A novel block to mouse mammary tumor virus infection of lymphocytes in B10.BR mice.
ABSTRACT 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-2(k)) 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.
- SourceAvailable from: Marisa N Madison[Show abstract] [Hide abstract]
ABSTRACT: Background Exosomes are membranous nanovesicles secreted into the extracellular milieu by diverse cell types. Exosomes facilitate intercellular communication, modulate cellular pheno/genotype, and regulate microbial pathogenesis. Although human semen contains exosomes, their role in regulating infection of viruses that are sexually transmitted remains unknown. In this study, we used semen exosomes purified from healthy human donors to evaluate the role of exosomes on the infectivity of different strains of HIV-1 in a variety of cell lines.ResultsWe show that human semen contains a heterologous population of exosomes, enriched in mRNA encoding tetraspanin exosomal markers and various antiviral factors. Semen exosomes are internalized by recipient cells irrespective of cell type and upon internalization, inhibit replication of a broad array of HIV-1 strains. Remarkably, the anti-HIV-1 activity of semen exosomes is specific to retroviruses because semen exosomes blocked replication of the murine AIDS (mAIDS) virus complex (LP-BM5). However, exosomes from blood had no effect on HIV-1 or LP-BM5 replication. Additionally, semen and blood exosomes had no effect on replication of herpes simplex virus; types 1 and 2 (HSV1 and HSV2). Mechanistic studies indicate that semen exosomes exert a post-entry block on HIV-1 replication by orchestrating deleterious effects on particle-associated reverse transcriptase activity and infectivity.Conclusions These illuminating findings i) improved our knowledge of the cargo of semen exosomes, ii) revealed that semen exosomes possess anti-retroviral activity, and iii) suggest that semen exosome-mediated inhibition of HIV-1 replication may provide novel opportunities for the development of new therapeutics for HIV-1.Retrovirology 11/2014; 11(1):102. · 4.77 Impact Factor
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ABSTRACT: JF1/Ms mice, an inbred strain derived from Japanese wild mice, carry a germline hypomorphic mutation in the endothelin receptor type B gene (Ednrb). We observed that the JF1/Ms mice develop various spontaneous tumors at a high incidence late in life. The aim of this study was to elucidate the mechanism responsible for spontaneous tumors in these mice. Possible relevance of milk-borne mammary tumor virus and gene alterations in Ednrb to tumorigenesis was explored. Expression and methylation status of Ednrb were quantitatively analyzed in normal and cancer tissues of mammary gland, liver, submandibular gland as well as in a cultured cell line, MW1, established from a submandibular gland adenocarcinoma. The biological effects of EDNRB were examined in the MW1 cells transfected with wild-type Ednrb. Transcripts of Ednrb were barely detectable, and the promoter region of Ednrb was hypermethylated in tumor tissues and the MW1 cells. In contrast, normal counterpart tissues showed positive expression of Ednrb transcripts and had unmethylated promoter regions. Treatment of the MW1 cells with 5-Aza-dC restored transcription of Ednrb to normal levels. Transfection of the MW1 cells with Ednrb1 (MW1-Ednrb1) resulted in lower growth rates and morphological changes compared with the mock-transfected MW1 cells (MW1-mock1). Furthermore, the MW1-Ednrb1 cells transplanted in syngeneic mice showed a lower proliferation rate than the MW1-mock1 cells. Germline mutation and subsequent promoter methylation of Ednrb may be relevant to cancer susceptibility in the JF1/Ms mice. These data indicate that Ednrb acts as a tumor suppressor, as reported in human prostate, bladder, and clear cell renal carcinomas.Journal of Cancer Research and Clinical Oncology 11/2013; · 2.91 Impact Factor
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ABSTRACT: BST-2 is a virus restriction factor whose expression is principally induced by IFNα through the type I IFN receptor. However, expression of BST-2 is modulated by mitogens, notably the TLR4 agonist - LPS, via mechanisms that are poorly understood. In this study, the role of TLR4 pathway on BST-2 expression was examined. We demonstrate that the TLR4/PI3K signaling pathway regulates both constitutive and LPS-induced BST-2 expression. LPS stimulation induces BST-2 expression in a manner dependent on TLR4/TRIF/IRF3 pathway. Genetic deletion or pharmacological inhibition of signaling through TLR4, as well as, the deletion of the TRIF and IRF3 genes blunts BST-2 induction by LPS. However, MYD88-/- cells have enhanced BST-2 levels and respond to LPS-mediated induction of BST-2. High level of BST-2 in MYD88 null cells is dependent on IFNβ since antibody-mediated neutralization of IFNβ synthesis results in reduced BST-2 levels in these cells. Similar to the effect of MYD88, inhibition of PI3K activity elevates basal BST-2 level and augments LPS-induced BST-2 expression. Importantly, BST-2 regulation via TLR4 and PI3K is transcriptionally controlled. We discovered that actinomycin D-mediated blocking of gene transcription and inhibition of protein synthesis with cycloheximide result in impairment of BST-2 mRNA expression. Taken together, our results demonstrate that activation of TLR4 results in TRIF/IRF3-mediated positive regulation of BST-2 or MYD88/PI3K-directed negative regulation of BST-2. Thus, our findings enlist BST-2 as one of the genes regulated by PI3K downstream of TLR4 and identify the TLR4/PI3K signaling as a novel pathway that controls BST-2 expression.Cellular Signalling 09/2013; · 4.47 Impact Factor
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 MMTV 1315
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.
1318 OKEOMA ET AL.J. VIROL.