JOURNAL OF VIROLOGY, Feb. 2008, p. 1314–1322
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 3
A Novel Block to Mouse Mammary Tumor Virus Infection of
Lymphocytes in B10.BR Mice?
Chioma M. Okeoma, Ming Shen, and Susan R. Ross*
Department of Microbiology and Abramson Family Cancer Center, University of Pennsylvania School of
Medicine, Philadelphia, Pennsylvania 19104
Received 22 August 2007/Accepted 1 November 2007
Classic studies on C57BL-derived mouse strains showed that they were resistant to mouse mammary tumor
virus (MMTV) infection. Although one form of resistance mapped to the major histocompatibility complex
(MHC) locus, at least one other, unknown gene was implicated in this resistance. We show here that B10.BR
mice, which are derived from C57BL mice but have the same MHC locus (H-2k) as susceptible C3H/HeN mice,
are resistant to MMTV, and show a lack of virus spread in their lymphoid compartments but not their
mammary epithelial cells. Although in vivo virus superantigen (Sag)-mediated activation of T cells was similar
in C3H/HeN and B10.BR mice, T cell-dependent B-cell and dendritic cell activation was diminished in the
latter. Ex vivo, B10.BR T cells showed a diminished capacity to proliferate in response to the MMTV Sag. The
genetic segregation of the resistance phenotype indicated that it maps to a single allele. These data highlight
the role of Sag-dependent T-cell responses in MMTV infection and point to a novel mechanism for the
resistance of mice to retroviral infection that could lead to a better understanding of the interplay between
hosts and pathogens.
Many studies in human and animal populations have shown
a genetic component to susceptibility to viruses. For example,
there are a number of loci in mice, termed Fv, that confer
resistance to infection by murine leukemia virus (14). Similarly,
there are individuals who remain resistant to human immuno-
deficiency virus (HIV) type 1 in spite of multiple exposures;
included in this group are individuals with germ line mutations
in the gene encoding the chemokine receptor that functions as
a coreceptor for HIV type 1 (27). It is clear that determining
the genetic basis for resistance leads to elucidation of the
infection pathway, as well as to the creation of novel treatment
paradigms for viruses and other pathogens.
The mouse is particularly well suited for such genetic anal-
ysis because of the large number of genetically well-character-
ized inbred strains and the ability to generate transgenics and
targeted germ line mutations. Mouse mammary tumor virus
(MMTV), an endemic betaretrovirus found in many mouse
strains, has been used extensively in a large number of genetic
models to dissect its in vivo infection pathway (38). Genetic
crosses performed early in the last century indicated that for
some mouse strains, only females transmitted a trait of high
breast cancer incidence. The classic studies of Bittner showed
that this transmission was not genetic but due to a milk-borne
agent acquired in the first week of life from females with high
mammary tumor incidence (6).
It is now known that there are two mechanisms of MMTV
acquisition, the milk-borne exogenous pathway and the inher-
itance of germ line copies of endogenous virus, termed Mtv
loci. Like other retroviruses, the genome of MMTV includes
gag, pol, and env genes, as well as a recently described rem gene
involved in RNA export (29). In addition, the long terminal
repeat (LTR) of both exogenous and endogenous MMTVs
encodes a superantigen (Sag), a cell surface protein presented
by major histocompatibility complex (MHC) class II proteins
of antigen-presenting cells (APCs), such as B cells and den-
dritic cells (DCs), to CD4-positive (CD4?) T cells bearing
specific T-cell receptor (TCR) V? chains. Sag presentation
causes activation of specific V?-bearing T cells when it is rec-
ognized as foreign and deletion of such T cells when it is
recognized as self (i.e., when expressed by endogenous provi-
ruses or as a transgene) (37). Different proviruses cause the
deletion or stimulation of different classes of V?-bearing T
cells because they encode Sag proteins with different C-termi-
nal amino acid sequences (termed the hypervariable region);
this region of the Sag protein contacts the TCR V? molecule.
MMTV uses this Sag activity to amplify in lymphoid cells.
MMTV first infects APCs in Peyer’s patches, including den-
dritic and B cells (3, 7, 10, 28, 42). The infected APCs then
present Sag to cognate CD4?T cells, causing their stimulation
and subsequent bystander B-cell activation that is dependent
on CD40-CD40L interactions (9). This bystander activation
sets up a reservoir of dividing, infection-competent cells; thus,
Sag-dependent lymphocyte activation is critical for efficient
virus spread (16). Virus infection spreads to other lymphoid
organs, and B, T, and dendritic cells become MMTV infected
(13, 28, 42). T and B cells, as well as DCs, are capable of
producing infectious virus (10, 13), and infected lymphoid cells
are required for virus spread within the mammary gland (18).
Thus, MMTV represents a model system for the study of
milk-borne retroviruses, such as HIV and human T-cell leuke-
mia virus type 1, that initially infect lymphocytes in the gut
mucosa (39, 40, 45).
Though MMTV is endemic in mice, mouse strains vary
greatly in their susceptibilities to MMTV infection, and the
* Corresponding author. Mailing address: Room 313 BRBII/III,
University of Pennsylvania, 421 Curie Blvd., Philadelphia, PA 19104-
6142. Phone: (215) 898-9764. Fax: (215) 573-2028. E-mail: rosss@mail
?Published ahead of print on 14 November 2007.
level of infection ultimately affects both mammary tumor inci-
dence and latency (2, 11). Several mechanisms of resistance
have been identified. They include deletion of Sag-cognate T
cells caused by Mtv loci; in this case, the retention of endoge-
nous sag genes with the same V? specificity as those encoded
by infectious virus greatly diminishes infection because the
mice delete Sag-responsive T cells during the shaping of the
immune repertoire (38). Similarly, C57BL/6 mice and related
strains lack the appropriate MHC class II protein (I-E) re-
quired for Sag presentation, thereby abrogating the in vivo
infection process at an early step (4, 23, 34). Other strains, such
as I/LnJ mice and BALB/c congenic mice, lacking endogenous
Mtv loci are also resistant to MMTV infection (8, 35).
Previous genetic studies mapped one major resistance gene
to the MHC locus in C57BL mice and an additional resistance
locus that could be genetically segregated from the MHC locus
(11, 30). Here, we show that B10.BR mice, which are derived
from C57BL mice but carry the same MHC class II allele
(H-2k) as highly susceptible C3H/HeN mice, are resistant to
MMTV infection. In vivo studies indicated that the block to
MMTV infection was the result of decreased virus spread in
the lymphoid compartment. Although Sag-induced T-cell stim-
ulation was not diminished in B10.BR mice in vivo, subsequent
Sag-dependent APC activation was dramatically reduced in
B10.BR mice compared to C3H/HeN susceptible mice. More-
over, ex vivo B10.BR CD4?T-cell proliferation was signifi-
cantly diminished in response to MMTV Sag. These data sug-
gest a defect in the CD4?T-cell response to Sag that
ultimately leads to diminished infection and mammary tumor-
igenesis in B10.BR mice.
MATERIALS AND METHODS
Mice. C57BL/6, C3H/HeN MMTV-negative (MMTV?), and C3H/HeN
MMTV?mice were purchased from the National Cancer Institute, and B10.BR
H2kH2-T18a/SgSnJ, C58J, and C57BR/cDJ mice were from The Jackson Lab-
oratory. To examine milk-borne transmission in the mice, C3H/HeN MMTV?
females were used as foster mothers. All mice were housed according to the
policies of the University of Pennsylvania.
Detection of integrated exogenous viral DNA by PCR. To detect newly inte-
grated copies of exogenous MMTV(C3H), splenic and thymic DNAs were am-
plified by semiquantitative PCR using LTR-specific primers, as previously de-
scribed (18). These primers also amplify some endogenous MMTVs. To
distinguish endogenous from exogenous MMTV sequences, each PCR amplifi-
cation reaction mixture was incubated with MfeI restriction enzyme (New En-
gland Biolabs, Beverly, MA), as indicated in the figure legends, and the resulting
products were analyzed on 1.5% agarose gels.
Detection of integrated exogenous viral DNA by RT-qPCR. Levels of inte-
grated MMTV(LA) DNA in infected mouse tissues were determined by Sybr
green real-time quantitative PCR (RT-qPCR) performed with primers specific to
the MMTV(LA) LTR and to a single-copy mouse glyceraldehyde-phosphate-3-
dehydrogenase (GAPDH) gene. Reactions were performed in triplicate using
Sybr green 1 master mix and run on an ABI Prism model 7900HT, as previously
described (32). Data are presented as relative levels of MMTV normalized to the
single-copy GAPDH gene.
MMTV-XC cell injection. Three- to 4-week-old female mice were injected with
107XC cells expressing the MMTV hybrid provirus (HP) construct, a gift from
Jaquelin Dudley, as described by Shackleford and Varmus (41). All injected
females were bred, and RNA extracted from milk at their first pregnancy was
subjected to RNase protection analysis.
RNase protection assay. RNase T1 protection assays were performed as pre-
viously described using a probe specific for MMTV (C3H) viral transcripts (19).
Forty micrograms of total RNA isolated from the lactating mammary glands and
5 ?g of RNA isolated from the milk were used. Forty micrograms of Saccharo-
myces cerevisiae tRNA was used as a negative control.
Fluorescence-activated cell sorting (FACS). The following monoclonal anti-
bodies (conjugated with phycoerythrin, fluorescein isothiocyanate, or allophyco-
cyanin; BD Bioscience, Inc.) were used: anti-CD71 (C2), anti-CD69 (H1.2F3),
anti-B220 (RA3-6B2), anti-CD4 (RM4-5), anti-CD11c (HL3), anti-CD80 (16-
10A1), anti-CD86 (MR1), anti-CD25 (PC61), and anti-CD40L (7D4). Cells were
acquired on a FACS Calibur cytometer (Becton Dickinson) and analyzed using
CellQuest software (Becton Dickinson Immunocytometry Systems).
Western blots. Sera were obtained from infected and uninfected B10.BR and
C3H/HeN mice, diluted 1:100, and used to probe Western blots of MMTV(LA)
viral particles (1 ?g/lane). Anti-mouse antibody conjugated to horseradish per-
oxidase (Amersham BioSciences) was used as the secondary antibody and was
detected using ECL kits (Amersham BioSciences).
Virus isolation and injection. Virus was purified from tumors, lactating mam-
mary glands, or milk from MMTV(LA)- or MMTV(FM)-infected C3H/HeN
mice, as previously described (19). MMTV(FM) or MMTV(LA) was diluted in
sterile phosphate-buffered saline and injected into the right hind footpads of 1-
to 2-month-old mice. Twenty-four and 96 hours later, the draining (right) and
nondraining (left) popliteal lymph nodes were harvested, and the cells were
analyzed by FACS. Dilutions of purified virus were tested for B-cell and Sag-
mediated T-cell activation in C3H/HeN mice in vivo, and the highest dilution
giving the maximum Sag-dependent stimulation (usually 1:200) was used for
subsequent experiments. All virus preparations were also tested for lipopolysac-
charide contamination, as previously described (7, 10, 36).
Mixed lymphocyte cultures. Total lymphocytes were isolated from the lymph
nodes of naı ¨ve B10.BR and C3H/HeN mice. CD4?T cells were purified using a
CD4?T Cell Isolation Kit (Miltenyi Biotec, Inc.); the purity of the populations
was determined by FACS analysis using anti-CD4 antibodies and was ?96% (not
shown). Unprimed B10.BR or C3H/HeN CD4?T cells (1 ? 106) were cultured
in triplicate with 2 ? 106splenocytes isolated from HYB PRO transgenic mice
(17) in 0.2 ml of RPMI 1640 complete medium (10% heat-inactivated fetal calf
serum, 0.05 mM 2-mercaptoethanol), or 5 ?g/ml concanavalin A (ConA) for the
indicated times. T cells cultured alone or with autologous APCs served as
controls. In some experiments, allogeneic splenocytes from C57BL/6 mice were
also cocultured with lymphocytes from B10.BR and C3H/HeN mice. During the
last 18 h of incubation, the cultures were pulsed with 1.0 ?Ci/well of [3H]thymi-
dine (GE Healthcare, Inc.). The cells and supernatants were harvested, and
thymidine incorporation was quantified.
Statistical analysis. Statistical analysis was performed with a two-sample un-
equal-variance/two-tail distribution t test.
B10.BR mice are resistant to MMTV infection. MMTV is
naturally acquired through milk when neonates nurse on in-
fected mothers. To determine whether B10.BR mice, which
are H-2kand express the same MHC class II proteins as
MMTV-susceptible C3H/HeN mice, were resistant to MMTV
infection, B10.BR pups were foster nursed on C3H/HeN
(MMTV?) mothers from 1 to 2 days after birth until they were
weaned. C3H/HeN pups that had nursed on the same mothers
served as controls. Female foster-nursed offspring of both
strains were tested for infection when they reached adulthood.
The mice were mated and sacrificed after the second preg-
nancy, and RNA isolated from lactating mammary glands and
milk was subjected to RNase protection analysis to determine
the virus load; we had previously shown that this is an accurate
measure of the level of infection (21). B10.BR lactating mam-
mary glands (Fig. 1A) and milk (Fig. 1B) had much lower
levels of MMTV RNA than those of C3H/HeN mice. These
data demonstrated that B10.BR mice have a block to infection
that is MHC independent.
B10.BR mammary tissue is susceptible to infection. As de-
scribed above, milk-borne MMTV infection initiates in lym-
phoid cells, at least in part through the action of its Sag, and
then spreads to the mammary epithelia during puberty and
pregnancy. To determine whether the block to infection in
B10.BR mice was due to a defect in mammary epithelial cell
VOL. 82, 2008GENETIC RESISTANCE TO MMTV1315
infection, we injected 3-week-old B10.BR and C3H/HeN fe-
males with rat XC cells expressing high levels of a molecular
clone of MMTV, HYB PRO, that carries the MMTV(C3H)
sag; this mode of infection is Sag independent (43). Following
infection, the mice were bred, and RNA isolated from their
milk after the first pregnancy was subjected to RNase protec-
tion analysis for viral sequences. The B10.BR and C3H/HeN
mice injected with the MMTV-producing XC cells shed similar
levels of virus in milk (Fig. 2), indicating that there was no
block to infection of mammary epithelial cells. This was in
contrast to mice that were infected by milk-borne transmission
(Fig. 1 and 2). Moreover, examination of virus production after
the second and third pregnancies revealed no differences in
infection (not shown). Thus, the mammary epithelial cells of
B10.BR mice showed no block to direct infection.
B10.BR lymphocytes show lower levels of MMTV infection.
We also examined lymphocyte infection via milk-borne infec-
tion. First, we examined Sag-mediated deletion of V?14 cog-
nate T cells (21) in B10.BR and C3H/HeN mice that were
foster nursed on C3H/HeN MMTV?mothers. Deletion of
these T cells was slightly delayed in B10.BR mice relative to
C3H/HeN mice, although they still showed substantial loss of
this T-cell population, indicating that Sag presentation did
occur (not shown). Next, we directly examined infection of
lymphoid tissues from these mice. DNA was isolated from the
spleens and thymi of age-matched B10.BR and C3H/HeN mice
nursed on the same C3H/HeN MMTV?mothers and was
subjected to PCR analysis for integrated exogenous viral DNA,
as previously described (13). Shown in Fig. 1C is a represen-
tative PCR from one set of mice. In all cases, the B10.BR
lymphoid tissue showed much lower levels of virus infection
than did the C3H/HeN tissue. These data indicated that the
block to infection in B10.BR mice was due to decreased virus
spread in the lymphoid compartment.
B10.BR mice show diminished Sag-dependent APC activa-
tion in vivo. It is well established that efficient infection of
lymphocytes by MMTV requires Sag-dependent T-cell activa-
tion (37). We next investigated whether lymphoid cell re-
sponses were affected in B10.BR mice. MMTV has two phases
of lymphocyte activation. At early times after infection, virus
binds to and activates APCs, at least in part through interac-
tion with toll-like receptor 4 (TLR4) (7, 10, 36). To determine
if initial APC activation occurred in B10.BR mice, we per-
formed subcutaneous injection of either MMTV(LA) or
MMTV(FM) into adult mice and determined whether the
CD69 activation marker was up-regulated on CD11c?DCs
and B220?B cells in the draining lymph node. B-cell and DC
activation in resistant B10.BR mice at 18 h after injection was
similar to that seen in susceptible C3H/HeN mice, indicating
that the TLR4-mediated activation by MMTV was not altered
in B10.BR mice (Fig. 3A and B). In support of this, we also
found that the responses to the TLR4 ligand lipopolysaccha-
FIG. 1. B10.BR mice show lower levels of virus infection in their
mammary and lymphoid tissues and shed less virus in milk than C3H/
HeN mice. (A and B) RNase protection analysis of RNA isolated from
the lactating mammary glands at the second pregnancy (A) and milk at
the first and third pregnancies (B). C3, C3H/HeN; B10, B10.BR; F1,
C3H/HeN ? B10.BR F1females at their second pregnancies; M,
MMTV-specific probe (17); a, mouse ?-actin-specific probe. (C) PCR
analysis of genomic DNAs from the spleens (S) and thymi (T) of
milk-borne MMTV(C3H)-infected B10.BR and C3H/HeN mice to
detect integrated exogenous MMTVs. The primers used amplified
both endogenous and exogenous MMTVs. Following amplification,
the amplicons were digested (?) with MunI, which restricts only the
amplification products of exogenous MMTV (EXO) (13). The endog-
enous band after MunI digestion served as a control for DNA integrity.
FIG. 2. B10.BR mammary glands are susceptible to infection. Vi-
rus RNA was isolated from the milk of B10.BR and C3H/HeN mice
that received mammary gland injections of MMTV-producing XC cells
at 3 weeks of age at the first pregnancy and subjected to RNase
protection analysis using a probe specific for exogenous MMTV (XC).
Shown for comparison is RNase protection analysis of RNA isolated
from the milk from mammary glands of age- and pregnancy-matched
C3H/HeN and B10.BR mice that nursed on MMTV-infected C3H/
HeN mothers (milk).
FIG. 3. Early activation of B cells and DCs is similar in B10.BR and
C3H/HeN mice. B10.BR (filled bars) and C3H/HeN (open bars) mice
received subcutaneous injections of MMTV(FM) in their footpads,
and at 18 h, the lymphocytes from their draining lymph nodes were
analyzed by FACS for CD69 on B220?B cells (A) and CD69 on
CD11c?cells (B). The data presented are the averages of three mice
and are representative of at least 10 independent experiments. The
error bars indicate standard deviations.
1316 OKEOMA ET AL.J. VIROL.
ride were equivalent in B10.BR and C3H/HeN mice (not
After their initial activation, infected APCs present the viral
Sag to cognate CD4?T cells. These T cells in turn provide
costimulation to the APCs, causing their activation and migra-
tion into the lymph node; activation peaks at days 3 and 4 after
inoculation and declines thereafter. To determine whether
(FM), both of which encode Sags that mediate a robust T-cell
response, and examined a number of activation markers on
DCs and B and T cells at 4 and 6 days postinoculation. Sag-
mediated activations of T cells were similar in B10.BR and
C3H/HeN mice, using CD69 (Fig. 4A) or CD40L and CD25
(Table 1) as markers. Moreover, the characteristic MMTV
Sag-mediated increases in V?2-, V?6-, and V?14-bearing
[MMTV(LA)] or V?8.1-bearing [MMTV(FM)] T cells were
similar in the draining lymph nodes of C3H/HeN and B10.BR
mice (not shown). In contrast, activation of B220?B cells was
significantly reduced in B10.BR draining lymph nodes at both
4 and 6 days postinoculation, using CD69 (Fig. 4D) or CD80
and CD86 (Table 1) as the markers. CD69 up-regulation on
CD11c?DCs was also reduced in response to MMTV(LA)
(Fig. 4B), as was the recruitment of CD11c?DCs into the
lymph node (Fig. 4C); similar results were obtained when
MMTV(FM) was injected into C3H/HeN and B10.BR mice
(not shown). These results indicated that Sag-dependent acti-
vation of APCs was diminished in B10.BR mice and that this
diminution was independent of the particular class of V?-
bearing T cells activated by the Sag.
To ensure that the diminished lymphocyte activation in
FIG. 4. Sag-dependent B-cell and DC activation is impaired in B10.BR mice. B10.BR (filled bars) and C3H/HeN (open bars) mice received
subcutaneous injections of MMTV(LA) in their footpads, and after 4 days (A to D) or 6 days (D), the lymphocytes from their draining lymph nodes
were analyzed by FACS for CD69 on CD4?T cells (A), CD69 on CD11c?DCs (B), the increase in the percentage of CD11c?cells in the draining
compared to the nondraining contralateral lymph node (C), and CD69 on B220?B cells (D). A representative FACS plot of cells from the draining
lymph nodes of B10.BR and C3H/HeN mice stained with anti-CD69 and -B220 is also shown. D, draining lymph node; ND, contralateral
nondraining lymph node. The data presented are the averages of three mice and are representative of at least 10 independent experiments with
MMTV(LA) or MMTV(FM). The error bars indicate standard deviations.
TABLE 1. Activation marker expression on CD4?T and B220?
B cells in response to MMTV Saga
Cell type Marker
D NDD ND
29.8 ? 2.9
15.6 ? 2.9
10.0 ? 1.8
7.1 ? 0.6
16.7 ? 2.2
9.2 ? 0.7
9.3 ? 0.6
7.64 ? 0.6
aMice were injected with MMTV(FM), and 4 days later, lymphocytes in the
draining (D) and contralateral nondraining (ND) lymph nodes were examined by
FACS for the different cell surface markers.
bShown are the percentages of total B220?B cells or CD4?T cells that
expressed each marker; n ? 3 mice per group. Nondraining lymph nodes from
the three mice were pooled for analysis.
VOL. 82, 2008 GENETIC RESISTANCE TO MMTV1317
B10.BR mice reflected the level of infection, we also per-
formed RT-qPCR on lymphocytes isolated from mice that
received subcutaneous injections of MMTV(LA), using prim-
ers that specifically detect the exogenous viral sequences. At 4
days postinoculation, little viral DNA was detected in the
draining lymph nodes of either C3H/HeN or B10.BR mice
(Fig. 5). By 6 days postinoculation, the level of infection in
C3H/HeN mice had increased dramatically, while no increase
was seen in B10.BR mice. Thus, the diminished Sag-dependent
APC activation in B10.BR mice was paralleled by a lack of
B10.BR T cells showed diminished T-cell responses ex vivo.
Sag-mediated CD4?T-cell activation occurs when APCs
present this virus protein. The activated T cells then provide
help and interact in turn with additional APCs. The diminished
activation of B10.BR APCs in response to T cells could be due
to cell-intrinsic differences in the ability to respond to signals
from the Sag-activated T cells or to differences in the ability of
B10.BR T cells to provide these signals after Sag activation.
We therefore tested in an ex vivo mixed lymphocyte culture
assay whether CD4?T cells from B10.BR mice responded to
Sag to the same extent as those isolated from C3H/HeN mice.
We used splenocytes from HP transgenic mice expressing the
Sag from MMTV(C3H) as APCs, which we had previously
demonstrated were able to activate Sag-responsive T cells (17).
Sag-mediated induction of the CD69 activation marker on T
cells was similar for both B10.BR and C3H responder cells
(Table 2), as was seen in vivo (Fig. 4A). However, Sag-medi-
ated T-cell proliferation was dramatically reduced with re-
sponders isolated from B10.BR mice in comparison to those
from C3H mice, even after 4 days of coculture (Fig. 6A);
similar results were obtained when purified T cells were used
as responders (Fig. 6B). Additionally, B10.BR T cells showed
a diminished proliferative response to allogeneic APCs (Fig.
6A). This was not due to a general defect in B10.BR T cells,
since their response to the T-cell mitogen ConA was similar to
that seen with C3H T cells (Fig. 6A, B, and C). While C3H/
HeN CD4?T-cell proliferation in response to the MMTV Sag
FIG. 5. In vivo infection of lymphocytes from B10.BR mice is lower
than in those from C3H/HeN mice. B10.BR (filled bars) and C3H/
HeN (open bars) mice received subcutaneous footpad injections of
MMTV(LA), and at 4 and 6 days postinoculation, the lymphocytes
from their draining lymph nodes were analyzed for MMTV(LA) se-
quences by RT-qPCR. MMTV signals were normalized to GAPDH.
D, draining lymph node; ND, contralateral nondraining lymph node.
FIG. 6. B10.BR T cells show lower proliferation than C3H/HeN T
cells in response to MMTV Sag. (A) Responder cells from the lymph
nodes of B10.BR (filled bars) or C3H/HeN (open bars) mice were
cocultured for 4 days alone (?), with mitomycin-treated splenocytes
from MMTV transgenic mice (HP) or C3H/HeN or C57BL/6 mice, or
in the presence of ConA. (B) T cells purified from B10.BR or C3H/
HeN mice were cocultured as in panel A for the indicated times.
(C) Responder cells from the lymph nodes of B10.BR (filled bars) or
C3H/HeN (open bars) mice were cocultured with splenocytes from
mitomycin-treated MMTV transgenic mice (HP) for the indicated
times or with ConA for 2 days. During the last 18 h of culture, the cells
were pulsed with 1.0 ?Ci/well of [3H]thymidine at the indicated times
(days). The error bars indicate standard deviations.
TABLE 2. Ex vivo T-cell activation by MMTV Saga
% CD69?CD4?T cells for activatorb:
NothingConA C3H/HeN HP
7.78 ? 0.48
7.63 ? 0.51
55.36 ? 3.23
65.27 ? 1.19
8.22 ? 1.49
7.64 ? 0.36
69.81 ? 3.19
76.37 ? 4.20
aFour days after coculture of lymph node lymphocytes with the indicated cells
or treatments, the cells were stained for the CD69 activation marker and CD4.
bShown are the percentages of CD69?CD4?T cells of the total population
of CD4?T cells. The cultures were done in triplicate.
1318OKEOMA ET AL.J. VIROL.
increased during up to 6 days of coculture with HP APCs, there
was no change in B10.BR T-cell responses (Fig. 6C).
B10.BR mice do not make increased protective humoral
antibody responses. Previously it was demonstrated that
adult mice immunized with one MMTV strain are resistant
to challenge with a second strain because they make a pro-
tective humoral immune response (26). Moreover, at least
one mouse strain, I/LnJ, is resistant to MMTV because
infected mice make a hyper-humoral immune response to
virus after milk-borne transmission (35). To test if B10.BR
mice made a stronger initial or challenge humoral immune
response to MMTV than C3H/HeN mice, which protected
them from infection, we performed subcutaneous injection
of MMTV(RIII) or MMTV(LA) into naive B10.BR and
C3H/HeN adult mice. Four days after injection, the mice were
sacrificed. A second set of mice received an initial inoculation
with MMTV(RIII) and at day 4 were challenged with
MMTV(LA). At day 12 after the primary infection, the mice
were sacrificed (Fig. 7A).
The sera from these mice were then used to probe Western
blots of MMTV virions to determine if antiviral antibodies
were produced. C3H/HeN, but not B10.BR, mice that received
a single inoculation of either MMTV(LA) or MMTV(RIII)
made anti-TM antibodies (Fig. 7B). When the mice were
challenged with a second injection of virus, both strains
made anti-TM antibodies, although C3H/HeN mice ap-
peared to make a stronger response (Fig. 7C). These results
indicate that resistance to MMTV infection by B10.BR mice
is not the result of greater humoral immune response. In-
deed, these data support previous observations that the hu-
moral immune response to MMTV relies on Sag-mediated
T-cell activation (25).
Genetic analysis of B10.BR resistance. We next analyzed the
segregation patterns of resistance to MMTV infection in F1
and G2backcrosses. B10.BR males were crossed with C3H/
HeN (MMTV?) females, and F1females were generated. The
MMTV(C3H)?F1females were then crossed with B10.BR
males, and mammary gland infections in their female offspring
were determined by RNase protection analysis of virus in milk.
Figure 8A shows a representative analysis of 6 mice out of 151
mice analyzed; 68 G2females showed low levels of infection
FIG. 7. B10.BR mice do not make a more robust humoral immune
response to MMTV. (A) Scheme of virus injections. (B) C3H/HeN and
B10.BR mice (three each) received subcutaneous injections of either
MMTV(LA) or MMTV(RIII). Four days after injection, the mice were
bled and their sera were tested for anti-MMTV antibodies by Western
blot analysis of virion proteins. The blots incubated with C3H/HeN sera
were exposed for ?5 s; the blots incubated with B10.BR sera were ex-
posed for 30 s. As controls, the blots were stripped and incubated with
mouse monoclonal antibodies against the MMTV SU or TM protein; ?,
naı ¨ve mice. (C) C3H/HeN or B10.BR mice were inoculated with
MMTV(RIII). Four days after injection, the mice were inoculated with
MMTV(LA). Eight days later, the mice were bled and their sera were
tested for anti-MMTV antibodies. The arrowheads point to background
bands used to align the blots. The open arrow indicates the TM protein.
FIG. 8. The B10.BR resistance cell phenotype segregates as a single gene. (A) Milk RNAs isolated from G2backcross, C3H/HeN, and B10.BR mice
that had nursed on MMTV?mothers were subjected to RNase protection analysis for exogenous MMTV. Shown above the G2lanes are the presumed
genotypes of the mice (S/s, susceptible; s/s, resistant). Shown are the data for six representative mice. (B) B-cell activation in B10.BR (filled bars), C3H
(open bars), F1(hatched bars), and eight G2backcross mice. The mice received subcutaneous injections of MMTV, and after 4 days, the lymphocytes
from their draining lymph nodes were analyzed by FACS for CD69 on B220?B cells. The data are the averages of three mice each for the C3H/HeN,
F1, and B10.BR samples. The error bars indicate standard deviations. Shown below the line are the presumed genotypes of the G2mice.
VOL. 82, 2008GENETIC RESISTANCE TO MMTV1319
similar to those in B10.BR females, while the remainder
showed high levels, similar to those in C3H/HeN mice. F1
females showed high levels of mammary gland infection, sim-
ilar to that seen with C3H/HeN susceptible mice (Fig. 1A).
These data indicated that resistance to MMTV infection seg-
regated as a recessive trait and were consistent with its map-
ping to a single genetic locus.
To test whether the Sag-dependent activation of APCs also
showed similar segregation, we generated a cohort of 85 un-
infected G2backcrossed mice. B-cell and DC activation levels
for C3H/HeN, B10.BR, F1, and G2mice were compared 4 days
after subcutaneous virus inoculation. We found that F1mice
routinely showed virus-dependent B-cell activation that was
intermediate between those seen in susceptible C3H/HeN and
resistant B10.BR mice (Fig. 8B), indicating that the C3H allele
was semidominant, using lymphocyte activation as the pheno-
type; similar segregation was also seen using DC activation as
the phenotype readout (not shown). When this assay was per-
formed with the G2backcross mice, the mice showed B-cell
and DC (not shown) stimulation similar to that seen with
either the F1or B10.BR mice (shown for eight G2offspring in
Fig. 8B). Of the 85 G2mice analyzed, 41 showed the low
B10.BR B-cell/DC activation phenotype (not shown). This sug-
gests that the lymphocyte activation phenotype also maps to a
Finally, to determine whether the defect in lymphocyte
activation was unique to B10.BR mice or was shared by
other strains derived from a C57 background, we tested
MMTV-mediated B-cell and T-cell activation in C57BR/cdJ
and C58/J mice, both of which are also H-2k. We also tested
lymphocyte activation in B10.BR ? C57BL/6 F1 mice;
C57BL/6 mice, which are H-2b, lack the MHC class II I-E
molecule required for efficient presentation of most MMTV
Sags (34). All the C57-derived strains showed lower levels of
B-cell (Table 3) activation than did C3H/HeN mice; in con-
trast, T-cell activation was similar to that in C3H/HeN mice,
except for C58/J mice, in which it was lower (not shown).
C57BL/6 mice showed no lymphocyte activation, as previ-
ously described (4, 23, 34). Interestingly, the B10.BR ?
C57BL/6 F1mice showed low-level B-cell activation similar
to that seen in B10.BR mice, indicating that the two strains
might contain the same allele responsible for this pheno-
The genetics of susceptibility to MMTV infection has been a
subject of investigation since the 1930s, when Bittner described
a milk-borne transmissible agent that causes breast cancer in
mice (6). Much has been learned about the genes that confer
resistance or susceptibility to infection during the 70 years
following his initial description. For example, early studies had
shown that C57BL and derivative strains were resistant to
tumor induction by most MMTV strains (11). With the discov-
ery of the virus-borne sag, it was recognized that C57BL and
H-2bstrains, which genetically lack the MHC class II I-E gene,
had poor presentation of this antigen to cognate CD4?T cells,
leading to resistance to infection (4, 34). Similarly, mice con-
taining germ line MMTVs with the same Sag as exogenous
MMTV, either as a transgene or as an endogenous provirus,
delete Sag-cognate T cells during the shaping of the immune
repertoire, rendering them resistant to infection by exogenous
MMTVs carrying sag genes with the same T-cell specificity,
and V?8.2 TCR transgenic mice cannot be infected by exoge-
nous MMTVs whose Sag proteins do not bind V?8.2-bearing T
cells (16, 22).
Other mechanisms of resistance to MMTV infection also
occur. For example, I/LnJ mice show wild-type levels of lym-
phocyte infection but little or no transfer of virus to mammary
tissue or to subsequent generations (15). These mice develop
high-titer anti-MMTV antibodies as they age, which coat the
virions and thereby block mammary gland infection and milk-
borne transmission to the next generation (35). It has also
recently been shown that BALB/c congenic mice lacking en-
dogenous Mtv loci are resistant to infection; the mechanism of
this resistance is not yet known (5). These and other genetic
studies have led to an understanding of the pathway of virus
infection and mammary tumor induction in vivo.
Early genetic studies also indicated that C57BL-derived
mice might have resistance alleles in addition to the MHC class
II restriction. The block to infection in this mouse strain ap-
peared to occur prior to mammary gland infection, since
C57BL mammary tissue transplanted into the cleared fat pads
of susceptible hosts developed into tumors with the same ki-
netics and frequency as tissue transplanted from susceptible
mice (12, 31). Indeed, we show here that in B10.BR mice,
which have the same MHC allele as susceptible C3H/HeN
mice, resistance to MMTV infection resides at a step prior to
mammary gland infection. By systematically examining the
steps of the in vivo infection pathway, we were able to dem-
onstrate that B10.BR CD4?T cells were activated by the
MMTV Sag, indicating that initial infection of APCs by
MMTV was not affected. Instead, although B10.BR mice have
APCs with the appropriate MHC class II molecules and have
T cells bearing TCRs capable of interacting with MMTV Sags,
the subsequent Sag-dependent T-cell help to B cells and DCs
was greatly diminished. This was seen at the level of B-cell and
DC activation and in the ability of B10.BR mice to make
anti-MMTV antibodies; it had been previously established that
the anti-MMTV humoral immune response requires Sag-me-
diated T-cell help (25, 26).
Neither Sag-mediated T-cell deletion after milk-borne infec-
tion nor activation of T cells after virus inoculation was signif-
icantly diminished in B10.BR mice. This may be due to the lack
TABLE 3. Lack of B-cell response in B10.BR ? C57BL/6 F1micea
C3H/HeN...............................................................................63.5 ? 6.2
B10.BR...................................................................................37.8 ? 3.8
C57BL/6................................................................................. 6.2 ? 0.9
C57BL/6 ? B10.BR F1........................................................30.9 ? 3.9
aMice were injected with MMTV(LA), and 4 days later, lymphocytes in the
draining lymph nodes were examined by FACS for CD69 and B220 cell surface
bShown are the percentages of CD69?B220?B cells out of the total popu-
lation of B220?B cells; n ? 3 mice for the B10.BR, C3H/HeN, and C57BL/6
mice; n ? 4 for the F1mice; n ? 2 for the C58J and C57BR/cDJ mice.
1320 OKEOMA ET AL.J. VIROL.
of sensitivity of Sag-mediated effects as a readout assay; we
have previously shown that very low levels of Sag expression on
APCs can produce maximum T-cell stimulation (17). Although
their in vivo activation was not reduced, ex vivo proliferation of
B10.BR T cells was defective compared to that of C3H/HeN T
cells, suggesting that it is the T cells themselves, rather than the
B cells and DCs, that are unable to respond appropriately to
the MMTV Sag. Indeed, we also found that the B10.BR T cells
showed a decreased ex vivo allogeneic response compared to
those from C3H/HeN mice. Taken together, these data suggest
that defective Sag-mediated T-cell stimulation in B10.BR mice
accounts for the lack of efficient virus spread in the lymphocyte
compartment and thus results in lower levels of lymphocyte
infection after both experimental inoculation and milk-borne
MMTV infection. However, we cannot rule out the possibility
that B10.BR B cells and DCs are also deficient in the ability to
respond to T-cell help.
Sag proteins typically stimulate up to 20% of all CD4?T
cells in a given mouse by directly binding to both MHC class II
and particular TCR V? chains. In this study, we used four
different MMTV strains, MMTV(C3H) for milk-borne trans-
mission and MMTV(FM), MMTV(RIII), and MMTV(LA) for
experimental inoculation. The different MMTVs encode Sag
proteins with different V? specificities: MMTV(C3H) interacts
with V?14-bearing, MMTV(FM) with V?8.1-bearing, MMTV
(RIII) with V?2-bearing, and MMTV(LA) (a mixture of three
viruses) with V?2-, V?14-, and V?6-bearing T cells (20, 24, 32,
44). We showed that B10.BR mice were resistant to milk-borne
infection by MMTV(C3H) and that this resistance occurred in
the lymphoid compartment. We have also found that milk-
borne infection with MMTV(LA) is reduced in B10.BR mice
(not shown). Because the MMTV(C3H) Sag is weaker than
those encoded by other MMTVs, we could not use an acute-
infection assay to functionally dissect the block to infection in
lymphocytes. Instead, using either MMTV(LA) or MMTV(FM),
we showed that B10.BR mice had altered Sag-dependent B-cell
and DC stimulation in vivo. That several different viruses en-
coding different Sags showed similar phenotypes in B10.BR
mice argues that the lack of response and subsequent ampli-
fication of MMTV infection in lymphocytes is not specific to a
particular strain of MMTV but represents a generalized resis-
tance to infection by the virus. This is in contrast to recent work
demonstrating that in some cases, MMTVs encoding “strong”
Sag proteins can infect mice in the absence of a robust Sag-
mediated T-cell response (33).
Although we have yet to determine the precise molecular
process leading to resistance to MMTV in B10.BR mice, we do
have evidence suggesting that resistance to MMTV infection in
this mouse strain segregates as a recessive autosomal allele in
crosses with C3H/HeN mice. Interestingly, when we used
mammary gland infection as the readout, F1mice were fully
susceptible to infection (Fig. 1A). In contrast, when lympho-
cyte activation was used as the assay, the F1phenotype was
intermediate between susceptible C3H/HeN and B10.BR mice
(Fig. 8B), indicating that the gene determining the Sag-medi-
ated lymphocyte response to MMTV in the former is semid-
ominant. These data indicate that even though F1mice have
lower levels of lymphocyte infection than do C3H/HeN mice,
this level of lymphocyte infection was sufficient to achieve the
maximum mammary tissue infection, at least at the time point
we studied (2 to 3 months of age). It is also possible that
additional alleles that restrict or enhance virus replication in
vivo beyond the initial steps of lymphocyte activation deter-
mine the level of mammary gland infection in F1mice. Further
studies are in progress to determine the exact mechanism of
resistance to MMTV observed in the B10.BR mouse strain and
to map the allele that affects Sag-mediated lymphocyte activa-
It is likely that B10.BR mice will show differential suscepti-
bilities to other pathogens, in addition to MMTV. Indeed,
B10.BR mice are known to be resistant to staphylococcal en-
terotoxin B (SEB)-induced lethal shock, while C3H/HeJ mice,
genetically similar to the C3H/HeN mice used here, are sus-
ceptible (1). Although T cells were required for disease induc-
tion in the SEB model, a non-T-cell compartment was impli-
cated in the B10.BR resistance. Whether the resistance to
MMTV is also the result of a non-T-cell compartment and
whether the MMTV and SEB-induced disease resistance phe-
notypes map to the same genetic locus will be the subjects of
future experiments. Importantly, B10.BR mice were not bred
for any particular mutation but represent a “normal” genetic
variant. Identification of the gene(s) involved in resistance to
the viral and bacterial pathogens in different inbred mice, like
B10.BR, is likely to lead to greater understanding of the role
genetics plays in the response to infectious disease and to help
develop new treatment paradigms.
We thank Tatyana Golovkina and Wayne Clemmons for the early
work that led to these studies and Jennifer Meyers for technical assis-
tance. The HP-transfected XC cells were a gift from Jaquelin Dudley,
and the anti-MMTV SU and TM hybridomas were a gift from Tatyana
This work was supported by NIH RO1CA45954 (to S.R.R.); C.M.O.
was supported by training grant NIH/NCI T32-CA9140.
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