JEM Viral-Immunity Special Issue

THE
JOURNAL OF
EXPERIMENTAL
MEDICINE
SELECTED ARTICLES JANUARY 2015 www.jem.org
VIRAL IMMUNITY
Welcome
V i r a l I m mu n i t y
The Journal of Experimental Medicine now prints topic-specific mini collections
to showcase a handful of our recent publications. In this installment, we
highlight papers focusing on host-virus interactions and their implications for
disease outcome.
Our collection begins with Insights and articles relevant to the pathogenesis
of influenza viral infections in mice. Flu infection can cause respiratory as well
as gastrointestinal symptoms, even though the virus only exhibits tropism for
respiratory tissues. An Insight from Carolina Amezcua, Nicola Gagliani, and Richard Flavell highlights findings from Wang et al.,
who provide an explanation for gut inflammation in the absence of detectable virus in the gastrointestinal tract. They find that
infection in mice recruits lung-derived IFNg secreting CCR9+CD4+ T cells into the small intestine that alter the composition
of the gut microbiota. Th17 cells then expand in the small intestine and neutralization with IL-17A or antibiotic treatment
reduces intestinal injury.
In another flu study, Heaton et al. demonstrate how club cells in the respiratory tract are infected by influenza, survive
acute infection, and establish a proinflammatory environment that contributes to lung pathology. Depletion of club cells reduces
lung tissue damage associated with the infection. An accompanying Insight by Thomas Braciale and Taeg Kim discusses this
mechanism of flu pathogenesis and poses questions about viral and immune evasion strategies as well as therapeutic potential.
An article by Woodruff et al. relies on imaging techniques and examines aspects of flu infection relevant to vaccination. The
authors describe how during immunization, resident lymph node dendritic cells can rapidly relocate to sites of viral influenza
antigen, driving early activation of T cells, and contributing to germinal center formation and B cell memory to establish an
appropriate immune response.
In a human study of hepatitis B and C, Kurktschiev et al. implicate the transcription factor T-bet in viral clearance. The
authors find that acute resolving infections are characterized by high expression of T-bet in CD8+ T cells which is correlated
with enhanced IFNg production, while absence of T-bet is more often seen in patients whose infections become chronic. IFN-g
induction and T-bet expression are restored in dysfunctional T cells upon IL-2 and IL-12 supplementation.
Rapid and effective adaptive immune responses to viral pathogens rely on immunological memory and are curtailed by
lymphocyte exhaustion. An article by Penaloza-Macmaster et al. investigates how regulatory T cells (T reg) can modulate CD8+
T cell exhaustion during chronic lymphocytic choriomeningitis virus infection in mice. Their findings show that depletion of
T regs can expand functional virus-specific CD8+ T cells, rescuing exhausted CD8+ T cell subpopulations. T reg depletion also
upregulates the programmed-death ligand 1 receptor (PD-L1) on CD8+ T cells, but it is a combination of PD-L1 blockade
and T reg depletion that is able to reduce viral load. These findings suggest that T regs have the ability to contribute to the
maintenance of exhausted CD8+ T cells during chronic infection.
Collectively, the presented articles identify pathways and processes related to viral pathogenesis and immunity that may
contribute to therapeutic approaches to combat viral infection and disease. We hope you enjoy this complimentary copy of our
Viral Immunity collection. We invite you to explore additional collections at www.jem.org and to follow JEM on Facebook,
Google+, and Twitter.
Selected Articles January 2015

FLUshing in the bathroom
Carolina Amezcua, Nicola Gagliani, and Richard Flavell
Respiratory influenza virus infection induces intestinal immune injury via microbiota-mediated
Th17 celldependent inflammation
Jian Wang, Fengqi Li, Haiming Wei, Zhe-Xiong Lian, Rui Sun, and Zhigang Tian
Influenza pathogenesis: Club cells take the cure
Thomas Braciale and Taeg Kim
Long-term survival of influenza virus infected club cells drives immunopathology

Nicholas S. Heaton, Ryan A. Langlois, David Sachs, Jean K. Lim, Peter Palese, and Benjamin R. tenOever
Trans-nodal migration of resident dendritic cells into medullary interfollicular regions initiates immunity
to influenza vaccine
Matthew C. Woodruff, Balthasar A. Heesters, Caroline N. Herndon, Joanna R. Groom, Paul G. Thomas,
Andrew D. Luster, Shannon J. Turley, and Michael C. Carroll
Dysfunctional CD8+ T cells in hepatitis B and C are characterized by a lack of antigen-specific
T-bet induction
Peter D. Kurktschiev, Bijan Raziorrouh, Winfried Schraut, Markus Backmund, Martin Wchtler,
Clemens-Martin Wendtner, Bertram Bengsch, Robert Thimme, Gerald Denk, Reinhart Zachoval, Andrea Dick,
Michael Spannagl, Jrgen Haas, Helmut M. Diepolder, Maria-Christina Jung, and Norbert H. Gruener
Interplay between regulatory T cells and PD-1 in modulating T cell exhaustion and viral control
during chronic LCMV infection
Pablo Penaloza-MacMaster, Alice O. Kamphorst, Andreas Wieland, Koichi Araki, Smita S. Iyer, Erin E. West,
Leigh OMara, Shu Yang, Bogumila T. Konieczny, Arlene H. Sharpe, Gordon J. Freeman, Alexander Y. Rudensky,
and Rafi Ahmed
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INSIGHTS
Seasonal influenza represents a contagious family of respiratory viruses that infect

530% of the global population yearly and account for as many as 500,000 deaths
annually. Flu infection is characterized by symptoms such as fever, cough, sore throat,
runny nose, body aches, and fatigue. Interestingly, some people also develop diarrhea,
even though the virus tropism is for respiratory tissue. Despite being a well-studied
viral infection, the underlying mechanisms involved in the development of gastroenteritis-like syndrome with flu infection are poorly understood.
Now, Wang et al. have used a mouse model to show that influenza infection Insight from (left to right) Carolina Amezcua,
not only causes lung inflammation but also causes intestinal inflammation, even Nicola Gagliani, and Richard Flavell
though influenza virus was not detectable in the gastrointestinal tract. They showed
that during intranasal flu infection, CCR9+CD4+ T cells migrate from the lung into the intestinal mucosa in a CCL25/CCR9dependent manner and alter the composition of the gut microbiota by secreting IFN-g. Homeostasis of the intestinal microbiota
was altered, and increased numbers of Escherichia coli were detected after flu infection. Antibiotic treatment of intranasal flu-infected
mice protected them against infection-induced diarrhea. These data suggest that the dysbiosis generated after flu infection leads
to intestinal injury. The changes generated in the gut microbiota induced the production of IL-15 by intestinal epithelial
cells. IL-15 induced the expansion of Th17 cells in the small intestine, which then mediated intestinal immune injury.
It is noteworthy that not all flu-infected patients develop gastroenteritis-like symptoms. Why is this? One possibility could be
that the migration of pathogenic cells into the intestine depends on the severity of the infection. Consequently only highly infected
patients may get diarrhea. Alternatively, the specific microbial landscape in some individuals and the existence of regulatory bacteria,
could obstruct the growth of E. coli or promote regulatory T cells which in turn can control intestinal inflammation.
The data presented in this paper corroborate
the hypothesis that the intestine is a suitable place
to defuse an immune response. The abundance of
antiinflammatory cytokines, such as IL-10 and
TGF-b, regulatory cells and continuous regeneration of the tissue predispose the intestine with an
ability to control effector cells. Moreover, if effector
T cells escape all possible regulatory mechanisms,
diverting them to the lumenin essence flushing
them awaycould still be less dangerous than
allowing them to remain in situ and risking lung
tissue damage.
Wang, J., et al. 2014. J. Exp. Med. http://dx.doi.org/10.1084/
jem.20140625.
Working model of intestinal injury induced by respiratory influenza virus infection
Carolina Amezcua, Nicola Gagliani, and Richard Flavell; Howard Hughes Medical Institute, Yale School of Medicine: richard.flavell@yale.edu, caro.amezcua@yale.edu,
and nicola.gagliani@yale.edu
4
2348
INSIGHTS | The Journal of Experimental Medicine
Adapted from an illustration by the authors
FLUshing in the bathroom
Article
Respiratory influenza virus infection induces

intestinal immune injury via microbiotamediated Th17 celldependent inflammation
Jian Wang,1* Fengqi Li,1* Haiming Wei,1,2 Zhe-Xiong Lian,1,2 Rui Sun,1,2
and Zhigang Tian1,2,3
1Institute
of Immunology and CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences
and Medical Center, University of Science and Technology of China, Hefei, Anhui 230027, China
2Hefei National Laboratory for Physical Sciences at Microscale, Hefei, Anhui 230027, China
3Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis
and Treatment of Infectious Diseases, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang
310003, China
Influenza in humans is often accompanied by gastroenteritis-like symptoms such as diarrhea, but the underlying mechanism is not yet understood. We explored the occurrence of
gastroenteritis-like symptoms using a mouse model of respiratory influenza infection. We
found that respiratory influenza infection caused intestinal injury when lung injury occurred, which was not due to direct intestinal viral infection. Influenza infection altered the
intestinal microbiota composition, which was mediated by IFN- produced by lung-derived
CCR9+CD4+ T cells recruited into the small intestine. Th17 cells markedly increased in the
small intestine after PR8 infection, and neutralizing IL-17A reduced intestinal injury.
Moreover, antibiotic depletion of intestinal microbiota reduced IL-17A production and
attenuated influenza-caused intestinal injury. Further study showed that the alteration of
intestinal microbiota significantly stimulated IL-15 production from intestinal epithelial
cells, which subsequently promoted Th17 cell polarization in the small intestine in situ.
Thus, our findings provide new insights into an undescribed mechanism by which respiratory
influenza infection causes intestinal disease.
CORRESPONDENCE
Zhigang Tian:
tzg@ustc.edu.cn
Abbreviations used: BALF,
bronchoalveolar lavage fluid;
IBD, inflammatory bowel disease; IEC, intestinal epithelial
cell; i.g., intragastrical(ly); i.n.,
intranasal(ly); SFB, segmented
filamentous bacteria.
Influenza is an infectious respiratory disease affecting many bird and mammal species (Laver
and Webster, 1979; Reid et al., 1999). Clinically,
the most common symptoms include cough,
fever, headache, and weakness (Monto et al.,
2000). These symptoms are often accompanied
by gastroenteritis-like symptoms in many influenza patients, such as abdominal pain, nausea,
vomiting, and diarrhea, especially in young children (Baden et al., 2009; Shinde et al., 2009;
Dilantika et al., 2010). However, the immune
mechanisms underlying these clinical manifestations in the intestine during a lung-tropic
viral influenza infection remain unclear.
The intestinal tracts in humans and other
animals are inhabited by hundreds of diverse
species of commensal bacteria, which are essential in shaping intestinal immune responses during both health and disease (Hooper and Gordon,
*J. Wang and F. Li contributed equally to this paper.

The Rockefeller University Press $30.00
J. Exp. Med. 2014 Vol. 211 No. 12 23972410
www.jem.org/cgi/doi/10.1084/jem.20140625
2001; Chervonsky, 2009). Distinct components

of commensal bacteria were associated with special status of the immune system. Although most
commensal bacteria are beneficial (Ichinohe et al.,
2011), a few can be potentially harmful in some
conditions; for example, some commensal bacteria have been suggested to influence susceptibility to inflammatory bowel disease (IBD; Garrett
et al., 2007; Mazmanian et al., 2008).Thus, when
conditions in the host are unfavorable, such as
during infection, the resulting changes within
the intestinal tract environment may promote
growth of the harmful bacteria that induce intestinal disease.
It is well known that the respiratory and intestinal tracts are both mucosal tissues. Over 30 years
ago, John Bienenstock hypothesized that the
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2397
Figure 1. Respiratory influenza virus

infection causes lung and intestinal immune injury. C57BL/6 mice were i.n. infected
with saline or 0.1 HA of PR8. (A) Body weight
was monitored after PR8 infection. (B) The
pathology of lung and small intestine was
assayed after PR8 infection. (C) The length of
colon was recorded after PR8 infection.
(D) The severity of the diarrhea was scored
after PR8 infection (0, normal stool or absent;
1, slightly wet and soft stool; 2, wet and unformed stool with moderate perianal staining
of the coat; and 3, watery stool with severe
perianal staining of the coat). (E) The pathology of liver and kidney was assayed after PR8
infection. (F) Serum ALT and BUN levels were
measured after PR8 infection (dashed lines
represent damage threshold). All tissue sections were stained with H&E. Bars, 100 m.
Data represent three independent experiments with at least five mice/group in A, C,
and D or three mice/group in B, E, and F. Data
are expressed as mean SEM by a Students
t test. ***, P < 0.001.
immune cells and structures contained in mucosal tissues were

universally connected within the whole body. This common
mucosal immune system concept speculated that the mucosal
immune system was itself an organ in which the mucosal
immune cells distributed throughout the body could interplay between or among different mucosal tissues or organs
(McDermott and Bienenstock, 1979; McDermott et al., 1980).
Although this term was coined three decades ago, appreciation
of its importance is only just beginning. Much was learned
from the numerous studies conducted on the mucosal immune
system during this time, which mainly focused on understanding its individual components (Holmgren and Czerkinsky,
2005; Sato and Kiyono, 2012). Although a few studies have
suggested that the mucosal immune system is a system-wide
organ (Gallichan et al., 2001; Sobko et al., 2010), some questions
still need to be clarified. For example, how do the different components affect each other, and how is cross talk achieved among
the various mucosal sites (Gill et al., 2010)?
In this study, we found that lymphocytes derived from the
respiratory mucosa specifically migrated into the intestinal
mucosa during respiratory influenza infection by the CCL25
CCR9 chemokine axis and destroyed the intestinal microbiota
homeostasis in the small intestine, finally leading to intestinal
immune injury. Our findings may provide new insights into not
only the mechanisms underlying intestinal immune injury induced by influenza infection of the lung but also the interplay
of immune cells between or among different mucosal sites.
2398
RESULTS
Intranasal (i.n.), but not intragastric (i.g.), infection
with influenza virus causes intestinal immune injury
To test whether intestinal injury was also a feature in a mouse
model of influenza, we infected mice i.n. with the A/PR/8/34
(PR8) influenza virus strain. Indeed, their body weight gradually decreased from days 2 to 9 as compared with saline-treated
controls, which maintained their body weight over the same
period (Fig. 1 A). Furthermore, both the lung and small intestine had severe injury after PR8 infection (Fig. 1 B). Colon
length was shortened (Fig. 1 C) and mild diarrhea occurred
(Fig. 1 D), further indicating intestinal injury (Zaki et al., 2010;
Murray and Rubio-Tapia, 2012). In contrast, nonmucosal liver
and kidney tissues appeared normal after PR8 infection (Fig. 1 E),
which was also supported by ALT and BUN analysis (Fig. 1 F).
Together, these data indicate that respiratory influenza infection causes severe immune injury not only in the lung but also
in the intestine.
To rule out the possibility that the influenza virus entered
the gastrointestinal tract and directly caused immune injury
at this site, we tested for the presence of virus within the small
intestine after i.n. infection and found that the influenza virus
could not be detected at this site (Fig. 2 A). To test this possibility in a more rigorous way, we i.g. infected mice with PR8
and found that live virus could be detected in the intestinal
contents and intestinal tissues in a short time after infection, and
virus was completely cleared from these sites 3 d after infection
Microbiota cause intestine injury during influenza | Wang et al.
Ar ticle
Figure 2. Influenza virus does not infect the

small intestine directly. (A) C57BL/6 mice were i.n.
infected with 0.1 HA of PR8. The levels of the influenza virusderived matrix protein gene in lung and
small intestine were detected by PCR. (BE) C57BL/6
mice were i.g. infected with saline or 0.1 HA of PR8.
Viral titer in intestinal contents was determined by
50% tissue culture infective dose (TCID50) assay after
PR8 infection (B). The levels of the influenza virus
derived matrix protein gene in small intestine were
detected by PCR after PR8 infection (C). The pathology of lung and small intestine was assayed after
PR8 infection, and tissue sections were stained with
H&E. Bar, 100 m (D). The length of colon was recorded after PR8 infection (E). Data represent three
independent experiments with at least three mice/
group in AE. Data are expressed as mean SEM by a
Students t test. NS: not significant.
(Fig. 2, B and C). However, pathological injury was not found

in any of the examined tissues (Fig. 2, D and E). These results
collectively suggest that influenza infection does not directly
cause immune injury in the small intestine.Thus, we unexpectedly observed that influenza infection induced severe immune
injury within the intestine only when the virus infected the respiratory tract and immune injury occurred in the lung.
Intestinal microbiota is required for influenza-induced
intestinal immune injury
Changes in intestinal microbiota are often involved in the occurrence of intestinal inflammation in many mouse models
(Lupp et al., 2007; Maslowski et al., 2009). To determine
whether intestinal microbiota was involved in influenza
induced intestinal immune injury, we first assayed whether viral
infection affected the relative composition of several major
bacterial groups within the intestinal microbiota. Although
the number of total bacteria remained the same after infection as quantified by both real-time PCR and selective culture (Fig. 3 A), the numbers of segmented filamentous bacteria
(SFB) and Lactobacillus/Lactococcus decreased after PR8 infection, whereas the number of Enterobacteriaceae increased; moreover, the numbers of mouse intestinal Bacteroides, Eubacterium
rectale/Clostridium coccoides, and Bacteroides were unchanged
(Fig. 3 B). We next administered combinatorial antibiotics to
the mice via their drinking water to deplete intestinal microbiota (Ichinohe et al., 2011) 4 wk before infecting them with
PR8. In antibiotic-treated mice, the lungs still sustained severe
immune-mediated injury after PR8 infection, but the small
intestine and colon were protected (Fig. 3, C and D). In another
way, transferring intestinal microbiota from PR8-infected mice
JEM Vol. 211, No. 12
into healthy WT mice increased the number of Enterobacteriaceae

and caused intestinal immune injury in recipient mice even
in the absence of viral infection as compared with the intestinal microbiota from saline-treated mice (Fig. 3, E and F).Thus,
these data suggest that respiratory influenza infection induces
intestinal immune injury by altering the composition of intestinal microbiota.
Escherichia coli is an important component of Enterobacteriaceae, and pathogenic E. coli infection often causes vomiting and
diarrhea in humans (Ochoa and Contreras, 2011).The number
of E. coli in the intestinal tract significantly increased after PR8
infection (Fig. 3 G). Treating mice with streptomycinan
antibiotic to which E. coli is sensitiveprotected mice against
PR8 infection-induced immune injury to the small intestine
by inhibiting the increase of Enterobacteriaceae (Fig. 3, H and I).
Furthermore, directly infecting mice i.g. with E. coli caused
immune injury in the small intestine (Fig. 3 J).Thus, these data
suggest that the increase of E. coli may be the primary cause for
intestinal immune injury during influenza infection.
Th17 cells mediate influenza-induced intestinal immune injury
To explore the mechanism by which intestinal bacteria caused
intestinal immune injury during influenza infection, many different types of proinflammatory cells involved in intestinal
inflammation (Zhou et al., 2007a; Kleinschek et al., 2009;
Leppkes et al., 2009) were examined. Depletion of NK1.1+
by specific antibodies or T cell deficiency could not reduce the PR8 infection-induced intestinal immune injury in
our study (Fig. 4, A and B). However, no intestinal injury was
observed in IL-17A/ mice after PR8 infection (Fig. 4 C),
2399
Figure 3. Antibiotic treatment reduces influenza-induced intestinal immune injury. (A) Bacteria in the small intestine were assayed by real-time
PCR and selective culture in blood plate 7 d after PR8 infection. (B) Several major bacterial groups in intestinal microbiota were assayed by real-time PCR
7 d after PR8 infection. (C and D) C57BL/6 mice were subjected to a 4-wk oral treatment of combinatorial antibiotics in drinking water, followed by
i.n. infection with saline or 0.1 HA of PR8. The pathology of lung and small intestine was assayed 7 d after PR8 infection (C). The length of colon was
recorded 7 d after PR8 infection (D). (E and F) Transfer of intestinal microbiota from saline-treated or PR8-infected mice into healthy WT mice by the
i.g. route. Major bacterial groups in the intestinal microbiota (E) and the pathology of small intestine were assayed 6 d later (F). (G) The number of E. coli
in stool was detected by E. coli/Coliform Count Plates 6 d after PR8 infection. (H and I) C57BL/6 mice were subjected to a 1-wk oral treatment of streptomycin
in their drinking water and then were i.n. infected with 0.1 HA of PR8. The pathology of lung and small intestine (H) and major bacterial groups in intestinal microbiota (I) were assayed 6 d after PR8 infection. (J) C57BL/6 mice were i.g. infected with saline or 5 108 E. coli, and the pathology of small intestine was assayed 3 d later. All tissue sections were stained with H&E. Bars, 100 m. Data represent two independent experiments with three mice/group
in I and J or three independent experiments with at least three mice/group in AH. Data are expressed as mean SEM by a Students t test. *, P < 0.05;
**, P < 0.01; NS: not significant.
2400
Ar ticle
Figure 4. IL-17A deficiency reduces

influenza-induced immune injury in small
intestine but not in lung. (A) The pathology
of lung and small intestine from control and
PK136-treated mice was assayed 6 d after
PR8 infection. (B) The pathology of lung and
small intestine from WT and Tcrd/ mice
was assayed 6 d after PR8 infection. (C) The
pathology of lung and small intestine from
WT and IL-17A/ mice was assayed 6 d after
PR8 infection. (D) IL-17A and IL-17F expressions in the lung from WT mice were detected by real-time PCR 6 d after PR8
infection. (E) Body weight of WT and
IL-17A/ mice was monitored after PR8 infection. (F) Evans blue dye concentration in BALF
from WT and IL-17A/ mice was determined
by spectrophotometer 6 d after PR8 infection. (G and H) Total protein (G) and lactate
dehydrogenase (H) levels in BALF from WT
and IL-17A/ mice were determined by
ELISA 6 d after PR8 infection. All tissue sections were stained with H&E. Bars, 100 m.
Data represent two independent experiments
with five mice/group in EH or three mice/
group in AD. Data are expressed as mean
SEM by a Students t test. *, P < 0.05; **,
P < 0.01; ***, P < 0.001; NS: not significant.
suggesting that Th17 cells might be involved in influenzainduced intestinal immune injury.
To rule out the possibility that lung injury might also be
reduced in IL-17A/ mice after influenza infection, which
subsequently resulted in reducing the small intestinal injury
indirectly, we compared the degree of the lung injury after
influenza infection between WT and IL-17A/ mice.The results showed that both IL-17F and IL-17A expressions in lung
from WT mice were increased after PR8 infection (Fig. 4 D).
Compared with WT mice, IL-17A/ mice exhibited reduced body weight loss during PR8 infection (Fig. 4 E). However, the degree of lung leak and the levels of total protein and
lactate dehydrogenase in bronchoalveolar lavage fluid (BALF)
were not significantly different between WT and IL-17A/
mice (Fig. 4, FH), suggesting that the lung injury did not reduce in IL-17A/ mice after influenza infection when compared with WT mice.Thus, these data suggest that the decrease
of immune injury in the small intestine from IL-17A/ mice
after influenza infection is independent of the decrease of
lung injury.
To further determine that Th17 cells were responsible for
influenza-induced intestinal immune injury, we detected the
expression of Th17-specific transcription factor RORt and
IL-17A and found that their expressions increased in the small
intestine after PR8 infection (Fig. 5 A). The percentage and
number of Th17 cells increased in the small intestine and colon
after PR8 infection (Fig. 5, B and C), but not in the liver or kidney (Fig. 5 D), consistent with previous observations (Esplugues
et al., 2011). Furthermore, treating mice i.p. with a neutralizing

antiIL-17A antibody during PR8 infection effectively reduced
intestinal injury (Fig. 5 E). Together, these data suggest that
influenza infectioninduced intestinal immune injury is dependent on Th17 cells.
Because we observed that influenzainduced intestinal immune injury is dependent on both intestinal microbiota and
Th17 cells, we wondered whether there was an association
between intestinal bacteria and Th17 cells.The results showed
that the percentage and number of Th17 cells in the small intestine were unchanged in antibiotic-treated mice after PR8
infection as compared with uninfected control mice (Fig. 5 F);
transferring intestinal microbiota from PR8-infected mice into
healthy WT mice promoted IL-17A expression in the small
intestine of recipient mice (Fig. 5 G); and streptomycin treatment inhibited IL-17A expression in the small intestine during PR8 infection (Fig. 5 H). Collectively, these data suggest
that changes in intestinal microbiota induced by influenza infection promote Th17 cell production, which subsequently
causes intestinal immune injury.
CCL25/CCR9 mediates the recruitment of lung-derived
CD4+ T cells into the small intestine
Because respiratory influenza infection influences the composition of intestinal microbiota, which subsequently promotes
Th17 cell production and causes intestinal immune injury, we
wanted to know how respiratory influenza infection destroyed
the microecological homeostasis of the intestinal microbiota.
2401
Figure 5. Increased Th17 cells occur in the

small intestine during influenza virus infection. (A) RORt and IL-17A expressions in the
small intestine were detected by real-time PCR
7 d after PR8 infection. (B) The percentage and
number of Th17 cells in intestinal IEL and LPL
were detected 7 d after PR8 infection. (C) The
percentage and number of Th17 cells in colonic
LPL were detected 7 d after PR8 infection. (D) The
number of Th17 cells in liver and kidney was detected 7 d after PR8 infection. (E) C57BL/6 mice
were i.p. treated with a neutralizing antiIL-17A
antibody during PR8 infection. The pathology of
lung and small intestine was assayed 6 d after
PR8 infection, and tissue sections were stained
with H&E. Bars, 100 m. (F) The percentage and
number of Th17 cells in IEL and LPL were detected
7 d after PR8 infection in antibiotic-treated mice.
(G) Transfer of intestinal microbiota from salinetreated or PR8-infected mice into healthy WT
mice by the i.g. route. IL-17A expression in the
small intestine was detected by real-time PCR 6 d
later. (H) IL-17A expression in the small intestine
was detected by real-time PCR at day 6 after PR8
infection in streptomycin-treated mice. Data
represent two independent experiments with
three mice/group in A, D, E, G, and H or three
independent experiments with three mice/group
in B, C, and F. Data are expressed as mean SEM
by a Students t test. *, P < 0.05; **, P < 0.01;
***, P < 0.001; NS: not significant.
Given that influenza infection specifically caused immune injury in the respiratory and intestinal mucosal tissues, but not in
the nonmucosal liver or kidney in our study, an interconnected
relationship existed between them was intriguing according
to the common mucosal immune system theory (McDermott
and Bienenstock, 1979; McDermott et al., 1980). The CCL25
chemokine is expressed by intestinal epithelial cells (IECs) and
functions to specifically guide CCR9-expressing effector lymphocytes into the small intestine as a homing mechanism
(Campbell and Butcher, 2002). Consistent with previous observations, CCL25 expression in the small intestine tissue was
much higher than any other tissues, including liver, kidney, and
lung (Fig. 6 A). Treating mice i.v. with a neutralizing antiCCL25 antibody during PR8 infection reduced intestinal
immune injury (Fig. 6 B), inhibited the changes in intestinal
microbiota (Fig. 6 C), and reduced the number of Th17 cells
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in the small intestine (Fig. 6 D). These results suggest that the
CCL25CCR9 axis contributes to altering the composition
of the intestinal microbiota after influenza infection and the
subsequent development of intestinal inflammation via recruiting effector lymphocytes into the intestinal mucosa.
Next, we explored which lymphocyte subsets were recruited
by CCL25 in our influenza model. Although the total number
of T cells increased in the LPL after PR8 infection, the total
number of B cells decreased (Fig. 6 E).Within the T cell population, the CCR9+CD4+ T cell subset increased (Fig. 6 F), whereas
the CCR9+CD8+ T cell subset remained unchanged (Fig. 6 G),
indicating that CCR9+CD4+ T cells might play a key role in altering the intestinal microbiota. Evaluating this subpopulation
in other tissues revealed that the number of CCR9+CD4+ T cells
was significantly increased in the lung and in the mediastinal
LNs after PR8 infection, but not in the mesenteric LNs (Fig. 6 G),
Ar ticle
Figure 6. Anti-CCL25 antibody treatment reduces influenzainduced intestinal immune injury. (A) CCL25 expression in various tissues was detected by real-time PCR 4 d after PR8 infection. (BD) C57BL/6 mice were i.v. treated with a neutralizing anti-CCL25 antibody during PR8 infection. The
pathology of lung and small intestine (B), major bacterial groups in intestinal microbiota (C), and the number of Th17 cells in IEL and LPL were assayed 7 d
after PR8 infection (D). (EG) C57BL/6 mice were i.n. infected with saline or 0.1 HA of PR8. The number of T and B cells in LPL (E), the percentage and
number of CCR9+CD4+ T cells in small intestine (F), and the number of CCR9+CD8+ T cells in LPL and CCR9+CD4+ T cells in lung, mediastinal LNs, and mesenteric LNs were assayed 7 d after PR8 infection (G). (H) ALDH1A2 expression in lung was detected by real-time PCR 6 d after PR8 infection. (I) CD4+
T cells from the lungs of saline- or PR8-infected CD45.1+ mice were adoptively transferred into WT CD45.2+ mice, and the percentage of CD45.1+CD4+
T cells in total CD4+ T cells in LPL from recipient CD45.2+ mice was detected by flow cytometry 48 h later. (J) C57BL/6 mice were i.n. infected with saline or
0.1 HA of PR8. CD4+ T cells in the lung and LPL were purified 6 d later by MACS and then co-cultured with antigen-presenting cells and heat-killed PR8
in an IFN- ELISPOT plate. The number of positive spots was counted 20 h later. (K) Parabiotic pairs of WT mice were established first, and the left partner
was i.n. infected with PR8 2 wk later. The pathology of small intestine was assayed 6 d after PR8 infection. All tissue sections were stained with H&E.
Bars, 100 m. Data represent three independent experiments with three mice/group in AH and K or three wells/treatment in J. Data are expressed as
mean SEM by a Students t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS: not significant.
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Figure 7. Lung-derived CD4+ T cells influence microbiota and intestine injury by secreting IFN-. (A) IFN- expression in CD4+ T cells from lung
was detected by flow cytometry 6 d after PR8 infection in WT mice. (B) The pathology of small intestine was assayed at day 7 after PR8 infection in WT
and IFN-/ mice, and tissue sections were stained with H&E. Bars, 100 m. (C and D) IL-17A expression in the small intestine (C) and major bacterial
groups in intestinal microbiota (D) were assayed at day 7 after PR8 infection in IFN-/ mice. (E and F) IL-17A expression in CD4+ T cells from lung (E)
and the percentages of CCR9 Th17 cells and CCR9+ Th17 cells in lung and LPL (F) were detected 6 d after PR8 infection in WT mice. (G) IL-17A expression
in CD4+ T cells from mesenteric LNs, Peyers patches, and blood was detected by flow cytometry 6 d after PR8 infection in WT mice. (H) IL-17A level in
serum was detected by ELISA 6 d after PR8 infection in WT mice. (I) LPL from WT mice at day 6 after PR8 infection was stimulated by heat-killed E. coli
in vitro; 24 h later, the expression of IL-17A in CD4+ T cells was detected by flow cytometry. (J) Lung lymphocytes and LPL from WT mice at day 6 after PR8
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Ar ticle
suggesting that the lung and mediastinal LNs might be the main
sources of CCR9+CD4+ T cells recruited to the small intestine
during PR8 infection. Retinoic acid is reported to promote the
expression of CCR9 on T cells (Ohoka et al., 2011), and the
production of retinoic acid is regulated by the aldehyde dehydrogenase (ALDH) 1A2 (Yokota et al., 2009). In our study, the
expression of ALDH1A2 in lung increased after influenza infection (Fig. 6 H), suggesting that the increase of retinoic acid in
lung after influenza infection might be responsible for promoting the CCR9 expression on lung CD4+ T cells.
To determine whether influenza infectionactivated lung
CD4+ T cells tended to migrate into the small intestine, we
adoptively transferred lung CD4+ T cells from saline- or PR8infected CD45.1+ mice into recipient WT CD45.2+ mice and
found that LPL in recipient mice contained a higher frequency
of CD45.1+CD4+ T cells from PR8-infected CD45.1+ mice
(Fig. 6 I). Moreover, PR8-specific CD4+ T cells were detected
not only in lung but also in the small intestine after PR8 infection, as assessed by the IFN- ELISPOT plate (Fig. 6 J),
and, in a parabiotic mice model, PR8 infection in one partner
caused small intestinal injury to occur in a noninfected partner
(Fig. 6 K). Thus, these data suggest that the CCL25CCR9
axis mediates the recruitment of lung-derived effector CD4+
T cells into the small intestine as well as the alterations to the
intestinal microbiota composition during influenza infection.
Lung-derived CD4+ T cells destroy microbiota homeostasis
and promote resident Th17 cell polarization
As we found that lung-derived effector CD4+ T cells are
recruited into the small intestine and alter the intestinal microbiota during influenza infection, we wondered how they
influenced the intestinal microbiota composition and whether
Th17 cells in the small intestine originated from the polarization of them. For the first question, IFN- expression was found
to be significantly increased in lung CD4+ T cells after PR8
infection (Fig. 7 A). When IFN- was deficient, the mice exhibited reduced intestinal immune injury, normal IL-17A expression, and unchanged intestinal microbiota in the small
intestine after PR8 infection (Fig. 7, BD). Thus, these data
suggest that lung-derived effector CD4+ T cells destroy the
homeostasis of intestinal microbiota by secreting IFN-. For
the second question,Th17 cells were not found to be increased
in lung after PR8 infection (Fig. 7 E) and, although some CCR9+
Th17 cells were present in the small intestine, most Th17 cells
(90%) exhibited a CCR9 phenotype (Fig. 7 F). Meanwhile,Th17 cells were also not found increased in the mesenteric LNs, Peyers patches, and blood (Fig. 7 G), and IL-17A
levels in blood did not increased after PR8 infection (Fig. 7 H).
More convincing evidences showed that E. colispecific Th17
cells could be detected in the small intestine (Fig. 7 I), but PR8specific Th17 cells could not be detected both in the lung and
small intestine (Fig. 7 J). Thus, these data suggest that Th17 cell
polarization, but not recruitment, occurs in the small intestine
in situ during influenza infection.
Intestinal microbiotainduced IL-15 production
promotes intestinal Th17 cell polarization
Because Th17 cell polarization occurs in the small intestine in
situ during influenza infection, we next explore what kind of
factors mediated this process. IL-6 expression in the small intestine was increased after PR8 infection, but IL-23 and TGF-
expressions were unchanged (Fig. 8 A). However, treating mice
i.v. with a neutralizing antiIL-6 antibody during PR8 infection
could not reduce intestinal immune injury (Fig. 8 B). Thus, the
increase of IL-6 is not the main reason for Th17 cell polarization
in our study. IL-15 has been reported to contribute to intestinal
inflammation in various mouse models (Zhou et al., 2007b;
Schulthess et al., 2012) and, importantly, it has been shown to induce IL-17A expression in both mice and human CD4+ T lymphocytes (Ziolkowska et al., 2000; Ferretti et al., 2003). In our
study, IL-15 expression in the small intestine, but not in serum,
was up-regulated after PR8 infection (Fig. 8 C).Transferring intestinal microbiota from PR8-infected mice also increased IL-15
expression in the small intestine of recipient mice (Fig. 8 D). To
explore whether IL-15 contributed to Th17 cell polarization in
our study, we first assayed the expression of IL-15 receptor and
found that intestinal CD4+ T cells expressed the IL-15 receptor
after PR8 infection (Fig. 8 E). Next, treating mice with a neutralizing antiIL-15 antibody during PR8 infection effectively
reduced intestinal immune injury (Fig. 8 F).Thus, IL-15, which
was induced by intestinal bacteria, contributes to intestinal immune injury during influenza infection. Further experiments
showed that IL-15 neutralization inhibited IL-17A and IL-6 expression in the small intestine after PR8 infection (Fig. 8 G) and,
consistent with the previous observations (Ziolkowska et al.,
2000; Ferretti et al., 2003), exogenous IL-15 promoted IL-17A
secretion in purified CD4+ T cells from LPL in vitro (Fig. 8 H),
suggesting that intestinal bacteriainduced IL-15 might promote
Th17 cell polarization in the small intestine in situ by a direct
and/or indirect way. However, IL-15 neutralization did not influence the changes of the intestinal microbiota (Fig. 8 I), suggesting that IL-15 functioned upstream of IL-17A production but
downstream of the change in microbiota after PR8 infection.
Exploring the in vivo cellular sources of IL-15, high IL-15
expression was detected in IECs after PR8 infection (Fig. 8 J),
suggesting that IECs might be an important source of IL-15
in the small intestine during influenza infection.
DISCUSSION
Mucosal tissues, including the gastrointestinal, respiratory, and
urogenital tracts, etc., are the first line of host defense against external invaders. Although much has been learned from studying
infection were stimulated by heat-killed PR8 in vitro; 24 h later, the expression of IL-17A in CD4+ T cells was detected by flow cytometry. Data represent
three independent experiments with three mice/group in AH or three wells/treatment in I and J. Data are expressed as mean SEM by a Students t test.
*, P < 0.05; **, P < 0.01; ***, P < 0.001; NS: not significant.
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Figure 8. Intestinal microbiota induces

Th17 cell polarization in situ via triggering IL-15 production. (A) IL-6, IL-23, and
TGF- expressions in the small intestine were
detected by real-time PCR 6 d after PR8 infection. (B) The pathology of lung and small
intestine from control and antiIL-6treated
mice was assayed 6 d after PR8 infection.
(C) IL-15 expression in the small intestine and
serum was detected 6 d after PR8 infection.
(D) Transfer of intestinal microbiota from
saline-treated or PR8-infected mice into
healthy WT mice by the i.g. route. IL-15 expression in the small intestine was detected
6 d later. (E) IL-15R expression on CD4+ T cells
in LPL from WT mice was detected at day 6
after PR8 infection. (F and G) C57BL/6 mice
were i.p. treated with a neutralizing antiIL-15
antibody during PR8 infection. The pathology
of lung and small intestine (F) as well as
IL-17A and IL-6 expressions in the small intestine were assayed 6 d after PR8 infection
(G). (H) MACS-purified CD4+ T cells from LPL
were stimulated by IL-15 in vitro, and IL-17A
levels in supernatant were measured at days 2
and 3 by ELISA. (I) Major bacterial groups in
the intestinal microbiota from control and
antiIL-15treated mice were assayed by realtime PCR 6 d after PR8 infection. (J) IL-15
expression in IECs was detected 6 d after PR8
infection in WT mice. All tissue sections were
stained with H&E. Bars, 100 m. Data represent three independent experiments with
three mice/group in AG, I, and J or three
wells/treatment in H. Data are expressed as
mean SEM by a Students t test. *, P < 0.05;
**, P < 0.01; ***, P < 0.001; NS: not significant.
each of these components individually, the mucosal immune

system has not yet been examined from a holistic point of view
as a system-wide organ (Gill et al., 2010), as conceptualized by
the common mucosal immune system hypothesis. Unexpectedly, we observed that respiratory influenza infection in mice
caused immune injury not only in the lung but also specifically
in the intestine, as it had no influence on the pathology in nonmucosal organs such as the liver or kidney. Because this resembles the symptoms exhibited by humans after influenza infection,
these influenza virusinfected mice provide a good model in
which to study the mechanisms underlying how respiratory influenza infection causes intestinal immune injury; furthermore,
these observations provide further evidence to support the existence of a common mucosal immune system.
Pathogens extensively disseminate beyond the limits of the
primary infection site in almost all cases of infectious diseases,
2406
particularly at the early stage of infection. Although some studies suggest that influenza virus disseminates into extrapulmonary tissues or organs during infection (Korteweg and Gu,
2008), others contradict this finding (Mauad et al., 2010), and
direct evidence for viral replication in extrapulmonary tissues
or organs has not yet been shown (Kuiken and Taubenberger,
2008). It therefore remains a mystery how influenza infection
can be associated with immune injury to extrapulmonary tissues or organs if these injuries are not induced by direct virus
infection of these tissues or organs (Polakos et al., 2006; Mauad
et al., 2010). In our mouse model of respiratory influenza infection, no influenza virus was detected in the small intestine,
and i.g. administration of the influenza virus directly into the
intestine did not lead to intestinal immune injury. Thus, the
intestinal immune injury observed in our study was not directly
caused by influenza infection of the intestine.
Ar ticle
An explosive increase in neutrophils is responsible for

influenza-induced acute lung injury and death. IL-17 is a potent regulator for the neutrophils recruitment. Previous studies
have shown that respiratory influenza infection increases the
IL-17A and IL-17F expressions in lung, and IL-17RA/
mice exhibit reduced lung injury and higher survival rates
after influenza infection (Crowe et al., 2009; Li et al., 2012).
In our mouse model of respiratory influenza infection, IL-17A
and IL-17F expressions in lung also increased after infection,
but IL-17A deficiency could not reduce influenza-induced
lung injury. Thus, based on above results, we speculated that
IL-17A and IL-17F played the same function during influenza infection, and IL-17F alone might be enough to function
to activate IL-17RA and recruit neutrophils when IL-17A
was deficiency.
Recruitment and infiltration of inflammatory cells into the
gastrointestinal mucosa critically regulates the development as
well as progression of IBD (Wurbel et al., 2011). Differential
expression of chemokine receptors and adhesion molecules
on lymphocytes not only determine their migration into different tissues but also their localization within these tissues.
CCL25 is constitutively expressed by the epithelium of the small
intestine (Papadakis et al., 2000), and the CCL25CCR9 chemokine axis is considered to be one of the few non-promiscuous
chemokine/receptor pairs involved in gut-specific migration
of lymphocytes (Stenstad et al., 2006). In our study, the percentage and number of CCR9+CD4+ T cells in both lung and
small intestine increased after influenza infection, and neutralizing CCL25 with antibody treatment reduced CCR9+CD4+
T cell recruitment and intestinal immune injury. Thus, these
data might explain why influenza infection specifically caused
immune injury in the intestine, but not the liver or kidney, in
our study.
IBD is a common disease characterized by severe inflammation of the intestine (Hooper and Macpherson, 2010). However, the exact causes of this disease remain unclear. Some studies
suggest that IBD arises from dysregulated control of host
microorganism interactions. For example, patients with this
disease have an increased number of epithelial cell surface
associated bacteria (Swidsinski et al., 2005), suggesting the failure of a mechanism designed to limit direct contact between
the epithelium and the microbiota. Similarly, in our study, we
also found that CCR9+CD4+ T cell recruitment correlated
to intestinal inflammation by the following mechanism: the
CCR9+CD4+ T cells destroyed the homeostasis of the intestinal
microbiota, the altered microbiota then promoted Th17 cell polarization in the small intestine in situ, and the resulting IL-17A
secretion finally mediated the intestinal immune injury.
This concept of the common mucosal immune system proposed by John Bienenstock suggests that the mucosal immune
system may be considered as one large interconnected network
(McDermott and Bienenstock, 1979; McDermott et al., 1980),
which is supported by some recent researches. For example,
i.n. immunization results in vaginal protection against genital
HSV-2 infection (Neutra and Kozlowski, 2006), and antibiotics used in neonates increases the risk to develop asthma (Sobko
et al., 2010). These findings suggest that potential exists for an

undetermined link between mucosal immune components and
that each component is efficient at sharing information distally
(Gill et al., 2010). In our study, we found that lung-derived virusspecific effector CCR9+CD4+ T cells were recruited into the
small intestine and destroyed the homeostasis of intestinal microbiota by secreting IFN- after influenza infection. Thus,
we speculated that the effector CCR9+CD4+ T cells might enter
into the small intestine by a special way as described above and
remained in the active state to secrete IFN- even in the absence of antigen stimulation.
The intestinal microbiota is extensively accepted in the
field as a virtual metabolic organ in and of itself (OHara and
Shanahan, 2006). Beyond this role in metabolism, the intestinal
microbiota has a conspicuous effect on host immune functions,
as indicated by comparing immune responses between germfree and conventional animals. A previous study showed that
commensal SFBs induce IL-6 and IL-23 production to stimulate Th17 cell polarization (Ivanov et al., 2009). However, in
our mouse model of respiratory influenza infection, the number of SFB decreased while the number of E. coli increased in
the intestinal tract after influenza infection; E. coli promoted
IL-15 expression in IECs, and this IL-15 then promoted Th17
cell polarization. Moreover, overgrowth of existing strain
and/or acquisition of new pathogenic strain are involved in
E. colicaused gastrointestinal symptoms (Nguyen et al., 2006;
Ochoa and Contreras, 2011). In our study, considering that the
mice live in an SPF environment and that intestinal inflammation occurs in different kinds of mice, we think that the overgrowth of existing E. coli in the gut may be the primary cause
for intestinal immune injury during influenza infection.
The function of IL-15 in regulation of Th17 responses has
been studied extensively, but there are still some controversies.
Some studies found that IL-15 induces IL-17A expression in
both mice and human CD4+T lymphocytes directly (Ziolkowska
et al., 2000; Ferretti et al., 2003), which was also demonstrated
in our study. However, another study showed that IL-15 inhibits Th17 cell polarization in a mouse model of EAE (Pandiyan
et al., 2012). We thought that there were two main reasons to
explain why IL-15 played the opposite effect in different mouse
model: (1) the immuno-microenvironment in different mouse
model is different; (2) IL-15 is reported to activate both STAT3
and STAT5 (Johnston et al., 1995), which is inferred either to
inhibit or to promote Th17 cells.
MATERIALS AND METHODS
Mice, virus, and bacteria. Male C57BL/6 mice were purchased from
the Shanghai Laboratory Animal Center, Chinese Academy of Sciences.
IFN-/, Tcrd/, and IL-17A/ mice were purchased from The Jackson
Laboratory. All mice were housed under specific pathogen-free conditions at
the School of Life Sciences, University of Science and Technology of China
(USTC), and were used at 610 wk of age. Animal care and experimental
procedures were followed in accordance with the experimental animal guidelines at USTC. Mouse-adapted influenza A/PR/8/34 strain (H1N1) was a
gift from H. Meng (Institute of Basic Medicine, Shandong Academy of Medical Sciences, Shandong, China). For influenza infection studies, mice were
anesthetized and infected i.n. with 0.1 HA of PR8 in 50 l sterile saline.
2407
E. coli strain was isolated from stool of PR8-infected mice by the 3M Petrifilm E. coli/Coliform Count Plate and was cultured in broth medium for
amplification. For E. coli infection, mice were anesthetized and infected i.g.
with 5 108 E.coli in 500 l sterile saline.
Histopathology. Lung, small intestine, liver, and kidney tissues were removed and fixed immediately in 10% neutral-buffered formalin in PBS for
>24 h, embedded in paraffin, and cut into 57-m sections. The sections
were deparaffinized and stained with hematoxylin and eosin (H&E) to determine histological changes.
Analysis of lung injury. Lung leakage: 1 h before sacrificing mice, 20 mg/kg
Evans blue dye was administered i.v. The lung was instilled with 1 ml of
saline, and the BALF was collected. After centrifugation, Evans blue dye concentration in supernatant was determined by spectrophotometer at 620 nm.
Total protein and lactic dehydrogenase in BALF: The lung was instilled with
1 ml saline, and the BALF was collected. After centrifugation, the level of
total protein in supernatant was assayed by the BCA protein assay kit, and the
level of lactic dehydrogenase in supernatant was assayed by ELISA kit (CloudClone Corp).
Analysis of liver and kidney function. Serum from infected mice or
control mice were collected and stored at 80C until analysis. Liver function was determined by measuring serum ALT (alanine aminotransferase) using
a commercially available kit (Rong Sheng). Kidney function was assessed by
measuring serum BUN (blood urea nitrogen) using a commercially available
kit (Jiancheng Bioengineering Institute).
Determination of virus and bacteria. Influenza virus in the lung and
small intestine were detected by PCR. The primer sequences to detect the
gene encoding the matrix protein within the influenza virus were as follows:
5-GGACTGCAGCGTAGACGCTT-3 (forward) and 5-CATCCTGTTGTATATGAGGCCCAT-3 (reverse). Intestinal bacterial genomic DNA
was extracted from the stool using a stool kit (QIAGEN) according to the
manufacturers instruction (the optional high-temperature step was performed). The abundance of total and specific intestinal bacterial groups was
measured by real-time PCR with corresponding 16S rDNA gene primers
(Sangon Biotech; Table S1). The number of E. coli in stool was detected by
the 3M Petrifilm E. coli/Coliform Count Plate according to the manufacturers instructions.
Isolation of IEC, IEL, and LPL. IECs were isolated as described in a previous study (Zhou et al., 2007a). IELs and LPLs were isolated as previously
described with minor modifications (Das et al., 2003; Kamanaka et al., 2006;
Esplugues et al., 2011). In brief, small intestines were harvested and washed
with PBS, and mesentery and Peyers patches were carefully dissected out. Intestines were opened longitudinally and then cut into 1-cm pieces. Intestinal
pieces were incubated in 10 ml of extraction buffer (5% FCS, 1 mM DTT,
and 5 mM EDTA in PBS) at 37C for 30 min.The released cells were loaded
onto a Percoll gradient and centrifuged.The cells at the interface of a 40/70%
Percoll solution were collected and used as IELs. The remaining segments
were incubated twice in extraction buffer to remove IELs and isolate LPLs.
The tissue was digested with prewarmed complete RPMI1640 containing
2 mg/ml collagenase IV at 37C for 60 min, loaded onto a Percoll gradient,
and centrifuged. The cells at the interface of a 40/70% Percoll solution were
collected and used as LPLs.
Flow cytometry. After blocking the Fc receptor with anti-CD16/CD32,
single-cell suspensions were incubated with the fluorescently labeled mAbs
at 4C for 30 min in PBS and then washed twice. For intracellular cytokine
staining, cells were first stimulated for 4 h at 37C with 50 ng/ml PMA,
1 g/ml ionomycin, and 10 g/ml monensin (all from Sigma-Aldrich); cells
were then stained for extracellular markers, fixed, permeabilized, and stained
with the fluorescently labeled mAbs against the indicated intracellular cytokines
2408
or isotype control Abs. Samples were collected by a flow cytometer (LSR II;
BD) and analyzed by FlowJo and WinMDI 2.9 software.
Real-time PCR. Total RNA was extracted from tissues using TRIzol (Invitrogen), and cDNA was then synthesized. Real-time PCR was performed
according to the manufacturers instructions using a SYBR Premix Ex Taq
(Takara Bio Inc.). For analysis, target gene expression was normalized to the
housekeeping gene -actin. Gene expression values were then calculated
using the mean from the control samples as a calibrator. Real-time PCR
primers were synthesized by Sangon Biotech (Table S1).
Neutralizing antibodies and antibiotic treatment. For in vivo neutralization, the following neutralizing antibodies were administered: 100 g/
mouse antiIL-17A (TC11-18H10.1), 100 g/mouse anti-CCL25 (89818),
100 g/mouse antiIL-6 (MP5-20F3), or 100 g/mouse antiIL-15 (AIO.3)
were administered into mice at days 0, 2, and 4 after PR8 infection. For
in vivo cell depletion, 200 g/mouse anti-NK1.1 was administered i.v. into
mice 2 d before PR8 infection. For intestinal microbiota depletion, mice
were treated with a mixture of antibiotics (1 mg/ml ampicillin, 0.5 mg/ml
vancomycin, 1 mg/ml neomycin sulfate, and 1 mg/ml metronidazole [Sangon Biotech]) added to their drinking water beginning 4 wk before PR8 infection and continuing until sacrifice, as previously described (Ichinohe et al.,
2011). For intestinal E. coli depletion, mice were treated with 1 mg/ml streptomycin (Sangon Biotech) added to their drinking water beginning 1 wk
before PR8 infection and continuing until sacrifice. Antibiotic-containing
water was changed twice a week.
Microbiota transplantation. Cecal contents from saline- or PR8-infected
mice were suspended in 1 ml saline and were administered (0.5 ml per mouse)
immediately to WT mice by the i.g. route.Transplanted mice were maintained
in sterile cages and detected intestinal immune injury 7 d later.
Transfer of T cells and PR8-specific CD4+ T cells assay. For T cells
transfer, 5 105 CD4+ T cells from the lungs of saline- or PR8-infected
CD45.1+ mice were adoptively transferred i.v. into WT CD45.2+ mice, and
the percentage of CD45.1+CD4+ T cells in total CD4+ T cells in LPL from
recipient CD45.2+ mice was detected 48 h later by flow cytometry. For
PR8-specfic CD4+ T cells assay, CD4+ T cells in the lung and LPL from
saline-treated and PR8-infected mice were purified 6 d later by MACS and
then co-cultured with antigen-presenting cells and heat-killed PR8 in an
IFN- ELISPOT plate. The number of positive spots was counted 20 h later
according to the manufacturers instructions.
Statistical analysis. A two-tailed Students t test was used for statistical
analyses. Data were expressed as the mean SEM, and the data were considered statistically significant when differences achieved values of P < 0.05.
Online supplemental material. Table S1 shows primers used for realtime PCR. Online supplemental material is available at http://www.jem
.org/cgi/content/full/jem.20140625/DC1.
We thank H. Meng (Shandong Academy of Medical Sciences) for the influenza
A/PR/8/34 strain.
This work was supported by Ministry of Science & Technology of China
(#2010CB911901 and #2013CB530506), Natural Science Foundation of China
(#31300753 and #31400783), Fundamental Research Funds for the Central
Universities (#WK2070000039), and China Postdoctoral Science Foundation
(#2013M531532 and #2014T70599).
Author contributions: J. Wang and F. Li performed experiments. J. Wang, F. Li,
H. Wei, Z.-X. Lian, R. Sun, and Z. Tian designed the research. J. Wang, F. Li, and Z. Tian
wrote the manuscript. Z. Tian supervised the project. J. Wang and F. Li contributed
equally to this paper.
Submitted: 3 April 2014
Accepted: 14 October 2014
Ar ticle
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INSIGHTS
Influenza pathogenesis: Club cells take the cure
The illness that follows influenza A virus (IAV) infection results from both direct effects of
IAV replication in the respiratory tract (RT) and from sequelae of the host immune response. The virus directly induces apoptosis or necrosis of infected RT cells and NK cells,
CD8 T cells lyse infected cells, and damaging inflammatory mediators are produced by various infiltrating immune cells. Accordingly, the degree of RT pathology is believed to reflect
both the extent of virus replication and the magnitude of the host immune response. In this
issue, however, Heaton et al. provide evidence for a novel mechanism of IAV pathogenesis.
Insight from Thomas Braciale (left)
They find that a small fraction of a particular RT cell type, the club cell, can cure itself of
and Taeg Kim
IAV but continue to produce inflammatory mediators, which may contribute to sustained
RT inflammation and injury following IAV clearance.
Heaton et al. adapted a strategy to indelibly mark IAVinfected cells where any cell infected by the virus will express
RFP. Contrary to expectation, they detected RFP+ cells in
the RT up to day 21 post-infection (p.i.), long after virusinfected cells are cleared. The authors identify these residual
RFP+ cells as club cellsbronchiolar (small airway) exocrine cells (formerly known as Clara cells), which secrete
products that protect the small airways.
Remarkably, by day 10 p.i. the residual RFP+ club cells
were cured, that is, they no longer expressed detectable
viral RNA. But they retained the type I interferon stimulated gene expression signature of infected cells and production of inflammatory chemokines, suggesting that these
cells may contribute to RT pathology. Supporting this idea,
selective depletion of the residual RFP+ cells diminished
epithelial cell damage in the RT. However, severe RT
injury with lethal outcome is associated with infection of
alveolar epithelial cells, which are not cured in this
model, so the contribution of club cells in IAV pathogenesis
remains to be determined.
These provocative findings raise many questions, notably,
how do infected club cells escape destruction by IAV and
recognition by NK cells or CD8 CTL? Also, what sustains
the inflammatory signature of club cells after viral RNA
elimination? Nevertheless, these results provide a potential
IAV replicates primarily in the respiratory tract epithelium, resulting
explanation for persistent RT inflammation following virus
in cell death via direct- or immune-mediated lysis (A). A small fraction
clearance and may presage the development of new strategies
of club cells can cure themselves of IAV but continue to produce
interferon-stimulated gene (ISG) products (B).
to treat the sequelae of IAV infection.
Heaton, N.S., et al. 2014. J. Exp. Med. http://dx.doi.org/10.1084/jem.20140488.
Thomas Braciale and Taeg Kim, University of Virginia: tjb2r@virginia.edu
JEM Vol. 211, No. 9
1705
Brief Definitive Report
Long-term survival of influenza virus

infected club cells drives immunopathology
Nicholas S. Heaton,1 Ryan A. Langlois,1,2 David Sachs,3 Jean K. Lim,1
Peter Palese,1 and Benjamin R. tenOever1,2
1Department
of Microbiology, 2Global Health and Emerging Pathogens Institute, and 3Department of Genetics and Genomic
Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029
Respiratory infection of influenza A virus (IAV) is frequently characterized by extensive

immunopathology and proinflammatory signaling that can persist after virus clearance.
In this report, we identify cells that become infected, but survive, acute influenza virus
infection. We demonstrate that these cells, known as club cells, elicit a robust transcriptional response to virus infection, show increased interferon stimulation, and induce high
levels of proinflammatory cytokines after successful viral clearance. Specific depletion of
these surviving cells leads to a reduction in lung tissue damage associated with IAV
infection. We propose a model in which infected, surviving club cells establish a proinflammatory environment aimed at controlling virus levels, but at the same time contribute to
lung pathology.
CORRESPONDENCE
Peter Palese:
peter.palese@mssm.edu
OR
Benjamin tenOever:
benjamin.tenOever@mssm.edu
Abbreviations used: IAV,
influenza A virus; ISG,
interferon-stimulated gene.
Influenza A virus (IAV) is a seasonal pathogen

with the capacity to cause devastating pandemics. IAV infects a variety of cells within the respiratory tract, including ciliated epithelial cells,
type I and II alveolar cells, and immune cells
(Matrosovich et al., 2004; Manicassamy et al.,
2010; Shieh et al., 2010; Langlois et al., 2012;
Smed-Srensen et al., 2012). Classically, IAVinfected cells are tracked through detection of
virus-derived products or reporters (e.g., virus
RNA or protein), all of which have short halflives and are therefore incapable of defining
infected cell types in the long-term. Ultimately,
acute IAV infections are resolved within 2 wk
post-infection (Carrat et al., 2008).
Infected cells are eliminated through two
major mechanisms, apoptosis/necrosis driven by
virus replication (Sanders et al., 2011;Yatim and
Albert, 2011) or clearance mediated through the
innate and adaptive arms of the immune system
(Zinkernagel and Doherty, 1979; Eichelberger
et al., 1991; Julkunen et al., 2001; Takeuchi and
Akira, 2009). Clearance of IAV infections can come
at the cost of aberrant immune-mediated disease
(Damjanovic et al., 2012). Therefore, a balance
between virus clearance and immune-mediated
N.S. Heaton and R.A. Langlois contributed equally to

this paper.
R.A. Langloiss present address is Department of Microbiology, University of Minnesota, Minneapolis, MN 55455.
J. Exp. Med. 2014 Vol. 211 No. 9 1707-1714
tissue damage is important for recovery from

IAV infections.
In this study, we define the long-term fate of
virus-infected cells within the lung through an
IAV expressing Cre recombinase and transgenic
reporter mice (Nagy, 2000). This experimental
model system allows for the indelible labeling
of virus-infected cells, even at time points well
after replication has ceased and virus has been
cleared. Surprisingly, despite a potent viral lytic
phase and generation of antiviral immune responses, we demonstrate that a small population
of cells that were infected by IAV persist after
virus clearance. Furthermore, using a combination of next-generation mRNA sequencing
and flow cytometry, we determine that in
fected long-term surviving cells were comprised
mainly of a single cell lineage, club cells (formerly
termed Clara cells; Winkelmann and Noack,
2010), and that these cells have heightened interferon stimulated gene (ISG) levels. Specific
depletion of surviving cells results in increased
pulmonary pathology, suggesting a proinflammatory role in recovery. This study provides
evidence of cellular survival from acute virus
infection and details new cellular mechanisms
of immunopathology.
2014 Heaton et al. This article is distributed under the terms of an Attribution
Share Alike 3.0 Unported license, as described at http://creativecommons
.org/licenses/by-nc-sa/3.0/).
1707
RESULTS AND DISCUSSION

To identify and characterize cells that are productively infected by IAV but go on to survive infection, we generated an
H1N1 strain (A/Puerto Rico/8/1934) expressing the bacteriophage protein Cre recombinase after a PTV-1 self-cleavage
site with a glycine-serine linker (Kim et al., 2011) on the viral
PB2 protein (Fig. 1 A). By infecting mice harboring the appropriate transgenic fluorescent reporter cassette, the expression of Cre leads to the excision of a stop cassette (Madisen
et al., 2010). After the stop element is removed, the cells will
constitutively express the red fluorescent protein tdTomato
(Fig. 1 B). Because the host cell harbors the tdTomato expression cassette, the cells continue to express the reporter protein
even if viral replication is stalled or eliminated.
To characterize the system, we performed ex vivo experiments on mouse lung fibroblasts isolated from the transgenic
tdTomato reporter animals. Wild-type IAV or mock-infected
fibroblasts failed to express tdTomato; however, upon infection with IAV-Cre, we observe red fluorescence (Fig. 1 C).To
demonstrate that viral replication is required to activate the
reporter, we pretreated cells with IFN-/ and infected with
IAV-Cre. Under these conditions, we observed no red signal,
indicating that viral RNA replication and protein expression
are required (Fig. 1 C). Finally, to determine if phagocytosis of
infected cellular extract was sufficient for tdTomato expression, we applied lysed cell debris from IAV-Cre infections
in the presence of a neutralizing antibody but found no
evidence for fluorescence (Fig. 1 C). Collectively, these data
Figure 1. Generation of influenza A virus expressing Cre recombinase. (A) Schematic showing insertion of Cre recombinase (Cre) downstream of a
PTV-1 2A site at the 3 end of PB2 segment. (B) Model depicting Cre mediated excision of tdTomato reporter stop cassette. (C) Lung fibroblast generated
from ROSA26 tdTomato lox-stop mice were mock infected or infected with WT IAV or IAV-Cre at an MOI of 5 (top three panels). Reporter fibroblasts were
treated with 100 U of IFN-/ for 6 h and infected with IAV-Cre at MOI of 5 (fourth panel). MDCK cells were infected with IAV Cre at an MOI of 5 for 24 h.
Cell debris were treated with anti-HA antibodies for 30 min and placed on reporter fibroblast (bottom). All images were taken 36 hpi. Bar, 50 m. Data are
representative of two independent experiments. (D) WT C57BL/6 mice were infected with WT IAV (left) or IAV-Cre (right) at the indicated doses and monitored for morbidity and mortality. Calculated LD50 values are 50 PFU for WT IAV and 240PFU for IAV-Cre. n = 5 mice per group. The experiment was
performed once.
1708
Surviving flu infected club cells drive pathology | Heaton et al.
Br ief Definitive Repor t
suggest that activation of the tdTomato cellular reporter requires active viral replication.
We next characterized the virulence of IAV-Cre in vivo
to ensure that the pathogenesis of this recombinant virus was
maintained. To this end, C57BL/6 mice were infected with
either wild-type IAV or IAV-Cre at a range of infectious doses,
and body weight and survival were monitored over time
(Fig. 1 D). Excitingly, despite the insertion of Cre recombinase, intranasal inoculation of IAV-Cre was sufficient to induce morbidity in a manner comparable to the parental IAV
stain (Fig. 1 D). Furthermore, mortality upon infection with
IAV-Cre was only mildly attenuated when compared with
the parental strain, with the median lethal dose (LD50) shifting
from 50 to 240 PFU (Fig. 1 D). These data suggest that
IAV-Cre retains the pathogenic properties associated with
IAV disease and justifies its utilization as a tool to probe for
cells that survive replicating virus in vivo.
Although IAV infection and replication generally result in
the induction of cell death, here we chose to determine whether
any cell types could successfully clear virus and survive. We
therefore infected tdTomato reporter mice with IAV-Cre and
assessed the presence of reporter-positive cells using flow

cytometry at various times after infection (Fig. 2 A). While
no tdTomato+ cells were identified in uninfected mice or reporter mice infected with WT PR8 virus (Fig. 2, A and B), we
observed a population of reporter-positive cells during active
viral replication (5 d post-infection) as expected. Surprisingly,
we also observed a population of tdTomato+ cells at 10 and
21 d post-infection (Fig. 2 A), time points that exceed the
physiological course of IAV replication ( Eichelberger et al.,
1991; Carrat et al., 2008).
To formally demonstrate that the observed tdTomato+ cells
were not a reflection of prolonged replication of IAV-Cre, we
determined the amount of recoverable virus from lungs over
the same time course as the FACS experiments. While high
titers were recovered at 2 and 5 post-infection, no virus was
detected at day 10 post-infection (Fig. 2 C). To identify where
in the lung architecture these surviving cells exist, we infected
reporter animals and collected lungs for histological analysis at
10 d post-infection. Upon examination of the lung sections,
tdTomato+ cells were only found in the epithelial layer of
larger airway spaces (bronchi), never in the alveoli (Fig. 2 D).
Figure 2. IAV-Cre demonstrates long-term cell

survival in vivo. (A) tdTomato reporter mice were
infected with 500PFU of IAV-Cre and live CD45 cells
were analyzed for tdTomato expression by FACS.
Values represent percent live, CD45 tdTomato+ cells.
Data are representative of five independent experiments. (B) The presence of surviving tdTomato+ cells
was measured 10 d post-infection from mice infected
with WT IAV or IAV-Cre. (C) Mice were infected with
IAV-Cre and lungs were removed at indicated time
points and virus titers from total lung homogenates
were assessed by plaque assay. Dotted line represents
the limit of detection. ND, not detected; NT, not titered. n = 3 mice per time point. Data are representative of two independent experiments. (D) Mice were
infected as in A and lungs were harvested at 10 d
post-infection. Frozen lung sections were counter
stained with DAPI and analyzed for tdTomato expression. Insets are enlargements of the white-boxed
areas. Bars, 100 m. Data are representative of two
independent experiments.
JEM Vol. 211, No. 9
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In an effort to better characterize and identify the surviving cells type(s), we profiled the transcriptome of tdTomato+
cells. To this end, reporter mice were infected with IAV-Cre
and over a time course we performed fluorescence-activated
cell sorting (FACS). At each time point (days 0, 2, 5, 10,
21 post-infection), we separated live, CD45, tdTomato+ and
negative cells and collected RNA from the different populations. Next-generation RNA-seq analysis was then performed
to define the transcriptional profiles of each population.Transcriptome analyses of these two cellular cohorts demonstrated
that despite deriving from comparable environments, there
were significant differences in the gene expression profiles
(Fig. 3 A). Results from replicate tdTomato+ cell isolation and
RNA-seq experiments were highly reproducible (Fig. 3 B).
Thus, cells that will go on to survive infection have a distinct
transcriptional profile relative to uninfected cells.
Within the RNA-seq data, we assessed IAV-specific
mRNA reads. In agreement with data in Fig. 2 C, we observed high levels of viral mRNA at early time points (days
2 and 5 post-infection), but viral mRNA is lost from tdTomato+ cells by day 10 post-infection (Fig. 3 C). Recovered viral
mRNA reads from day 5 infected cells mapped to all 8 viral
segments (Fig. 3 D) indicating that activation of the reporter
is unlikely to be a reflection of defective interfering particle
entry but instead competent viral infection and replication.
To further demonstrate that tdTomato+ cells were the result
of productive viral infection, we injected FACS-purified
tdTomato+ cells from day 5 post-infection animals into
embryonated chicken eggs, for which 11/12 eggs became
positive for virus (Fig. 3 E). Conversely, eggs injected with
tdTomato+ cells from 10 d post-infection animals failed to induce viral amplification (Fig. 3 E). Together, these data argue
that the observed tdTomato+ cells are the result of cells surviving and successfully clearing a productive IAV infection.
In an effort to define the cell lineage capable of surviving a
productive IAV infection, we analyzed the RNA-seq data for
cell type-specific markers over a 21-d time period.At early time
points during active IAV replication, tdTomato+ cells demonstrate high levels of Sftpc and Cc10 (Fig. 3 F), markers specific
for type II alveolar cells and club cells, respectively (Glasser
et al., 1990; Hackett et al., 1992; Kalina et al., 1992; Daly et al.,
1997). Interestingly, time points as early as day 5 post-infection
show almost a complete loss of Sftpc in tdTomato+ cells while
the levels of Cc10 are maintained (Fig. 3 F). Furthermore, with
the exception of Cc10, no cell-specific marker was detected in
appreciable amounts in the surviving cell population implicating club cells as the long-term cell survivors of IAV infection
(Table S1).This hypothesis is further supported by data demonstrating that club cells are restricted to the larger airways of
the respiratory tract, the same physiological location where
tdTomato+ expression was observed (Fig. 2 D).
We next turned our attention to understanding the mechanism by which tdTomato+ cells resisted the cytopathic effects
of IAV. The mammalian response to infection is mediated
by the type I IFN (IFN-I) response and the transcriptional
up-regulation of a barrage of antiviral genes (Bowie and
1710
Unterholzner, 2008). Strikingly, we observed an increase in

magnitude of ISGs in tdTomato+ cells relative to tdTomato
cells in the same lungs (Fig. 3 G). Furthermore, this enhanced
antiviral transcriptional signature persisted for the duration
of the time course (Table S1). Given the capacity of ISGs to
block IAV replication (Fig. 1 C), we postulate that this transcriptional signature is responsible for allowing cellular survival and clearance of viral replication.
To determine if club cells are more responsive to IFN-I,
as suggested by in vivo RNA-seq data, we treated a murine
club cell line (mtCC10-1; Magdaleno et al., 1997) and a lung
epithelial cell line (MLE-15; Wikenheiser et al., 1993) with
either IFN-I or IAV (Fig. 3, H and I). In agreement with the
in vivo transcriptome analyses, club cells had significantly
higher expression of ISGsincluding the master regulator of
the responseIrf 7 (Honda et al., 2005). Although the relationship between ISG up-regulation and cellular survival is
only correlative, we favor the hypothesis that the robust IFN-I
response in club cells permits a subpopulation of cells to successfully clear infection despite active virus replication.
Finally, we wanted to ascertain the function of club cells
as they relate to the pathogenesis of IAV infection. To define
a role for surviving cells, we used another Cre-responsive
transgenic mouse strain, one in which instead of turning on
tdTomato expression, the diphtheria toxin receptor is expressed (Buch et al., 2005). By using these mice, we can administer diphtheria toxin and selectively deplete only those
cells that have been infected by IAV. To this end, we infected
wild-type or the diphtheria receptor transgenic mice with
500 PFU of IAV-Cre as in previous studies with the tdTomato
reporter mice (Fig. 2). After allowing the virus to replicate
for 5 d, we administered diphtheria toxina time where
diphtheria toxin receptor expression should be limited to
long-term survivors of viral infection (Fig. 3 F). At 10 d postinfection (5 d after diphtheria toxin administration), lung
sections were processed for histological analysis. Sections
were then analyzed and scored by an independent pathologist. Counterintuitively, there was a noticeable improvement
in lung pathology under conditions where surviving cells
were depleted (Fig. 4 A). Although the overall cell infiltration
scores were not significantly different between the treatment
groups, there was a significant reduction in bronchiolar epithelial pathology, the exact localization of club cells (Fig. 4 B).
Specifically, the epithelium in nondepleted mice was observed to frequently be necrotic and attenuated in a segmental manner that affected most of the airways. Rarely, a few
normal segments were found at the terminal bronchioles. In
contrast, the bronchiolar epithelium in mice where surviving
club cells were depleted showed only minor necrotic lesions
and less of the airway epithelium was affected. Frequently,
the terminal bronchioles were completely unaffected (Fig. 4,
A and B). Although the interpretation of these data are
complicated by the possible contribution of surviving nonclub cells, the data show that surviving cells do significantly
contribute to the lung pathology in the larger airways after
IAV infection.
Figure 3. Surviving cells have an altered transcriptional profile and represent a unique cellular lineage. Live, CD45 tdTomato+, and
tdTomato cells were sorted at the indicated time points and transcriptional profile assessed by mRNA-seq. (A) The 300 most differentially expressed transcripts mapping to the mouse genome were ranked by magnitude of induction (at 2 d post-infection) and represented as fold change
relative to mock-infected lung cells. Data from 2, 5, 10, and 21 d post-infection are represented. (B) Replicate sequencing experiments of 10 d
post-infection tdTomato+ samples were plotted against each other to demonstrate reproducibility. Data are representative of all time points ana
lyzed. R 2 indicates the coefficient of determination and the p-value indicates the significance of the association between the two variables.
(C) Transcripts from tdTomato+ and tdTomato cells that mapped to IAV mRNA were analyzed at the indicated time points. (D) Transcripts that
mapped to each of the eight IAV segments were analyzed from tdTomato+ cells at 5 d post-infection. (E) FACS sorted cells from tdTomato+ or
tdTomato populations at the indicated time points were injected into embryonated chicken eggs. 48 h after injection, the detection of amplified
virus (via hemagglutination assay) was scored as positive. (F) mRNA reads were analyzed for cell typespecific signatures, Cc10 and Sftpc at the
indicated time points. Data are representative of two independent experiments. (G) The fold induction of interferon stimulated genes in tdTomato+
and tdTomato cells at 5 d post-infection were ranked and plotted by largest change in tdTomato+ cells. (H) Club or mouse lung epithelial cell
lines infected with IAV (MOI = 1) or treated with IFN-/ and analyzed for Irf7 expression. (I) Cells were infected/treated as in H and analyzed for
Isg15 expression. Data in H and I are representative of two independent experiments. Significance is based on an unpaired Students t test.
*, P 0.05; **, P 0.001.
JEM Vol. 211, No. 9
1711
To understand the mechanism of how surviving cells were

negatively influencing the bronchiolar epithelium, we looked
for highly induced transcripts that were also differentially expressed between tdTomato+ and tdTomato cells. Many of
the genes that fit these criteria were proinflammatory cytokines and chemokines, most strikingly: Cxcl10, Ccl20, and
Ccl5 (Fig. 4 C). Interestingly, it does not appear that pro
inflammatory mediators are universally up-regulated, as the
monocyte chemoattractant Ccl2 was not appreciably induced
in surviving tdTomato+ cells (Fig. 4 C). To validate secretion
of the chemokines identified from our in vivo RNA-seq data,
we infected murine club cells in vitro and quantified CXCL10,
CCL20, CCL5, and CCL2 protein levels in the supernatant
(Fig. 4 D). In agreement with our RNA data, infection of
club cells led to a significant increase of secreted CXCL10,
CCL20, and CCL5 (Fig. 4 D). No significant changes were
observed in CCL2 levels, as expected (Fig. 4 D).
Finally, we wanted to ensure that the club cell biology
described here was not murine specific.We therefore infected
the human club cell line H441 (Brower et al., 1986) with IAV
and measured release of a panel of proinflammatory factors.
Similar to mouse club cells, CXCL10, CCL20, and CCL5
were significantly up-regulated upon infection (Fig. 4 E). CCL2
was not reproducibly induced by viral infection as expected
(Fig. 4 E). These data suggest that human club cells and murine club cells respond similarly during viral infection.
In summary, we demonstrate that despite IAV causing an
acute and lytic infection, a subpopulation of lung epithelial
cells survive viral replication. Long-term surviving cells were
identified to be predominantly club cells that expressed a robust ISG response. Furthermore, surviving club cells demonstrate elevated levels of proinflammatory mediators relative to
uninfected cells in the same lungs, which ultimately leads to
increased lung pathology in the bronchi. We therefore propose a model in which club cells that are directly infected by
IAV but survive that infection provide a local proinflammatory
environment in the bronchi.Although club cells are likely critical for the initial up-regulation of an antiviral response, their
prolonged activation appears to be detrimental to bronchus remodeling after the pathogen has been cleared. This finding is
particularly relevant given that the significant mortality in the
most severe influenza infections (recently with the 2009 swine
Figure 4. Surviving cells contribute to IAV-induced immuno

pathology. Wild-type (C57BL/6) or diphtheria toxin receptor Cre inducible
(C57BL/6 Cre-DTR) mice were infected with 500 PFU of IAV-Cre. 5 d postinfection, mice were administered diphtheria toxin to deplete surviving
club cells. At 10 d post-infection (5 d after toxin depletion), lungs were
collected, and lung pathology was assessed. (A) Representative images of
lung sections from uninfected mice, C57BL/6 mice infected and toxin
treated (nondepleted), and C57BL/6 Cre-DTR mice infected and toxin treated
(depleted for surviving club cells). Enlargements (bottom) show a reduction
in bronchiolar epithelial necrosis when surviving club cells are depleted,
arrows indicate the epithelial cell layer. Bar, 200 m. (B) Independent
1712
pathologist scoring of virus induced bronchiolar epithelial cell necrosis

(Bronch. Nec.), bronchiolar infiltration (Bronch. Infilt.), and alveolar infiltration (Aveo. Infilt.). Data are representative of two independent experiments. (C) RNA-seq reads of chemokine transcripts highly induced in
tdTomato+ surviving cells relative to uninfected mice. The dashed line
indicates the reproducible level of detection for RNA transcripts based on
Fig 3 B. (D) The murine club cell line mtCC10-1 was mock or IAV-infected
at an MOI of 2 for 24 h. The concentration of the secreted chemokines in
the cellular supernatant is shown. (E) The human club cell line H441 was
mock or IAV infected at an MOI of 2 for 24 h. The concentrations of the
secreted chemokines in the cellular supernatant are indicated. Data in
D and E are representative of two independent experiments. Significance
is based on an unpaired Students t test. *, P 0.05; **, P 0.001.
H1N1 virus [Gao et al., 2013] and avian H5N1 viruses [de Jong
et al., 2006]) is attributed to a sustained proinflammatory response after infection. Although it is unlikely that the small
numbers of surviving club cells contribute significantly to the
global cytokine profile of an infected host, the localized effect
on bronchiolar epithelial layer integrity may be an important
component of IAV-related immunopathology.
Cell lines and in vitro infections. 293T, H441, and MDCK cells (American type Culture Collection) were used in this study. mtCC10-1 and MLE-15
cells were gifts from F. DeMayo (Baylor College of Medicine, Houston, TX)
and J. Whitsett (University of Cincinnati, Cincinnati, OH), respectively. All
cells were maintained in DMEM supplemented with 10% FCS, l-glutamine, and
Pen/Strep with the exception of the H441 cells, which were maintained in
RPMI-1640 and supplemented with 10% FCS and Pen/Strep. For infection,
virus was diluted to the appropriate concentration in PBS supplemented
with 3% BSA and used to infect cells for 1 h at 37C, followed by replacement of culture media. Virus was titered on MDCK cells as previously described (Langlois et al., 2013).
Generation of Cre recombinase expressing influenza virus and virus
quantification. The plasmid encoding PB2-Cre in the pDZ vector, which
expresses negative sense vRNA and positive sense mRNA. PB2 with silent
mutations in the packaging signal was amplified from the previously published
PB2-GLuc plasmid (Heaton et al., 2013a) with the following primers: forward
5-CTCCGAAGTTGGGGGGGAGCGAAAGCAGG-3 and reverse 5-GTT
AATAGCCATACGGATCCTCTTAG-3.We synthesized a sequence encoding a PTV-1 2A site, a codon-optimized Cre recombinase with an N-terminal
SV40 NLS and a duplicated PB2 packaging signal.The Cre region was amplified
with: forward 5-ATCCGTATGGCTATTAACGGAAGCGGAGCAACAAATTTCAGC-3 and reverse 5-TGGGCCGCCGGGTTATTAGTAGAAACAAGG-3. The two PCR fragments were recombined into pDZ
via Infusion HD cloning (Takara Bio Inc.). Clones were sequence verified and
rescued via 293T transfection and amplification in embryonated chicken eggs
as previously described (Heaton et al., 2013b). Rescued virus was dilution purified and titered on MDCK cells. For all experiments, viral titer was determined via standard plaque assay on MDCK cells.
Mice, virus infection, and diphtheria treatment. Wild-type C57BL/6,
B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze/J and C57BL/6-Gt(ROSA)
26Sor tm1(HBEGG)Awai/J mice were purchased from The Jackson Laboratory.
Mice were anesthetized with ketamine/xylazine and infected i.n. with
500 PFU of IAV-Cre unless otherwise indicated. 5 d post-infection, mice
were administered 100 ng diphtheria toxin (Sigma-Aldrich) i.p. and
10 ng i.n. Body weight was monitored over the course of infection, and 80%
initial body weight was designated as the humane endpoint. All experiments involving animals were performed in accordance with the Icahn
School of Medicine Animal Care and Use Committee.
Lung sectioning and histology. Mice were euthanized via CO2 inhalation and lungs were inflated before removal. For H&E staining, lungs were
inflated and fixed with 4% PFA in PBS. Lungs were paraffin embedded and
5-m sections were cut. For pathological scoring, two lung sections 100 m
apart were analyzed. For frozen sectioning and tdTomato+ cell localization,
lungs were inflated with 10% OCT in PBS. Lungs were embedded in OCT
and frozen at 80C. 5 m sections were dried on slides, fixed for 3 min in
1% PFA, and mounted in Prolong Gold with DAPI (Invitrogen).
Flow cytometry. Lungs were digested with Type-IV collagenase (Worthington) for 30 min with agitation, and then processed into single-cell suspension
and incubated with a rat antimouse CD45 antibody (30-F11; BD). Live/dead
violet dye was used as per the manufacturers instructions (Life Technologies).
JEM Vol. 211, No. 9
All flow cytometry data were acquired on a LSR II (BD), sorting performed
on a FACS Aria (BD), and analyzed using FlowJo software (Tree Star).
Next generation mRNA sequencing. Mice were infected and cells
FACS sorted as described above. 500 of the indicated cells were sorted into a
96-well plate, containing 20 l of RNA reaction buffer. RNA was extracted
and reverse transcribed via SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara Bio Inc.) as per the manufacturers instructions. cDNA was
sheared via Covaris sonication and prepared for sequencing using the TruSeq
DNA sample Preparation kit (Illumina) as per the manufacturers instructions. Samples were sequenced via 100 nt single-end run on a HiSeq 2000.
The raw data are available under accession nos: GSM1422381, GSM1422382,
GSM1422383, GSM1422384, GSM1422385, GSM1422386, GSM1422387,
GSM1422388, GSM1422389. The reads were mapped to the mouse transcriptome and the influenza genome using Bowtie. All software was parallelized and run on an internal high performance-computing cluster at Mount
Sinai. Heat map visualization of the sequencing data were generated using
the method described by Pavlidis and Noble (2003).
Microscopy. All images of tdTomato+ cells in cell culture and lung sections
were captured on an Olympus IX-70 camera. All H&E-stained sections were
captured on an Axioplan 2 camera (Carl Zeiss). Images were captured with
the same exposures and processed with ImageJ (National Institutes of Health).
The same thresholds were applied to all images in a given experiment with
the exception of the DAPI channel, which due to nonuniform staining was
adjusted to be visible in all sections.
qRT-PCR. RNA was extracted, reverse transcribed and assessed by qPCR as
previously described (Langlois et al., 2012). In brief, cDNA was amplified using
primers specific for tubulin, IRF-7, and ISG15 with KAPA SYBR FAST
qPRC Master Mix (KAPA Biosystems) and analyzed on a realplex2 (Eppendorf). Delta delta cycle threshold (CT) values were calculated with replicates
over tubulin.Values represent the fold change over mock-infected samples.
Cytokine and chemokine quantification. Protein levels of cytokines/
chemokines in culture medium were evaluated using a multiplex bead array
assay. All the antibodies and cytokine standards were purchased as antibody
pairs from R&D Systems or PeproTech. Individual bead sets (Luminex) were
coupled to cytokine-specific capture antibodies according to the manufacturers recommendations and as previously described (Biancotto et al., 2007).
Conjugated beads were washed and kept at 4C until use. Biotinylated polyclonal antibodies were used at twice the concentrations recommended for a clas
sical ELISA according to the manufacturer. All assay procedures were performed
in assay buffer containing PBS supplemented with 1% normal mouse serum
(Invitrogen), 1% normal goat serum (Invitrogen), and 20 mM Tris-HCl
(pH 7.4). The assays were run using 1,500 beads per set of each of cytokines
measured per well in a total volume of 50 l. The plates were read on a Luminex MAGPIX platform. For each bead set, >50 beads were collected. The
median fluorescence intensity of these beads was recorded and used for analysis
with the Milliplex software using a 5P regression algorithm.
Statistical analysis. Statistical analysis between datasets was performed using
a one- or two-tailed Students t test where appropriate. Differences were con
sidered to be statistically significant at P values at or below 0.05.
Online supplemental material. Table S1 shows normalized RNA transcript numbers detected in FACS purified tdTomato and tdTomato+ cell
populations over a 21-d time course. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20140488/DC1.
We would like to thank Virginia Gillespie for performing the lung pathological
scoring, Omar Jabado and the Genomics core facility, Carlos A. Rodriguez, and
the Mount Sinai Microscopy Shared Resource Facility for technical assistance. We
also thank Adeeb Rahman and the Flow Cytometry Shared Resource Facility for
assistance in FACS experiments.
1713
N.S. Heaton is a Merck fellow of the Life Sciences Research Foundation.

R.A. Langlois is supported by the Research Training Program in Molecular and Cell
ular Hematology (T32-HL094283). This work was partially supported by a Centers for
Excellence for Influenza Research and Surveillance grant (HHSN26620070010C to
P. Palese), NIH program project grant (1P01AI097092 to P. Palese), PATH (P. Palese),
and by the NIH (grant number A1093571 to B.R. tenOever).
The authors declare no competing financial interests.
Submitted: 14 March 2014
Accepted: 23 July 2014
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Article
Trans-nodal migration of resident dendritic

cells into medullary interfollicular regions
initiates immunity to influenza vaccine
Matthew C. Woodruff,1,4 Balthasar A. Heesters,4,5 Caroline N. Herndon,4
Joanna R. Groom,6 Paul G. Thomas,7 Andrew D. Luster,1,8 Shannon J. Turley,1,3,9
and Michael C. Carroll1,2,4
1Graduate
Program in Immunology, 2Department of Pediatrics, and 3Department of Microbiology and Immunobiology,

Harvard Medical School, Boston, MA 02115
4The Program in Cellular and Molecular Medicine, Childrens Hospital Boston, Boston, MA 02115
5Department of Medical Microbiology, University Medical Center Utrecht, 3584 CX Utrecht, Netherlands
6Department of Medical Biology, The Walter and Eliza Hall Institute of Medical Research, University of Melbourne,
Parkville, Victoria 3052, Australia
7Department of Immunology, St. Jude Childrens Research Hospital, Memphis, TN 38105
8Division of Rheumatology, Allergy, and Immunology, Center for Immunology and Inflammatory Diseases,
Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129
9Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02215
Dendritic cells (DCs) are well established as potent antigen-presenting cells critical to
adaptive immunity. In vaccination approaches, appropriately stimulating lymph node
resident DCs (LNDCs) is highly relevant to effective immunization. Although LNDCs have
been implicated in immune response, their ability to directly drive effective immunity to
lymph-borne antigen remains unclear. Using an inactive influenza vaccine model and whole
node imaging approaches, we observed surprising responsiveness of LNDC populations to
vaccine arrival resulting in a transnodal repositioning into specific antigen collection sites
within minutes after immunization. Once there, LNDCs acquired viral antigen and initiated
activation of viral specific CD4+ T cells, resulting in germinal center formation and B cell
memory in the absence of skin migratory DCs. Together, these results demonstrate an
unexpected stimulatory role for LNDCs where they are capable of rapidly locating viral
antigen, driving early activation of T cell populations, and independently establishing
functional immune response.
CORRESPONDENCE
Michael C. Carroll:
michael.carroll@
childrens.harvard.edu
Abbreviations used: ALN,
auricular LN; cDC, conventional DC; cIFR, cortical IFR;
IFR, interfollicular region;
LNDC, LN-resident DC; mDC,
migratory DC; mIFR, medullary
IFR; MP-IVM, multiphoton
intravital microscopy; MPM,
multiphoton microscopy; PLN,
popliteal LN; vG, van Gogh.
Since early descriptions of DCs as primary stimulators of adaptive immunity (Steinman, 1991),
their role in establishing and regulating immune
responses has been central to diverse immunological fields such as transplantation (Larsen et al.,
1990; Hill et al., 2011), autoimmunity (Llanos
et al., 2011), infectious disease (Poudrier et al.,
2012), and vaccinology (Arnason and Avigan,
2012). As critical mediators of antigen presentation, significant effort has been spent describing
activation of conventional DCs (cDCs) in peripheral tissue (Moodycliffe et al., 1994; Austyn,
1996; Rescigno et al., 1997) and characterization
of their subsequent migration to secondary lymphoid organs (Itano et al., 2003; Randolph et al.,
2005; Alvarez et al., 2008; Braun et al., 2011; Tal
et al., 2011). Once in peripheral LNs, migratory
DC (mDC) populations from the injection site

J. Exp. Med. 2014 Vol. 211 No. 8 1611-1621
present antigen to cognate T and B cells and

stimulate adaptive immunity (Qi et al., 2006).
The activation and maturation of mDCs is
thought to follow a three-stage process. First, immature DCs encounter antigen in the periphery, leading to up-regulation of MHC class II and
co-stimulatory molecules with a concomitant
reduction in phagocytic capacity (Rescigno
et al., 1997). Second, antigen-loaded DCs acquire migratory capacity through the expression
of matrix metalloproteases (Yen et al., 2008), migratory adhesion molecules (Acton et al., 2012),
and rapid actin treadmilling to enter and migrate
2014 Woodruff et al. This article is distributed under the terms of an Attribution
1611
along lymphatic vessels (Lmmermann et al., 2008). Finally, LNbound mDCs cross the subcapsular sinus floor into the paracortical region and interact with cognate T cells and LN-resident
DCs (LNDCs) within the draining LN (Allan et al., 2006; Braun
et al., 2011) to establish protective downstream immunity.
After antigen capture in peripheral tissues, the activation
and migration of mDCs into draining LNs is delayed for up
to 1824 h to allow for transcriptional and translational modification and a crawling migration sometimes representing distances of thousands of cell body lengths of the mDC. In the
case of vaccination, however, arrival of injected antigen is rapid,
with detectable antigen arriving in the draining LN via the
afferent lymphatics within minutes (Roozendaal et al., 2009;
Gonzalez et al., 2010).This timing discrepancy between antigen
arrival in the LN and the migration of DCs from the periphery leaves open a potential window whereby targeting a vaccine
to a nondegradative, immunostimulatory compartment within
the LN could have important humoral immune ramifications.
Several studies have focused on the drainage of lymph-borne
antigen from the afferent lymph into the subcapsular sinus of
the draining LN (Szakal et al., 1983; Carrasco and Batista, 2007;
Junt et al., 2007; Phan et al., 2007; Roozendaal et al., 2009;
Gonzalez et al., 2010). A current view is that subcapsular sinus
macrophages rapidly capture antigen from the lymph and participate in its active transport to the B cell follicle. Less well
described is the downstream filtration of the lymph within the
medulla by medullary sinus-lining macrophages (Gray and
Cyster, 2012) and LNDCs (Gonzalez et al., 2010). Historically,
DCs residing in the LN (LNDCs) have been described as relatively sessile at steady-state, (Steinman et al., 1997; Lindquist et al.,
2004) and insufficient to drive effective immunity after direct
antigen acquisition (Itano et al., 2003; Allenspach et al., 2008).
However, the recent observation of direct viral capture in the medulla by the LNDC population suggested they may have a more
active role in the establishment of downstream immune response
in the case of influenza vaccination (Gonzalez et al., 2010).
To extend our understanding of the role of LNDCs in establishing immune response to influenza vaccination, resident
DCs were characterized at a whole-LN level. Unexpectedly,
a major trans-nodal repositioning of LNDCs from the T cell
cortex to the afferent medulla was observed within minutes
of viral antigen arrival from the afferent lymphatics, areas recently shown to be important in vaccine efficacy (Liu et al.,
2014). This migration leads to rapid viral acquisition by
LNDCs and stimulation of viral-specific naive CD4+ T cells.
Furthermore, total elimination of skin mDCs had a negligible
effect on the generation of a protective humoral response in
mice vaccinated with UV-inactive virus. Collectively, the results suggest a model in which LNDCs are fully competent in
establishing robust, long-term viral immunity, even in the absence of mDCs from the injection site.
RESULTS
Activation of LNDCs after influenza vaccination
To characterize LNDC response after vaccination, CD11ceYFP C57BL/6 mice (Lindquist et al., 2004; Hickman et al.,
1612
2008; Sung et al., 2012; Kastenmller et al., 2013) were immunized s.c. in the footpad with UV-inactivated influenza A
virus strain PR8 (UV-PR8). DCs were tracked by multiphoton intravital microscopy (MP-IVM) of the popliteal LN (PLN)
by surgically exposing the node in live, anesthetized mice
(Gonzalez et al., 2010). Continuous imaging for 40 min after
UV-PR8 vaccination revealed an influx of LNDCs proximal to the collagen capsule (<150 m; Fig. 1 a and Video 1).
Quantitation of cellular trafficking over this period identified
a three- to fourfold increase in the number of YFP+ DCs
within this region (3.23 0.24; P < 0.001), suggesting a rapid
repositioning to the periphery of the PLN.
In addition to increased cell number within the LN periphery, LNDCs exhibited extensive morphological changes
over the 40-min imaging period (Fig. 1 a, inset). Measured in
bulk, DCs within these regions increased in surface area by
almost 50% after vaccination and experienced a concomitant
increase in volume and decrease in spherical index, a measure
of the spherical nature of an object (Fig. 1 c and not depicted).
Importantly, the fluorescence intensity of individual DCs did
not change over this time period, indicating that the observed
phenotypic changes were not caused by changes in YFP expression. Together, these data suggest an unexpected accumulation and activation of resident DCs in the PLN periphery
immediately after vaccination.
Repositioning of LNDCs to the medulla
To address the origin of the accumulating DCs, in vivo cellular tracking was applied to the live imaging model. By tracking individual DCs, it was observed that rather than infiltrating
from an outside source, the cells originated from the interior
of the PLN (>150 m from the collagen capsule), which is
beyond the imaging depth threshold for our imaging system
(Fig. 1 b and Video 1). Pretreatment of mice with CD62L
blocking antibody or local administration of Pertussis toxin,
two approaches which limit influx of leukocytes from the vasculature or lymphatics, had negligible effects on DC accumulation (not depicted).These results provide additional evidence
that the activated DCs originated within the PLN before vaccination and confirmed their identity as LNDCs.
To assess the overall movement of LNDCs after vaccination, 50-m serial cryosections of PLNs were imaged by multiphoton microscopy (MPM) and serially reconstructed for
analysis of whole PLNs. Similar approaches were reported by
Grigorova et al. (2010) using confocal microscopy for partial
PLN imaging. By in vivo labeling medullary macrophages
through preinjection of an -F4/80 mAb into the footpad of
CD11c-eYFP reporter mice, the medulla could be outlined and
shown to include relatively sparse populations of LNDCs in
resting naive LNs (Fig. 1 d). In agreement with the live imaging data, injection of UV-PR8 into the footpad of CD11ceYFP mice stimulated a visible shift of the LNDC population
into the medullary compartment by 60 min after vaccination
(Fig. 1 d). Flow cytometric analysis of single cell suspensions
of PLN indicated no appreciable increase of LNDCs at these
time points. This global repositioning of the LNDCs can be
LNDCs initiate humoral immunity to flu vaccination | Woodruff et al.
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Figure 1. LNDCs migrate to the medulla after influenza vaccination. (a) MP-IVM of DC arrival in the PLN periphery after UV-PR8 vaccination.
Snapshots were taken at 0 and 36 min after injection and are representative of three independent surgeries (three mice) from different imaging sessions.
(inset) High magnification of two individual LNDCs. (b, left) Real-time tracking of LNDCs from panel a. LNDC tracks are highlighted (white) and final destinations marked (closed circles). (right) Vector representation of complete tracks. Green and red vectors represent LNDCs with migration paths toward or
away from the PLN capsule, respectively. Data are representative of three independent surgeries (three mice) from different imaging sessions. (c) Quantitation of bulk DCs from live imaging in panel a. DC spherical index (red) and surface area (black) are displayed. ANOVA (black), P < 0.005; ANOVA (red),
P < 0.005. (d) Fluorescent reconstructions of in vivolabeled CD11c-eYFP reporter PLNs. PLNs were collected at 60 min after PBS or UV-PR8 vaccination.
Data are representative of three reconstructed PLNs. B, follicles; M, medulla; MM, medullary macrophage; T, T cell cortex. (e) Quantitation of the percentage of LNDCs inside or outside of the medulla as in d (n = 3 PLNs). *, P < 0.05. Mean SEM.
quantified through a shift in the ratio of medulla-occupying

versus total LNDCs (Fig. 1 e). Migration data were further
verified through MP-IVM, through which the rapid arrival
of LNDCs into the medulla could be observed (Video 2).
Surprisingly, this change in both LNDC morphology and localization could not be identified after the injection of traditional adjuvants such as alum and MF59 despite extensive
inflammation of the PLN, suggesting that this response may
be specific toward viral antigen or endosomal TLR signaling.
Capture of viral antigen by LNDCs
within interfollicular regions (IFRs)
Recent studies have identified interactions of DCs and T cells
outside of the T cell area within IFRs in the stimulation of
memory CD8+ T cell responses (Hickman et al., 2008; Len
et al., 2012; Sung et al., 2012). In these studies, central memory T cells were generated after viral infection with the purpose of tracking those cells in the LN after secondary challenge.
Although there is debate on the resting location within the
LN, it is clear that after secondary challenge, central memory
CD8+ T cells are rapidly recruited to the IFRs where they
undergo activation by antigen-loaded APCs. Furthermore, a
JEM Vol. 211, No. 8
study by Hickman et al. (2008) suggested a primary role for

these sites in priming CD8 T cell immunity.
We hypothesized that these sites may serve as destinations
for migrating LNDCs. To characterize the architectural identity of the IFRs, PLNs were in vivo labeled with antibodies
against the lymphatic endothelium (a-Lyve-1) and subcapsular sinus macrophages (a-CD169) and then optically cleared
for MP imaging. Projections of whole nodes after optical
clearing (Ertrk et al., 2012) identified long extensions of the
medulla that protruded extensively between B cell follicles
and merged with the subcapsular sinus (Video 3). This observation identified two different types of IFR structure: cortical
IFRs (cIFRs), which represent chemokine boundaries between the paracortex and B cell follicles, and medullary IFRs
(mIFRs), which represent the most afferent connection points
between the medulla and the subcapsular sinus (Fig. 2 a). Interestingly, these regions have recently been targeted by Liu
et al. (2014) in vaccination attempts, resulting in greatly enhanced T cell priming. Confocal imaging of mIFRs in PBS or
UV-PR8vaccinated PLNs identified YFP+ clusters similar to
those identified in reconstruction analysis, identifying these
sites as destinations for migrating LNDCs (Fig. 2 b). Histological staining confirmed that both CD11bhi and CD8a+ LNDC
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Figure 2. LNDCs infiltrate mIFRs and acquire viral antigen. (a) Schematic diagram of the architecture of a PLN. mIFRs and cIFRs are highlighted in red and
blue, respectively. (b) Confocal imaging of mIFRs in CD11c-eYFP PLNs 40 min after vaccination with PBS or UV-PR8. Blue arrowheads: CD8a+ DCs; red arrowheads:
CD11bhi DCs. Images are representative of three independent trials; three mice/trial. B, follicles; M, medulla; T, T cell cortex. (c) Reconstructions of in vivolabeled
C57BL/6 PLNs vaccinated with A633UV-PR8 and collected at 0.5 or 6 h after injection. White arrowheads: IFRs. SS, subcapsular sinus. Images are representative
of three independent experiments; two mice per time point per experiment. (d) MPM of in vivolabeled CD11c-eYFP PLNs 60 min after PBS or A633UV-PR8
vaccination. Dashed lines: IFRs. Images are representative of four independent experiments; two mice per experiment. (e) LNDC capture of A488UV-PR8 by
flow cytometry 60 min after injection. (left) Cells displayed are pregated to be CD11chi (n = 4 PLNs). Mean SEM. (f) MPM of in vivolabeled CD11c-eYFP
PLNs 6 h after A633UV-PR8 vaccination. White arrowhead: UV-PR8 patch on the LNDC. Image is representative of four independent experiments; two
mice per experiment.
populations had accumulated in mIFRs within 60 min of

vaccination, suggesting a multisubset responsiveness to inactivated influenza (Fig. 2 b).
Afferent lymph flowing through the medullary sinus is
constitutively filtered by sinus-lining macrophages (Gray and
Cyster, 2012), and this process predicts a natural gradient of
viral antigen after vaccination. Thus, it is proposed that the
highest antigen concentrations would reside at the tips of
these mIFRs (Video 4). To test this possibility, mice were injected s.c. with UV-PR8 labeled with Alexa Fluor 633. Fluor
escent PLN reconstructions at various time points identified
accumulation of viral antigen within mIFRs over 6 h after
vaccination (Fig. 2 c). In contrast, lower levels of virus were
retained in the subcapsular sinus and the deeper medullary
compartments connecting with the efferent lymphatics. It
was hypothesized that these antigen-rich regions might serve
as a destination for migrating LNDCs. High-resolution imaging of IFRs bearing dense deposits of viral antigen showed
large numbers of infiltrating LNDCs within 60 min of viral
arrival at the node. Analysis of LNDCs by flow cytometry
confirmed viral capture by both CD11bhi and CD8a+ LNDCs
within 30 min after injection (Fig. 2 e), which was verified by
MPM identifying viral patches on the surface of LNDCs at
later time points (Fig. 2 f, inset).
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LNDCs present viral antigen to CD4+ T cells near IFRs

Previous work by Itano et al. (2003) interrogated the role of
mDCs versus LNDCs in CD4+ T cell stimulation. In that study,
soluble peptide designed for presentation on MHC II was injected s.c., and downstream T cell responses were tracked in
the draining LN. The authors observed two waves of peptide
presentation, one at 6 h, which resulted in only transitory activation, and one at 18 h, which stimulated a more robust, longterm T cell response. The authors concluded that although
resident DCs can activate T cells, the resident cells induce
only a limited response with mDCs from the injection site
required for robust immunity. To determine whether LNDCs
participate in activation of viral-specific CD4+ T cells within
this study, CD11c-eYFP mice were adoptively transferred with
labeled, ova-specific CD4+ OT-II T cells 24 h before vaccination with a UV-inactive recombinant strain of PR8 (UV-PR8OTII), which was engineered to express the OT-II epitope
(Thomas et al., 2006).
As predicted, vaccination with UV-PR8-OTII stimulated
activation of cognate T cells within 6 h, as indicated by CD69
up-regulation (Fig. 3 a). Although individual DCT cell contacts could be observed as early as 6 h, extensive interaction
between LNDCs and CD4+ OT-II T cells was observed near
IFRs by 12 h after vaccination (Fig. 3 b). Additionally, small
Ar ticle
clusters of viral-specific T cells could be identified surrounding LNDCs in these regions. To quantitate clustering, labeled,
naive OT-II T cells were transferred into CD11c-eYFP recipients, which were subsequently vaccinated with UV-PR8OTII 12 h before LN harvesting.The LNs were serially imaged
for reconstruction, and single plane images were captured from
each reconstruction, representing 400 m of vaccinated versus control LNs. Using histocytometric analysis (Gerner et al.,
2012), OT-II T cells were identified within each plane, and
each T cell was assessed for the number of T cell neighbors
present in the image within one T cell diameter.The resulting
measure of T cell clustering revealed significant increases in
the mean number of T cell neighbors and increases in the
absolute number of T cells with multiple neighbors within
vaccinated versus nonvaccinated LNs (Fig. 3 c). Significantly,
heat mapping the number of T cell neighbors onto the ori
ginal images identified the border between the paracortex
and IFRs as the most densely populated by clustered T cells
(Fig. 3 d). Analysis of T cell migration at 24 h after immunization
identified discreet clusters at the extremes of the paracortex,
with specific colocalization of T cell clusters at the mIFR
cortical junctions (Fig. 3, e and f).
Clustering of viral-specific CD4 T cells is chemokine dependent
Previously mentioned studies describing central memory
CD8 T cell responses within cIFRs have suggested the chemokine CXCR3 as a critical mediator of cellular retention in
these regions. The ligands for CXCR3, CXCL9 and CXCL10,
are produced by both hematopoietic and stromal cells in response to viral challenge and are critical to CD8 T cell recruitment to these sites (Sung et al., 2012; Kastenmller et al.,
2013). Additionally, Groom et al. (2012) have recently described the up-regulation of CXCR3 as an early first step in
the generation of robust Th1 CD4 T cell responses. Thus, we
hypothesized that the clustering of CD4+ OT-II T cells with
LNDCs and virus in the IFR after immunization with UVPR8-OTII could be CXCR3 dependent.To examine whether
CD4+ OT-II T cells become activated and differentiate to
CXCR3+ T cells, LNs were harvested from immunized mice,
and CD4+ T cells were analyzed by flow cytometry for expression of cell surface markers of activation. Notably, an increase in CD69 expression on CD4+ OT-II T cells was observed,
followed by a corresponding increase in CD44 (Fig. 4 a and
not depicted). By gating on newly activated CD69+ cells, increases in CXCR3 expression could be identified as early as
12 h after vaccination and continued to increase over several
days (Fig. 4, a and b).
As recent reporting has shown that activated DCs arriving from the periphery express CXCL10 and that direct
injection of LPS/Poly I:C can induce CXCL10 expression
within 12 h in the draining LN (Groom et al., 2012), it was
predicted that LNDCs might express this chemokine after immunization with UV-PR8-OTII. Thus, the release of CXCL10
by activated LNDCs within the IFR could explain the extensive
clustering of CD4+ T cells observed within the antigen-rich
IFRs. To test this possibility, CXCL9/10 reporter mice (REX3;
JEM Vol. 211, No. 8
Figure 3. Cognate CD4+ T cells relocate to mIFRs after vaccination. (a) C57BL/6 mice received naive OT-II T cells and were vaccinated
with UV-PR8-OTII. OT-II T cell activation in PLNs was analyzed by flow
cytometry (n = 4 mice). *, P < 0.05; ***, P < 0.001. Mean SEM. (b) CD11ceYFP mice received labeled naive OT-II T cells and were vaccinated
with UV-PR8-OTII. MPM of a PLN 12 h after vaccination. White arrowheads:
LNDC/OT-II contacts. Images are representative of three PLNs; two
independent trials. (c) OT-II neighbor analysis of PLNs as shown in b.
Each symbol type represents an individual PLN, with four XY planes
shown. **, P < 0.005. Horizontal bars indicate mean. (d) Heat map of
OT-II neighbor counts as shown in c. OT-II cells were identified from
images (left) and displayed with color indicative of the number of
corresponding OT-II neighbors (right). B, follicles; M, medulla; T,
T cell cortex. (e) CD11c-eYFP recipients received labeled naive OT-II T cells
and were vaccinated with UV-PR8-OTII. Fluorescent reconstruction of
a PLN 24 h after vaccination is shown. Per, PLN periphery. Image is representative of three independent trials; two mice/trial. (f) C57BL/6 mice
received OT-II T cells and were vaccinated with UV-PR8-OTII. PLNs were
collected at 24 h after vaccination, optically cleared, and imaged. Data are
represented with medulla isosurfacing to aid visual interpretation. White
arrowheads: mIFR/T cell cluster colocalization. Image is representative
of two trials; two mice/trial.
1615
Groom et al., 2012) were vaccinated with UV-PR8, and LNs

were harvested after 24 h for analysis by flow cytometry and
immunohistochemistry. Flow analysis identified the expression of CXCL10 by both CD11bhi and CD8a+ LNDCs after
vaccination (Fig. 4 c), although the CD11bhi population appeared more robustly responsive. Fluorescent reconstructions
of vaccinated REX3 PLNs identified high CXCL10 expression on dendritic-looking cells within mIFRs after vaccination, suggesting LNDC expression (Fig. 4 d). Confocal analysis
positively identified these cells as DCs through CD11c expression and confirmed clustering of CXCL10-positive DCs
within IFRs by 24 h after vaccination (Fig. 4 e). The timing
of this event corresponded with both LNDC migration and
formation of T cell clusters.
To confirm the importance of CXCL10 in attracting viralspecific T cells to the IFRs, mice were adoptively transferred
with differentially labeled CXCR3+/+ and CXCR3/ OT-II
T cells and subsequently immunized with UV-PR8-OTII virus.
Characterization of the vaccinated recipients revealed a significant defect in the clustering of CXCR3/ OT-II cells at
24 h after vaccination, confirming the importance of CXCR3
in T cell migration (Fig. 4 f). Additionally, flow cytometric
analysis of CXCR3/ OT-II T cells displayed a profound
deficiency in both CD69 activation and CD40L expression
(Fig. 4, g and h). These results replicate the study by Groom
et al. (2012), which described early activation defects, followed by a deficient Th1 response in the absence of CXCR3.
Together, these data suggest a model whereby LNDC activation of CD4+ T cells at early time points leads to expression of
CXCR3 and contributes to the efficient localization of cognate cells into mIFRs, resulting in efficient early activation.
Figure 4. CXCR3-dependent clustering of viral-specific CD4+

T cells in mIFRs. (a) Flow cytometry of OT-II T cell activation after UVPR8-OTII vaccination at the indicated time points (n = 4). (b) Expression
of CD69 (black) and CXCR3 (red) by OT-II T cells at the indicated time
points as in panel a. CD69+ OT-II cells were gated for CXCR3 expression
analysis. Symbols represent individual mice. ANOVA (CD69), P < 0.0005;
ANOVA (CXCR3), P < 0.005 (n = 4). Mean SEM. (c) CXCL10 expression
in vaccinated REX3 mice or unvaccinated REX3 controls by flow cytometry. (left) Displayed cells gated on CD11chi cDCs, followed by CD11bhi or
CD8a+ as indicated. (right) Percentage of CXCL10-positive DCs by subset
after vaccination (n = 4). ***, P < 0.001. Horizontal bars indicate mean.
(d) MP imaging of REX3 PLN 24 h after UV-PR8 vaccination. Dashed
line: medulla (M). B, follicles; T, T cell cortex. Images are representative
of four PLNs from two independent trials. (e) Confocal imaging of REX3
PLN 24 h after UV-PR8 vaccination. Arrowheads: CXCL10-positive DCs.
Images are representative of four PLNs; two independent trials. (f) C57BL/6
recipients received differentially labeled WT or CXCR3-deficient naive
OT-II T cells. Recipients were vaccinated with UV-PR8-OTII, and PLNs
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LNDC-dependent T cell activation

Although the activation of T cells within 6 h of vaccination suggested a role for LNDCs in early activation of CD4+
T cell responses to UV-PR8, it remained unclear whether
skin-resident mDCs were required to establish an enduring humoral response. Using a system developed by Itano et al. (2003),
the contribution of the mDC population could be effectively
removed through resection of the injection site within 30 min
after immunization (Kissenpfennig et al., 2005). By administering UV-PR8 intradermally in the ear and removing the ear
shortly after administration, the relative contribution of mDC
and LNDC populations could be evaluated in the auricular
LN (ALN).
were collected at 24 h. Quantitation of clustering efficiency of WT or

CXCR3-deficient OT-II T cells is shown (n = 5). (g and h) Adoptive transfers were performed as in f. Recipients were vaccinated, and PLN
suspensions were collected 60 h after vaccination for flow analysis.
Individual T cell populations were compared with the same population
in unvaccinated contralateral controls. CD40L (g) and CD69 (h) acquisition is expressed as fold change (FC) in vaccinated versus unvaccinated
population controls (n = 5). **, P < 0.005; ***, P < 0.001.
Ar ticle
Using this approach, C57BL/6 mice were vaccinated with

PBS or UV-PR8 intradermally, with half of the vaccinated
group undergoing ear removal (here referred to as the van Gogh
[vG] group) within 30 min after vaccine administration. Using
REX3 reporter mice, expression of CXCL10 by both CD11bhi
and CD8a+ in WT or vG-vaccinated animals was assessed 24 h
after injection/resection. Interestingly, although CXCL10 expression was still robust in both LNDC populations, suggesting
normal responsiveness to vaccination despite a lack of mDCs
in the vG group (Fig. 5 a), there was a small decrease in expression by the CD8a+ subset, perhaps relating to its known reliance
on incoming mDCs from the periphery (Allan et al., 2006).
By transferring CFSE-labeled naive OT-II T cells into B6
recipients and using the vG vaccination system, potential deficiencies in T cell proliferation and activation could be assessed
in the absence of mDCs from the injection site. Surprisingly,
vG group OT-II T cells proliferated similarly to the WT group
(Fig. 5 b) and could be tracked through seven divisions in 60 h.
Tracking T cell activation by division status, cells could be
observed acquiring both CXCR3 and CD40L as they proliferated in both the WT and vG groups (Fig. 5, c and d). Interestingly, although CD40L expression appeared identical in
both groups, there was a slight delay in CXCR3 acquisition
between the WT and vG group. These findings may also suggest collaboration with mDCs in reinforcing Th1 immunity
development as they arrive from the periphery.
LNDC-dependent B cell memory
As LNDC and T cell activation appeared to be intact despite
the absence of mDCs in the vG model, it was hypothesized
that LNDCs may be sufficient to drive downstream protective
humoral immunity despite the absence of mDCs. To test this
hypothesis, the vG vaccination model was used in C57BL/6
mice alongside PBS-vaccinated controls. ALNs were collected
at day 7 after vaccination, and germinal center staining appeared
normal in both WT and vG groups, confirming effective T cell
help despite the absence of mDCs (Fig. 6 a). Antibody titers
collected at day 10 after vaccination showed normal increases
in IgM, IgG1, and IgG2b in vG versus WT vaccination groups
in comparison with unvaccinated controls, suggesting normal
class switching and germinal center development (Fig. 6 b).
To test the functionality of this antibody response, vaccinated, or unvaccinated controls were challenged intratracheally with a lethal dose of live PR8 on day 21 after vaccination.
Morbidity was monitored for 9 d after infection, at which point
the unvaccinated control group was sacrificed as a result of
excessive weight loss. Neither WT nor vG groups experienced
significant weight loss over the course of infection, indicating
protective immunity had been generated with or without mDC
arrival from the injection site (Fig. 6, c and d). Finally, antibody
titers were assessed at day 9 and compared with both unvaccinated and uninfected controls. As expected, antibody titers
showed extensive class switching in both WT and vG groups,
and protective IgG2b responses were identical between groups.
It is worth noting that although not required for protection,
IgG1 antibody titers were slightly, but insignificantly decreased
JEM Vol. 211, No. 8
Figure 5. mDC-independent LNDC/CD4+ T cell activation. Ear resection in the vG model 30 min after vaccination. (a) Percentage of CD8a+
or CD11bhi cDCs in the ALN 24 h after ear vaccination. PBS, WT, and vG
groups are displayed. ANOVA (light gray), P < 0.0001; ANOVA (dark gray),
P < 0.0001 (n = 4). *, P < 0.05; ***, P < 0.001. (b) CFSE-labeled OT-II T cells
were transferred into C57BL/6 recipients. Recipients were vaccinated in
the ear and separated into WT and vG groups. ALNs were isolated at 60 h
after injection and analyzed by flow cytometry. (left) Percentage of OT-II
T cells by division number as assessed by CFSE dilution. ANOVA, P > 0.1.
(right) Representative plot of CFSE dilution in WT and vG groups (n = 4
mice/group). (c and d) Adoptive transfers were established as in c. OT-II
T cell expression (MFI) of CXCR3 (c) and CD40L (d) was assessed by T cell
division to track acquisition over time. (c) Two-way ANOVA, P < 0.0001
overall, P < 0.05 between groups. (d) Two-way ANOVA, P < 0.0001 overall,
P > 0.1 between groups (n = 4 mice/group). Mean SEM.
in the vG group, providing additional evidence for LNDC/

mDC collaboration in a vaccination setting. Altogether, these
data demonstrate that LNDCs are capable of stimulating
CD4+ Th1-dependent protective humoral responses to influenza vaccination.
DISCUSSION
An earlier study identifying capture of lymph-borne inactive
influenza virus by LNDCs raised the question of the relative
importance of this population in overall humoral immunity
(Gonzalez et al., 2010). To gain a broader understanding of
LNDCs in response to vaccination with inactive influenza,
a whole-node imaging approach was developed, and mice
bearing a fluorescent reporter for DCs (CD11c+) were vaccinated with UV-PR8. After s.c. vaccination, viral antigen was
observed within the draining LN within minutes, as expected.
Surprisingly, however, the virus accumulated over the first 6 h
within specialized sites identified as mIFRs.
1617
Figure 6. mDC-independent protection from influenza. (a) Confocal

imaging of ALN follicles at day 10 after UV-PR8 vaccination in WT and vG
mice. Dashed line: B cell follicle. Images are representative of two independent trials; five mice/group. (b) ELISA analysis of PR8-specific serum
antibodies at day 10 after UV-PR8 vaccination. Relative titers of PR8specific IgM, IgG1, and IgG2b are shown. Symbols represent individual
animals. (c) Unvaccinated or vaccinated WT or vG groups were challenged
with LD80 PR8 21 d after vaccination. Morbidity (left) and mortality
(right) are displayed as percentage of body weight or percent survival,
respectively. Red dashed line: morbidity/euthanasia experimental cutoff.
Six mice/group. (d) ELISA analysis of PR8-specific serum antibodies at day
9 after PR8 challenge as in c, compared with naive C57BL/6 littermates.
Relative titers of PR8-specific IgM, IgG1, and IgG2b are shown. Symbols
represent individual animals. *, P < 0.05; ***, P < 0.001. Mean SEM.
Using fluorescent reconstructions of full PLNs isolated at

early time points after vaccination, we observed a major repositioning of LNDCs from the T cell cortex to the mIFR where
they acquired viral antigen and became activated. This was
unexpected because LNDCs are reported to be relatively sessile at steady-state.The capture of virus by the resident DC was
biologically relevant because they became positive for the chemokine CXCL10 and formed clusters with viral-specific CD4+
1618
T cells that became activated based on expression of CD69,

CD44, and CXCR3. Thus, three lines of evidence are presented
that support a role for LNDCs in promoting a humoral response to inactive PR8 independent of mDCs: (1) activation
of viral-specific CD4+ T cells before expected arrival of skin
mDCs, (2) activation of viral-specific CD4+ T cells within
PLNs in the absence of mDCs, and (3) a normal humoral memory response despite surgical removal of the injection site
within 30 min of vaccination. Notably, although these findings clearly demonstrate sufficiency of the resident DC population in establishing humoral immunity, they do not address
the requirement of mDCs under conditions where lymphborne antigen is not readily available.
Although several groups have previously described resident DC populations in skin-draining LNs (Lindquist et al.,
2004), few have directly assessed their potential in presenting
antigen acquired directly from the lymph. In one study, Itano
et al. (2003) describe a two-step process of antigen presentation
where resident DCs in the LN were capable of acquisition and
presentation of soluble peptides, but skin-resident mDCs were
required for effective immunity. In another study, Allenspach
et al. (2008) found that in the case of soluble peptide administered with CFA, resident DCs were similarly deficient in T cell
presentation. Our findings are not inconsistent with these
results, as the robust activation/migration of resident DCs
cannot be identified after traditional adjuvant administration
(alum or MF59). The profound differences in these responses
also raise interesting questions about a potential gap between
traditional vaccination approaches and natural response to viral
antigen. As medullary macrophages rapidly degrade protein
antigen as a part of normal lymphatic filtration, it is important
to understand how to maximize antigen exposure to efficient
antigen presenters, especially when antigen concentration is
highest immediately after vaccination. Data from this study
suggest that LNDCs are capable of effectively using antigen
when appropriately stimulated and could represent an efficient target for vaccination approaches.
An unexpected observation was the rapid kinetics of LNDC
repositioning from the paracortical region to the mIFR in response to localization of viral antigen. Although large deposits
of LNDCs were observed in viral-loaded IFRs within hours
of vaccination, individual LNDC morphological changes
could be observed within 12 min of vaccination. The highly
directional migration pattern of LNDCs to the sites of viral
accumulation suggests a response to chemotactic mediators
released from a prestored source after innate sensing of viral
antigen, although this source remains yet unclear.
A recent study has identified CXCR3 and its ligands
CXCL9 and CXCL10 as required for migration of CD8
memory T cells into IFRs, where they make contact with
both antigen and APCs (Sung et al., 2012). Although the distinction between mIFRs and cIFRs was not made in these
studies, it is clear that this chemoattractant is important in
various conditions of immune response. In our study, formation
of clusters of viral-specific CD4+ T cells with LNDCs was
significantly reduced in CXCR3/ OT-II T cells. Because
Ar ticle
CXCL10 expression is not restricted to LNDCs, this pathway

might provide an efficient mechanism for collaboration between LNDCs and incoming mDCs, whereby early activation of naive CD4 T cells, and concomitant expression of
CXCR3, allows activated T cells to more easily find mDCs
from the injection site, which also express CXCL10. Additionally, it is possible that mDCs might be able to locate hot
spots of immune activation within the LN through their own
expression of CXCR3.
In summary, using novel whole-node imaging approaches,
we observed a multistep system of immune activation whereby
viral antigen, LNDCs, and cognate naive CD4+ T cells rapidly
assemble in highly organized environments within the draining LNs known to be relevant to vaccine design (Liu et al.,
2014). In addition to identifying an unexpected repositioning
of LNDCs to specialized sites within the draining LN, these
findings clarify the importance of CXCR3 in establishing
immunostimulatory microenvironments in three dimensions
and establish LNDCs as biologically relevant cell populations,
sufficient to drive humoral memory response to an influenza
vaccine in the absence of mDCs.
Animals. Mice used in this study were bred and housed in standard conditions
and used between 6 and 12 wk of age. All experimental protocols were approved
through Harvard Universitys Institutional Animal Care and Use Committee.
In vivo labeling. In vivo labeling of the PLN subcapsular sinus and medulla
was achieved through injection of Alexa Fluorconjugated (A488, A568,
A633) antibody targeting stromal (aLYVE-1) or macrophage populations
(a-CD169, a-SIGNR1, a-F4/80) s.c. in the footpad 4 h before PLN imaging
or tissue harvest.
PLN live imaging. PLN live imaging was performed through surgical exposure of the PLN in anesthetized mice and MPM. Temperature was monitored using a digital thermometer embedded in the imaging chamber created
around the PLN and controlled using a closed-circuit water circulation system (Lindquist et al., 2004; Gonzalez et al., 2010).
Three-dimensional reconstruction. Collected organs were fixed in 4%
PFA, embedded in OCT, and serially cryosectioned (50 m). PLN sections
were serially imaged by MPM and assembled using Imaris software (Bitplane)
to obtain three-dimensional reconstructions. Final analysis was performed
using Volocity image analysis software (PerkinElmer).
Whole-organ imaging. LNs were dissected and fixed overnight in 4%
paraformaldehyde. The sample was then incubated in increasing concentrations of tetrahydrofuran followed by dichloromethane. Finally, the PLN was
immersed in BABB (benzyl alcohol/benzyl benzoate, mix 1:2) for final clearing and imaging (Lindquist et al., 2004; Ertrk et al., 2012).
Viral propagation/labeling. Influenza virus was grown in live chicken
eggs, purified over sucrose gradients (Szretter et al., 2006; Gonzalez et al.,
2010), and labeled using standard Alexa Fluorlabeling protocol (Invitrogen).
1020-l s.c. injections were given in the footpad at 106 PFU.
T cell isolation. LNs of OT-II mice were processed using Liberase DH
(Sigma-Aldrich) digestion, and T cells were isolated through MACS negative
selection (CD11c, CD11b, NK1.1, B220, GR-1, CD69, CD8). T cells were
labeled in 10 m CMTMR for imaging or 5 M CFSE for imaging and flow
JEM Vol. 211, No. 8
cytometric identification. 1 or 5 106 cells were adoptively transferred into

naive recipients for flow cytometric or imaging experiments, respectively.
Three-dimensional image analysis. Images were processed and
analyzed using Volocity imaging software. Histocytometric analysis of
reconstructions was performed with CellProfiler (Carpenter et al., 2006;
Grigorova et al., 2010), and CellProfiler Analyst (Hickman et al., 2008;
Jones et al., 2008; Len et al., 2012). Analysis of reconstructions was performed with individual XY imaging planes, which were representative of
at least 400 m of reconstructed LNs.
T cell clustering. CXCR3/ and WT OT-II cells were isolated as above,
differentially labeled, and adoptively transferred into C57BL/6 recipients.
PLNs were collected, processed, and serially imaged 24 h after vaccination
with UV-PR8-OTII. T cell clusters were identified using a blinded approach,
and the percentage of each population within these clusters was measured.
Ear resections. Ear removal was performed 30 min after injection of 10 l
PBS or UV-PR8 s.c. between the ear dermal layers. ELISA analysis of serum
was performed through immobilization of UV-PR8 on the plate, addition of
serum, and probing for specific binding of IgM, IgG1, or IgG2b.
Statistics. All statistics/graphical representations of collected data were assembled through Prism (GraphPad Software). Mean and SEM are displayed
where applicable. Results from Students t tests or Tukeys post tests are indicated by asterisks in the figures (*, P < 0.05; **, P < 0.005; ***, P < 0.001).
One- or two-way ANOVA testing is indicated in the legends.
Online supplemental material. Video 1 shows LNDC migration in response to influenza vaccination. Video 2 shows that LNDCs infiltrate the
medulla after UV-PR8 vaccination. Video 3 shows reconstruction of a PLN.
Video 4 shows lymph flow through the PLN. Online supplemental material
is available at http://www.jem.org/cgi/content/full/jem.20132327/DC1.
We thank J. Campbell for providing CCR7-deficient mice, N. Anandasabapathy and
S. Jones for critical discussion and review of this manuscript, and H. Leung, E.
Carroll, S. Lavoie, and M. Ma for technical support.
This work was supported by the National Institutes of Health (NIH)National
Institute of Allergy and Infectious Diseases (grants 1 P01 AI078897, R37 AI054636,
and R01 AI039246 to M.C. Carroll; R01CA069212 to A.D. Luster; RO1 DK074500 and
PO1 AI045757 to S.J. Turley; and AI107625 to P.G. Thomas), NIH T32 Training Grant
in Transplantation (T32 AI007498 to M.C. Woodruff), the American Lung Association
(grant RT-224269-N to C.N. Herndon), and the National Health and Medical
Research Council, Australia (fellowship 516791 to J.R. Groom).
The authors declare no competing financial interests.
Author contributions: M.C. Woodruff, B.A. Heesters, and C.N. Herndon performed all
experiments and analysis described in the text. P.G. Thomas, J.R. Groom, and A.D. Luster
developed critical reagents for the completion of the study. M.C. Woodruff, S.J. Turley,
and M.C. Carroll designed this study and developed the manuscript for publication.
Submitted: 6 November 2013
Accepted: 19 June 2014
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1621
Article
Dysfunctional CD8+ T cells in hepatitis

B and C are characterized by a lack
of antigen-specific T-bet induction
Peter D. Kurktschiev,1,2 Bijan Raziorrouh,1,2 Winfried Schraut,1,2
Markus Backmund,2,3 Martin Wchtler,4 Clemens-Martin Wendtner,4
Bertram Bengsch,5 Robert Thimme,5 Gerald Denk,2 Reinhart Zachoval,2
Andrea Dick,6 Michael Spannagl,6 Jrgen Haas,7 Helmut M. Diepolder,2
Maria-Christina Jung,8 and Norbert H. Gruener1,2
1Institute
for Immunology, Ludwig-Maximilians-University, 80539 Munich, Germany

of Medicine II, University Hospital Munich, 80539 Munich, Germany
3PiT Praxis im Tal, 80331 Munich, Germany
4Department of Medicine, Klinikum Schwabing, 81925 Munich, Germany
5Department of Medicine II, University Hospital Freiburg, 79106 Freiburg, Germany
6Laboratory of Immunogenetics and Molecular Diagnostics, 80539 Munich, Germany
7Division of Infection and Pathway Medicine, University of Edinburgh, Edinburgh EH16 4SB, Scotland, UK
8Leberzentrum Mnchen, 80336 Munich, Germany
2Department
The transcription factor T-bet regulates the production of interferon- and cytotoxic
molecules in effector CD8 T cells, and its expression correlates with improved control of
chronic viral infections. However, the role of T-bet in infections with differential outcome
remains poorly defined. Here, we report that high expression of T-bet in virus-specific CD8
T cells during acute hepatitis B virus (HBV) and hepatitis C virus (HCV) infection was associated with spontaneous resolution, whereas T-bet deficiency was more characteristic of
chronic evolving infection. T-bet strongly correlated with interferon- production and
proliferation of virus-specific CD8 T cells, and its induction by antigen and IL-2 stimulation
partially restored functionality in previously dysfunctional T-betdeficient CD8 T cells.
However, restoration of a strong interferon- response required additional stimulation with
IL-12, which selectively induced the phosphorylation of STAT4 in T-bet+ CD8 T cells. The
observation that T-bet expression rendered CD8 T cells responsive to IL-12 suggests a
stepwise mechanism of T cell activation in which T-bet facilitates the recruitment of
additional transcription factors in the presence of key cytokines. These findings support a
critical role of T-bet for viral clearance and suggest T-bet deficiency as an important
mechanism behind chronic infection.
CORRESPONDENCE
Peter Kurktschiev:
peter.kurktschiev@
med.uni-muenchen.de
Abbreviations used: acHCV,
acute chronic-evolving HCV;
arHBV, acute resolving
HBV; arHCV, acute resolving
HCV; cHBV, chronic HBV;
cHCV, chronic HCV; EBV,
Eppstein-Barr virus; Eomes,
Eomesodermin; Flu, Influenza
A virus; HBV, hepatitis B virus;
HCV, hepatitis C virus; PBSE,
Pacific Blue succinimidyl ester;
rHBV, long-term resolved HBV;
rHCV, long-term resolved
HCV; STAT4, signal transducer
and activator of transcription 4.
The transcription factor T-bet (T-box expressed

in T cells; Tbx21) is a crucial regulator of T cell
immunity. It mediates the differentiation of
CD4 T cells into Th1 cells and of CD8 T cells
into Tc1 cells (Szabo et al., 2000; Mullen et al.,
2001; Sullivan et al., 2003). In effector CD8
T cells, T-bet is an activator of interferon-
production and correlates with increased cytotoxic activity (Szabo et al., 2000; Cruz-Guilloty
et al., 2009). A recent study has found that T-bet
is highly expressed in HIV-specific CD8 T cells
of HIV elite controllers who control viral load
to very low levels without therapy (Hersperger
et al., 2011). Correspondingly, its loss has been

J. Exp. Med. 2014 Vol. 211 No. 10 2047-2059
observed in dysfunctional CD8 T cells of chronic

HIV patients and in the murine LCMV model
of chronic viral infection (Kao et al., 2011;
Ribeiro-dos-Santos et al., 2012). Furthermore,
it has been shown that T-bet and the homologous transcription factor Eomesodermin (Eomes)
define two distinct states of virus-specific CD8
T cells and their balance plays an important
role in the control of chronic viral infection
(Paley et al., 2012). Interestingly, retroviral
2014 Kurktschiev et al. This article is distributed under the terms of an Attribution
2047
overexpression of T-bet prevented CD8 T cell exhaustion in

chronic LCMV infection, demonstrating the therapeutic potential of T-bet modulation (Kao et al., 2011). However, the
role of T-bet in human viral infections with dichotomous outcome remains to be determined. Because HIV and LCMV
clone13 establish chronic infection in all infected subjects,
other pathogens would be more suitable to dissect the differences between successful versus failing immune response during acute infection.
Human hepatitis B virus (HBV) and hepatitis C virus
(HCV) infection can both either resolve spontaneously or
establish chronic infection. Virus-specific CD8 T cells play a
causal role in the clearance of both infections, as demonstrated
by in vivo CD8 T cell depletion in the chimpanzee model
where all subjects challenged with HBV or HCV developed
chronic infection (Shoukry et al., 2003; Thimme et al., 2003).
In chronic HBV and HCV infection, virus-specific CD8
T cells gradually lose their effector functions and become increasingly dysfunctional (Lechner et al., 2000a; Gruener et al.,
2001; Boni et al., 2007). One hallmark of severe dysfunction
is the lack of antigen-specific interferon- production by
T cells (Lechner et al., 2000b). Although the molecular mechanisms behind T cell dysfunction are the focus of intensive
research (Bowen et al., 2004; von Hahn et al., 2007; Wherry,
2011) it is yet unknown how far impaired regulation of
T-bet might be involved in the development of chronic HBV
and HCV infection.
In this study, we determined the expression of T-bet
in virus-specific CD8 T cells during acute HBV and HCV
infection and examined its correlation with the clinical
outcome. T-bet was highly expressed in spontaneously resolving but deficient in chronic-evolving infection. When
we further characterized the functional correlates behind
these differential expression patterns, we found a strong association of T-bet with antigen-specific proliferation and
interferon- production by virus-specific CD8 T cells. Induction of T-bet by antigen or IL-2 recovered antigenspecific proliferation but was not sufficient to restore
interferon- expression. However, restoration of a strong
interferon- response in previously dysfunctional CD8
T cells was achieved by additional stimulation with IL-12,
which selectively induced phosphorylation of STAT4
(pSTAT4) in T-bet+ CD8 T cells. This is consistent with
previous findings that T-bet and STAT4 cooperate in the
transcriptional control of interferon- (Thieu et al., 2008).
The observation that T-bet rendered CD8 T cells susceptible to IL-12 suggests a stepwise mechanism of T cell activation in which T-bet facilitates the recruitment of additional
transcription factors in the presence of key cytokines, and
thus contributes to the adjustment of an appropriate
T cell response.
These findings indicate a critical role of T-bet for a successful CD8 T cell response against HBV and HCV infection
and suggest that impaired induction of T-bet could be an important mechanism involved in CD8 T cell dysfunction during chronic viral infections.
2048
RESULTS
T-bet is highly expressed during
acute resolving HBV infection
Acute HBV infection resolved spontaneously in all enrolled
patients. Therefore, we would expect up-regulation of T-bet
in HBV-specific CD8 T cells of these individuals, in case it
plays an important role in viral clearance. Ex vivo expression
of T-bet was determined by intracellular flow cytometry
combined with MHC-I pentamers detecting HBV core 1827
(c18-27)specific CD8 T cells, which react with a major
immunodominant epitope in MHC-I A0201 background.
The frequencies of virus-specific CD8 T cells are shown
in Table S1.
When we compared T-bet expression in patients with
acute resolving HBV (arHBV), chronic HBV (cHBV) and
resolved HBV (rHBV) infection, we found a significantly
higher mean percentage of T-bet+ c18-27specific CD8
T cells during arHBV (57.9%) compared with cHBV (10%)
and rHBV (0.7%; Fig. 1, A and C). Our subanalysis of antiHBe+ and anti-HBe cHBV patients showed no significant
difference in T-bet expression (unpublished data). In healthy
controls T-bet+ CD8 T cells comprised 10.3% (mean) of
total CD8 T cells (unpublished data). Elevated T-bet expression during arHBV was confirmed for 2 additional HBVspecific epitopes (HBV envelope 183191 and HBV polymerase
573581; Fig. 1, D and E). To rule out nonspecific bystander
up-regulation of T-bet in CD8 T cells, we determined its
expression in non-HBV-specific CD8 T cells during acute
HBV infection. We chose EBV-specific CD8 T cells because
they are broadly detectable. There was no significant increase
in T-bet+ EBV-specific CD8 T cells (mean 10.2%; unpublished data). As a proof of principle, we determined antigenspecific T-bet up-regulation in EBV-specific CD8 T cells
(EBV BMLF-1 259267) during acute (aEBV) and latent
persisting (pEBV) EBV infection. 62.7% (mean) of EBVspecific CD8 T cells were T-bet+ in aEBV, whereas only
10.2% were T-bet+ in pEBV. Additionally, we found a mean
of 7.5% T-bet+ CD8 T cells specific for Influenza A (Flu; Influenza A MP 5866) in healthy controls with previously resolved infection, which served as control for a memory CD8
T cell response (Fig. 1, A and C).
Strong T-bet expression in acute HCV infection
correlates with spontaneous resolution
Whereas acute HBV infection in adults is almost universally
cleared, acute HCV infection becomes chronic in the majority of cases (Wright and Lau, 1993; Lauer and Walker, 2001).
In our study, 50% of the patients with acute HCV developed
chronic infection. HCV-specific CD8 T cells were detected
by HCV-specific pentamers covering several epitopes with
different MHC-I backgrounds. T-bet expression was determined during the earliest available time points of acute HCV
infection that was either cleared (arHCV) or became chronic
(acHCV) later on. All analyzed patients were viremic at this
time. Consistent with our results found in arHBV infection, frequencies of T-bet+ HCV-specific CD8 T cells were
Dysfunctional CD8+ T cells in HBV/HCV lack T-bet | Kurktschiev et al.
Ar ticle
Figure 1. HBV- and HCV-specific CD8

T cells express high amounts of T-bet during acute spontaneously resolving infection. PBMCs were isolated from (A) patients
with arHBV, cHBV, rHBV (top), aEBV, pEBV, or
resolved Flu infection (bottom) and (B) patients with arHCV (top left), rHCV (top right),
acHCV (bottom left), or cHCV infection (bottom right) and analyzed by flow cytometry.
The outlined areas indicate the population of
pentamer+ CD8 T cells and the numbers indicate the percentage of T-bet+ (above) and
T-bet (below) cells among total pentamer+
CD8 T cells. (C) Quantification of ex vivo T-bet
expression in virus-specific CD8 T cells of the
subjects described in A and B. Bars represent
the mean percentage of T-bet+ pentamer+
CD8 T cells among total pentamer+ CD8 T cells
of patients with arHBV (n = 19), cHBV (n = 24),
rHBV (n = 5), arHCV (n = 7), acHCV (n = 7),
cHCV (n = 7), rHCV (n = 7), aEBV (n = 3), pEBV
(n = 6), and Flu (n = 6). Error bars indicate the
SEM. *, P < 0.05; ***, P < 0.001 (Mann-WhitneyU test). (D) Representative ex-vivo flow
cytometry plots showing expression of T-bet
in HBV envelope (183191)specific (left) and
HBV polymerase (573581)specific CD8
T cells (right) of patients with arHBV. The
numbers indicate the percentage of T-bet+/
and pentamer+/ CD8 T cells among total CD8
T cells. (E) Scatter dot plot showing the percentage of T-bet+ envelope (183191)specific
(filled circles; n = 5) or polymerase (573581)specific (open circles; n = 5) CD8 T cells among
total pentamer+ CD8 T cells of patients with
arHBV. The data are representative of one
experiment due to limited patient material.
significantly higher in patients with spontaneous resolution

of HCV infection (arHCV; mean, 66.3%) compared with
patients with chronic-evolving infection (acHCV; mean, 14%;
Fig. 1, B and C). The frequencies of T-bet+ HCV-specific
CD8 T cells in long-term chronic (cHCV; mean, 4.5%) and
long-term resolved (rHCV; mean, 6.9%) HCV infection were
comparable to those found in cHBV and rHBV.
T-bet deficiency in early acute HCV
precedes chronic-evolving infection
To characterize the duration of T-bet up-regulation and
fluctuations in its expression levels in virus-specific CD8
T cells we performed longitudinal measurements of T-bet

expression in 5 patients with arHBV, 4 patients with acHCV,
and 4 patients with arHCV. This kinetics profile contained
data from 3 different time points within 6 mo after symptom
onset. The first time point (t1) was defined as the earliest
available patient sample obtained within 1 mo after onset of
acute symptoms. The following analyzed time points were
13 mo (t2) and 36 mo (t3) after symptom onset, respectively. In arHBV, we observed a high percentage of T-bet+
c18-27specific CD8 T cells at t1 (mean, 73.1%), which
gradually decreased to 40.3% at t2 and 21.8% at t3 (Fig. 2,
left). In arHCV infection, we found a similar pattern as seen
2049
Figure 2. T-bet expression is lost early in acute chronic-evolving HCV infection. Percentage of T-bet+ pentamer+ CD8 T cells among total pentamer+ CD8 T cells of patients with arHBV (n = 5; left), arHCV (n = 4; middle), and acHCV (n = 4; right) was determined by ex vivo flow cytometry at the
indicated time points. Data show the mean percentages and are representative of one experiment due to limited patient material. Error bars indicate SEM.
in HBV-specific CD8 T cells during arHBV (mean t1 = 67.1%;

t2 = 54.5%; and t3 = 29.2%) with high initial expression of
T-bet in HCV-specific CD8 T cells, which slowly decreased
over the 6 mo of follow up (Fig. 2, middle). In contrast, in
acHCV T-bet expression was low right from the beginning
(mean: t1 = 14.8%; t2 = 12.6%, and t3 = 11.9%; Fig. 2, right).
The 4 patients with acHCV were further differentiated into
partial (n = 3) versus no controllers of viremia (n = 1) according to the evolution of viral load between t1 and t3 (Table 1).
No correlation was found between T-bet or Eomes expression and quality of viral control. However, studies on larger
cohorts would be required to confirm this observation.
Eomes and T-bet can be coexpressed and define
different subsets of virus-specific CD8 T cells
As recent literature has reported that Eomes, which bears
strong homology with T-bet, can compensate for T-bet deficiency in CD8 T cells of T-bet/ mice, we investigated
if Eomes is associated with a successful immune response in
human HBV and HCV infection as well (Intlekofer et al.,
2005). Furthermore, one study has demonstrated that the
balance of two subsets of CD8 T cells, T-bet (high) and
Eomes (high), respectively, is important for the control of
murine LCMV and chronic HCV infection (Paley et al.,
2012). Therefore, we examined the co-expression of T-bet
and Eomes in virus-specific CD8 T cells under several conditions (Fig. 3, A and D). We found that Eomes+/T-bet
CD8 T cells did not show any significant difference between arHBV (10%, mean), cHBV (8.9%), arHCV (3%),
Table 1. Kinetics of viral load in acute chronic-evolving HCV

infection
Patient
P1
P2
P3
P4
2050
t1 viral load [IU/ml]
t3 viral load [IU/ml]
1,300
2,980
110,000
66,000
14
15
150
9,350,000
acHCV (10.5%), and cHCV (8.7%). In contrast, T-bet+/

Eomes cells, which comprised the majority of virus-specific
CD8 T cells, were significantly more frequent during
arHBV (mean, 38.5%) and arHCV (54.6%) as compared
with cHBV (9.2%), acHCV (11.7%), and cHCV (13.4%).
Interestingly, we consistently observed a population of Eomes+/
T-bet+ cells, which were significantly increased in arHBV
(19.4%, mean) and arHCV (11.7%) compared with cHBV
(0.8%), acHCV (2.3%), and cHCV (1.1%).
T-bet and PD-1 are coexpressed during acute
but not in chronic HBV or HCV infection
PD-1 is the hallmark inhibitory receptor found on exhausted
CD8 T cells. As recent publications have shown that T-bet can
suppress PD-1 we examined the coexpression of both factors in
HBV and HCV infection ex vivo. T-bet and PD-1 are highly
coexpressed in virus-specific CD8 T cells during resolving
arHBV (mean 51.2%) and arHCV (45%), whereas the frequencies of T-bet+/PD-1+ virus-specific CD8 T cells were significantly lower in cHBV (11%), acHCV (7.6%), and cHCV
(0.7%; Fig. 3, B and E). In contrast, the mean percentage of
PD-1+/T-bet virus-specific CD8 T cells was highest in cHBV
(65.7%) and cHCV (50.9%) as compared with acute arHBV
(37.7%), arHCV (19.2%), and acHCV (19%) infection.
Mutually exclusive expression of T-bet and CD127
In the murine LCMV infection model, it has been shown
that T-bet promotes the differentiation of effector and effector
memory CD8 T cells at the cost of centralmemory cells by
repression of IL-7R (CD127; Intlekofer et al., 2007). We
investigated if T-bet can repress CD127 expression on virusspecific CD8 T cells during HBV and HCV infection, as this
could affect the generation of memory CD8 T cells. T-bet+/
CD127+ virus-specific CD8 T cells were rare (means: arHBV,
3.7%; cHBV, 7.7%; arHCV, 4.9%; acHCV, 3.1%; and cHCV,
5.9%). The T-bet+/CD127 population showed higher frequencies in resolving (means: arHBV, 61.8%; arHCV, 48.9%)
as compared with chronic-evolving infections (cHBV, 5.8%;
Ar ticle
Figure 3. Expression of T-bet is associated with distinct phenotypes of virus-specific CD8 T cells. Ex vivo expression of T-bet, Eomes, PD-1, and
CD127 in PBMCs from patients with acute HBV or HCV infection was analyzed by flow cytometry on the earliest available samples obtained within 3 wk
of acute symptom onset. The data on cHBV and cHCV patients were obtained at any time point during chronic infection. (A) Contour plots show ex vivo
coexpression of T-bet and Eomes in virus-specific CD8 T cells of patients with arHBV, cHBV, arHCV, acHCV, and cHCV infection. The numbers indicate the
percentage of T-bet+/ and Eomes+/ CD8 T cells among pentamer+ CD8 T cells. (B) Representative contour plots of T-bet and PD-1 coexpression as
described in (A). (C) Representative contour plots of T-bet and CD127 coexpression as described in A. (D) Mean percentage of pentamer+ CD8 T cells with
a T-bet+/Eomes, T-bet/Eomes+, and T-bet+/Eomes+ phenotype as determined by flow cytometry. Data were obtained from arHBV (n = 19), cHBV (n = 24),
arHCV (n = 7), acHCV (n = 7), and cHCV (n = 7) patients. (E) Mean percentage of pentamer+ CD8 T cells with T-bet+/ PD-1+ and T-bet/ PD-1+ phenotype
found in the patients described in D. (F) Mean percentage of T-bet+/ CD127+, T-bet/ CD127+, and T-bet+/CD127 analogous to D. All error bars indicate
SEM. ***, P < 0.001 (Mann-Whitney-U test). Data are representative of one experiment due to limited patient material.
2051
acHCV, 13.5%; cHCV, 10.8%). On the contrary, T-bet/

CD127+ cells were more frequent in chronic-evolving
(means: cHBV, 69.8%; acHCV, 29.5%; cHCV, 50.4%) than
in resolving infection (arHBV, 4.8%; arHCV, 10.4%; Fig. 3,
C and F).
Induction of T-bet by IL-2 facilitates antigenspecific interferon- production in HBV-specific
CD8 T cells in cooperation with IL-12
To examine the effects of T-bet on CD8 T cell functionality,
we compared its coexpression with interferon- and perforin
in CD8 T cells of healthy controls, which were nonspecifically
activated by TCR-stimulation with CD3+CD28. The vast
majority of interferon- and perforin-producing stimulated
CD8 T cells were T-bet+ (unpublished data). Next, we addressed the question of whether specific stimulation with antigen (c18-27) or cytokines described as T-bet inducers in
literature (Lighvani et al., 2001; Ylikoski et al., 2005) could increase T-bet and interferon- expression in virus-specific CD8
T cells of chronic HBV patients. We tested several cytokines in
different concentrations (see Materials and methods) for their
potential to induce T-bet in CD8 T cells (Fig. 4 B). The only
cytokine that significantly induced T-bet was IL-2. Therefore,
we wanted to examine if T-bet induction by IL-2 was directly
coupled with interferon- production. We found a significant
but weak increase of T-bet+/interferon-+ CD8 T cells after
stimulation with IL-2+c18-27 (mean, 0.154%) compared with
stimulation with c18-27 antigen alone (mean, 0.012%) or the
control (mean, 0.008%). However, we observed that stimulation with IL-2+IL-12+c18-27 resulted in a much stronger
increase of T-bet+/interferon-+ CD8 T cells (mean 1.981%),
whereas IL-12+c18-27 was not able to induce the same effect
in the absence of IL-2 (0.014%). IL-2+IL-12 induced considerable background stimulation (1.191%; Fig. 4, A and C).
Figure 4. Antigen-specific interferon- production correlates with T-bet and can be restored by IL-2+IL-12 co-stimulation. PBMCs of patients with cHBV (n = 11) were cultured for 3 d in culture medium (control) containing either HBV-c18-27 antigen (Ag), c18-27 antigen+IL-2 (Ag+IL-2),
c18-27 antigen+IL-12 (Ag+IL-12), IL-2+IL-12, c18-27 antigen+IL-2+IL-12 (Ag+IL-2+IL-12), or CD3+CD28. On day 3 antigen-treated groups were
restimulated with antigen and all groups were incubated for 6 h in the presence of Brefeldin A before intracellular flow cytometry was performed.
(A) Expression of T-bet and interferon- by CD8 T cells. The numbers indicate the percentage of T-bet+/ and interferon-+/ CD8 T cells among total CD8
T cells. (B) Mean induction of T-bet by treatment with the respective cytokines. Induction was defined as the percentage of T-bet+ CD8 T cells after stimulation subtracted by the percentage of T-bet+ CD8 T cells in unstimulated controls. Error bars represent the SEM. (C) Mean percentage of T-bet+ interferon-+
CD8 T cells after stimulation. Error bars indicate the SEM. Data are representative of one experiment due to limited patient material. ***, P < 0.001 (MannWhitney-U test).
2052
Ar ticle
High expression of T-bet is associated

with strong antigen-specific proliferation
Antigen-specific proliferation of CD8 T cells is key to the generation of sufficient amounts of effector cells for control of the
virus.We analyzed the correlation between T-bet expression and
expansion of epitope-specific CD8 T cells upon stimulation.
PBMCs of patients with chronic HBV were stimulated with
either antigen c18-27 and/or cytokines IL-2 and IL-12 for 7 d
as described in the Materials and methods. The expansion of
c18-27specific CD8 T cells and the correlation between T-bet
expression and proliferation was determined by flow cytometry
with the proliferation marker Pacific Blue succinimidyl ester
(PBSE).The strongest expansion of c18-27specific CD8 T cells
was observed in the groups stimulated with antigen c18-27
(mean, 0.7% of total CD8 T cells), IL-2+c18-27 (mean, 1.83%),
and IL-2+IL-12+c18-27 (mean, 1.93%), which was a significant
induction compared with controls (mean, 0.1%). Of note, IL-12
did not significantly increase proliferation when added to
IL-2+Ag. PBMCs stimulated with IL-2 (mean, 0.1%), IL-12
(mean, 0.09%), or IL-2+IL-12 (mean, 0.15%) showed no increased
frequencies of virus-specific CD8 T cells (Fig. 5,A and B). Next,
we divided proliferating PBSE- and c18-27specific CD8
T cells in a T-bet+ and a T-bet subset to determine if T-bet was
preferentially expressed in proliferating cells. We found significantly higher frequencies of T-bet+ CD8 T cells in the groups
with strong proliferation, whereas T-bet CD8 T cells showed
low frequencies (Ag mean: T-bet+, 0.29%; T-bet, 0.09%;
IL-2+Ag T-bet+, 1.12%; T-bet, 0.07%; and IL-2+IL12+Ag
T-bet+, 1.04%;T-bet, 0.05%; Fig. 5, C and D). Interestingly, we
observed strong induction of T-bet+ virus-specific CD8 T cells
in the groups stimulated with antigen c18-27 (mean, 82.6% of
specific cells) or IL-2 (mean, 56.2%) compared with IL-12
(mean, 12.5%), and control (mean, 8.5%; unpublished data).
IL-12 selectively induces phosphorylation
of STAT4 in T-bet+ CD8 T cells
We sought to determine the mechanism behind the much
stronger induction of interferon- after additional stimulation
with IL-12. STAT4 is of crucial importance for signal transduction by the IL-12 receptor and is known to cooperate with
T-bet in the regulation of the interferon- gene (Thieu et al.,
2008).Therefore, we investigated if phosphorylation of STAT4
might be a possible explanation. PBMCs were either pretreated
with IL-2 to induce T-bet or left unstimulated as control. On
day 3, cells were restimulated with either IL-12 which should
induce STAT4 phosphorylation or IL-2 as negative control.
We found that IL-12 preferentially induced pSTAT4 in T-bet+
CD8 T cells (mean, 2.6%) as compared with T-bet cells
(0.38%; Fig. 6, A and B). IL-2 restimulation minimally induced
pSTAT4 in T-bet+ (0.3%) and T-bet CD8 T cells (0.34%)
compared with controls (T-bet+, 0.03%; T-bet, 0.05%).
DISCUSSION
In this study, we investigated in how far T-bet is involved in
the early events during acute infection, which are critical for
viral clearance. Although previous studies have discovered the
importance of T-bet in HIV and LCMV clone 13 that universally establish chronic infection, we assessed its role in human
HBV and HCV infection that allowed us the direct comparison of successful versus failing CD8 T cell response against
the same pathogen (Hersperger et al., 2011; Kao et al., 2011;
Ribeiro-dos-Santos et al., 2012). Our most important finding
is that expression of T-bet in virus-specific CD8 T cells was
strongly associated with clearance of acute HCV infection.
HCV-specific CD8 T cells in acute chronic-evolving HCV
infection, though broadly detectable, failed to clear HCV infection and were deficient in T-bet expression. In acute HBV
infection, increased expression levels of T-bet were observed
in patients who later resolved the disease, whereas expression
was lost in dysfunctional CD8 T cells in spite of viral persistence during long-term chronic infection. As acute HBV infection rarely takes chronic course in adults, we were not able
to confirm T-bet deficiency in HBV-specific CD8 T cells of
such patients.Therefore, further studies will be required to establish a definitive association between T-bet expression and
clinical course of HBV infection. All patients who later controlled viral replication had high expression of T-bet in virusspecific CD8 T cells, and we confirmed the same pattern in
acute and latent persisting EBV infection. These data provide
further evidence for a central role of T-bet for a successful
virus-specific CD8 T cell response in self-limiting human
viral infections whereas T-bet deficiency was associated with
chronic infection. Several studies have provided insights into
the mechanisms behind T-bet deficiency in chronic infections. In the murine LCMV infection model, antigen persistence and high viral load lead to reduced T-bet levels in CD8
T cells, possibly by impaired T cell receptor signaling (Kao
et al., 2011). Direct inhibition of CD8 T cell signaling by interaction with viral proteins can occur as well. For example,
HCV core protein inhibits proliferation and interferon- production of HCV-specific T cells by blocking their C1q complement receptor (Kittlesen et al., 2000). Generation of escape
mutations can also lead to reduced T cell receptor stimulation
(Chang et al., 1997). Interestingly, we did not observe ex vivo
expression of T-bet and Eomes on HCV-specific CD4 T cells
of patients with acute HCV infection, which warrants further
investigation (Fig. S1). Of note, T-bet was readily inducible in
CD4 T cells under Th1 culture conditions.
Our longitudinal measurements demonstrate that T-bet
expression levels are stable over longer time spans and are not
affected by rapid fluctuations. In acute resolving HBV and
HCV infection, the highest expression of T-bet was observed
at the earliest available time points and remained elevated
compared with controls for at least 46 mo. In acute chronicevolving HCV infection, however, T-bet remained low at all
analyzed time points. Although very early loss of previously
induced T-bet might be one explanation, our observations of
T-bet kinetics in resolving infection suggest that this elevation
should be detectable for several months. As this was not the
case, an alternative explanation could be that T-bet is only
weakly induced and thus lost at earlier time points or is not
induced at all.
2053
Figure 5. Induction of T-bet by IL-2 and antigen is associated with antigen-specific proliferation. PBMCs of patients with cHBV (n = 10) were
labeled with the proliferation marker PBSE and cultured for 7 d in culture medium in the presence or absence of HBV-c18-27 antigen (Ag), IL-2, or IL-12.
Antigen c18-27 was added on day 0, and cytokines were administered on day 4 in the designated groups. Negative controls were cultured in medium
without further supplements, while positive controls were stimulated with CD3+CD28. On day 7, cells were stained for flow cytometry. (A) Frequencies of
pentamer+ CD8 T cells among total CD8 T cells and their PBSE labeling intensity. (B) Expression of T-bet and the frequency of pentamer+ CD8 T cells after
stimulation. (C) Mean percentage of c18-27specific CD8 T cells among total CD8 T cells. (D) Mean percentage of PBSE/T-bet (white) and mean percentage of PBSE/T-bet+ specific CD8 T cells (black) among total CD8 T cells. Error bars represent the SEM. Data are representative of one experiment due
to limited patient material. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Mann-Whitney-U test).
Eomes can trigger antigen-specific interferon- production in CD8+ T cells of T-bet/ mice and, together with
T-bet, it defines different subsets of virus-specific CD8 T cells
2054
that play an important role in the control of chronic viral infection (Pearce et al., 2003; Paley et al., 2012). As Eomes might
compensate for the lack of T-bet in HBV and HCV infection,
Ar ticle
Figure 6. IL-12 selectively induces STAT4

phosphorylation in T-bet+ CD8 T cells. PBMCs
of patients with cHBV (n = 7) were cultured for 3 d
with either IL-2 or medium as control. On day 3
cells were restimulated for 20 min with either
IL-2 or IL-12. Controls were left unstimulated.
Cells were then analyzed by flow cytometry.
(A) Coexpression of T-bet and pSTAT4 in CD8
T cells of an unstimulated control (left), PBMCs
cultured with IL-2 and restimulated with IL-2
(middle), and PBMCs cultured in IL-2 and restimulated with IL-12 (right). Representative plots
demonstrate the percentage of T-bet+/ and
pSTAT4+/ CD8 T cells among total CD8 T cells.
(B) Mean frequency of T-bet pSTAT4+ (white)
and mean frequency of T-bet+ pSTAT4+ (black)
CD8 T cells among total CD8 T cells after stimulation with the respective cytokines. Error bars represent the SEM. Data are representative of one
experiment due to limited patient material.
**, P < 0.01 (Mann-Whitney-U test).
we further analyzed its coexpression with T-bet in virus-specific

CD8 T cells under different conditions. In contrast to the predominant T-bet+ Eomes population, which was significantly
more frequent in acute resolving versus chronic-evolving infection and correlated with viral clearance, we did not observe
any up-regulation of Eomes+ T-bet CD8 T cells in acute
resolving infection. Of note, this was true for patients with
cHBV and cHCV as well, which differs from previous studies
that have suggested up-regulation of Eomes in exhausted cells
of patients with chronic infection (Paley et al., 2012). This
might be explained by the fact that we focused on HBVand HCV-specific peripheral blood lymphocytes, whereas the
cited study analyzed HCV-specific intrahepatic lymphocytes
or LCMV-specific lymphocytes in murine infection. Furthermore, a subset of T-bet+ Eomes+ cells was consistently detectable and showed significantly elevated frequencies in resolving
versus chronic-evolving infection, as described for the T-bet+
Eomes subset. The finding that Eomes was preferentially expressed in T-bet+ CD8 T cells supports the notion of a dominant role of T-bet for a functional CD8 T cell response during
acute infection.
One recent study has shown that the expression of PD-1,
the hallmark inhibitory receptor of exhausted CD8 T cells,
can be repressed by T-bet in chronic murine LCMV infection. Retroviral overexpression of T-bet in CD8 T cells led
to a sustained antiviral CD8 T cell response and down-regulation
of several inhibitory surface receptors (Kao et al., 2011). In
another study, induction of T-bet by third signal cytokines
decreases PD-1 levels in chronic HBV infection and restored
interferon- production (Schurich et al., 2013). We found
that T-bet and PD-1 are highly coexpressed in acute infection but later on T-bet is lost while PD-1 levels remain high.
The lack of T-bet might facilitate the high expression of
PD-1 on dysfunctional CD8 T cells during chronic infection.
However, as T-bet was not expressed at later stages of infection in our analyzed samples we could not assess possible suppressive effects on PD-1 ex vivo.
A successful immune response also depends on the balanced differentiation of CD8 T cells into terminally differentiated effector CD8 T cells and self-renewing centralmemory
CD8 T cells. Former studies demonstrated that T-bet drives
effector CD8 T cell differentiation at the expense of central
memory cells. Our finding that T-bet and CD127 are expressed in a mutually exclusive way fits into this hypothesis.
It remains to be determined how far T-bet expression during
acute infection could impair the differentiation of memory
CD8 T cells. Of note, T-bet was down-regulated in memory CD8 T cells of patients with resolved HBV, HCV, and
Flu infection. This could be explained by a lack of TCR
stimulation after antigen elimination. Another possible explanation is that T-bet+ CD8 T cells are short-lived effector cells
and are lost over time (Intlekofer et al., 2005; Joshi et al.,
2007). In summary, the factor that shows the strongest correlation with viral clearance is expression of T-bet. Although
significant, the correlation of T-bet+Eomes+ or T-bet+PD-1+
HCV-specific CD8 T cells and viral clearance was less pronounced. Interestingly, CD127 expression on virus-specific
CD8 T cells during acute HCV infection has shown a negative correlation with viral clearance.
Because our data suggested that the association of T-bet
with clearance of infection is not just a secondary phenomenon
but causally involved in the mechanisms behind a successful
immune response, we further investigated the effects of T-bet on
proliferation and interferon- production of virus-specific CD8
T cells.We observed that virtually all interferon-producing
CD8 T cells expressed T-bet as well, whereas T-betexpressing
CD8 T cells did not necessarily produce interferon-. We
tested several cytokines and cytokine combinations, including
2055
interferon-, interferon-, IL-2, and IL-12, which have been

used for induction of T-bet in recent literature and observed
the most striking effect on T-bet induction when stimulating
with IL-2 (Lighvani et al., 2001; Ylikoski et al., 2005). Sub
sequent experiments revealed a similar effect by stimulation with
antigen, which had an additive effect when combined with
IL-2. It was surprising that antigen-stimulated proliferation in
exhausted CD8 T cells. However, it has been previously
shown that exhausted T cells can proliferate extensively upon
antigen exposure without producing cytokines (Shin et al.,
2007). This fits with our experience that T cells require antigen for in vitro expansion. Although IL-2 plays an important
role for in vitro expansion, our results suggest that antigen itself
can trigger proliferation to some extent, too. We used low
doses of antigen because higher doses can induce anergy. In
addition, antigen concentration was kept constant. This is different from the in vivo setting, where antigen levels are fluc
tuating and can reach much higher levels as used in our
experiments. Induction of T-bet by IL-2 and/or antigen
strongly induced proliferation of HBV-specific CD8 T cells
but did not induce interferon-. Restoration of a strong interferon- response required additional stimulation with IL-12,
which could be explained by the finding that T-bet can induce
the IL-12R2 receptor, and thus make T cells more susceptible to IL-12 (Liao et al., 2011). We sought to clarify the mechanism behind the enhanced IFN- induction in IL-12stimulated
CD8 T cells and examined if STAT4, which is known as specific signal transducer of the IL-12 receptor, might be involved.
Interestingly, we found that IL-12 selectively induced STAT4
phosphorylation in T-bet+ CD8 T cells, which is consistent
with previous studies that describe cooperation of T-bet and
pSTAT4 in the induction of IFN- (Thieu et al., 2008). IL-12
single treatment induced neither T-bet up-regulation nor
interferon- secretion or proliferation. We observed background
stimulation of CD8 T cells in the group stimulated with
IL-2+IL-12 but without antigen, which can be explained in part
by TCR-independent reactivation of CD8 T cells by cytokines, as described for memory CD8 T cells (Rau et al.,
2013). One recent study has found that IL-12 induces T-bet
and interferon- in CD8 T cells of patients with chronic
HBV infection (Schurich et al., 2013). However, as IL-2 was
universally added to cell culture medium in the cited study,
considering our observations, we suggest that the observed
effects occur only under combined treatment with both cytokines. This is important, as the synergistic effects of both cytokines allowed us a 200-fold reduction of the effective IL-12
dose, thus making it more approachable for potential clinical
trials. Although previous trials with single cytokine treatment of chronic HBV and HCV infection found no significant improvement of the clinical outcome (Pardo et al., 1997;
Artillo et al., 1998; Zeuzem et al., 1999; Carreo et al., 2000),
a combination treatment with reduced cytokine doses might
be more promising.
Viral clearance requires a balanced interplay of virusspecific CD4 T cells, CD8 T cells and APC. The various patterns of viral replication seen during acute infection could
2056
result from impairment at different levels of this regulatory network during its adjustment. Loss of HCV-specific CD4 T cell
responses and IL-2 production as consistently observed in
patients developing chronic infection is one mechanism that
could interfere with proper induction of T-bet in CD8 T cells
(Diepolder et al., 1995; Gerlach et al., 1999; Urbani et al.,
2006). Furthermore, several studies have reported that HCV
core protein disturbs APC maturation during acute HCV infection, leading to decreased levels of IL-12, which could contribute to loss of interferon- production (Auffermann-Gretzinger
et al., 2001; Eisen-Vandervelde et al., 2004).
Our data provide for the first time evidence for the central role of T-bet in self-limiting human viral infections and
for deficient T-bet induction in virus-specific CD8 T cells as
a mechanism associated with viral persistence. T-bet induction by IL-2 and co-stimulation with IL-12 restored function
in previously exhausted virus-specific CD8 T cells and could
be a potential target for future therapies.
Study subjects. Peripheral blood was obtained from patients and controls at
the University Hospital Munich. We examined HBV-specific CD8 T cell
responses in patients with MHC-I A0201 background and arHBV, cHBV, or
rHBV infection. The HCV-specific CD8 T cell responses were analyzed in
patients with arHCV, acHCV, cHCV, and rHCV HCV infection. HCV patients had MHC-I backgrounds A0101, A0201, A0301, B0701, or B3501. All
HCV genotypes were included. Patients with acute EBV infection and
healthy subjects served as controls.The patient characteristics are summarized
in Table 2.
Ethics statement. This study was conducted in conformity with the ethical
guidelines of the Declaration of Helsinki. Written informed consent was
obtained from all patients. Approval for this study was obtained from the Institutional Review Board of the medical faculty of the Ludwig-MaximiliansUniversity (Munich).
Diagnostic criteria. Acute hepatitis was defined as acute onset of nonspecific Flu-like symptoms and jaundice in previously healthy persons with peak
GPT elevation 10 times above the upper limit of normal. Acute HBV was
confirmed by concomitant detection of HBsAg, HBV-DNA, or anti-HBcIgM and acute HCV by detection of HCV-RNA or seroconversion of antiHCV. Other possible causes of acute hepatitis, like autoimmune hepatitis,
alcoholic liver disease, or toxins were excluded. Resolution of acute hepatitis
B was confirmed by seroconversion of anti-HBs. Acute-resolving HCV was
defined as spontaneous loss of initially detectable HCV-RNA, which remained negative for at least 12 mo. In chronic-evolving acute HCV infection, HCV-RNA remained detectable for longer than 6 mo after symptom
onset. Resolved HCV was defined as a previously cleared HCV infection
without detection of HCV-RNA for at least 12 mo before enrollment. Diagnosis of chronic HCV infection was based on elevated serum GPT levels
for at least 6 mo and the consistent detection of HCV-RNA. Chronic HBV
was defined by detection of HBV-DNA or HBsAg for more than 6 mo.
Acute EBV was diagnosed by acute clinical symptoms, typical hematologic
findings, and detection of EBV-DNA.
Isolation of PBMCs. Human PBMCs were isolated from heparinized
blood by Ficoll-Paque density-gradient centrifugation as described earlier
and were either analyzed directly or cryopreserved (Perlmann et al., 1976).
HLA typing. DNA was extracted from PBMCs with the QIAamp DNA
Blood Mini kit (QIAGEN) following the manufacturers instructions. HLA
typing was performed as described previously (Witt et al., 2002).
Ar ticle
Table 2. Patient characteristics

Group
arHBV
cHBV
aHCV
cHCV
rHBV
rHCV
aEBV
healthy
n
19
24
14
7
5
7
3
9
Female /male
6/13
7/17
6/8
3/4
0/5
3/4
2/1
8/1
Age (mean)
38.4
44.6
53.5
44.8
47.4
53.3
44.3
39.1
GPT(U/l; mean)
Viral load (mean)
HCV GT1
2,567
59.5
984
81.9
normal
normal
n.d.
normal
cop/ml
19.6
18.3 103 cop/ml
6.9 106 IU/ml
0.8 106 IU/ml
negative
negative
negative
negative
9
n.d.
n.d.
-
Antibody reagents and viability dyes. The following antibodies were

used for flow cytometry: FITC antiT-bet (clone 4B10), Pacific Blue antiCD45RA (HI100), APC-H7 antiinterferon- (4S.B3), and Pacific Blue
anti-CD57 (HCD57; BioLegend); APC anti-Eomes(WD1928) and eFluor780
anti-CD127 (eBioRDR5; eBioscience); APC-H7 anti-CD27 (M-T271),
APC antiPD-1 (MIH4), Alexa Fluor 647 anti-STAT4 (pY693; 38/pStat4),
PerCP anti-CD3 (SK7), V500 anti-CD8 (SK1), V450 anti-CD8 (RPA-T8),
PerCP anti-CD14 (MP9), and PerCP anti-CD19 (SJ25C1; BD). 7-AAD
(BD) was used as viability dye in ex vivo stainings, whereas FVD eFluor450
(eBioscience) was used for fixed cells.
Synthetic peptides, pentamers, and cytokines. The following MHC-I
pentamers were used for the detection of epitope-specific CD8 T cells: HBV
core antigen 1827 FLPSDFFPSV, HBV polymerase 573581 FLLSLGIHL,
HBV envelope 183191 FLLTRILTI, HCV-NS3 14061415 KLVALGINAV,
EBV BMLF-1 259267 GLCTLVAML, and Flu MP 5866 GILGFVFTL (all
with MHC-I A0201 background); HCV-NS3 14361444 ATDALMTGF
(HLA-A0101); HCV-NS5 25882596 RVCEKMALY (HLA-A0301); HCV
core 4149 GPRLGVRAT (HLA-B0701) and HCV-NS3 13591367 HPNIEEVAL (HLA-B3501). All MHC-I pentamers were PE-labeled and obtained
from ProImmune. All corresponding peptides were synthesized by EMC microcollections and used for the in vitro stimulation of specific CD8 T cells. For
stimulation experiments, recombinant human IL-2 (R&D Systems) and IL-12
p70 (eBioscience) were used.The following cytokines were screened for induction of T-bet in CD8 T cells: IL-18 (10 ng/ml), IL-21 (25 ng/ml), IL-23 (50 ng/ml),
interferon- (2,000 IU/ml), interferon- (2,000 IU/ml; eBioscience).
PBMC stimulation. PBMCs were cultured for 3 d in RPMI 1640 medium
(Invitrogen) containing 2 mM l-glutamine, 1 mM sodium pyruvate,
100 U/ml of penicillin, 100 g of streptomycin/ml and 5% human AB
serum. In the cytokine-stimulated groups, IL-2 (20 IU/ml) and/or IL-12
(50 pg/ml) were added on day 0. Antigen-stimulated groups received 5 g/ml
antigen on day 0 and were restimulated with the same dose of antigen on day 3.
On day 3, cells were incubated for 6 h under stimulating conditions in the
presence of Brefeldin A (eBioscience). After stimulation, cells were prepared
for flow cytometry by intracellular cytokine staining.
PBSE proliferation assay. 500,000 PBMCs per condition were stained in
120 M PBSE (Life Technologies). Cells were distributed on a 96-well
round-bottomed cell culture plate in 100 l of culture medium and were
cultured in the presence or absence of antigen (5 g/ml) for 7 d. On day 4,
IL-2 (20 IU/ml) and/or IL-12 (50pg/ml) were added. On day 7, cells were
collected and stained for flow cytometry.
Flow cytometry. 23 106 PBMCs were stained with MHC-I pentamers
according to the manufacturers instructions. After staining with viability dyes
and antibodies specific for surface markers, cells were fixed with intracellular
fixation buffer and permeabilized with permeabilization buffer (both from
eBioscience). After the permeabilization step, cells were stained with intracellular markers (T-bet, Eomes, and interferon-). For ex vivo staining, pentamer+
106
Anti-HBe n.d.
8
13
6
5
-
5
6
cells were enriched by anti-PE MACS-beads, directed against PE-labeled

pentamers. Calculation of pentamer+ CD8 T cells was performed as pre
viously described (Lucas et al., 2007). In brief, samples were divided in a
preenrichment probe (10% of sample cells), which was used to determine the
input number of CD8 T cells and a post-enrichment probe (90% of sample
cells), which was run through miniMACS MS separation columns (Miltenyi
Biotec). Frequencies of pentamer+ CD8 T cells were calculated by dividing
the absolute number of pentamer+ CD8 T cells from the postenrichment
probe by the number of CD8 T cells in the preenrichment probe 9. Samples were acquired on a FACSCanto II flow cytometer (BD). Data were analyzed with FlowJo 9.6.1. software (Tree Star). Gating strategy excluded
monocytes (CD14+), B-lymphocytes (CD19+), and dead cells (7-AAD+) by
a dump channel.
Detection of phosphorylated STAT4 by intracellular phospho-flow
cytometry. 2 106 PBMCs per well were incubated for 3 d with either
IL-2 or medium as control. On day 3, cells were stimulated for 20 min with
either IL-2 or IL-12. After fixation and permeabilization with Perm buffer
III (BD) cells were stained with anti-pSTAT4 and antiT-bet before analysis
by flow cytometry.
Statistical analysis. Statistical analysis was performed with Prism 5.0 software (GraphPad) and included 2-sided Wilcoxon matched pairs test and
Mann-Whitney-U test for unpaired samples. Statistical significance was defined as P < 0.05.
Online supplemental material. Table S1 shows the ex vivo frequencies of
pentamer+ CD8 T cells of the patients described in Fig. 1. Fig. S1 demonstrates flow cytometry plots and gating procedure of CD4 tetramer stainings
of 2 patients with acute HCV (central plots). Online supplemental material
http://www.jem.org/cgi/content/full/jem.20131333/DC1.
We thank Michaela Zankl for excellent technical assistance and Jutta Drrmann for
her continuous support.
This work was supported by the Research Network Host and viral
determinants for susceptibility and resistance to hepatitis C virus infection from
the German Federal Ministry of Research, and the European Research Council
funded Research Network HepaCute: Host and viral factors in acute hepatitis C.
The authors have no competing financial interests.
Author contributions: P.D. Kurktschiev, B. Raziorrouh, W. Schraut, H.M. Diepolder,
and N.H. Gruener designed the experiments and analyzed the data; M. Spannagl
and A. Dick did the HLA typing; B. Bengsch and R. Thimme performed analysis of
cHCV patients and critically reviewed the manuscript; M. Wchtler, M. Backmund,
R. Zachoval, and G. Denk provided patient samples and clinical information;
M.C. Jung and J. Haas provided patient samples and clinical information and
critically reviewed the manuscript; and P.D. Kurktschiev and N.H. Gruener wrote
the manuscript.
Submitted: 26 June 2013
2057
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2059
Article
Interplay between regulatory T cells

and PD-1 in modulating T cell
exhaustion and viral control
during chronic LCMV infection
Pablo Penaloza-MacMaster,1 Alice O. Kamphorst,1 Andreas Wieland,1
Koichi Araki,1 Smita S. Iyer,1 Erin E. West,1 Leigh OMara,1 Shu Yang,1,2
Bogumila T. Konieczny,1 Arlene H. Sharpe,3 Gordon J. Freeman,4
Alexander Y. Rudensky,5,6,7 and Rafi Ahmed1
1Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322
2Xiangya
School of Medicine, Central South University, Changsha, Hunan Province, 410013, China
of Microbiology and Immunology, and 4Department of Medical Oncology and Dana Farber Cancer Institute,
Department of Medicine, Harvard Medical School, Boston, MA 02115
5Howard Hughes Medical Institute, 6Immunology Program, Sloan-Kettering Institute for Cancer Research, and 7Ludwig Center
at Memorial Sloan-Kettering Cancer Center, New York, NY 10065
3Department
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 10100-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.
CORRESPONDENCE
Rafi Ahmed:
rahmed@emory.edu
OR
Alexander Rudensky:
rudenska@mskcc.org
Abbreviations used: Arm, Armstrong; cl-13, clone 13; DT,
diphtheria toxin; LCMV, lymphocytic choriomeningitis virus;
MFI, mean fluorescence intensity; PD-1, programmed cell
death-1; PD-L1, programmed
cell death ligand-1.
Regulatory T cells expressing transcription factor Foxp3 are indispensable for preventing immune responses to self, and their absence results
in multi-organ autoreactivity and death (Kim
et al., 2007; Sakaguchi et al., 2008). In addition
to their major role in maintaining peripheral
tolerance, T reg cells also control immune responses to infections. During acute infection,
T reg cells can promote migration of effector
immune cells to infection sites by modulating
chemokine production (Lund et al., 2008), and
prevent the activation of low avidity CD8
T cells (Pace et al., 2012). However, in cancer
and persistent infections, T reg cells may expand
Pablo Penaloza-MacMaster and Alice O. Kamphorst contributed equally to this paper.

J. Exp. Med. 2014 Vol. 211 No. 9 1905-1918
and facilitate disease progression due to inhibition of T cell responses (Zou, 2006; Li et al.,
2008; Belkaid and Tarbell, 2009; Dietze et al.,
2011; Punkosdy et al., 2011).
In cancer and persistent infections, chronic
antigenic stimulation causes deterioration of
T cell responses.T cell exhaustion is manifested
by progressive loss of proliferative potential,
cytokine production, and for CD8 T cells, killing
capability (Zajac et al., 1998;Wherry, 2003, 2011).
This progressive T cell dysfunction is associated
with expression of programmed cell death-1
2014 Penaloza-MacMaster et al. This article is distributed under the terms of an
AttributionNoncommercialShare AlikeNo Mirror Sites license for the first six months
1905
Figure 1. T reg cells have an activated phenotype during chronic viral infection. Analysis was done on splenocytes from naive uninfected mice,
mice infected with LCMV Arm, and analyzed at least 100 d after acute infection and LCMV cl-13 chronically infected mice (mice transiently depleted of CD4
T cells and infected with LCMV cl-13, with analysis done at least 40 d after infection). (A) Percentage of T reg cells (Foxp3+) among CD4 T cells. (B) Absolute
number of T reg cells in spleen. (C) Frequency of V5+ cells among T reg cells. Mice infected with LCMV cl-13 and analyzed 21 d later were included as positive controls (2240% V5+ cells). (D) Graph shows MFI of CD25 expression on T reg cells. (E) Histograms show representative expression of different
markers by T reg cells and numbers represent MFI. Data are a compilation of 4 experiments (A and B) or show representative results from 3 experiments
(CE; n = 34 mice per group). Error bars indicate SEM. Non-parametric Mann Whitney test, where *, P < 0.05; **, P < 0.01; NS = not significant.
(PD-1) and other inhibitory receptors such as Tim-3 and LAG-3

(Barber et al., 2006; Blackburn et al., 2009; Jin et al., 2010). Importantly, proliferation and function of exhausted T cells can be
rescued by blockade of inhibitory pathways, which can result in
restoration of effective immune responses that control infections and tumors (Barber et al., 2006; Fourcade et al., 2010;
Sakuishi et al., 2010; Butler et al., 2012; Topalian et al., 2012).
Multiple pathways contribute to T cell dysfunction. Besides expression of inhibitory receptors by T cells, extrinsic
factors such as cytokines also play a fundamental role in T cell
exhaustion (Wherry, 2011). In addition, lack of CD4 help
exacerbates CD8 T cell exhaustion (Matloubian et al., 1994;
Zajac et al., 1998; Lichterfeld et al., 2004), and its restoration
via adoptive transfer of CD4 T cells can reinvigorate virusspecific responses in mice chronically infected with lymphocytic choriomeningitis virus (LCMV; Aubert et al., 2011).
IL-21 produced by CD4 T cells plays an important role in
sustaining CD8 T cells during chronic infection (Elsaesser
et al., 2009; Frhlich et al., 2009; Yi et al., 2009), and it was
recently reported that IL-21 may also aid CD8 T cells by
restricting T reg cell expansion (Schmitz et al., 2013). Thus,
conventional CD4 T cells have a positive impact on modulating CD8 T cell function during persistent antigenic stimulation. In contrast, it has been described that T reg cells are
detrimental to virus-specific T cell responses during persistent infection in mice (Dittmer et al., 2004; Dietze et al.,
2011; Schmitz et al., 2013); nevertheless, the role of T reg cells
in maintaining T cell exhaustion has not been well characterized or fully explored as a therapeutic approach.
To analyze the effects of T reg cells on exhausted virusspecific CD8 T cells, we used LCMV clone 13 (cl-13) infected
1906
Foxp3DTR knock-in mice in which Foxp3+ T reg cells express

the human diphtheria toxin (DT) receptor under control of
the endogenous Foxp3 locus and can be efficiently and specifically deleted by administration of DT (Kim et al., 2007).
Using this approach, we found that T reg cell ablation in
chronically infected mice leads to a striking rescue of exhausted
viral-specific CD8 T cells. Restoration of antiviral CD8 T cell
responses was dependent on cognate antigen, B7 costimulation, and conventional CD4 T cells. Interestingly, viral control
was not achieved unless T reg cell depletion was combined to
blockade of the PD-1 pathway. Thus, we propose that even
though T reg cells maintain CD8 T cells in an exhausted state
during persistent infections, the PD-1 inhibitory pathway
further operates by inhibiting the cytotoxic activity of rescued CD8 T cells toward target cells expressing high levels of
programmed cell death ligand-1 (PD-L1).
RESULTS
Regulatory T cells modulate exhausted
CD8 T cells during chronic viral infection
To establish a lifelong viral infection of multiple organs accompanied by exhaustion of virus-specific CD8 T cells, we in
fected mice with LCMV cl-13 after antibody-mediated transient
depletion of CD4 T cells (Matloubian et al., 1994). By day 45
after infection, CD4 T cells bounced back to normal numbers
in infected mice. When compared with naive or LCMV Armstrong (Arm)infected mice that had cleared virus, LCMV
chronically infected mice had increased frequency of T reg
cells (Fig. 1 A). However, due to decreased splenic cellularity,
the absolute numbers of T reg cells were actually reduced in
chronically infected mice (Fig. 1 B). Punkosdy et al. (2011)
T reg cells and PD-1 modulate T cell exhaustion | Penaloza-MacMaster et al.
Ar ticle
Figure 2. Exhausted CD8 T cells expand and undergo phenotypic changes upon T reg cell ablation. (A) Experimental outline. (B) Total numbers
of activated T cells in the spleen of PBS-treated or T reg celldepleted (DT) mice chronically infected with LCMV. (C) Number of LCMV-specific (Db GP276)
CD8 T cells among PBMCs before and after DT treatment. (D and E) Absolute numbers (D) and frequency (E) of LCMV-specific (Db GP276) CD8 T cells
within total CD8 T cell population. (F) Absolute numbers of LCMV DbGP276-specific CD8 T cells in the spleen at different days after T reg cell ablation.
(G) Proliferative activity of LCMV-specific (Db-GP276) CD8 T cells. Numbers show MFI of Ki67 expression. (H) Phenotype of LCMV-specific (Db GP276) CD8
T cells in spleen. Numbers show MFI of different CD8 T cell markers. Data are a compilation of 5 independent experiments with 35 mice per group. Error
bars indicate SEM. Non-parametric Mann Whitney test, where *, P < 0.05; ***, P < 0.001; NS = not significant.
have reported a transient expansion of V5+ T reg cells recognizing Mtv-9 endogenous retrovirus that peaks at day 17 of chronic
LCMV infection in B6 mice. Hence, the frequency of V5+ T reg
cells was not significantly increased in LCMV chronically infected
mice analyzed at 40 or more days after infection (Fig. 1 C).
To further characterize the T reg cell population in LCMV
chronically infected mice, we assessed the expression of several cell surface molecules. CD25 expression was not significantly different between T reg cells of chronically infected
mice, naive mice, or LCMV Arminfected mice that had
cleared virus (Fig. 1 D). However, based on the expression
levels of several other markers, such as CD69, CD44, CD62L,
ICOS, GITR, and CTLA-4, T reg cells from chronically infected mice appeared more activated (Fig. 1 E).
To examine the role of T reg cells during chronic infection, we infected Foxp3DTR knock-in mice with LCMV cl-13
after antibody-mediated transient depletion of CD4 T cells,
JEM Vol. 211, No. 9
and at least 45 d after infection, T reg cells were depleted by

DT administration (Fig. 2 A). T reg cell ablation in LCMV
chronically infected mice led to a marked increase in absolute
numbers of activated T cells (Fig. 2 B) and LCMV-specific
CD8 T cells (Fig. 2, CE). There was a >50-fold increase in
Db GP276 LCMV-specific CD8 T cells in blood (Fig. 2 C),
10-fold increase in the spleen, and up to 100-fold increase in
multiple nonlymphoid tissues (Fig. 2, D and E). This striking
reversal of CD8 T cell exhaustion occurred between day 5 and 7
after T reg cell depletion (Fig. 2 F) and was accompanied by
restoration of proliferative capacity assessed by the expression
of the cell cycle progression marker Ki67 (Fig. 2 G).These results show that during chronic infection, T reg cell depletion
enables expansion of previously exhausted T cells.
In addition to expansion, the surface phenotype of exhausted LCMV-specific CD8 T cells was also altered by T reg
cell ablation manifested by up-regulation of IL-7 receptor
(CD127), and increased CD44 and granzyme B expression
1907
Figure 3. LCMV-specific CD8 T cells regain effector function upon T reg cell ablation in chronically infected mice. LCMV
chronically infected Foxp3DTR knock-in mice
were depleted of T reg cells for 10 d by DT
administration as in Fig. 2. (A) Absolute numbers of CD8 T cells in spleen producing IFN-.
(B) MFI of IFN- production by CD8 T cells
after in vitro restimulation with various LCMV
peptides. (C) Percentage among DbGP276specific cells that express IFN- in spleen.
(D) Frequency of CD8 T cells producing both
IFN- and TNF after in vitro restimulation with
LCMV peptides. (E) Degranulation and surface
expression of CD107 a/b in CD8 T cells after
in vitro restimulation with GP276 LCMV peptide.
(F) Granzyme B expression on LCMV DbG276specific CD8 T cells in spleen. (G) Ex vivo cytotoxic activity of splenic CD8 T cells measured
by 51Cr release from MC57 target cells unpulsed (control) or pulsed with a mix of LCMV
peptides (GP33, GP276, and NP396). Data are
a compilation of 5 independent experiments
with 35 mice per group. Error bars indicate
SEM. Non-parametric Mann Whitney test,
where *, P < 0.05; ***, P < 0.001.
(Fig. 2 H). CD127 is expressed on naive and memory cells to

promote their long-term survival but is down-regulated on
exhausted T cells (Kaech et al., 2003; Wherry and Ahmed,
2004). Interestingly, a recent report described that IL-2 therapy
induces CD127 expression on exhausted CD8 T cells during

chronic LCMV infection (West et al., 2013). Hence, it is possible that an increase in IL-2 availability upon T reg cell ablation triggers CD127 up-regulation on exhausted cells.
Figure 4. Rescue of LCMV-specific CD8 T cell

responses by T reg cell ablation is greater than
by blockade of the PD-1 pathway. LCMV chronically infected Foxp3DTR knock-in mice were depleted of T reg cells for 10 d by DT administration
as in Fig. 2 or mice received 3 doses of PD-L1
blocking antibody, every 3 d. (A and B) Dot plots
(A) show frequency in the same mouse and graphs
(B) show number of LCMV-specific (Db GP276)
CD8 T cells among PBMCs before and after treatment. (C and D) Dot plots show frequency (C) and
graphs show number (D) of LCMV-specific (Db
GP33) CD8 T cells in different organs 11 d after
treatment. Data are a compilation of 3 independent experiments with 35 mice per group. Error
bars indicate SEM. Non-parametric Mann Whitney
test, where *, P < 0.05; **, P < 0.01; ***, P < 0.001;
NS = not significant.
1908
Ar ticle
Figure 5. Cognate antigen is necessary for activation of antiviral T cells after T reg cell depletion. (A) Experimental outline. (B) Total numbers
of activated T cells in the spleen of PBS-treated or T reg celldepleted (DT) mice 100 d after infection with LCMV Arm. (C) Phenotype of total CD8 T cells
in spleen. (D) Absolute numbers of LCMV-specific (Db GP276) CD8 T cells. (E) Absolute numbers of cells in the spleen that produce IFN- after in vitro
restimulation with various LCMV peptides. (F) Phenotype of splenic LCMV-specific (Db GP276) CD8 T cells. A representative of 3 independent experiments
is shown, with 56 mice per group. Error bars indicate SEM. Non-parametric Mann Whitney test, where ***, P < 0.001; NS = not significant.
Next, we wanted to ascertain whether T reg cell ablation

in chronically infected mice restored functionality of exhausted
T cells. We observed a marked increase in frequency and absolute numbers of virus-specific CD8 T cells that could produce IFN- upon stimulation with LCMV peptides (Fig. 3 A).
T reg cell depletion not only affected the number of LCMVspecific functional T cells but also substantially increased the
amount of IFN- produced per cell as indicated by the mean
fluorescence intensity (MFI) of intracellular IFN- staining
(Fig. 3 B). After T reg cell depletion, >75% of Db GP276 LCMVspecific T cells became functionally competent and acquired
ability to produce IFN- (Fig. 3 C).We also observed increased
TNF production to all LCMV epitopes tested and even severely exhausted CD8 T cells specific for the NP396 LCMV
epitope were rescued upon T reg cell ablation (Fig. 3 D).
Furthermore, after T reg cell depletion, LCMV-specific
CD8 T cells showed heightened ability to degranulate upon
cognate peptide stimulation as measured by surface expression
of the lysosomal marker CD107a/b (Fig. 3 E). As much as 73%
of all CD8 T cells expressed granzyme B (Fig. 3 F), and rescued LCMV-specific T cells were capable of in vitro killing of
antigen-bearing target cells (Fig. 3 G). Hence,T reg cell ablation
in LCMV chronically infected mice rescued virus-specific
CD8 T cells to proliferate and recover function.
The impressive increase in LCMV-specific responses after
T reg cell depletion was of a much higher magnitude than the
one caused by other means of rescue of exhausted CD8 T cells.
JEM Vol. 211, No. 9
For example, blockade of the PD-1 inhibitory pathway increased the frequency of LCMV-specific CD8 T cells in blood
by an average of fivefold, whereas T reg depletion led to an
increase of almost 100-fold (Fig. 4, A and B). Similarly, the
frequency and total number of LCMV-specific CD8 T cells in
spleen, lung, and liver achieved by T reg cell depletion was
higher than by blockade of the PD-1 inhibitory pathway in
LCMV chronically infected mice (Fig. 4, C and D).Thus,T reg
cells play a prominent role in the maintenance of CD8 T cell
exhaustion during chronic viral infection.
Cognate antigen is necessary for activation
of antiviral T cells after T reg cell depletion
Our results so far revealed that T reg cell ablation leads to a
striking rescue of virus-specific CD8 T cells during chronic
LCMV infection.To explore if cognate antigen was necessary
for T cell activation that ensues after T reg cell depletion, we
examined antiviral CD8 T cells after acute infection when
antigen has been cleared. We infected Foxp3DTR knock-in
mice with LCMV Arm, and T reg cells were depleted by DT
administration at least 100 d after infection (Fig. 5 A). LCMV
Arm causes an acute infection that is cleared within 810 d,
and generates virus-specific long-lived memory CD8 T cells
that persist in the absence of cognate antigen (Lau et al., 1994;
Murali-Krishna et al., 1999;Wherry and Ahmed, 2004). Similar to mice chronically infected with LCMV, T reg cell ablation triggered expansion of total activated CD4 and CD8
1909
Figure 6. Co-stimulation and CD4 T cells are

required for the rescue of exhausted CD8 T cells
upon T reg cell depletion. Foxp3DTR knock-in mice
chronically infected with LCMV cl-13 received DT for 10 d
(as in Fig. 2). CTLA-4 Ig was administered every other
day during T reg cell depletion starting at day 2,
and CD4 T cells were depleted upon administration of
CD4 antibody on days 2 and 1 of T reg cell ablation. (A) Representative dot plots show expression of
B7.1 and B7.2 on splenic DCs after 8 d of T reg cell
ablation. (B and C) Frequency of splenic CD8 T cells
that co-express IFN- and TNF (B) and absolute
numbers of CD8 T cells that produce IFN- (C) after
in vitro restimulation with LCMV peptides. (D) Frequency
of LCMV-specific (Db-GP276) and CD127-expressing
splenic CD8 T cells. (E) Graphs show B7.1 and B7.2 MFI
on CD11b+ splenic DCs after 8 d of T reg cell ablation
preceded or not by conventional CD4 T cell depletion.
The data are representative of 3 independent experiments with 35 mice per group. Error bars indicate
SEM. Non-parametric Mann Whitney test, where
*, P < 0.05; **, P < 0.01; ***, P < 0.001.
T cells (Fig. 5 B). After T reg cell depletion, CD8 T cells showed
extensive phenotypic changes associated with activation such
as down-regulation of CD62L and CD127 (Fig. 5 C). However,
despite massive activation of the bulk CD4 and CD8 T cell
subsets upon T reg cell ablation, the absolute numbers of LCMVspecific CD8 T cells remained unchanged in the spleen and
nonlymphoid organs in mice that had cleared LCMV (Fig. 5 D).
Analysis of T cell responses to multiple LCMV epitopes also
showed no alterations in the functionality of LCMV-specific
memory CD8 T cells after T reg cell depletion (Fig. 5 E). In
addition, the phenotype associated with long-lived memory
CD8 T cells was unaltered by depletion of T reg cells (Fig. 5 F).
Thus, T reg cells do not play a significant role in regulating
the homeostasis of memory T cells in the absence of cognate
1910
antigen. Our results show that bystander effects upon T reg

cell ablation do not impact memory T cells once the antigen
has been cleared and indicate that T reg cells mostly suppress
antigen-driven proliferation of T cells.
Rescue of exhausted CD8 T cells by T reg ablation is dependent
on B7 costimulation and conventional CD4 T cells
To further explore potential mechanisms of T reg cell
dependent enforcement of an exhausted state of T cells, we
looked at DC activation status. Under steady-state conditions,
T reg cells control expression of costimulatory molecules on
DCs (Kim et al., 2007; Wing et al., 2008; Schildknecht et al.,
2010; Qureshi et al., 2011). Consistent with previous reports,
we noticed increased expression of costimulatory molecules
Ar ticle
Figure 7. Inability to clear virus coincides with PD-L1 up-regulation. Foxp3DTR knock-in mice chronically infected with LCMV received DT for 10 d
(as in Fig. 2). (A) Viral load after 10 d of T reg cell depletion. (B) Dot plots show PD-1 expression on DbGP276-specific CD8 T cells. (C) PD-L1 expression on
splenic DCs after 7 d of T reg cell ablation. (D) Graph shows IFN- in serum before and at day 5 after initiation of DT treatment. Dashed line indicates the
limit of detection of this assay. (E) Representative dot plots show intracellular staining of LCMV nucleoprotein (NP) and PD-L1 expression on splenic DCs.
Staining of DCs in uninfected mice is shown as a control for specificity of NP staining and for basal PD-L1 expression. PD-L1 expression is shown as MFI
on infected (NP positive) or noninfected (NP negative) DCs. Data are a compilation of 3 experiments (A) or show representative results of 23 experiments
(BE) with 36 mice per group. Error bars indicate SEM. (A) Non-parametric Mann Whitney test, where NS = not significant. (D) Wilcoxon matched pairs
test, where *, P < 0.05. (E) Students t test, where **, P < 0.01; ***, P < 0.001.
B7-1 (CD80) and B7-2 (CD86) on CD11b+ and CD8+

DC subsets after T reg cell depletion (Fig. 6 A). To explore
whether increased costimulation was necessary to rescue
virus-specific CD8+ T cells, we treated T reg cell ablated
mice with CTLA-4 Ig, a fusion protein which binds B7-1
and B7-2 and thus blocks the B7/CD28 costimulatory pathway. In mice subjected to T reg cell depletion in combina
tion with CTLA4-Igmediated B7 blockade, the number of
functionally competent LCMV-specific CD8 T cells (Fig. 6,
B and C) and their phenotype (Fig. 6 D) remained similar to
that of untreated mice. Thus, B7 costimulation blockade prevented the increase in the response of virus-specific CD8
T cells that ensues upon T reg cell ablation in chronically infected mice. These results imply a critical role for costimulation in the rescue of exhausted T cell responses after T reg cell
depletion and suggest an important role of DC activation in
this process.
It was previously proposed that conventional CD4 T cells
mediate the DC activation that ensues after T reg cell depletion (Kim et al., 2007). To address the role of conventional
JEM Vol. 211, No. 9
CD4 T cells in our model, we depleted total CD4 cells in

concert with T reg cell depletion. We observed that CD4
T cell depletion reduced DC activation, especially of the
CD11b+ DCs (Fig. 6 E), a DC subset which efficiently presents antigens to CD4 T cells (Dudziak et al., 2007). Importantly, CD4 T cell depletion completely abrogated the rescue
of LCMV-specific CD8 T cells observed upon T reg cell ablation in chronically infected mice (Fig. 6, BD).These findings
suggest that in chronically infected mice,T reg cells modulate
DC activity and conventional CD4 T cells to maintain an
exhausted state of virus-specific CD8 T cells. In the chronic
viral infection model used in our studies, CD4 T cells were
transiently depleted before infection with LCMV cl-13 and
even after restoration of CD4 T cell numbers, LCMV-specific
CD4 T cells were not detected (unpublished data), as it was
expected due to the infection of the thymus and negative selection (Matloubian et al., 1994). Therefore, it is highly unlikely that cognate CD4 T cell help is involved in the rescue
of CD8 T cells upon T reg cell depletion, at least in this model
of chronic LCMV infection.
1911
Figure 8. Essential role for PD-L1 blockade in viral control after T reg cell depletion. Foxp3DTR knock-in mice chronically
infected with LCMV received DT for 10 d (as in
Fig. 2) in combination with PD-L1 blocking
antibody. PD-L1 antibody was administered
on days 1, 4, and 7 of DT treatment. (A) Viral
titers in serum before and after treatment.
(B) Fold reduction in serum viral titer after
treatment. (C) Absolute numbers of splenic
CD8 T cells that produce IFN- after in vitro
restimulation with LCMV peptides. (D) Absolute numbers of CD8 T cells co-expressing
IFN- and TNF after in vitro restimulation
with GP276 LCMV peptide. (E) Representative
dot plots showing frequency of CD8 T cells
producing cytokines as in D. Data are a compilation (A and B) or show representative
results of 3 independent experiments, with
46 mice per group. Error bars indicate SEM.
Non-parametric Mann Whitney test, where
*, P < 0.05; ***, P < 0.001; NS = not significant.
Failure to control virus by T reg cell ablation, despite

striking rescue of LCMV-specific CD8 T cell responses,
may be due to PD-L1 up-regulation on infected cells
We then tested whether the functional rescue of LCMVspecific CD8 T cells was associated with viral control. Unexpectedly, we did not observe a significant reduction in viral
load in chronically infected mice (Fig. 7 A) despite the impressive rescue of LCMV-specific CD8 T cell responses induced by T reg cell depletion. Antiviral T cells are known to
control virus by direct killing of infected cells and by production of cytokines (Kaech et al., 2002). Because T reg cell
depletion resulted in a substantial increase in the number of
LCMV-specific functional CD8 T cells (Fig. 3), we reasoned
that the lack of viral control might be due to an inhibitory
process that limits killing of infected cells in vivo.
Although LCMV-specific T cells were rescued by T reg
cell ablation and regained function, they still remained PD-1+
1912
(Fig. 7 B). PD-1 is triggered by TCR signaling; thus, as long

as virus persists, LCMV-specific CD8 T cells maintain PD-1
expression (Blattman et al., 2009). Furthermore, T reg cell
ablation also led to increased expression of PD-L1 in both
CD11b+ and CD8+ DCs (Fig. 7 C). To gain an insight into
potential reasons for PD-L1 up-regulation, we assessed inflammatory cytokine levels in the serum of LCMV chronically infected mice 5 d after T reg cell depletion. Most notably,
we found increased levels of serum IFN- (Fig. 7 D), which
has been described to modulate PD-L1 expression (Dong
et al., 2002). Interestingly, we found higher PD-L1 expression
on infected, LCMV nucleoprotein-positive DCs in comparison with their uninfected counterparts (Fig. 7 E). In addition
to DCs, LCMV cl-13 replicates extensively in nonhematopoietic cells, such as fibroblastic reticular cells, endothelial
cells, and diverse parenchymal cell types, and PD-L1 expression in these cells is known to restrict viral control (Mueller
Ar ticle
Figure 9. Transient T reg cell depletion

improves CD8 T cell rescue and viral control when combined with PD-L1 blockade
without causing overt disease in LCMV
chronically infected mice. (A) Foxp3DTR
knock-in mice chronically infected with LCMV
received continuous DT (on days 0, 1, 4, and 7)
and/or PD-L1 antibody (on days 1, 4, and 7),
as in Fig. 8. Mice weight after 11 d of treatment, relative to initial weight before treatment. (B) As in A, but mice received transient
DT treatment (on days 0, 1, and 4). Mice
weight after 12 d of treatment. (C) Representative dot plots show frequency of T reg cells
in PBMC before and after continuous or transient DT treatment. (D) Absolute numbers of
LCMV-specific (Db GP276) CD8 T cells within
the total splenic CD8 T cell population.
(E and F) Frequency of CD8 T cells producing
both IFN- and TNF (E) and absolute numbers
of CD8 T cells in the spleen that produce IFN-
after in vitro restimulation with LCMV peptide
GP276 (F). (G) Viral titers in serum before and
after treatment. (H) Fold reduction in serum
viral titer after treatment. A representative of
3 independent experiments is shown, with
6 mice per group. Error bars indicate SEM.
Non-parametric Mann Whitney test, where
*, P < 0.05; **, P < 0.01; ***, P < 0.001;
NS = not significant.
et al., 2007, 2010; Frebel et al., 2012). Thus, we reasoned that

PD-1 interactions with the increased levels of PD-L1 expressed by infected cells might oppose viral clearance by functionally competent CD8 T cells in T reg celldepleted mice.
Combining T reg cell depletion to blockade
of the PD-1 pathway results in viral control
To test the idea that PD-L1 expression could be protecting
LCMV-infected cells from cytotoxic T cells, we administered
PD-L1 blocking antibodies to LCMV chronically infected
Foxp3DTR knock-in mice in conjunction with T reg cell depletion. Combining blockade of the PD-1 pathway with
T reg cell ablation resulted in a significant reduction of viral
load (Fig. 8 A), which was more pronounced than that upon
blockade of the PD-1 pathway alone (Fig. 8 B). These results
demonstrated an essential role for the PD-1PD-L1 pathway
in limiting viral control after CD8 T cell rescue. Importantly,
the overall magnitude of multiple LCMV-specific CD8 T cell
responses observed after T reg cell depletion alone or in combination with the PD-1 pathway blockade was similar (Fig. 8,
CE), with the exception of an additive increase in the frequency of IFN- and TNF double-producing cells (Fig. 8 E).
Thus, these results demonstrated an essential role for the
PD-1PD-L1 pathway in limiting viral control after CD8 T cell
JEM Vol. 211, No. 9
rescue. Because T reg cell depletion resulted in a greater number of functional virus-specific T cells when compared with
PD-1 blockade alone (Fig. 8, CE), and the combined therapy further improved viral control (Fig. 8, A and B), our data
support the use of combination therapies based on modulation of T reg cell function and the PD-1 pathway to rescue
exhausted T cell responses. Moreover, our data indicate that
the magnitude of virus-specific CD8 T cell responses and decrease in viral load are not always directly correlated. These
results show that target cell elimination is affected both by intrinsic cytotoxic potential of antigen-specific CD8 T cells and
by inhibitory ligands such as PD-L1 displayed by target cells
that may limit cytotoxicity.
Transient T reg cell depletion improves antiviral T cell
responses when combined to blockade the PD-1 pathway
In adult healthy mice, chronic T reg cell ablation triggers
autoimmunity as early as 10 d after the start of DT administration, and all mice succumb to overt disease by 3 wk of sustained
absence of T reg cells (Kim et al., 2007). Not unexpectedly,
sustained T reg cell ablation in LCMV chronically infected
mice also induced immune-mediated wasting disease, which
was further exacerbated by blockade of the PD-1 pathway
(Fig. 9 A). To establish the therapeutic utility of our approach,
1913
we subjected chronically infected mice to a transient T reg cell

depletion regimen where mice received three DT doses instead
of four doses. This regimen of DT administration did not
cause an overt disease since these mice showed only minimal
weight loss (Fig. 9 B). Although continuous DT treatment resulted in nearly complete absence of T reg cells in Foxp3DTR
knock-in mice, upon transient depletion the T reg cell subset
had mostly recovered by the time of analysis (day 12; Fig. 9 C).
To ensure that wasting disease and mortality were associated
with the chronic T reg cell depletion rather than nonspecific
DT toxicity, we treated LCMV chronically infected B6 mice
with DT as a control and did not observe significant weight
loss or mortality.
Transient T reg cell ablation in LCMV chronically infected
mice did not significantly improve antiviral responses as a single therapy, but improved T cell rescue and viral control when
combined to blockade of the PD-1 pathway (Fig. 9, DH).
Most importantly, combination treatment was more effective
for viremia control than antiPD-L1 treatment alone (Fig. 9,
G and H). Additionally, even partial (fivefold lower DT dose)
and transient T reg cell depletion was able to improve LCMVspecific T cell responses obtained by PD-L1 blockade, and mice
remained healthy (unpublished data).Thus, careful optimization
of the dose and timing of immunotherapy based on a combination of T reg cell and PD-1 signaling pathway can maximize
the desired T cell responses while minimizing adverse events.
DISCUSSION
In this study, we show that T reg cells play a major role in
T cell exhaustion during chronic viral infection. T reg cell
ablation in LCMV chronically infected mice led to a striking
rescue of exhausted CD8 T cells. Virus-specific CD8 T cells
expanded 10100-fold after depletion of T reg cells, and rescued CD8 T cells were able to degranulate, produce IFN-,
TNF, and granzyme B, and also kill LCMV peptide-pulsed
cells. Notably, the anti-LCMV CD8 T cell response obtained
upon T reg cell ablation was of much higher magnitude than
the rescue obtained in LCMV chronically infected mice by
PD-1PD-L1 blockade (Barber et al., 2006) or even combination of PD-1 pathway blockade with IL-2 therapy (West
et al., 2013). In spite of this, the antiviral CD8 T cell responses
that ensued after T reg cell depletion were ineffective to reduce viral burden. When we tried to understand this unexpected lack of viral control, we detected up-regulation of the
inhibitory molecule PD-L1 after T reg cell ablation, especially
in infected cells. Blockade of the PD-1 pathway in combination with T reg cell depletion resulted in significant control of
viral load, despite minimal improvement of CD8 T cell rescue
when compared with T reg cell ablation alone. We propose
that the increased PD-L1 on target cells binds PD-1 on the
rescued exhausted CD8 T cells and inhibits cytotoxic activity.
These results support the hypothesis that T reg cells modulate
T cell exhaustion, but the PD-1PD-L1 pathway operates to
prevent destruction of target cells. Our data show that rescue
of effective T cell responses can be improved by combining
blockade of the PD-1 pathway to T reg cell depletion.
1914
In this study, we uncovered a major role of T reg cells in

T cell exhaustion. Interestingly,T reg cells from LCMV chronically infected mice were phenotypically different from mice
that had cleared LCMV infection or naive mice. During chronic
infection, T reg cells displayed an activated phenotype, as pre
viously reported (Punkosdy et al., 2011), with lower CD62L
and higher CD44 and CD69 expression, as well as an increase
in the inhibitory molecule PD-1. T reg cells from persistently
infected animals also had increased expression of CD103,
an important molecule for lymphocyte retention in skin and
other epithelial rich sites (Suffia et al., 2005). Furthermore, we
observed increased expression of molecules that have been
associated with T reg suppressive activity, such as ICOS (Chen
et al., 2012), granzyme B (Gondek et al., 2005; Cao et al.,
2007), and CD39, an ectoenzyme which catabolizes proinflammatory extracellular ATP (Borsellino et al., 2007).
Most notably,T reg cells in chronically infected mice showed
a twofold increase in the levels of CTLA-4, an inhibitory
protein implicated in T reg cellmediated suppressive function (Wing et al., 2008). It has been shown that CTLA-4 interacts with and internalizes B7 molecules by trans-endocytosis
from the surface of the interacting cells, which results in APCs
with lower stimulatory capacity for effector T cells (Qureshi
et al., 2011). In our model,T reg cell depletion leads to increased
B7 expression on DCs and we demonstrate that B7 stimulation is essential for rescue of exhausted LCMV-specific CD8
T cells. T reg cells may affect DC activation status directly
(possibly through CTLA-4 interactions with B7 molecules)
and also indirectly (possibly through conventional CD4
T cells). It is important to point out that during chronic
LCMV infection CTLA-4 blockade does not rescue exhausted CD8 T cells (Barber et al., 2006), suggesting that
CTLA-4 does not constitute a major nonredundant suppressive mechanism in this model.
We found that T reg cell control of CD8 T cell exhaustion
operates by restraining activation of DCs and conventional
CD4 T cells. After depletion of T reg cells we observed generalized activation of CD4 T cells but no LCMV-specific CD4
T cells that could provide cognate help to virus-specific CD8
T cells. Most likely, when autoreactive CD4 T cells are released
from T reg cell inhibition, they become activated and then
trigger activation of DCs presenting autoantigens, as it has been
previously suggested (Kim et al., 2007). Consistent with the
concept that CD4 T cells activate DCs, we found that depletion of conventional CD4 T cells abrogated activation of splenic
CD11b+ DCs triggered by T reg cell ablation in LCMV chronically infected mice. Nonetheless, T reg cells might also affect
DC activation status by other mechanisms as we observed that
depletion of conventional CD4 T cells could not completely
abrogate activation of splenic CD8+ DCs after T reg cell ablation. There might be important differences between DC subsets with regard to interaction with T reg cells and how distinct
DC subsets respond to the lack of T reg cells.
Even though our results suggest that DC activation plays
an important role in the rescue of exhausted CD8 T cells, it is
important to take into account that the presence of conventional
Ar ticle
CD4 T cells was required for the rescue of exhausted CD8

T cells after T reg cell depletion. CD4 T cells can directly provide
factors for the rescue of exhausted CD8 T cells, such as IL-2
(Blattman et al., 2003; West et al., 2013) and IL-21 (Elsaesser
et al., 2009; Frhlich et al., 2009;Yi et al., 2009). Activation of
CD4 T cells and DCs constitute a very intricate relationship
and interfering with one will affect the other, thus, in our
study we could not determine exactly the factors necessary
for the rescue of exhausted CD8 T cells. Our data highlight
that manipulation of costimulatory pathways and identification of activating signals from CD4+ T cells would provide
pivotal knowledge for treating chronic infections and cancer.
During chronic LCMV infection, antigen is abundant, yet
virus-specific CD8 T cells are suppressed and maintained by
minimal proliferation. Our data indicate that in chronic viral
infection T reg cell ablation promotes T cell activation but does
not elicit de novo T cell responses. Consistent with this notion,
T reg cell depletion did not generate LCMV-specific CD8
T cell responses in carrier mice infected with LCMV at birth
in which there are no LCMV-specific T cells in the periphery
due to negative selection in the thymus (Pircher et al., 1989;
King et al., 1992). After 11 d of sustained T reg cell depletion
in LCMV carrier Foxp3DTR knock-in mice (>10,000 PFU/ml
in serum), despite prominent T cell activation (85.73%, SD =
2.55 CD44hi CD8 T cells in DT treated vs. 34.4%, SD =
2.81 in untreated mice), we were unable to detect any
LCMV-specific CD8 T cells (DbGP33+ or DbGP276+). This
important observation implies that antigen-specific T cells must
be present to elicit T cell responses by T reg cell manipulation.
In addition, our data suggest that the T cell activation that
occurs upon T reg cell ablation is antigen-driven. In contrast
to the impressive rescue of virus-specific CD8 T cells in LCMV
chronically infected mice, T reg cell depletion in mice that
had cleared the infection did not affect the numbers and the
activation state of virus-specific CD8 T cells.This observation
reveals that T reg cells do not control maintenance of memory cells and suggests that T reg cell manipulation would not
affect homeostasis of memory cells generated by vaccination
or acute infection.
In this study, we compared the effect of T reg cell depletion and blockade of the PD-1 pathway on the antiviral response in chronically infected mice. T reg cell depletion in
LCMV chronically infected mice resulted in an unprecedented
rescue of antiviral CD8 T cells. Distinctly, after T reg cell ablation, LCMV-specific CD8 T cells re-expressed CD127, which
may be indicative of increased IL-2 signals (West et al.,
2013). T cells integrate signals received from APCs and the
environment to determine their differentiation program. Besides increased systemic IFN-, we also observed an increase
in TNF (10140 pg/ml), MCP-1 (45360 pg/ml), and IL-6
(655 pg/ml), as it has been previously reported after T reg
cell depletion in naive mice (Chinen et al., 2010). Interestingly, we could not detect any significant amount of inflammatory cytokines and chemokines (IFN-,TNF, MCP-1, and
IL-6) in the serum of LCMV chronically infected mice
treated with antiPD-L1 blocking antibodies for 5 or 8 d.
JEM Vol. 211, No. 9
Thus, the presence of inflammatory cytokines in the serum is

most likely the result of autoreactivity unleashed by T reg cell
depletion and not a measure of antiviral T cell responses.Thus,
it is likely that LCMV-specific CD8 T cells rescued by T reg
cell ablation might be quite different from CD8 T cells rescued
by blockade of the PD-1 pathway, and better understanding of
these differences might help shed light into potential molecular mechanisms involved in rescue of T cell exhaustion.
Importantly, our study shows that even a 10100-fold increase in the number of functional viral-specific CD8+ T cells
does not necessarily correlate with a decline in viral load. We
show that the PD-1 pathway plays a fundamental role in viral
clearance and we propose that PD-L1 expression in virusinfected cells is a potent inhibitor of target cell elimination.We
observed an increase in B7 and PD-L1 expression on DCs
after T reg cell depletion.We propose that LCMV-specific CD8
T cells are rescued because there is a net positive balance of
signals from APCs presenting viral antigens to exhausted CD8
T cells. However, the bulk viral burden in chronically infected
mice is largely due to LCMV infection of nonhematopoietic
cells that express PD-L1 but no B7 molecules (Mueller et al.,
2007, 2010). Thus, even though LCMV-specific CD8 T cells
regain function after T reg cell depletion, there is no improvement in viral control because rescued CD8 T cells maintain
PD-1 expression and their cytotoxic activity is inhibited by
PD-L1expressing infected cells (Mueller et al., 2010; Frebel
et al., 2012). Notably, PD-L1 also protects tumor cells from
CD8 T cellmediated killing (Iwai et al., 2002). When T reg
cell depletion was combined with PD-L1 blocking antibodies, rescue of exhausted T cells was only marginally improved
as reflected by higher frequency of LCMV-specific CD8
T cells coproducing IFN- and TNF. However, T reg cell depletion in combination with blockade of the PD-1 pathway
resulted in marked improvement of viral control. In summary,
our data show that the magnitude of T cell responses and
effective control of infected cells (and possibly tumors) are not
always coupled together.
PD-L1 is ubiquitously expressed and its expression is further increased by several cytokines, including IFN- (Dong
et al., 2002; Keir et al., 2008).We showed that T reg cell depletion in LCMV chronically infected mice causes a systemic
increase in IFN- as early as day 5 after treatment, which may
modulate PD-L1 expression. In addition, functional rescue of
CD8 T cells might directly induce PD-L1 up-regulation, especially on infected cells presenting LCMV antigens due to
cognate interactions with T cells and local IFN- production.
It is important to point out that LCMV cl-13 and other viruses
that cause chronic infections are relatively resistant to IFNs
(Moskophidis et al., 1994) and IFNs may even have a detrimental effect on the course of the infection (Teijaro et al.,
2013; Wilson et al., 2013). Thus, therapies that promote cytotoxic T cell responses still need to ensure that further inhibitory mechanisms will not hamper destruction of target cells.
Reversal of T cell exhaustion is the ultimate goal of immunotherapies aiming to treat chronically infected patients as
well as cancer patients.There have been many recent advances
1915
in this field, and although single therapies such as blockade

of the PD-1 pathway and antiCTLA-4 antibody treatment
have achieved success in clinical trials with advanced cancer
patients, most studies suggest that a combination of therapies
may achieve better results. Rational combination of therapies
that operate through different mechanisms will probably improve synergy to achieve better response rates as well as longevity of the immune response. Our study uncovers a potential
mechanism for synergistic effects of T reg cell depletion and
blockade of the PD-1 pathway.
Recent evidence suggests that antiCTLA-4 may function
by depletion of T reg cells at the tumor site (Bulliard et al.,
2013; Selby et al., 2013; Simpson et al., 2013). Importantly, it
was recently shown that combining antiCTLA-4 to PD-1
blockade resulted in >40% objective responses in advanced
melanoma patients (Wolchok et al., 2013). In this study, among
responders, most patients had >80% tumor regression, which
exceeds the rate of responses reported previously in monotherapy trials. Thus, our results provide important concepts that
may have direct relevance to cancer treatment. Nevertheless, additional studies need to be done to determine which chronic
infections and cancers would be responsive to this combinatorial immunotherapy involving T reg cell depletion and
blockade of the PD-1 pathway because there may be heterogeneity in the response to such therapies (Bos et al., 2013).
In LCMV chronically infected mice, virus is widespread and
there is a delicate balance between an effective immune response
that controls viral load and overt immunopathology. Using
Foxp3DTR knock-in mice, we were able to achieve very efficient
depletion of T reg cells.Although the complete absence of T reg
cells may increase T cell rescue, serious adverse effects may limit
its application in a clinical setting. We show that transient T reg
cell depletion, as a single therapy, failed to rescue antiviral responses,
but when combined to PD-1 pathway blockade, transient T reg
cell depletion improved both CD8 T cell responses and viral control.Thus, milder T reg cell depletion regimens, which could be
achieved in patients with antibody-based depletion strategies,
might improve immune responses induced by other therapies
without overt adverse effects. Alternative approaches that would
preferentially affect a subset of T reg cells, such as CCR4+
T reg cells which are predominant at the tumor site (Sugiyama
et al., 2013) or Nrp1+ T reg cells which are recruited to VEGFproducing tumors (Hansen et al., 2012), have also been proposed as potential strategies that would evoke immune responses
while minimizing autoimmunity.
In summary, our studies demonstrate that T reg cells control
chronically stimulated exhausted CD8 T cells during persistent
viral infection.We show that this T reg cellmediated control of
exhausted CD8 T cells involves restraint of B7 costimulation
and of help from conventional CD4 T cells. The findings presented here also underscore a critical role of PD-L1 in determining viral control after T cell rescue from an exhausted state.
Mice and infections. 48-wk-old homozygous Foxp3DTR knock-in mice
on a C57BL/6 background (Kim et al., 2007) or C57BL/6 (The Jackson
Laboratory) were infected with LCMV Arm or LCMV cl-13. Memory T cell
1916
responses were generated by i.p. injection with 2 105 PFU of LCMV Arm,
which results in an acute infection that is cleared within 8 d. Chronic infections with exhausted T cell responses and lifelong viremia were induced by
antibody-mediated transient depletion of CD4 T cells with 2 doses of 500 g
GK1.5 (Bio X Cell) i.p., followed by i.v. injection with 2 106 PFU LCMV
cl-13 as described previously (Matloubian et al., 1994). To analyze memory
T cell responses, we waited at least 100 d after LCMV Arm infection, and for
exhausted T cell responses, we waited at least 45 d after LCMV cl-13 infection. Titration of virus was performed on Vero cell monolayers as described
previously (Ahmed et al., 1984). All mouse experiments were performed according to institutional guidelines and were approved by the Emory University Institutional Animal Care and Use Committee.
Antibody treatments and T reg cell depletion. 500 g CTLA-4 Ig (gift
from the Ford and Larsen laboratory, Emory University, Atlanta, GA) was
injected i.p., on days 2, 0, 2, 4, 6, 8, and 10 of T reg cell ablation. CD4+
T cell depletion was achieved by i.p. injection of 500 g GK1.5 on days 2
and 1 of T reg cell ablation. 200 g PD-L1 blocking antibody (10F.9G2)
was administered i.p. on days 1, 4, and 7 after T reg cell ablation. Each
mouse received 1 g DT (Sigma-Aldrich) i.p. (50 g/kg).
Cytotoxicity activity by 51 chromium-release assay. MC57 mouse fibroblast cell targets were coated with a mix of GP33, GP276, and NP396
peptides or no peptide, and labeled with 350 Ci 51-Cr. Target cells were
incubated for 6 h with different amounts of effector splenic CD8+ T cells
enriched using mouse CD8 beads (Miltenyi Biotec). Absolute numbers of
viral-specific CD8+ T cells was retroactively calculated and plotted. E:T = effector to target ratio. MC57 cells with 1% Triton X-100 were used as positive
control (total release), and MC57 cells without effectors were used to calculate spontaneous release.
Antibodies and flow cytometry. Single cell suspensions were obtained
from blood, spleen, lungs, liver, kidney, and gut as previously described
(Masopust et al., 2001). For analysis of dendritic cells, spleens were digested
with 0.4 U/ml collagenase D (Roche) for 30 min at 37C. Single cell suspensions were stained with anti-CD8 (536.7), -CD4 (RM4-5), -CD25
(PC61), CD40 (3/23), CD80 (16-10A1), CD86 (GL1), -CD107a (1D4B),
-CD107b (ABL-93),V 5.1 5.2 (MR9-4), and Ki67 (B56; from BD); -ICOS
(7E.17G9), PD-L1 (MIH5), -CD11c (N418), -CD11b (M1/70), -CD127
(A7R34), -MHCI (AF6-88.5.5.3), and -Foxp3 (FJK-16s; from eBioscience);
-CD44 (IM7) and PD-1 (RMP1-30; from BioLegend); and granzyme B
(MHGB04; Invitrogen). Anti-LCMV NP antibody was a gift from the
Buchmeier Laboratory (University of California, Irvine, Irvine, CA). Dead
cells were excluded by gating out cells positive for Live/Dead fixable dead cell
stain (Invitrogen). LCMV MHC class I tetramers were prepared and used as
previously described (Wherry et al., 2003). The LCMV MHC class II tetramer (I-Ab GP61-80) was obtained from the National Institutes of Health
tetramer facility and stains were performed at 37C for 3 h, gently mixing
cells every 30 min. LCMV-specific responses were assessed by restimulating
splenocytes with 0.1 g/ml GP33, NP396, GP276, NP118, or NP235 LCMV
peptides in the presence of brefeldin and monensin for 5 h at 37C. Intracellular staining for IFN-, TNF, Ki67, and granzyme B were performed with
the Cytofix/Cytoperm kit (BD). Intracellular staining of Foxp3 was performed according to manufacturers instructions (eBioscience). Serum cytokines were measured by cytometric bead array according to manufacturers
instructions (BD). Samples were acquired with a FACSCanto or LSRII (BD)
and analyzed using FlowJo (Tree Star).
Statistical analysis. Statistical analysis was performed on Prism software
(GraphPad Software).
We thank Dr. Rouse, Dr. Pulendran, and members of the Ahmed laboratory for
helpful discussion.
Ar ticle
This work was supported by National Institutes of Health grants R01 AI3004
(R. Ahmed), P01 AI080192 (R. Ahmed and G.J. Freeman), P01 AI056299 (R. Ahmed,
G.J. Freeman, and A.H. Sharpe), and R37 AI034206 (A.Y. Rudensky), by the Ludwig
Cancer Research (A.Y. Rudensky), and by the Cancer Research Institutes Irvington
Institute Fellowship Program (A.O. Kamphorst). A.Y. Rudensky is an investigator with
the Howard Hughes Medical Institute.
R. Ahmed, A.H. Sharpe, and G.J. Freeman hold patents and receive patent royal
ties related to the PD-1 inhibitory pathway. A.H. Sharpe and G.J. Freeman are scien
tific founders of Costim Pharmaceuticals. R. Ahmed, A.H. Sharpe, and G.J. Freeman
declare no additional financial interests. The remaining authors declare no
competing financial interests.
Submitted: 13 December 2013
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THE

Selected Articles January 2015

Long-term survival of influenza virus infected club cells drives immunopathology

phone (212) 327-8575

Seasonal influenza represents a contagious family of respiratory viruses that infect

INSIGHTS | The Journal of Experimental Medicine

Adapted from an illustration by the authors

FLUshing in the bathroom

Respiratory influenza virus infection induces

*J. Wang and F. Li contributed equally to this paper.

2001; Chervonsky, 2009). Distinct components

Figure 1. Respiratory influenza virus

immune cells and structures contained in mucosal tissues were

Figure 2. Influenza virus does not infect the

(Fig. 2, B and C). However, pathological injury was not found

into healthy WT mice increased the number of Enterobacteriaceae

Microbiota cause intestine injury during influenza | Wang et al.

Figure 4. IL-17A deficiency reduces

et al., 2011). Furthermore, treating mice i.p. with a neutralizing

Figure 5. Increased Th17 cells occur in the

Microbiota cause intestine injury during influenza | Wang et al.

Figure 8. Intestinal microbiota induces

each of these components individually, the mucosal immune

An explosive increase in neutrophils is responsible for

et al., 2010). These findings suggest that potential exists for an

Kleinschek, M.A., K. Boniface, S. Sadekova, J. Grein, E.E. Murphy, S.P.Turner,

fine-tuning IL-17A production in CD4 T lymphocytes. J. Immunol.

Stenstad, H., A. Ericsson, B. Johansson-Lindbom, M. Svensson, J. Marsal, M.

Microbiota cause intestine injury during influenza | Wang et al.

JEM Vol. 211, No. 9

Brief Definitive Report

Long-term survival of influenza virus

Respiratory infection of influenza A virus (IAV) is frequently characterized by extensive

Influenza A virus (IAV) is a seasonal pathogen

N.S. Heaton and R.A. Langlois contributed equally to

tissue damage is important for recovery from

RESULTS AND DISCUSSION

Surviving flu infected club cells drive pathology | Heaton et al.

Br ief Definitive Repor t

assessed the presence of reporter-positive cells using flow

Figure 2. IAV-Cre demonstrates long-term cell

Unterholzner, 2008). Strikingly, we observed an increase in

Br ief Definitive Repor t

To understand the mechanism of how surviving cells were

Figure 4. Surviving cells contribute to IAV-induced immuno

pathologist scoring of virus induced bronchiolar epithelial cell necrosis

Br ief Definitive Repor t

N.S. Heaton is a Merck fellow of the Life Sciences Research Foundation.

Julkunen, I., T. Sareneva, J. Pirhonen, T. Ronni, K. Meln, and S. Matikainen.

Trans-nodal migration of resident dendritic

Program in Immunology, 2Department of Pediatrics, and 3Department of Microbiology and Immunobiology,

The Rockefeller University Press $30.00

present antigen to cognate T and B cells and

quantified through a shift in the ratio of medulla-occupying

study by Hickman et al. (2008) suggested a primary role for

populations had accumulated in mIFRs within 60 min of

LNDCs present viral antigen to CD4+ T cells near IFRs

Groom et al., 2012) were vaccinated with UV-PR8, and LNs

Figure 4. CXCR3-dependent clustering of viral-specific CD4+

LNDC-dependent T cell activation

were collected at 24 h. Quantitation of clustering efficiency of WT or

Using this approach, C57BL/6 mice were vaccinated with

in the vG group, providing additional evidence for LNDC/

Figure 6. mDC-independent protection from influenza. (a) Confocal

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