Wild-derived inbred mice have a novel basis of susceptibility to polyomavirus-induced tumors.

Abstract

Polyomavirus induces a broad array of tumors when introduced into newborn mice of certain standard inbred strains, notably those bearing the H-2(k) 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 Vbeta deletion. C57BR/cdJ (BR) mice, which are H-2(k) 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 F(1)-derived tumor cells in vitro. The expected precursors of polyomavirus-specific cytotoxic T cells are present in both the wild inbred animals and their F(1) progeny. These findings indicate a novel basis of susceptibility that operates independently of endogenous superantigen and prevents the development of tumor immunity.

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JOURNAL OF VIROLOGY,
0022-538X/99/$04.00?0
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Dec. 1999, p. 10079–10085 Vol. 73, No. 12
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
Polyomavirus induces a broad array of tumors when introduced into newborn mice of certain standard
inbred strains, notably those bearing the H-2khaplotype. 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-2kbut 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 F1progeny. These findings indicate a novel basis of
susceptibility that operates independently of endogenous superantigen and prevents the development of tumor
immunity.
The murine polyomavirus can be a powerful oncogenic
agent in its natural host, as evidenced by the rapid develop-
ment 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 determi-
nants in the viral T (tumor) antigens involved in cell transfor-
mation (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 histocom-
patibility 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 re-
sistance and become susceptible after radiation or neonatal
thymectomy (1, 9, 26). A radiation-resistant or nonimmuno-
logical 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 car-
ried by the susceptible strain (28). C57BR/cdJ (BR) mice, used
as the resistant parent, generate virus-specific cytotoxic T lym-
phocytes (CTLs) with a V? specificity (V?6) that would be
deleted by the Mtv-7 superantigen (sag) present in all highly
susceptible H-2kstrains (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 im-
mune evasion by rare tumors that arise in this resistant strain.
We describe a genetic and immunological basis of susceptibil-
ity 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 suscepti-
bility in a dominant fashion. Tumor cells derived from F1
animals are killed by CTLs from strain BR mice, demonstrat-
ing that these mice are able to process and present the appro-
priate 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?V?6?T cells that
are the expected precursors of polyomavirus-specific CTLs in
this system. These results suggest a novel basis of tumor sus-
ceptibility involving a mechanism that interferes with the de-
velopment 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 ?l of virus containing 2 ? 106to
10 ? 106PFU 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 tu-
mors that arose in normal (unirradiated) virus-infected BR mice (28). Cultures
of tumor cells from PE, CZ, and F1animals 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 ?l of a reaction mixture contain-
ing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2, 100 ?g of gelatin per
* 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
@hms.harvard.edu.
10079

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ml, 200 ?M concentrations of each of the deoxynucleoside triphosphates, and 0.5
?M 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?-TTACATCTACAGACCAAC
AGATGCCCCGT-3?) and reverse primer Mtv-7-1 (5?-GAAGCCAACGCGAC
CCCC-3?) based on published sequences (5, 35). Primers for conserved mouse
mammary tumor virus (MMTV) sag sequences were forward MTV-cons (5?-G
GGAATTCTCGAGATGCCGCGCCTGCAG-3?) and reverse MTV-cons (5?-G
GGGATCCTCTAGAGGGAACCGCAAGGTTGGG-3?) (21). For polyomavi-
rus 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?-AAATGGTGCTGCGGTTA
CAA-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 Mar-
tin E. Dorf, Department of Pathology, Harvard Medical School, Boston, Mass.)
and then with fluorochrome-labeled second antibodies (Jackson Immunore-
search, 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 chlo-
ride lysis of erythrocytes, followed by gravity sedimentation to remove cell
clumps and debris. One million cells were stained with 1 ?g of fluorescein
isothiocyanate (FITC)-conjugated anti-H-2Kkor anti-H-2DkMAbs (Pharmin-
gen, 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 ?g/50 million cells). Responder (2 ? 105cells/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 humid-
ified chamber at 37°C with 7% CO2for 4 days and subsequently pulsed with 1
?Ci 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 ?l of A2 virus containing 108PFU/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 with51Cr-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
wereradiolabeledbytheadditionof200?CiofNa251CrO4(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 free51Cr.
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 ?M 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 then51Cr-labeled as
described above and used as targets at 16 to 20 h postinfection.
The radiolabeled targets (5 ? 103cells/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 ?M
2-mercaptoethanol. After incubation for 4 h at 37°C, the supernatant (100 ?l)
from each well was collected and counted on a gamma counter (Wallac, Inc.,
Gaithersburg, Md.). Spontaneous and total51Cr 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?V?6?T cells (28).
These CTLs have been shown to be H-2Dk-restricted and spe-
cific 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 immuno-
dominant epitope. Three tumors were tested: A-6215, a posi-
tive 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 ex-
press 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 sponta-
neous origin or possibly a virus-induced tumor that subse-
quently lost the viral DNA. Sequencing of the PCR product
from the DNA-positive tumor A-6689 showed a wild-type mid-
dle T sequence, including an unaltered epitope. Immunopre-
cipitation 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 demon-
strate that A-6689 did not escape recognition due to a failure
to express middle T or to antigenic variation.
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.
10080VELUPILLAI ET AL.J. VIROL.

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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 vari-
ant 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-2Dkbut not
anti-H-2KkMAb. In addition, pulsing of normal adherent
spleen cells from BR mice with the H-2Dkimmunodominant
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-2Kkepitope) had no effect (Table 2). LN-13.1
cells thus resemble the virus-specific CTLs previously de-
scribed 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 condi-
tions. However, when pretreated with IFN-?, A-6689 became
susceptible, a result consistent with evasion based on low con-
stitutive 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 stan-
dard 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 suscep-
tibility in a dominant fashion when crossed with BR mice.
Results with F1mice 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%
TABLE 1. MHC class I expression on polyomavirus tumor
cell surface
Cell linea
IFN-?
% Fluorescence-positive cellsb
Anti-Kk
Anti-Dk
A-6215
?
?
?
?
?
?
80
98
88
79
14
81
58
76
A-6241
A-668921
97 59
aPolyomavirus tumor cell lines A-6215, A-6689, and A-6241 were described
previously (28). Cells were treated with either medium alone or IFN-? for 36 h.
bFluorescent staining (FITC-labeled anti-H-2Kkor anti-H-2DkMAb) and
FACS analysis were carried out. Values represent one of three experiments with
similar results. The percent positive cells with mouse IgG1 isotype control MAb
was less than 3%.
TABLE 3. Susceptibility to CTL killing of “immune escape” tumor
cell variants
Tumor cellsa
Treatment
% Specific lysisb
Expt 1Expt 2
A-6215 Medium
IFN-?
Medium
IFN-?
Medium
IFN-?
mT-R389
mT-E328
36
49
37
50
A-668970
4442
A-62413
2
9
0
A-6241
A-6241
3941
79
aTumor cells were treated with either medium alone or IFN-? (100 U/ml) for
36 h. A-6241 cells (without IFN-? treatment) were pulsed for 1 h at 37°C with
either R389 (RRLGRTLLL) or E328 (EEQVPQLI) peptide (1 ?M) derived
from middle-T (mT) antigen.
bThe CTL assay was carried out with the LN-13.1 CTL line at a 5:1 effector/
target ratio. The percent specific lysis was calculated.
TABLE 4. Inheritance of tumor susceptibility of wild inbred
mouse strainsa
Mouse strain
Fraction of mice
with tumor(s) (%)
Parental
PE ..................................................................................... 41/42 (97)
CZ..................................................................................... 30/30 (100)
BR..................................................................................... 2/47 (4)b
F1
(PE ? BR)F1................................................................... 36/40 (90)
(CZ ? BR)F1.................................................................. 28/30 (93)
Backcross
(PE ? BR) ? BR........................................................... 64/121 (53)
(CZ ? BR) ? BR.......................................................... 26/70 (37)
aNewborn mice were inoculated intraperitoneally with either the A2 or A2?
strain of polyomavirus. These two viral strains differ with respect to a 40-bp
duplication in the noncoding region which promotes gross thymic tumor devel-
opment (15). No differences were noted among susceptible mice in terms of their
responses to the A2 or A2?strain, apart from those expected for thymic tumor
development. Mice were monitored for development of all tumor types, and the
data in each group for the two viruses were pooled.
bData from Lukacher et al. (28).
TABLE 2. Specificities of LN-13.1 CTL cell line
Type of specificity
and antibody or
peptide used
% Specific lysis
MHC restriction specificitya
mIgG...................................................................................
Anti-H-2Kk........................................................................
Anti-H-2Dk........................................................................
35
25
0
Peptide specificityb................................................................
mT-E328.............................................................................
mT-R389............................................................................
11
92
aTumor cells (A-6215) derived from an irradiated, virus-infected BR mouse
were used as targets. These cells were pretreated with either anti-H-2Kkor
anti-H-2DkMAbs (5 ?g/well). A monoclonal mouse Ig6 (mIgG; specific for
TNP) was used as a control.
bPlastic-adherent cells from naı ¨ve BR spleens were pulsed with middle-T
peptides, and the CTL assay was carried out at a 5:1 effector-to-target ratio.
VOL. 73, 1999SUSCEPTIBILITY TO POLYOMAVIRUS TUMORS IN MICE10081

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[(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, F1, and backcross mice were 4.4, 2.6, and 1.3, respec-
tively. For the CZ cross, the TFIs were 3.6, 2.3, and 1.7, re-
spectively. The drop in TFI between parental and F1mice
indicates codominance with a clear dosage effect of the sus-
ceptibility 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 (F1s) to
112 days (affected backcross animals). Although “age at nec-
ropsy” is a crude estimate of tumor latency, the trend of in-
creasing time for tumor development with dilution of the ge-
netic 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 in-
creasing latencies in F1and 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 pub-
lished sequences (5, 35). The other pair was designed to am-
plify a 1,018-bp fragment by using a forward primer comple-
mentary 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.
Splenic T cells from parental and F1mice 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 endog-
enous 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 17a(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 per-
centages, a finding consistent with its more limited repertoire.
The percentages in the F1s were intermediate and close to the
average of the parental values, indicating the absence of de-
leting 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 measure-
ment of CD8?V?6?cells showed these to be present at 4 to
5% in F1mice, 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 F1s 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-2khaplotype common
to highly susceptible standard inbreds, splenic leukocytes were
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.
TABLE 5. T-cell receptor V? representation in splenic T cellsa
TCR antibody
% Fluorescence-positive cells (mean ? SE)
BR PECZ (PE ? BR)F1
(CZ ? BR)F1
??
V?2
V?3
V?4
V?5
V?6
V?7
V?8
V?10
V?14
73 ? 2
19 ? 0
7 ? 0
10 ? 1
0
15 ? 1
8 ? 0
0
0b
11 ? 1
74 ? 3
4 ? 0
3 ? 0
3 ? 0
8 ? 1
5 ? 0
3 ? 0
15 ? 1
3 ? 0
4 ? 1
72 ? 5
4 ? 0
1 ? 0
6 ? 1
3 ? 0
4 ? 0
4 ? 0
17 ? 0
3 ? 0
4 ? 0
76 ? 4
11 ? 0
4 ? 1
7 ? 0
0
11 ? 1
4 ? 1
12 ? 1
3 ? 0
7 ? 1
73 ? 5
12 ? 0
3 ? 0
10 ? 1
0
11 ? 2
4 ? 1
13 ? 0
3 ? 0
7 ? 0
aEnriched splenic T cells were screened for the V? T-cell receptor (TCR)
profile by using clonotypic specific MAbs. Positive cells were enumerated in
spleens of different mouse strains as indicated. Fluorochrome-stained cells were
analyzed on a Coulter Profile II flow cytometer. Each datum point represents the
mean ? the standard error of four animals in each group. Zero corresponds to
?0.2% positive cells.
bV?10 shows considerable diversity among inbred strains and the antibody
used here may react poorly with the V?10apresent in BR mice (31).
10082VELUPILLAI ET AL.J. VIROL.

Page 5

analyzed by FACS with MAbs specific for H-2Dkand 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 neg-
ative. Similar results were found using antibody specific for
class II (anti-IEk[data not shown]).
Mixed lymphocyte reactions were also carried out as a func-
tional 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-2khaplotype and that
CZ mice do not.
Antigen processing and presentation by PE and CZ mice.
Adherent cells from spleens of parental and F1mice were
pulsed with middle T-derived peptides and incubated with
LN-13.1 virus-specific CTLs (Table 7). The R389 peptide sen-
sitized 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 F1mice are able to present the immuno-
dominant viral peptide on H-2Dkclass I molecules.
To test directly for a possible defect in antigen processing,
adherent spleen cells from parental and F1mice 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 re-
sults, achieved with both normal cells infected in vitro and cells
derived from primary tumors as targets, establish that neither
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-2bclass I MAb compared to ?90% with anti-H-2kMAb (BR and PE mice).
TABLE 6. Mixed lymphocyte reactionsa
Stimulator
lymphocyte group
[3H]thymidine uptake (cpm)
Expt 1Expt 2
Medium
PE
CZ
BR
B6
2,228
1,052
28,523
950
37,304
2,191
1,776
28,749
931
37,882
aStimulator lymphocytes from spleens of different mouse strains were treated
with mitomycin C. Responder cells were splenocytes from BR mice throughout.
Reactions were performed in 96-well plates containing 4 ? 105stimulator lym-
phocytes plus 2 ? 105responder lymphocytes per well. The cells were incubated
for 4 days and then for an additional 16 h in the presence of 1 ?Ci of [3H]thy-
midine per well. Thymidine incorporation was measured on a beta scintillation
counter. Values are the means of triplicate cultures.
TABLE 7. Killing by CTLs of middle T peptide-pulsed targetsa
Host strain
% Specific lysis of cells pulsed with
peptide:
mT-R389mT-E328
PE
CZ
(PE ? BR)F1
(CZ ? BR)F1
825
0
2
0
0
74
93
aPlastic-adherent cells from naive spleens were prepared from different
mouse strains, pulsed with middle T (mT) peptides, and used as targets in the
CTL assay at a 5:1 effector/target ratio.
VOL. 73, 1999SUSCEPTIBILITY TO POLYOMAVIRUS TUMORS IN MICE 10083