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
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1322OKEOMA ET AL.J. VIROL.