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Departments of
*
Neurology,
Pathology, and
Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ELISPOT analysis. Although
overall T cell recruitment into the CNS of µMT mice was not impaired,
discrepancies in frequencies of virus-specific CD8+ T cells
were most severe during acute infection. Impaired ex vivo cytolytic
activity of µMT CNS mononuclear cells, concomitant with reduced
frequencies, implicated IFN- as the primary anti viral factor early
in infection. Reduced virus-specific CD8+ T cell responses
in the CNS coincided with poor peripheral expansion and diminished
CD4+ T cell help. Thus, in addition to the lack of Ab,
limited CD8+ and CD4+ T cell responses in µMT
mice contribute to the ultimate loss of control of CNS infection. Using
a model of virus infection restricted to the CNS, the results provide
novel evidence for a role of B cells in regulating T cell expansion and
differentiation into effector cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The contribution of B cells and Ig in viral clearance as well as activation and maintenance of Ag-specific CD4+ and CD8+ T cells has been studied in several viral infection models using C57BL/6-Igh-6tm1Cgm B cell-deficient (µMT) (3) mice (7, 9, 10, 11, 12, 13, 14, 15, 16, 17). These mice lack mature B220+ cells and consequently both germinal center formation and the ability to secrete Ig (9). Trapping of Ag-Ab immune complexes on follicular dendritic cells potentially maintaining T cell memory is thereby abrogated. However, no differences in viral replication or clearance were observed comparing infection of µMT and wild-type (wt)3 mice with lymphocytic choriomeningitis virus (LCMV), influenza virus, or murine cytomegalovirus (7, 8, 10, 11, 14). CD8+ T cell function appeared to be independent of B cells during numerous acute infections (7, 8, 10, 13). Furthermore, the CD8+ memory population remained stable in the absence of B cells or Ab following viral clearance (10, 13), and immune µMT mice effectively clear challenge virus (10, 15). Similarly, the frequency of CD4+ T cells was not altered significantly during acute influenza virus infection nor in the memory phase comparing wt and µMT mice (11). Thus, for these primary viral infections, CD8+ and CD4+ T cell activation and memory were independent of B cells.
By contrast, B cells and/or Ab play a critical role in controlling
recrudescence and spread of viruses establishing persistent or latent
infections (7, 13, 14, 17, 18, 19, 20). Uncontrolled LCMV
infection in µMT mice is associated with CD8+ T
cell exhaustion, similar to high-dose exhaustion in wt mice (8, 13). Furthermore, LCMV immune T cells from µMT mice adoptively
transferred into LCMV-infected recipients were less efficient in
clearing infectious virus compared with immune splenocytes from wt mice
(16). Despite containing similar frequencies of
Ag-specific T cells, splenocytes from immune µMT mice had a reduced
capacity to secrete both IL-2 and IFN-, suggesting impaired T cell
effector function (16). Contrasting the majority of data
implicating no role of B cells in T cell expansion and differentiation,
the latter data suggested that B cells do participate in the generation
and maintenance of fully competent CD4+ and
CD8+ T cells during systemic LCMV infection.
Infection with the neurotropic JHM strain (JHMV) of mouse hepatitis virus provides a model of localized infection in which the absence of B cells results in uncontrolled, persistent CNS infection despite initial viral reduction by T cells (17). In wt mice, a potent regional CD8+ T cell response is sufficient to completely eliminate infectious virus from the CNS (21, 22, 23, 24). There is little mortality and the clinical symptoms of disease partially resolve following acute infection (25, 26). Nevertheless, viral Ag and RNA persist exclusively within the CNS (25, 27, 28). Persistence is associated with ongoing demyelination, but little or no clinical abnormalities, thus providing a model for the human demyelinating disease multiple sclerosis (25, 26). Humoral immune responses were thought to play a minor role based on observations that infectious virus is eliminated from the CNS before detection of neutralizing anti-viral Ab (29). Consistent with a primary role of cell-mediated immunity in controlling acute JHMV replication, infectious virus declines with similar kinetics in JHMV-infected µMT and wt mice (17). However, in contrast to wt mice, infectious virus recrudesces in the CNS of µMT mice following initial control of replication. Concomitant with virus reactivation, clinical symptoms continue to worsen and the majority of mice succumb to infection by days 30–45 postinfection (p.i.). Importantly, passive transfer of polyclonal anti-JHMV Ab, but not control Ab from naive mice, following initial clearance prevents virus reactivation (17). These data suggest that although T cell-mediated immunity is sufficient to reduce infectious virus during acute disease, immunological control is not maintained in the absence of humoral immunity.
Recrudescence of infectious virus in µMT mice could not be associated with selection of CTL escape variants nor an apparent loss of T cells in the CNS by immunohistological examination (17). However, decreased CD8+ T cell cytotoxicity from splenocytes of recrudescing µMT mice (17) suggested the possibility of CD8+ T cell anergy or exhaustion. To explore the mechanisms underlying the inability of T cells to control persistent CNS infection in the absence of humoral immunity, CNS mononuclear cells (CMC) from infected mice were examined for the frequency and function of virus-specific T cells. Although total CMC CD8+ populations were similar in magnitude in µMT and wt mice, class I tetramer analysis revealed reduced numbers of CD8+ T cells specific for the immunodominant S510 epitope throughout the entire disease course in µMT mice. Lower numbers of virus-specific CD8+ T cells in the CNS coincided with impaired expansion in the periphery and was linked to reduced frequencies of virus-specific CD4+ T cells. The results further indicate that while minimal T cell responses initially suffice to control CNS infection, viral replication outruns T cell replenishment, potentially leading to peripheral T cell exhaustion. Compared with other well-described viral infections of µMT mice (7, 8, 10, 11, 13), limited CD4+ and CD8+ T cell function apparent during the primary response appears to be unique to infection of the CNS. Consistent with recent evidence of impaired function of LCMV-specific memory T cells primed in the absence of B cells (16), the present findings thus provide direct support for a role of B cells in regulating CD8+ T cells directly or via CD4+ T cells in a model of viral infection confined to the CNS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male C57BL/6 (H-2b) mice were purchased from the National Cancer Institute (Frederick, MD) at 6 wk of age and certified naive to prior mouse hepatitis virus exposure. µMT mice, obtained as breeding pairs from The Jackson Laboratory (C57BL/6-Igh-6tm1Cgn; Bar Harbor, ME), were bred at the University of Southern California Keck School of Medicine under pathogen-free conditions. Mice were housed in microisolator cages in an accredited animal facility at the University of Southern California and infected at 6–7 wk of age. CNS infections were induced by intracranial injection of 30 µl containing 200–500 PFU of the 2.2v-1 mAb-derived variant of JHMV as previously described (30). This variant produces paralysis associated with demyelination and replicates predominantly in oligodendrocytes. Virus was propagated in the presence of neutralizing mAb J.2.2 and quantified by plaque assay using monolayers of the murine delayed brain tumor astrocytoma cell line (17). Intraperitoneal injections were conducted with 4 x 106 PFU of the parental JHMV isolate designated DM variant (31).
Mice were depleted of CD4+ T cells by i.p. injection of 250 µg of anti-CD4 mAb GK1.5 at days -2, 0, and +2 relative to virus infection as described elsewhere (22, 32); control mice received PBS injections at the same time points. This treatment resulted in >98% depletion of CD4+ T cells at day 8 p.i. as analyzed by flow cytometry.
Tissue sampling and isolation of lymphocytes
CMC were obtained from pooled brains and spinal cords of 6–10 mice/group at various time points p.i. as described previously (33, 34). Briefly, tissues were minced and homogenized in Tenbrock homogenizers. Cell suspensions were adjusted to 30% Percoll (Pharmacia, Uppsala, Sweden) and concentrated onto a 70% Percoll cushion by centrifugation at 800 x g at 4°C for 20 min, washed, and resuspended in RPMI 1640 medium. Yields varied from 0.8 to 2.0 x 106 cells/mouse, with maximum yields between days 8 and 12 p.i. Single-cell suspensions were prepared from the spleens and cervical lymph nodes (CLN) from identical groups of mice as previously described (35).
CTL assays and synthetic peptides
CTL assays were performed as described elsewhere (24, 36). Briefly, EL-4 (H-2b) target cells were labeled with 100 µCi of Na51CrO4 (New England Nuclear, Boston, MA) and S510 peptide was added to washed target cells at a final concentration of 1 µM before addition of CTL at the indicated E:T ratios. Supernatants (100 µl) were removed after 4 h of incubation and specific 51Cr release was determined. Specific lysis was defined as 100 x (experimental release - spontaneous release)/(detergent release - spontaneous release). Maximum spontaneous release values were <10% of the total release values.
The Db-restricted S510 peptide (37, 38) was synthesized by the University of Southern California Norris Cancer Center Microchemistry Laboratory and purity assessed by HPLC and mass spectrometry. The I-Ab-restricted M133 peptide (39) was purchased from Genemed Synthesis (South San Francisco, CA). Peptides were solubilized at 1 mM in DMSO and diluted in sterile PBS.
ELISPOT assays
ELISPOT assays to measure the frequency of Ag-specific
IFN--secreting cells were conducted as described previously
(24). Serial 2.5-fold dilutions of cells were plated in
triplicate into 96-well plates supporting cellulose ester membranes
(MultiScreen HA; Millipore, Bedford, MA) precoated with R4-6A2 mAb (BD
PharMingen, San Diego, CA) and stimulated in the presence of irradiated
(25 Gy) splenocytes from naive mice (5 x
105/well), either pulsed with 1 µM class
I-restricted S510 peptide, class II-restricted M133 peptide, or left
untreated. EL-4 supernatant was added as a source of IL-2 to a final
concentration of 2.5%. Cultures were incubated for 36 h at
37°C. Bound IFN- was detected by overnight incubation at 4°C
with biotinylated anti-IFN- mAb (0.5 µg/ml, XMG1.2; BD
PharMingen), followed by consecutive incubations with
streptavidin-peroxidase (Sigma, St. Louis, MO) and
3,3'-diaminobenzidine as substrate (Sigma).
Flow cytometry
Single-cell suspensions were blocked with purified
anti-mouse CD16/CD32 (2.4G2; BD PharMingen) and stained with FITC-,
PE-, or CyChrome-conjugated mAb specific for CD8
(7), CD4 (GK1.5), CD62L (MEL-14), CD43 (1B11), CD44
(IM7), CD69 (H1.2F3), CD25 (PC61), CD11a (2D7), CD49d (R1-2; BD
PharMingen), and PE-conjugated Db-S510 tetramer
(0.1–0.2 µg/0.5–1.0 x 106 cells) in
various combinations. Cells were typically stained at 4°C with mAb
for 15–20 min before incubation with tetramer for 30 min in PBS
containing 0.1% BSA. The generation of Db-S510
tetramers, composed of Db class I H chains,
2-microglobulin, and the S510 peptide, was
previously described (24). Samples were analyzed by flow
cytometry on a FACStar (BD Biosciences, Mountain View, CA). Forward and
side scatter signals obtained in linear mode were used to establish a
gate that contained intact lymphocytes, while excluding remaining
tissue debris. A minimum of 4 x 105 viable
cells were stained and 5 x 104–1 x
105 events were analyzed per sample.
Synthesis of intracellular IFN- in response to Ag stimulation was
determined by incubating 4–5 x 105 CMC or
1 x 106 splenocytes in 100 µl of RPMI
1640 complete supplemented with 10% FCS, 1 µM peptide, and 1 µg/ml
monensin (BD PharMingen) for 5 h at 37°C (3).
Peptide was omitted in negative control samples. Cells were then
stored at 4°C overnight and stained 14–16 h later using the
Cytofix/Cytosperm kit (BD PharMingen). Following surface staining,
cells were fixed, permeabilized, and stained with mAb specific for
IFN- (XMG1.2) as recommended by the supplier (BD PharMingen).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies of acute JHMV infection in µMT mice suggested
that T cell responses within the CNS were comparable to those in wt
mice (17). This interpretation was based on 1) similar
kinetics of initial virus clearance, 2) no apparent differences in the
number of CNS infiltrating CD8+ or
CD4+ T cells determined by immunohistochemistry,
and 3) similar CTL responses of in vitro-restimulated splenocytes
harvested at day 7 p.i. However, the inability of µMT mice to
control the infection beyond day 10 p.i. implied that T cells were
rendered unresponsive and/or underwent activation-induced cell death
due to increasing viral load. To assess the possibility of
CD8+ T cell exhaustion, the frequencies of
CD8+ T cells specific for the immunodominant S510
epitope were determined at times when virus has been cleared from the
CNS of wt mice, but re-emerged in µMT mice (17). CMC
from wt and µMT-infected mice were initially analyzed at days 14 and
21 p.i. by flow cytometry and IFN- ELISPOT assay (Fig. 1
). The percentage of total
CD8+ T cells in CMC was similar or elevated in
µMT mice (Fig. 1A), consistent with immunohistochemical
analysis (17). Nevertheless, as determined by tetramer
staining, the population of virus-specific cells within the
CD8+ T cell compartment never exceeded 12% in
µMT-derived CMC, whereas it ranged between 30 and 40% in wt-derived
CD8+ CMC (Fig. 1A). Consistent with a
relative decrease in tetramer staining cells, the frequencies of
CD8+ T cells secreting IFN- at day 14
p.i. were at least 3-fold lower in µMT- compared with wt-derived CMC
(Fig. 1B). In wt mice, frequencies of IFN--secreting
cells further declined by day 21 p.i., consistent with the
decline in viral Ag (17). Decreased
frequencies of IFN--secreting CD8+ T cells
were also evident in CMC from µMT mice, despite viral recrudescence.
This confirmed an inability to respond to increasing virus load and
suggested either limited resources of peripheral
Ag-specific T cells to replenish the CNS, inhibited infiltration,
down-regulation of type 1 CD8+ T cell effector
function, or a combination of these factors.
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). At day 5 p.i., percentages of
total CD8+ T cells were higher in CMC from µMT
mice, suggesting that T cell recruitment into the CNS was not impaired
in µMT mice; tetramer+ cells within the CMC
population of either group were below detection limits at this time
(Fig. 2A). Surprisingly however, at day 8 p.i., when
viral clearance was similar in both groups (17),
CD8+ T cells within CMC derived from µMT mice
only contained 4–9% tetramer+ cells, whereas
CD8+ T cells from wt mice typically contained
33% tetramer+ cells (Fig. 2A).
Despite similar overall CD8+ T cell infiltration,
tetramer+ cells constituted a significantly
reduced percentage within the CNS of µMT mice compared with wt mice.
Overall yields of CMC from either infected group ranged from 1.5 to
2.0 x 106/mouse during acute infection, and
differences never exceeded 25% between the groups. These data implied
that few of the CD8+ T cells recruited into the
CNS of JHMV-infected µMT mice are indeed S510 specific or,
alternatively, TCR down-regulation due to high stimulation. Frequencies
of S510-specific IFN--secreting cells were also at least 3-fold
lower in µMT-derived CMC (Fig. 2B), confirming reduced
numbers of S510-specific T cells at the functional level. Nevertheless,
similar or lower ratios of tetramer+ to
IFN--secreting CD8+ T cells in CMC from µMT
mice compared with wt mice suggests that µMT-derived
CD8+ T cells are not inherently impaired in
cytokine-mediated effector function.
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). Even
when E:T ratios were adjusted to reflect only
tetramer+ CD8+ T cells,
cytolysis on a per cell basis was severely reduced.
Furthermore, re-emerging viral replication did not trigger increased
cytolytic activity (Fig. 3). Thus, similar to wt mice
(24), CNS-resident CD8+ T cells in
µMT mice were rendered noncytolytic after initial viral reduction.
These data support IFN- secretion as a major antiviral mechanism
(23), as this function did not appear impaired on a per
cell basis (Fig. 2
) in contrast to cytolytic function (Fig. 3).
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).
The majority of CNS-infiltrating CD8+ T cells at
day 8 p.i. were CD44high and
CD62-, CD11ahigh and
CD49d+, characteristic of an activated/memory
phenotype (40). However, µMT-derived CMC consistently
contained slightly increased populations of naive
CD8+ T cells characterized by the
CD44low/int and CD62high,
CD11alow, and CD49d-
phenotype. CD69 and CD25 expression were similar or slightly elevated
on the CD8+ populations of µMT-derived CMC
(Table I); no differences in CD69 or CD25 expression was evident in
CD4+ T cells derived from either CMC group
(
21–23% CD69+ and 15–16%
CD25+; data not shown). The only distinct
difference between µMT and wt mice was a reduced frequency of
CD43highCD8+ T cells
reaching only 43% in µMT CD8+ populations,
compared with 76% in wt CD8+ T cells (Table I).
CD43 up-regulation has recently been identified as marking cytolytic
CD8+ T cells (41). Phenotypic
analysis thus confirmed poor cytolysis exhibited by µMT-derived CMC
rather than potential heterologous responses to other viral
epitopes.
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To determine whether reduced frequencies of virus-specific
CD8+ T cells in the CNS coincided with reduced
numbers in the periphery, splenocytes and CLN T cells were analyzed for
S510-specific TCR expression and S510-induced IFN- secretion at days
5 and 8 p.i. Tetramer+
CD8+ T cells were readily detectable in the
spleen and CLN of wt mice, but were severely reduced in µMT mice,
despite higher CD8+ T cell percentages arising
from the lack of B cells (Fig. 4A). Analysis of CMC from the
same groups of mice confirmed accumulation of Ag-specific cells
specifically at the site of infection. These data suggested that
reduced S510-specific CD8+ T cells in the CNS of
µMT mice resulted from impaired peripheral expansion following CNS
infection. Consistent with these data, the frequency of
IFN--secreting S510-specific CD8+ T cells in
both spleens and CLN was 2- to 3-fold lower in µMT mice (Fig. 4B). However, each group of mice revealed discrepancies
between tetramer staining and ELISPOT analysis. Although splenocytes of
wt mice contained higher frequencies of tetramer+
T cells than CLN, the frequency of IFN--secreting cells was higher
in the CLN. This inability to detect tetramer+
cells may reside in down-regulation of TCR due to more potent
Ag-specific stimulation in the CLN. The same observation was made in
µMT mice, despite overall lower frequencies of IFN--secreting
cells at both sites. Enhanced frequencies of IFN--secreting
cells in the CLN compared with spleen were already pronounced at day
5 p.i., when tetramer+ cells were below
detection. These data thus support CLN as the primary site
of T cell priming in both wt and µMT mice during CNS infection, as
suggested for CNS infection induced by LCMV
(42). The lack of higher frequencies of virus-specific
CD8+ T cells in the spleen of wt mice compared
with µMT mice at day 5 p.i. further indicates that sequestration
of virus-natural Ab immune complexes to the spleen (43)
plays a minor role in T cell priming in this model of infection.
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For some viral infections, CD8+ T cell
expansion, function, and survival is tightly linked to the
CD4+ T cell compartment (22, 44, 45, 46, 47). During acute JHMV infection, survival and function of
CD8+ T cells in the CNS, but not entry, is
dependent on CD4+ T cells (22).
Furthermore, CD4+ T cells appear to enhance
peripheral CD8+ T cell expansion, as indicated by
decreased cytolytic activity of splenocytes from infected mice depleted
of CD4+ T cells (22). ELISPOT
analysis was therefore used to analyze expansion of
CD4+ T cells specific for the dominant M133
epitope located within the viral matrix protein (39). At
day 8 p.i., both splenocytes and CMC from µMT mice contained
severely reduced frequencies of IFN--secreting
CD4+ T cells (Fig. 5). Reduced frequencies of M133-specific
CD4+ T cells in the CNS, despite similar total
CD4+ populations, again implied a defect in
virus-specific CD4+ T cell expansion rather than
recruitment, similar to CD8+ T cell expansion.
This was indeed supported by the low frequencies of splenic
virus-specific CD4+ T cells. Reduced
CD8+ T cell responses in µMT mice following
JHMV infection may thus be a direct consequence of impaired
CD4+ T cell expansion, rather than a
CD8+ T cell-specific defect.
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by peptide-stimulated
CD8+ T cells (Fig. 6). Intracellular IFN- staining
revealed that CD4+ T cell depletion reduced the
frequency of S510-responsive CD8+ T cells to 60%
of that in untreated wt mice. However, only 4% of
CD8+ T cells from µMT mice produced IFN-
following stimulation; this represents only 19% of the wt value.
Nevertheless, the mean fluorescence intensities of IFN-+ cells from µMT and CD4-depleted mice were only slightly
reduced, indicating no significant impairment in IFN- production.
These data suggest that although CD4+ T cells
enhance CD8+ T cell responses during JHMV
infection, deprivation of CD4+ T cell help alone
does suffice to account for the impaired CD8+ T
cell response in µMT mice. Transfer of 2.5 x
107 splenic B cells from naive wt mice into µMT
mice a day before infection did not affect priming/expansion of either
CD4+ or CD8+ T cells (data
not shown). There were no differences in the frequencies of S510
CD8+ T cells or M133 CD4+ T
cells at 5 or 8 days p.i. in either the spleen, CLN, or CNS. However,
CD19+ B cells only comprised 1–2% in splenic or
CLN populations at the time of T cell analysis. The inability to rescue
T cell expansion presumably resides in the previously reported rapid
disappearance of B cells from µMT recipients (48). Thus,
unlike other peripheral infections studied in µMT mice
(10, 11, 12, 13, 14, 15), T cell priming and expansion following infection
of the CNS appears to require the physical presence of B cells and/or a
B cell-dependent component absent in µMT mice.
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). Percentages of
both tetramer+ and IFN--producing cells in the
CD8+ population were reduced at least 5-fold in
µMT mice. These data confirm a role of B cells in priming
CD8+ T cell responses to JHMV, independent of the
complexities associated with the lack of CNS lymphatic drainage
(42, 49, 50).
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The effects of impaired Ag-specific CD8+ T
cell expansion in µMT mice are compounded by reduced cellularity of
both CLN and spleen (11). Although relative percentages of
CD4+ and CD8+ T cells are
increased, cell yields of CLN and spleen per mouse in JHMV-infected
µMT mice at day 8 p.i. were typically 2- to 3- and 4- to 6-fold
reduced compared with wt mice, respectively. Because
tetramer+ cells were below detection limits in
spleen and CLN after d 8 p.i. in both wt and µMT mice,
frequencies of S510-specific IFN--secreting T cells were used to
calculate S510-responding T cells per spleen to adjust for the reduced
cellularity in µMT mice throughout the course of infection (Fig. 8). A reduction of splenic S510-specific
CD8+ T cells in µMT mice was most dramatic on
day 8 p.i., but was evident throughout the infection in µMT
mice; at day 21 p.i., the frequency of S510-specific IFN- spots
was below 1 in 106 splenocytes (Fig. 8). This
supports the notion that few available circulating memory cells may
lead to CD8+ T cell exhaustion, even when chronic
Ag stimulation is confined to the CNS. These data suggest that in
addition to the lack of Ab, inefficient CD8+ T
cell expansion and concomitant exhaustion may contribute to viral
recrudescence within the CNS of µMT mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The reactivity of virus-specific T cells was examined to determine the
fate of initially protective CD8+ T cells during
viral recrudescence in µMT mice. The CNS of µMT mice contained
comparable numbers of CD8+ T cells relative to wt
mice, but surprisingly lower frequencies of virus-specific
CD8+ T cells throughout the entire course of
infection. In addition, severely compromised cytolytic function by
µMT-derived CMC during the acute response raises the question as to
the mechanism which initially controls virus replication. Although
perforin-mediated cytolysis clears virus from microglia and astrocytes
(29), IFN- acts as the dominant antiviral cytokine in
oligodendrocytes (23). However, studies of pathogenesis in
infected perforin- and IFN--deficient mice indicate that IFN-
plays a more prominent role in protection (23, 29). By
contrast, Fas-Fas ligand interactions do not contribute to viral
clearance or pathogenesis (51). Initial viral clearance in
µMT mice may thus be predominantly controlled by antiviral IFN-
rather than perforin-mediated cytolysis. An ineffective cytolytic
response in vivo is supported by the similar distribution of viral Ag
during viral recrudescence compared with acute infection, i.e., in
microglia, astrocytes, and oligodendrocytes (17).
Nevertheless, impaired cytotoxicity is not intrinsic to µMT-derived
CD8+ T cells, as cytolytic function is readily
restored upon restimulation with infected wt splenocytes in vitro
(17). Furthermore, a pronounced defect in IFN-
secretion by µMT-derived CD8+ T cells was
inapparent taking the overall lower frequencies of virus-specific
CD8+ T cells into account. This is supported by
high levels of IFN- mRNA in the CNS of µMT mice
(17), similar
IFN-+:tetramer+ T cell
ratios, and only moderate reduction in IFN- production as measured
by intracellular staining (see Figs. 2 and 6). Since NK cells
constitute similar percentages of CMC from both groups of mice early
during infection (4–5% at day 8 p.i.) and drop below detection
by day 14 p.i. (data not shown), they are not believed to
contribute significantly to altered pathogenesis in µMT mice. Initial
viral clearance in µMT mice, despite reduced T cell effector
function, suggests that the response in wt mice by far surpasses a
minimal threshold required for antiviral control. Nevertheless, this
apparently excessive response may be critical in prolonging viral
control and limiting virus spread within the CNS until the emergence of
protective Ab.
Reduced primary virus-specific T cell responses in the absence of B
cells were not evident following a number of other virus infections
(7, 8, 10, 11, 14). Effector function of virus-specific
CD8+ T cells, as well as
CD8+ memory populations (10, 13),
and frequencies of virus-specific memory CD4+ T
cells (11) were not altered significantly in µMT mice
compared with wt mice. However, impaired antiviral T cell function in
µMT mice was suggested by the inability of adoptively transferred T
cells primed in these mice to control LCMV infection (16).
A reduced capacity of splenocytes from LCMV-immune µMT mice to
secrete both IL-2 and IFN-, but not reduced frequencies of
Ag-specific T cells, contrasts sharply with reduced virus-specific
CD4+ and CD8+ T cells but
no significant reduction in IFN- production observed during JHMV
infection. The reasons underlying impaired T cell function in
JHMV-infected µMT mice compared with other viral infections is
unclear. Ineffective recruitment of T cells into the CNS can be ruled
out because total percentages of CD4+ and
CD8+ T cells are similar in the CNS of wt and
µMT mice. Reduced frequencies of virus-specific
CD8+ T cells in both spleen and CLN during the
acute response rather implicates inefficient priming/expansion. The
fact that CD4+ T cell expansion is also severely
limited in infected µMT mice suggests that reduced
CD4+ T cell help diminishes
CD8+ T cell expansion and differentiation.
CD4+ T cell responsiveness was also decreased in
B cell-deficient mice early during ocular HSV infection
(52), and expansion, but not function, was also impaired
in CD4+ T cells from µMT mice immunized with
keyhole limpet hemocyanin (53, 54). The defects in
keyhole limpet hemocyanin-responding T cells were attributed to
ineffective Ag presentation due to the lack of immune complex formation
(54). Nevertheless, B cells appear to provide a more
critical component than CD4+ Th cells in
regulating CD8+ T cell expansion during JHMV
infection, as virus-specific CD8+ T cells in
peripheral lymphoid organs as well as in CMC were more severely reduced
in the absence of B cells than in CD4+ T
cell-depleted mice. Efforts to enhance T cell responsiveness via
transfer of B cells from naive mice to µMT recipients failed. No
differences in virus-specific CD4+ or
CD8+ T cells frequencies were evident 5 or 8 days
p.i. in these recipients compared with untreated µMT mice (data not
shown). B cell transfers also had no effect on virus titers in the CNS
at days 5 or 8 p.i. (data not shown). The degree to which the
absence of B cells or Ab alone, the lack of CD4+
T cell-B cell interactions, or inhibited follicle formation and
disrupted lymphoid architecture in µMT mice contribute to
CD8+ T cell expansion therefore remains
unclear.
Exclusive neurotropism of JHMV may compound inefficient Ag-specific
CD4+ and CD8+ T cell
expansion in JHMV-infected µMT mice. Although the CNS is efficient in
supporting secondary activation of primed T cells, it is poor in
priming naive T cells (49, 50, 55). T cell priming during
a parenchymal CNS infection is believed to take place in peripheral
lymphoid organs where B cells may contribute to activation. The absence
of B cell follicle formation is associated with reduced follicular
dendritic cell networks in µMT mice and diminished spleen and CLN
cellularity (11). Thus, the restricted nature of JHMV
replication in the CNS, coincident with limited peripheral Ag
presentation, may render T cell activation more dependent on a
component provided by B cells compared with other viral infections
previously studied. However, perturbed T cell expansion even following
peripheral JHMV immunization suggested a mechanism involving Ag
presentation rather than CNS infection as responsible for diminished T
cell responsiveness. There is no evidence that B cells themselves act
as APC during JHMV infection. However, an indirect regulatory role for
B cells during priming cannot be ruled out. Although a role for Ab and
complement in enhancing Ag presentation in wt mice via sequestration of
immune complexes to the spleen cannot be ruled out (43, 54), there was no evidence for enhanced splenic vs CLN priming
in wt mice compared with µMT mice (Fig. 4). The observation that JHMV
replication in µMT mice follows similar kinetics and never exceeds
titers in wt mice also negates a role of immune complexes in delaying
CNS infection. Interestingly, a significant contribution of B cells to
effector CD8+ T cells was recently uncovered in
the clearance of, and recovery from, influenza virus infection
(56). However, in contrast to JHMV infection, the as yet
unidentified B cell activity was Th cell independent and did not appear
to enhance CD8+ T cell function. Furthermore, the
lack of Th cell-B cell interaction may indirectly dysregulate APC
function. B cells have indeed been shown to affect dendritic cell
function by regulating cytokine production. Upon Ag stimulation,
dendritic cells from µMT mice produce higher levels of IL-12p70 and
fail to induce IL-4 secretion, resulting in potential
CD4+ Th1 cell deviation (57).
Although there is no evidence for this pathway during JHMV infection,
alternative default interactions of CD4+ T cells
with APC rather than B cells may conceivably result in diminished T
cell activation.
In summary, the data indicate that not only the lack of Ab, but severely reduced frequencies of virus-specific T cells contribute to viral recrudescence in the CNS of µMT mice. Impaired CD4+ and CD8+ T cell expansion appears to be unique to JHMV infection of the CNS parenchyma and results in reduced T cell effector function within the CNS. Although the limited T cell response is sufficient to reduce viral replication, virus is not cleared below detection and recrudesces. Premature loss of effector function below a minimal threshold may thus provide a window for virus reactivation. An inability to replenish effector cells concomitant with increasing Ag load in the CNS appears to ultimately lead to exhaustion. Reduced numbers of splenic T cells in µMT mice (10, 11) and inadequate CD4+ T cell help may facilitate this process, analogous to high-dose LCMV-induced loss of specific CD8+ T cells (1, 2, 3, 7). Thus, a vigorous early T cell response may tip the balance between viral replication and immunity to viral persistence, which is ultimately controlled by Ab.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Cornelia C. Bergmann, 1333 San Pablo Street, MCH 142, Los Angeles, CA 90033. E-mail address: cbergman{at}hsc.usc.edu
3 Abbreviations used in this paper: wt, wild type; CLN, cervical lymph node; CMC, CNS-derived mononuclear cells; JHMV, JHM strain of mouse hepatitis virus; LCMV, lymphocytic choriomeningitis virus; p.i., postinfection.
Received for publication February 2, 2001. Accepted for publication May 30, 2001.
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, wt CMC; 
,
µMT CMC. Data from two representative experiments of four, designated
a and b, are shown. B, The
frequency of cells responding to S510-coated feeder splenocytes was
measured by IFN-
