis was detected
with the terminal deoxynucleotidyl transferase-mediated, dUTP-biotin
nick end-labeling method. Magnification, x110 (inset, x440).
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Discussion
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CD8+ CTL are major components of many antiviral
immune responses. They not only reduce virus load but also correlate
directly with protection from otherwise fatal outcomes (1, 2, 8, 46).
The control of many viral infections by CTL is consistent with
expression of their recognition elements, MHC class I molecules, on
virtually all somatic cells. This contrasts with antiviral
CD4+ T cell responses, which appear to be more dependent on
the tropism or route of entry due to the restricted expression of MHC
class II molecules on professional APC (1). Many acute infections by
both cytopathic and noncytopathic viruses occur predominantly in highly
vascularized tissues such as lung, spleen, and liver, in which the
access of CTL does not appear to be limited by a tight endothelial cell
barrier. By contrast, brain, eye, and testis are considered to be at
least partially immunologically privileged, resulting in the apparent
sequestration of Ag from immune recognition (14). The basis of
immunologic privilege is not completely clear; however, a lack of MHC
expression by specific cell types, induction of regulatory cells,
expression of Fas/Fas ligand, and/or local production of cytokines that
limit immunologic reactivity and tissue damage have all been suggested
to play roles (47, 48, 49, 50, 51). Limited access of effectors provides an
important structural barrier to a vigorous immune response to
infection. Viral infections, either those limited to these organs or
infections that disseminate into these organs, appear to transiently
disrupt these structural barriers, allowing entry of immune effectors
(15). In addition to disruption of structural barriers, expression of
adhesion molecules is critical for access across these barriers and
subsequent expression of effector function (52, 53). For example,
during JHMV infection, CD49d is expressed on approximately 40% of
CD8+ T cells obtained from the CNS parenchyma. Passive
transfer of CD49d-specific mAb to infected mice inhibits both
CTL-mediated virus clearance and protection (our unpublished
observations). These data suggested that the CD4+ T cells
might facilitate CNS access either directly by participation in the
disruption of the blood-brain barrier or indirectly via an increase in
expression of adhesion molecules on CNS endothelial cells. However,
during the natural course of infection, CD4 cell activation may
contribute to a more differentiated CD8 T cell state, potentially
enhancing both cytolytic and trafficking functions. By tracking
BrdU-labeled, activated, pN-specific CTL, this report demonstrates that
CD4+ T cell activation is not required for the entry of
activated CTL into the CNS. Although virus-specific CTL clearly
influence the outcome of JHMV CNS infection (27), no antiviral CTL
activity is detectable in the peripheral lymphoid organs at any time
during JHMV infection (38, 39, 40, 41). One interpretation of these data is
that the JHMV-specific CD8+ T cells that express
perforin-mediated cytolytic activity are either expanded within the CNS
and/or mature into lytic CTL within the microenvironment of the CNS
(38). The extent of CD4 T cell involvement is unknown. Comparison of
mononuclear cell infiltrates in the CNS of JHMV-infected untreated and
CD4-depleted mice showed that, in contrast to the adoptive transfer of
activated CTL, the number of CD8+ T cells within the
parenchyma was significantly reduced in the absence of CD4+
T cells. These data are consistent with the idea that CD4+
T cells support the entry of CD8+ T cell into the CNS
parenchyma from the perivascular or subarachnoid spaces. However, a
dramatic increase in the number of apoptotic cells was noted in
infected CD4-depleted mice. We have been unable to visualize surface
markers on BrdU-labeled apoptotic cells by immunohistochemistry, which
may be due to decreased CD8 expression on Ag-reactive cells (54).
However, these data suggest that CD8+ T cells that express
CTL activity in vitro enter the CNS in a CD4+ T
cell-independent manner, similar to the ability of CTL to enter other
highly vascularized tissues (15, 16, 17). In contrast to the maintenance of
CTL effector function in peripheral tissues (15, 16, 17, 29), the effector
function of CTL within the CNS parenchyma of the CD4-depleted,
JHMV-infected mice appears compromised as measured by their inability
to clear virus from the CNS. This loss of function appears to correlate
with increased apoptosis in the absence of CD4+ T
cells.
Whether the increased apoptosis is due to the absence of a tropic
factor(s) required for CTL survival or increased Ag load within the CNS
of CD4-depleted mice is currently under investigation. However, reduced
CTL activity in splenocytes from CD4-depleted infected mice and the
recovery of cytolytic activity by IL-2 support its role in enhancing
CTL survival and optimal induction of effector function (4, 23, 24).
These data suggest that increased apoptotic lymphocytes within the CNS
of CD4-depleted recipients may reflect depletion of cytokines or growth
factors provided by CD4+ T cells. Indeed, CTL induction is
independent of IL-2 (23); however, expression of cytolytic activity is
supported by an autocrine loop of IL-2 secreted by activated
CD8+ T cells (18, 19, 23). Our data support the idea that
CD8+ T cells within the CNS parenchyma may be unable to
induce or maintain lytic function due to a defect in the autocrine IL-2
secretion supplemented by the CD4+ T cell population.
However, the present data cannot exclude the possibility that the
effect on CTL activity is indirect and derived from other cytokines or
is a factor(s) derived from endogenous CNS cells (46, 51, 55). For
example, CD4+ T cells may secrete or induce the secretion
of anti-inflammatory cytokines, such as IL-4, IL-7, IL-10, and
IL-15, which maintain or augment CTL activity (55, 56, 57, 58, 59). Both IL-4 and
IL-10 mRNA levels increase within the CNS of JHMV-infected mice at the
time of viral clearance, although very little IL-4 mRNA can be detected
(60). In this regard it is interesting that virus replicates to a
higher titer in the CNS of mice rendered deficient in IL-10 by
homologous recombination compared with that in control mice (our
unpublished observations). Although this may suggest that IL-10
contributes to the CTL activity in the CNS, IL-10 is secreted from
CD4- endogenous CNS cells during inflammatory responses
(56, 61), and few Th2 type CD4+ T cells are present within
the CNS of JHMV-infected mice, as judged by the absent (62) or low (60)
levels of IL-4 mRNA detected.
JHMV infection of cells in vitro results in cytopathic effects
characterized by giant cell formation. However, in vivo there are few
giant cells or apoptotic cells in the absence of an immune response.
This suggests that in vivo JHMV infection may be more closely related
to infections by noncytopathic viruses, such as LCMV and HIV. Analysis
of mice infected with high doses of LCMV suggested that
CD4+ T cells may be important in preventing CTL exhaustion
due to a high Ag load (2, 3, 22, 23). However, this interpretation is
controversial, since other data suggest that CD4+ T cells
are required to sustain CTL activity and CTL memory during the high Ag
load associated with persistent LCMV infection (18, 21, 22, 63).
Whether the requirement for CD4+ T cells is related to
their ability to assist in the reduction of viral load during JHMV
infection via secretion of antiviral cytokines or the provision of B
cell help is not clear. However, CD4+ T cells may also
contribute directly to the prevention of CTL exhaustion by the
provision of cytokines, such as IL-2. In contrast to JHMV infection of
the CNS, the reduction in LCMV-specific CTL activity does not occur
until after acute infection, and neither CTL-mediated viral clearance
or protection from acute infection is compromised via elimination of
CD4+ T cells (18, 21, 22, 29, 63). The JHMV Ag load within
the CNS appears sufficient for CD8+ T cells to overcome a
requirement for CD4-derived IL-2 during induction (4, 19); however,
cytokine secreted by activated CD4+ T cells may be required
to maintain the lytic activity or viability of virus-specific CTL
within the CNS. In addition to the requirement(s) for CD4+
T cells to secrete cytokines, the ability to inhibit virus replication
and mediate protection from JHMV infection may also be attributed to
differences in the access of CD8+ T cells to peripheral
tissues vs the CNS parenchyma (15).
In the present report the host immune response assisting CTL-mediated
clearance of JHMV from the CNS was dependent on CD4+ T
cells. The data show that the CTL response induced by JHMV infection in
the absence of CD4+ T cells is reduced, but not abrogated,
compared with that in normal mice. Therefore, JHMV infection, although
restricted to the CNS, is similar to the majority of other viruses
examined (1) in that CTL are initially induced in a CD4+ T
cell-independent manner. However, in determining the basis for the
requirement for CD4+ T cells in the antiviral activity of
CTL within the CNS, the data show that activated CTL can access the CNS
in the absence of CD4+ T cells. These CTL appear to be
nonfunctional, since there is little or no evidence for
CTL-mediated virus clearance in CD4+ T
cell-depleted mice even following the adoptive transfer of highly
activated CTL. This interpretation is consistent with histologic
analysis of the CNS of mice depleted of CD4+ T cells, which
exhibits a reduction in mononuclear cell infiltration, yet an increase
in the number of virus-infected cells. These data suggest that the CNS
differs in a fundamental aspect from peripheral organs, which allow
ready access to activated CTL that appear to be able to recognize
Ag-bearing target cells and clear virus (15).
 |
Acknowledgments
|
|---|
The authors appreciate the excellent technical assistance of
Wenqiang Wei, Qin Yao, and Chung Kang Ho.
 |
Footnotes
|
|---|
1 This work was supported by U.S. Public Health Service Grants NS18146 and AI33314 and by U.S. Public Health Service Training Grant NS7149 (to D.C.). 
2 Address correspondence and reprint requests to Dr. S. Stohlman, University of Southern California, MCH 142, 1333 San Pablo St., Los Angeles, CA 90033. 
3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; CNS, central nervous system; JHMV, JHM strain of mouse hepatitis virus; N, nucleocapsid; BrdU, bromodeoxyuridine; RCS, rat concanavalin A supernatant; low (superscript), low level; high (superscript), high level. 
Received for publication August 20, 1997.
Accepted for publication November 19, 1997.
 |
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