Previous Article | Next Article 
Journal of Virology, December 1999, p. 10079-10085, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Wild-Derived Inbred Mice Have a Novel Basis of
Susceptibility to Polyomavirus-Induced Tumors
Palanivel
Velupillai,
Izumi
Yoshizawa,
Dilip C.
Dey,
Sharon
R.
Nahill,
John P.
Carroll,
Roderick T.
Bronson, and
Thomas L.
Benjamin*
Department of Pathology, Harvard Medical
School, Boston, Massachusetts 02115
Received 18 June 1999/Accepted 25 August 1999
 |
ABSTRACT |
Polyomavirus induces a broad array of tumors when introduced into
newborn mice of certain standard inbred strains, notably those bearing
the H-2k haplotype. Susceptibility in these
mice is conferred by an endogenous mouse mammary tumor virus
superantigen (Mtv-7 sag) that acts to delete T cells
required for polyomavirus-induced tumor immunosurveillance. In the
present study we show that mice of two wild-derived inbred strains,
PERA/Ei (PE) and CZECH II/Ei (CZ), are highly susceptible to
polyomavirus but carry no detectable Mtv sag-related sequences and show
no evidence of V
deletion. C57BR/cdJ (BR) mice, which are
H-2k but lack the endogenous Mtv-7, are highly
resistant based on an effective anti-polyomavirus tumor immune
response. When crossed with BR, both PE and CZ mice transmit their
susceptibility in a dominant fashion, indicating a mechanism(s) that
overrides the immune response of BR. Susceptibility in PE and CZ mice
is not based on interference with antigen processing or presentation since cytotoxic T cells from BR can efficiently kill
F1-derived tumor cells in vitro. The expected precursors of
polyomavirus-specific cytotoxic T cells are present in both the wild
inbred animals and their F1 progeny. These findings
indicate a novel basis of susceptibility that operates independently of
endogenous superantigen and prevents the development of tumor immunity.
| |
INTRODUCTION |
The murine polyomavirus can be a
powerful oncogenic agent in its natural host, as evidenced by the rapid
development of multiple solid tumors after inoculation of newborn
animals (11, 19). Genetic backgrounds of both virus and host
play important roles in determining the tumor response. By using a
highly susceptible host, the effects of various determinants in the
viral T (tumor) antigens involved in cell transformation (6, 14,
16, 38), as well as ones in the viral structural proteins with
effects on receptor binding, cell penetration, and spread (2, 14, 36), have been investigated.
The role of the host genetic background is complex and less well
understood. Earlier studies of susceptible and resistant strains
established an important role of the major histocompatibility complex
(MHC) type and the ability to generate antitumor cellular immune
responses (4, 17, 25, 27). Most resistant mouse strains show
a radiation-sensitive form of resistance and become susceptible after
radiation or neonatal thymectomy (1, 9, 26). A
radiation-resistant or nonimmunological form of host resistance that
acts by curtailing virus spread has also been described (9).
In crosses between susceptible and resistant mice of the same MHC type
(H-2k), susceptibility is inherited as a single
dominant Mendelian trait (17, 30). This gene segregates with
the endogenous mouse mammary tumor provirus Mtv-7 carried by
the susceptible strain (28). C57BR/cdJ (BR) mice, used as
the resistant parent, generate virus-specific cytotoxic T lymphocytes
(CTLs) with a V specificity (V6) that would be deleted by the
Mtv-7 superantigen (sag) present in all highly susceptible
H-2k strains (28, 29).
In the present study we first isolated and characterized a
polyomavirus-specific CTL line from a virus-infected BR mouse and used
it to investigate possible mechanisms of immune evasion by rare tumors
that arise in this resistant strain. We describe a genetic and
immunological basis of susceptibility to polyomavirus tumors manifested
by two wild-derived inbred mouse strains. These highly susceptible wild
inbreds are shown to be free of endogenous Mtvs and to express no
sag(s) and yet, in crosses with BR mice, they transmit their
susceptibility in a dominant fashion. Tumor cells derived from
F1 animals are killed by CTLs from strain BR mice,
demonstrating that these mice are able to process and present the
appropriate viral epitope and that the tumor cells themselves are not
intrinsically resistant to CTL killing. The wild-derived inbreds and
their F1s have normal levels of CD8+
V6+ T cells that are the expected precursors of
polyomavirus-specific CTLs in this system. These results suggest a
novel basis of tumor susceptibility involving a mechanism that
interferes with the development of tumor immunity.
| |
MATERIALS AND METHODS |
Tumor studies.
C57BR/cdJ, PERA/Ei (PE), CZECH II/Ei (CZ),
and C57BL/6J (B6) mice were purchased from the Jackson Laboratory (Bar
Harbor, Maine). C3H/BiDa mice were obtained from Clarence Reeder at the National Cancer Institute, Frederick, Md. All mice were bred and maintained in our virus antibody-free (VAF) barrier facility prior to
virus inoculation. The A2 and A2+ high-tumor strains of
polyomavirus were used (15). Newborn animals (<18 h old)
were inoculated intraperitoneally with ~50 µl of virus containing
2 × 106 to 10 × 106 PFU and were
monitored for up to 6 months for tumor development. Animals were
sacrificed when moribund and then necropsied; gross tumors, as well as
apparently normal tissues, were examined histologically as described
earlier (10). Tumor-derived cell lines were as follows:
A-6215 is immunogenic, derived from a salivary gland tumor that arose
in an irradiated virus-infected BR mouse; A-6241 and A-6689 are
nonimmunogenic, derived from mammary tumors that arose in normal
(unirradiated) virus-infected BR mice (28). Cultures of
tumor cells from PE, CZ, and F1 animals were prepared by
collagenase treatment of primary tumors and plating cells in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum.
Detection of antigens by PCR.
Genomic DNAs were prepared
from tail tissue as described previously (24). PCR was
conducted in a PTC-100 thermocycler (MJ Research, Inc., Watertown,
Mass.). Briefly, 100 µl of a reaction mixture containing 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2, 100 µg of
gelatin per ml, 200 µM concentrations of each of the deoxynucleoside
triphosphates, and 0.5 µM concentrations of each primer was mixed
with 2.5 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer, Foster City,
Calif.) and 200 ng of genomic DNA. The PCR mixture was subjected to
predenaturation at 94°C (5 min), followed by 35 cycles of 94°C (45 s), 58°C (45 s), and 72°C (1.5 min) and a final postcycling
extension at 72°C for 5 min. PCR amplification of Mtv-7
sag-specific sequences was carried out with forward primer Mtv-7-2
(5'-TTACATCTACAGACCAACAGATGCCCCGT-3') and reverse primer Mtv-7-1 (5'-GAAGCCAACGCGACCCCC-3') based on published
sequences (5, 35). Primers for conserved mouse mammary tumor
virus (MMTV) sag sequences were forward MTV-cons
(5'-GGGAATTCTCGAGATGCCGCGCCTGCAG-3') and reverse MTV-cons
(5'-GGGGATCCTCTAGAGGGAACCGCAAGGTTGGG-3') (21).
For polyomavirus sequences in tumor cell DNAs, primers were used to
give a 542-bp product covering overlapping sequences for the large and
middle T antigens: forward, 5'-GTATTTGGACATCCTAC-3'; and
reverse, 5'-AAATGGTGCTGCGGTTACAA-3'. The annealing
temperature for polyomavirus PCR was 55°C; other parameters were the
same as those given above. As a PCR control, mouse-specific DNA was amplified by using Map Pair D8 Mit 223 from Research Genetics, Inc.
(Huntsville, Ala.).
Cytofluorometric analysis: screening for V expression in T
cells from spleen.
Leukocytes from the spleen were isolated by
centrifugation of a single cell suspension over Ficoll-Hypaque
gradients (Accurate Chemicals, Westbury, N.Y.). T cells were enriched
by passage through CD3 T-cell enrichment columns (R&D, Minneapolis,
Minn.). The enriched T cells were first incubated with V T-cell
receptor-specific monoclonal antibodies (MAbs) (kindly provided by
Martin E. Dorf, Department of Pathology, Harvard Medical School,
Boston, Mass.) and then with fluorochrome-labeled second antibodies
(Jackson Immunoresearch, West Grove, Pa.). The stained cells were
analyzed by using a Coulter Profile II flow cytometer (Coulter,
Hialeah, Fla.).
Analysis of MHC haplotype.
Splenocytes were prepared by
ammonium chloride lysis of erythrocytes, followed by gravity
sedimentation to remove cell clumps and debris. One million cells were
stained with 1 µg of fluorescein isothiocyanate (FITC)-conjugated
anti-H-2Kk or anti-H-2Dk
MAbs (Pharmingen, San Diego, Calif.). A monoclonal rat immunoglobulin (immunoglobulin G1 [IgG1]) was used as a control. Flow cytometry was
performed on a Becton Dickinson (Mountain View, Calif.) FACScan by
using LYSIS II software, and the data were analyzed with CellQuest software.
In vitro one-way mixed lymphocyte reaction.
Splenocytes were
prepared by ammonium chloride lysis of erythrocytes. Responder cells
were from BR (H-2k) mice. Stimulator cells from
different mouse strains were treated with mitomycin C (50 µg/50
million cells). Responder (2 × 105 cells/well) and
stimulator (4 × 105 cells/well) cells were added
together in wells of a 96-well tissue culture plate in DMEM containing
10% fetal calf serum. The cells were incubated in a humidified chamber
at 37°C with 7% CO2 for 4 days and subsequently pulsed
with 1 µCi of [3H]thymidine per well for an additional
16 h. Cells were collected on a glass fiber filter and counted in
a beta scintillation counter.
In vitro CTL assay: generation of polyomavirus-specific CTL line
LN-13.1.
Cell line LN-13.1 was generated from a virus-immunized BR
mouse. A newborn mouse was infected in the footpad with 50 µl of A2
virus containing 108 PFU/ml. Two weeks later, lymphocytes
from the draining lymph nodes were harvested and cultured in a 24-well
plate in complete Iscove modified Dulbecco medium (IMDM) with 10%
fetal bovine serum. Lymphocytes were stimulated weekly with A2
virus-infected and gamma-irradiated (2,000 rads) BR splenocytes. After
three cycles of restimulation, bulk cultures were further established
by using mitomycin-treated immunogenic BR salivary gland tumor cell
line A-6215 and naive BR splenocytes as stimulator and feeder cells, respectively. The CTL line LN-13.1 thus generated was used along with
51Cr-labeled target cells in all CTL assays.
Polyomavirus tumor cell targets.
Cells derived from
virus-induced tumors were maintained in DMEM containing 10% fetal
bovine serum. The target cells were radiolabeled by the addition of 200 µCi of Na251CrO4 (NEN, Boston,
Mass.) per ml in DMEM with 5% fetal bovine serum. Cells were incubated
at 37°C for 1 h and washed three times in the same medium to
remove the free 51Cr.
Targets prepared from normal cells by viral infection or peptide
pulsing.
Adherent cells from the spleen were harvested from
different mouse strains and selected by incubating the cell suspensions on a polystyrene plastic surface. Polyomavirus middle T peptide R389
(RRLGRTLLL) or E328 (EEQVPQLI) was added to the adherent cells at a
concentration of 1 µM in DMEM with 10% fetal bovine serum, incubated
at 37°C for 1 h, and then washed in the same medium three times.
Alternatively, adherent cells were infected by virus at a multiplicity
of infection of 1 to 2 PFU/cell; cells were then
51Cr-labeled as described above and used as targets at 16 to 20 h postinfection.
The radiolabeled targets (5 × 103 cells/well) were
distributed into wells of 96-well U-bottom tissue culture plates along
with effector CTLs (25 × 103 cells/well) at a 5:1
effector/target ratio. The assay medium was complete IMDM containing
10% fetal calf serum, 6% rat T-stimulation medium (Collaborative
Research, Bedford, Mass.), 4 mM glutamine, 2 mM sodium pyruvate, and 50 µM 2-mercaptoethanol. After incubation for 4 h at 37°C, the
supernatant (100 µl) from each well was collected and counted on a
gamma counter (Wallac, Inc., Gaithersburg, Md.). Spontaneous and total
51Cr release were counted from supernatants of targets
incubated with medium alone and 1% Triton X-100, respectively. The
spontaneous release in all assays was less than 20% of the total
release. The percent specific lysis was calculated as follows: [(lysis with effector cells 
spontaneous lysis)/(total lysis spontaneous lysis)] × 100. Each assay contained triplicate wells, and
each experiment was repeated at least twice, with identical results.
| |
RESULTS |
Immune surveillance and evasion in the BR mouse.
The
resistance of BR mice to tumor induction by polyomavirus is based on
effective immune surveillance and the elimination of virus-induced
tumors. BR mice show the radiation-sensitive form of resistance
(9, 28) and mount an effective antitumor immune response
dominated by CD8+ V6+ T cells
(28). These CTLs have been shown to be
H-2Dk-restricted and specific for an
immunodominant epitope derived from the middle T protein, the product
of the major transforming gene of the virus (29). Rare
tumors that arise in polyomavirus-infected BR mice appear to be
"immune escape" variants by virtue of being transplantable in
polyomavirus-immunized syngeneic hosts (28). The
mechanism(s) of escape is unknown.
To investigate possible mechanisms of immune evasion, we first sought
to recover viral DNA sequences by PCR. Primers were
specific to a
region of middle T containing the immunodominant
epitope. Three tumors
were tested: A-6215, a positive control,
was derived from an irradiated
virus-infected BR mouse and is
known to express virus-specific
transplantation antigen(s); and
two variant tumors, A-6241 and A-6689,
which arose in nonirradiated
virus-infected BR mice and fail to express
virus-specific transplantation
antigen(s) (
28). A-6215 and
one of the variants were positive
for viral DNA, while the other
variant was negative (Fig.
1).
The
variant that failed to show viral DNA sequences (A-6241) is
considered
to be of spontaneous origin or possibly a virus-induced
tumor that
subsequently lost the viral DNA. Sequencing of the
PCR product from the
DNA-positive tumor A-6689 showed a wild-type
middle T sequence,
including an unaltered epitope. Immunoprecipitation
of tumor cell
extracts showed that A-6215 and A-6689 were both
positive and A-6241
was negative for middle T protein expression
(data not shown). These
results demonstrate that A-6689 did not
escape recognition due to a
failure to express middle T or to
antigenic variation.
View larger version (62K):
[in this window]
[in a new window]
|
FIG. 1.
Test for polyomavirus DNA sequences in tumors from BR
mice. A-6215 is an immunogenic tumor from an irradiated virus-infected
mouse, A-6241 is a nonimmunogenic tumor from an unirradiated
virus-infected mouse, and A-6689 is another nonimmunogenic tumor from
an unirradiated virus-infected mouse. On the left are molecular size
markers. The arrow on the right indicates the position of the expected
542-bp DNA product. PCR was carried out for polyomavirus middle
T-coding sequences. A PCR control is shown at the bottom of the
figure.
|
|
Expression of MHC class I products by each of these tumor-derived cell
lines was measured (Table
1). A-6215 and
A-6241 showed
moderate to high levels either with or without gamma
interferon
(IFN-
) pretreatment. The viral DNA-positive variant
A-6689, however,
showed a nearly complete absence of expression without
IFN-
,
although class I expression could be induced to high levels
after
the treatment. This finding raises the possibility that A-6689
escaped immune recognition by virtue of a failure to express class
I
adequately.
As a tool for further studies, we isolated and characterized a
virus-specific CTL line from polyomavirus-immunized BR mice
(see
Materials and Methods). LN-13.1 cells were greater than 98%
CD8
+ V
6
+. The results in Table
2 show that LN-13.1 killed A-6215 tumor
cells and that killing could be prevented by pretreatment of the
cells
with anti-
H-2Dk but not
anti-
H-2Kk MAb. In addition, pulsing of normal
adherent spleen cells from
BR mice with the
H-2Dk immunodominant middle T peptide R389
sensitized the cells to
killing by LN-13.1, while the control middle T
peptide E328 (with
a sequence bearing an
H-2Kk
epitope) had no effect (Table
2). LN-13.1 cells thus resemble
the
virus-specific CTLs previously described for this system (
28,
29).
When tested for their susceptibility to killing by LN-13.1, only the
immunogenic control tumor A-6215 was killed in the absence
of added IFN
(Table
3). Neither the spontaneous viral
DNA-negative
tumor A-6241 nor the escape variant A-6689 expressing
wild-type
middle T was killed under these conditions. However, when
pretreated
with IFN-
, A-6689 became susceptible, a result consistent
with
evasion based on low constitutive expression of class I. As
expected,
exposure of A-6241 to IFN-
had no effect on its
resistance, although
these cells could be sensitized to killing by
exposure to the
R389 peptide.
Dominant susceptibility to polyomavirus tumors in PE and CZ
mice.
PE and CZ mice are highly susceptible to tumor induction by
polyomavirus, with 97 to 100% of the animals developing multiple tumors by 3 to 4 months of age (Table 4).
Neither of these strains develops spontaneous tumors in the relatively
short time period of studies with the virus. With respect to the range
and histological features of tumor types and the time required for
tumor development, these wild-derived inbred mice resemble strain
C3H/BiDa and other standard inbreds (CBA/J, AKR, RF/J, and C58), which
possess an H-2k/Mtv-7 basis of susceptibility
(10, 16, 28). Also similar to the standard inbreds, PE and
CZ mice transmit their susceptibility in a dominant fashion when
crossed with BR mice. Results with F1 mice were independent
of the mother-father pairing of the parental strains, indicating an
autosomal basis of susceptibility.
In backcross mice, 53% [(PE × BR) × BR] and 37%
[(CZ × BR) × BR] developed at least single, and most
often multiple, tumors
(Table
4). These frequencies are consistent with
a single dominant
susceptibility gene in PE mice and one or two genes
in CZ mice.
Susceptibility is also reflected in the average number of
tumors
per affected animal, referred to as the tumor frequency index
(TFI). In the PE cross, the TFIs for parental, F
1, and
backcross
mice were 4.4, 2.6, and 1.3, respectively. For the CZ cross,
the
TFIs were 3.6, 2.3, and 1.7, respectively. The drop in TFI between
parental and F
1 mice indicates codominance with a clear
dosage
effect of the susceptibility gene(s). The further drop in TFI
in
the affected backcross animals suggests possible additive or
interactive gene effects. The latencies of tumor development are
reflected in the average ages at which tumor-bearing animals come
to
necropsy. In both crosses, latencies were found to increase
from an
average of 68 days (parental mice) to 95 days (F
1s) to
112 days (affected backcross animals). Although "age at necropsy"
is a
crude estimate of tumor latency, the trend of increasing
time for tumor
development with dilution of the genetic contribution
from the PE and
CZ parental backgrounds is consistent with gene
dosage and possible
gene interaction. A similar pattern of inheritance
with decreasing TFIs
and increasing latencies in F
1 and backcross
animals was
observed previously in crosses with mice carrying
Mtv-7 as
the dominant susceptibility factor (
28).
Absence of endogenous superantigens in PE and CZ mice.
It was
important in the present study to establish whether either PE or CZ
mice carried endogenous MMTV sag sequences or showed any evidence of
V deletion that might account for their susceptibility. CZ mice have
previously been screened for endogenous MMTV sequences by Southern
hybridization and found to be negative, although MMTV-related sequences
were detected under low-stringency conditions (7). A
milk-borne MMTV was transmitted in CZ mice originally (18) but was no longer carried by CZ mice used in this study. PE mice have
not been characterized for endogenous MMTVs to the best of our knowledge.
PCR was carried out on genomic DNAs by using primers specific for MMTV
sag sequences (Fig.
2). Two primer pairs
were used.
The first was designed to amplify a 319-bp fragment specific
to
the carboxy terminus of
Mtv-7 sag based on published
sequences
(
5,
35). The other pair was designed to amplify a
1,018-bp
fragment by using a forward primer complementary to a
conserved
or common region among MMTV sags and a reverse primer outside
the coding region in the 3' long terminal repeat (
21). PE
and
CZ mice proved to be negative with both sets of probes. The
standard
susceptible C3H/BiDa strain used as a positive control gave
products
of the expected size with both probes. BR mice were positive
for
the conserved region probe and negative for the
Mtv-7-specific
probe, as expected. These results indicate
that neither PE nor
CZ mice carry detectable
Mtv-7 sag or
other MMTV sag sequences.
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 2.
Tests for Mtv-7 Sag and other MMTV sag
sequences in genomic DNAs of various mouse strains. (Left) PCR product
for Mtv-7 sag-specific sequences. Lanes (left to right):
C3H/BiDa, BR, PE, CZ, and markers. The arrow indicates the position of
the predicted 319-bp product. (Right) PCR product for common MMTV sag
sequences. Lanes (left to right): markers, C3H/BiDa, BR, PE, and CZ.
The arrow indicates the position of the predicted 1,018-bp product. A
PCR control is shown at the bottom of the figure. PCR was carried out
as described in Materials and Methods.
|
|
Splenic T cells from parental and F
1 mice were examined for
V
gene expression by fluorescence-activated cell sorter (FACS)
analysis by using a series of V
-specific antibodies (Table
5).
BR mice express a limited repertoire
of V
genes and are therefore
useful as a "sag indicator" strain
in genetic crosses with strains
of uncertain status with respect to
endogenous MMTVs. BR mice
carry a large genomic deletion of V
genes,
including V
genes
5, 8, 9, 11, 12, and 13 (
3). They also
carry several endogenous
Mtvs which cause somatic deletion of V
genes 5, 11, 12, 16, and
17
a (
13,
22,
37). All
V
genes expressed by BR mice were found
to be expressed by PE and CZ
mice, with BR mice expressing most
types at higher percentages, a
finding consistent with its more
limited repertoire. The percentages in
the F
1s were intermediate
and close to the average of the
parental values, indicating the
absence of deleting elements introduced
by either the PE or CZ
parents. This is particularly clear for V
6,
which is expected
to be of critical importance in this virus-host
system. Direct
measurement of CD8
+ V
6
+ cells
showed these to be present at 4 to 5% in F
1 mice, compared
to 2% in PE and CZ mice and 7% in BR mice. The results shown in
Table
5 confirm the absence of V
5 and V
8 in T cells from BR
mice and
their presence in PE and CZ mice. In the F
1s with BR
mice,
V
5 is absent due to somatic deletion, while V
8 is present
as
expected. Of the remaining V
genes expressed by BR mice, only
two
(V
1 and -15) could not be examined due to the absence of
available
antibody reagents. These data show that neither PE nor
CZ mice carry
endogenous sags encoded by MMTVs or indeed by any
other agent(s).
MHC typing of PE and CZ mice.
To determine whether either PE
or CZ mice might share the H-2k haplotype common
to highly susceptible standard inbreds, splenic leukocytes were
analyzed by FACS with MAbs specific for H-2Dk
and H-2Kk class I molecules (Fig.
3). Leukocytes from PE mice stained well with both antibodies, with results resembling the positive control from
BR mice. Those from CZ mice were clearly negative. Similar results were
found using antibody specific for class II (anti-IEk [data
not shown]).
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 3.
FACS analysis of H-2 haplotype in
wild-derived mouse strains. Splenocytes from BR, PE, and CZ mice were
stained with the FITC-labeled anti-MHC class I
(H-2k) MAb. A monoclonal mouse IgG1 was used as
a negative control. Spleen cells of BR mice served as a positive
control. The analysis was performed by using a flow cytometer after
gating on the viable cell population as determined by forward- and
side-scatter analysis. Positive staining and fluorescence intensity
(log scale) for 10,000 events are shown. The histograms represent one
of three experiments. Similar results were obtained by using anti-class
II (H-2k) MAb. The levels of surface positive
cells were <1% with anti-H-2b class I MAb
compared to >90% with anti-H-2k MAb (BR and PE
mice).
|
|
Mixed lymphocyte reactions were also carried out as a functional test
of histocompatibility (Table
6). In
one-way tests,
splenocytes from PE mice were unable to stimulate BR
mice while
those splenocytes from CZ and B6 mice, as a known
MHC-incompatible
strain (
H-2b), were both
positive. These data indicate that PE mice carry
the
H-2k haplotype and that CZ mice do not.
Antigen processing and presentation by PE and CZ mice.
Adherent cells from spleens of parental and F1 mice were
pulsed with middle T-derived peptides and incubated with LN-13.1 virus-specific CTLs (Table 7). The R389
peptide sensitized normal cells from PE, PE × BR, and CZ × BR mice to
killing by LN-13.1 but did not sensitize those from CZ mice. The E328
peptide had no effect. Similar results were obtained by pulsing
peritoneal exudate cells with peptides (data not shown). These results
are in line with those of MHC typing (Fig. 3 and Table 6) and
demonstrate that killing is restricted to cells expressing
H-2k. They also show that normal cells from
tumor-susceptible F1 mice are able to present the
immunodominant viral peptide on H-2Dk class I
molecules.
To test directly for a possible defect in antigen processing, adherent
spleen cells from parental and F
1 mice were infected
by
polyomavirus in vitro and incubated with LN-13.1 cells (Table
8). Infected target cells from BR, PE, PE × BR, and CZ × BR mice
were killed, while those from CZ mice were
not, a finding consistent
with the known pattern of MHC expression.
Tumor-derived cells
were also used as targets and found to follow the
same pattern.
Cells derived from a variety of different tumors (mammary
gland,
salivary gland, kidney, and fibrosarcoma) were tested; killing
was MHC restricted but independent of the tissue origin or type
of
tumor. These results, achieved with both normal cells infected
in vitro
and cells derived from primary tumors as targets, establish
that
neither PE nor CZ mice (the latter deduced from results with
CZ × BR
mice) are defective in antigen processing and presentation
with respect
to the immunodominant viral peptide.
| |
DISCUSSION |
As a general rule, inheritance of immune responsiveness to a
defined antigen is expected to show dominance over immune
nonresponsiveness. With respect to polyomavirus-specific
transplantation antigen(s), host resistance based on an effective
immune response to virus-induced tumors should be dominant over
susceptibility. An exception to this rule was first noted in standard
inbred mouse strains where an endogenous superantigen gives rise to
dominant nonresponsiveness by preventing the generation of polyomavirus
tumor immunity (28, 30).
Here we describe a distinctly different genetic and immunological basis
of susceptibility in two unrelated strains of wild-derived inbred mice.
These mice are highly susceptible to tumor induction by polyomavirus
but show no evidence of endogenous sags. In crosses with
immunologically resistant BR mice, the wild-derived inbreds transmit
their susceptibility in a dominant manner. We conclude that these mice
have a sag-independent mechanism(s) operating to suppress the
generation of polyomavirus tumor immunity.
No gross features indicative of immunological abnormality
(splenomegaly, lymphadenopathy, or absence of thymic involution) in
either PE or CZ mice have been observed by us, and none have been
reported by others. A defect(s) in CTL effector functions (e.g.,
granzyme or perforase) could in principle explain the failure of PE and
CZ mice to eliminate viral tumors. A simple absence of these functions,
however, would not be transmitted as a dominant susceptible trait.
Several additional observations rule against a defect at the level of
CTL-tumor cell interaction in these mice. Tumor cells derived from PE,
PE × BR, and CZ × BR F1 mice (but not from CZ mice which
are non-H-2k) are susceptible to killing by
virus-specific CTLs from BR mice. Normal cells from PE, PE × BR, and
CZ × BR mice that are either infected in vitro by polyomavirus or
incubated with specific viral peptide also become susceptible targets
for these CTLs. Tumor cells derived from these mice thus express normal
costimulatory functions and are not intrinsically resistant to T cell
killing. Processing and presentation of viral antigen also appear to
proceed normally. An absence of appropriate CTL precursors is ruled out since PE and CZ mice and their F1 progeny all show normal
levels of CD8+ T cells bearing V6.
A mechanism leading to CTL tolerance to viral antigen could account for
the dominant susceptibility of PE and CZ mice. This could arise in
principle by molecular mimicry in which a self-antigen effectively
tolerizes the host against the virus, although the likelihood is remote
that this would arise in two unrelated mouse strains. Alternatively,
tolerance to viral antigen could be induced by cross-presentation
(8, 23, 34), aided by virus-induced cytolysis and the high
viral antigen load present in neonatally infected mice. Virus-specific
CTLs without effector function can arise in chronically infected hosts
under certain conditions (39). Other mechanisms not based on
tolerance or nonresponsiveness are also possible. For example,
expression of Fas ligand by tumor cells can lead to induction of
apoptosis in tumor infiltrating lymphocytes (TIL) and thus to immune
escape (20, 32, 33). Although not apparent in our assays of
tumor cell killing in vitro, Fas ligand expression in vivo by tumors in
PE and CZ mice, or other mechanisms leading to TIL dysfunction
(12), may occur. Further experiments employing immunological
as well as genetic approaches to map and identify the gene(s) should
help to clarify the basis of susceptibility to virus-induced tumors in
these mice.
| |
ACKNOWLEDGMENTS |
P.V. and I.Y. have contributed equally to this work.
We thank Martin Dorf for providing antibody reagents, for the use of a
FACS, and for helpful discussions during preparation of the manuscript.
This work has been supported by a grant from the National Cancer
Institute (R35 CA44343).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pathology, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-1960. Fax: (617) 277-5291. E-mail:
Thomas_Benjamin{at}hms.harvard.edu.
| |
REFERENCES |
| 1.
|
Allison, A. C.,
J. N. Monga, and V. Hammond.
1974.
Increased susceptibility to virus oncogenesis of congenitally thymus-deprived nude mice.
Nature (London)
252:746-747[Medline].
|
| 2.
|
Bauer, P. H.,
C. Cui,
T. Stehle,
S. C. Harrison,
J. A. DeCaprio, and T. L. Benjamin.
1999.
Discrimination between sialic acid-containing receptors and pseudoreceptors regulates polyomavirus spread in the mouse.
J. Virol.
73:5826-5832[Abstract/Free Full Text].
|
| 3.
|
Behlke, M. A.,
H. S. Chou,
K. Huppi, and D. Y. Loh.
1986.
Murine T-cell receptor mutants with deletions of -chain variable region genes.
Proc. Natl. Acad. Sci. USA
83:767-771[Abstract/Free Full Text].
|
| 4.
|
Berke, Z.,
T. Wen,
G. Klein, and T. Dalianis.
1996.
Polyoma tumor development in neonatally polyoma-virus-infected CD4 / and CD8/ single knockout and CD4/8/ double knockout mice.
Int. J. Cancer
67:405-408[Medline].
|
| 5.
|
Beutner, U.,
W. N. Frankel,
M. S. Cote,
J. M. Coffin, and B. T. Huber.
1992.
Mls-1 is encoded by the long terminal repeat open reading frame of the mouse mammary tumor provirus Mtv-7.
Proc. Natl. Acad. Sci. USA
89:5432-5436[Abstract/Free Full Text].
|
| 6.
|
Bronson, R.,
C. Dawe,
J. Carroll, and T. Benjamin.
1997.
Tumor induction by a transformation-defective polyoma virus mutant blocked in signaling through Shc.
Proc. Natl. Acad. Sci. USA
94:7954-7958[Abstract/Free Full Text].
|
| 7.
|
Callahan, R.,
W. Drohan,
D. Gallahan,
L. D'Hoostelaere, and M. Potter.
1982.
Novel class of mouse mammary tumor virus-related DNA sequences found in all species of Mus, including mice lacking the virus proviral genome.
Proc. Natl. Acad. Sci. USA
79:4113-4117[Abstract/Free Full Text].
|
| 8.
|
Carbone, F. R.,
C. Kurts,
S. R. M. Bennett,
J. F. A. P. Miller, and W. R. Heath.
1998.
Cross-presentation: a general mechanism for CTL immunity and tolerance.
Immunol. Today
19:368-373[Medline].
|
| 9.
|
Carroll, J. P.,
J. S. Fung,
R. T. Bronson,
E. Razvi, and T. L. Benjamin.
1999.
Radiation-resistant and radiation-sensitive forms of host resistance to polyomavirus.
J. Virol.
73:1213-1218[Abstract/Free Full Text].
|
| 10.
|
Dawe, C. J.,
R. Freund,
G. Mandel,
K. Balmer-Hofer,
D. A. Talmage, and T. L. Benjamin.
1987.
Variations in polyoma virus genotype in relation to tumor induction in mice: characterization of wild type strains with widely differing tumor profiles.
Am. J. Pathol.
127:243-261[Abstract].
|
| 11.
|
Eddy, B. E.
1969.
Polyoma virus, p. 1-114.
In
S. Gard, C. Hallawer, and K. F. Meyer (ed.), Virology monograph 7. Springer-Verlag, New York, N.Y
|
| 12.
|
Finke, J.,
S. Ferrone,
A. Frey,
A. Mufson, and A. Ochoa.
1999.
Where have all the T cells gone? Mechanisms of immune evasion by tumors.
Immunol. Today
20:158-160[Medline].
|
| 13.
|
Frankel, W. N.,
C. Rudy,
J. M. Coffin, and B. T. Huber.
1991.
Linkage of Mls genes to endogenous mammary tumour viruses of inbred mice.
Nature (London)
349:526-528[Medline].
|
| 14.
|
Freund, R.,
A. Calderone,
C. J. Dawe, and T. L. Benjamin.
1991.
Polyomavirus tumor induction in mice: effects of polymorphisms of VP1 and large T antigen.
J. Virol.
65:335-341[Abstract/Free Full Text].
|
| 15.
|
Freund, R.,
C. J. Dawe, and T. L. Benjamin.
1988.
Duplication of noncoding sequences in polyomavirus is required for the development of thymic tumors in mice.
J. Virol.
62:3896-3899[Abstract/Free Full Text].
|
| 16.
|
Freund, R.,
C. J. Dawe,
J. P. Carroll, and T. L. Benjamin.
1992.
Changes in frequency, morphology and behavior of tumors induced in mice by a polyoma virus mutant with a specifically altered oncogene.
Am. J. Pathol.
141:1409-1425[Abstract].
|
| 17.
|
Freund, R.,
T. Dubensky,
R. Bronson,
A. Sotnikov,
J. Carroll, and T. Benjamin.
1992.
Polyoma tumorigenesis in mice: evidence for dominant resistance and dominant susceptibility genes of the host.
Virology
191:724-731[Medline].
|
| 18.
|
Gallahan, D., and R. Callahan.
1987.
Mammary tumorigenesis in feral mice: identification of a new int locus in mouse mammary tumor virus (Czech II)-induced mammary tumors.
J. Virol.
61:66-74[Abstract/Free Full Text].
|
| 19.
|
Gross, L. G.
1983.
Oncogenic viruses, 3rd ed., vol. 2. , p. 737-828.
Pergamon Press, Inc., Oxford, England
|
| 20.
|
Hahne, M.,
D. Rimoldi,
M. Schroter,
P. Romero,
M. Schreier,
L. E. French,
P. Schneider,
T. Bornand,
A. Fontana,
D. Lienard,
J.-C. Cerottini, and J. Tschopp.
1996.
Melanoma cell expression of Fas (Apo-1/CD95) ligand: implications for tumor immune escape.
Science
274:1363-1366[Abstract/Free Full Text].
|
| 21.
|
Ignatowicz, L.,
J. W. Kappler,
P. Marrack, and M. T. Scherer.
1994.
Identification of two V7-specific viral superantigens.
J. Immunol.
152:65-71[Abstract].
|
| 22.
|
Kozak, C.,
G. Peters,
R. Pauley,
V. Morris,
R. Michalides,
J. Dudley,
M. Green,
M. Davisson,
O. Prakash,
A. Vaidya,
J. Hilgers,
A. Verstraeten,
N. Hynes,
H. Diggelmann,
D. Peterson,
J. C. Cohen,
C. Dickson,
N. Sarkar,
R. Nusse,
H. Varmus, and R. Callahan.
1987.
A standardized nomenclature for endogenous mouse mammary tumor viruses.
J. Virol.
61:1651-1654[Abstract/Free Full Text].
|
| 23.
|
Kurts, C.,
J. F. A. P. Miller,
R. M. Subramaniam,
F. R. Carbone, and W. R. Heath.
1998.
Major histocompatibility complex class I-restricted cross-presentation is biased towards high dose antigens and those released during cellular destruction.
J. Exp. Med.
188:409-414[Abstract/Free Full Text].
|
| 24.
|
Laird, P. W.,
A. Zijderveld,
K. Linders,
M. Rudnicki,
R. Jaenisch, and A. Berns.
1991.
Simplified mammalian DNA isolation procedure.
Nucleic Acids Res.
19:4293[Free Full Text].
|
| 25.
|
Law, L. W.
1966.
Immunologic responsiveness and the induction of experimental neoplasms.
Cancer Res.
26:1121-1132[Abstract/Free Full Text].
|
| 26.
|
Law, L. W., and C. J. Dawe.
1960.
Influence of total body X-irradiation on tumor induction by parotid tumor agent in adult mice.
Proc. Soc. Exp. Biol. Med.
105:414-419.
|
| 27.
|
Law, L. W.,
R. C. Ting, and E. Leckband.
1967.
Prevention of virus-induced neoplasms in mice through passive transfer of immunity by sensitized syngeneic lymphoid cells.
Proc. Natl. Acad. Sci. USA
57:1068-1075[Free Full Text].
|
| 28.
|
Lukacher, A. E.,
Y. Ma,
J. P. Carroll,
S. R. Abromson-Leeman,
J. C. Laning,
M. E. Dorf, and T. L. Benjamin.
1995.
Susceptibility to tumors induced by polyoma virus is conferred by an endogenous mouse mammary tumor virus superantigen.
J. Exp. Med.
181:1683-1692[Abstract/Free Full Text].
|
| 29.
|
Lukacher, A. E., and C. S. Wilson.
1998.
Resistance to polyoma virus-induced tumors correlates with CTL recognition of an immunodominant H-2Dk-restricted epitope in the middle T protein.
J. Immunol.
160:1724-1734[Abstract/Free Full Text].
|
| 30.
|
Lukacher, A. E.,
R. Freund,
J. P. Carroll,
R. T. Bronson, and T. L. Benjamin.
1993.
Pyvs: a dominantly acting gene in C3H/BiDa mice conferring susceptibility to tumor induction by polyoma virus.
Virology
196:241-248[Medline].
|
| 31.
|
Necker, A.,
N. Rebai,
M. Matthes,
E. Jouvin-Marche,
P. A. Cazenave,
P. Swarnworawong,
E. Palmer,
H. R. MacDonald, and B. Malissen.
1991.
Monoclonal antibodies raised against engineered soluble mouse T cell receptors and specific for V 8-, V 2- or V10-bearing T cells.
Eur. J. Immunol.
21:3035-3040[Medline].
|
| 32.
|
O'Connell, J.,
M. W. Bennet,
G. C. O'Sullivan,
J. K. Collins, and F. Shanahan.
1999.
The fas counterattack: cancer as a site of immune privilege.
Immunol. Today
20:46-52[Medline].
|
| 33.
|
O'Connell, J.,
G. C. O'Sullivan,
J. K. Collins, and F. Shanahan.
1996.
The fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing fas ligand.
J. Exp. Med.
184:1075-1082[Abstract/Free Full Text].
|
| 34.
|
Rolph, M. S.,
K. I. Matthaei,
F. R. Carbone,
W. R. Heath, and I. A. Ramshaw.
1998.
Loss of antiviral cytotoxic T-lymphocyte activity during high level antigen stimulation.
Viral Immunol.
11:183-195[Medline].
|
| 35.
|
Rudy, C. K.,
E. Kraus,
E. Palmer, and B. T. Huber.
1992.
Mls-1-like superantigen in the MA/MyJ mouse is encoded by a new mammary tumor provirus that is distinct from Mtv-7.
J. Exp. Med.
175:1613-1621[Abstract/Free Full Text].
|
| 36.
|
Sahli, R.,
R. Freund,
T. Dubensky,
R. Garcea,
R. Bronson, and T. Benjamin.
1993.
Defect in entry and altered pathogenicity of a polyoma virus mutant blocked in VP2 myristylation.
Virology
192:142-143[Medline].
|
| 37.
|
Sherer, M. T.,
L. Ignatowicz,
G. M. Winslow,
J. W. Kappler, and P. Marrack.
1993.
Superantigens: bacterial and viral proteins that manipulate the immune system.
Annu. Rev. Cell Biol.
9:101-128.
|
| 38.
|
Talmage, D. A.,
R. Freund,
A. T. Young,
J. Dahl,
C. J. Dawe, and T. L. Benjamin.
1989.
Phosphorylation of middle T by pp60c-src: a switch for binding of phosphatidylinositol 3-kinase and optimal tumorigenesis.
Cell
59:55-65[Medline].
|
| 39.
|
Zajac, A. J.,
J. N. Blattman,
K. Murali-Krishna,
D. J. D. Sourdive,
M. Suresh,
J. D. Altman, and R. Ahmed.
1998.
Viral immune evasion due to persistence of activated T cells without effector function.
J. Exp. Med.
188:2205-2213[Abstract/Free Full Text].
|
Journal of Virology, December 1999, p. 10079-10085, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Velupillai, P., Garcea, R. L., Benjamin, T. L.
(2006). Polyoma Virus-Like Particles Elicit Polarized Cytokine Responses in APCs from Tumor-Susceptible and -Resistant Mice. J. Immunol.
176: 1148-1153
[Abstract]
[Full Text]
-
Velupillai, P., Carroll, J. P., Benjamin, T. L.
(2002). Susceptibility to Polyomavirus-Induced Tumors in Inbred Mice: Role of Innate Immune Responses. J. Virol.
76: 9657-9663
[Abstract]
[Full Text]
-
Li, D., Dower, K., Ma, Y., Tian, Y., Benjamin, T. L.
(2001). A tumor host range selection procedure identifies p150sal2 as a target of polyoma virus large T antigen. Proc. Natl. Acad. Sci. USA
98: 14619-14624
[Abstract]
[Full Text]
-
Moser, J. M., Altman, J. D., Lukacher, A. E.
(2001). Antiviral Cd8+ T Cell Responses in Neonatal Mice: Susceptibility to Polyoma Virus-Induced Tumors Is Associated with Lack of Cytotoxic Function by Viral Antigen-Specific T Cells. JEM
193: 595-606
[Abstract]
[Full Text]
Top
Abstract
Introduction
