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Rebuilding an Immune-Mediated Central
Nervous System Disease: Weighing the
Pathogenicity of Antigen-Specific versus
Bystander T Cells
Dorian B. McGavern and Phi Truong
J Immunol 2004; 173:4779-4790; ;
doi: 10.4049/jimmunol.173.8.4779
http://www.jimmunol.org/content/173/8/4779
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References
The Journal of Immunology
Rebuilding an Immune-Mediated Central Nervous System
Disease: Weighing the Pathogenicity of Antigen-Specific versus
Bystander T Cells1
Dorian B. McGavern2 and Phi Truong
T
he exquisite specificity of the adaptive immune system is
exemplified by the ability of the host to display both foreign and self-derived peptides in a molecular scaffold,
referred to as the MHC (1, 2). In most instances, the context within
which these peptides are displayed permits T lymphocytes to make
a critical distinction between self and nonself, thus averting undesired immunopathological consequences. Unfortunately, this distinction is occasionally associated with a lapse in judgment that
results in the adaptive immune response becoming misdirected toward self. This aberrant T cell response, now defined by its specificity toward self-proteins, can be triggered by several established
mechanisms, including bystander activation (3– 6), epitope spreading (7, 8), and molecular mimicry (9 –15). In each scenario the
factors governing the maintenance of tolerance (16) are overridden, resulting in the expansion of an activated population of selfreactive T lymphocytes capable of directing (or participating in)
immunopathogenesis.
The fact that self-reactive T cells when mistakenly activated can
contribute to tissue breakdown and subsequent disease seems intuitive and fits appropriately within the self/nonself framework that
has become so prominent in immunology. Along this continuum of
T lymphocyte-induced immunopathology, it has been recognized
repeatedly that an adaptive immune response directed against in-
Department of Neuropharmacology, Division of Virology The Scripps Research Institute, La Jolla, CA 92037
Received for publication January 7, 2004. Accepted for publication July 23, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants AI09484 and
NS048866-01.
2
Address correspondence and reprint requests to Dr. Dorian B. McGavern, 10550
North Torrey Pines Road, La Jolla, CA 92037. E-mail address: [email protected]
Copyright © 2004 by The American Association of Immunologists, Inc.
fectious agents can also play an irreducible role in human disease
(17). The immense clonal expansion of T lymphocytes that often
follows infection (18, 19) probably represents an effort by the host
to completely overwhelm an invading pathogen, thus minimizing
the amount of tissue pathology that develops. However, other factors, such as the anatomical positioning of a pathogen during clearance (20) or the ability of a pathogen to persist (21, 22), can shift
the balance in favor of dire immunopathological consequences.
Because a subset of T lymphocytes is armed with the capacity to
lyse targets (23–25), immunopathology is an unfortunate side effect associated with the eradication of pathogens from the host.
Therefore, it is imperative that several dampening mechanisms
converge to thwart this ill-favored side effect. Specificity (1, 26)
and tight regulation (27, 28) lie at the heart of such dampening
mechanisms.
An issue central to the ability of both self- and pathogenspecific T lymphocytes to cause immunopathology is that of
specificity. Both populations recognize cognate peptides presented in MHC complexes on the surface of cells residing in
various tissue compartments. This evolutionary checkpoint limits the degree of tissue destruction by ensuring that T lymphocytes use effector mechanisms only after target cell recognition
is achieved. However, recent studies focusing on viral infection
of the CNS (29, 30) and the eye (4, 31, 32) have revealed that
T lymphocytes under certain circumstances are able to bypass
recognition of peptide-MHC complexes and mediate tissue destruction without using TCR engagement. The T lymphocytes
studied in the aforementioned model systems lack apparent
specificity to self or foreign Ags and thus are appropriately
referred to as bystander T cells. The precise mechanism by
which these bystander T cells cause immunopathology is not
known; however, their participation in disease appears to defy
important evolutionary constraints imposed by the host.
0022-1767/04/$02.00
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Although both self- and pathogen-specific T cells can participate in tissue destruction, recent studies have proposed that after viral
infection, bystander T cells of an irrelevant specificity can bypass peptide-MHC restriction and contribute to undesired immunopathological consequences. To evaluate the importance of this mechanism of immunopathogenesis, we determined the relative
contributions of Ag-specific and bystander CD8ⴙ T cells to the development of CNS disease. Using lymphocytic choriomeningitis
virus (LCMV) as a stimulus for T cell recruitment into the CNS, we demonstrate that bystander CD8ⴙ T cells with an activated
surface phenotype can indeed be recruited into the CNS over a chronic time window. These cells become anatomically positioned
in the CNS parenchyma, and a fraction aberrantly acquires the capacity to produce the effector cytokine, IFN-␥. However, when
directly compared with their virus-specific counterparts, the contribution of bystander T cells to CNS damage was insignificant
in nature (even when specifically activated). Although bystander T cells alone failed to cause tissue injury, transferring as few as
1000 naive LCMV-specific CD8ⴙ T cells into a restricted repertoire containing only bystander T cells was sufficient to induce
immune-mediated pathology and reconstitute a fatal CNS disease. These studies underscore the importance of specific T cells in
the development of immunopathology and subsequent disease. Because of highly restrictive constraints imposed by the host, it is
more likely that specific, rather than nonspecific, bystander T cells are the active participants in T cell-mediated diseases that afflict
humans. The Journal of Immunology, 2004, 173: 4779 – 4790.
4780
Materials and Methods
Mice
C57BL/6, C57BL/6 OT-I TCR-transgenic (tg) (36, 37), C57BL/6 gp33
TCR-tg (38), and C57BL/6-TgN(ACTbEGFP)10sb (39) mice were bred
and maintained in the closed breeding facility at The Scripps Research
Institute. All strains are on a pure C57BL/6 background. The gp33 TCR-tg
mice were crossed with GFP mice to generate a GFP-labeled population of
Dbgp33– 41-specific T cells (40). The handling of all mice conformed to the
requirements of the National Institutes of Health and The Scripps Research
Institute animal research committee.
Virus
The parental Armstrong 53b (Arm) of LCMV, which is a triple-plaquepurified clone from Armstrong CA 1371 (41), was used for all experiments.
Mice at 8 –10 wk of age were injected either intracerebrally with 103 PFU
or i.p. with 105 PFU of LCMV Arm.
Intracellular cytokine analyses
Splenocytes or brain-infiltrating lymphocytes were cultured (5 h, 37°C,
5% CO2) in RPMI 1640 (Life Technologies, Eggenstein, Germany)
supplemented with 10% FBS, 1% penicillin/streptomycin, 1% L-glutamine, 1% HEPES, 1% nonessential amino acids, 1% sodium pyruvate,
1 ␮g/ml brefeldin A, 100 U/ml IL-2, and 1 (or 2) ␮g/ml of the indicated
peptides. The following peptide sequences were purchased from PeptidoGenic Research (Livermore, CA): gp33– 41 (KAVYNFATC), gp276 –286
(SGVENPGGYCL), gp118 –125 (ISHNFCNL), gp92–101 (CSANNAHHYI),
NP396 – 404 (FQPQNGQFI), NP205–212 (YTVKYPNL), gp61– 80 (GLNgpDIYKGVYQFKSVEFD), NP309 –328 (SGEGWPYIACRTSVVGRAWE),
and OVA257–264 (SIINFEKL). After the 5-h in vitro stimulation, surface as
well as intracellular cytokine stains were performed to evaluate the percentage of CD8⫹ or CD4⫹ T cells producing IFN-␥. Cells from uninfected
mice or cultures stimulated with a mismatched peptide were used as a
negative control.
Immunizations
OT-I mice were immunized 1 day before infection by i.p. injecting 100 ␮g
of cognate peptide (OVA257–264) emulsified in CFA (Difco, Detroit, MI).
Control mice received CFA containing HBSS buffer only.
Flow cytometry
The following Abs purchased from BD Pharmingen (La Jolla, CA) were
used to stain splenocytes and brain-infiltrating lymphocytes: anti-CD83
Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; Arm,
Armstrong 53b; DAPI, 4⬘,6⬘-diamidino-2-phenylindole; GFAP, glial fibrillary acidic
protein; MHV, mouse hepatitis virus; NeuN, neuronal nucleus; tg, transgenic; NP,
nucleoprotein.
FITC, anti-CD8-PE, anti-CD8-PerCP, anti-CD8-allophycocyanin, antiCD4-PE, anti-CD44-allophycocyanin, anti-V␤5.1-PE, anti-V␣2-FITC, and
anti-IFN-␥-allophycocyanin. Cells were acquired using the FACSCalibur
flow cytometer (BD Biosciences, Mountain View, CA) and were analyzed
with the FlowJo (Treestar) software.
Mononuclear cell isolations and tissue processing
To obtain cell suspensions for flow cytometric analyses/peptide stimulation
cultures, spleens and CNS were harvested from mice after an intracardiac
perfusion with a 0.9% saline solution to remove the contaminating blood
lymphocytes. Splenocytes and brain-infiltrating leukocytes were collected
after mechanical disruption of the respective tissues through a 70-␮m filter.
Splenocytes were treated with RBC lysis buffer (0.14 M NH4Cl and 0.017
M Tris-HCl, pH 7.2), washed, and then analyzed. To extract brain-infiltrating leukocytes, homogenates were resuspended in 90% Percoll (4 ml),
which was overlaid with 60% Percoll (3 ml), 40% Percoll (4 ml), and
finally 1⫻ HBSS (3 ml). The Percoll gradients were then centrifuged at
1500 rpm for 15 min, after which the band corresponding to mononuclear
cells was carefully extracted, washed, and ultimately analyzed. The number
of mononuclear cells was determined from each organ preparation and
used to calculate the absolute number of CD8⫹ T cells. PBMC were obtained by centrifugation after incubating (1 h on ice) a capillary of blood
(⬃210 ␮l) with 10 ml of RBC lysis buffer. To visualize GFP⫹Dbgp33– 41specific T cells in situ, mice received an intracardiac perfusion of 4%
paraformaldehyde. Brains were then removed, incubated for 24 h in 4%
paraformaldehyde and for an additional 24 h in 30% sucrose. After freezing
tissues in OCT (Tissue-Tek), 6-␮m frozen sections were cut and stained
with 4⬘,6⬘-diamidino-2-phenylindole (DAPI; 1 mg/ml, 5 min) to visualize
nuclei. For all other immunohistochemical analyses, fresh, unfixed brains
were submerged in OCT and frozen using dry ice.
Adoptive transfer of GFP⫹Dbgp33– 41 T cells
CD8⫹ T cells were purified from gp33 TCR-tg ⫻ GFP mouse splenocytes
by negative selection (StemCell Technologies, Cambridge, MA). The enrichment procedure resulted in a purity of ⬎98%. Concentrations ranging
from 101–105 GFP⫹Dbgp33– 41 T cells were transferred i.v. into naive
C57BL/6 or OT-I TCR-tg recipients. Two days after the adoptive transfer,
mice were infected intracerebrally with 103 PFU or i.p. with 105 PFU of
LCMV Arm. To enumerate the absolute number of GFP⫹Dbgp33– 41 CD8⫹
T cells present in OT-I mice reconstituted with 103 naive precursors,
splenocytes and CNS (brain/spinal cord)-infiltrating leukocytes were harvested at precisely the time point when overt neurologic dysfunction became apparent. Each cell population was stained with antiCD8-allophycocyanin and analyzed by flow cytometry to establish the
percentage of the population that was both CD8⫹ and GFP⫹. This percentage was then multiplied by the number of cells harvested from each
organ to obtain values approximating the absolute number of
GFP⫹Dbgp33– 41 CD8⫹ T cells required in the spleen and CNS to reconstitute disease in OT-I mice (see Fig. 9D).
Immunohistochemistry
To visualize LCMV, neurons, astrocytes, and CD8⫹ T cells, 6-␮m frozen sections were cut, fixed with 2% formaldehyde, blocked with 10% FBS
(1 h, at room temperature), and stained (1 h, at room temperature) with
guinea pig anti-LCMV (1/1000), mouse anti-neuronal nuclei (anti-NeuN;
1.25 ␮g/ml; Chemicon International, Temecula, CA), rabbit anti-glial
fibrillary acidic protein (anti-GFAP; 1/800; DakoCytomation, Carpinteria,
CA), and rat anti-CD8 (1.0 ␮g/ml; BD Pharmingen, La Jolla CA) Abs,
respectively. To block endogenous mouse Abs, sections stained with
mouse anti-NeuN were preincubated (1 h, at room temperature) with a Fab⬘
anti-mouse H and L chain Ab (35 ␮g/ml; Jackson ImmunoResearch Laboratories, West Grove, PA). After the primary Ab incubation, sections
stained with Abs directed against NeuN, GFAP, or CD8 were washed,
stained with a biotinylated secondary Ab (1/400; 1 h, at room temperature;
Jackson ImmunoResearch Laboratories), washed, and then stained with
streptavidin-rhodamine red-X (1/400; 1 h, at room temperature; Jackson
ImmunoResearch Laboratories). The polyclonal anti-LCMV Ab was detected using an anti-guinea pig secondary conjugated to FITC (1/800; 1 h,
at room temperature). All sections were costained with DAPI (1 ␮g/ml; 5
min, at room temperature) to visualize nuclei. We calculated the percentage
of LCMV-infected cells accounted for by neurons (NeuN) or astrocytes
(GFAP) using frozen brain sections from persistently infected OT-I mice
costained with the above-mentioned Abs (see Fig. 3). Eighteen random
⫻40 fields were captured using a confocal microscope (see Microscopy
section) and analyzed to determine the number LCMV-infected cells in
each field that overlapped with NeuN or GFAP. The resultant fractional
value was ultimately represented as a percentage.
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In the present study we used a well-defined model of meningitis
induced by lymphocytic choriomeningitis virus (LCMV)3 (20, 33)
in an attempt to further explore as well as generalize the importance of bystander T cells in the development of CNS pathology.
A strong base of literature indicates that the lethal meningitis initiated after an intracerebral challenge with LCMV bears the hallmarks of specificity. CD8⫹ T cells play an indispensable role in
the disease pathogenesis, which becomes lethal at 6 –7 days postinfection (34). Moreover, it has been touted that the effector molecule perforin, which is secreted by CD8⫹ T cells, is also important
in the acute disease process (23). However, at the end stage of the
disease when neurologic deficits become apparent, we were unable
to account for the specificity of most CD8⫹ T cells residing in the
CNS. This led us to theorize that bystander CD8⫹ T cells of an
irrelevant specificity might indeed contribute to the development
of pathology if retained in the CNS over an extended time period.
Furthermore, the ability of a seemingly endless supply of pathogens to replicate within the CNS (35) may serve as a stimulus to
recruit and activate bystander T cells. Consequently, we set out to
address several important issues regarding bystander T cell activation, recruitment, and potential effector mechanisms within the
CNS after viral infection.
REBUILDING AN IMMUNE-MEDIATED CNS DISEASE
The Journal of Immunology
4781
Analysis of apoptosis
Analysis of cell death was performed on 6-␮m frozen brain sections using
the ApopTag Red apoptosis detection kit according to the manufacturer’s
instructions (Chemicon International). Sections were costained with DAPI
(1 ␮g/ml; 5 min) to visualize nuclei. The number of apoptotic cells per
square centimeter of brain tissue was calculated from brain reconstructions
(see Fig. 7 and Microscopy section) using a program written for the KS300
image analysis software. After segmentation of the regions representing
apoptotic cells from each image, the average area of a single apoptotic cell
as well as the total area occupied by apoptotic cells were calculated. The
absolute number of apoptotic cells was then determined by dividing the
total area occupied by apoptotic cells by the average area of a single apoptotic cell. The data were ultimately represented as the absolute number
of apoptotic cells per square centimeter of brain tissue sampled.
Microscopy
Statistical analyses
Data handling, analysis, and graphic representation were performed using
Microsoft Excel and SigmaPlot. Statistical differences were determined by
Student’s t test or one-way ANOVA ( p ⬍ 0.05) using SigmaStat.
Results
Bystander T cells fail to drive an acute meningitis
Because of the rapidity with which disease presents in mice infected intracerebrally with LCMV Arm (i.e., 6 –7 days), it is reasonable to hypothesize that virus-specific T cells play a pivotal role
in pathogenesis. Supporting this hypothesis are several irrefutable
studies demonstrating that LCMV-specific CD8⫹ T cells are required for the lethality of the disease process (34, 42). However, at
the precise time point when mice succumb to the meningitis, no
more than 20.4 ⫾ 2.5% of the CNS-infiltrating CD8⫹ T cells could
be tallied as those capable of responding to one of seven different
MHC class I-restricted peptides thought to represent the entire
LCMV-specific T cell response (43– 44). These data were obtained
by analyzing six symptomatic B6 mice on day 6 postinfection.
Splenocytes and CNS-infiltrating mononuclear cells were incubated with a peptide pool containing the seven known class I-restricted LCMV epitopes and then assayed for IFN-␥ production
(see Materials and Methods). At this time point postinfection, a
similar frequency of LCMV-specific CD8⫹ T cells was observed
in the spleen (19.5 ⫾ 5.0%).
Considering that we could not account for ⬃80% of the CD8⫹
T cells residing in the CNS at the peak of disease, we surmised that
T cells of an irrelevant specificity (i.e., bystander T cells) were
recruited into the CNS and may indeed participate in disease
pathogenesis. To test this supposition, we intracerebrally inoculated a TCR-tg mouse strain (hereafter referred to as an OT-I mice)
in which ⬎90% of the CD8⫹ T cells recognized aa 257–264 of
OVA presented in the H-2Db molecule (36, 37). Because there is
no overlap between this portion of OVA and any LCMV protein,
KbOVA257–264-specific CD8⫹ T cells are truly bystander T cells of
an irrelevant specificity. When OT-I mice were infected intracerebrally with LCMV Arm, the majority of the mice failed to succumb to the infection (Fig. 1A). In fact, for the entire observation
period (⬎150 days), OT-I mice remained completely asymptom-
FIGURE 1. Analysis of survival and the peripheral T cell compartment
in OT-I mice. Wild-type (n ⫽ 5) and OT-I mice (n ⫽ 5) were infected
intracerebrally with 103 PFU of LCMV Arm and monitored for survival.
The plot in A illustrates an observation period of 55 days, which is representative of the entire 150-day window of the study. The OT-I curve shows
that one of the five mice succumbed to the intracerebral challenge. This
probably resulted from the intracerebral injection procedure, because no
additional OT-I mice succumbed to infection in subsequent experiments
(see Fig. 9B for examples). PBMC harvested from uninfected wild-type
and OT-I mice were analyzed flow cytometrically to establish the frequency of CD8⫹, CD4⫹, and CD8⫹CD4⫹ cells in the T cell compartment
(B). Gray boxes are used to highlight each of the aforementioned T cell
populations. Note the significant skewing of the repertoire in OT-I mice:
28% CD8⫹ and only 1.8% CD4⫹.
atic. In contrast, wild-type C57BL/6 mice showed signs of neurologic dysfunction (i.e., ruffled fur, hunched back, and tremors) and,
as expected, succumbed to the meningitis precisely on day 6
postinfection. These data indicate that the restricted, largely monospecific T cell repertoire present in OT-I mice was incapable of
recapitulating the overt disease observed in wild-type mice.
OT-I mice do not mount an LCMV-specific T cell response
Having established that signs reminiscent of LCMV-induced meningitis were not observed in OT-I mice, we next set out to define
the parameters underlying this benign disease course and gather
information regarding the migration, activation status, and cytokine production of bystander T cells. Because the LCMV-specific
T cell response is thought to drive the disease process through the
engagement of virus-infected targets residing in the meninges,
ependyma, and choriod plexus, we initially evaluated whether
OT-I mice were indeed capable of mounting an LCMV-specific T
cell response despite their restricted T cell repertoire. To maximize
the probability of detecting such a response, we infected OT-I and
wild-type control mice i.p. with 105 PFU of LCMV Arm and analyzed the entire known MHC class I- and class II-restricted immune response (Fig. 2). Splenocytes were harvested on day 8
postinfection (the established peak of the virus-specific immune
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Two-color reconstructions to visualize the distribution of LCMV, apoptotic
cells, or GFP⫹Dbgp33– 41-specific T cells on coronal brain sections were
obtained using an Axiovert S100 immunofluorescence microscope (Zeiss,
Oberkochen, Germany) fitted with an automated xy stage, an Axiocam
color digital camera, and a ⫻5 objective. Registered images were captured
for each field on the coronal brain section, and reconstructions were performed using the MosaiX function in the KS300 image analysis software.
Higher resolution images were captured with an MRC1024 confocal microscope (Bio-Rad, Hercules, CA) fitted with a krypton/argon mixed gas
laser (excitation at 488, 568, and 647 nm) and a ⫻40 oil objective. All
two-dimensional confocal images illustrate a single z section captured at a
position approximating the midline of the cell.
4782
REBUILDING AN IMMUNE-MEDIATED CNS DISEASE
response (18)), stimulated with the indicated LCMV peptides, and
evaluated for the synthesis of IFN-␥. As anticipated, OT-I mice,
when compared with wild-type controls, failed to mount a CD8⫹
T cell response to any of the known LCMV peptides ( p ⬍ 0.05;
Fig. 2, A and C). However (and perhaps more surprisingly), a
population of LCMV-specific CD4⫹ T cells also failed to expand
in OT-I mice ( p ⬍ 0.05; Fig. 2, B and C). This can probably be
explained by the skewing in the CD4⫹ T cell compartment that
occurs in TCR-tg mice (37). Because of alterations in thymic selection, a substantially reduced number of CD4⫹ T cells were observed in the peripheral tissues of naive OT-I mice (Fig. 1B). In
concert, these data suggest that the absence of an acute LCMVspecific T cell response explains the failure of OT-I mice to present
with the rapid and overt neurologic complications observed in
wild-type mice. However, the reason behind the lack of neurologic
complications over the entire observation period as well as the
status of the bystander T cells remained open-ended questions.
Viral tropism shifts in the brains of OT-I mice
Because the lethal disease process observed after infection of wildtype mice depends not only on the expansion of LCMV-specific T
cells, but also on the anatomical positioning of the virus within the
CNS, we next addressed whether LCMV replicated in the meninges, ependyma, and choriod plexus of infected OT-I mice. At the time
point when wild-type mice normally succumb to the infection (i.e.,
day 6 postinfection), the tropism of LCMV was indistinguishable
from that observed in wild-type mice (Fig. 3A) (see Ref. 40 for example of viral tropism in wild-type mice), indicating that a failure to
develop neurologic complications could not be explained by an aberrant anatomical positioning of the LCMV. Moreover, the lack of
neurologic complications over time could also not be explained by the
inability of LCMV to establish persistence in the CNS. Interestingly,
when the brains of OT-I mice were examined on day 55 postinfection,
it was found that LCMV persisted in the meninges and ependyma as
well as the brain parenchyma (Fig. 3B). Within the brain parenchyma,
LCMV preferred to persist in astrocytes and neurons, because 72%
(or 354 of 492) of all LCMV-infected cells were found to colocalize
with GFAP staining (Fig. 3, F–H), and an additional 17% (or 71 of
410) colocalized with NeuN staining (Fig. 3, C–E). The presence of
LCMV in astrocytes is supported by recent studies demonstrating that
astrocytes are among the first cells that become infected in the developing rat brain (46).
Bystander T cells traffic to the CNS, but with limited efficiency
Knowing that LCMV could indeed replicate as well as persist in
the brains of OT-I mice, we focused our next line of experimentation on whether this could serve as a stimulus to recruit a monospecific population of bystander T cells. Because OT-I mice did
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FIGURE 2. Quantification of the
LCMV-specific T cell response in
wild-type vs OT-I mice. Splenocytes
from wild-type (n ⫽ 4) and OT-I
(n ⫽ 4) mice infected i.p. with 105
PFU of LCMV Arm were analyzed
on day 8 postinfection for CD8⫹ (A)
or CD4⫹ (B) T cell responses to eight
known class I- and class II-restricted
peptides that map to the NP and glycoprotein (GP) of LCMV Arm (see
Materials and Methods). The peptides are shown in order of immunodominance. Splenocytes were incubated in the presence of the
indicated peptides for 5 h in vitro and
then were analyzed for the production of IFN-␥. A summation of the
results is shown in C. Data are represented as the mean ⫾ SD. Statistical differences denoted by asterisks
were determined using Student’s t
test (p ⬍ 0.05).
The Journal of Immunology
4783
not develop overt disease over time, it remained a distinct possibility that bystander T cells were simply not recruited into the
CNS. To address this possibility, we isolated mononuclear cells
from the brains of wild-type and OT-I mice on day 6 after an
intracerebral infection with LCMV. Quantification of the absolute
number of mononuclear (Fig. 4A) as well as CD8⫹ (Fig. 4B) T
cells in the brains at this time point revealed that inflammatory
cells could traffic to the CNS of OT-I mice, but to a markedly
limited degree ( p ⬍ 0.05). Of the few CD8⫹ T cells residing in the
CNS on day 6, the majority were deemed KbOVA257–264 specific
(data not shown) after analysis of V␣2 and V␤5.1 expression (the
TCR present on transgenic cells). Therefore, despite extensive
LCMV replication in the CNS on day 6 postinfection (Fig. 3A),
there was an inherent failure to efficiently recruit bystander
KbOVA257–264-specific CD8⫹ T cells.
Throughout the CNS literature, the dogma states that only activated T cells are permitted access to the CNS (47, 48). However,
some have called this view into question, indicating that naive T
cells also share this property, which is thought to be unique to
activated T cells (49, 50). Based on the prevailing dogma, we
surmised that the reduced ability of bystander KbOVA257–264-specific CD8⫹ T cells to migrate into the CNS could be explained by
their failure to become activated after LCMV infection. Thus, we
quantitatively evaluated CD44 expression (an established activation marker) in the CD8⫹ T cell compartment of wild-type and
OT-I mice on day 6 after intracerebral infection. As expected, the
expression of CD44 was markedly increased on CD8⫹ T cells
obtained from the spleen and CNS of wild-type mice (Fig. 4, C and
D). In fact, ⬎93% of all CD8⫹ T cells residing in the CNS ex-
pressed CD44, confirming that T cells with an activated surface
phenotype have preferential access to this tissue. In contrast,
splenic CD8⫹ T cells (⬎90% of which are KbOVA257–264 specific)
from OT-I mice showed an activation profile identical with that
observed in naive mice (Fig. 4, C and D), and those that were
recruited to the CNS were largely CD44high. These data suggest
that the limited trafficking of KbOVA257–264-specific CD8⫹ T cells
to the CNS in OT-I mice probably results from a failure of the bulk
population to become activated.
Chronic viral persistence enhances bystander T cell recruitment
into the CNS as well as cytokine production
KbOVA257–264-specific CD8⫹ T cells fail to efficiently migrate to
the CNS 6 days after intracerebral infection with LCMV. Based on
these findings, it could be argued that this is an insufficient time
period to recruit a nonactivated population of bystander T cells
into the CNS. Furthermore, the chronicity of a disease process as
well as the continued stimulation of APCs from viral persistence
may, in fact, drive aberrant T cell activation, thus promoting CNS
recruitment and tissue pathology. To address these issues, we
quantified the absolute number of CD8⫹ T cells in the brain on day
55 postinfection. Compared with wild-type mice on day 6, the
absolute number of CD8⫹ T cells (⬎90% of which are
KbOVA257–264 specific) extracted from the brain remained significantly reduced ( p ⬍ 0.05; Fig. 5A), and as before, the majority of
these cells were CD44high (Fig. 5B). Nevertheless, the absolute
number of bystander T cells in the CNS of OT-I mice at this time
point was indeed higher than that observed on day 6 (see Fig. 4B).
These data indicate that over time an elevated number of bystander
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FIGURE 3. Evaluation of viral tropism in OT-I mice. Six-micron coronal brain sections from OT-I mice on day 6 (n ⫽ 3; A) and day 55 (n ⫽ 3; B) postinfection
were stained with a polyclonal anti-LCMV Ab (green) and a nuclear dye (red). Brain reconstructions were performed to illustrate the anatomical distribution of
LCMV. On day 6 after an intracerebral inoculation, LCMV localizes to the meninges, ependyma, and choriod plexus. At later time points, the virus also establishes
persistence in the brain parenchyma. C–H, High resolution analyses of LCMV-infected cells (green; D and G) that colocalize with neuronal (red; C) or astrocyte
(red; GFAP; F) staining on day 55 postinfection. Overlapping fluorescence illustrated in the merged panels (E and H) appears in yellow. The percentages shown
on the merged images indicate the frequency of LCMV-infected cells that can be accounted for by neuronal (17%) or astrocyte staining (73%).
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T cells are retained in the CNS, although the efficiency of recruitment remains diminished compared with that in wild-type mice
infected with LCMV.
Because mononuclear cell preparations from the CNS require
mechanical disruption of the tissue, the aforementioned analyses of
absolute cell numbers provide no information regarding the anatomical positioning of bystander T cells within the CNS. Consequently, we next addressed whether bystander T cells in the brains
of OT-I mice were anatomically poised to drive tissue pathology.
Coronal brain reconstructions were performed on day 55 postinfection to examine the localization of CD8⫹ T cells within the
brain (Fig. 5C). The results revealed that the bystander T cells
were widely distributed throughout the brain parenchyma and
meningeal surface, which are both sites of viral persistence in OT-I
mice (see Fig. 3B). Thus, bystander T cells are not only retained in
the CNS over time, but are anatomically positioned to facilitate
tissue pathogenesis if triggered to release effector molecules.
The ability of a bystander T cell population to drive tissue pathology is predicated on the supposition that the population in
question has been nonspecifically (or aberrantly stimulated) to engage effector mechanisms. Therefore, we addressed whether
KbOVA257–264-specific CD8⫹ T cells could produce IFN-␥ in response to their cognate Ag ex vivo despite having never been
challenged with this Ag in vivo. To ensure that an appropriate
number of cells could be analyzed, splenocytes (instead of braininfiltrating mononuclear cells) were harvested from acutely (day 6)
and persistently (day 40) infected OT-I mice, stimulated with peptide for 5 h in vitro, then analyzed for IFN-␥ production. On day
6 postinfection, the percentage of KbOVA257–264-specific CD8⫹ T
cells that produced IFN-␥ in response to OVA257–264 was not statistically increased over that observed with an irrelevant peptide
(i.e., gp33– 41; Fig. 6, A and C). When the same population was
analyzed in persistently infected OT-I mice on day 40, 1.0% of all
bystander T cells responded to their cognate Ag (a statistically
significant increase over the irrelevant peptide control, p ⬍ 0.05),
which was not observed in age-matched uninfected OT-I mice
(Fig. 6C). Interestingly, this frequency increased to 4.8% on day
230 postinfection (Fig. 6, A and C). However, the response was
both qualitatively and quantitatively diminished when compared
with that of a dominant LCMV-specific T cell population (i.e.,
Dbgp33– 41-specific cells; Fig. 6, A and B). The geometric mean
fluorescent intensity of IFN-␥-producing, Dbgp33– 41-specific T
cells in wild-type mice was 810.2 ⫾ 29.3 (mean ⫾ SD). In contrast, the geometric mean fluorescent intensity for KbOVA257–264specific T cells from persistently infected mice was only 80.7 ⫾
10.2 on day 40 and 129.6 ⫾ 22.3 on day 230, which resembled the
low producing, Dbgp33– 41-specific cells observed in wild-type
mice (Fig. 6B). Collectively, these data demonstrate that bystander
T cells are stimulated (perhaps aberrantly) to produce the effector
cytokine, IFN-␥, in persistently infected OT-I mice, although the
quality of this response on a per cell basis is markedly reduced
compared with that in LCMV-specific CD8⫹ T cells.
Bystander T cells induce minimal apoptosis in the brains of
persistently infected mice
Having established that bystander T cells are anatomically positioned in the CNS parenchyma and harbor the potential to produce
cytokines (albeit to a reduced degree) in persistently infected OT-I
mice, we theorized that the retention of this population of cells
within the CNS over time could potentially have a detrimental
impact. Although overt disease was not noted in persistently infected OT-I mice, we opted to evaluate a more sensitive readout
for tissue pathology (i.e., apoptosis). The number of apoptotic cells
per square centimeter of tissue was analyzed quantitatively on
coronal brain reconstructions from naive, persistently infected
OT-I (days 31 and 150), and acutely infected (day 6) wild-type
mice (Figs. 7 and 8A). The number of apoptotic cells per square
centimeter was similar between uninfected (137 ⫾ 55) and persistently infected OT-I mice on day 31 (609 ⫾ 383) and day 150
(116 ⫾ 48) postinfection, although a slight, but not statistically
significant, difference was noted on day 31 ( p ⫽ 0.329). In stark
contrast, the number of apoptotic cells in the brains of wild-type
mice on day 6 (57,283 ⫾ 18,541) was substantially elevated ( p ⬍
0.05), which accurately reflects both the severity and the outcome
of the pathologic process (i.e., death). Importantly, quantitative
analysis revealed that 78.0 ⫾ 4.7% (or 384 of 490) of the apoptotic
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FIGURE 4. Quantitative analysis of bystander T cell trafficking to the
brain. The total number of mononuclear cells (A) and CD8⫹ T cells (B)
residing in the brain was determined for wild-type (n ⫽ 5) and OT-I (n ⫽
4) mice on day 6 postinfection (d6). Statistical differences denoted by asterisks were determined using Student’s t test (p ⬍ 0.05). Flow cytometric
analyses were used to evaluate the expression of CD44 on CD8⫹ T cells
obtained from the spleen or brain of mice at the indicated time point (C and
D). A representative histogram gated on CD8⫹ T cells is shown for both
tissue compartments (C). A summation of the results that illustrates the
percentage of CD8⫹ T cells deemed CD44high is shown in D. For splenic
CD8⫹ T cells, CD44 expression was compared statistically by one-way
ANOVA (p ⬍ 0.05) using the values obtained from uninfected wild-type
(n ⫽ 4), uninfected OT-I (n ⫽ 4), day 6 wild-type (n ⫽ 5), and day 6 OT-I
(n ⫽ 4) mice. Asterisks indicate a statistically significant reduction compared with day 6 wild-type mice. Uninfected wild-type and day 6 OT-I
mice were not statistically different from one another. Because no CD8⫹ T
cells were extracted from the brains of uninfected mice, analysis of
CD44high expression on brain-infiltrating CD8⫹ T cells was restricted to
infected wild-type and OT-I mice. Compared with wild-type mice, a slight
reduction in the percentage of CD8⫹CD44high T cells was observed in OT-I
mice; however, this percentage was significantly higher than that in the
splenic compartment (p ⬍ 0.05).
REBUILDING AN IMMUNE-MEDIATED CNS DISEASE
The Journal of Immunology
4785
cells colabeled with LCMV Ab staining. Because LCMV is a noncytopathic virus, these results signify that the majority of the apoptosis results from immune targeting of virus-infected cells (presumably by CD8⫹ T cells). Only a minor fraction (⬍22%) of the
cellular death can be accounted for by infiltrating inflammatory
cells. In concert, these results demonstrate that bystander T cells
induce minimal CNS pathology despite their chronic retention,
activation status, and potential to produce an effector cytokine.
Because the OT-I mice used in the previous studies were not
primed with cognate Ag before infection with LCMV, the potential
for CNS pathology relied solely on the recruitment of an existing
population of bystander T cells that aberrantly acquired the capacity to produce cytokines and effector molecules. Consequently, it
could be argued that the failure to observe CNS pathology in OT-I
mice results from an inability to recruit a minimum number of
activated bystanders. To address this fundamental concern, OT-I
mice were immunized with cognate peptide (OVA257–264) emulsified in CFA and analyzed on day 17 after an intracerebral challenge with LCMV (mirroring previous studies that achieved bystander CNS damage) (29, 30). Despite the enhanced recruitment
( p ⬍ 0.05) of mononuclear cells (Fig. 8B) as well as bystander
CD8⫹ T cells (Fig. 8C) to the CNS after immunization with OVA
peptide, the level of apoptosis observed in brains of OVA/CFA
mice (214 ⫾ 99) was statistically similar ( p ⬍ 0.05) to that observed in HBSS/CFA controls (238 ⫾ 102) and naive B6 mice
(Fig. 8A). Thus, specific activation of bystander T cells did not
enhance their ability to mediate damage in the CNS.
Naive, monospecific Dbgp33– 41-specific T cells completely
reconstitute disease in OT-I mice
The inability of OT-I mice to mount an LCMV-specific immune
response (see Fig. 2) indicates that the T cell repertoire is remarkably restricted, especially when one considers that the number of
T cell precursors required to spawn an Ag-specific immune response is exceedingly low (51). Knowing this, we next addressed
whether it was possible to “rebuild” the disease process in OT-I
mice by reconstituting with naive Dbgp33– 41-specific precursors.
To establish the validity of the transfer model system, we first
evaluated the relationship between precursor frequency and the
magnitude of the virus-specific immune response in wild-type
mice infected i.p. with LCMV Arm. Mice were infected by this
traditional route to ensure survival as well as to maximize the
efficiency of the LCMV-specific immune response. Before infection, mice received log-serial dilutions of naive GFP⫹Dbgp33– 41specific (40) CD8⫹ T cells (ranging from 105–100). At the peak of
the virus-specific immune response (day 8 postinfection), splenocytes were harvested and analyzed for the frequency of CD8⫹ T
cells that were also GFP⫹ (i.e., Dbgp33– 41 specific). A perfect sigmoidal relationship (r ⫽ 1.0) between the two variables was observed, demonstrating that a plateau in the response was reached
when 105 naive precursors were injected (Fig. 9A). Because the
burst size of the immune response was indeed impacted by the
precursor frequency, we surmised that this experimental paradigm
could be used to calculate the number of Ag-specific T cells required to rebuild disease in OT-I mice. To this end, OT-I mice
received log-serial dilutions (ranging from 104–100) of naive
GFP⫹Dbgp33– 41-specific CD8⫹ T cells and were then infected intracerebrally with LCMV Arm (Fig. 9B). As expected, all wildtype control mice succumbed to the disease by day 7 postinfection,
whereas OT-I mice receiving no transfer (i.e., 100 cells) survived
for the entire observation period (⬎100 days). Interestingly, we
established that as few as 103 (or 1000) naive GFP⫹Dbgp33– 41specific T cells were able to reconstitute disease in OT-I mice,
although the kinetics of the disease process were delayed compared with those in wild-type controls.
Because naive LCMV-specific CD8⫹ T cells undergo massive
clonal expansion after infection (18, 19), the quantity of precursors
injected does not necessarily represent the absolute number of
effector cells required in the CNS to drive pathogenesis. Thus,
we addressed this question by calculating the number of
GFP⫹Dbgp33– 41-specific CD8⫹ T cells in the brain/spinal cord
and spleen of OT-I mice receiving the minimum number of
precursors required to reconstitute disease (i.e., 103). At the
time when overt neurologic dysfunction became apparent,
GFP⫹Dbgp33– 41-specific T cells localized to the meninges,
ependyma, and choroid plexus of all reconstituted OT-I mice (Fig.
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FIGURE 5. Enumeration and anatomical localization
of bystander T cells in persistently infected OT-I mice.
The total number of brain-infiltrating CD8⫹ T cells (A)
as well the percentage of CD8⫹CD44high T cells (B)
were compared between acutely infected (day 6 (d6))
wild-type mice (n ⫽ 5) and persistently infected (day 55
(d55)) OT-I mice (n ⫽ 5). Statistical differences denoted
by asterisks were determined using Student’s t test (p ⬍
0.05). The anatomical distribution of CD8⫹ T cells in
persistently infected OT-I mice (n ⫽ 3) was evaluated
by performing reconstructions of 6-␮m coronal brain
sections stained with an anti-CD8 Ab (green) and a nuclear dye (blue). Anti-CD8 (instead of a V␤5.1) Ab was
used because flow cytometric analyses revealed that
⬎90% of brain-infiltrating CD8⫹ T cells at this time
point coexpressed V␣2 and V␤5.1, confirming that they
were KbOVA257–264 specific. A representative coronal
brain reconstruction is shown in C. The diameter of each
green dot, which represents an individual CD8⫹ T cell,
was digitally enhanced by one pixel to facilitate visualization of these cells on the reconstruction.
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9C). Quantitatively, it was determined at this time that an average
of 1,287,000 LCMV-specific T cells were present in the spleen,
and 51,647 cells were present in the brain/spinal cord (Fig. 9D).
Because the SD for the collective group was surprisingly small
(Fig. 9D), these values provide a fair approximation of the number
of monospecific Dbgp33– 41-specific T cells required to drive pathogenesis and enhance mortality in OT-I mice.
Dbgp33– 41-specific T cells only modestly enhance the
recruitment of bystander T cells into brain
In our final line of experimentation using this reductionist model
system, we evaluated the influence of LCMV-specific T cells on
bystander T cell recruitment and activation. Because the biologic
system in question (i.e., reconstituted OT-I mice) contained primarily only two CD8⫹ T cells populations (i.e., Dbgp33– 41-specific
effectors and KbOVA257–264-specific bystanders), it became possible to address the impact that one monospecific T cell population
had on the other. OT-I mice were reconstituted with 104 naive
GFP⫹Dbgp33– 41 T cells to restore the normal disease kinetics observed in wild-type mice (see Fig. 10B) and compared directly to
OT-I mice receiving no transfer. At 7 days after intracerebral in-
FIGURE 7. Apoptosis in the brains of infected wild-type and OT-I
mice. An ApopTag detection kit (see Materials and Methods) was used to
visualize apoptotic cells (green) on 6-␮m frozen brain sections from uninfected wild-type (n ⫽ 3; A), day 31 after infection (d31) OT-I (n ⫽ 3; B),
and d6 wild-type (n ⫽ 5; C) mice. Sections were counterstained with a
nuclear dye (blue). The diameter of each green dot, which represents an
individual apoptotic cell, was digitally enhanced by 1 pixel to facilitate
visualization of these cells on the reconstruction. Note the marked increase
in the number of apoptotic cells in the brain of symptomatic B6 mice on d6
after an intracerebral infection.
fection with LCMV Arm, mononuclear cells were extracted from
the brains of both groups and enumerated. As expected, two populations of Ag-specific CD8⫹ T cells were observed in the brains
of symptomatic OT-I mice that received an adoptive transfer (Fig.
10A). However, Dbgp33– 41-specific T cells had only a small influence on bystander T cell recruitment into the brain. The number of
KbOVA257–264-specific T cells in the brain increased by 2-fold in
transfer recipients ( p ⬍ 0.05; Fig. 10B, inset), although this number remained markedly reduced compared with that of Dbgp33– 41
T cells ( p ⬍ 0.05; Fig. 10B). In addition, the activation status of
bystander T cells (as measured by CD44 expression) was statistically identical between transfer recipients and controls. Thus, in
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FIGURE 6. Analysis of IFN-␥ production by bystander T cells. Splenocytes from intracerebrally inoculated wild-type (day 6 (d6); n ⫽ 3), OT-I
(d6; n ⫽ 4), OT-I (d40; n ⫽ 3), and OT-I (d230; n ⫽ 3) mice were analyzed
for the ability to produce IFN-␥ after a 5-h in vitro stimulation with either
gp33– 41 or OVA257–264 peptide. Representative flow cytometric plots gated
on CD8⫹ T cells are shown in A. The percentage of CD8⫹ T cells that
produce IFN-␥ in response to the indicated peptide is shown on each plot
(f). V␤5.1 expression was evaluated to ensure that the CD8⫹ T cells
analyzed from OT-I mice were, in fact, KbOVA257–264 specific. B, Representative histogram (gated on CD8⫹ T cells) comparing the intensity of
IFN-␥ production between Dbgp33– 41-specific (d6) and KbOVA257–264-specific (d40) T cells. C, Summary of the percentage of IFN-␥ expressing
CD8⫹ T cells to cognate peptide shown in A. This panel also includes
uninfected OT-I (n ⫽ 3) mice as a control. Asterisks denote a statistical
difference, as determined by Student’s t test (p ⬍ 0.05), between cells
stimulated with cognate vs irrelevant peptide. For example, a statistically
significant increase in IFN-␥ production was observed for OT-I mice on
d40 when OVA257–264 peptide (cognate) was compared with gp33– 41
(irrelevant).
REBUILDING AN IMMUNE-MEDIATED CNS DISEASE
The Journal of Immunology
the acute timeframe analyzed (i.e., 7 days), LCMV-specific effector cells only modestly influence bystander T cell trafficking into
the brain.
Discussion
When researchers are faced with immunopathology in human disease, the next challenge is to elucidate the potential mechanisms by
which this immunopathology develops, with the ultimate intention
of devising more efficacious treatment modalities. Although it is
widely appreciated that T lymphocytes (both CD4 and CD8) directed against self- or pathogen-specific Ags can mediate tissue
destruction, support for the participation of bystander T cells in
such processes remains limited (4, 29) and appears to be in contradiction with an important constraint (i.e., peptide-MHC recognition) imposed by the host. At the outset of the present study, we
surmised that bystander T cells, if stimulated (either aberrantly or
specifically) and recruited to the CNS for a chronic time window
(as might be expected to occur in a chronic human disease such as
multiple sclerosis), would contribute to nonspecific tissue destruction and subsequent neurologic dysfunction.
Our results revealed, however, that the final component of this
hypothesis did not occur, and that specificity is greatly favored
over nonspecificity where tissue destruction is concerned. In support of this statement is the fact that bystander CD8⫹ T cells were
unable to reconstitute the fatal meningitis observed in wild-type
mice. During an acute time window (i.e., 6 –7 days), this observation was perhaps expected given that the prevailing literature
supports a notable role for LCMV-specific CD8⫹ T cells in the
lethality of this disease process (34, 42). However, it was anticipated that if recruited into the CNS, bystander CD8⫹ T cells would
have an impact over a chronic time window (i.e., ⬎30 days), especially when considering that LCMV established persistence
throughout the brain parenchyma. Indeed, over time, a population
of KbOVA257–264-specific T cells with an activated surface phenotype (i.e., CD44high) was recruited from the periphery into the
CNS. These cells were anatomically poised in the brain parenchyma and thus had the potential to mediate immunopathology.
Moreover, it was revealed that as a consequence of persistent infection, a fraction of these bystander T cells was relieved of their
naive status and acquired the ability to release an effector cytokine
(i.e., IFN-␥), albeit to a reduced degree compared with LCMVspecific T cells. This observation is in line with several studies
demonstrating that bystander activation can occur after viral infection (3– 6), although the relevance of such activation has been
called into question (52). Regardless, in the present study, activation status, cytokine-producing ability, and retention of bystander
T cells within the CNS over an extended time frame appeared to
have no bearing on the ability of these cells to participate in immunopathogenesis. Infected OT-I mice remained completely
asymptomatic, and there was no evidence of enhanced apoptosis in
the CNS despite the sustained presence of bystander T cells. Even
specific activation of bystander T cells with cognate peptide did
not enhance their ability to mediate CNS damage, which is in
opposition with previous studies that achieved bystander-mediated
damage in the CNS (29, 30).
In our model system, significant CNS pathology depended entirely on the activities of Ag-specific CD8⫹ T cells. Wild-type
mice containing a diverse repertoire of LCMV-specific T cells
showed a prodigious amount of CNS apoptosis in a relatively confined time window (⬃6 days), which underscores the severity with
which CD8⫹ effector T cells can mediate tissue destruction when
directed by specific peptide-MHC interactions. Also supporting
this point was the ability of 1000 naive Dbgp33– 41-specific CD8⫹
T cell precursors to become activated, clonally expand, and completely reconstitute disease in OT-I mice. Thus, one monospecific
T cell population can drive an entire disease process with lethal
consequences.
Although the participation of bystander T lymphocytes in
immunopathogenesis remains a theoretical possibility, it seems
more plausible that self- or pathogen-specific T cells represent
the major driving force behind most T cell-mediated disorders
afflicting humans. However, under certain circumstances an aberrant (and perhaps uncontrolled) proinflammatory response
may give rise to global activation of the T cell compartment,
permitting bystander pathology to occur. For example, an ocular infection of TCR-tg mice with HSV results in the development of lesions in the absence of HSV-specific T cells (4, 31,
32). Curiously, bystander T cell activation and pathology were
observed when CD8⫹ or CD4⫹ TCR-tg mice were used (53),
indicating that either T cell population is capable of performing
the task in this model system. Although the specific mechanism
by which bystander T cells induce ocular lesions after HSV
infection has not been determined, the authors favor the generation of a cytokine storm as the underlying cause (4). Thus, it
could be argued that LCMV is not a potent inducer of such
cytokines (which presently have not been identified in the HSV
system), explaining the inability of bystander T lymphocytes to
mediate tissue destruction in our study.
Damage mediated by bystander T cells has also been observed
in the CNS white matter of TCR-tg mice infected with mouse
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FIGURE 8. Quantitative analysis of apoptosis and bystander T cell recruitment in the brains of infected wild-type and OT-I mice. A, Number of
apoptotic cells per square centimeter quantified on coronal brain reconstructions (see Fig. 7 for examples) using an image analysis program (see
Materials and Methods). The data are represented as the mean ⫾ SD for
the following experimental groups: uninfected wild-type (n ⫽ 3), day 31 of
infection (d31) OT-I (n ⫽ 3), d150 OT-I (n ⫽ 3), d17 HBSS/CFA OT-I
(n ⫽ 4), d17 OVA/CFA OT-I (n ⫽ 3), and d6 wild-type (n ⫽ 5). Numbers
on the graph denote the mean for each group. Similar results were obtained
when OT-I mice were analyzed on d55. B and C, Absolute number of
mononuclear (B) and activated (i.e., CD44⫹) KbOVA257–264-specific T
cells (C) in CNS of the indicated groups on d6 postinfection. V␤5.1 expression was evaluated in C to ensure that the CD8⫹ T cells analyzed from
the CNS of OT-I mice were, in fact, KbOVA257–264 specific. Data are
represented as the mean ⫾ SD. Statistical differences between all groups
(denoted by asterisks) were determined by one-way ANOVA (p ⬍ 0.05).
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FIGURE 9. Reconstitution of acute disease in OT-I mice. A, The perfect sigmoidal relationship (r ⫽ 1.0) between the number of naive GFP⫹Dbgp33– 41-specific
CD8⫹ T cells injected and the magnitude of the virus-specific immune response on day 8 after an i.p. infection of wild-type mice with 105 PFU of LCMV Arm.
Two days before infection, mice received log-serial dilutions of naive GFP⫹Dbgp33– 41-specific CD8⫹ T cells ranging from 105–100. B, Survival curve of OT-I
mice (n ⫽ 5/group) reconstituted with log-serial dilutions of naive GFP⫹Dbgp33– 41-specific CD8⫹ T cells ranging from 104–100. Wild-type mice (n ⫽ 5) served
as a positive control for this experiment. Two days after the adoptive transfer, mice received intracerebral infection with 103 PFU of LCMV Arm. C, Representative
example (n ⫽ 3) of a coronal brain reconstruction from a symptomatic OT-I mouse (day 8) receiving the minimum number of naive GFP⫹Dbgp33– 41-specific
CD8⫹ T cells (i.e., 103) required to reconstituted disease. Green dots represent individual GFP⫹Dbgp33– 41-specific CD8⫹ T cells in the brain. Sections were
costained with a nuclear dye (blue). D, Quantification of the absolute number of GFP⫹Dbgp33– 41-specific CD8⫹ T cells present in the spleen and CNS of
symptomatic OT-I mice (n ⫽ 4) on day 8 postinfection, receiving the minimum number of cells required to reconstitute disease. Data are represented as the mean ⫾
SD. The average for each tissue is shown on the graph.
hepatitis virus (MHV) (29, 30) and, unlike the HSV model, occurred only with transgenic CD8⫹ (not CD4⫹) T cells (54). Furthermore, aggressive immunization strategies (i.e., injection of
cognate peptide in CFA) were used to fully activate the transgenic
bystander T cell population before infection with MHV, and even
then the pathology was never as severe as that observed in wildtype mice containing MHV-specific T cells. Because TCR-tg mice
have a reduced number of regulatory CD4⫹ T cells (especially
when crossed on a background deficient in RAG-1) (55–57), activation of an entire monospecific T cell population in the absence
The Journal of Immunology
4789
of sufficient regulation may inadvertently favor the desired outcome (i.e., bystander T cell-mediated pathology). Although studies
of regulatory cells in transgenic mice have focused mainly on
those containing a population of monospecific CD4⫹ T cells, alterations in thymic selection also occur in CD8 TCR-tg mice that
significantly reduce the number of CD4⫹ T cells in the periphery
(see Fig. 1B). This consequently diminishes the absolute number
of CD4⫹CD25⫹ regulatory T cells in the circulation. For example,
a comparison between wild-type (n ⫽ 4) and OT-I (n ⫽ 4) mice
revealed a 73% reduction ( p ⬍ 0.05) in the absolute number of
splenic regulatory T cells (1,855,400 ⫾ 344,289 vs 494,113 ⫾
41,210). Thus, it is important to note that studies reporting pathology induced by bystander T cells have used TCR-tg mice devoid
of all other B or T cells (i.e., RAG-1-deficient or SCID mice) (4,
29 –32), which, unlike the immunocompetent OT-I mice used in
the present study, should have no CD4⫹CD25⫹ regulatory T cells.
Although it is clear that pathology can be observed under such
circumstances, it remains to be determined whether this is a commonly used mechanism of tissue destruction in a physiologic microenvironment replete with a diverse repertoire of B and T lymphocytes. This caveat should be carefully considered when
conducting and interpreting studies performed in immunodeficient
TCR-tg mice. In fact, the presence of regulatory cells in the OT-I
mice used in the present study (although reduced compared with
that in wild-type mice) may explain our inability to induce bystander pathology after immunization with cognate Ag.
In conclusion, our study clearly demonstrate that a population of
pathogen-specific T cells is a far more potent mediator of tissue
destruction in the CNS than bystander T cells of an irrelevant
specificity (even when the time window of study is extended to
favor bystander T cell activation and pathology). It has been proposed (29) that during a chronic disease process, such as human
multiple sclerosis, activation and recruitment of bystander T cells
into the CNS may explain the established link between microbial
infections and relapses (58). However, given that specific T cells
may heavily outweigh their nonspecific counterparts in the ability
to efficiently mediate tissue destruction, the aggravation of preexisting self (or pathogen)-specific T cells provides a more suitable
explanation for such a link. There appears to be a surprising degree
of overlap between heterologous viral infections (59) that may
indeed prime cross-reactive responses capable of causing pathology. Furthermore, triggering of self-reactive T cells by molecular
mimicry (9 –15), bystander activation (3– 6), or epitope spreading
(7, 8) may also play a fundamental role in lesion exacerbation. In
each of the aforementioned scenarios, specificity is a vital component of the pathologic outcome. Because such a high threshold
has been imposed by the host for the activation and subsequent
release of effector molecules from T cells of an irrelevant specificity, it is likely that bystander T cells are of minimal importance
in T cell-mediated disease pathogenesis.
Acknowledgments
We thank Michael B. A. Oldstone for his generous support and insightful
discussions. Phi Truong participated in this study as a scientific intern from
the University of California-San Diego.
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FIGURE 10. The influence of Dbgp33– 41-specific T cells on bystander T cell recruitment and
activation. Representative dot plots of brain-infiltrating leukocytes (gated on CD8) from OT-I mice
receiving no transfer (n ⫽ 5) or a transfer of 104
GFP⫹Dbgp33– 41-specific CD8⫹ T cells (n ⫽ 5) are
shown in A. Both groups (day 7 postinfection) received intracerebral infection with 103 PFU of
LCMV Arm. Note the absence of GFP⫹ T cells in
mice not receiving a transfer, whereas the V␤5.1expressing bystander T cell population is roughly
equivalent in both groups. B, Quantification of the
absolute number of brain-infiltrating GFP⫹ (transfer recipients only) and V␤5.1⫹ (transfer vs no
transfer) CD8⫹ T cells. Data are represented as the
mean ⫾ SD. Asterisks denote a statistically significant decrease (as determined by one-way
ANOVA, p ⬍ 0.05) from the number of GFP⫹ T
cells in the brain. B, Inset, The number of V␤5.1⫹
T cells on a smaller scale. Student’s t test was used
to determine that the values are, in fact, statistically
different (p ⬍ 0.05). C, The percentage of the aforementioned T cell populations that is CD44high. Data
are represented as the mean ⫾ SD. Statistical decreases (asterisks) from GFP⫹ T cells were determined by one-way ANOVA (p ⬍ 0.05).
4790
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REBUILDING AN IMMUNE-MEDIATED CNS DISEASE