Regulatory T-cell expansion during chronic viral infection is dependent on endogenous retroviral superantigens.
ABSTRACT Regulatory T cells (Treg) play critical roles in the modulation of immune responses to infectious agents. Further understanding of the factors that control Treg activation and expansion in response to pathogens is needed to manipulate Treg function in acute and chronic infections. Here we show that chronic, but not acute, infection of mice with lymphocytic choriomeningitis virus results in a marked expansion of Foxp3(+) Treg that is dependent on retroviral superantigen (sag) genes encoded in the mouse genome. Sag-dependent Treg expansion was MHC class II dependent, CD4 independent, and required dendritic cells. Thus, one unique mechanism by which certain infectious agents evade host immune responses may be mediated by endogenous Sag-dependent activation and expansion of Treg.
- [Show abstract] [Hide abstract]
ABSTRACT: It is still unclear whether expanded and activated regulatory T cells (Tregs) in chronic viral infections can influence primary immune responses against superinfections with unrelated viruses. Expanded Tregs found in the spleens of chronically FV-infected mice decreased mCMV-specific CD8(+) T cell responses during acute mCMV superinfection. This suppression of mCMV-specific T cell immunity was only found in organs with FV-induced Treg expansion. Surprisingly, acute mCMV infection itself did not expand or activate Tregs.Journal of Virology 09/2014; · 4.65 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Regulatory T (T reg) cells are critical for preventing autoimmunity mediated by self-reactive T cells, but their role in modulating immune responses during chronic viral infection is not well defined. To address this question and to investigate a role for T reg cells in exhaustion of virus-specific CD8 T cells, we depleted T reg cells in mice chronically infected with lymphocytic choriomeningitis virus (LCMV). T reg cell ablation resulted in 10-100-fold expansion of functional LCMV-specific CD8 T cells. Rescue of exhausted CD8 T cells was dependent on cognate antigen, B7 costimulation, and conventional CD4 T cells. Despite the striking recovery of LCMV-specific CD8 T cell responses, T reg cell depletion failed to diminish viral load. Interestingly, T reg cell ablation triggered up-regulation of the molecule programmed cell death ligand-1 (PD-L1), which upon binding PD-1 on T cells delivers inhibitory signals. Increased PD-L1 expression was observed especially on LCMV-infected cells, and combining T reg cell depletion with PD-L1 blockade resulted in a significant reduction in viral titers, which was more pronounced than that upon PD-L1 blockade alone. These results suggest that T reg cells effectively maintain CD8 T cell exhaustion, but blockade of the PD-1 inhibitory pathway is critical for elimination of infected cells.Journal of Experimental Medicine 08/2014; · 13.91 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: In multicellular organisms, specialized functions are delegated to distinct cell types whose identity and functional integrity are maintained upon challenge. However, little is known about the mechanisms enabling lineage inheritance and their biological implications. Regulatory T (Treg) cells, which express the transcription factor Foxp3, suppress fatal autoimmunity throughout the lifespan of animals. Here, we show that a dedicated Foxp3 intronic element CNS2 maintains Treg cell lineage identity by acting as a sensor of the essential Treg cell growth factor IL-2 and its downstream target STAT5. CNS2 sustains Foxp3 expression during division of mature Treg cells when IL-2 is limiting and counteracts proinflammatory cytokine signaling that leads to the loss of Foxp3. CNS2-mediated stable inheritance of Foxp3 expression is critical for adequate suppression of diverse types of chronic inflammation by Treg cells and prevents their differentiation into inflammatory effector cells. The described mechanism may represent a general principle of the inheritance of differentiated cell states.Cell. 08/2014; 158(4):749-63.
is dependent on endogenous retroviral superantigens
George A. Punkosdya, Melissa Blaina, Deborah D. Glassa, Mary M. Lozanob, Leigh O’Marac, Jaquelin P. Dudleyb,
Rafi Ahmedc,1, and Ethan M. Shevacha,1
aLaboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;bSection of Molecular
Genetics and Microbiology and Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712; andcEmory Vaccine Center and
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322
Contributed by Rafi Ahmed, January 10, 2011 (sent for review October 11, 2010)
Regulatory T cells (Treg) play critical roles in the modulation of
immune responses to infectious agents. Further understanding of
the factors that control Treg activation and expansion in response
to pathogens is needed to manipulate Treg function in acute and
chronic infections. Here we show that chronic, but not acute,
infection of mice with lymphocytic choriomeningitis virus results
in a marked expansion of Foxp3+Treg that is dependent on retro-
viral superantigen (sag) genes encoded in the mouse genome. Sag-
dependent Treg expansion was MHC class II dependent, CD4 inde-
pendent, and required dendritic cells. Thus, one unique mechanism
by which certain infectious agents evade host immune responses
may be mediated by endogenous Sag-dependent activation and
expansion of Treg.
nisms controlling this outcome begin during T-cell development
in the thymus. T cells expressing a fully rearranged T-cell re-
ceptor (TCR) with relatively high affinity to antigens presented
in the thymus are deleted or rendered anergic during the process
of negative selection. As a result, mature T cells in the periphery
are largely specific for nonself antigens. Because this process is
not completely efficient, additional mechanisms are required in
the periphery to maintain homeostasis. Regulatory T cells (Treg)
are a subset of CD4+T cells that express the lineage commitment
transcription factor Foxp3 and are critical in controlling self-
tolerance in the periphery (1, 2). Treg use a number of mecha-
nisms to suppress the activation and effector function of con-
ventional Foxp3−T cells (Tconv) (3), and their importance is
multiorgan autoimmunity early in life (4–6).
Treg originate from at least two sources. First, T cells
expressing TCR with intermediate affinity to thymic antigens es-
cape negative selection and develop into Foxp3-expressing Treg
(natural Treg). It has been shown that natural Treg share a non-
overlapping TCR repertoire with Tconv, suggesting that the nat-
ural Treg population may be biased toward self-antigens (7–9).
Additionally, Treg can differentiate from Foxp3−precursors in
the periphery in response to TCR stimulation and TGF-β (in-
cells is less clear, these cells may recognize a combination of self
and nonself antigens. Nevertheless, the relationship between the
antigen specificity of Treg and their effector function is not clear.
After an infection, the balance between Tconv and Treg is
critical. Pathogen-specific CD4+and CD8+T cells rapidly ex-
pand after infection and, in most cases, clear the infection. Treg
may play a role during acute infection, but their importance is
still unclear (11). However, during chronic infection, Treg can
play critical roles by limiting excessive immune activation and
tissue damage, while at the same time facilitating pathogen
persistence and maintenance of immunity (12). Further un-
derstanding of the role of Treg during chronic infection has been
limited by our lack of knowledge regarding the stimuli that drive
Treg activation and expansion in these situations. Not only are
hallmark of the mammalian immune system is the ability to
distinguish between self and nonself antigens. The mecha-
chronic infection, but tissue damage may also result in the pre-
sentation of self-antigens. Some studies suggest that Treg that
expand after chronic infection can be pathogen specific (13–15),
but these findings are not universal (16). Thus, additional in-
formation regarding the mechanism of Treg activation and ex-
pansion during chronic infection is important.
To examine the role of Treg after infection, we have used the
mouse model of infection with lymphocytic choriomeningitis vi-
rus (LCMV). Using different strains of the virus, this model
allows differentiation between the effects of acute and chronic
viral infection. The Armstrong strain of LCMV causes an acute
infection that is cleared within 1 wk, whereas the variant clone 13
establishes a chronic infection whereby the mice remain infected
virtually for life (17). Treg activation and expansion after clone
13 infection in C57BL/6 mice was most prominent among a
subpopulation of cells expressing the TCR Vβ5 segment. These
cells expanded from a preexisting population of Treg and dis-
played characteristics of a superantigen (Sag)-mediated re-
sponse. In addition to the Vβ specificity, the Treg response was
MHC class II dependent, CD4 independent, and entirely de-
pendent on retrovirus-encoded Sags in the mouse genome. Our
results describe a unique mechanism by which “self-reactive”
Treg expand after chronic infection.
Vβ5+Treg Expand During Chronic Viral Infection. We compared the
frequency and phenotype of Treg in C57BL/6 mice during the
course of Armstrong or clone 13 LCMV infections. Treg dra-
matically increased in frequency among splenic CD4+CD8−T
cells during clone 13, but not Armstrong, infection. This in-
creased frequency of Foxp3+Treg was transient and peaked
at ≈17 dpi (days post infection) (Fig. 1A). The increase in fre-
quency also translated to an approximately threefold increase
in absolute numbers of splenic Treg at the peak. Furthermore,
Treg from clone 13-infected mice displayed a more activated
phenotype than those from Armstrong-infected animals (Fig.
1B). Specifically, Treg from clone 13-infected mice down-
regulated the lymphoid homing marker CD62L and up-regulated
the activation marker CD69. Two cell-surface markers that have
been described as in vivo activation markers of Treg, CD101
(18) and CD103 (19), were also expressed on a higher percent-
age of Treg from the clone 13-infected group. In addition,
Treg from the clone 13-infected mice had higher levels of the
costimulatory/inhibitory markers Inducible T cell Costimulator
M.M.L., and L.O. performed research; G.A.P. analyzed data; and G.A.P. and E.M.S. wrote
The authors declare no conflict of interest.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| March 1, 2011
| vol. 108
| no. 9
(ICOS), Programmed Death 1 (PD-1), OX-40 (CD134), and
We next asked whether the increased frequency and number
of Treg observed during clone 13 infection was the result of an
expansion of a subpopulation of Treg that could be detected by
a skewing of the TCR Vβ repertoire. The percentage of Treg
expressing different mouse TCR Vβ segments was determined by
flow cytometry, and a marked increase in the frequency of Treg
expressing TCR Vβ5 was observed specifically during clone 13
infection (Fig. 1C). The percentage of CD4+Foxp3+Vβ5+cells
increased in frequency from 7% in uninfected and Armstrong-
infected mice to ≈25% in clone 13-infected mice. No change in
the percentage of Treg expressing any other Vβ subset was ob-
served, and the frequency of CD4+Vβ5+Foxp3−T cells was un-
altered during either Armstrong or clone 13 infections. At 17 dpi
with clone 13, the number of Vβ5+Treg approximated the total
number of splenic Foxp3+Treg in an uninfected mouse (Fig. 1D).
A higher percentage of the Vβ5+Treg were also positive for the
Treg activation markers CD101 and CD103 compared with Vβ5−
Treg, and ICOS and PD-1 were up-regulated on both Vβ5+and
Vβ5−Treg (Fig. 1E). In a standard in vitro suppression assay, the
expanded Vβ5+Treg maintained their anergic status and were as
suppressive as the Vβ5−Treg (Fig. S1), indicating that the ex-
panded Vβ5+Treg were functional.
The failure to observe Treg expansion during Armstrong in-
fection correlated with the absence of viral chronicity and not
with the minor sequence differences between the two strains.
When perforin−/−mice that lacked functional cytotoxic T cells
were infected with Armstrong to establish a persistent infection
(20), the magnitude of Vβ5+Treg expansion was similar to that
of wild-type mice infected with clone 13 (Fig. S2A). Conversely,
when IL-10−/−mice were infected with clone 13 to allow rapid
viral clearance (21), no expansion of Vβ5+Treg was observed
(Fig. S2B), suggesting that chronic infection was both necessary
and sufficient to drive Vβ-specific Treg expansion.
Vβ5+Treg Expand from a Preexisting Pool of Treg. The observation
that the Vβ5+T-cell expansion was Treg specific raised the
question of the origin of these cells in vivo. To determine whether
the expanded Treg were induced in the periphery from Foxp3−
precursors, we transferred Foxp3−cells [FACS sorted from
Foxp3/GFP reporter mice (22)] into naïve congenic C57BL/6
mice 1 d before clone 13 infection (Fig. 2A). After infection,
endogenous and transferred CD4+T cells were analyzed to de-
termine the percentage of cells expressing Foxp3 (Fig. 2B).
Among the endogenous CD4+T cells, the percentage of cells
expressing Foxp3 increased to levels observed previously. How-
ever, no induction of Foxp3 was detected in the transferred CD4+
T cells, indicating that Treg are not generated de novo under
these conditions. Because LCMV infects the thymus, it remained
possible that the increase in number of Vβ5+Treg was due to an
increase in thymic generation. However, Vβ5+Treg expansion
was completely normal when adult thymectomized mice were
infected with clone 13 (Fig. S3).
To test whether the expanded Vβ5+Treg were derived from
the endogenous Treg pool, we transferred GFP/Foxp3+T cells
into congenic mice 1 d before clone 13 infection. At 15 dpi, en-
dogenous and transferred Treg were analyzed to determine the
percentage of cells expressing Vβ5 (Fig. 2C). The increase in
frequency of Vβ5+Treg was the same among endogenous and
transferred Foxp3+cells (2.8- and 2.5-fold, respectively), consis-
tent with the preferential expansion of a preexisting population of
Treg. To further test this hypothesis, we examined cell division of
the different CD4+T-cell populations early in infection. At 7 dpi,
the CD4+Foxp3+Vβ5+subset had the highest percentage cells
incorporating BrdU and expressing the cell cycle marker Ki-67,
compared with all other CD4+T cell populations (Fig. 2D).
Vβ5+Treg Expand in Response to Mtv Sag. Studies in other in-
fectious disease models suggest that Treg in infected animals can
be specific for the infectious organism. We obtained no evidence
for LCMV-specific reactivity of Treg from clone 13-infected
mice, including the failure to bind a peptide (GP66-77)-MHC
class II tetramer specific for an immunodominant LCMV epi-
tope (Fig. S4).
Because themajor fraction ofTreg that expanded afterclone 13
infection was restricted to a specific Vβ subset, we hypothesized
that a Sag was responsible for proliferation. We initially analyzed
and CD4 molecules. Both C57BL/6 Abβ−/−and C57BL/6 CD4−/−
mice have a population of TCR+Foxp3+cells that have charac-
teristics of Treg (23). We infected both of these strains with clone
13 and analyzed the percentage of CD3+Foxp3+cells that express
Vβ5+. For the Abβ−/−mice, no change in the percentage of CD4+
single-positive (or CD8+single-positive) Foxp3+Vβ5+cells was
observed (Fig. 3A). In the CD4−/−mice, the expansion of Vβ5+
cells among the CD4−CD8−Foxp3+population was completely
unaffected (Fig. 3B), together suggesting that the expansion of the
chronic LCMV infection. (A) Kinetics of Foxp3 ex-
pression on C57BL/6 splenic CD4+CD8−T cells after
LCMV Armstrong or clone 13 infection. Error bars
represent the SD of the mean of at least four animals
per time point. (B) Surface expression of activation or
costimulatory/inhibitory markers on CD4+Foxp3+cells
from uninfected mice (gray) and mice infected with
LCMV Armstrong (blue) or clone 13 (red) (22 dpi). (C)
Expression of TCR Vβ segments on CD4+Foxp3+or
CD4+Foxp3−T cells after infection with LCMV Arm-
strong and clone 13 (25 dpi). (D) Absolute number of
CD4+Foxp3+T cells in the spleen of uninfected and
clone 13-infected mice (17 dpi) showing the number
of Vβ5+and Vβ5−cells. (E) Surface expression of ac-
CD4+Foxp3+Vβ5+(green) or Vβ5−(purple) T cells after
clone 13 infection (22 dpi).
Vβ5+Foxp3+Treg expand specifically during
| www.pnas.org/cgi/doi/10.1073/pnas.1100213108Punkosdy et al.
Vβ5+subset was MHC class II dependent and CD4 independent.
This pattern is consistent with Sag-mediated activation of CD4+T
indicating that CD8 cannot substitute for CD4 as a coreceptor in
this mechanism of Treg expansion.
BecauseLCMVhasnot beenpreviouslyshowntoexpress aSag,
a likely explanation for our results was that the expansion of the
Vβ5+Treg was secondary to stimulation by an endogenous Sag,
such as mouse mammary tumor virus (Mtv)-encoded Sag. Most
common strains of laboratory mice carry endogenous Mtv provi-
Sag expression in mice expressing MHC Class II I-E leads to
intrathymic deletion of specific Vβ subsets in a strain-dependent
manner (27). We infected several strains of mice expressing I-E
with clone 13 and analyzed the effects of infection on the Vβ rep-
ertoire.In everystrain,infection with clone13resulted in selective
expansion of a Vβ-expressing CD4+Foxp3+population that was
normally deleted by Mtv-encoded Sag in the thymus (Table S1).
Specifically in the case of BALB/c (H-2d, I-E+) mice, we observed
a significant increase in the percentage of CD4+Foxp3+Vβ5+and
a dramatic increase in Foxp3+Vβ12+Treg, but no change in the
percentage of CD4+Foxp3−Vβ5+or Foxp3−Vβ12+T cells, after
clone 13 infection (Fig. 4A). No changes in the percentages of the
otherVβ populationsweredetected.These Vβ5+andVβ12+Treg
displayed an activated phenotype similar to that seen in the ex-
panded Vβ5+Treg in the C57BL/6 mice (Fig. 4B).
To rule out the possibility that the differences observed be-
tween C57BL/6 and BALB/c mice were due to the different ge-
netic backgrounds, we also infected B10.D2 (H-2d, I-E+) and
BALB/b (H-2b, I-E−) mice and performed the same Vβ repertoire
analysis. The pattern of Treg Vβ expansion in infected BALB/
b mice was the same as in C57BL/6 mice, and B10.D2 mice be-
haved identically to BALB/c mice (Fig. S5). These results are
consistent with an Mtv Sag-mediated increase in Treg cells after
Treg Do Not Expand After Infection of BALB/c Mtv-Null Mice. To di-
rectly prove that expansion of the Vβ5 and Vβ12 Foxp3+subsets
in BALB/c mice was secondary to activation by endogenous Mtv
Sag, we infected congenic BALB/c mice lacking all three of the
endogenous Mtv proviruses (Mtv-null) (28) and analyzed their
Vβ repertoire. Consistent with a lack of Mtv Sag, these mice had
a substantial population of T cells that expressed either Vβ5 or
Vβ12 in Foxp3+and Foxp3−subsets. However, we observed no
change in the frequencies of either Vβ5+or Vβ12+Treg in these
mice after clone 13 infection (Fig. 5A), suggesting that the ob-
served Vβ-specific Treg expansion was due to stimulation by an
endogenous retroviral Sag. We also infected F1 crosses between
the Mtv-null mice and either wild-type C57BL/6 or BALB/c mice.
Vβ5 and Vβ12 T-cell deletion occurred as expected in the F1
hybrids, and Vβ5+and Vβ12+Treg expanded to the same extent
as seen in the wild-type BALB/c mice (Fig. S6), thus demon-
strating that the presence of the Mtv Sag is dominant.
To determine the relative contribution of each of the Mtv Sag
to Treg expansion, we infected congenic BALB/c strains carrying
single Mtv proviruses (called BALB/Mtv6, BALB/Mtv8, and
BALB/Mtv9). The BALB/Mtv6 animals failed to delete both Vβ5
and Vβ12 T cells, and no expansion of Treg in either population
was observed after infection (Fig. 5B). Uninfected BALB/Mtv8
animals had normal numbers of Vβ5+and ≈50% of Vβ12+T
cells (compared with Mtv-null and BALB/Mtv6 mice), and only
the Vβ12+Treg expanded after clone 13 infection (Fig. 5C).
However, the BALB/Mtv9 animals were similar to wild-type
BALB/c (both in terms of T-cell deletion and Treg expansion
after infection) (Fig. 5D), suggesting that Mtv9 Sag is the major
contributor to the expansion of Treg observed in wild-type mice.
ulation of Treg after LCMV infection. (A) CD4+GFP/Foxp3+or GFP/Foxp3−
T cells were FACS sorted from naïve CD45.2 reporter mice and transferred
into naïve CD45.1 recipients 1 d before clone 13 infection. Control un-
infected mice were transferred with identical numbers of cells. (B) GFP and
Foxp3 expression on splenic CD4+
CD45.2+CD4+GFP/Foxp3−T cells (15 dpi). Endogenous (Left) or transferred
(Right) cells are shown. Numbers represent the percentage of cells in each
quadrant. (C) GFP and Vβ5 expression on splenic CD4+Foxp3+T cells from
mice that received CD45.2+CD4+GFP/Foxp3+T cells (15 dpi). Endogenous
(Left) or transferred (Right) are shown, and numbers represent the per-
centage of GFP−or GFP+cells, respectively. (D) Adult C57BL/6 mice were
injected with 1 mg BrdU 7 dpi with clone 13, and splenic CD4+T cells were
analyzed 24 h later. Numbers represent percentage of cells in each quadrant.
Vβ5+Foxp3+Treg preferentially expand from a preexisting pop-
T cells from mice that received
independent. Adult C57BL/6 Abβ−/−or CD4−/−mice were infected with LCMV
clone 13, and spleens were harvested 18 dpi. (A) Top: Percentage of
CD3+Foxp3+T cells in Abβ−/−mice. Middle: Gated on the CD3+Foxp3+cells
from Top. Bottom: Vβ5 expression on CD4+CD8−(Left) and CD4−CD8+(Right)
cells from Middle. (B) Analysis of cells from CD4−/−mice. Top and Middle:
Gated as in A. Bottom: Vβ5 expression on CD4−CD8−(Left) and CD4−CD8+
(Right) cells from Middle. Histograms show cells from uninfected (gray) or
clone 13-infected (red) mice. Numbers represent percentage of cells in each
region or quadrant.
Expansion of Vβ5+Foxp3+Treg is MHC class II dependent but CD4
Punkosdy et al.PNAS
| March 1, 2011
| vol. 108
| no. 9
Mtv Sag-Dependent Treg Expansion Requires Dendritic Cells. To
address the mechanism by which chronic infection results in Mtv
Sag-dependent Treg expansion, we first asked whether LCMV
infection resulted in increased expression of the endogenous sag
genes. Because Mtv8 and Mtv9 Sag seemed to be responsible for
the Vβ-specific Treg expansion, we determined the relative ex-
pression of these genes in splenic MHC class II (I-A/I-E)-
enriched cells from BALB/c mice by quantitative real-time PCR
using primers specific for the intragenic env promoter and 3′
LTR sequence (Mtv6 does not contain an env gene and therefore
could not be detected using these primers). At 8 dpi, Mtv8 and/or
Mtv9 sag expression was up-regulated ≈2- and 25-fold in Arm-
strong- and clone 13-infected mice, respectively, compared with
uninfected controls (Fig. 6A). The inability to detect a product in
cells isolated from the Mtv-null mice demonstrated that the
primers were specific.
Because the I-A/I-E enriched cells were a heterogeneous pool,
we sought to further define the type of antigen-presenting cells
responsible. Previously, both B cells and dendritic cells (DC)
have been shown to be the primary antigen-presenting cells that
express several endogenous Mtvs in the periphery (29). We ex-
amined the role of each of the cell types initially using genetically
deficient mice. Infection of C57BL/6 B cell-deficient mice (μMT)
with clone 13 resulted in Vβ5+Treg expansion similar to that in
wild-type mice. However, Flt3L−/−mice (which are defective in
several hematopoietic cell lineages, but primarily DC) infected
with clone 13 displayed only a modest (approximately twofold)
increase in frequency of Vβ5+Treg (Fig. 6B), suggesting that Sag
presentation by B cells was not necessary and that DC were
primarily responsible for the Treg expansion.
To further define the role of DC in the expansion of Treg, we
used the CD11c-diphtheria toxin receptor (DTR) mice (30).
Lethally irradiated C57BL/6 mice reconstituted with CD11c-
DTR bone marrow were infected with clone 13 and injected with
DT or saline as a control every other day throughout the course
of infection. At 17 dpi, DT-treated mice did not contain CD11c/
GFP+cells in their spleens (Fig. 6C), and viral titers in the DT-
treated mice were similar to those in controls (Fig. 6D). These
results indicated that the DC depletion was efficient and that DT
treatment did not alter the course of infection. However, mice
treated with DT had significantly fewer Vβ5+Treg than controls
(25.5% reduction; Fig. 6E). Finally, Mtv8 and/or Mtv9 sag ex-
pression was assessed in purified splenic DC (defined as
highest in DC from clone 13-infected animals (Fig. 6F), suggesting
that modulation of Sag gene expression in DC may be important
for Vβ-specific Treg expansion.
These studies define a unique mechanism of Treg activation and
expansion after chronic viral infection. Infection of C57BL/6
mice with LCMV clone 13 resulted in the selective expansion
of a preexisting population of Treg expressing TCR Vβ5,
whereas infection of mice expressing MHC class II I-E resulted
in the selective expansion of Treg expressing particular Vβ sub-
sets that were deleted in the thymus. Taken together, we believe
that the Vβ-specific Treg expansion observed after chronic
LCMV infection is mediated by Mtv-encoded Sag proteins be-
cause (i) the Treg response is Vβ specific and corresponds with
specific Sag-expressing Mtvs found in the germline of multiple
mouse strains, (ii) the Treg response is MHC class II dependent
and CD4 independent, (iii) the use of a congenic BALB/c mouse
strain lacking all endogenous Mtvs abolished Vβ-specific Treg
expansion, and (iv) F1 hybrids between Mtv-null and BALB/c
mice revealed that Treg expansion is dominant, typical of Sag-
mediated effects. Although C57BL/6 mice do not delete Vβ
subsets in the thymus because of the lack of I-E expression, Mtv-
dependent abortive activation and deletion of Vβ5+T cells in the
Treg populations that are thymically deleted in response to Mtv/I-E recog-
nition. (A) TCR Vβ analysis of splenic CD4+T cells from adult BALB/c mice
infected with LCMV clone 13. (B) Surface expression of activation or cos-
timulatory/inhibitory markers on CD4+Foxp3+Vβ12+(green) or Vβ12−(pur-
ple) T cells after clone 13 infection (17 dpi).
Chronic infection of BALB/c mice results in the expansion of Foxp3+
Sag dependent. (A) TCR Vβ analysis of splenic CD4+T cells from adult BALB/c
Mtv-null mice infected with LCMV clone 13. (B) TCR Vβ analysis of splenic
CD4+T cells from adult BALB/Mtv6 mice infected with LCMV clone 13. (C)
TCR Vβ analysis of splenic CD4+T cells from adult BALB/Mtv8 mice infected
with LCMV clone 13. (D) TCR Vβ analysis of splenic CD4+T cells from adult
BALB/Mtv9 mice infected with LCMV clone 13. All mice were analyzed at 17
dpi. Numbers represent percentages of Foxp3+or Foxp3−cells in each plot.
Expansion of thymically deleted Foxp3+Treg in BALB/c mice is Mtv
| www.pnas.org/cgi/doi/10.1073/pnas.1100213108Punkosdy et al.
periphery of older mice is observed (31). Therefore, we suggest
that a similar Mtv Sag-dependent mechanism occurs during
chronic viral infection in C57BL/ 6 mice that leads to the expan-
sion of Treg.
The T-cell repertoire of almost all inbred mouse strains is
influenced by the expression of various Sags encoded by Mtv
proviruses (26, 27). In the present study, we define a major role
for Mtv9 in causing expansion of Treg after chronic LCMV in-
fection. However, it is interesting to note that not all Foxp3+Vβ
subpopulations recognized by an Mtv-encoded Sag expanded
after infection. Although the reason for this observation is not
clear, it may be related to several factors. The levels of sag
mRNA produced by different Mtv proviruses are known to differ
and may be dependent on the mouse strain. Further, the cell
types for Mtv Sag expression vary according to the provirus, likely
owing to variations in their transcription control regions (32, 33).
The levels of MHC class II necessary for Sag expression on DC
also may vary after LCMV infection. MHC haplotype and the
presence or absence of I-E play a role, because Vβ12 expansion
occurs in BALB/c and B10.D2 strains that express H2d, I-E+, but
not in congenic strains that express H2b, I-E−, even though the
same Mtv proviruses are present.
The mechanism by which chronic LCMV infection results in
Mtv Sag-dependent Treg activation and expansion seems to, at
least partially, require DC presentation of the endogenous Sag.
Although B cells and DC have been shown to express endogenous
Mtv genes in the periphery (29), mice genetically lacking DC
showed only a modest level of Vβ-specific Treg expansion after
infection. The observation that Vβ-specific Treg expansion was
limited in mice depleted of DC by DT treatment provides further
evidence that DC are important. However, the fact that Vβ5+
Treg were 75% of the levels of controls in the latter experiment
suggests that other cells types may contribute. Although the
presence of B cells was not necessary to drive Vβ-specific Treg
expansion, B cells may contribute under normal circumstances.
Indeed, sag gene expression levels were highly up-regulated in
total MHC class II-enriched cells (a population that largely
consists of B cells). The failure to observe Treg expansion after
acute LCMV infection is likely due to much lower expression of
Mtv8 and/or Mtv9 sag mRNA and, presumably, Sag presentation
on DCs compared with that observed during chronic infection
by clone 13.
It is unclear from these studies why Mtv Sag-specific T cell
expansion was confined to the Foxp3+subset, because Sags have
not previously been shown to differentiate between different
populations of Vβ-specific cells. One explanation may be that
someexpansion of Foxp3−Vβ5+T cells may have occurred during
the course of the chronic infection but was balanced by deletion
similar to that seen by exogenous mouse mammary tumor virus
(MMTV) Sag after infection (34). Previous studies have also
shown that Treg are less susceptible to Sag-dependent deletion
(35). Alternatively, activated Foxp3+T cells could have func-
tioned to suppress any activation of Foxp3−Vβ5+T cells. Further,
Sag activation, although occurring in a TCR Vβ-specific fashion,
may be enhanced by the presence of costimulatory/adhesion
molecules that are not involved in the activation of T cells by
conventional antigens (36). Foxp3+Treg may preferentially use
such unique costimulatory molecules.
Various chronic infections, including some retroviruses, have
been correlated with modest increases in Treg numbers and, in
some cases, Treg have been implicated in determining the out-
come of infection (37). We have as yet been unable to directly
determine whether Mtv Sag-mediated Treg expansion is involved
in the establishment of chronic infection in this model. Viral
titers in wild-type and Mtv-null BALB/c mice were similar
throughout the course of infection with clone 13. However, the
expansion of Treg in BALB/c mice was modest, and many other
factors may play roles in the control of viral chronicity. Never-
theless, the absence of Mtv proviruses has been linked to altered
outcomes to disparate infectious agents. For example, infection
Relative gene expression of Mtv sag genes in splenic MHC class
II-enriched cells from uninfected BALB/c mice, BALB/c mice 8
dpi with either LCMV Armstrong or clone 13, and uninfected
BALB/c Mtv-null mice. N.D., not detected. Error bars represent
the SD of the mean of at least four animals per group. (B)
Analysis of Vβ5-expressing Treg from B cell- (μMT) and Flt3L-
deficient mice. Histograms are gated on CD4+Foxp3+T cells
from uninfected mice (gray) and mice infected with LCMV
clone 13 (red; 17–20 dpi). (C) Depletion of DC in CD11c-DTR
TCRβ−CD49b−splenocytes. (D) Serum viral titers in clone 13-
infected CD11c-DTR mice (17 dpi). Each circle represents
a single mouse and the horizontal bar the mean. (E) Vβ5
analysis of splenic CD4+Foxp3+cells from CD11c-DTR mice.
Each circle represents a single mouse and the horizontal bar
the mean. *P < 0.05. (F) Relative gene expression of Mtv sag
genes in purified DC from uninfected BALB/c mice and BALB/c
mice 8 dpi with either LCMV Armstrong or clone 13.
Mtv Sag-dependent Treg expansion requires DC. (A)
Punkosdy et al. PNAS
| March 1, 2011
| vol. 108
| no. 9
of Mtv-null mice with MMTV or a thymotropic variant prevents
breast cancer or leukemia (28, 38). It is possible that these results
are related to Mtv Sag effects on Treg.
Infection with EBV results in the expression of an endogenous
retroviral Sag that is encoded in the envelope gene of an en-
dogenous human retrovirus, HERV-18, localized to a region of
DNA in the vicinity of an EBV-inducible enhancer (39). Al-
though Vβ-specific T-cell activation was observed in these
experiments, it did not seem to be Treg specific. Additionally,
because HERV-18 is normally transcriptionally silent and only
expressed after infection (in contrast to Mtv Sags that are con-
stitutively expressed), it is unlikely that these phenomena are
related mechanistically. However, ≈8% of the human genome
corresponds to endogenous retroviral or retrovirally related
sequences that can encode Sags (40), many with unknown
functions. Therefore, the possibility that other HERV-encoded
Sags may exert similar effects on Treg activation and expansion
in man exists.
Mice and Infection. Commercially available mice were obtained from Jackson
Laboratories or Taconic. Mtv-null mice and Mtv single-positive mice were
generated as previously described (28). Infection of adult mice (6–8 wk old)
with LCMV Armstrong or clone 13 was performed as previously described (17).
Adoptive Transfer and Flow Cytometry. Cells for adoptive transfer experi-
ments were FACS sorted using a FACSAria cell sorter (BD Bioscience) and
transferred i.v. into congenic recipients 1 d before infection. Spleen samples
were harvested at the indicated dpi and stained with appropriate fluo-
rescently labeled antibodies. Flow cytometry data were collected using
a FACSCalibur or LSR II cytometer (BD Bioscience), and data were analyzed
using Flowjo (Treestar).
37 °C. Splenic MHC class II+cells were enriched by magnetic-activated cell
magnetic beads (Miltenyi), and purified DC were FACS sorted. Total RNA was
purified from each population of cells, and cDNA was synthesized using RT
(Invitrogen). RT-PCR for Mtv Sags was performed using SYBR-green (Applied
Biosystems) and the following primers: 5′ intragenic env promoter, ATCGC-
CTTTAAGAAGGACGCCTTCT; and 3′ LTR sequence, GCAAAGCAGAGCTAT-
GCC. A dissociation curve was run at the conclusion of the reaction to verify
a single product. Control RT-PCR was performed using the 18S rRNA reaction
kit (Applied Biosystems).
ACKNOWLEDGMENTS. We thank Y. Belkaid [National Institute of Allergy
and Infectious Diseases (NIAID)] for critical reading of the manuscript. This
work was supported by the Intramural Research Program of the National
Institutes of Health (NIH), NIAID, the Bill and Melinda Gates Foundation, and
NIH Grant R01-CA116813.
1. Fontenot JD, Rudensky AY (2005) A well adapted regulatory contrivance: Regulatory
T cell development and the forkhead family transcription factor Foxp3. Nat Immunol
2. Sakaguchi S (2004) Naturally arising CD4+ regulatory t cells for immunologic self-
tolerance and negative control of immune responses. Annu Rev Immunol 22:531–562.
3. Shevach EM (2009) Mechanisms of foxp3+ T regulatory cell-mediated suppression.
4. Bennett CL, et al. (2001) The immune dysregulation, polyendocrinopathy, enteropathy,
X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet 27:20–21.
5. Fontenot JD, Gavin MA, Rudensky AY (2003) Foxp3 programs the development and
function of CD4+CD25+ regulatory T cells. Nat Immunol 4:330–336.
6. Wildin RS, et al. (2001) X-linked neonatal diabetes mellitus, enteropathy and
endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet 27:
7. Hsieh CS, Zheng Y, Liang Y, Fontenot JD, Rudensky AY (2006) An intersection
between the self-reactive regulatory and nonregulatory T cell receptor repertoires.
Nat Immunol 7:401–410.
8. Pacholczyk R, et al. (2007) Nonself-antigens are the cognate specificities of Foxp3+
regulatory T cells. Immunity 27:493–504.
9. Wong J, et al. (2007) Adaptation of TCR repertoires to self-peptides in regulatory and
nonregulatory CD4+ T cells. J Immunol 178:7032–7041.
10. Curotto de Lafaille MA, Lafaille JJ (2009) Natural and adaptive foxp3+ regulatory
T cells: More of the same or a division of labor? Immunity 30:626–635.
11. Rouse BT, Suvas S (2007) Regulatory T cells and immunity to pathogens. Expert Opin
Biol Ther 7:1301–1309.
12. Belkaid Y, Tarbell K (2009) Regulatory T cells in the control of host-microorganism
interactions (*). Annu Rev Immunol 27:551–589.
13. Cabrera G, et al. (2008) Early increases in superantigen-specific Foxp3+ regulatory
T cells during mouse mammary tumor virus infection. J Virol 82:7422–7431.
14. McKee AS, Pearce EJ (2004) CD25+CD4+ cells contribute to Th2 polarization during
helminth infection by suppressing Th1 response development. J Immunol 173:
15. Suffia IJ, Reckling SK, Piccirillo CA, Goldszmid RS, Belkaid Y (2006) Infected site-
restricted Foxp3+ natural regulatory T cells are specific for microbial antigens. J Exp
16. Antunes I, et al. (2008) Retrovirus-specificity of regulatory T cells is neither present nor
required in preventing retrovirus-induced bone marrow immune pathology.
17. Wherry EJ, Blattman JN, Murali-Krishna K, van der Most R, Ahmed R (2003) Viral
persistence alters CD8 T-cell immunodominance and tissue distribution and results in
distinct stages of functional impairment. J Virol 77:4911–4927.
18. Fernandez I, et al. (2007) CD101 surface expression discriminates potency among
murine FoxP3+ regulatory T cells. J Immunol 179:2808–2814.
19. Rötzschke O, Borsellino G, Battistini L, Falk K, Kleinewietfeld M (2009) In vivo-
activated CD103+ Foxp3+ Tregs: Of men and mice. Blood 113:2119–2120, author reply
20. Walsh CM, et al. (1994) Immune function in mice lacking the perforin gene. Proc Natl
Acad Sci USA 91:10854–10858.
21. Brooks DG, Lee AM, Elsaesser H, McGavern DB, Oldstone MB (2008) IL-10 blockade
facilitates DNA vaccine-induced T cell responses and enhances clearance of persistent
virus infection. J Exp Med 205:533–541.
22. Korn T, et al. (2007) Myelin-specific regulatory T cells accumulate in the CNS but fail to
control autoimmune inflammation. Nat Med 13:423–431.
23. Stephens GL, Andersson J, Shevach EM (2007) Distinct subsets of FoxP3+ regulatory
T cells participate in the control of immune responses. J Immunol 178:6901–6911.
24. Borrero H, Collazo L, Macphail S (1993) The role of class II molecules in Mls 1a
recognition by CD4+ T cells is independent of the CD4 molecule. Cell Immunol 152:
25. Penninger JM, Schilham MW, Timms E, Wallace VA, Mak TW (1995) T cell repertoire
and clonal deletion of Mtv superantigen-reactive T cells in mice lacking CD4 and CD8
molecules. Eur J Immunol 25:2115–2118.
26. Cohen JC, Varmus HE (1979) Endogenous mammary tumour virus DNA varies among
wild mice and segregates during inbreeding. Nature 278:418–423.
27. Scherer MT, Ignatowicz L, Winslow GM, Kappler JW, Marrack P (1993) Superantigens:
Bacterial and viral proteins that manipulate the immune system. Annu Rev Cell Biol 9:
28. Bhadra S, Lozano MM, Payne SM, Dudley JP (2006) Endogenous MMTV proviruses
induce susceptibility to both viral and bacterial pathogens. PLoS Pathog 2:e128.
29. Ardavín C, et al. (1996) Expression and presentation of endogenous mouse mammary
tumor virus superantigens by thymic and splenic dendritic cells and B cells. J Immunol
30. Sapoznikov A, Jung S (2008) Probing in vivo dendritic cell functions by conditional cell
ablation. Immunol Cell Biol 86:409–415.
31. Blish CA, et al. (1999) Chronic modulation of the TCR repertoire in the lymphoid
periphery. J Immunol 162:3131–3140.
32. Barnett A, Mustafa F, Wrona TJ, Lozano M, Dudley JP (1999) Expression of mouse
mammary tumor virus superantigen mRNA in the thymus correlates with kinetics of
self-reactive T-cell loss. J Virol 73:6634–6645.
33. Xu L, Wrona TJ, Dudley JP (1997) Strain-specific expression of spliced MMTV RNAs
containing the superantigen gene. Virology 236:54–65.
34. Held W, et al. (1993) Superantigen-induced immune stimulation amplifies mouse
mammary tumor virus infection and allows virus transmission. Cell 74:529–540.
35. Papiernik M, de Moraes ML, Pontoux C, Vasseur F, Pénit C (1998) Regulatory CD4
T cells: Expression of IL-2R alpha chain, resistance to clonal deletion and IL-2
dependency. Int Immunol 10:371–378.
36. Bueno C, et al. (2006) Bacterial superantigens bypass Lck-dependent T cell receptor
37. Lund JM, Hsing L, Pham TT, Rudensky AY (2008) Coordination of early protective
immunity to viral infection by regulatory T cells. Science 320:1220–1224.
38. Bhadra S, Lozano MM, Dudley JP (2009) BALB/Mtv-null mice responding to strong
mouse mammary tumor virus superantigens restrict mammary tumorigenesis. J Virol
39. Sutkowski N, Conrad B, Thorley-Lawson DA, Huber BT (2001) Epstein-Barr virus
a superantigen. Immunity 15:579–589.
40. Larsson E, Andersson G (1998) Beneficial role of human endogenous retroviruses:
facts and hypotheses. Scand J Immunol 48:329–338.
| www.pnas.org/cgi/doi/10.1073/pnas.1100213108Punkosdy et al.