From the Nuffield Department of Medicine, University of Oxford, John Radcliffe Hospital,
Headington, Oxford OX3 9DU, United Kingdom
Mice intranasally inoculated with influenza A/X-31 are protected against a subsequent intracerebral challenge with the neurovirulent influenza A/WSN and this heterotypic protection is
mediated by CD8+ cytotoxic T lymphocytes. We have studied the kinetics of this secondary
immune response and found that despite the elimination of replication-competent virus by day
10, we were able to recover activated influenza-specific cytotoxic T lymphocytes (CTLs) that killed freshly ex vivo from the brains of mice for at least 320 d after the intracerebral inoculation. The activated antiviral CTLs expressed high levels of the early activation marker CD69, suggesting continuing TCR signaling despite a lack of viral protein and major histocompatibility complex staining by immunohistochemistry in the brain parenchyma and barely detectable
levels of viral nucleic acid by single and two-step reverse transcription PCR. Local persistence
of activated lymphocytes may be important for efficient long-term responses to viruses prone
to recrudesce in sites of relative immune privilege.
Key words:
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Introduction |
One outcome of inefficient central nervous system
(CNS)1 immunity is virus persistence (for review see
reference 1) and the diseases increasingly associated with it
(2, 3). Terminally differentiated cells such as neurons are
good long-term hosts for viruses since they do not readily
undergo either viral or CTL-mediated apoptosis (4) and
they can escape CTL detection due to low levels of surface
MHC molecules (5). We have recently shown that although influenza virus infection confined to the brain parenchyma is associated with marked inflammation, it is weakly immunogenic even if the virus actively replicates
(6, 7). The lack of lymphoid tissue and resident dendritic
cells (for review see reference 8), the exclusion of lymphocytes with a naive phenotype (9, 10), and the lack of well-defined lymphatic drainage (for review see reference 11) all
reduce the efficiency of immune responses to virus infection in the CNS parenchyma. Control of virus infection
largely depends on an influx into the brain of cellular effectors and/or a specific antibody that have been primed systemically. Systemic activation of antiviral CTLs would be
expected in acute encephalitis since systemic infection usually precedes or accompanies infection in the CNS. But
what provides the stimulus for CNS migration after the peripheral infection subsides is unknown; the activation state
of most circulating antiviral effectors wanes after systemic
virus has been cleared (indeed some memory cells revert to
a more naive phenotype; reference 12).
Persistent antiviral immunity in the CNS could result either from continuing lymphocyte recirculation into the
CNS or from persistence of lymphocytes at the site of initial inflammation. Recently, memory CTLs have been
shown to persist locally in the lungs after intranasal infection (13). We now show that after an acute monophasic viral encephalitis, activated CTLs remain localized to the site
of virus infection long after effective viral clearance. The
persistent CD8+ lymphocytes are not proliferating yet express high levels of the early activation marker CD69, suggesting continuing lymphocyte interaction with specific
MHC-peptide complexes, despite an absence of detectable
viral protein and undetectable levels of viral nucleic acid.
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Materials and Methods |
Mice, Viruses, and Inoculation.
6-8-wk-old C57BL/10 mice were
used for experiments. Influenza viruses A/WSN (H1N1) and A/X31
(H3N2) were propagated, titered, and inoculated as previously
described (14).
Virus Titers and Viral RNA.
Replication-competent A/WSN
virus was recovered from infected organs as previously described
(14). cDNA was synthesized from total brain RNA by standard
methods. A 489-bp fragment of the influenza A nucleoprotein (NP)
gene was amplified using the following oligonucleotide primer
pairs and conditions: NP1, 5'-GCATGCAATTCTGCTGCAT-3'; NP2, antisense 5'-CTCTGCATTGTCTCCGAAG-3'; 5 min at 95°C, followed by 30 1-min cycles of 57, 70, and 94°C.
As a positive control for the cDNA synthesis, we also amplified a
214-bp fragment of murine hypoxanthine guanosine phosphoribosyl
transferase cDNA using the following primer pairs and conditions: 5'-GTAATGATCAGTCAACGGGGGAC-3'; 5'-CCAGCAAGCTTGCAACCTTAACCA-3' at 95°C, followed by 30 1-min cycles of 55, 70, and 94°C. A 293-bp fragment was also
amplified from one fifth of each of the first set of NP reactions
and their negative, using an additional internal NP primer (NP4,
5'-ATCAACAGAGGGCCTCTGC-3', with the above NP2
primer) as for the first round reaction.
Lymphocyte Cultures.
Lymphocyte cultures were maintained
as in reference 6. The NP 366-374-specific CTL clone was derived from an A/X-31-stimulated short-term line by limiting dilution using standard methods (15). It responded to influenza A/
WSN and to picomolar concentrations of its NP 366-374 peptide, ASNENMETM.
Isolation of Lymphocytes from the Brain and Lung.
Terminally anesthetized mice were perfused with heparinized PBS (1 U/ml) via
the left (for brain) or right (for lungs) cardiac ventricles, and the
organs were harvested into ice-cold 25 mM Hepes-buffered F-10
medium (Life Technologies, Paisley, UK)/10% FCS. After premincing the lung samples with scissors to assist homogenization,
the brain and lung samples were homogenized by gentle trituration through 19- and then 21-gauge needles. Homogenates (derived from single animals) were then centrifuged in 15-ml conical tubes at 400 g (at 4°C) for 7 min and the pellets were resuspended in 4 ml of Percoll (Pharmacia, Milton Keynes, UK) solution containing 10% RPMI × 10 (Life Technologies) and 0.2% bicarbonate (taken as 100% Percoll). Dilutions of RPMI containing 60, 40, and 0% of the Percoll solution were overlaid. The discontinuous gradients were centrifuged for 15 min at 1, 000 g (at 4°C) and
lymphocytes were harvested from the 40-60% interface after first
removing the myelin layer (at the 0-40% interface). In preliminary experiments, >98% of the total recoverable intracerebral T
cells were found at the 40-60% interface (not shown).
CTL Assays.
Cytotoxic activity was tested in standard 4-5-h
51Cr-release assays as previously described (6, 14). The percentage
specific lysis was calculated as 100 × (release by CTL
release
by targets alone)/(release by 2% Triton X-100
release by targets alone). 51Cr release from the targets alone was 5-15% of 51Cr
release with Triton X-100.
Limiting Dilution Analysis.
Established methods were used
for limiting dilution analysis (12). Varying numbers of responder
cells (12-24 replicates) pooled from two to six mice were restimulated in IL-2-supplemented medium for 8 d using 1 µM NP
366-374-pulsed irradiated splenic feeder cells (3-4 × 105 cells/
200-µl well). 51Cr-labeled, peptide-pulsed EL-4 (H-2b) targets
were added to 100 µl of each resuspended replicate well for 5 h at
37°C and the supernatants were assayed for 51Cr release as above.
We designated wells as positive if the cpm released was greater
than the mean plus three standard deviations of the feeder cell
replicates that had only received targets and no effector cells. The
log10 of the proportion of negative wells was plotted against the
input cell number and a line of best fit drawn using the least
squares method. r2 values were all >0.93.
Flow Cytometry, Cell Sorting, and Immunohistochemistry.
Cell surface staining was performed as described in reference 6 using the
following primary antibodies: rat anti-mouse: anti-CD4, KT6-PE, IgG2a (Serotec, Kidlington, UK); anti-CD8, KT15-FITC, IgG2a (Serotec); anti-CD62L, biotinylated MEL-14, IgG2a; anti-CD44, biotinylated IM7.8.1, IgG2b; anti-CD49d, biotinylated
PS/2, IgG2b (16); anti-Mac-1, biotinylated M1/70, IgG2b; anti-CD25, Quantum red-conjugated 3C7 (Sigma Immunochemicals,
Poole, UK); hamster anti-mouse: anti-CD69, biotinylated H1.2F3,
IgG (PharMingen, San Diego, CA), anti-
/
TCR mAb, biotinylated GL3, IgG (Serotec). After a 30-min primary incubation
and a wash, the appropriate samples were incubated for 15 min
with streptavidin Quantum red (Sigma Immunochemicals). After
a further wash the samples were immediately analyzed on a Becton Dickinson (Mountain View, CA) FACSort® instrument using
Cellquest V1.1 software (Becton Dickinson). The combinations
of primary antibodies used provided internal specificity controls.
M1/70 and GL3 staining were negative on brain and spleen samples (not shown). Changes in cell phenotype consequent upon
the process of cell purification were excluded by mixing brain
cells and splenocytes at the time of homogenization and then
demonstrating that naive cells could be purified by density gradient centrifugation (Hawke, S., unpublished data; reference 10). In
vivo administration and internal staining of bromodeoxyuridine (BRDU) with anti-BRDU antibody (Becton Dickinson) was
performed as described previously (17). After perfusion with heparinized PBS, brains from A/X-31 immune animals were cryopreserved at the following time points after intracranial challenge
with A/WSN on days 2, 5, 7, 21, 42, 115, 212, and 320 (three
mice at each time point). Immunohistochemistry was performed
exactly as in references 6, 7, and 14 using the following primary
antibodies: rat anti-mouse CD8, YTS 169.4; CD4, GK1.5;
MHC class I (H-2K), M1-42.3.9.8; MHC class II, M5/114.15.2;
B220, RA3-6B2; and rabbit antiinfluenza ribonucleoprotein
(sourced as in reference 7).
 |
Results and Discussion |
After direct inoculation into the lateral cerebral ventricle
of the mouse, the neurovirulent influenza A/WSN (H1N1)
replicates in ependymal cells and then spreads into the brain
parenchyma where it infects neurons (18). In nonimmune
mice, the resulting encephalitis leads to death within 6-8 d.
Earlier immunization with intranasal influenza A/X-31
(H3N2) protects 80% of mice and this "heterotypic" protection is mediated by CD8+ lymphocytes (14). A sustained
clinical recovery occurs after day 5, and this is coincident
with a marked reduction in recoverable replication-competent virus (14).
We purified lymphocytes from the brains of immune
mice challenged with A/WSN and found that there was
rapid recruitment of CD4+ and CD8+ lymphocytes (Fig. 1
a), initially (days 0 and 4) in proportions similar to those
found in peripheral blood or spleen. But from day 4 to day
21 there was a 300-fold increase in the number of CD8+
lymphocytes, which then declined exponentially, never
reaching normal levels during the follow-up period (320 d).
Histological analysis, even as late as 320 d after the intracerebral challenge showed CD8+ lymphocytes in the brain,
both within or adjacent to the choroid plexus and scattered
throughout the brain parenchyma (Fig. 2 g). In contrast,
numbers of CD4+ cells returned to normal within 28 d, although rare CD4+ staining cells were evident in the choroid plexus and even in the brain parenchyma at later time
points (Fig. 2 h). Whether CD4+ lymphocytes undergo apoptosis in the brain (19), or eventually migrate out of the
brain is unknown. CD4+ cells do not contribute to protection in this system (14). In the acute phase, large aggregates
of B220+ cells were present in the choroid plexus and some
were scattered throughout the brain parenchyma (Fig. 2 e).
At later time points, small aggregations of B220+ cells were
evident adjacent to or within the choroid plexus, but not in
the brain parenchyma (Fig. 2 k compared with reference 20). MHC class I and II molecules were rapidly upregulated in acute A/WSN encephalitis (Fig. 2, b and c, and reference 14) and then declined to normal levels by days 115 and 320 (Fig. 2, i and j) after inoculation.

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Fig. 1.
High frequencies of CD8+ T cells are recoverable from the brain for long after A/WSN infection. (a) Cells were purified from the brains of
A/X-31-primed A/WSN-challenged mice at various time points as described in Materials and Methods and used directly in CTL assays without restimulation. Total cell counts (broken line, circles), CD4 (triangles), and CD8 (squares) frequencies are plotted. Each data point represents the means of two separate trials with two to eight mice at each time point, except for day 320, which represents a single assay on brain lymphocytes pooled from eight mice.
Standard deviations <10% are not shown. (b) Lysis of virus-infected (circles) and peptide-pulsed (triangles) EL-4 target cells is titratable and antigen specific
(squares, untreated target cells). Variation between duplicates was <15%. E/T ratios refer to the ratio of CD8+ cells to target cells.
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Fig. 2.
Cryostat coronal brain sections from mice inoculated 5 (a-f ) and 320 (g-l) d previously with intracerebral A/WSN. Day 5: (a) CD8+ cells
infiltrating the choroid plexus of the third ventricle (IIIv) and the periaqueductal parenchyma. (b) CD4+ cells (c) markedly increased MHC class I expression in brain parenchyma, (d) increased MHC class II expression predominantly within the inflammation obliterating the lateral ventricle (Lv), (e) B220+
cells in the lateral recess of the third ventricle, and (f ) influenza ribonucleoprotein (RNP) expression surrounding the cerebral aqueduct (Aq). Arrow, virus-infected cell. Day 320: (g) groups of CD8+ cells adjacent to the corpus callosum, (h) parenchymal CD4+ cell, single CD4+ cells in the brain parenchyma
were evident on occasional sections. Lack of MHC class I (i) and II (j) staining in the brain parenchyma. (k) Accumulation of B220+ cells within the
choroid plexus of the lateral ventricle, (j) absence of antiinfluenza RNP staining (periaqueductal region). The ependymal cell loss seen in h, k, and l is
consequent on viral cytopathology. Bar: 50 µm in g and h and 250 µm in the other panels.
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We tested the ability of lymphocytes purified from the
brain to kill peptide-pulsed and virus-infected target cells.
By day 7 when the mice were clinically recovering, we
found consistent cytotoxicity immediately ex vivo against
the immunodominant Db-restricted influenza A NP 366-274
peptide (21) and virus-pulsed targets. Surprisingly, this fresh
killing ex vivo was still demonstrable 320 d after clinical recovery, which was the last time point examined (Fig. 1 b
and Table 1). This fresh killing was also remarkably consistent when sufficient cells were recovered to test the response from individual mice. To compare the secondary
CTL response in another organ, we intranasally inoculated
A/X-31-primed mice with 0.2 hemagglutinating units A/
WSN and purified lung lymphocytes by the same method.
In contrast to the brain, fresh killing by lung lymphocytes
ex vivo was demonstrable from day 4 to day 18, but was
absent at day 33 (Table 1), even though previous work has
established that memory cells can be detected at mucosal
sites (after in vitro restimulation) for >1 yr after primary
infection (13). Presumably, the factors that promoted continuing lymphocyte activation were present in the brain
and not in the lung. Fresh killing wanes at other sites in the
absence of continuing infection (22). We measured the frequency of NP 366-374-specific CTLs at various times after
the intracerebral challenge and found, as expected, a high
frequency of NP-reactive brain lymphocytes as the immune response evolved. (Table 2). Comparison of the NP
366-374-specific CD8+ cell frequency in the brain and
splenic CD8+CD44hiCD62Llo populations indicated that
the brain was enriched with virus-specific precursors.
Since the frequency of NP 366-374-reactive lymphocytes in the brain was high even at day 7, it was possible
that persistently activated extracerebral CD44hiCD62Llo
cells in the A/X-31-primed mice not challenged intracerebrally might also exhibit fresh killing, and that the activated
cells selectively migrated into the brain, i.e., without requiring activation (9). We therefore cytofluorometrically
sorted CD8+ CD62Llo cells from the A/X-31 spleens directly into microtiter plates and then added the antigen-pulsed or unpulsed EL-4 targets. No fresh killing of either
was apparent (not shown). This suggests that intracerebral
challenge with A/WSN had led to the reactivation of the
memory population either in the periphery due to the 5-µl volume of injection used (6) or at the sites of intracerebral inflammation (cells similarly cloned from the brains of A/
X-31-primed A/WSN-challenged mice killed peptide and
virus-pulsed targets; not shown).
As expected (10), we found that most lymphocytes recruited to the brains of A/X-31-primed A/WSN challenged mice had a memory/activated phenotype (CD44hi
CD62Llo), whether assessed early or late in the immune response. We also found high levels of CD49d on all cells.
CD25 and CD71 were only very weakly expressed on
CD4+ or CD8+ lymphocytes, if at all. Mac-1 expression
has been reported to be increased, but like others, we
found no evidence of this (10). The activation marker that
most clearly discriminated between peripheral and CNS
lymphocytes was CD69 (Fig. 3 a). Remarkably, CD69 expression remained high 6 mo after the intracerebral challenge (Fig. 3 c), indicating that specific viral MHC-peptide
complexes persisted and/or that the activation state had not
been turned off. Only a small proportion of the CD8+ cells
were dividing (as measured by BRDU uptake) whether assessed at 3 (not shown) or 6 (Fig. 3 c) mo, possibly because
levels of cytokines such as type 1 interferon (23) had fallen.
This contrasted with the large proportion of CD8+ cells
that took up BRDU when pulsed for 6 d after the intracerebral challenge with A/WSN (Fig. 3 b).

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Fig. 3.
(a) CD8+ cells purified from day 7 (early response)
and day 180 (late response) have
a similar activated/memory phenotype and CD69 is a better activation marker than CD25. (b)
In vivo proliferation of brain
CD8+ cells early in the response
to A/WSN. (c) Brain CD8+ cells
are not proliferating late in the
response. A representative one of
three experiments is shown. Comparison of BRDU uptake by the
splenic and brain CD8+CD44hi
populations showed that there
was a 2.9 ± 0.17-fold enrichment of BRDU+ cells at day 6 in
the brain compared with a 0.48 ± 0.22-fold enrichment at day 180 (mean ± SEM; P < 0.005 using
an unpaired t test). A significant
reduction in CD8+BRDU+ cells
in the late brains compared with
their CD8+CD44hi counterparts
was also evident. Quadrant frequencies are shown (%).
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Previous work had demonstrated that after intracerebral
inoculation of A/WSN into X-31-primed mice, the virus
replicated to high titers for 5 d, but thereafter declined rapidly and was unrecoverable after 6 d (14). In this study we
were again unable to recover replication-competent virus
from brain homogenates on day 7 and 10 by plaque assay
or brain coculture on MDCK cells or by inoculation into
embryonated eggs. Finally, infectious virus was also unrecoverable from the brains of lethally irradiated (1,000 cGy)
mice 1 and 6 mo after the intracerebral A/WSN challenge.
In the lung, replication-competent virus was recoverable
from only 30% of mice 3 d after the challenge.
What maintains the persistently activated NP-specific
CTL in the brain? Viral antigens have been reported to
persist in the CNS (24, 25), but in our model, viral ribonucleoproteins were undetectable immunohistochemically after 21 d (days 5 and 320 shown in Fig. 2). We sought viral
mRNA from whole brain cDNA using single-step PCR
and were consistently unable to amplify the influenza nucleoprotein cDNA after 23 d (Fig. 4). This was in contrast
to the slower clearance of viral nucleic acid in other experimental CNS virus infections after adoptive transfer of immune effectors (24). To increase the sensitivity of detection, we performed two-step seminested PCR (Fig. 4)
and identified viral nucleic acid in 7 of 24 mice tested from
day 30. At day 115, three of nine mice were positive.
However, NP cDNA was never detected in brain cDNA
by the two-step, seminested PCR in animals sampled after
day 115 or from mice that had received intranasal A/X-31 alone (Table 3). Both products included the NP 366-374 epitope, to which persisting CTLs were reactive. In the
heterotypic lung infection, viral nucleic acid was undetectable with single-step PCR after day 9 (not shown). Others
similarly found that viral nucleic acid is not amplifiable
with single step PCR 2 wk after primary influenza A/X-31
in the lung (27).

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Fig. 4.
PCR amplification of nucleoprotein cDNA from mouse brain. Numbers refer to day after intracerebral inoculation. M, HaeIII-digested x
174 markers. R1 is a positive from the NP1/NP2 reaction run for size comparison. Representative gels are shown; each lane represents the result from an
individual mouse. With seminested PCR, nucleoprotein cDNA was amplified from three of nine animals at day 115 and from none of six animals at day
196 (Table 3). Hypoxanthine guanosine phosphoribosyl transferase PCR was positive in all samples indicating that the cDNA synthesis had been satisfactory. PCR products were visualized on 2% agarose ethidium bromide gels under UV light and photographed on Polaroid film.
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In previous work from other groups, CTLs have been
recovered from the brains of animals infected with persistent CNS viruses (28). However, our results indicate that
activated specific antiviral CTLs can persist in the brain
long after viral clearance. Recently, a population of activated antiviral lymphoblasts was recovered from the spleens
of mice after clearance of systemic lymphocytic choriomeningitis virus (LCMV; reference 29). It remains to be
determined if our persistent antiviral CTLs in the CNS are
analogous to this population, but collectively these data indicate that a component of long-term memory may be persistently activated cells at the site of initial infection. If this
population is capable of recirculation after LCMV infection, they may have an important role in immune surveillance in nonlymphoid organs where their state of activation
would favor endothelial transmigration. However, in our
model where systemic infection is minimal, it is more likely
that CTLs recovered from the brain are not recirculating into it, but given the extreme sensitivity of CTLs (30),
have been retained by persisting specific MHC class I-viral
peptide complexes on neural cells that are below the
threshold of detection by immunohistochemistry. It would
be of interest if the persistence of CTLs specific for influenza virus reflects virus persistence at the DNA level as has
recently been demonstrated for LCMV (31). The persistence of activated CTLs at sites of previous viral infection may be an important component of the response to virus
recrudescence.
Address correspondence to S. Hawke, Division of Neuroscience and Psychological Medicine, Level 10E,
Imperial College School of Medicine at Charing Cross, St. Dunstan's Rd., London W6 8RP, UK. Phone:
44-181-8467686; Fax: 44-181-8467715; E-mail: s.hawke{at}cxwms.ac.uk
Received for publication 10 September 1997 and in revised form 9 February 1998.
P.G. Stevenson's current address is the Department of Immunology, St. Jude Children's Research Hospital,
Memphis, TN. C.R.M.This work was supported by project grants from the Multiple Sclerosis Society of Great Britain and Northern Ireland. P.G. Stevenson was a Medical Research Council Clinical Training Fellow.
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