of June 13, 2013.
This information is current as
in Susceptible and Resistant Hosts
of Mouse Hepatitis Virus-1
T Cell Epitope Specificity and Pathogenesis
Nhat-Long Pham, Dilea Tifrea, Steven M. Varga and John
Aaruni Khanolkar, Ross B. Fulton, Lecia L. Epping,
2010; 185:1132-1141; Prepublished online 16
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The Journal of Immunology
by guest on June 13, 2013
The Journal of Immunology
T Cell Epitope Specificity and Pathogenesis of Mouse
Hepatitis Virus-1–Induced Disease in Susceptible and
Aaruni Khanolkar,* Ross B. Fulton,* Lecia L. Epping,* Nhat-Long Pham,†
Dilea Tifrea,* Steven M. Varga,*,†and John T. Harty*,†
Intranasal mouse hepatitis virus-1 (MHV-1) infection of susceptible mouse strains mimics some important pathologic features ob-
served in the lungs of severe acute respiratory syndrome (SARS)-coronavirus–infected humans. The pathogenesis of SARS remains
poorly understood, although increasing evidence suggests that immunopathology could play an important role. We previously
reported that the adaptive immune response plays an important protective role in MHV-1–infected resistant B6 mice and that
both CD4 and CD8 T cells play a significant role in the development of morbidity and lung pathology following intranasal MHV-1
infection of susceptible C3H/HeJ and A/J mice. In this study, we have identified novel CD4 and CD8 epitopes in MHV-1–infected
susceptible and resistant strains of mice. Susceptible C3H/HeJ mice mount robust and broad MHV-1–specific CD4 T cell responses,
whereas in resistant B6 mice, Ag-specific CD8 T cell responses dominate. We also show that previously immunized susceptible
C3H/HeJ mice do not develop any morbidity and are completely protected following a lethal-dose MHV-1 challenge despite
mounting only a modest secondary T cell response. Finally, we demonstrate that the resistance displayed by B6 mice is not solely
accounted for by the elaboration of a broad and vigorous MHV-1–specific CD8 T cell response, as MHV-1 infection of C3.SW-H2b/
SnJ mice, which mount an equally robust CD8 T cell response of the same specificity, is still associated with significant morbidity.
Thus, identification of novel CD4 and CD8 T cell epitopes for MHV-1 permitted high-resolution analyses of pulmonary T cell
responses in a mouse model of SARS.The Journal of Immunology, 2010, 185: 1132–1141.
establishing a systemic infection, the most striking pathology is
observed in the lungs (1). There appears to be an emerging con-
sensus that immunopathology may play an important role in
mediating the morbidity and mortality associated with SARS-CoV
infection (2–4). The early phase of SARS-CoV infection is asso-
ciated with elevated levels of proinflammatory cytokines (2, 3),
however, the exact role of specific components of the immune
response to SARS-CoV infection that could be involved in the
development of pathology is still unclear and remains an area of
active investigation. Although some reports have suggested a link
between the severity of disease and an inability of the host to
mount an optimal adaptive immune response following infection,
others have reported a correlation between increased disease se-
verity and robust Ag-specific CD4 and CD8 T cell responses (2,
3). In addition, one of the major limitations of human studies is
their restriction to analyzing responses in PBMCs and the inability
to sample immune responses occurring in the tissue of interest.
evere acute respiratory syndrome (SARS) is a clinical
manifestation of infection by a human respiratory coro-
navirus (CoV) (1). Although the virus is capable of
Mouse hepatitis virus-1 (MHV-1) infection of susceptible strains
of mice has been shown to be a clinically relevant model to study
SARS (5–7). Using this model, we have recently shown that both
CD4 and CD8 T cells contribute to the development of morbidity
and lung pathology in susceptible strains of mice following
primary sublethal MHV-1 infection (6). Additionally, we see en-
hancement of disease in naive recipients that are adoptively trans-
ferred with purified memory CD4 and CD8 T cells obtained from
donors previously immunized with MHV-1 (6). In concordance
with other published reports (8–11), we have also shown that
primary MHV-1 infection of susceptible strains of mice induces
robust neutralizing Ab responses, and transfer of immune serum
into naive recipients that are subsequently challenged with
MHV-1 reduces systemic viral burden and morbidity (6). In con-
trast, B6 mice that are resistant to MHV-1–induced disease
experience increased morbidity in the absence of T and B cells
(6). Collectively, our data from MHV-1 infection of susceptible
and resistant strains of mice suggest that the adaptive immune
response can play an important role in infection control but can
also mediate immunopathology in hosts with specific genotypes.
In this study, we have delineated the Ag-specific CD4 and CD8
T cell responses to MHV-1 structural proteins in susceptible
C3H/HeJ and resistant B6 mice to define the role of the T cell re-
MHV-1 infection. In addition, we have also examined the impor-
tance of non–MHC-linked genes in mediating susceptibility to
MHV-1–induced respiratory disease.
Materials and Methods
A total of 420 peptides encompassing the spike (S), nucleocapsid (N),
*Department of Microbiology and†Interdisciplinary Graduate Program in Immunol-
ogy, University of Iowa, Iowa City, IA 52242
Received for publication August 20, 2009. Accepted for publication May 6, 2010.
This work was supported by National Institutes of Health Program Project Grant PO1
AI-060699 (to J.T.H. and S.M.V.).
Address correspondence and reprint requests to Dr. John T. Harty, University of Iowa,
51 Newton Road, 3-530 Bowen Science Building, Iowa City, IA 52242. E-mail
Abbreviations used in this paper: CoV, coronavirus; E, envelope; M, membrane;
MHV-1, mouse hepatitis virus-1; N, nucleocapsid; Penh, enhanced pause; S, spike;
SARS, severe acute respiratory syndrome.
by guest on June 13, 2013
of 5 aa. The crudepeptides (.65% pure by HPLC)were reconstituted using
100% sterileDMSO (Sigma-Aldrich, St. Louis, MO) to a concentration of 2
mM. A substock was prepared that was diluted to a concentration of 25 mM
using sterile 13 Dulbecco’s PBS (Life Technologies, Grand Island, NY).
Peptides were screened for potential epitopes by performing standard in-
tracellular cytokine staining for IFN-g production from splenocytes
obtained from mice 7 d after i.p. infection with MHV-1 (described below).
Following identification of the relevant epitopes, independent synthesis of
candidate peptides was undertaken (Biosynthesis, Lewisville, TX).
Five- to 7-wk-old female A/J (H-2a), C3H/HeJ (H-2k), BALB/c (H-2d), and
C57BL/6J (H-2b) were purchased from the National Cancer Institute
(Frederick, MD). C3.SW-H2b/SnJ mice were obtained from The Jackson
Laboratory (Bar Harbor, ME). Kb2/2mOva mice were obtained from Dr.
Stephen P. Schoenberger (La Jolla Institute of Allergy and Immunology,
San Diego, CA). All mice were housed under specific pathogen-free con-
ditions at the University of Iowa (Iowa City, IA) animal care unit until the
time of infection, at which point the mice were transferred to housing at the
appropriate biosafety level. All animals were maintained in accredited
facilities at the University of Iowa and used in accordance with the guide-
lines established by the University of Iowa animal care and use committee.
Mice were infected at 8 to 9 wk of age.
Parent stock of MHV-1 was obtained from the American Type Culture Col-
lection, (Manassas, VA). Thevirus was propagatedand titered as previously
Virus infection of mice
For intranasal infections, mice were anesthetized with avertin (2,2,2-
tribromoethanol; Aldrich,Milwaukee, WI) i.p.and administered a sublethal
dose of MHV-1 intranasally in a volume of 50 ml. The sublethal dose used
for infecting C3H/HeJ mice was 5 3 103PFU/mouse and 105PFU/mouse
for B6 mice. This dosing was based on morbidity and mortality patterns
described for susceptible and resistant strains previously (6). For screening
the peptide library, splenocytes were obtained from mice that had been pre-
viously infected with 2 3 105PFU MHV-1 i.p.
Analysis of FoxP3+CD4+T cells
Single-cell suspensions obtained from spleens, lungs, and draining lymph
nodes of naive B6 and C3H/HeJ control mice and B6 and C3H/HeJ mice
infected intranasally with MHV-1 were stained for Foxp3 using the mouse
to the manufacturer’s instructions. Briefly, following cell surface staining
and fixation, cells were stained with optimal concentrations of mAb spe-
cific to Foxp3 (clone FJK-16s; eBioscience). Cells were then washed twice
with 13 permeabilization buffer and resuspended in staining buffer.
Intracellular cytokine staining
Intracellular cytokine staining was performed as previously described (12,
13). For the peptide library screen and analysis of Kb2/2mOva mice,
single-cell suspensions from the spleens of MHV-1–infected mice were
either left untreated or stimulated with MHV-1–derived peptide epitopes
at a concentration of 1 mM for 5 h at 37˚C. The intracellular accumulation
of IFN-g was facilitated by the addition of brefeldin A (GolgiPlug; BD
Pharmingen, San Jose, CA). Infected mice were perfused with 10 ml PBS,
and explanted lungs were treated with collagenase (100 U/ml) (Life
Technologies)/DNAse I (1 mg/ml) (Sigma-Aldrich) for 45 min. Single-
cell suspensions were prepared using wire mesh screens, and erythrocytes
were removed by treatment with ACK lysis buffer and counted using 0.1%
trypan blue. APCs were left either untreated or incubated with appropriate
MHV-1–derived peptides at a concentration of 1 mM for CD8 peptide
epitopes and 10 mg/ml for CD4 peptide epitopes at 37˚C for 1 h, washed,
and added to each tube along with the lung cell suspensions followed by
incubation for 5 h at 37˚C. For B6 mice, the mouse B cell line CHB3 was
used as APC. In the case of C3H/HeJ mice, APCs were derived from
splenocytes of naive syngeneic mice that were left either untreated or
incubated with appropriate MHV-1 peptide epitopes at 37˚C for 1 h,
washed, and then labeled with 2 mM CFSE for 10 min at 37˚C in PBS to
facilitate discrimination from lung cells during flow cytometric analysis.
For assays describing the identification of MHC restriction element for CD8
peptide epitopes in B6 mice, APCs used were either P815 cells (H-2dMHC)
or P815 cells transfected with cDNA encoding either the Dbor Kb
molecules. Total numbers of Ag-specific CD4 and CD8 T cells in the
spleens and lungs were determined by multiplying the frequency of
IFN-g+cells with total numbers of CD4 or CD8 T cells present in that
tissue. Surface and intracellular staining was performed using the following
mAbs: anti-CD8 (clone 53-6.7), anti-CD4 (clone RM4-5), anti-CD90.2
(clone 30-H12), anti–IFN-g (clone XMG1.2), anti–TNF-a (clone MP6-
XT22), and anti–IL-2 (clone JES6-5H4). Samples were acquired using an
FACSCalibur and FACSCanto (BD Biosciences, San Jose, CA) and ana-
lyzed using FlowJo software (Tree Star, Ashland, OR).
Evaluation of morbidity and mortality
normalized to 100% at the time they were infected (day 0), and subsequent
weight measurements were recorded at defined time points postinfection.
Weight data are presented as the mean percentage of the starting weight 6
SEM. Survival data for mice are represented by Kaplan-Meier curves in-
dicating the percentage of mice that survived the viral challenge.
Baseline airway resistance as determined by the measurement of the pa-
rameter enhanced pause (Penh) was evaluated using a whole-body plethys-
mograph from Buxco Electronics (Troy, NY).
All statistical analyses were performed using GraphPad software (Graph-
Pad, San Diego, CA). For weight loss and cytokine analysis data, statistical
significance was determined using an unpaired Student t test. A p value
,0.05 was considered statistically significant. For survival data, statistical
significance was determined using a two-tailed Fisher’s exact test. Once
again, a p value ,0.05 was considered statistically significant.
Identification of MHV-1–specific CD4 and CD8 T cell epitopes
in susceptible and resistant mice
Previously published data from our laboratory showed that T cells
contribute to morbidity and lung pathology observed in MHV-1–
susceptible strains of mice (6). We have also demonstrated that
resistance to MHV-1–induced disease is compromised in B6-
Rag1-KO mice, indicating the importance of the adaptive immune
response in infection control (6). Based on these data, wewanted to
examine Ag-specific T cell responses to MHV-1 in susceptible and
resistant strains of mice. T cell epitopes in other CoVs have been
primarily localized to structural proteins (3, 14–20), thus we
obtained protein sequence information for the S, N, M, and E struc-
tural proteins of MHV-1 from the National Center for Biotechnol-
ogy Information database (accession number EF682498) to screen
a peptide library consisting of 420 overlapping peptides encom-
passing the four proteins mentioned above. Each peptide was
a 15-mer with an overlap of 10 aa and an offset of 5 aa. To identify
MHV-1–specific T cell epitopes, each peptide was individually
screened in an intracellular cytokine staining assay to determine
IFN-g production by peptide-pulsed splenocytes harvested and
pooled from mice 7 d after i.p. MHV-1 infection. The large number
of peptides to be screened necessitated the use of splenocytes in-
stead of lung cells in our initial analysis. Responses to peptide
stimulation that resulted in IFN-g production at least 2.5-fold
over that observed in no-peptide controls were considered positive.
in both susceptible (C3H/HeJ) and resistant (B6) mice (Fig. 1,
Table I). In the susceptible C3H/HeJ strain of mice, we identified
to the N protein, and one to the M protein. The screen revealed
a single CD8 epitope in these susceptible mice, and it was localized
in the N protein. Based on the analysis of the peptide library screen
for the resistant B6 mice, we identified four CD8 epitopes and three
CD4 epitopes. Once again, the majority of CD4 and CD8 epitopes
were found in the S protein. None of the epitopes identified in the
resistant B6 mice mapped to the N protein. In addition, we also
The Journal of Immunology1133
by guest on June 13, 2013
identified several novel MHV-1–specific CD4 and CD8 T cell
epitopes in susceptible A/J mice and BALB/c mice that display
an intermediate susceptibility phenotype (data not shown). These
analyses revealed that MHV-1 infection induced a more robust and
broader virus-specific CD4 T cell response in the susceptible
C3H/HeJ mice, whereas the resistant B6 mice mounted a broad
and vigorous virus-specific CD8 T cell response with the S587–
594-specific response clearly being the most dominant.
Prediction of MHC restriction elements and minimal epitopes
of newly identified MHV-1–specific epitopes
further analyzedusingtheSYFPEITHIepitope-prediction software
todetermine putative MHC-restriction elements (21).Based on this
analysis, all of the CD4 epitopes identified in susceptible C3H/HeJ
mice were predicted to bind to I-Ek, and because the resistant B6
mice lack the I-Eballele, we expected all three CD4 epitopes
identified in these mice to be I-Ab-restricted, and this was also
confirmed using the software program. Given the variability in
the lengths of CD4 epitopes in general, further definition of the
CD4 epitopes was not attempted.
The CD8 peptides identified as those containing potential
epitopes by the peptide library screen were similarly analyzed by
the SYFPEITHI epitope-prediction software program (21). The
lone CD8 epitope in the C3H/HeJ was determined to be an
octamer expressed in the context of Dkbased on the presence of
the arginine residue at position 2 and leucine at position 8 (22).
The four CD8 peptides that came up positive for potential epitopes
in B6 mice were similarly predicted to be octamers restricted by
Kb. These predictions regarding MHC restriction for CD8 epitopes
identified in B6 mice were formally confirmed using P815 cells
transfected with either the Dbor KbcDNA in intracellular cyto-
kine staining assays where P815-Dbor P815-Kbcells coated with
15-mers were used to stimulate splenocytes obtained from MHV-
1–infected B6 mice (Fig. 2A). These results were also separately
confirmed by intracellular cytokine staining for IFN-g production
using splenocytes from MHV-1–infected wild-type B6 and Kb2/2
mOva B6 mice (Fig. 2B) that do not express the KbMHC mole-
cule on the surface of their cells but are fully capable of presenting
epitopes that bind to the Dbmolecule (N.L. Pham and J.T. Harty,
unpublished observations). Based on these data, we obtained the
minimal predicted CD8 epitopes for both C3H/HeJ and B6 mice.
Kinetics of MHV-1–specific CD4 and CD8 T cell responses in
the lungs of susceptible C3H/HeJ and resistant B6 mice
As mentioned above, our analysis of the peptide library was
performed using splenocytes from mice infected i.p. with MHV-1.
Intraperitoneal MHV-1 infection of mice is asymptomatic, whereas
intranasal infection is associated with pulmonary pathology and
morbidity that is most severe in susceptible C3H/HeJ mice and
minimal in the resistant B6 mice (6, 7). Given these differences
in morbidity after intranasal MHV-1 infection, we were interested
in determining Ag-specific T cell responses in the lungs of both
susceptible and resistant strains of mice after sublethal infections,
as previously defined for each mouse strain (6). Virus-specific
CD4 and CD8 T cell responses in the lungs of C3H/HeJ mice
(Fig. 3) and B6 mice (Fig. 4) were determined by intracellular
cytokine staining following peptide stimulation at the indicated
time points after MHV-1 infection, and total numbers of Ag-
specific T cells in the lungs were also enumerated. MHV-1 in-
fection induced a broad virus-specific CD4 T cell response in the
lungs of C3H/HeJ mice. Between days 8 and 11 postinfection,
roughly 16–19% of the total CD4 T cells in the lungs of these
mice were specific for MHV-1. The response to all six CD4 epit-
opes was similar in magnitude, and we did not observe any sig-
nificant difference in the numbers of Ag-specific CD4 T cells
between days 8 and 11 postinfection. A slight reduction in num-
bers was discernible at day 15 postinfection, and these numbers
continued to decline up to day 66 postinfection, the last time point
measured for this analysis. The CD8 T cell response in the lungs
of the C3H/HeJ mice was more narrowly focused targeting the
N421 epitope. This response peaked at day 11 postinfection, after
which there was a clearly defined contraction phase, and thereaf-
ter, similar to the CD4 T cells, the numbers of N421-specific CD8
T cells displayed a downward trend.
Clearly measurable responses to all three CD4 epitopes iden-
tified in the peptide library scan were also detected in the lungs of
the resistant B6 mice, with ∼5% of the total CD4 T cells in the
were infected with MHV-1 i.p. (2 3 105PFU/mouse), and 7 d postinfec-
tion, spleens were harvested and processed to generate single-cell suspen-
sions and pooled to be used as effectors in an intracellular cytokine assay.
To induce IFN-g production, effector splenocytes were stimulated with
each of the 420 peptides separately. These 420 peptides encompassed
the four main structural proteins, S (peptides 1–271), N (peptides 272–
360), M (peptides 361–404), and E (peptides 405–420). Data are shown as
percentage of CD4+Thy1.2+splenocytes producing IFN-g for C3H/HeJ
mice (A) and B6 mice (B) and percentage of CD8+Thy1.2+splenocytes
producing IFN-g for C3H/HeJ mice (C) and B6 mice (D). Responses that
were at least 2.5-fold above those of no-peptide controls were scored as
positive (indicated by an asterisk). The line parallel to the x-axis indicates
the magnitude of the background response observed in no-peptide controls.
Screening of the peptide library. C3H/HeJ and B6 mice
1134Ag-SPECIFIC T CELL RESPONSES TO MHV-1
by guest on June 13, 2013
lungs of these mice being specific for MHV-1 at day 8 postinfec-
tion (Fig. 4). Calculation of total numbers revealed that B6 mice
had ∼4.3-fold lower numbers of Ag-specific CD4 T cells (∼7 3
104cells) in the lungs compared with C3H/HeJ mice (∼3 3 105
cells) at the peak of the response. The magnitude of the responses
of all three CD4 epitopes in B6 mice were similar to each other,
and their numbers peaked at day 8 postinfection, after which they
declined and by day 58 postinfection were at levels similar to
those observed in the lungs of C3H/HeJ mice. B6 mice also
responses with the S587-specific response being clearly immuno-
dominant, accounting for 72% of the total Ag-specific CD8 T cell
response in the lungs of these mice. The responses directed against
the other three CD8 epitopes were similar to each other in mag-
nitude and ∼8-fold lower than that measured for the S587 epitope.
Overall, the MHV-1–specific CD8 T cell response in the B6 mice
(∼7.4 3 105cells) was ∼11-fold greater than the corresponding
CD8 T cell response measured in the lungs of the susceptible
C3H/HeJ mice (∼7 3 104cells). The CD8 T cell response also
peaked at day 8 postinfection, after which it contracted and grad-
ually declined. However, the S587-specific CD8 T cells were
maintained at higher levels in the lungs of infected B6 mice than
the other specificities.
Collectively, these data suggest that either qualitative (ratios of
CD4/CD8 T cells) or quantitative (absolute numbers of CD4 or
CD8 T cells) differences in the MHV-1–specific T cell response
could dictate the development of lung disease and morbidity
following intranasal MHV-1 infection of mice.
MHV-1–specific CD8T cell
Evaluation of cytokine production by Ag-specific T cells and
determining FoxP3+CD4+T cell responses in MHV-1–infected
B6 and C3H/HeJ mice
cytokine production by MHV-1–specific T cells might also account
for some of the differences in morbidity between MHV-1–infected
B6 and C3H/HeJ mice. To address this possibility, we used
intracellular cytokine staining to analyze the ability of MHV-1–
specific CD8 and CD4 T cells to coproduce IFN-g and TNF-a
following MHV-1 infection of B6 and C3H/HeJ mice (Fig. 5A,
5B). We observed a significant reduction in the frequency of
N421-specific CD8 T cells (C3H/HeJ) coproducing IFN-g and
TNF-a in comparison with S587-specific CD8 T cells (B6) in the
lungs. No differences were observed in the spleens and draining
lymph nodes of the infected mice. CD4 T cell responses were
analyzed using pooled APCs separately pulsed with M131, S361,
and S766 peptides (B6) or M196, S171, and S921 peptides (C3H/
HeJ). In contrast to the CD8 T cell data, we observed a significantly
greater fraction of IFN-g–TNF-a–coproducing CD4 T cells in all
Table I.Identification of MHV-1–specific T cell epitopes in susceptible C3H/HeJ and resistant B6 mice
15-merCD4 or CD8 Epitope Predicted 8-mer
identified in MHV-1–infected B6 mice. A, 15-mers identified by the pep-
tide library screen to contain potential epitopes recognized by CD8 T cells
from MHV-1–infected mice were further analyzed to identify the
restricting MHC molecule. Single-cell suspensions derived from the
spleens of B6 mice infected with MHV-1 (2 3 105PFU/mouse i.p.) 7 d
previously were used as effectors in an intracellular cytokine assay. To
induce IFN-g production by the splenocytes, we used peptide-pulsed P815
cells (H-2d) that expressed either Dbor Kbas APCs. Nontransfected P815
cells (H-2d) were included in the assay as controls. IFN-g production
against non–peptide-pulsed P815, P815-Db, and P815-Kbcells served as
additional background controls (not shown). Representative contour plots
gated on CD8+T cells are shown, and the numbers over each plot depict
the magnitude of the IFN-g+response to the indicated peptide. B,
Octamers identified as minimal epitopes based on the SYFPEITHI
epitope-prediction software program were additionally tested to conclu-
sively identify the MHC-restriction element. Wild-type B6 mice and Kb2/2
mOva mice that lack the Kbmolecule were infected intranasally with
MHV-1 (1 3 105PFU/mouse), and 1 wk postinfection, the mice were
sacrificed, and spleens were harvested and processed to generate single-
cell suspensions that were assessed for IFN-g production in an intra-
cellular cytokine assay. Representative contour plots gated on CD8+
T cells are shown, and the numbers over each plot depict the magnitude
of the IFN-g+response to the indicated octamer.
Identification of MHC-restriction element for CD8 epitopes
The Journal of Immunology1135
by guest on June 13, 2013
three tissues analyzed in the susceptible C3H/HeJ mice versus the
resistant B6 mice.
In addition to looking at TNF-a production, a preliminary
examination of IL-2 production by MHV-1–specific CD4 T cells
showed that although the C3H/HeJ mice had a higher fraction of
IFN-g–IL-2 coproducers in the spleens and draining lymph nodes
versus the B6 mice, this trend was reversed in the lungs (data not
In an effort to further dissect the qualitative properties of the
immune response to MHV-1 in the resistant B6 and susceptible
C3H/HeJ mice, we also analyzed FoxP3+CD4+regulatory T cells.
Published evidence from animal models and human studies of viral
infections suggest a crucial role for FoxP3+CD4+T cells in the
development of symptomatic disease (23, 24). Therefore, we
wanted to determine if differences in FoxP3+CD4+T cell responses
might account for the difference in morbidity between the resistant
B6 and susceptible C3H/HeJ mice following intranasal MHV-1 in-
fection. We examined the spleens, draining lymph nodes, and lungs
of infected B6 and C3H/HeJ mice for the presence of FoxP3+CD4+
T cells at day 8 postinfection, a time point at which morbidity
differences between the two strains are clearly evident (Fig. 5C).
Our data revealed no significant differences in the total numbers of
FoxP3+CD4+T cells detected in all three of the tissues examined
between the two strains of mice.
Overall, these results demonstrate the presence of subtle yet
significant differences in the cytokine profiles of MHV-1–specific
CD8 and CD4 T cells that might potentially influence development
of symptomatic disease in the susceptible C3H/HeJ mice following
intranasal MHV-1 infection. Furthermore, MHV-1–induced lung
disease in susceptible C3H/HeJ mice is not due to numerical
deficiencies in FoxP3+regulatory CD4 T cells.
Previously immunized C3H/HeJ mice display no morbidity and
are completely protected following a lethal MHV-1 challenge
Intranasal MHV-1 infection of naive C3H/HeJ mice induces vigor-
ous Ag-specific T cell responses in the lungs as well as significant
morbidity (6). Additionally, we have previously shown that naive
C3H/HeJ mice adoptively transferred with memory CD4 or CD8
T cells obtained from previously MHV-1–immunized syngeneic
donors display enhanced morbidity and mortality following
intranasal MHV-1 infection (6). Given these data, we were also
interested in determining the outcome of an MHV-1 challenge in
previously immunized C3H/HeJ mice that have an established pool
of memory T cells. Significant lethality is observed in naive
in the lungs of resistant B6 mice. Naive B6 mice were intranasally infected
with MHV-1 (1 3 105PFU/mouse), and at the indicated time points
following infection, lung cells were obtained as described in Materials
and Methods. MHV-1–specific CD4 and CD8 T cells in the bulk lung
population were identified by peptide-stimulated intracellular cytokine
staining. A, Representative contour plots are gated on CD4 or CD8
T cells. Background staining is represented by no-peptide controls. The
numerical values above each contour plot depict the frequency of the
epitope-specific T cell response. The line graphs enumerate the total num-
bers of Ag-specific CD4 (B) and CD8 (C) T cells identified in the lungs of
MHV-1–infected B6 mice at the indicated time points.
Kinetics of MHV-1–specific CD4 and CD8 T cell responses
in the lungs of susceptible C3H/HeJ mice. Naive C3H/HeJ mice were
intranasally infected with MHV-1 (5 3 103PFU/mouse), and at the in-
dicated time points following infection, lung cells were obtained as de-
scribed in Materials and Methods. MHV-1–specific CD4 and CD8 T cells
in the bulk lung population were identified by peptide-stimulated intracel-
lular cytokine staining. A, Representative contour plots are gated on CD4
or CD8 T cells. Background staining is represented by no-peptide controls.
The numerical values above each contour plot depict the frequency of the
epitope-specific T cell response. The line graphs enumerate the total num-
bers of Ag-specific CD4 (B) and CD8 (C) T cells identified in the lungs of
MHV-1–infected C3H/HeJ mice at the indicated time points.
Kinetics of MHV-1–specific CD4 and CD8 T cell responses
1136Ag-SPECIFIC T CELL RESPONSES TO MHV-1
by guest on June 13, 2013
C3H/HeJ mice at doses .5 3 103PFU/mouse of MHV-1 admin-
istered intranasally (6, 7). Thus, we administered a 10-fold higher
dose to C3H/HeJ mice that had previously been immunized with
a sublethal dose of MHV-1. As controls, we also included naive
C3H/HeJ mice that also received the same high-dose challenge as
the immunized mice. Both sets of mice were followed for the de-
velopment of morbidity following infection. As expected, naive
controls progressively lost weight following infection, and all mice
in this group died by day 8 postinfection. In contrast, previously
immunized mice experienced no morbidity, and all mice survived
the lethal MHV-1 challenge (Fig. 6). This result was noteworthy
considering the propensity of adoptively transferred memory T cell
responses to enhance morbidity and pathology in naive mice fol-
lowing exposure to MHV-1 and prompted us to examine secondary
T cell responses in previously immunized C3H/HeJ mice following
a lethal MHV-1 challenge.
Suboptimal expansion of secondary T cell responses in lethally
challenged immune C3H/HeJ mice
mortality, leading us to speculate that the memory T cells in these
mice were unable to exert their pathologic effects. To examine this
issue, we evaluated Ag-specific T cell responses in the lungs of pre-
viously immunized C3H/HeJ following rechallenge with a lethal
dose (5 3 104PFU/mouse) of MHV-1. As controls, we included
naive C3H/HeJ mice that received a sublethal dose of MHV-1
(5 3 103PFU/mouse). This difference in viral input doses was
necessary to ensure survival of naive mice to allow us to evaluate
secondary S171-specific CD4 and N421-specific CD8 T cell
responses revealed that their numbers remained fairly static be-
tween days 5 and 8 postrechallenge, whereas enumeration of cells
participating in a primary response in naive control mice that re-
ceived a 10-fold lower dose of MHV-1 displayed a marked expan-
sion in numbers between days 5 and 8 postinfection (Fig. 7). In
addition, the control mice developedcharacteristic signs and symp-
toms of MHV-1–induced disease (weight loss, ruffled fur, hunched
posture, reduced activity), whereas no morbidity was observed in
CD8 and CD4 T cells and analysis of FoxP3+CD4+T cells in MHV-1–
infected B6 and C3H/HeJ mice. Naive B6 and C3H/HeJ mice were
intranasally infected as described above. At day 8 postinfection, single-
cell suspensions were prepared from the spleens, mediastinal lymph nodes
and lungs. A, S587-specific (B6) and N421-specific (C3H/HeJ) CD8
T cells were evaluated for IFN-g–TNF-a coproduction by intracellular
cytokine staining. B, For examining IFN-g–TNF-a coproduction by
MHV-1–specific CD4 T cells, single-cell suspensions were stimulated us-
ing the following peptide epitope pools: M131-145 + S361-375 + S766-
780 (B6) and M196-210 + S171-185 + S921-935 (C3H/HeJ). C, FoxP3+
CD4+cells were enumerated in the spleens, mediastinal lymph nodes, and
lungs following the staining protocol outlined in the Materials and Meth-
ods. These results are representative of two independent experiments, and
a minimum of three mice per group was analyzed in each experiment.
Statistically significant differences between the two groups were deter-
mined using an unpaired Student t test and are indicated by asterisks.
pp , 0.05; ppp , 0.01.
Comparison of IFN-g–TNF-a coproduction by Ag-specific
munized C3H/HeJ mice rechallenged with a lethal dose of MHV-1. C3H/
HeJ mice that had been intranasally infected with MHV-1 (5 3 103PFU/
mouse) at least 3–6 mo previously were rechallenged with a lethal dose
(5 3 104PFU/mouse) of MHV-1 delivered intranasally. Naive C3H/HeJ
mice infected with 5 3 104PFU/mouse of MHV-1 were included as con-
trols. A, Development of morbidity was monitored by tracking changes in
body weight at the indicated time points postinfection. Statistically signif-
icant differences between the mean weights of rechallenged mice and
those undergoing a primary infection were determined using an unpaired
Student t test and are indicated by an asterisk. B, Kaplan-Meier survival
curves depict the percentage of surviving mice in each group at the in-
dicated time points following infection. Statistically significant differences
between the survival rates of rechallenged mice and those undergoing
a primary infection were determined using a Fisher’s exact test and are
indicated by an asterisk. Five mice per group were analyzed, and data are
representative of two independent experiments. pp , 0.05.
Assessment of morbidity and mortality in previously im-
The Journal of Immunology1137
by guest on June 13, 2013
the rechallenged group. The numbers for all of the remaining five
specificities of MHV-1–specific CD4 T cells tested were also 5–10-
fold higher for the primary responders at day 8 in comparison with
the secondary responders (data not shown). C3H/HeJ mice exposed
to MHV-1 develop high-titer neutralizing Abs 15–20 d after pri-
mary MHV-1 infection (6, 8–11). The neutralizing Ab response
likely accounts for the attenuated secondary T cell response in
rechallenged mice, and a consequence of this is the reduced mor-
bidity observed in rechallenged mice. Control mice undergoing
a primary MHV-1 infection lack pre-existing neutralizing Abs
and mount a vigorous T cell response that helps control the infec-
tion but also induces significant immunopathology (6).
Elaboration of a broad and vigorous Ag-specific CD8 T cell
response to MHV-1 does not prevent the development of
morbidity in susceptible C3.SW-H2b/SnJ mice
One of the key differences we observed between the susceptible
C3H/HeJ mice and the resistant B6 micewas that MHV-1 infection
elicited a broad and vigorous virus-specific CD8 T cell response in
the lungs of the resistant B6 mice. Thus, we were interested in de-
termining if elaboration of a similarly broad and vigorous virus-
minimize morbidity. Hence, we examined morbidity and Ag-
specific T cell responses in the lungs of congenic C3.SW-H2b/
SnJ mice that express the H-2bMHC-haplotype of the B6 mice
(25) following infection with a sublethal dose of MHV-1 (5 3 103
PFU/mouse). As controls, we included wild-type B6 mice that
were similarly infected. Analysis of the Ag-specific T cell re-
sponses in the lungs of the congenic C3.SW-H2b/SnJ mice at
day 8 postinfection demonstrated that they elaborated strong Ag-
specific CD8 and CD4 T cell responses that were not significantly
different from the responses observed in the lungs of similarly
infected control B6 mice (Fig. 8A–C). Interestingly, however, de-
velopment of morbidity assessed in terms of weight loss and de-
velopment of airway resistance revealed that, despite elaboration
of Ag-specific T cell responses similar to those observed in the
resistant B6 mice, the C3.SW-H2b/SnJ mice nonetheless lost
a significant amount of weight and showed significantly elevated
airway resistance compared with controls following intranasal
MHV-1 infection (Fig. 8D, 8E). These data demonstrate that
previously immunized C3H/HeJ mice rechallenged with a lethal dose of
MHV-1. C3H/HeJ mice that had been intranasally infected with MHV-1
(5 3 103PFU/mouse) at least 6 mo previously were rechallenged with
a lethal dose (5 3 104PFU/mouse) of MHV-1 delivered intranasally. Naive
C3H/HeJ mice undergoing a primary MHV-1 infection (5 3 103PFU/
mouse) were included as controls. Ag-specific CD4 and CD8 T cell
responses in the lungs of both groups of mice (2˚ = secondary infection;
1˚ = primary infection) were evaluated by peptide-stimulated intracellular
cytokine staining on days 5 and 8 postinfection. A, Representative contour
plots are gated on CD4 or CD8 T cells. Background staining is represented
by no-peptide controls. The numerical values above each contour plot
depict the frequency of the epitope-specific T cell response. The line
graphs enumerate the total numbers of S171-specific CD4 T cells (B)
and NP421-specific CD8 T cells (C) identified in the lungs of MHV-1–
infected C3H/HeJ mice at the indicated time points.
Measurement of secondary T cell responses in the lungs of
the lungs of MHV-1–infected C3.SW-H2b/SnJ mice. Congenic C3.SW-
H2b/SnJ mice that express the H-2bMHC locus were intranasally
infected with 5 3 103PFU/mouse of MHV-1. Similarly infected wild-
type B6 mice were included as controls in the analysis. Ag-specific CD4
and CD8 T cell responses in the lungs of both groups of mice were
evaluated by intracellular cytokine staining on day 8 postinfection. A,
Representative contour plots are gated on CD4 or CD8 T cells. Back-
ground staining is represented by no-peptide controls. The numerical val-
ues above each contour plot depict the frequency of the epitope-specific
T cell response. The bar graphs enumerate the total numbers of MHV-1–
specific CD4 T cells (B) and CD8 T cells (C) identified in the lungs of
monitored by tracking changes in body weight (D) and airway
resistance (Penh) (E) at the indicated time points postinfection. Ten to
20 mice per group were evaluated at each time point, and statistically
significant differences between the mean weights and Penh values of the
C3.SW-H2b/SnJ mice and wild-type B6 mice were determined using an
unpaired Student t test and are indicated by an asterisk (p).
Evaluation of morbidity and Ag-specific T cell responses in
1138Ag-SPECIFIC T CELL RESPONSES TO MHV-1
by guest on June 13, 2013
elaboration of vigorous Ag-specific CD8 T cell responses on their
own are insufficient to confer a resistant phenotype following in-
tranasal MHV-1 infection.
In this report, we have expanded on the findings of our previous
study in which we examined the protective and pathologic roles of
the immune response to MHV-1 in resistant and susceptible strains
of mice (6). In the previous report, we had demonstrated that the
adaptive immune response plays a protective role in the resistant
B6 mice, whereas conversely, both CD4 and CD8 T cells contrib-
uted significantly to the development of lung pathology following
intranasal MHV-1 infection of susceptible C3H/HeJ mice (6).
Given these contrasting roles of the adaptive immune response
to MHV-1 infection, we were interested in carrying out a more
comprehensive examination of the T cell response to MHV-1.
Screening of an overlapping peptide library encompassing the four
main structural MHV-1 proteins led to the identification of several
novel CD4 and CD8 T cell epitopes in both susceptible and re-
sistant strains of mice. Identification of these epitopes allowed us
to examine the kinetics of the Ag-specific T cell responses in the
lungs of these mice following both a primary as well as a second-
ary infection. One of the differences between the susceptible and
resistant strains was the fact that MHV-1 infection induced robust
and broad Ag-specific CD4 T cell responses in the susceptible
mice, whereas the Ag-specific CD8 T cell responses dominated
in terms of magnitude and breadth in the resistant B6 mice. Al-
though primary MHV-1 infection was associated with significant
morbidity and T cell-mediated immunopathology in susceptible
strains (6), re-exposure to lethal doses of MHV-1 following recov-
ery from primary infection was marked by the complete absence of
any morbidity. The rechallenged mice were fully protected, and
analysis of the secondary T cell responses in these mice revealed
a lack of robust expansion of these cells. Finally, we also demon-
strate that vigorous expansion and elaboration of a broad set of
MHV-1–specific CD8 T cells, which is the hallmark of the resistant
B6 mice, fail to induce a similar resistant phenotype in C3H mice
engineered to express the B6 haplotype.
for MHV-1 necessitated the generation and screening of a peptide
library encompassing the four main viral structural proteins, S,
epitopesforcoronavirusesresideprimarily instructural proteins(3,
14–20). This also appears to be the case for SARS-CoV, as a re-
cently published report showed 70% of the responses were local-
ized to the four main structural proteins, with the S protein being
highly immunogenic and accounting for 41% of the total Ag-
specific T cell response (3). In contrast, the nonstructural replicase
protein that spans two-thirds of the viral proteome accounted for
only 13% of the total Ag-specific response (3). Screening of our
peptide library revealed that resistant B6 mice had a broader MHV-
1–specific CD8 T cell response, whereas the susceptible C3H/HeJ
mice had a broader MHV-1–specific CD4 T cell response with
a majority of the epitopes mapping to the S protein. Following
the identification of the epitopes, we ran the sequences of the
specific 15-mers through the SYFPEITHI epitope-prediction soft-
ware program (21) to identify the minimal CD8 epitopes as well as
the MHC restriction element for the particular epitope. MHC re-
striction element binding was also separately confirmed as de-
scribed in the Results. Overall, these results seem consistent with
those observedfor SARS-CoVin which analysis of PBMC samples
from SARS-CoV–infected patients revealed that patients that had
more severe disease, like the susceptible C3H/HeJ mice in our
analysis, tended to have stronger CD4 T cell responses that were
primarily directed against epitopes identified in the S protein (3).
feature of intranasal MHV-1 infection of mice (6), we were inter-
ested in examining the magnitude and kinetics of the Ag-specific
T cell responses in the lungs of the infected mice. Although a num-
ber of studies have examined T cell responses to SARS both in
human and murine systems, they are beset with certain limitations
(2, 3, 14, 26–31). One of the major limitations of human studies is
that they are often restricted to analyzing immune responses from
PBMC samples that may not reflect the quality and magnitude of
the immune response occurring in situ at the primary site of in-
fection or pathology, and the limitation of a majority of small
animal studies examining immune responses to SARS is the fact
that the human SARS-CoV infection of mice does not recapitulate
all of the pathologic features of the disease observed in humans (1,
32).Hence, measurementofimmune responsesinthelungsofmice
following human SARS-CoVinfection may not providea complete
picture of the immunopathology that is believed to play an impor-
tant role in the pathogenesis of the disease. However, the use of
MHV-1 infection of susceptible and resistant mouse strains (5–7)
a useful tool to carry out rapid throughput analyses to examine
pathology and the role of the immune response in the pathogenesis
of respiratory coronaviral diseases such as SARS. Our analysis
revealed that intranasal MHV-1 infection of susceptible C3H/HeJ
mice resulted in the induction of a robust Ag-specific CD4 T cell
response in the lungs of these mice and between days 8 and 11
postinfection. Overall, based on the available epitope information,
the total numbers of Ag-specific CD4 T cells (∼3 3 105) far out-
numbered the Ag-specific CD8 T cells (7 3 104) in the lungs of the
C3H/HeJ mice. In contrast to this picture, intranasal MHV-1 in-
fection of the resistant B6 mice induced a very strong and multi-
epitope specific CD8 T cell response in the lungs of these mice,
with S587–594 clearly emerging as the immunodominant epitope.
The S587-specific CD8 T cells also had a significantly greater pro-
specific CD8 T cell response elaborated in the lungs of the suscep-
tible C3H/HeJ mice. This trend was reversed for the CD4 T cells in
which the susceptible C3H/HeJ mice had a significantly greater
proportion of IFN-g–TNF-a coproducers in the lungs, spleen,
and mediastinal lymph nodes in comparison with the resistant B6
mice. Our preliminary data also indicate the presence of fewer
IFN-g–IL-2–coproducing CD4 T cells in the lungs of the
susceptible C3H/HeJ mice. We also examined the role of FoxP3+
CD4+T cells in regulating MHV-1 infection-associated morbidity
in the susceptible and resistant strains. We had speculated that
susceptible strains would mount a suboptimal regulatory T cell
response, thereby allowing the development of immunopathology,
whereas the resistant strains of mice would mount an optimal regu-
latory T cell response that keeps T cell-mediated immunopathology
lymph nodes measured at day 8 postinfection, indicating that
MHV-1 infection-associated disease in susceptible mouse strains
is not due to decreased regulatory CD4 T cell responses. The data
up until this point suggested that differences in magnitude and cy-
tokine profile of the Ag-specific CD4 and CD8 T cell responses to
MHV-1 might account for the differences in morbidity observed
between the susceptible and resistant mouse strains.
important role in mediating acute disease and lung pathology ob-
served in the susceptible C3H/HeJ mice following primary intra-
nasal infection (6). Moreover, these T cells retain their pathologic
The Journal of Immunology1139
by guest on June 13, 2013
properties as they differentiate into memory T cells (6). Adoptive
transfer of these memory T cells into naive syngeneic hosts that are
subsequently intranasally infected with MHV-1 results in greater
morbidity and mortality than that observed during primary
MHV-1 infection (6). These findings suggested that re-exposure to
the virus could potentially amplify the pathologic behavior of the
MHV-1–specific T cells. To examine this issue in a system that
would be more representative of what would occur in a natural
setting, we rechallenged C3H/HeJ mice that had been previously
immunized with MHV-1 at least 3–6 mo previously with a lethal
points following reinfection. In contrast to what we had observed in
the adoptive transfer experiment, interestingly, the rechallenged
mice did not develop any morbidity, and all of the mice survived
the lethal dose challenge. We have previously shown that primary
MHV-1 infection of susceptible C3H/HeJ mice induces robust neu-
tralizing Ab responses by days 15–20 postinfection, and adoptive
transfer of immune serum into naive syngeneic recipients is very
effective at minimizing systemicviral burden andmorbidity follow-
previously immunized C3H/HeJ that were subsequently rechal-
lenged with MHV-1. Given this information, we wanted to closely
examine the magnitude of the secondary T cell response in the
rechallenged mice. One of the hallmarks of a secondary T cell re-
sponse is to respond very vigorously and rapidly following re-
exposure to the pathogen (34). Our analysis of the secondary T cell
response in the lungs of the rechallenged mice at days 5 and 8
postinfection revealed that the memory T cells failed to expand
optimally, and their numbers remained unchanged between these
two time points. Overall, these findings suggested a putative link
between that the absence of morbidity in the previously immunized
C3H/HeJ mice that were rechallenged with a lethal dose of
MHV-1 and the blunted secondary T cell response. Although it is
formally possible that the secondary T cell response may have
peaked prior to day 5, the pathologic properties of these T cells
and the failure to detect any morbidity during the course of this
analysis argue more in favor of a weak secondary T cell response.
It is likely that neutralizing Ab responses dominate the recall re-
sponse in previously immunized C3H/HeJmice after re-exposure to
the virus, effectively and rapidly controlling the rechallenge inocu-
lum, thereby reducing the numbers of memory T cells that get
recruited into the secondary response. This could be a mechanism
to try and curb T cell-mediated immunopathology in susceptible
hosts that survive primary MHV-1 infection-induced disease.
Our data show that C3H/HeJ mice that are highly susceptible to
MHV-1–induced disease mount a vigorous and Ag-specific CD4
T cell response directed against at least six epitopes in the lungs,
whereas B6 mice that are resistant to MHV-1–induced disease
generate a strong and multiepitope-specific CD8 T cell response
in the lungs following intranasal MHV-1 infection. These findings
led us to ask the question if the susceptible phenotype could be
modulated by inducing a strong, multiepitope-specific CD8 T cell
response similar to that observed in the resistant B6 mice. We
addressed this question by analyzing T cell responses in the lungs
of C3.SW-H2b/SnJ mice following intranasal MHV-1 infection.
This congenic strain of mice expresses the H-2bMHC haplotype
similar to the B6 mice instead of the H-2khaplotype of the parent
strain, C3H/HeSnJ, which is also shared by the susceptible
C3H/HeJ mice (25). As we expected, the C3.SW-H2b/SnJ
mounted an Ag-specific T cell response very similar in magnitude
to that of wild-type B6 controls characterized by a strong and
broadly defined Ag-specific CD8 T cell response that was domi-
nated by the S587-specific CD8 T cells. However, these mice did
experiencemorbidity, as evidenced by weight loss anddevelopment
of airway resistance following primary MHV-1 infection. These
results suggest that the fine specificity of the Ag-specific T cell
response does not control disease phenotype in the context of
MHV-1 infection. In the larger context, these findings additionally
suggest that host background genes play a significant role in de-
termining host resistance/susceptibility to disease. It is formally
possible that qualitative differences in innate immune responses
and functional properties of FoxP3+CD4+T cells influenced by host
background genes could affect development of disease following
MHV-1 infection. The cross-talk between background genes and
theimmuneresponsegenesmay becrucial inprogramming the host
immune response into one that induces pathology and morbidity (as
observed in the C3H/HeJ mice and C3.SW-H2b/SnJ mice) versus
one that does not (as observed in the B6 mice).
In conclusion, in this report, we have identified novel CD4 and
CD8 T cell epitopes specific to MHV-1 in both susceptible and re-
sistant strains of mice. Our data demonstrate the divergence in the
qualityandquantity ofthe Ag-specificTcellresponsebetween sus-
ceptible and resistant hosts following primary MHV-1 infection.
This report also shows that re-exposure to MHV-1 in susceptible
hosts will most likely be associated with minimal morbidity and
a favorable outcome for the host. Reduced morbidity in this setting
will most likely be due to a dominant neutralizing Ab response that
reduces viral burden and curtails an overwhelming and potentially
lethal secondary T cell response. Finally, our data also highlight the
potentially important contribution that host background genes
make in shaping the host response to infection that alters the bal-
ance between disease and lack thereof. Overall, these findings have
important implications for trying to understand the complex, multi-
factorial nature of coronaviral disease pathogenesis and suggest the
importance of focusing on an Ab-based versus a T cell-based vac-
cine approach to combat respiratory coronaviral infections.
We thank Stanley Perlman for critical review of the manuscript.
The authors have no financial conflicts of interest.
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