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* Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97201;
Max von Pettenkofer Institute, Munich, Germany; and
Corixa Corporation, Seattle, Washington 98104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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m152 made similar responses to
the M45 Ag. Moreover, we saw no skewing of the proportion of
M45-specific CD8 T cells within the total MCMV-specific response after
infection with MCMV with m152. Despite the profound
effect m152 has on presentation of M45 in vitro, it does
not affect the immunodominance of M45 in the CTL response in
vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most CD8 T cell responses to virus infections focus on very few epitopes, even though a much larger pool of peptides is processed and presented; this phenomenon is known as immunodominance (reviewed in Ref. 11). What causes some epitopes to become immunodominant over others is poorly understood, and studies are limited for large complex viruses such as herpesviruses. We would predict that the immune response to a herpesvirus would be constrained to detect the Ags least affected by its immunomodulatory genes. In contrast, the immune response to a virus lacking immunomodulatory genes should be free to focus on more abundantly presented epitopes. As a result, we and others (11) have predicted that immunomodulatory genes would affect the immunodominance hierarchy of the CTL response.
We would expect that the Ags least affected by the immunomodulatory
genes would be those that are expressed in the infected cell before
expression of the immunomodulatory genes. These could be structural
virion proteins introduced into the cytosol at the time of viral entry.
Alternately, more abundant material should be provided by the first
viral gene products synthesized. As with other herpesviruses, MCMV gene
expression occurs as a regulated cascade; genes are classified as
immediate early (IE), early (E), and late (L). The immunomodulatory
genes m4, m6, and m152 are all E
genes. An epitope from the IE1 gene product pp89 is
recognized by a substantial number of CD8 T cells in BALB/c mice
(12, 13, 14). Because pp89 is expressed before m4,
m6, and m152, it is presumably less affected than
E Ags are by these immunomodulatory genes. The pp89 Ag is presented if
gene expression is limited to IE genes, but once E genes are expressed
pp89 presentation is abolished due to the action of immune evasion
genes (15). However, the addition of IFN-
enables
pp89-specific CTL to recognize infected cells even after E gene
expression (16); in contrast, the E-specific CTL clones
that we have isolated from C57BL/6 (B6) mice are not able to recognize
wild-type MCMV-infected targets even with the addition of IFN-
(3, 4, 5). The fact that inhibition of pp89 presentation is
readily overcome may indicate that a significant amount of Ag
presentation has already occurred before the immune evasion genes are
expressed.
For many years, pp89 was the only MCMV CTL Ag identified, and in consequence it has been widely studied. However, most CTL in both BALB/c and B6 mice actually recognize E and not IE Ags (15, 17, 18, 19). Thus, paradoxically, the Ags that would seem most likely to be affected by immunomodulatory genes actually elicit the majority of the immunodominant responses. This led us to question what effect the viral immune evasion genes, specifically m152, which has the most profound effect, have on the immunodominance of E Ags. Until recently, no immunodominant E Ags for MCMV had been described. Three CTL epitopes from E genes (m4, M83, and M84) for BALB/c mice were identified by Holtappels and colleagues (20, 21, 22), but these are recognized by only a small number of CD8 T cells. Very recently, Holtappels et al. (23) identified a Dd-restricted epitope in m164, which is as immunodominant as pp89. There is at present no information on the effect of immunomodulatory genes on the immunodominance of these epitopes.
In the current paper we describe the use of CTL clones and a genomic expression library to identify a Db-restricted epitope encoded by the MCMV E gene M45. These clones were isolated from mice infected with a mutant MCMV lacking m152 and recognize viruses lacking m152 but are unable to detect wild-type virus in 51Cr release assays. We compared the M45-specific CTL response in mice infected with MCMV with and without m152. Surprisingly, the percentage of CD8 T cells recognizing M45 was similar in mice infected with wild-type virus or with viruses lacking m152. These data indicate that while m152 affects M45 Ag presentation in vitro it does not affect the immunodominance of M45 in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two MCMV expression libraries representing the MCMV genome were constructed using MCMV DNA that was originally cloned as HindIII fragments into pUC-based plasmids (known as plasmids A–P) (24). Library I was constructed using equimolar amounts of HindIII fragments A, G–K, and M–P (equivalent to 99,563 bp of the MCMV genome) purified by agarose gel electrophoresis of the respective HindIII-digested plasmids. Similarly, library II (equivalent to 106,408 bp of the MCMV genome) was constructed using equimolar amounts of HindIII-purified fragments B–D, F, and L. Fragment E DNA (22,749 bp) was not included in the libraries. For expression library construction, the purified HindIII fragments 066–33(1,066–33,141 bp) were further fragmented by partial restriction enzyme digestion using a combination of BclI, BglII, BamHI (library I), or Sau3AI (library II) to generate BamHI compatible ends and ligated to BamHI cut pcDNA4 HisMax (Invitrogen, Carlsbad, CA). The pcDNA4 HisMax A, B, and C vectors allow expression of DNA in each reading frame by staggered insertion of DNA fragments downstream of a QBI SP163 translational enhancer. Characterization of a subset of clones showed that insert frequencies were 92 (I) and 83% (II), and the average insert sizes were 1,130 (I) and 530 bp (II). Approximately 30,000 independent clones (library I) and 25,000 independent clones (library II) were arrayed in pools of 50 clones per pool. DNA for transfections was prepared using Qiagen 96 Turbo-Prep plates (Qiagen, Valencia, CA).
Library screening
The libraries were screened using K41 cells, an SV-40
transformed H-2b fibroblast line (gift of M.
Michalek, University of Alberta, Edmonton, Alberta, Canada), that were
seeded at 5000 cells/well in a 96-well plate 24 h before
transfection. For transfections, 100 ng (per pool) of library DNA was
mixed with OptiMem (Invitrogen) and 1 µl of the transfection reagent
Fugene-6 (Roche, Basel, Switzerland) and added to plated K41 cells
using standard protocols. Wells were supplemented with medium 2 h
after transfection and incubated at 37°C. At 48 h, 2000 T
cells/well (clones 3 and 55 derived as previously described in Ref.
3) were added to the transfected K41s. After 8 h,
supernatants were collected and assayed for TNF-
by bioassay using
the TNF--sensitive indicator cell line WEHI164/clone 20, derived
(S. P. Fling, unpublished data) from WEHI164 (CRL-1751; American
Type Culture Collection, Manassas, VA) that is similar to that
described by Khabar et al. (25).
Epitope identification
One pool of 50 DNA clones from library II was confirmed to elicit a positive response by two Db-restricted MCMV-specific CTL clones (clones 3 and 55). From the positive pool, individual bacterial clones were derived and DNA was prepared and screened as described above. Individual bacterial clones that stimulated the CTL clones were sequenced. DNA was confirmed to be MCMV sequence. Peptides were synthesized based on an algorithm of optimal Db peptide-binding motifs (http://bimas.dcrt.nih.gov/molbio/hla_bind).
CTL line and clones
Generation and maintenance of CTL clones 3 and 55 have been previously described (3). A polyclonal M45-specific CTL line was generated by methods previously described (23). Briefly, 1.5 x 107 splenocytes from Kb-/- mice chronically infected with MCMV were incubated for 4 days with clone medium (RPMI with 10% FBS, 10-5 M 2-ME, and 10% rat con A supernatant) and the M45 peptide at a concentration of 10-10 M. Recombinant human IL-2 (100 U) was added on day 4 and the medium was replaced with fresh clone medium. The cells were used 7 days after one round of stimulation with the M45 peptide.
Viruses
MCMV Smith was purchased from American Type Culture Collection
(1399-VR). MC96.24 (m152) and rMC96.27 (wild-type rescuant)
(10), MS94.5 (lacking open reading frames
m151–165) (9), BAC-derived wild-type virus
MW97.01 (26), and m152-MW99.05 (3) have
been described.
Mice
Six-week-old female C57BL/6 mice were purchased from Simonsen Laboratories (Gilroy, CA). Kb-/- mice, the gift of F. Lemmonier (Institute Pasteur, Paris, France), were maintained in our animal facilities at Oregon Health and Science University (Portland, OR) and were used in experiments no earlier than 6 wk postbirth.
CTL assays
Either IFN--pretreated, B6 mouse embryo fibroblasts (MEF) or
untreated JAWS II cells (CRL-11904; American Type Culture Collection)
were used as virus-infected targets. The addition of IFN- to JAWS II
cells does not alter Ag presentation to MCMV-specific CD8s and
therefore was not added to the cultures (M. C. Gold, unpublished
data). Cells were infected with 50 PFU (except when noted) of virus for
16 h in the presence of 300 µg/ml phosphonoacetic acid (PAA;
Sigma-Aldrich, St. Louis, MO) to prevent L gene expression. IE only or
IE and E gene expression were enhanced as previously described
(15). Briefly, to selectively enhance IE gene expression,
B6 targets were infected with MCMV in the presence of cycloheximide (50
µg/ml; Sigma-Aldrich) followed by actinomycin D (5 µg/ml;
Sigma-Aldrich). For IE and E gene expression, cells were infected in
the presence of cycloheximide alone followed by a 3-h incubation
without drugs. For peptide screening, 104
51Cr-labeled RMA-S target cells were incubated with peptide
at the concentrations indicated and plated with CTL clones at an E:T
ratio of 15:1. After 5 h, the amount of radioactivity (cpm) in the
supernatant was counted using a TopCount (Packard Instrument, Meriden,
CT). The percentage of specific lysis was determined from the following
equation: ((cpm experimental release - cpm medium release)/(cpm
total release - cpm medium release)) x 100.
ICS assay
Splenocytes were isolated from MCMV-infected mice. APC (JAWS II
cells) were infected with MCMV (m152-MW99.05) at an MOI of 100 for
16 h in the presence of PAA. Effector splenocytes were incubated
with peptide at various concentrations, or at a ratio of 1:1 with
infected or uninfected JAWS II cells for 5 h in the presence of
brefeldin A (GolgiPlug; BD PharMingen, Franklin Lakes, NJ). Cells were
washed, incubated with FcBlock (BD PharMingen), and surface stained
with an Ab to the CD8 chain (BD PharMingen). Cells were then fixed
and permeabilized using BD PharMingen’s Cytofix/Cytoperm kit
before staining with an Ab to IFN- (BD PharMingen).
CD8+ T cells were analyzed by flow cytometry
using FACS in conjunction with CellQuest software (BD PharMingen).
All further analyses were performed using FlowJo software (Treestar,
San Carlos, CA).
RMA-S stabilization assay
HGIRNASFI and KHGIRNASFI peptides were titrated from 10-6 to 10-13 M in RPMI supplemented with 10% FBS, added to RMA-S cells, and incubated overnight at 23°C. The next day, the cells were washed four times in PBS (pH 7.4) at 23°C to remove excess peptide and incubated at 37°C for 4 h. Only peptide-loaded class I MHC is stable at the cell surface at this temperature (27). Cells were washed, then surface stained using the Db-specific mAb B22.249 (American Type Culture Collection) followed by FITC-conjugated goat anti-mouse IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD), and analyzed by flow cytometry as above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A panel of CTL clones was isolated from B6 mice infected with
various MCMV strains (3) and was used to screen an
expression library of random MCMV DNA fragments, as described in
Materials and Methods. Two CTL clones (clones 3 and 55) were
isolated from mice infected with the MCMV mutant MS94.5, which lacks
14 genes including m152 (9). Clones 3 and 55
have been previously shown to be Db restricted
(3). Drug blockade experiments either to limit herpesvirus
gene expression to IE genes or to allow E gene expression while
blocking L gene expression indicated that clone 3 was specific for an E
Ag (Fig. 1
A). E gene
expression occurs in waves, with m152 expression occurring
in the earliest wave, 2 h postinfection (5, 20). We
predicted that Ags expressed after m152 is expressed would
be most susceptible to the effects of m152. To determine
when in the infectious cycle the Ag recognized by clone 3 is expressed,
we asked when it could be presented if m152 were absent. To
this end, we infected cells with virus lacking m152 and used
intracellular cytokine staining (ICS) to determine how many hours of
infection were required for Ag recognition by clone 3. The presence of
brefeldin A in the ICS assay precludes further export of MHC class I in
the infected cells and thus restricts the MHC-peptide complexes
detected to those already at the cell surface at the indicated time
postinfection. Fig. 1B shows that clone 3 first detected Ag
6 h postinfection, and that recognition increased thereafter (Fig. 1B). This is well after m152 expression, which
commences 2 h postinfection (5, 20). To confirm that
the assay was capable of detecting Ag expression earlier in the
infectious cycle we included for comparison another MCMV-specific CTL
clone. Fig. 1B shows that clone 11 detected Ag by 4 h
postinfection and showed quite different kinetics. Therefore, clone 3
detects an Ag expressed in the infectious cycle after m152
is active. We concluded that the Ag recognized by clone 3 would be a
useful indicator with which to investigate the effect of the
immunomodulatory genes on immunodominance.
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From the MCMV genomic expression library, we identified a plasmid that both CTL clones 3 and 55 recognized. This plasmid encoded a small fragment of the M45 gene (28). Based on the published MCMV sequence (28) and the corrected addition of a cytosine at nucleotide position 61918 of the MCMV genome (29), M45 encodes a 1174-aa protein that is homologous to human CMV UL45 and to the large subunit of the class Ia murine ribonucleotide reductase gene (28).
From the translated sequence of the expressed M45 fragment,
we predicted four potential Db-binding peptides
that were synthesized and used to sensitize RMA-S cells for lysis by
clones 3 and 55 in a 51Cr release assay. Fig. 2 shows that both the 9-mer HGIRNASFI (aa
985–993) and the 10-mer KHGIRNASFI (aa 984–993) were equally able to
sensitize targets for killing by clones 3 and 55. Both peptides
sensitized targets down to 10-12 M. As a measure
of the relative affinity of HGIRNASFI and KHGIRNASFI, we compared their
ability to stabilize cell surface Db in the
TAP-deficient cell line, RMA-S (27). As shown in Fig. 3, the 9-mer and the 10-mer were equally
effective at stabilizing cell surface Db.
It is likely that the 9-mer represents the minimal epitope with the
histidine residue occupying the A pocket of Db
(30).
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We next determined whether we could detect HGIRNASFI-specific CD8
T cells directly ex vivo. Because of the low frequency of CD8s
responding to previously identified E Ags, we optimized our chances of
detecting (K)HGIRNASFI-specific CD8 T cells in vivo by looking at the
peak of the response in mice whose only class I molecule is
Db. Kb-/- mice were
infected with the MCMV mutant MS94.5 (which lacks 14 genes,
including m152). The ICS assay was used to quantify the
frequency of M45-specific CD8+ spleen cells from
acutely infected mice. Over 20% of all CD8+
splenocytes from Kb-/- mice infected for 6 days
with MS94.5 were (K)HGIRNASFI-specific (Fig. 4). We were encouraged by the strength of
this response to look at the CD8 T cell response in chronically
infected B6 mice. Fig. 4 shows the result from a representative mouse
infected with MCMV-m152 for 1.5 years, in which 2.5% of
CD8+ splenocytes recognized the (K)HGIRNASFI
peptides (Fig. 4). Finally, no response to HGIRNASFI was detected in a
naive B6 mouse, while in the same assay 4% of CD8s from a mouse
infected with MS94.5 for 12 wk made IFN- in response to HGIRNASFI
(Fig. 5). In another experiment with four
B6 mice chronically infected with viruses lacking m152,
between 1 and 4% of CD8s were specific for HGIRNASFI, whereas <0.3%
of CD8s were positive in two naive mice (data not shown). Other
experiments suggest that the difference in the magnitude of the
HGIRNASFI-specific responses seen in Fig. 4 is most likely to be due to
differences between the acute and chronic phase of the CD8 response
rather than a difference between Kb-/- mice and
B6 mice. Future studies are planned to determine whether the
HGIRNASFI-specific response changes in its relative proportion to the
total MCMV-specific response over time in mice chronically infected
with MCMV.
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The effects of the immunomodulatory gene m152 on M45
presentation in vitro are profound. Fig. 6A shows that clones 3 and 55
were unable to kill fibroblasts infected with three different wild-type
MCMV strains. However, this defect was reversed if m152 was
absent: clones 3 and 55 were able to kill fibroblasts infected with
three different MCMV mutants lacking m152. m152
was equally effective at inhibiting presentation of M45 in MEFs and the
dendritic cell (DC) line, JAWS II, that was used as the APC in the ICS
assays. Fig. 6B shows a CTL assay using a polyclonal
M45-specific CTL line: wild-type infected targets were not recognized,
whereas m152-infected targets were readily lysed. We also tested
this line for IFN- release by ICS, using both MEFs and JAWS II cells
as APCs. Wild-type MCMV-infected MEFs and JAWS II cells failed to
stimulate IFN- secretion, whereas both cell types infected with
MCMV-m152 did stimulate IFN- secretion (data not shown). We
conclude that m152 effectively inhibits presentation of
HGIRNASFI in both MEFs and a DC line.
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Because m152 appeared able to completely prevent
presentation of M45, we hypothesized that mice infected with a
wild-type (m152+) MCMV would not develop an
M45-specific CTL response. To test this prediction, we infected
Kb-/- mice with wild-type MCMV (Smith),
MS94.5, MC96.24 (m152), and the rescued virus rMC96.27
(MC96.24 with m152 restored). Fig. 7 shows that, surprisingly, the
percentage of CD8s recognizing M45 was very similar in each infection:
between 12 and 13% of CD8+ splenocytes on day 8
postinfection were specific for M45.
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m152 (MC96.24) or the
rescuant (rMC96.27) MCMV strains for 8 days. Fig. 8 shows that CD8 T cell responses to MCMV
(m152)-infected DCs ranged from 3 to 11%. No remarkable differences
were seen in the CD8 T cell responses to virus-infected APC after MCMV
infection with or without m152. This indicated that
m152 did not affect the total numbers of MCMV-specific CD8 T
cells that were generated in vivo. This assay also allowed a rough
assessment of the immunodominance of M45 within the total MCMV-specific
CD8 response. While the HGIRNASFI peptide stimulation probably detects
all CD8s of this specificity, it is likely that simulation with
virus-infected DCs underestimates the total number of MCMV-specific
CD8s. Therefore, we conclude that, while a substantial number of
CD8s recognized HGIRNASFI, other specificities exist. Finally, we
compared the HGIRNASFI-specific response to the detectable response to
whole virus after infection with MCMV with or without m152.
Although there was mouse to mouse variation, the ratio of the
peptide-specific response to the total MCMV-specific response did not
differ significantly (p = 0.39). The ratio of
the M45-specific CD8 T cell response to the total MCMV-specific CD8
response averaged among five mice (expressed as a percentage) was
62.1% after MCMVm152 infection vs 72.8% after infection with the
rescuant (Fig. 8 and Table I). These data
again indicate that m152 did not alter the immunodominance
of the M45 Ag.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this paper we describe the use of CTL clones and a genomic
fragment library to identify an immunodominant epitope in MCMV.
Identification of CTL epitopes in large viruses remains a difficult
procedure, and several points in the method described here are worthy
of note. To initially isolate the CTL clones we screened clones for
their ability to kill targets infected with a virus (MS94.5) that
lacks the key immune evasion gene, m152. The CTL clones used
in this paper were generated from m152-infected mice, but we have
also generated clones from wild-type-infected mice (3);
CTL clones from both types of infection were able to lyse
MS94.5-infected targets but not wild-type-infected targets. Thus,
the use of a virus lacking immune evasion genes to screen CTL clones
was critical for isolation of these clones. M45 is immunodominant in
wild-type infection, but clones of this specificity would not have been
identified had we used wild-type virus in our initial screen. The use
of viruses lacking immune evasion genes for screening clones may also
be useful in identifying new CTL epitopes in other viruses such as
human CMV, HSV, and Kaposi’s sarcoma-associated herpesvirus.
The second feature of the strategy described here was the use of a viral genomic fragment library. This strategy has previously been applied successfully to identify epitopes in Chlamydia and HSV (S. P. Fling, unpublished results). If a pathogen’s genomic DNA can be obtained independently of the host genome this approach can be useful, because it eliminates contaminating host transcripts that would be expressed in a cDNA library and which would increase the clonal complexity of the library. Another advantage of expression cloning in general is the potential to identify both dominant and subdominant epitopes. We do not yet know whether genomic libraries constructed by restriction digest express all possible epitopes. Clearly, if a restriction site coincides with the portion of DNA encoding an epitope, representation may be limited. However, this problem is abrogated to some extent by partial restriction digests of input DNA.
The M45 epitope is clearly recognized by a substantial fraction of
MCMV-specific CD8s in B6 mice. Identification of other epitopes is
necessary before we can determine whether it is the most immunodominant
epitope in this strain. The only other immunodominant MCMV epitopes
identified so far are for the H-2d haplotype:
epitopes from pp89 (Ld-restricted) (12, 13) and m164 (Dd-restricted)
(23) have been described. Reddehase and colleagues
(21, 23) injected MCMV into the footpad, measured the CTL
response using an ELISPOT assay, and determined that 
0.7% of CD8s
in the spleens of chronically infected BALB/c mice were specific for
each of these epitopes. These authors used the number of CD8s that made
IFN- in response to anti-CD3 (2%) to identify previously
activated CD8s and argued that this number approximated the total
MCMV-specific memory population. Based on this assumption they
concluded that most MCMV-specific CD8s in BALB/c mice are specific for
either pp89 or m164. Very recently, Ye et al. (14) used a
protocol similar to the one we used in the current paper and determined
that pp89-specific CD8s made up close to 3% of CD8s 1 mo after
infection. We measured a similar response to pp89 (4% of CD8s) in one
experiment in BALB/c mice (M. C. Gold, unpublished observation).
We think it more likely that differences in infectious route and method
of detecting the response, rather than between BALB/c and B6 mice per
se, will be found to account for these differences in the measured size
of the response. It will be interesting to perform side by side
comparisons using the same methods to determine the relative size of
the overall MCMV-specific response in both mouse strains, as well as
the proportion of that response that can be attributed to the currently
identified epitopes.
m152 affects presentation of M45 without affecting its immunodominance
The hypothesis that m152 would affect the hierarchy of
immunodominant CD8 epitopes is based on the fact that m152
severely impairs Ag processing and presentation in fibroblasts.
However, our work demonstrates that the CD8 T cell response to M45 is
not affected by the presence of m152 in vivo even though its
presentation is severely affected by m152 in vitro. We can
think of two possible explanations for this surprising result. First,
Ag presentation in some important cell types in vivo, such as
macrophages or DCs, may not be affected by m152 in the same
way that fibroblasts are affected in vitro. It has been reported that
macrophages can overcome the effect of immune evasion genes and
effectively present the IE pp89 Ag (31). However, our
experience is that M45-specific CTL clones are unable to kill wild-type
virus-infected macrophages, even though they readily kill
m152-infected macrophages (D. LoPicollo, M. Gold, D. Kavangh, M.
Wagner, U. Koszinowski, and A. Hill, manuscript in preparation).
Similarly, we show in this paper that m152 effectively
prevents Ag presentation in a DC line. Thus, while it remains possible
that presentation of M45 by an infected cell in vivo is not affected by
m152, there is currently no evidence to support that
hypothesis.
A second explanation for the lack of effect of m152 on the immunodominance of M45 could be that cross-presentation is the principle mechanism for priming the CD8 T cell population to M45 in vivo. Cross-presentation is the process by which DCs take up Ag from infected cells and present it on newly synthesized class I molecules. Presumably m152, a glycoprotein that acts in the ER, only affects Ag presentation in infected cells and does not affect cross-presentation. Cross-presentation is believed to be an important mechanism by which CD8 T cells are primed in vivo (32, 33), and it is thus perhaps not surprising that M45-specific CD8 T cell responses would be elicited in wild-type MCMV-infected mice. Nevertheless, if cross-presentation is the main mechanism used for priming the CTL response, this could potentially be problematic for the immune response. If the Ags presented by cross-priming are different from those presented directly by virus-infected cells, then the immune response may be fooled into making a CTL response that is ineffective in clearing virus-infected cells. Indeed, we had assumed the CD8 T cell response would focus on Ags that are efficiently presented by virus-infected cells in vivo. In an attempt to reduce in vitro artifact, we assessed the efficacy of immune evasion genes using virus infections (rather than transfections) of primary untransformed murine fibroblasts, as well as a DC line. In these assays m152 prevented recognition of the M45-encoded epitope. However, we do not yet know how effective M45-specific CD8s are in vivo against wild-type virus or, indeed, for which infected cell types in vivo CD8 control is most important. However, we do note that MCMV is effectively controlled by the immune response and that CD8 T cells play an important role in this control. Future studies will address these issues.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ann B. Hill, Department of Molecular Microbiology and Immunology, L220, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97201. E-mail address: hillan{at}ohsu.edu
3 Abbreviations used in this paper: MCMV, murine CMV; DC, dendritic cell; PAA, phosphonoacetic acid; ER, endoplasmic reticulum; ICS, intracellular cytokine staining; MEF, mouse embryo fibroblast; IE, immediate early; E, early; L, late.
Received for publication December 20, 2001. Accepted for publication April 30, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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interferon combats viral escape. J. Virol. 68:289.This article has been cited by other articles:
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  V. Bohm, C. K. Seckert, C. O. Simon, D. Thomas, A. Renzaho, D. Gendig, R. Holtappels, and M. J. Reddehase Immune Evasion Proteins Enhance Cytomegalovirus Latency in the Lungs J. Virol., October 1, 2009; 83(19): 10293 - 10298. [Abstract] [Full Text] [PDF]
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R. Holtappels, D. Thomas, and M. J. Reddehase The Efficacy of Antigen Processing Is Critical for Protection against Cytomegalovirus Disease in the Presence of Viral Immune Evasion Proteins J. Virol., September 15, 2009; 83(18): 9611 - 9615. [Abstract] [Full Text] [PDF]
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 A. C. Jirmo, C.-H. Nagel, C. Bohnen, B. Sodeik, and G. M. N. Behrens Contribution of Direct and Cross-Presentation to CTL Immunity against Herpes Simplex Virus 1 J. Immunol., January 1, 2009; 182(1): 283 - 292. [Abstract] [Full Text] [PDF]
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V. Bohm, C. O. Simon, J. Podlech, C. K. Seckert, D. Gendig, P. Deegen, D. Gillert-Marien, N. A. W. Lemmermann, R. Holtappels, and M. J. Reddehase The Immune Evasion Paradox: Immunoevasins of Murine Cytomegalovirus Enhance Priming of CD8 T Cells by Preventing Negative Feedback Regulation J. Virol., December 1, 2008; 82(23): 11637 - 11650. [Abstract] [Full Text] [PDF]
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R. Holtappels, C. O. Simon, M. W. Munks, D. Thomas, P. Deegen, B. Kuhnapfel, T. Daubner, S. F. Emde, J. Podlech, N. K. A. Grzimek, et al. Subdominant CD8 T-Cell Epitopes Account for Protection against Cytomegalovirus Independent of Immunodomination J. Virol., June 15, 2008; 82(12): 5781 - 5796. [Abstract] [Full Text] [PDF]
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R. Arens, P. Wang, J. Sidney, A. Loewendorf, A. Sette, S. P. Schoenberger, B. Peters, and C. A. Benedict Cutting Edge: Murine Cytomegalovirus Induces a Polyfunctional CD4 T Cell Response J. Immunol., May 15, 2008; 180(10): 6472 - 6476. [Abstract] [Full Text] [PDF]
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M. S. Hossain, J. D. Roback, F. Wang, and E. K. Waller Host and Donor Immune Responses Contribute to Antiviral Effects of Amotosalen-Treated Donor Lymphocytes following Early Posttransplant Cytomegalovirus Infection J. Immunol., May 15, 2008; 180(10): 6892 - 6902. [Abstract] [Full Text] [PDF]
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 B. Bolinger, P. Krebs, Y. Tian, D. Engeler, E. Scandella, S. Miller, D. C. Palmer, N. P. Restifo, P.-A. Clavien, and B. Ludewig Immunologic ignorance of vascular endothelial cells expressing minor histocompatibility antigen Blood, May 1, 2008; 111(9): 4588 - 4595. [Abstract] [Full Text] [PDF]
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M. W. Munks, A. K. Pinto, C. M. Doom, and A. B. Hill Viral Interference with Antigen Presentation Does Not Alter Acute or Chronic CD8 T Cell Immunodominance in Murine Cytomegalovirus Infection J. Immunol., June 1, 2007; 178(11): 7235 - 7241. [Abstract] [Full Text] [PDF]
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M. S. Hossain, J. D. Roback, B. P. Pollack, D. L. Jaye, A. Langston, and E. K. Waller Chronic GvHD decreases antiviral immune responses in allogeneic BMT Blood, May 15, 2007; 109(10): 4548 - 4556. [Abstract] [Full Text] [PDF]
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D. N. Streblow, K. W. R. van Cleef, C. N. Kreklywich, C. Meyer, P. Smith, V. Defilippis, F. Grey, K. Fruh, R. Searles, C. Bruggeman, et al. Rat Cytomegalovirus Gene Expression in Cardiac Allograft Recipients Is Tissue Specific and Does Not Parallel the Profiles Detected In Vitro J. Virol., April 15, 2007; 81(8): 3816 - 3826. [Abstract] [Full Text] [PDF]
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K. L. Hokeness, E. S. Deweerd, M. W. Munks, C. A. Lewis, R. P. Gladue, and T. P. Salazar-Mather CXCR3-Dependent Recruitment of Antigen-Specific T Lymphocytes to the Liver during Murine Cytomegalovirus Infection J. Virol., February 1, 2007; 81(3): 1241 - 1250. [Abstract] [Full Text] [PDF]
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S. Bozza, F. Bistoni, R. Gaziano, L. Pitzurra, T. Zelante, P. Bonifazi, K. Perruccio, S. Bellocchio, M. Neri, A. M. Iorio, et al. Pentraxin 3 protects from MCMV infection and reactivation through TLR sensing pathways leading to IRF3 activation Blood, November 15, 2006; 108(10): 3387 - 3396. [Abstract] [Full Text] [PDF]
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A. K. Pinto, M. W. Munks, U. H. Koszinowski, and A. B. Hill Coordinated Function of Murine Cytomegalovirus Genes Completely Inhibits CTL Lysis. J. Immunol., September 1, 2006; 177(5): 3225 - 3234. [Abstract] [Full Text] [PDF]
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R. Holtappels, D. Gillert-Marien, D. Thomas, J. Podlech, P. Deegen, S. Herter, S. A. Oehrlein-Karpi, D. Strand, M. Wagner, and M. J. Reddehase Cytomegalovirus encodes a positive regulator of antigen presentation. J. Virol., August 1, 2006; 80(15): 7613 - 7624. [Abstract] [Full Text] [PDF]
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M. W. Munks, M. C. Gold, A. L. Zajac, C. M. Doom, C. S. Morello, D. H. Spector, and A. B. Hill Genome-Wide Analysis Reveals a Highly Diverse CD8 T Cell Response to Murine Cytomegalovirus J. Immunol., March 15, 2006; 176(6): 3760 - 3766. [Abstract] [Full Text] [PDF]
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M. Lotzerich, Z. Ruzsics, and U. H. Koszinowski Functional Domains of Murine Cytomegalovirus Nuclear Egress Protein M53/p38 J. Virol., January 1, 2006; 80(1): 73 - 84. [Abstract] [Full Text] [PDF]
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S. K. Tripathy, H. R. C. Smith, E. A. Holroyd, J. T. Pingel, and W. M. Yokoyama Expression of m157, a Murine Cytomegalovirus-Encoded Putative Major Histocompatibility Class I (MHC-I)-Like Protein, Is Independent of Viral Regulation of Host MHC-I J. Virol., January 1, 2006; 80(1): 545 - 550. [Abstract] [Full Text] [PDF]
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 J. D. Roback Vaccine-Enhanced Donor Lymphocyte Infusion (veDLI) Hematology, January 1, 2006; 2006(1): 486 - 491. [Abstract] [Full Text] [PDF]
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C. S. Morello, M. Ye, S. Hung, L. A. Kelley, and D. H. Spector Systemic Priming-Boosting Immunization with a Trivalent Plasmid DNA and Inactivated Murine Cytomegalovirus (MCMV) Vaccine Provides Long-Term Protection against Viral Replication following Systemic or Mucosal MCMV Challenge J. Virol., January 1, 2005; 79(1): 159 - 175. [Abstract] [Full Text] [PDF]
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 A. B. Kamath, J. Woodworth, X. Xiong, C. Taylor, Y. Weng, and S. M. Behar Cytolytic CD8+ T Cells Recognizing CFP10 Are Recruited to the Lung after Mycobacterium tuberculosis Infection J. Exp. Med., December 6, 2004; 200(11): 1479 - 1489. [Abstract] [Full Text] [PDF]
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M. Ye, C. S. Morello, and D. H. Spector Multiple Epitopes in the Murine Cytomegalovirus Early Gene Product M84 Are Efficiently Presented in Infected Primary Macrophages and Contribute to Strong CD8+-T-Lymphocyte Responses and Protection following DNA Immunization J. Virol., October 15, 2004; 78(20): 11233 - 11245. [Abstract] [Full Text] [PDF]
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T. J. Manley, L. Luy, T. Jones, M. Boeckh, H. Mutimer, and S. R. Riddell Immune evasion proteins of human cytomegalovirus do not prevent a diverse CD8+ cytotoxic T-cell response in natural infection Blood, August 15, 2004; 104(4): 1075 - 1082. [Abstract] [Full Text] [PDF]
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M. C. Gold, M. W. Munks, M. Wagner, C. W. McMahon, A. Kelly, D. G. Kavanagh, M. K. Slifka, U. H. Koszinowski, D. H. Raulet, and A. B. Hill Murine Cytomegalovirus Interference with Antigen Presentation Has Little Effect on the Size or the Effector Memory Phenotype of the CD8 T Cell Response J. Immunol., June 1, 2004; 172(11): 6944 - 6953. [Abstract] [Full Text] [PDF]
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T. Storni and M. F. Bachmann Loading of MHC Class I and II Presentation Pathways by Exogenous Antigens: A Quantitative In Vivo Comparison J. Immunol., May 15, 2004; 172(10): 6129 - 6135. [Abstract] [Full Text] [PDF]
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S. Khan, A. Zimmermann, M. Basler, M. Groettrup, and H. Hengel A Cytomegalovirus Inhibitor of Gamma Interferon Signaling Controls Immunoproteasome Induction J. Virol., February 15, 2004; 78(4): 1831 - 1842. [Abstract] [Full Text] [PDF]
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R. Holtappels, J. Podlech, M.-F. Pahl-Seibert, M. Julch, D. Thomas, C. O. Simon, M. Wagner, and M. J. Reddehase Cytomegalovirus Misleads Its Host by Priming of CD8 T Cells Specific for an Epitope Not Presented in Infected Tissues J. Exp. Med., January 5, 2004; 199(1): 131 - 136. [Abstract] [Full Text] [PDF]
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J. D. Roback, M. S. Hossain, L. Lezhava, J. W. Gorechlad, S. A. Alexander, D. L. Jaye, S. Mittelstaedt, S. Talib, J. E. Hearst, C. D. Hillyer, et al. Allogeneic T Cells Treated with Amotosalen Prevent Lethal Cytomegalovirus Disease without Producing Graft-versus-Host Disease Following Bone Marrow Transplantation J. Immunol., December 1, 2003; 171(11): 6023 - 6031. [Abstract] [Full Text] [PDF]
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M. N. Starnbach, W. P. Loomis, P. Ovendale, D. Regan, B. Hess, M. R. Alderson, and S. P. Fling An Inclusion Membrane Protein from Chlamydia trachomatis Enters the MHC Class I Pathway and Stimulates a CD8+ T Cell Response J. Immunol., November 1, 2003; 171(9): 4742 - 4749. [Abstract] [Full Text] [PDF]
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M. Lodoen, K. Ogasawara, J. A. Hamerman, H. Arase, J. P. Houchins, E. S. Mocarski, and L. L. Lanier NKG2D-mediated Natural Killer Cell Protection Against Cytomegalovirus Is Impaired by Viral gp40 Modulation of Retinoic Acid Early Inducible 1 Gene Molecules J. Exp. Med., May 19, 2003; 197(10): 1245 - 1253. [Abstract] [Full Text] [PDF]
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M. C. Ramirez and L. J. Sigal Macrophages and Dendritic Cells Use the Cytosolic Pathway to Rapidly Cross-Present Antigen from Live, Vaccinia-Infected Cells J. Immunol., December 15, 2002; 169(12): 6733 - 6742. [Abstract] [Full Text] [PDF]
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D. M. LoPiccolo, M. C. Gold, D. G. Kavanagh, M. Wagner, U. H. Koszinowski, and A. B. Hill Effective Inhibition of Kb- and Db-Restricted Antigen Presentation in Primary Macrophages by Murine Cytomegalovirus J. Virol., December 6, 2002; 77(1): 301 - 308. [Abstract] [Full Text] [PDF]
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